Healthy Preterm Newborns Show an Increased Frequency of CD4(+) CD25(high) CD127(low) FOXP3(+) Regulatory T Cells with a Naive Phenotype and High Expression of Gut-Homing Receptors.

Treg cells are crucial to prevent immune dysregulation, but little is known about the frequency of these cells in neonates, particularly in very/moderate and late preterm newborns studied as separate groups. The CD4(+) CD25(hi) CD127(lo) FOXP3(+) Treg population was phenotypically characterized to assess maturation markers and gut-homing integrins by flow cytometry in the cord blood of healthy preterm newborns born at 30-33(6/7) gestation weeks (Group 1), at 34-36(6/7) gestation weeks (Group 2) and term newborns born at 37-41 gestation weeks (Group 3), compared to healthy adults. An inverse correlation of the Treg percentage and gestational age was found, with significantly higher frequencies in Group 1 compared to Groups 2 and 3 and in Group 2 compared to Group 3, and significantly higher Treg frequencies and numbers in the neonates compared to the adults. All of the newborns exhibited increased Treg frequencies with a naive phenotype compared to adults. Cytotoxic T-lymphocyte-associated protein 4 CTLA-4 expression in the naive Treg was decreased in both preterm groups compared with those from term newborns and adults, and in the memory Treg from Group 1 compared with the other groups. The frequencies of Treg expressing α4ß7 and α4ß1 integrins were higher in both preterm groups, but significantly different only in Group 1, when compared with those from the term newborns and the adults. In conclusion, although a high frequency of Treg is present in newborns, an immature phenotype with a higher expression of CD45RA and α4ß7/α4ß1 and a lower expression of CTLA-4 is found, particularly in the very preterm group.

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.

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.

A model for preservation of thymocyte-depleted thymus.

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

Organ-specific autoantibodies and autoimmune diseases in juvenile systemic lupus erythematosus and juvenile dermatomyositis patients.

OBJECTIVES: To our knowledge, no study assessed simultaneously a variety of organ-specific autoantibodies and the prevalence of organ-specific autoimmune diseases in juvenile systemic lupus erythematosus (JSLE) and juvenile dermatomyositis (JDM). Therefore, the purpose of this study was to evaluate organ-specific autoantibodies and autoimmune diseases in JSLE and JDM patients. METHODS: Forty-one JSLE and 41 JDM patients were investigated for autoantibodies associated with autoimmune hepatitis, primary biliary cirrhosis, type 1 diabetes mellitus (T1DM), autoimmune thyroiditis (AT), autoimmune gastritis and coeliac disease (CD). Patients with positive antibodies were investigated for the respective organ-specific autoimmune diseases. RESULTS: Mean age at diagnosis was higher in JSLE compared to JDM patients (10.3±3.4 vs. 7.3±3.1years, p=0.0001). The frequencies of organ-specific autoantibodies were similar in JSLE and JDM patients (p>0.05). Of note, a high prevalence of T1DM and AT autoantibodies was observed in both groups (20% vs. 15%, p=0.77 and 24% vs. 15%, p=0.41; respectively). Higher frequencies of ANA (93% vs. 59%, p=0.0006), anti-dsDNA (61% vs. 2%, p<0.0001), anti-Ro, anti-Sm, anti-RNP, anti-La and IgG-aCL were observed in JSLE (p<0.05). Organ-specific autoimmune diseases were evidenced only in JSLE patients (24% vs. 0%, p=0.13). Two JSLE patients had T1DM associated with Hashimoto thyroiditis and another had subclinical thyroiditis. Another JSLE patient had CD diagnosis based on iron deficiency anaemia, anti-endomysial antibody, duodenal biopsy compatible to CD and response to a gluten-free diet. CONCLUSIONS: Organ-specific diseases were observed solely in JSLE patients and required specific therapy. The presence of these antibodies recommends the evaluation of organ-specific diseases and a rigorous follow-up.

Complement and antibody primary immunodeficiency in juvenile systemic lupus erythematosus patients.

