Joanna S. Hawkes

Cytokines (IL-1β, IL-6, TNF-α, TGF-β1, and TGF-β2) and prostaglandin E2 in human milk during the first three months postpartum

Postpartum changes in the concentrations of IL-1β, IL-6 tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), TGF-β2, and prostaglandin E2 in 257 human milk samples collected longitudinally from 49 healthy mothers during the first 12 wk of lactation were determined by ELISA or RIA. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were present in only a proportion of samples, and there was a wide range of concentrations detected at each time in the present study (IL-1β, <15-400 pg/mL; IL-6, <15-1032 pg/mL; TNF-α, <15-2933 pg/mL). Concentrations of prostaglandin E2 increased after the first week and remained elevated for the remainder of the study (range, <10-9966 pg/mL). The antiinflammatory cytokines TGF-β1 (range, 43-7108 pg/mL) and TGF-β2 (range, 208-57935 pg/mL) were present in substantial quantities in all samples, and there was little change in the mean concentration during 12 wk of lactation. The present study shows that immunomodulating agents are normally present in human milk in physiologically relevant quantities for at least the first 3 mo of the breast-fed infants life.

The Effect of Breast Feeding on Lymphocyte Subpopulations in Healthy Term Infants at 6 Months of Age

Breast milk contains many immunologically active components that influence the development of the immune system of the breast-fed infant. The purpose of this study was to investigate the difference in specific lymphocyte subsets between breast-fed and formula-fed 6-mo-old infants. Peripheral blood samples were collected from 79 breast-fed (<120 mL formula/wk) and 69 formula-fed (breast-fed < 4 wk) infants at 6 mo. All infants had been born at term and had no known illness at the time of blood collection. Packed cells from whole blood were incubated with fluorochrome-labeled monoclonal antibodies, followed by erythrocyte lysis. Washed lymphocytes were analyzed by two-color direct immunofluorescence on a flow cytometer. The percentage of T and B lymphocytes in the peripheral blood of 6-mo-old infants was the same, regardless of feeding regimen. However, the relative frequency of natural killer (NK) cells was greater in breast-fed infants than in formula-fed infants (9.7% vs 7.1%; p < 0.001). The percentage of cells expressing CD4 was lower in breast-fed infants than in formula-fed infants (47.3% vs 50.9%; p < 0.005), and that of cells expressing CD8 was greater (18.0% vs 16.4%; p < 0.05). As a result, the CD4:CD8 ratio in breast-fed infants was lower than that in formula-fed infants (2.8 vs 3.3; p < 0.005). The absolute size of the lymphocyte subpopulations T, B, and CD8+ was the same for each of the two populations of infants. However, breast-fed infants had fewer CD4+ T cells (p < 0.05) and a greater number of NK cells (p < 0.01) than the age-matched formula-fed infants. The immunophenotypic differences between breast-fed and formula-fed infants are consistent with reported age-related changes, suggesting greater maturity in the development of the immune system of breast-fed infants.

Interleukin-12 in Human Milk

The presence of interleukin-12 (IL-12) in 39 samples of human milk was investigated using the enzyme-linked immunosorbent assay. IL-12 (>40 pg/mL) was detected in 24 of the 39 samples collected (1408 ± 2256 pg/mL, mean ± SD, n = 24). A range of concentrations of IL-12 was observed in colostrum, transitional, and mature milk, with an apparent decrease in the mean concentration over time postpartum.

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.

Separation and quantification of PGE3 following derivatization with pancyl bromide by high pressure liquid chromatography with fluorometric detection

Separation of prostaglandin E3 (PGE3) from prostaglandin E2 (PGE2) and prostaglandin E1 (PGE1) was achieved following derivatization with p-(9-anthroyloxy)phenacyl bromide (panacyl bromide). The eicosanoid esters were analysed by reverse phase high pressure liquid chromatography with fluorometric detection (excitation 360nm and emission 470nm). Human, rat and mouse adherent cells were incubated overnight and the culture medium extracted, derivatized and analysed for PG production. PGE2 was detected from biological samples of each species tested. PGE2 synthesis was reduced when cells were incubated overnight with 5 microM eicosapentaenoic acid. PGE3 was not detectable under these experimental conditions. Studies were also undertaken using adherent cells from mice, rats and human subjects given dietary fish oil supplements rich in EPA. PGE3 production by these cells was not detected although the dietary regimens yielded substantial incorporation of EPA into cell membranes and leukocyte LTB5 production was demonstrable.

