Patient flowchart derivation and validation cohort.

<p>Patient flowchart derivation and validation cohort.</p

Distribution of Baseline Patient Characteristics in the Derivation and Validation Cohort (Number(percentage)).

*<p>No infants developed BPD ** either exclusive breastfeeding or mixed with formula feeding # predicted by parents at birth.</p

Distribution of potential predictors across cases and non-cases in the derivation and validation cohort.

*<p>either exclusive breastfeeding or mixed with formula feeding # predicted by parents at birth.</p

Results of the multivariable logistic regression analyses in the derivation cohort (n = 1227) and the performance of the model in the validation cohort (n = 1194): predictors for RSV hospitalization after bootstrapping.

*<p>either exclusive breastfeeding or mixed with formula feeding # predicted by parents at birth.</p

Operating Characteristics for Each Threshold of the RISK model in the validation cohort (n = 1194).

<p>Operating Characteristics for Each Threshold of the RISK model in the validation cohort (n = 1194).</p

Cord blood plasma polarizes cytokine responses to MyD88-dependent and independent TLR agonists.

<p>A–B; Agonist-induced production of IL-12p70 (A) and IL-10 (B) by adult PBMC stimulated in the presence of 10% cord blood plasma or adult plasma. PBMC were stimulated for 24 h with medium, poly I∶C (TLR3, 200 µg/mL) or CL-075 (TLR8, 10 µg/mL). After 24 h, supernatants were collected and cytokine concentrations were determined by ELISA. Bars represent mean+ SEM from five different plasma samples. C; TLR4-mediated production of IL-12p70 by adult PBMC stimulated in the presence of 10% cord blood plasma or adult plasma, with or without IL-10 neutralizing antibody (1 µg/mL). Each dot represents one individual plasma sample. Data are representative of three independent experiments. *; <i>p</i><0.05, **; <i>p</i><0.01.</p

Suppression of TLR4-mediated IL-12p70, but not induction of IL-10, by neonatal plasma is maintained up until the age of one month.

<p>Adult PBMC were stimulated with LPS (100 ng/mL) and IFN-γ (20 ng/mL) in the presence of plasma derived from cord blood, healthy newborns aged one month, or adult volunteers. After 24 h incubation, supernatants were collected and concentrations of IL-12p70 (A) and IL-10 (B) were determined by ELISA. Bars represent mean+SEM of three to five different plasma samples. α; <i>p</i><0.05 cord blood vs neonatal plasma, *; <i>p</i><0.05 neonatal vs adult plasma, &; <i>p</i><0.05 cord blood vs adult plasma.</p

Cord blood plasma suppresses TLR4-mediated IL-12p40 production by primary monocytes.

<p>Adult PBMC were stimulated with LPS (100 ng/mL) and IFN-γ (20 ng/mL) in the presence of 10% cord blood plasma or adult plasma. After 24 hours, intracellular levels of IL-12p40 in monocytes (CD14+/HLA-DR+), myeloid dendritic cells (mDC, CD3−/CD16−/CD56−/CD14−/HLA-DR+/CD11c+) and B-cells (CD3−/CD16−/CD56−/CD19+) were determined by flow cytometry. A; Proportion of monocytes, mDC and B-cells among the total population of IL-12p40⁺ cells. Bars represent means of five different plasma samples. B; Representative histograms of monocyte IL-12p40 production upon TLR4-stimulation in the presence of cord blood plasma or adult plasma. C; Mean fluorescence intensity (MFI) of IL-12p40 in monocytes and mDC upon TLR4-stimulation in the presence of cord blood plasma or adult plasma. Each dot represents one individual plasma sample. D; Correlation between TLR4-mediated monocyte IL-12p40 MFI and IL-12p70 protein production by PBMC. Each dot represents one individual plasma sample. All data are representative of three independent experiments. *; <i>p</i><0.05.</p

Human plasma suppresses TLR4-mediated production of IL-12p70 while inducing IL-10.

<p>A; TLR4-mediated release of IL-12p70 and IL-10 by cord blood mononuclear cells (CBMC) or adult peripheral blood mononuclear cells (PBMC) stimulated in the presence of 10% cord blood plasma or adult plasma. Bars represent mean+SEM of five independent cord blood or adult plasma donors. B; TLR4-mediated release of IL-12p70 and IL-10 by PBMC from three different adult donors stimulated in the presence of 10% cord blood plasma or adult plasma. C; Dose-response curves showing TLR4-mediated release of IL-12p70 and IL-10 by adult PBMC in the presence of increasing concentrations of cord blood plasma or adult plasma. Each dot represents mean ± SEM of five independent cord blood or adult plasma donors. Data are representative of three independent experiments. *; <i>p</i><0.05, **; <i>p</i><0.01.</p

Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk

<div><p>Background</p><p>Respiratory syncytial virus (RSV) infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins.</p><p>Objective</p><p>To investigate whether IgG purified from bovine milk (bIgG) can modulate immune responses against human RSV.</p><p>Methods</p><p>ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR) or bIgG-mediated binding of myeloid cells to hRSV respectively. <i>S. Epidermidis</i> and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated.</p><p>Results</p><p>bIgG recognised human RSV, influenza haemagglutinin and <i>Haemophilus influenza</i>. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV.</p><p>Conclusions</p><p>The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.</p></div