Magda Maria Sales Carneiro-Sampaio

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 (H1V)‐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 IgC 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.

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

Abstract 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 ∼ 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.

Early acquisition of serum and saliva antibodies reactive to enteropathogenic Escherichia coli virulence-associated proteins by infants living in an endemic area

Enteropathogenic Escherichia coli (EPEC) is the most common etiological agent of acute diarrhea among infants living in poor social conditions in Brazil and other developing countries. This infection is rare in breast‐fed infants, as well as in children older than 2 years. Over the past few years, our group has attempted to identify antibodies to EPEC virulence proteins in human milk and to establish the in vitro protective role of these antibodies. In the present study, we report the identification of antibodies to EPEC virulence proteins in sera and saliva from children of different ages, living in slums in the city of São Paulo, Brazil. Using EPEC and bacterial constructs (pET) for immunoblotting (IB) analysis, antibodies reacting to the main adhesins (intimin, bundle‐forming pilli) and cell‐signaling proteins (EPEC secreted proteins – Esp A, Esp B) were detected in sera from adults and children older than 1 year. Almost all children older than 1 year presented recognition patterns similar to those of adults in IB assays for serum IgG and secretory IgA antibodies, using EPEC outer membrane and other antigenic preparations. As previously observed for human milk, all samples from adults and older children recognized the 94 kDa molecular weight adhesin intimin strongly. In most children, previous EPEC symptomatic diarrhea could not be confirmed; however, almost all of them have presented one or more diarrhea episodes during their lifetime. These results suggest that reduction of EPEC infection frequency after 2 years of age may be associated with the development of anti‐EPEC antibody repertoires.

Elevated levels and different repertoire profile of colostral anti-LPS antibodies may have a significant role in compensating newborn immunity.

A high prevalence of systemic infections caused by enterobacteria such as Escherichia coli is observed during the neonatal period. Lipopolysaccharide (LPS) is one of the major factors responsible for septic shock caused by these Gram‐negative bacteria. We have recently demonstrated the presence of anti‐LPS immunoglobulin (Ig)G antibodies in cord blood with a repertoire identical to that found in maternal serum. In the present study, we analyzed anti‐LPS O111 antibody isotypes in maternal serum and colostrum from mothers and in cord serum from their respective full‐term (n = 30) and preterm (n = 13) neonate infants. The main isotype found in serum samples from mothers of term infants was IgM (range between 28 and 54 mg/l), followed by IgA (1–2 mg/l) and IgG (2–3 mg/l). The range of IgG antibody concentrations in cord blood was between 2 and 3 mg/l, as a result of placental transfer. A novel observation in our study was that the LPS bands recognized by colostral antibodies were completely different from those recognized by IgG in serum. Colostral IgA antibodies recognized several bands not bound by serum IgG antibodies from the respective maternal serum, independently of the antibody quantity. In addition, we verified the pattern of LPS recognition by serum IgA and colostral IgA antibodies was identical, what suggested that the antibody isotype found in serum could probably be derived from differentiated IgA‐positive cells which were homing to the mucosa through the mucosal homing mechanism. Identical pattern of recognition was obtained comparing the IgA and IgM isotypes in colostrum. Slight differences in the pattern of recognition were found between colostral and serum IgM antibodies. The fact that colostral antibodies recognize much more bands than serum antibodies may be important for the host to mount an effective immune response in the intestinal lumen, in order to prevent excessive absorption of LPS, reducing possible systemic effects caused by the molecule.

Passive immunity acquisition of maternal anti-enterohemorrhagic Escherichia coli (EHEC) O157:H7 IgG antibodies by the newborn

Enterohaemorrhagic Escherichia coli (EHEC) strains are among the main causes of haemorrhagic colitis (HC) and haemolytic-uremic syndrome (HUS) in industrialised countries. In Brazil, EHEC have been detected in the faeces of patients with non-bloody diarrhoea, though an association between EHEC and HUS has been detected recently. These observations suggest that there is a pre-existing immunity triggered by the contact with EHEC and other categories of bacteria, such as EPEC, that share similar virulence factors and to which our population is highly exposed. Our aim was to evaluate the placental transfer of IgG antibodies reactive to EHEC O157:H7 antigens. We evaluated 28 paired maternal and cord sera for the presence of IgG against EHEC O157:H7 protein antigens and IgG and IgM to O157 LPS employing ELISA and IB technique. Total IgG and IgM level analyses were also made. Anti-EHEC O157:H7 and anti-LPS O157 IgG antibody levels in cord sera were equivalent to those of their maternal sera. A good correlation between the mothers’ anti-LPS O157 IgM and total IgM levels was found. Anti-LPS O157 IgM levels were higher than anti-LPS O157 IgG levels in the same samples, and anti-LPS IgM antibodies were not detected in cord sera. Identical patterns of recognition of bacterial protein antigens by specific IgG were found in the paired samples and the recombinant purified variable region of γ intimin was specifically recognized by one paired maternal and cord sample. In conclusion, although the antibody profile varied among individuals, all paired cord and maternal serum samples showed an identical recognition pattern, indicating an efficient placental transfer of IgG antibodies reactive to EHEC O157:H7 antigens.

