5 Immunol of Mammary gland
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5 Immunol of Mammary gland






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    5 Immunol of Mammary gland 5 Immunol of Mammary gland Presentation Transcript

    • Session 5 Mucosal Immunology of the Mammary Gland Stephanie Dorosko, PhD Department of Microbiology & Immunology Dartmouth Medical School 1 Medical Center Drive Lebanon, NH 03756 USA
    • Topics • Anatomy and physiology review • Immune cells in breast milk • Non-cellular immune components in breast milk • Breast milk transmission: statistics, potential mechanisms, timing, sIgA, ART
    • Lawrence, RA.Breastfeeding: A Guide for the Medical Profession. Boston: Mosby, 1999, p.61
    • Lawrence, R.A. Breastfeeding: A Guide for the Medical Profession. Boston: Mosby, 1999, p. 62.
    • p.46 Lawrence, R.A. Breastfeeding: A Guide for the Medical Profession. Boston: Mosby, 1999, p.46
    • Lawrence, R.A. Breastfeeding: A Guide for the Medical Profession. Boston: Mosby, 1999, p.74
    • Mammary Alveoli – Cross Section Schackleton et al, Nature 2006, 439:84-88
    • Lymphocytes in breast milk: functions not well understood
    • Macrophages: phagocytosis, storage and transport of IgA, excrete factors to enhance maturation of intestinal epithelium
    • Neutrophils: mammary tissue defense against microorganisms
    • IgA: enteromammary link Brandtzaeg, P.Nutrition Reviews 1998,56:S5-18
    • Other immune factors • Lysozyme:enzyme secreted by neutrophils and some macrophages. Bacteriostatic against Enterobacteriaceae and gram-positive bacteria, digests part of cell wall proteoglycan • Lactoferrin: iron-binding protein, deprives many types of bacteria and yeast of iron (bacteriostatic) • Glycolipids, glycoproteins, free oligosaccharides: inhibit bacterial and viral adhesion to intestinal epithelium • Cell signalling factors: interleukins, cytokines
    • Breast milk transmission of HIV-1
    • Current AIDS Statistics on Children < 15 years of age Worldwide: 2005 • Living with HIV/AIDS 2.3 million • Newly infected 700,000 • Died from AIDS 570,000 • More than 90% infected by MTCT UNAIDS, AIDS Epidemic Update: 2005. www.UNAIDS.org
    • HIV in Tanzania • 7% of mainland adult population • 11% prevalence in cities and towns • 13% in women aged 30-34 years • Antenatal clinics: – 5% Kagera – 15% Mbeya (compared to >20% in 1994) • Lower prevalence than many of its neighboring countries
    • Timing and Rate of MTCT • 58% HIV+ before 4 weeks • 42% HIV+ after 2 months (breastfeeding) Coutsoudis et al, J Infect Dis 2004, 189:2154-66
    • MTCT of HIV-1 through Breast Milk • Mechanism of HIV transmission via this route is not fully understood • Previous studies have found several risk factors associated with MTCT through BF Clinical mastitis (Embree, 2000; John, 2001) Breast milk HIV-1 RNA viral load (Pillay, 2000; Rousseau 2003) Breast milk sodium level (Semba, 1999) Breast milk cell-associated DNA (Rousseau, 2004; Koulinska, 2006) Low maternal CD4+ T-cell count; high plasma HIV-RNA levels (Rousseau, 2004))
    • Definition of Mastitis • Inflammatory response of the mammary tissue in response to pathogenic bacteria which have entered the gland through the lactiferous ducts
    • From“Physiology of lactation,” Chap.3, from Lawrence RA and Lawrence RM, Breastfeeding: A Guide for the Medical Profession, 5th ed. Boston: Mosby, 1999.
