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Immunology & Immunological Disorders Poster Session






Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References




Discussion
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Bactericidal Activity of Human Milk Complement System


Contact Person: Michael O. Ogundele (mogundel@yahoo.com)


Results

EFFECT OF EDTA AND HEAT-INACTIVATION OF COMP0LEMENT ON BACTERICIDAL ACTIVITY OF HUMAN BREAST-MILK
Heating at 56C for 30 min and or addition of EDTA led to significant fall in the bactericidal effectof normal serum, colostrum and other milk samples (Fig. 1 and 2).

Figure 1. Effect of complement-inactivation and centrifugation (de-fattening process) on the bactericidal activities of different human breast-milk samples against E coli 0111.

Figure 2. Comparison of the effects of heat-inactivation and and addition of EDTA on the antibacterial activities of different human breast-ilkfractions.

EDTA is a potent ion chelator which inhibit the activation of C system in-vitro. It is used for preserving the native proteins in biological samples meant for subsequent laboratory analysis (18,21). It was observed that the inhibitory effects of EDTA on the bactericidal activities of most milk samples was lower than those produced by heat-inactivation of the C system. This might be attributable to the ability of EDTA to enhance the otherwise minimal bacteriolytic activities breast-milk lysozyme (22).The relative contribution of lysozyme to the bactericidal activities of breast-milk is assumed to be the observed difference between the fresh defatted fresh milk and that heated at 56C for 30 minutes, since the enzyme is known to be resistant to heating at this temperature (37).

C inactivation by heating at 56C either abolished the bactericidal effect of the serum and milk samples or even enhanced their ability to support growth of bacteria after 2 hours of incubation. This probably supports the opinion that the complement system is the one of the most important component of normal breast-milk samples effecting bactericiolysis in the absence of inflammation.

CORRELATION OF BACTERICIDAL ACYTIVITY OF HUMAN BREAST-MILK WITH THE LACTATION DURATION
All samples of normal colostrum (1-4 days postpartum) tested, showed evidence of C-dependent bacteriolysis. Transitional milk, up till 30 days postpartum, and mature milk showed progressively decreasing degrees of bacteriolysis (Fig. 1). While some samples of transitional (20%) and mature milk (50%) showed either no bacteriolysis or on the other hand, stimulated the growth of bacteria after a 2-hour incubation period, none of the colostrum samples lacked bacteriolytic activities. This could be attributable to the decreasing levels of C components in transitional and mature milk (10), the presence of a large number of "anti-complementary" factors and of a suitable micro-environment that favours bacterial growth-a neutral-alkaline pH, large quantities of carbohydrate and protein substrates in these milk samples.

The level of bactericidal activity of the HBM corresponds closely to the reported levels of major C factors, initially rapidly declining from up to twice the serum levels in early colostrum, and more gradually in the transitional milk to stable low levels in mature milk (10).

DILUTION LIMIT OF BACTERICIDAL ACTIVITY
The C-dependent bacteriolysis of human breast-milk could not be diluted beyond 1:4 while the bacteriolysis of the normal human serum on the other hand, could be diluted beyond 1:320 (Table 1).

Table 1. Effect of dilution and heat complement inactivation on the bactericidal activity of normal human serum (results expressed in % bacteriolysis)

  Degree of dilution  1:10   1:20    1:40    1:80    1:160   1:320
  Normal human serum  100    100     100      62      60      38
  Heateda NHS         nil**  nil***  nil     nil      nil     nil
NHS = Normal human serum
nil** = enhanced bacterial multiplication rather than bacteriolysis
a Heated at 56C for 30 minutes

BACTERICIDAL ACTIVITY
The clarified aqueous phase of breast-milk consistently showed a less bacteriolytic activity compared to undefattted whole-milk. This seems to strengthen the assumption that certain native C components might be closely bound to the milk lipid fraction, particularly the milk fat globule membrane, which are lost during high-speed centrifugation of HBM.

Correlation of bactericidal activity with opzonization of solid-phased killed bacteria
The levels of complement derived opsonins-activated C3 (C3b, iC3b, C3dg), estimated using a solid-phase killed bacteria to activate the C system, correlated closely with the bactericidal activities of the corresponding milk samples (Table 2). This further supports the conclusion that the bacteriolytic activities of milk is produced mainly by the C content (Table 3).

The maximal levels of C3 opsonins deposition from milk samples were obtained by dilution in detergent-containing buffer, compared to dilution in saline buffer, suggesting that the milk fat globule membrane (MFGM) strongly competes with the solid-phase bacteria for the deposition of opsonins. Fat clearance of the milk samples however failed to restore the deposition of opsonins to levels comparable to that of whole-milk in detergent buffer, suggesting that a significant portion of the native C components might also have been lost through centrifugation.

The opsonin deposition found in the milk that had been heated for 30 min at 56C probably demonstrates the presence of pre-formed opsonins in-vivo.

Table 2 Deposition of activated C3 opsoninsa on killed E. coli 0111 by human breast-milk b c
Colostrum Transitional milk
Post-partum duration of lactation 1-4 days 5-30 days
Heated whole milk 65 10* 70 5.5*
Wholemilk in PBS-Tw buffer 167 34 110 25
Wholemilk in GVB2+ buffer 3 1.2 --
Wholemilk in Mg-EGTA buffer & partially defatted milk -- 50 6.8
(x500-1500g) in GVB2+buffer 84 12 42+/-6
Partially defatted milk (x500-1500g) in Mg-EGTA buffer 14 5.6 64 8.4
a including C3b, iC3b, C3g and C3dg
* Real value of deposited opsonin with no substraction of the background opsonization.
b Values obtained after substraction of the background level of opsonization(opsonization by heated wholemilk)
c Results expressed in g/ml (Average SEM)
d Wholemilk heated to 56C for 30 minutes

Table 3. Relative contribution of different antibacterial components of human breast-milk to the observed lysis of E coli bacteria a b

  Antimicrobial
  component             Colostrum      Transitional milk    Mature milk


Complement                  83               13                 15
Lysozyme                    0                8                  0
Free fatty acid             13               0                  0
Total bactericidal
capacity                    95               19                 15
 
a Estimated from various results data b Results expressed as units of % Bacteriolysis

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Ogundele, M.O.; (1998). Bactericidal Activity of Human Milk Complement System. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/immunology/ogundele0172/index.html
© 1998 Author(s) Hold Copyright