Epicutaneous Administration of Papain Induces IgE and IgG Responses in a Cysteine Protease Activity-Dependent Manner

ABSTRACTBackground: Epicutaneous sensitization to allergens is important in the pathogenesis of not only skin inflammation such as atopic dermatitis but also "atopic march" in allergic diseases such as asthma and food allergies. We here examined antibody production and skin barrier dysfunction in mice epicutaneously administered papain, a plant-derived occupational allergen belonging to the same family of cysteine proteases as mite major group 1 allergens.Methods: Papain and Staphylococcus aureus V8 protease were patched on the backs of hairless mice. Tran- sepidermal water loss was measured to evaluate the skin barrier dysfunction caused by the proteases. Papain or that treated with an irreversible inhibitor specific to cysteine proteases, E64, was painted onto the ear lobes of mice of an inbred strain C57BL/6. Serum total IgE levels and papain-specific IgE and IgG antibodies were measured by ELISA.Results: Papain and V8 protease patched on the backs of hairless mice caused skin barrier dysfunction and increased serum total IgE levels, and papain induced the production of papain-specific IgG1, IgG2a, and IgG2b. Papain painted onto the ear lobes of C57BL/6 mice induced papain-specific IgE, IgG1, IgG2c, and IgG2b, whereas papain treated with E64 did not. IgG1 was the most significantly induced papain-specific IgG subclass among those measured.Conclusions: We demonstrated that the epicutaneous administration of protease not only disrupted skin barrier function, but also induced IgE and IgG responses in a manner dependent on its protease activity. These results suggest that protease activity contained in environmental sources contributes to sensitization through an epicutaneous route

Investigating the effects of perturbations to <it>pgi</it> and <it>eno</it> gene expression on central carbon metabolism in <it>Escherichia coli</it> using ¹³ C metabolic flux analysis

<p>Abstract</p> <p>Background</p> <p>It has long been recognized that analyzing the behaviour of the complex intracellular biological networks is important for breeding industrially useful microorganisms. However, because of the complexity of these biological networks, it is currently not possible to obtain all the desired microorganisms. In this study, we constructed a system for analyzing the effect of gene expression perturbations on the behavior of biological networks in <it>Escherichia coli</it>. Specifically, we utilized ¹³ C metabolic flux analysis (¹³ C-MFA) to analyze the effect of perturbations to the expression levels of <it>pgi</it> and <it>eno</it> genes encoding phosphoglucose isomerase and enolase, respectively on metabolic fluxes.</p> <p>Results</p> <p>We constructed gene expression-controllable <it>E. coli</it> strains using a single-copy mini F plasmid. Using the <it>pgi</it> expression-controllable strain, we found that the specific growth rate correlated with the <it>pgi</it> expression level. ¹³ C-MFA of this strain revealed that the fluxes for the pentose phosphate pathway and Entner-Doudoroff pathway decreased, as the <it>pgi</it> expression lelvel increased. In addition, the glyoxylate shunt became active when the <it>pgi</it> expression level was almost zero. Moreover, the flux for the glyoxylate shunt increased when the <it>pgi</it> expression level decreased, but was significantly reduced in the <it>pgi</it>-knockout cells. Comparatively, <it>eno</it> expression could not be decreased compared to the parent strain, but we found that increased <it>eno</it> expression resulted in a decreased specific growth rate. ¹³ C-MFA revealed that the metabolic flux distribution was not altered by an increased <it>eno</it> expression level, but the overall metabolic activity of the central metabolism decreased. Furthermore, to evaluate the impact of perturbed expression of <it>pgi</it> and <it>eno</it> genes on changes in metabolic fluxes in <it>E. coli</it> quantitatively, metabolic sensitivity analysis was performed. As a result, the perturbed expression of <it>pgi</it> gene had a great impact to the metabolic flux changes in the branch point between the glycolysis and pentose phosphate pathway, isocitrate dehydrogenase reaction, anaplerotic pathways and Entner-Doudoroff pathway. In contrast, the impact of perturbed <it>eno</it> expression to the flux changes in <it>E. coli</it> metabolic network was small.</p> <p>Conclusions</p> <p>Our results indicate that the response of metabolic fluxes to perturbation to <it>pgi</it> expression was different from that to <it>eno</it> expression; perturbations to <it>pgi</it> expression affect the reaction related to the Pgi protein function, the isocitrate dehydrogenase reaction, anaplerotic reactions and Entner-Doudoroff pathway. Meanwhile, <it>eno</it> expression seems to affect the overall metabolic activity, and the impact of perturbed <it>eno</it> expression on metabolic flux change is small. Using the gene expression control system reported here, it is expected that we can analyze the response and adaptation process of complex biological networks to gene expression perturbations.</p