    • Clinical Mastitis and Risk of MTCT • OR = 2.7 (95% CI, 1.1-6.7) – (Embree et al, AIDS, 2000) • RR = 3.9 (95% CI, 1.2-12.7) – (John et al, JID, 2001)
    • Subclinical Mastitis and Risk of MTCT: Sodium level Elevated Risk of MTCT Sodium (Na+) level OR (95% CI), P Univariate Multivariate Breast milk Na+ > 12 2.77 (1.52-5.04), 2.38 (1.26-4.42), mmol/L <0.0009 <0.0008 @ 6 weeks Breast milk Na+ > 12 2.95 (1.63-5.30), 2.31 (1.23-4.26), mmol/L <0.0004 <0.01 @ 12 months Semba et al, JID, 1999
    • Subclinical Mastitis Sodium Level & Viral RNA Semba et al. JID, 1999
    • Detectable Viral RNA and Risk of MTCT Postpartum Period Risk of MTCT OR (95% CI), P 6 weeks 2.97 (1.23-7.18),<0.016 Semba et al, JID, 1999 12 months 2.97 (1.25-7.04), <0.01 Semba et al, JID, 1999 1wk – 15 months 2.82 (1.22-6.51) Pillay et al, JAIDS, 2000
    • Breast milk HIV-1 RNA in transmitters versus non- transmitters Rousseau et al, J Infect Dis 2003, 187:741-7
    • Significance of cell-associated virus in breast milk • Proportion of breast milk HIV-infected cells: total cells association with breast milk RNA (Rousseau, J Infect Dis 2004, 190:1880-8) • Each log10 increase in infected cells/mL associated with 3-fold increase in transmission • Each 10-fold increase in breast milk HIV RNA or DNA (cell associated virus) associated with 3-fold increase in breast milk transmission (Koulinska J AIDS 2006, 41:93-9) • Breast milk cell-associated virus predictive throughout lactation; HIV RNA predictive of transmission only after 9 months
    • Mechanisms of HIV-1 intestinal transmission in vitro Hocini and Bomsel, J Infect Dis 1999, 179:S448-53
    • Possible anti-HIV immune factors in milk • CD 8+ T-lymphocytes • HIV-specific secretory IgA • Lactobaccillus • Lactoferrin • Alpha-defensins • SLPI • Vitamin A
    • CD8+ T-lymphocytes • Breast milk CD8+ T- lymphocytes with response to HIV-1 gag (Sabbaj et al, J Virol 2002, 76:7365-73) • Recognition of and responses to HIV-1 antigens gag and env greater in magnitude from breast milk CD8+ lymphocytes than from paired peripheral blood CD8+ cells (P< 0.05) (Lohman et al, J Infect Dis 2003, 188:1666-74) • Percentages of CD8+ cells were similar in both breast milk and blood • Selective transport to breast milk?
    • HIV-specific sIgA • HIV-specific sIgA, IgG and IgM have been found in colostrum and breast milk (Becquart, Bomsel) • Colostral sIgA has most efficient ability to neutralize/bind HIV (Bomsel) • No difference in quantity of HIV-specific sIgA or neutralizing activity in transmitters versus non-transmitters (Becquart) • However, Becquart’s study did not measure viral RNA or DNA in breast milk
    • Lactobaccillus ssp. • Naturally occuring bacteria found in gastrointestinal tract of breastfeeding infants • New data showing their presence in breast milk (Rodriguez) • Anti-HIV activity found in vitro (Connor, unpublished) exploring which of their products are responsible
    • Lactoferrin • Few studies specifically on lactoferrin’s effect on HIV-1 replication in breast milk • Lactoferrin demonstrated inhibition of HIV-1 replication at level of viral fusion/entry in vitro (Moriuchi and Moriuchi, 2001)
    • Alpha-defensins • Peptides produced by immune cells • Anti-HIV activity among HIV+ long-term nonprogressors • In breast milk, increased concentrations associated with increased HIV RNA (predictor of transmission) (Kuhn et al, J AIDS 2005, 39:138-42) • However, increased concentration associated with decreased risk of transmission during delivery and from breastfeeding
    • Vitamin A: mixed results Existing levels of Vitamin A in HIV-infected women: No association or increased risk with low maternal Vitamin A Study site and authors Population Parameters measured Outcome Statistical ssociations -Mean vitamin A in transmitters = 0.86, Malawi n=74 (Semba, 1994) 338 HIV-positive women Pre-partum serum Infant HIV infection at 1 -Mean vitamin A in nontransmitters = Vitamin A (µmol/L) yr of age 1.07, n=264 -Student’s t-test (P<0.0001) No association between vitamin A and presence of HIV-1 DNA in total sample Kenya 107 HIV-positive women Pre-partum serum Presence of HIV DNA in (Nduati, 1995) (212 breast milk samples Vitamin A (µg/dL) breast milk at various -If pre-partum maternal plasma CD4 < collected at various post- post-partum time points 400 cells/mm3, and vitamin A < 30, partum visits) presence of HIV DNA OR=3.1 (95% CI, 0.8-11.5) -If pre-partum maternal plasma CD4 < 400 cells/mm3, and vitamin A < 20, presence of HIV DNA OR=19.7 (95% CI, 2.1-188.5)
