Table 1.
Characteristics of breast milk donors.
Fig 1.
Inhibition of HIV-1 uptake by primary human intestinal epithelial cells (IECs).
(A) Breast milk inhibition of subtype A and D HIV-1 uptake by IECs. (B) Dose-dependent breast milk inhibition of IEC uptake of subtype A HIV-1 by breast milk from an HIV-1-infected Ugandan woman. Ugandan subtype A or D viruses were pre-incubated with breast milk from an HIV-1-infected Ugandan woman for 30 min and then incubated with isolated primary IECs for 2 hr. The uptake of virus by IECs was measured by p24 ELISA with the uptake of virus pre-incubated with media defined as 100%, i.e. no inhibition. The range of p24 in samples treated with breast milk was 622–1300 pg/mL. Results are the mean values ±SD using IECs isolated from 4 separate tissue donors. Differences in IEC uptake of virus pre-incubated with breast milk and virus pre-incubated with media was determined by non-parametric Mann-Whitney test with significance indicated by * (p < 0.05).
Fig 2.
Inhibition of IEC uptake of HIV-1.
Ugandan subtype A HIV-1 was pre-incubated with (A, E) breast milk or the corresponding (B, F) IgG, (C, G) IgA or (D, H) non-Ig components (diluted 1:4) and then incubated with isolated primary IECs for 2 hr. IEC uptake of virus was measured by p24 ELISA in homogenized IEC. Results in panels A-D are p24 values from a representative tissue donor. Values in panels E-H are the mean percent inhibition using IECs isolated from 3–5 tissue donors for each breast milk. The uptake of virus pre-incubated with media was defined as 100%, i.e. no inhibition. For control antibodies, each dot point represents a mean value from a separate tissue donor. Control IgG: human serum polyclonal IgG. b12: human IgG1 antibody against the CD4 binding site on gp120. Control IgA: human colostrum polyclonal IgA. 2F5 mIgA: Human cluster II monomeric IgA mAb to gp41 membrane proximal external region. Differences in the uptake for virus pre-incubated with breast milk, media or milk component was determined by the non-parametric Mann-Whitney test with significance indicated by NS (not significant), * (p < 0.05), or ** (p < 0.005).
Fig 3.
Breast milk impacts myeloid DC uptake and transport of HIV-1 through intestinal mucosa.
Ugandan subtype A HIV-1 was pre-incubated with skimmed breast milk (diluted 1:2) for 30 min, applied to the apical surface of explanted intestinal mucosa. After 2 hr, cells in the lower chamber of the explant system were harvested and analyzed by flow cytometry by gating on myeloid DCs (CD13+CD11c+) that contained HIV-1 using KC57-FITC. Results are representative of experiments with two tissue donors.
Fig 4.
Impact on myeloid DC uptake and transport of HIV-1 through intestinal mucosa.
Ugandan subtype A HIV-1 was pre-incubated with (A, E) skimmed breast milk or the corresponding (B, F) IgG, (C, G) IgA or (D, H) non-Ig component (diluted 1:2) for 30 min, applied to the apical surface of explanted intestinal mucosa, and 2 hr later cells in the lower chamber of the explant system were harvested, lysized and measured for HIV-1 by p24 ELISA. Results in panels A-D are p24 values from a representative tissue donor. Values in panels E-H represent the mean percent inhibition of HIV-1 transport by one breast milk through tissue from 3–5 separate tissue donors for unfractionated milk (E), purified milk IgG (F), purified milk IgA (G), and Non-Ig fractions (H). DC uptake and transport of virus pre-incubated with media was defined as 100%, i.e. no inhibition. For control antibodies, each dot point represents a mean value from a separate tissue donor. Differences in HIV-1 transport through the mucosa are noted by NS (not significant), * (p < 0.05), or ** (p < 0.005).
Fig 5.
HIV-1 replication in intestinal mucosa.
Ugandan subtype A HIV-1 was pre-incubated with (A, E) breast milk or the corresponding (B, F) IgG, (C, G) IgA or (D, H) non-Ig components (diluted 1:2) for 30 min and inoculated onto the apical surface of explanted intestinal mucosa. After 2 hr incubation, the mucosa was trypsinized, incubated for 3 days and HIV-1 replication was measured by p24 released into the media normalized to tissue weight. Results in panels A-D are p24 values from a representative tissue donor. Values in panels E-H represent the mean percent inhibition of HIV-1 infection in intestinal tissue from 3–5 different donors by each breast milk. For control antibodies and Nevirapine control, each dot point represents a mean value from a separate tissue donor. Infection with virus pre-incubated with media was defined as 100%, i.e. no inhibition. Differences in viral replication in the tissue when the virus was pre-incubated with breast milk, media or milk components was determined using the non-parametric Mann-Whitney test, and significance is indicated by NS (not significant), * (p < 0.05), ** (p < 0.005), or *** (p < 0.0005).