Donal M Roberton

Phenotype of T cells, their soluble receptor levels, and cytokine profile of human breast milk

Human breast milk has important immunoprotective and immunosuppressive functions for an infant. The purpose of this study was to extend the phenotype of milk cells and to measure soluble T cell receptor levels and cytokines in milk, and to compare these with neonatal and adult blood. Milk T cells had a more equivalent CD4: CD8 ratio than blood; milk CD4 T cells mainly expressed the CD45RO (antigen primed/memory) phenotype; milk CD8 cells had an equivalent CDllb:CD28 suppressor; cytotoxic phenotype; and milk T cells had 2‐3‐fold higher percentages of activated CD4 IL‐2R and CD8 HML‐1 or CD8 VLA‐1 cells than blood. Soluble IL‐2R, CD4 and CD8 concentrations were lower in milk than adult blood, although relatively increased when compared to the lower T cell concentration in milk. Breast milk contained high levels of IFN‐γ but low levels of other measured cytokines compared to blood. These distinct differences of T cells and their soluble products are likely to influence an infants immune system.

Effect of dietary nucleotide supplementation on growth and immune function in term infants: a randomized controlled trial.

Objective:To examine the effect of nucleotide (NT)-supplemented cows milk-based formula on growth and biochemical indices of immune function in healthy infants.Design:Randomized controlled trial (RCT) of formula-fed term infants allocated to control formula with an innate level of NT at 10 mg/l (n=102), or formula fortified with NT at 33.5 mg/l (n=98). A parallel group of 125 breastfed infants followed the same protocol as a reference.Outcome measures:Growth was assessed at enrolment, 7 weeks, 4 months and 7 months of age. Natural killer cell activity, cytokine production and lymphocyte subpopulations were assessed at 7 weeks of age. Antibody responses to diphtheria toxoid, tetanus toxoid and Haemophilus influenzae type b (Hib) immunizations were measured at 7 months of age.Results:NT supplementation did not influence the growth of formula fed infants or any markers of immunity measured at 7 weeks of age. Antibody responses to tetanus toxoid were higher in the NT-supplemented group (n=68) compared with the control group (n=70) at 7 months of age (median (5th, 95% percentile): 1.57(0.42, 3.43) vs 1.01(0.41, 4.66) IU/ml, P<0.03). A difference between treatments was seen in response to diphtheria toxoid but this effect disappeared when adjusted for hepatitis B immunization at birth. There was no effect of treatment on antibody responses to Hib immunization.Conclusions:Supplementation of formulas with NT at 33.5 mg/l resulted in a modest improvement in antibody response consistent with RCTs that used higher levels of NT supplementation. Whether this translates to clinical benefits in well-nourished infants requires further study.Sponsorship:Supported by a grant from Wyeth Nutrition. Dr Makrides was supported by an RD Wright Fellowship from the National Health and Medical Research Council of Australia and Dr Gibson was partially supported by the MS McLeod Research Trust and a Senior Research Fellowship from the National Health and Medical Research Council of Australia.

Somatic mutation of immunoglobulin VH6 genes in human infants

Infants respond to antigen by making antibody that is generally of low affinity for antigen. Somatic hypermutation of immunoglobulin genes, and selection of cells expressing mutations with improved affinity for antigen, are the molecular and cellular processes underlying the maturation of antibody affinity. We have reported previously that neonates and infants up to 2 months of age, including individuals undergoing strong immunological challenge, show very few mutated VH6 sequences, with low mutation frequencies in mutated sequences, and little evidence of selection. We have now examined immunoglobulin genes from healthy infants between 2 and 10 months old for mutation and evidence of selection. In this age group, the proportion of VH6 sequences which are mutated and the mutation frequency in mutated sequences increase with age. There is evidence of selection from 6 months old. These results indicate that the process of affinity maturation, which depends on cognate T–B cell interaction and functional germinal centres, is approaching maturity from 6 months old.

Immune activation during infancy in healthy humans.

Immune activity during infancy was investigated using blood samples from 30 neonates and 52 healthy infants between 2 and 15 months of age attending for immunization. The purpose of this study was to assess the total immune activity of T cells using soluble interleukin-2 receptor (IL-2R) and interferon-γ concentrations. These were compared with the proportion of CD4 CD45RO-, IL-2R (CD25)-, and transferrin receptor (CD71)-positive peripheral blood lymphocytes. The median duration of breast-feeding and of introduction of solid feeds was 4.2 and 4.0 months, respectively. Compared to neonates, the mean±SE soluble IL-2R concentration peaked at 4 months of age (1670±94 vs 3060±252 U/ml;P< 0.0001), as did pooled interferon-γ levels. The percentage of CD4 CD45RO T cells remained low and the proportion of activated peripheral blood lymphocytes decreased during infancy. We conclude that noncirculatory immune activity is increased during infancy and this is associated with weaning.

The antigen receptor complex on cord B lymphocytes

The neonatal immune system responds to a restricted range of antigens, producing largely IgM antibody of low affinity. Comparison of the components of the B‐cell antigen receptor complex shows significantly elevated membrane levels of IgM in neonatal B cells, compared with adult cells. CD79, which acts as the signal transducer for membrane immunoglobulin, is elevated in parallel with IgM, while IgD is elevated to a lesser degree. CD19, CD21, CD22 and CD81, which are all involved in transmitting activation signals when immunoglobulin is engaged, are not elevated. CD32, which is involved in negative regulation of activation, is present at reduced levels on cord B cells. The elevation of B‐cell membrane IgM persists during infancy. Neonatal B cells respond in vitro to interleukin‐4 (IL‐4) by further elevation of membrane IgM levels. The elevated level of membrane IgM may make neonatal B cells easier to trigger by low concentrations of antigen, but in vitro activation and immunoglobulin modulation experiments did not show significant differences between cord and adult B‐cell responses to anti‐IgM.