    • Vitamin A Intervention Studies: No difference or increased transmission Study site and authors Population Intervention Outcome Statistical ssociations 5000 IU retinyl palmitate South Africa and 30 mg beta-carotene Infant HIV infection at 3 No difference in risk of infant (Coutsoudis, 1999) 728 HIV-positive women daily during 3rd trimester, months of age HIV infection between and 200,000 IU retinyl intervention and placebo palmitate at delivery; or groups -Placebo 3 mg retinol, 30 mg iron, No difference in overall MTCT Malawi 697 HIV-positive pregnant 400 µg folate, or; Infant HIV infection at at 6wks, 12, and 24 mos (Kumwenda, 2002, CID) women at 18-28 wks - 30 mg iron, 400 µg folate 6wks, 12 and 24 mos of age between vitamin A and control gestation (daily until delivery) group -All women received 30 mg -Vitamin A group had less retinol at 6 wks postpartum MTCT between 6wks and 24 mos (P= 0.04) 5000 IU preformed vitamin A and 30 mg beta carotene Overall MTCT RR with or; Infant HIV infection vitamin A vs. placebo = 1.38 Tanzania 1078 HIV- positive women - Vitamins B1, B2, B6, including BF period (95% CI 1.09-1.76, P= 0.009) niacin, B12, C, E and folic -Infant HIV infection only -MTCT via breastfeeding RR (Fawzi,2002, AIDS) acid, or; through BF with vitamin A vs. placebo = -Both, or; 1.33 (95% CI 0.95-1.86, P = -Placebo 0.10) (from 2nd trimester through lactation)
    • Impact of Exclusive Breastfeeding versus Mixed Feeding Illif et al, AIDS 2005, 19:699-708
    • Antiretroviral Therapy and MTCT Shapiro et al, JID 2005, 192:720-7
    • Antiretroviral Therapy and MTCT Shapiro et al, JID 2005, 192:713-9
    • Resistance w/ single dose nevrapine • The majority of women (~65%) exposed to single-dose NVP have detectable NNRTI-associated resistance mutations 6-36 weeks post-partum (Johnson et al. JID 192: 16-23, 2005)
    • Resistance w/ single dose nevrapine • Using sensitive PCR-based techniques, NVP-resistance mutations can persist up to 12-14 months after treatment in both mothers and infants (Flys et al. JID 192: 24-29, 2005)
    • Resistance w/ single dose nevrapine • Women with detectable NNRTI-resistance mutations early in the postpartum period have inferior virologic response to subsequent NNRTI-based treatments (Jourdain et al. NEJM 351:229-40, 2004)
    • Resistance w/ single dose nevrapine • The frequency of NNRTI-associated resistance mutations is significantly higher in women infected with HIV-1 subtype C (62%) than HIV-1 subtype A (19%) or D (36%) (Eshleman et al.JID 192: 30-36, 2005)
    • Current Tanzanian policy • Single dose nevirapine 200 mg orally to mother at onset of labor • And single dose nevirapine 2 mg/kg to infant within 72 hours of delivery • Linking pregnant women to triple ART who have: – CD4+ count < 200 cells/mm3;or – WHO Stage 3 symptoms and CD4+ cell count ≤350; or – WHO Stage 4 regardless of CD4 + cell count United Republic of Tanzania Ministry of Health, National Guidelines for the Clinical Management of HIV and AIDS, 2005
    • Many questions • Final word on cell-free vs. cell-associated virus as major contributor to transmission (perhaps both) • Source of viral RNA and proviral DNA – all from plasma? • Role of infant intestinal inflammation (subclinical) in increased transmission during mixed feeding • Role of infant’s immune system • Proportions of milk anti-viral factors: viral load in breast milk? • Nevirapine- resistant virus from treatment at delivery transmitted to infant through breastfeeding? • Effect of HAART for mothers throughout lactation on breastfeeding transmission
    • Asanteni Sana Thank you Do you have questions?