Correlation of Lymphocyte Proliferating Cell Nuclear Antigen Expression with Dietary Cow’s Milk Antigen Load in Infants with Allergy to Cow’s Milk

Abstract

Background: Controversial results have been reported on the participation and diagnostic value of lymphocyte reactivity in cow’s milk (CM) allergy. In this study, we used a specific nuclear marker to evaluate lymphocyte proliferation in IgE–mediated CM allergy in infants, and examine its relation with diets containing different CM antigen loads. Methods: Infants with IgE–mediated CM allergy, as assessed by open provocation and RAST, were grouped according to their exclusive diet, either CM formulae, breast feeding, or hydrolysed whey formulae. A group of non–atopic infants receiving CM was also examined. Lymphocyte proliferation to β–lactoglobulin was evaluated by quantitation of the proliferating cell nuclear antigen (PCNA) expression in peripheral blood mononuclear cells, by flow cytometry. Immunophenotypic surface markers were also examined. Results: A marked difference of PCNA expression between CM–fed allergic infants and healthy controls was observed (p<0.001). In this setting, PCNA expression ≥10% was highly specific and sensitive as a marker of CM allergy in CM–fed infants. Moreover, a significant correlation (p<0.001) between antigen load and PCNA was established in CM–allergic infants under different diets, higher values obtained with increasing antigen loads. In addition, within the group fed hydrolyzed formulae, low–molecular–weight products resulted in marginally lower PCNA expression than higher–molecular–weight formulae. No differences in immunophenotype were found, with the exception of a higher CD23 expression in the breast–fed group. Conclusions: PCNA could be a useful marker in the assessment of lymphocyte proliferation to CM antigens. Low CM antigen diets are related with reduced lymphocyte reactivity, which may partly explain the clinical benefit observed with such diets.

Immunostimulatory potential of β-lactoglobulin preparations

“…This finding is significant for the conclusions of previously reported ex vivo studies using the Sigma β-LG, 5,10,11,13,[17][18][19][20][21][22][23]36,37 as the substantial immunostimulatory activity toward β-LG observed in those studies primarily might be a cause of nonspecific endotoxin-induced stimulation and therefore, not exclusively a cause of β-LG-specific activity. Especially the earlier findings of production of the T H 1-cytokine IFN-γ in CBMCs, 10,20 PBMCs, 20,22 lamina propria T cells, 19 and Peyer's patches 21 point to the presence of a T H 1-driving immunostimulatory component, like endotoxin, in the commercial β-LG preparations used for these studies.…”

“…6,7 Among the milk proteins, whey protein β-lactoglobulin (β-LG), 1 of the major allergens of cow's milk, 8 has been observed specifically to stimulate the proliferation of ex vivo cultured human cord blood mononuclear cells (CBMCs), [9][10][11][12][13][14][15] PBMCs, [15][16][17][18][19][20] lamina propria lymphocytes, 19 and Peyer's patches mononuclear cells, 21 as well as to induce specific proliferation and IgM production in murine immune cells. 5 In addition, cytokine production was observed in human CBMCs (IFN-γ 10 and IL-5, IL-10, IL-13, and IFN-γ 20 ), PBMCs (TNF-α; IL-10 19 ; and IL-5, IL-10, and IL-13 20 ; as well as IFN-γ 20,22 ), lamina propria T cells (IL-2, TNF-α, IL-10, and IFN-γ 19 ), and Peyer's patches (IFN-γ 21 ) after ex vivo exposure to commercial preparations of purified β-LG.…”

Rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells

“…At that time, as well as at 24, 48 and 72 hours later proliferation was estimated by staining with Proliferating Cell Nuclear Antigen (PCNA), a proliferation marker correlates with other markers of the S phase of cell cycle like tritiated thymidine and Bromodeoxyuridine labeling [ 24 ]. PCNA assessed with flow cytometry [ 25 ].…”

Lymphocyte Stimulation Test for the Diagnosis of Non-IgE-Mediated Cow’s Milk Allergy: A Step Closer to a Noninvasive Diagnostic Tool?

“…The consensus method usually involves isolating peripheral blood mononuclear cells, placing them in tissue culture with and without various stimuli, and measuring cell proliferation 5-7 days later [6][7][8] . The result, which is a function of the number of lymphocytes that are stimulated by a given antigen to produce a proliferative response, can be interpreted as the number of dividing cells and/or as the percentage of stimulated cells (stimulation index).…”