OBJECTIVE: To evaluate the frequency of primary immunodeficiencies (PID) in juvenile systemic lupus erythematosus (JSLE) patients. METHODS: Some 72 JSLE patients were analyzed for levels of immunoglobulin classes and IgG subclasses and early components of the classical complement pathway. Determination of C4 gene copy number (GCN) and detection of type I C2 deficiency (D) were also performed. RESULTS: PID was identified in 16 patients (22%): C2D in three, C4D in three, C1qD in two, IgG2D (<20 mg/dl) in four, IgAD (<7 mg/dl) in three, and IgMD (<35 mg/dl) in three; one of these patients presented IgA, C2 and C4D. Two patients had low C4 GCN and two had type I C2D. Demographic data, family history of autoimmune disease and PID, JSLE clinical findings, occurrence of infections, disease activity and therapies were similar in patients with and without PID (p > 0.05). Remarkably, the median of Systemic Lupus International Collaborating Clinics/ACR-damage index (SLICC/ACR-DI) was significantly higher in JSLE patients with PID compared with patients without these abnormalities (p = 0.0033), likewise the high frequency of SLICC/ACR-DI > 1 (p = 0.023). CONCLUSIONS: A high frequency of PID was observed in JSLE patients, suggesting that these defects may contribute to lupus development. Our findings indicate that these two groups of PID should be investigated in severe pediatric lupus.

Common variable immunodeficiency associated with hepatosplenic T-cell lymphoma mimicking juvenile systemic lupus erythematosus.

Common variable immunodeficiency (CVID) is a heterogeneous disorder with susceptibility to infections, autoimmune manifestations, and cancer. To our knowledge, CIVD with T-cell lymphoma mimicking juvenile systemic lupus erythematosus (JSLE) was not described in the literature, and one case was reported herein. An 8-year-old female was admitted in our Pediatric Immunology Unit with a clinical history of hypogammaglobulinemia, recurrent upper respiratory infections, and pneumonias. She had a marked decrease of three serum immunoglobulin isotypes, and the diagnosis of CVID was established. At the age of 17 years, she presented with oral ulceration, nonerosive arthritis, nephritis, serositis, cytopenia, positive antiphospholipid antibodies, and positive antinuclear antibody fulfilling the American College of Rheumatology (ACR) criteria for SLE. She was treated with intravenous methylprednisolone for three consecutive days, and intravenous immunoglobulin, and maintenance therapy of chloroquine, azathioprine and prednisone 40 mg/day. Two months later, she died of septic shock secondary to acute pneumonia. The necropsy showed hepatosplenic T-cell lymphoma with diffuse involvement of bone marrow, spleen, liver, and lungs. The lymphoma cells were positive for CD3 immunostaining and negative for CD20 and lysozyme. In conclusion, the association of CVID and hepatosplenic T-cell lymphoma may simulate JSLE diagnosis.

Passive acquisition of protective antibodies reactive with Bordetella pertussis in newborns via placental transfer and breast-feeding.

Although acquisition of anti-pertussis antibodies by the newborn via placental transfer has been demonstrated, a subsequent recrudescence of pertussis infection is often observed, particularly in infants. The present study investigated the passive transfer of anti-pertussis IgG and IgA antibodies to term newborns and their ability to neutralize bacterial pathogenicity in an in vivo experimental model using mice intracerebrally challenged with viable Bordetella pertussis. Forty paired samples of maternal/umbilical cord sera and colostrum were obtained. Anti-pertussis antibodies were analysed by immunoenzymatic assay and by Immunoblotting. Antibody neutralizing ability was assessed through intracerebral B. pertussis challenges in mice. Anti-pertussis IgG titres were equivalent in both maternal and newborn sera (medians = 1:225 and 1:265), with a transfer rate of 118%. The colostrum samples had variable specific IgA titres (median = 1:74). The immunoblotting assays demonstrated identical recognition profiles of paired maternal and newborn serum pools but different bacterial recognition intensities by colostrum pools. In the animal model, significant differences were always observed when the serum and colostrum samples and pools were compared with the positive control (P < 0.05). Unlike samples with lower anti-pertussis titres, samples with high titres showed protective capacities above 50%. Pertussis-absorbed serum and colostrum pools protected 30% of mice and purified IgG antibodies protected 65%. Both pooled and single-sample protective abilities were correlated with antibody titres (P < 0.01). Our data demonstrated the effectiveness of anti-pertussis antibodies in bacterial pathogenesis neutralization, emphasizing the importance of placental transfer and breast-feeding in protecting infants against respiratory infections caused by Bordetella pertussis.

T cell receptor excision circles as a tool for evaluating thymic function in young children.