Pneumonias de repetição em paciente com deficiência de anticorpos e imunoglobulinas normais

apos imunizacao para todos ossorotipos (1, 3, 5, 6, 9 e 14) testados, embora a paciente apresentasse niveis normais deimunoglobulinas. A avaliacao radiologica, no momento da admissao, demonstrou presenca deatelectasias difusas associadas a bronquiectasias. Apos inicio do tratamento com imunoglobulinaendovenosa e fisioterapia respiratoria houve esvaecimento gradual ate reversao das alteracoesradiologicas. Demonstrou-se, assim, a importância de um diagnostico preciso para inicio detratamento especifico, com melhora gradual do quadro clinico e radiologico, evitando sequelaspulmonares irreversiveis.

A Remarkable Depletion of Both Naïve CD4+ and CD8+ with High Proportion of Memory T Cells in an IPEX Infant with a FOXP3 Mutation in the Forkhead Domain

IPEX is a rare X‐linked syndrome, with immune dysfunction, polyendocrinopathy and enteropathy. We describe an infant who died at the age of 11 months after developing eczema, severe diarrhoea, diabetes, hypothyroidism, thrombocytopenia and four episodes of septicaemia. Immunophenotyping of peripheral blood at 8 months revealed normal CD3+ T, CD4+ T and CD8+ T cell numbers, with low NK and B cells. CD4+ and CD8+ T lymphocytes showed remarkably low numbers and percentages of naïve cells and high numbers of memory CD4 and CD8 cells. At autopsy, an intense depletion of immune cells in thymus, spleen and lymph nodes was observed. No Hassall’s corpuscles were found in thymus. Lymphocytic pancreatitis and intense villous atrophy with mucosal lymphocytic infiltration in small bowel were also seen. FOXP3 gene studies revealed a: C→G substitution 3 bp upstream of exon 10, which prevents splicing between exons 9 and 10, likely resulting in a functionally altered or deficient protein. Florid clinical findings are usually observed in association of forkhead DNA‐binding domain mutations. The intense depletion of naïve T cells we report suggest that depletion of immune cells might take place due to uncontrolled activation due to the absence of regulatory T cells.

Phenotypic Differences in Leucocyte Populations among Healthy Preterm and Full‐Term Newborns

The immune system of neonates has been considered functionally immature, and due to their high susceptibility to infections, the aim of this study was to analyse the phenotypic differences in leucocyte populations in healthy preterm and full‐term newborns. We evaluated the absolute numbers and frequencies of dendritic cells (DCs) and DC subsets, monocytes and T and B lymphocytes and subsets in the cord blood of healthy moderate and very preterm (Group 1), late preterm (Group 2) and full‐term (Group 3) newborns and in healthy adults, as controls, by flow cytometry. The analyses revealed statistically higher absolute cell numbers in neonates compared with adults due to the characteristic leucocytosis of neonates. We observed a lower frequency of CD80+ myeloid and plasmacytoid DCs in Group 1 and reduced expression of TLR‐4 on myeloid DCs in all neonates compared with adults. TLR‐2+ monocytes were reduced in Group 1 compared with Groups 2 and 3, and TLR‐4+ monocytes were reduced in Groups 1 and 2 compared with Group 3. The frequencies and numbers of naïve CD4+ T and CD19+ B cells were higher in the three groups of neonates compared with adults, while CD4+ effector and effector memory T cells and CD19+ memory B cells were elevated in adults compared with neonates, as expected. Our study provides reference values for leucocytes in cord blood from term and preterm newborns, which may facilitate the identification of immunological deficiencies in protection against extracellular pathogens.

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.

Human IgG but not IgM Antibodies can Protect Mice from the Challenge with Live O6 Escherichia coli

We evaluated the ability of human anti‐lipopolysaccharide (LPS) O6 immunoglobulin G (IgG) and IgM antibodies to protect mice challenged with Escherichia coli serotype O6:K2ac. Purified whole IgG, commercial gammaglobulin, whole IgM‐effluent, pool of normal human serum (NHS), agammaglobulinaemic serum (test groups) or phosphate‐buffered saline (control group) was injected into adult male 18 h before a challenge with viable O6 E. coli. The mortality rate was assessed over a period of 72 h. To determine the opsonic and phagocytic activity of the antibody isotypes, we incubated peritoneal macrophages from the control and test groups collected at different times after challenge with the live bacteria with acridine orange for fluorescent analysis. Tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 were quantified in serum of both the test and control groups. All mice that received commercial gammaglobulin or NHS survived. Purified whole IgG (containing 1.1 mg/l of anti‐LPS O6 IgG antibodies) protected 87.5% of the animals tested in this experiment, while whole IgM‐enriched effluent with 1.5 mg/l of anti‐LPS O6 IgM antibodies protected only 12.5%. The agamma serum showed no protective capacity compared with PBS (serving as control). The minimal concentration of anti‐LPS O6 IgG antibodies able to protect 50% of animals was 0.137 mg/l of purified whole IgG. Whole IgM‐enriched effluent showed no protective capacity independently of the concentration tested (0.048–17.0 mg/l of anti‐LPS O6 IgM antibodies). Fluorescent analysis of peritoneal macrophages from animals pretreated with purified whole IgG showed no bacteria at 8 h after the challenge. By contrast, whole IgM effluent showed an increasing number of live bacteria at the same time. Mice that had received whole IgM effluent (1.5 mg/l of anti‐LPS O6 IgM antibodies) before the challenge with LPS O6 presented 20.5 µg/l of IL‐6 and 1.5 µg/l of TNF‐α. Serum from animals pretreated with purified IgG did not present any detectable pro‐inflammatory cytokine. Our findings suggest that IgG but not IgM antibodies protect animals from a challenge with E. coli O6 serotype.