Biological activity of prostaglandin E3 with regard to oedema formation in mice

The biological activity of PGE3 with regard to oedema formation in mice was examined. Paw swelling was measured 30 minutes after injection of 10 μl PGE2 or PGE3 into the plantar region of the hind paw. Doses investigated ranged from 1 ng – 10 μg. Both PGE2 and PGE3 had substantial oedemogenic activity in this system.

Effect of dietary oils on the production of n-3 and n-6 metabolites of leukocyte 5-lipoxygenase in five rat strains.

We examined the influence of various dietary oils, including linseed and fish oil on the relative rates of leukotriene B4 (LTB4) and LTB5 production by rat peritoneal exudate cells in five rat strains. While there was an association between the membrane phospholipid levels of the fatty acid precursors (arachidonic acid (AA) and eicosapentaenoic acid (EPA)) and the rate of synthesis of their respective 5-lipoxygenase products (LTB4 and LTB5), the rate of LTB4 synthesis was a combined function of both AA and EPA levels. We observed a strong linear relationship (correlation coefficient = 0.99) between the ratio of EPA/AA in the cell membrane phospholipids and the ratio of LTB5/LTB4 produced by these cells in vitro; this association was independent of genetic (strain) variability and was independent of the source of EPA (dietary EPA or EPA endogenously synthesized from dietary alpha-linolenic acid).

Cytokine Production by Human Milk Cells and Peripheral Blood Mononuclear Cells from the Same Mothers

Samples of milk (n = 80) and venous blood were collected at 5 weeks postpartum from 82 lactating mothers. Human milk cells and peripheral blood mononuclear cells were isolated and the production of interleukin-1β, interleukin-6, and tumor necrosis factor-α in the absence and presence of lipopolysaccharide was determined by enzyme-linked immunosorbent assay. Human milk cells spontaneously produced significantly less interleukin-1β, interleukin-6, and tumor necrosis factor-α than peripheral blood mononuclear cells in the absence of stimulation. In vitro stimulation of human milk cells with lipopolysaccharide (500 ng/ml) for 24 hr increased cytokine production by approximately 40–50%, whereas peripheral blood mononuclear cells responded to lipopolysaccharide (200 ng/ml) with increased cytokine production of up to 350%. These observations suggest that cells in milk are capable of active involvement in the production of the interleukin-1β, interleukin-6, and tumor necrosis factor-α in the mammary gland and have the capacity to respond to further stimulation after leaving the breast.

Transforming growth factor beta in human milk does not change in response to modest intakes of docosahexaenoic acid.

Long-chain polyunsaturated fatty acids have been associated with aspects of immune regulation including cytokine production. The purpose of this study was to investigate the effect of maternal dietary supplementation with tuna oil, rich in docosahexaenoic acid (DHA), on the concentration of transforming growth factor beta 1 (TGFβ1) and TGFβ2 in breast milk. In this randomized, dietary intervention trial, mothers of term infants consumed a daily supplement of 2000 mg oil containing either placebo (n=40), 300 mg DHA (n=40), or 600 mg DHA (n=40). The DHA increase in milk and plasma was proportional to dietary DHA. There was no relationship between milk DHA status and TGFβ1 and TGFβ2 levels.

Effect of n-3 and n-6 dietary fats on the lipoxygenase products from stimulated rat neutrophils.

Fish oil was fed to rats in combination with an equal amount of olive, sunflower or linseed (flax) oil in semisynthetic diets for 3 weeks. Following stimulation of isolated neutrophils with calcium ionophore the levels of leukotrienes (LT) were determined by HPLC. Graphical presentation of the resultant data show a direct linear relationship between LTB production and substrate concentration with no preferential conversion of n-3 or n-6 substrates. In addition the results highlighted the greater conversion of eicosapentaenoic acid (EPA) and arachidonic acid (AA) to 5-hydroxy metabolites in stimulated neutrophils. There is no suggestion in our results of inhibition of any of the enzymatic conversion steps between EPA or AA and LTB production by any of the dietary fatty acids except by altering the EPA/AA ratio in neutrophil membranes.