The thymus is a primary lymphoid organ responsible for the maturation of T cells as well as the immunological central tolerance. It is in the antenatal period and infancy that it plays its major role. In clinical practice, T cell receptor excision circles (TRECs) are considered a direct and reliable measure of the thymic function. TRECs are a by-product of DNA formation in gene rearrangement of T cell receptors. They are stable and they do not duplicate during mitosis, representing the recent emigrant T cells from the thymus. Despite their importance, TRECs have been neglected by physicians and there is a lack of data regarding thymic function during infancy of healthy children. In order to evaluate thymic function in the first years of life, we propose measuring TRECs as a valuable tool. One hundred and three blood samples from children and adolescents between 3 months and 20 years of age were analyzed. The mean TRECs count was 136.77±96.7 copies of TRECs/µL of DNA. The individuals between 0 and 5 years of age had significantly higher TRECs values than those between 10 and 20 years of age. No significant difference was observed in TRECs values among age groups below 5 years of age. An inverse correlation between TRECs and age was found (r=0.3 P=0.003). These data highlight and validate the evidence of decreased thymus function with age, even during infancy. Awareness should be raised with this important albeit ignored organ.

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.

A model for preservation of thymocyte-depleted thymus

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

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.

Functional alterations of human neutrophils by medium-chain triglyceride emulsions: evaluation of phagocytosis, bacterial killing, and oxidative activity.

Medium-chain triglyceride (MCT) and long-chain triglyceride (LCT) emulsions currently used in nutritional therapy were evaluated for their in vitro effect on neutrophil oxidative metabolism, phagocytosis, and bacterial killing activities. Neutrophils from healthy adult male volunteers were assessed after blood incubation with commercially available fat emulsions containing LCT, MCT, or a mixture of 50% MCT and 50% LCT at a final triglyceride concentration of 20 mg/ml. It was observed that MCT-containing emulsions stimulated nitroblue tetrazolium (NBT) dye reduction by neutrophils as determined by a cytochemical NBT test performed directly on whole blood. This effect was dose dependent. However, after lipid removal by cell washing, the MCT-treated neutrophils showed decreased production of hydrogen peroxide (H2O2) and NBT reduction in response to bacterial lipopolysaccharide or phorbol myristate acetate stimuli as well as impaired phagocytosis and killing of Staphylococcus aureus. In contrast, the LCT emulsion did not alter any of the neutrophil functions evaluated. The present data suggest that MCTs elicit the oxidative metabolism of neutrophils, probably by phagocytosis of fat particles and, depending on the lipid concentration, this effect may not be reversible, leading to impairment of the cellular response to subsequent membrane stimuli.

Colostral neutrophils express Fc alpha receptors (CD89) lacking gamma chain association and mediate noninflammatory properties of secretory IgA.

Colostrum plays an important role in protecting newborn infants against acute gastrointestinal and respiratory infections. IgA antibodies have been considered the major effector component; however, the role of their receptors on colostral phagocytes, especially neutrophils, has not been studied. Here, we demonstrate that CD15+ colostrum neutrophils express IgA Fc receptors (Fc alphaR, CD89) at levels similar to those of blood neutrophils. Most colostral cells (70%) bear secretory IgA (SIgA) on their surface (and intracellularly), whereas blood cells do not. The Fc alphaR on colostral neutrophils was identified as the a.1 isoform with a similar molecular mass (55-75 kDa) as that identified for blood neutrophils. Removal of N-linked carbohydrates revealed a major protein core of 32 kDa for both cell types. In contrast, co-immunoprecipitation and immunoblot experiments using a mild detergent, digitonin, revealed a lack of gamma chain association with Fc alphaR (gamma-less) exclusively on colostral neutrophils. The functional role of these gamma-less Fc alphaR cells was evaluated by measuring superoxide release and killing of SIgA-coated enteropathogenic E. coli. No increase in superoxide release was observed in colostral cells compared with blood neutrophils, whereas optimal release was obtained with PMA stimulation. Furthermore, despite similar bacterial phagocytosis index between both cell types, IgA-mediated bacterial-killing was not detectable with colostral neutrophils, whereas killing was detectable on blood cells. These results reveal exclusive expression of gamma-less Fc alphaR on colostral neutrophils associated with receptor hyperoccupation by IgA and with low, bacterial-killing activity, which suggest that this receptor may mediate noninflammatory effects of SIgA.

Effect of therapy with recombinant human interferon-gamma on the release of nitric oxide by neutrophils and mononuclear cells from patients with chronic granulomatous disease.

