Placental transfer of IgG antibodies specific to Klebsiella and Pseudomonas LPS and to group B Streptococcus in twin pregnancies.

Group B Streptococcus (GBS), Klebsiella spp. and Pseudomonas spp. are important aetiological agents of neonatal infections in Brazil. There is a lack of data in the literature regarding the specific transport of immunoglobulin G (IgG) against these pathogens in multiple pregnancies. Maternal (n = 55) and umbilical cord (n = 110) blood samples were prospectively collected at birth from 55 twin pregnancies. The factors associated with cord levels and transfer ratios of IgG against GBS, Klebsiella and Pseudomonas were examined. The IgG umbilical cord serum levels specific to GBS, Klebsiella LPS and Pseudomonas LPS were significantly associated with maternal-specific IgG concentrations and the presence of diabetes. The anti-Klebsiella IgG cord serum concentrations were also related to birthweight and the presence of hypertension. The transfer ratios against GBS and Pseudomonas LPS were associated with maternal-specific IgG concentrations. The transfer ratios for GBS and Pseudomonas LPS were associated with gestational age at delivery and the presence of diabetes, respectively. None of the examined parameters were related to Klebsiella LPS transfer ratios. We conclude that in twin pregnancies, specific maternal IgG serum concentrations and diabetes were the parameters associated with umbilical cord serum IgG concentrations reactive with the three pathogens investigated. All the other parameters investigated showed different associations with neonatal-specific IgG levels according to the antigen studied. There was no uniformity of the investigated parameters regarding association with placental IgG transfer ratios against the GBS, Pseudomonas LPS and Klebsiella LPS.

Antibody response to pneumococcal capsular polysaccharide vaccine in Down syndrome patients.

The majority of children with Down syndrome (DS) tend to have frequent bacterial infections including recurrent respiratory infections. Our objective was to evaluate the production of antibodies to pneumococcal polysaccharide antigens after active immunization in DS subjects. IgG antibodies to pneumococcal serotypes (1, 3, 6B, 9V, and 14) were measured before and 6 weeks after immunization with a 23-valent pneumococcal vaccine (Pneumo23, Pasteur-Merrieux) in 6- to 13-year-old DS children (N = 17) and in aged-matched normal controls (N = 30). An adequate response was defined as a 4-fold increase over baseline or a post-immunization level of specific pneumococcal serotype antibody > or = 1.3 microg/mL. After immunization, all DS children had an increase in post-immunization levels against all serotypes analyzed. A 4-fold or more increase was observed in all DS children concerning serotypes 1 and 14, in 90% of subjects for serotypes 3 and 9V, and in 65% for serotype 6B. Regarding this increase, 8 of the 17 DS children had an adequate response to all serotypes analyzed, 8/17 patients to 4 serotypes and 1/17 to 3 serotypes. However, when we compared post-immunization levels between DS children and controls, we observed lower levels in the former group (P < 0.05) for all serotypes except serotype 3. We conclude that pneumococcal polysaccharide immunization could be beneficial for these DS children.

Antibody response to pneumococcal capsular polysaccharide vaccine in Down syndrome patients

The majority of children with Down syndrome (DS) tend to have frequent bacterial infections including recurrent respiratory infections. Our objective was to evaluate the production of antibodies to pneumococcal polysaccharide antigens after active immunization in DS subjects. IgG antibodies to pneumococcal serotypes (1, 3, 6B, 9V, and 14) were measured before and 6 weeks after immunization with a 23-valent pneumococcal vaccine (Pneumo23®, Pasteur-Merrieux) in 6- to 13-year-old DS children (N = 17) and in aged-matched normal controls (N = 30). An adequate response was defined as a 4-fold increase over baseline or a post-immunization level of specific pneumococcal serotype antibody > or = 1.3 æg/mL. After immunization, all DS children had an increase in post-immunization levels against all serotypes analyzed. A 4-fold or more increase was observed in all DS children concerning serotypes 1 and 14, in 90 percent of subjects for serotypes 3 and 9V, and in 65 percent for serotype 6B. Regarding this increase, 8 of the 17 DS children had an adequate response to all serotypes analyzed, 8/17 patients to 4 serotypes and 1/17 to 3 serotypes. However, when we compared post-immunization levels between DS children and controls, we observed lower levels in the former group (P < 0.05) for all serotypes except serotype 3. We conclude that pneumococcal polysaccharide immunization could be beneficial for these DS children.

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)-alpha 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 microg/l of IL-6 and 1.5 microg/l of TNF-alpha. 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.

Salivary lysozyme levels in patients with primary immunodeficiencies.

BACKGROUND: Lysozyme is a muramidase that acts on the peptideoglycan wall of Gram positive bacteria, causing cell death. It plays part in innate immunity and is present in blood, external fluid, as well in lysossomal granules of the phagocytes. Primary Immunodeficiencies are a diverse group of illnesses that, as a result of abnormalities of the immune system, increase susceptibility to infection. Among the examples of impaired natural immunity are defects in phagocytes and in the complement system. Innate immunity could be important in protecting mucosas against infections in patients with different forms of primary immunodeficiencies. The aim of this study was to investigate lysozyme concentrations in saliva from patients with primary immunodeficiencies. METHODS: Lysozyme levels in saliva samples from 34 patients with primary immunodeficiency (30 children and adolescents between the age of 3-13 years and 4 adults between the age of 20-33) and 60 age-matched healthy controls (49 children and adolescents between the ages of 3-15 and 11 adults between the ages of 22-42) were determined by the lysoplate method. RESULTS: There was no statistically significant difference between the lysozyme concentrations in the saliva of the immunodeficient subjects and those of the healthy controls. CONCLUSION: The results in the present work clearly show that salivary lysozyme levels in primary immunodeficient patients are equivalent to those found in healthy controls, suggesting that this enzyme still represents a remaining (but not a compensatory mechanism), contributing to the protection of there patients against infections.

