Methods for the in vitro determination of an individual disposition towards TH1- or TH2-reactivity by the application of appropriate stimulatory antigens

Abstract

SUMMARYIn this study we performed several methods for the determination of cytokines (RT-PCR for the demonstration of cytokine mRNA and flow cytometry for the analysis of intracellular cytokines) and compared them with a recently established test system stimulating peripheral blood mononuclear cells (PBMC) with TH1-and TH2-relevant recall antigens and analysing type 1 and type 2 cytokines by ELISA. Aim of the study was therefore to evaluate the reliability of TH1/TH2 cytokine profiles in two individuals with different types of an allergic/atopic disposition: one of them showed a strong TH1/type 1-mediated tuberculin-reaction (subject A), the other (subject B) revealed elevated IgE-levels and eosinophil counts (TH2/type 2-mediated). PBMC were incubated with the type 1-antigen purified protein derivative (PPD) and the type 2-antigen tetanus-toxoid (TT) for seven days. From the comparison of ELISA with RT-PCR and flow cytometry-analysis it became evident that all three methods allowed the definition of subject A as a 'type 1-responder'. Subject B showed a pure type 2-response in the ELISA method; PCR and flow cytometry analysis revealed the simultaneous production of type 1-and type 2-cytokines resulting in a mixed type 1/type 2-profile. Active immunization of subject A with TT at the end of the observation period of 12 months resulted in a transient shift from type 1-to a mixed type 1/type 2-profile (simultaneous PPD-induced IFN-g -and TT-induced IL-5 production). From this pilot study based on clear cut clinical criteria concerning either a humoral or cellular immunological reactivity towards allergens/antigens it is suggested that the determination of type 1/type 2-cytokines by ELISA in supernatants of PBMC stimulated with type 1/type 2-relevant antigens is a useful approach for a better classification of 'type1-' or 'type 2-responder'.Keywords type 1/ type 2 reactivity peripheral blood mononuclear cells purified protein derivative tetanus toxoid TH1/ TH2 cytokines

Modulation of intracellular expression of IFNγ and IL-2 in culture of splenic T lymphocytes by some flavonoid glycosides ofAlchornea floribunda

“…During type-1 immune response, the pro-inflammatory cytokines interferon-g (IFN-g) and interleukin-2 (IL-2) are produced by activated T-lymphocytes and natural killer (NK) cells to mount an effective response against pathogens and tumour cells, while type-2 cell responses are characterized by cytokines IL-4, IL-5 and IL-13 (Barth et al 2003;Romagnani 2006). In a cross-regulatory manner, type-2 cytokines may suppress type-1 immune response and vice versa (Fiorentino et al 1991;Lucey et al 1996).…”

Bedeutung von Zytokinbestimmungen in der umweltmedizinischen Praxis

“…Allerdings kann durch die intrazelluläre Zytokinmessung in Lymphozyten, ggf. auch nach Kultivierung der Zellen, eine Aussage über eine Allergie-begünsti-gende Reaktionslage des Organismus getroffen werden [34,35]. Zellkulturuntersuchungen gestatten zudem Hinweise auf die Reagibilität gegenüber Schadstoffen (s. auch die Stellungnahme zum Lymphozytentransformationstest von 2002 [36]).…”

Effect of buckminsterfullerenes on cells of the innate and adaptive immune system: an in vitro study with human peripheral blood mononuclear cells

“…Furthermore, BCG was used for the activation of macrophages/monocytes. 26,28,29 PPD was purchased from Statens Serum Institute (Copenhagen, Denmark); TT (40 IU) was obtained from Novartis Behring (Marburg, Germany); and BCG (2 × 10 8 bacteria/50 mL) was obtained from Medac (Wedel, Germany). Pokeweed mitogen (PWM) was purchased from Biochrom AG (Berlin, Germany) and was applied as a positive control nonspecifically stimulating several PBMC subpopulations.…”

“…25,26,[29][30][31][32] PBMC were adjusted to 1 Mio cells/mL in RPMI 1640 medium supplemented with 25% decomplemented autologous serum and gentamycin. 25,26,29,32,33 In order to determine the optimal conditions for the analysis, kinetics were performed prior to the experiments presented in this study. Best results for flow cytometry were observed after incubation of PBMC with the different antigens for 24 hours, incubation for 7 days revealing optimal results for proliferative responses and cytokine release.…”

“…In the first step we investigated their influence on unstimulated cells, and in the second step we mimicked inflammatory stages by preincubating the PBMC with TH1-(purified protein derivative, [PPD]), and TH2-cell activating antigens (tetanus toxoid [TT]) as well as BCG (bacillus Calmette-Guérin), stimulating mainly macrophages/monocytes. [25][26][27] It will be shown that these two fullerenes do not influence T cell reactivity but there is some evidence that they may activate cells of the innate immune system such as macrophages, dendritic cells (DC), or natural killer (NK) cells.…”