The aim of this study was to investigate the effect of recombinant human interferon-gamma (rHuIFN-gamma) therapy on the release of nitric oxide (NO) by neutrophils (NEU) and mononuclear cells (MON) from patients with chronic granulomatous disease (CGD). Five patients with this rare disease received rHuIFN-gamma (50 micrograms/m2 of body surface, given by subcutaneous injection three times a week) for 6 months. Clinical and laboratory evaluations were performed before and after 1 and 6 months of rHuIFN-gamma therapy. Nitric oxide release by NEU and MON was assessed by the ability of these cells to inhibit thrombin-induced washed platelet aggregation. The nitrite (NO2-) and nitrate (NO3-) levels in the supernatant of cultured NEU and MON, as well as in plasma and urine (24 h diuresis), were quantified by high-performance liquid chromatography (HPLC). Conventional immunologic tests for assessing phagocyte and lymphocyte functions and humoral immunity were also performed. Therapy with rHuIFN-gamma for 6 months did not enhance NO synthesis by NEU or MON from the patients with CGD. The urinary but not plasma levels of NO2- and NO3- were elevated after rHuIFN-gamma therapy. Phagocyte and lymphocyte functions as well as humoral immunity were not affected by rHuIFN-gamma therapy. Although few patients were available for the study, we conclude that therapy with rHuIFN-gamma for 6 months did not enhance the synthesis of NO by NEU and MON in CGD patients. Whether the increased excretion of NO2- and NO3- in the urine of CGD patients after rHUIFN-gamma therapy reflects an induction of NO-synthase in cells other than leukocytes remains to be investigated.

Evaluation of a fluorochrome assay for assessing the bactericidal activity of neutrophils in human phagocyte dysfunctions.

We evaluated a method for the assessment of the phagocytic and bactericidal activity of human peripheral neutrophils against Staphylococcus aureus Cowan I, which is a modified version of the acridine orange staining technique originally described by Smith and Rommel (1977). The modification consisted of the use of free leukocyte suspensions rather than coverglass adhered leukocytes in order to avoid two main problems: the inefficient neutrophil adherence to glass that can be observed in specimens from patients with certain functional phagocyte defects, and the risk of selecting among neutrophils. An additional advantage of the modified procedure is that it permits a uniform bacteria: phagocyte ratio in different cell samples. The method was tested on 25 healthy adults and on four children with functional phagocytic defects (chronic granulomatous disease of infancy, Chediak-Higashi syndrome, and Rothmund-Thomson syndrome associated to persistent neutropenia and low chemotactic response). The neutrophils of all four patients showed a low bactericidal activity, with percent values of intracellular killed bacteria below the mean +/- 2 SD range observed in the healthy population at all incubation times tested (5, 15 and 30 min). A significant reduction in phagocytosis index and in % killed unopsonized S. aureus was observed in relation to bacteria treated with a pool of normal human serum. These results demonstrate the high sensitivity of the method, which could be used to determine intrinsic and extrinsic functional alterations in human neutrophils.

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 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.

Functional evaluation of human neutrophils. Is the bactericidal activity correlated with nitroblue tetrazolium reduction?

The cytochemical nitroblue tetrazolium (NBT) reduction test continues to be used in clinical laboratories to detect defects in the oxidative metabolism of phagocytes. However, the specificity of the test is controversial, and it is not clear whether NBT reduction really reflects the microbicidal activity of these cells. In the present study, we evaluated the killing of Staphylococcus aureus by neutrophils from healthy adult individuals and from patients with phagocyte dysfunctions using a fluorochrome phagocytic assay, and compared the results with those obtained with a cytochemical NBT test performed simultaneously. The ability of neutrophils to reduce NBT (expressed as percent reducing neutrophils) with or without a lipopolysaccharide stimulus was not correlated with the bactericidal activity of these cells (expressed as percent killed bacteria per 100 neutrophils). The age and sex of the healthy adults did not influence the results of either assay. It seems that the superoxide anion played a small role in NBT reduction by normal neutrophils, since superoxide dismutase did not significantly inhibit this reaction. Only the absolute absence of NBT reduction reflected the low bactericidal activity of neutrophils, as seen in patients with chronic granulomatous disease (CGD). We conclude that the only clinical usefulness of the NBT test is for the screening of CGD, and that bacterial phagocytic assays are more appropriate for assessing the microbicidal function of neutrophils.