INTRODUCTION

The specific/adaptive arm of the immune response is governed by antigen-reactive lymphocytes. Because each lymphocyte has a different antigen-binding specificity, the fraction of lymphocytes that can bind and respond to any given antigen is very small. To generate sufficient specific effector lymphocytes to fight an infection, an activated lymphocyte must undergo mitosis and clonal proliferation before its progeny finally differentiate into effector cells. This clonal expansion is a feature of all adaptive

immune responses (J aneway et aI. , 1 999). Therefore, the proliferation of antigen­

specific lymphocytes in response to antigenic stimulation is an essential step in adaptive immunity allowing antigen-specific cells to increase in number so that they can effectively combat the antigen that originally elicited the response (Roitt, 1 996). In the absence of diseases where an enhanced proliferative response would be undesirable (such as auto immune disease), improving the ability of these cells to proliferate and differentiate into effector cells could feasibly enhance the immune system 's response to foreign antigenic challenge and hence (potentially) reduce susceptibility to infection.

Extracts of bovine whey protein (isolated by ultrafiltration) have been found to have an ability to induce or enhance mitosis in lymphocytes of many different specificities or clonal origins, and thus have mitogenic capabilities in vitro (Francis et al. , 1 995). �­ Lactoglobulin and the hydrophobic fraction of proteose-peptones have been

demonstrated to support the short-term growth of murine hybridomas in vitro (Mati et

al. , 1 993 ; Capiaumont et aI. , 1 994), and a glycophosphopeptide isolated by ultrafiltration from bovine cheese whey protein concentrate has been found to have strong mitogenic activity in murine splenocytes in vitro (Yun et al. , 1 996). The whey fractions from raw or uItrafiltrated pasteurised milk have also been shown to stimulate

DNA synthesis in hybridomas and fibroblasts (DameIji et aI. , 1 988). Fractions of dried

whey have been shown to facilitate the growth of hybridomas and increase their

antibody production by· 20% compared to hybridomas cultured with foetal calf serum

(Legrand et aI. , 1 993). Similar results concerning the proliferation of a CHO-K l cell line in a medium supplemented with industrial whey was also reported by Capiaumont et al. ( 1 996). Furthermore, a minor protein of whey, lactoferrin, has been shown to have

the ability to bind to human promonocytic cells and enhance their proliferation in vitro.

mild heat treatment of the type used in milk processing (Oria et al. , 1 993). Such findings strengthen the possibility of adding whey proteins in a biologically active form to dietary formulations based on cow's milk, suitable for both animal and human

consumption. Notably, Baruchel & Viau ( 1 996) found that a bovine whey protein

concentrate (0.01 - 1 00 llg/mL), exerted a dose-dependent stimulatory effect on the cell

proliferation of normal human peripheral blood leucocytes in vitro. On the contrary,

when .incubating human cancer cell lines (MATB or lurkat T cells) with bovine whey

protein concentrate (0.0 1 - 1 00 llg/mL), cellular proliferation was inhibited ( Baruchel &

Viau, 1 996). This finding demonstrates that bovine whey protein concentrate has a

differential effect on the proliferation of cells in vitro depending on whether normal

human or human cancer cells are used. This result is significant as the enhancement of

the proliferation of cancer cells by whey proteins (in vitro or in vivo) would be an

undesirable modulatory effect.

A further study ( Wong et ai. , 1 998) has provided additional evidence that bovine casein

and a whey mixture (consisting of a.-lactalbumin, bovine serum albumin, bovine gamma

globulin and p-Iactoglobulin) can stimulate IgM production and cell proliferation of

normal murine (BALB/c) spleen cells in vitro. p-Lactoglobulin was found to be the

most stimulatory of the proteins tested (Wong et ai. , 1 998). Reports of

immunostimulation in vitro by major whey proteins (such as p-Iactoglobulin (Mati et

ai., 1 993 ; Capiaumont et ai. , 1 994; Wong et al. , 1 998» and minor whey proteins (such as lactoferrin (Mincheva-Nilsson et ai. , 1 990; Oria et ai. , 1 993» support the screening of previously untested dairy-derived whey protein fractions for their immunomodulating

properties in vitro. Therefore, the aim of this chapter was to determine the

immunomodulatory potential of a previously untested non-commercially available

P­

lactoglobulin (NZDB, Wellington, New Zealand) and a.-lactalbumin (NZDB, Wellington, New Zealand) using the mitogenic activity of murine splenic lymphocytes as an index, both in the presence or absence of concanavalin A or phytohaemagglutinin (T cell mitogens) and lipopolysaccharide (a B cell mitogen). Evidence of

i mmunornodulatory function by these whey proteins in vitro may support the notion that

Different bovine whey protein fraction preparations have however been shown to have varying immunomodulatory effects (such as no effect, a stimulatory effect, or a

suppressive effect, reviewed in Section 1 .5 and 1 .6), possibly due to their different

processing treatments. Depending on origin and the methods used to isolate them, the

properties of commercial whey proteins vary appreciably (reviewed in Section 1 .4.3).

Several factors may contribute to the variability in functionality and immunomodulatory properties of different whey protein preparations. For example, whey protein preparations may contain different amounts of protein, lactose, fat and minerals. Differences in the ratios of the major proteins and the degree of protein denaturation and aggregation caused by processing may also contribute to the variability of commercially prepared whey protein. Commercially manufactured and dairy-derived (non­ commercial) whey protein fractions are rarely directly compared in the same study. Hence, this chapter also sought to directly compare the immunomodulatory effects of commercially prepared whey protein fractions (obtained from ICN) to dairy-derived whey protein fractions (obtained from NZDB).