Regulation of human neutrophil-mediated cartilage proteoglycan degradation by phosphatidylinositol-3-kinase

The ability of neutrophils to degrade cartilage proteoglycan suggests that the neutrophils that accumulate in the joints of rheumatoid arthritis patients are mediators of tissue damage. The regulatory mechanisms which are relevant to the proteoglycan‐degrading activity of neutrophils are poorly understood. Since phosphatidylinositol 3‐kinase (PI3‐K), protein kinase C (PKC), the extracellular signal‐regulated protein kinase (ERK)1/ERK2 and cyclic adenosine monophosphate (cAMP) have been reported to regulate neutrophil respiratory burst and/or degranulation, a role for these signalling molecules in regulating proteoglycan degradation was investigated. Preincubation of human neutrophils with GF109203X (an inhibitor of PKC), PD98059 (an inhibitor of MEK, the upstream regulator of ERK1/ERK2) or with forskolin or dibutyryl cAMP, failed to suppress proteoglycan degradation of opsonized bovine cartilage. In contrast, preincubation of neutrophils with wortmannin or LY294002, specific inhibitors of PI3‐K, inhibited proteoglycan degradation. Incubation of neutrophils with cartilage resulted in the activation of PI3‐K in neutrophils, consistent with a role for PI3‐K in proteoglycan degradation. Activation of PI3‐K and proteoglycan degradation was enhanced by tumour necrosis factor‐α. Degradation caused by neutrophils from the synovial fluid of rheumatoid arthritis patients was also inhibited by wortmannin. These data demonstrate that the proteoglycan degradative activity of neutrophils required PI3‐K but not PKC or the ERK1/ERK2/ERK5 cascades and was insensitive to increases in intracellular cAMP concentrations.

Interleukin 2 receptor regulation and IL-2 function in the human infant.

IL-2 receptor is expressed at low levels on adult blood lymphocytes, and at lower levels on cord blood cells. IL-2 receptor alpha and beta chain expression increases gradually from 0-18 months of age. The level of soluble CD25 (IL-2 receptor alpha chain) has been reported to be elevated in cord blood. Quantitative RT-PCR showed that adult cells express 10 times as much CD25 mRNA as cord cells. Cord plasma showed only a marginal ability to strip CD25 from the membrane. To assess the functional consequences of low IL-2 receptor expression, cord and adult cells were activated in vitro. The response was stimulus-dependent, but cord cells upregulated CD25 readily. Cord and adult cells proliferated in an IL-2-dependent assay to a similar extent. Infants suffering acute infection showed marginally higher levels of membrane CD25 expression than infants without overt infection. Thus neonatal and infant lymphocytes express lower levels of IL-2 receptors than adult cells, reflecting lower mRNA concentrations at least for CD25; they are able to up-regulate receptors in response to in vitro stimulation and are able to respond in vitro to IL-2-dependent stimulation; however in vivo there may be a dampening down of the IL-2 system in infancy.

The Fc Receptor for IgG (FcγRII; CD32) on human neonatal B lymphocytes

Abstract Abstract: B cells express an Fc receptor for IgG (FcγRII; CD32) which is involved in feedback inhibition of antibody production. Engagement of FcγRII during ligation of the antigen receptor provides an inhibitory signal. FcγRII exists as several isoforms, with FcγRIIb (which carries an immunoreceptor tyrosine-based inhibition motif; ITIM) being predominant form on adult B cells. The inhibitory role of FcγRIIb may be unhelpful to the infant, since primary exposure to infectious agents is likely to be in the presence of maternal IgG. We hypothesized that neonatal B cells would be less susceptible to feedback inhibition by antibody, either through the expression of activation-competent FcγRII isoforms (FcγRIIa and FcγRIIc) or through reduced expression of the inhibitory FcγRIIb isoforms. Cord and adult B cells were examined for expression of FcγRII isoforms using monoclonal antibodies and RT-PCR. In vitro assays were performed to assess susceptibility of cord and adult cells to FcγRII-mediated suppression. Although there is no phenotypic difference in FcγRII expression (FcγRIIb predominating on both adult and cord B cells), FcγRIIb is expressed at lower levels on cord cells. This quantitative difference in FcγRIIb expression may explain the reduced susceptibility of cord B cells to antibody-mediated inhibition observed in these experiments

Responses to immunisation with Hib conjugate vaccine in Australian breastfed and formula-fed infants.

Objective:  There are conflicting reports as to whether breastfed infants respond with higher antibody levels to conjugate Haemophilus influenzae type b (Hib) vaccine compared with formula‐fed infants. These observations prompted us to investigate the effect of feeding method on the antibody concentration to Hib polyribosylribitol (PRP) both prior to and 3 months after the primary course of immunisation with Hib (PRP‐OMP).

Medical student education in paediatrics and child health: where are we going?

In most undergraduate medical curricula, learning is becoming less content based and the emphasis is changing to problem based learning, continuing self directed learning, and the use of a wide range of learning resources. Particular needs in paediatrics and child health are an increasing emphasis on learning in ambulatory care and community based health facilities, and on assessment processes which are formative and reflect the learning objectives appropriately. A wide range of resources is needed for learning at a time when teaching hospital and health system facilities face significant financial restraints.