Immune-adjuvant activity of lentinan-modified calcium carbonate microparticles on a H5N1 vaccine.

In recent years, the high prevalence of avian influenza viruses especially H5N1 subtype isolated from poultry and human has become a major public health concern. Vaccination is still a major strategy for preventing H5N1 infections. Lentinan (LNT), a ß-1,3-glucohexaose with ß-1,6-branches, is extracted from Lentinus edodes and has been extensively studied for its immunoenhancement effects. In this study, we synthesized and characterized calcium carbonate (CaCO3) microparticles which modified with LNT as an adjuvant for H5N1 vaccine and investigated their ability to enhance immune responses. We prepared spherical and uniform CaCO3-LNT microparticles with a mean hydrodynamic size was around 2 µm. The H5N1 antigen-loaded CaCO3-LNT particles were injected into mice to evaluate their effectiveness as an adjuvant for H5N1 vaccines. The results demonstrated that CaCO3-LNT/H5N1 significantly enhanced the expression of MHC-II and CD86 in lymph node dendritic cells, and increased the ratio of CD4+ to CD8+ T cells in lymphocytes. Moreover, CaCO3-LNT/H5N1 surprisingly increased the HI titers and induced the secretion of IgG subtypes (IgG1 and IgG2b) and Th-associated cytokines (TNF-α, IFN-γ and IL-4) in immunized mice. Therefore, by combining with the immunostimulatory activity of LNT and the drug/antigen delivery capabilities of CaCO3, the CaCO3-LNT/H5N1 could induce a stronger cellular and humoral immune response and could be a potential adjuvant for the H5N1 vaccine.

Effect of calcium carbonate particle shape on phagocytosis and pro-inflammatory response in differentiated THP-1 macrophages.

Phagocytosis is a physiological process used by immune cells such as macrophages to actively ingest and destroy foreign pathogens and particles. It is the cellular process that leads to the failure of drug delivery carriers because the drug carriers are cleared by immune cells before reaching their target. Therefore, clarifying the mechanism of particle phagocytosis would have a significant implication for both fundamental understanding and biomedical engineering. As far as we know, the effect of particle shape on biological response has not been fully investigated. In the present study, we investigated the particle shape-dependent cellular uptake and biological response of differentiated THP-1 macrophages by using calcium carbonate (CaCO3)-based particles as a model. Transmission electron microscopy analysis revealed that the high uptake of needle-shaped CaCO3 particles by THP-1 macrophages because of their high phagocytic activity. In addition, the THP-1 macrophages exposed to needle-shaped CaCO3 accumulated a large amount of calcium in the intracellular matrix. The enhanced release of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) by the THP-1 macrophages suggested that the needle-shaped CaCO3 particles trigger a pro-inflammatory response. In contrast, no pro-inflammatory response was induced in undifferentiated THP-1 monocytes exposed to either needle- or cuboidal-shaped CaCO3 particles, probably because of their low phagocytic activity. We also found that phosphate-coated particles efficiently repressed cellular uptake and the resulting pro-inflammatory response in both THP-1 macrophages and primary peritoneal macrophages. Our results indicate that the pro-inflammatory response of macrophages upon exposure to CaCO3 particles is shape- and surface property-dependent, and is mediated by the intracellular accumulation of calcium ions released from phagocytosed CaCO3 particles.

Allergenicity of casein containing chalk in milk allergic schoolchildren.

BACKGROUND: Nondietary exposure to milk proteins may be a risk for children who do not outgrow milk allergy by school age. OBJECTIVE: To study the allergenicity of casein containing chalk. METHODS: A 6-year-old, milk allergic child developed asthma and rhinoconjunctivitis while in school. The suspected cause was dust-free chalk containing casein. To study the relationship of dust-free chalk containing casein with asthma and rhinoconjunctivitis, 13 additional milk allergic patients were studied: 3 school-aged children, 8 preschool-aged infants, and 2 children with outgrown milk allergy. Skin tests and/or specific IgE with chalk and casein were performed. A chalk use test was performed in older children. Milk allergens contained in chalk were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and IgE inhibition experiments. RESULTS: All school-aged, milk allergic children were exposed to chalk and reported symptoms attributed to chalk exposure. The skin test result to chalk was positive in 5 of 12 cases, and the specific IgE test result was positive in all 12 study participants in which it was performed. Casein strongly inhibited the binding of IgE to chalk. Chalk sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins with molecular weight similar to caseins. Immunoblot demonstrated strong binding of IgE to chalk in a blurred pattern and a band at 30 kDa, inhibited by casein. The chalk challenge test result was positive in 2 school-age children who had a positive skin test result to chalk. Their symptoms improved after avoidance of chalk in the school. In 2 other cases in which the challenge test result was negative, chalk was reintroduced without problems. CONCLUSION: Inhalation of chalk dust containing casein can induce asthma symptoms in milk allergic patients. Hidden and nondietary sources of exposure should always be considered in food allergic patients.