The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease

Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by a defective oxidative burst of phagocytes and subsequent impairment of their microbicidal activity. Mutations in one of the NADPH-oxidase components affect gene expression or function of this system, leading to the phenotype of CGD. Defects in gp91-phox lead to X-linked CGD, responsible for approximately 70 percent of CGD cases. Investigation of the highly heterogeneous genotype of CGD patients includes mutation analysis, Northern blot or Western blot assays according to the particular case. The aim of the present study was to use reverse transcription (RT)-PCR for the analysis of molecular defects responsible for X-linked CGD in eight Brazilian patients and to assess its potential for broader application to molecular screening in CGD. Total RNA was prepared from Epstein B virus-transformed B-lymphocytes and reverse transcribed using random hexamers. The resulting cDNA was PCR-amplified by specific and overlapping pairs of primers designed to amplify three regions of the gp91-phox gene: exons 1-5, 3-9, and 7-13. This strategy detected defective gp91-phox expression in seven patients. The RT-PCR results matched clinical history, biochemical data (nitroblue tetrazolium or superoxide release assay) and available mutation analysis in four cases. In three additional cases, RT-PCR results matched clinical history and biochemical data. In another case, RT-PCR was normal despite a clinical history compatible with CGD and defective respiratory burst. We conclude that this new application of RT-PCR analysis - a simple, economical and rapid method - was appropriate for screening molecular defects in 7 of 8 X-linked CGD patients.

The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by a defective oxidative burst of phagocytes and subsequent impairment of their microbicidal activity. Mutations in one of the NADPH-oxidase components affect gene expression or function of this system, leading to the phenotype of CGD. Defects in gp91-phox lead to X-linked CGD, responsible for approximately 70% of CGD cases. Investigation of the highly heterogeneous genotype of CGD patients includes mutation analysis, Northern blot or Western blot assays according to the particular case. The aim of the present study was to use reverse transcription (RT)-PCR for the analysis of molecular defects responsible for X-linked CGD in eight Brazilian patients and to assess its potential for broader application to molecular screening in CGD. Total RNA was prepared from Epstein B virus-transformed B-lymphocytes and reverse transcribed using random hexamers. The resulting cDNA was PCR-amplified by specific and overlapping pairs of primers designed to amplify three regions of the gp91-phox gene: exons 1-5, 3-9, and 7-13. This strategy detected defective gp91-phox expression in seven patients. The RT-PCR results matched clinical history, biochemical data (nitroblue tetrazolium or superoxide release assay) and available mutation analysis in four cases. In three additional cases, RT-PCR results matched clinical history and biochemical data. In another case, RT-PCR was normal despite a clinical history compatible with CGD and defective respiratory burst. We conclude that this new application of RT-PCR analysis--a simple, economical and rapid method--was appropriate for screening molecular defects in 7 of 8 X-linked CGD patients.

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.

Transfer of IgG subclasses across placenta in term and preterm newborns

In order to study placental transfer of IgG subclasses, paired blood samples were collected from mothers and umbilical cord of preterm (N = 69) and full-term (N = 68) newborns. The full-term group was further divided into 3 subgroups: appropriate for gestational age (AGA, N = 43), large for gestational age (LGA, N = 13) and small for gestational age (SGA, N = 12), according to birth weight. IgG subclasses (IgG1, IgG2, IgG3 and IgG4) were measured by the single radial immunodiffusion technique using monoclonal antibodies. IgG1 and IgG3 newborn subclass concentrations (10.17 and 0.57 g/l, respectively) increased with increasing gestational age and reached maternal levels (IgG1 = 8.86; IgG3 = 0.67 g/l) during the 37th week of pregnancy. Low levels of these subclasses were found in premature newborns. IgG2 from newborns were always lower than maternal levels (P<0.05). LGA and SGA newborns had equivalent levels of IgG1 and IgG2 compared with AGA. SGA newborns had higher levels of IgG3 and lower levels of IgG4 than LGA and AGA newborns.

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.

Effect of medium and long chain triglycerides on human neutrophil migration

There is some controversy concerning the effect of intravenous long-chain triglyceride (LCT) emulsions on the phagocytic system and little is known about the effect of medium-chain triglyceride (MCT) containing emulsions. We evaluated the chemotaxis and random migration of human neutrophils from 18 healthy adult after preincubation with the following fat emulsions: LCT, MCT and a mixture of 50% MCT and 50% LCT (MCT/LCT). Leukocyte-rich plasma (4 x 10 6 cells/ml) was diluted 4:1 (v/v) with commercial fat emulsions (LTC, MCT, or MCT?LCT, 1:1) or saline and tumbled at 20 cycles?min for 30 min at 37 grade C. The final composition or the emulsion was 20 mg/ml fat, 0.24% egg yolk lecithin, and 0.5% glycerol and the dispersion was made isotonic by adding NaCl. In a second set of experiments, the LCT and MCT concentrations were adjusted to be equimolar. Leukocyte viability was * 95% after exposure to the treatment with fat emulsions. For emulsions with the same weight of each fat, random migration and chemotaxis of neutrophils were unaffected by the LCT emulsion but there was a significant decrease in both chemotaxis and random migration in MCT- (79 and 74%) or MCT/LCT-treated (60 and 56%) neutrophils. Similar results were obtained when LCT and MCT were equimolar. These results demonstrate an inhibitory effect of MCT on two human neutrophil functions which may be dose dependent