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- Höglind, Ankie, et al.
(författare)
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Systematic evaluation of monoclonal antibodies and immunoassays for the detection of Interferon-gamma and Interleukin-2 in old and new world non-human primates
- 2017
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 441, s. 39-48
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Tidskriftsartikel (refereegranskat)abstract
- Non-human primates (NHP) provide important animal models for studies on immune responses to infections and vaccines. When assessing cellular immunity in NHP, cytokines are almost exclusively analyzed utilizing cross-reactive anti-human antibodies. The functionality of antibodies has to be empirically established for each assay/application as well as NHP species.A rational approach was employed to identify monoclonal antibodies (mAb) cross-reactive with many NHP species. Panels of new and established mAbs against human Interferon (IFN)-gamma and Interleukin (IL)-2 were assessed for reactivity with eukaryotically expressed recombinant IFN-gamma and IL-2, respectively, from Old (rhesus, cynomolgus and pigtail macaques, African green monkey, sooty mangabey and baboon) and New World NHP (Ma's night monkey, squirrel monkey and common marmoset).Pan-reactive mAbs, recognizing cytokines from all NHP species, were further analyzed in capture assays and flow cytometry with NHP peripheral blood mononuclear cells (PBMC). Pan-reactive mAb pairs for IFN-gamma well as IL-2 were identified and used in ELISA to measure IFN-gamma and IL-2, respectively, in Old and New World NHP PBMC supernatants. The same mAb pairs displayed high functionality in ELISpot and FluoroSpot for the measurement of antigen-specific IFN-gamma and IL-2 responses using cynomolgus PBMC Functionality of pan-reactive mAbs in flow cytometry was also verified with cynomolgus PBMC.The development of well-defined immunoassays functional with a panel of NHP species facilitates improved analyses of cellular immunity and enables inclusion in multiplex cytokine assays intended for a variety of NHP.
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- Masjedi, Khosro, 1969-
(författare)
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In vitro analyses of immune responses to metal and organic haptens in humans with contact allergy
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Contact allergy is one of the most common skin diseases with great social and economical impact. The origin and nature of contact allergens (haptens) capable of inducing T-cell mediated allergic reactions are diverse, ranging from organic molecules to metal ions. Most of the current knowledge on T-cell responses to haptens in humans with contact allergy have been established by studies on the metal ion nickel (Ni), the most common cause of contact allergy, whereas reactivity to the large group of organic haptens has been less studied.Haptens are not immunogenic by themselves but must bind carrier molecules prior to their presentation on MHC class I or II molecules and subsequent recognition by T cells. Due to differences in their chemical nature, haptens interact with host molecules by different mechanisms and differences in their solubility can influence their access to different antigen-presenting pathways.The aim of the present study was to define immune responses elicited by haptens of different chemical nature including Ni (hydrophilic metal ion), methylisothiazolinones (hydrophilic organic molecule) and parthenolide (lipophilic organic molecule). The immune response displayed by subjects with allergy to these substances, and non-allergic control subjects, was assessed by measuring hapten-induced cytokine production in peripheral blood mononuclear cells (PBMC) with a focus on ELISpot analysis of T-cell type 1 (e.g. IFN-g and IL-2) and type 2 (e.g. IL-4, IL-5 and IL-13) cytokines. For Ni and parthenolide, the phenotype of the hapten-reactive T cells was determined. The allergic status of subjects was defined by clinical history and patch testing. The latter is the established diagnostic method for contact allergy, based on applying various haptens to the subjects’ back and grading the skin reaction after 2-3 days.All three haptens elicited a concomitant T-cell type 1 and 2 response in subjects with contact allergy to the corresponding hapten, suggesting the induction of a functionally related cytokine profile, irrespective of the chemical character of the hapten. The cytokine response was related to the degree of the subjects’ patch test reactivity; PBMC from a vast majority of subjects with strong and moderate patch test reactivity displayed detectable cytokine responses to the corresponding haptens, whereas subjects with weak or no (controls) patch test reactivity did not. Despite the similar cytokine profile induced, the phenotype of the reactive T cells was found to differ between haptens with Ni eliciting CD4+ T cells and parthenolide eliciting CD8+ T cells. This difference may be explained by a better ability of a lipophilic hapten to gain access to the MHC class I-restricted antigen-presentation pathway. Moreover, the data suggest that analysis of cytokine responses to haptens may facilitate future development of in vitro-based diagnostics assay for contact allergy.Finally, the relationship between the variation over time in patch test reactivity and systemic reactivity to Ni, in terms of cytokine responses to Ni in vitro, was investigated. The degree of patch test reactivity is known to vary over time, in particular in subjects with weak reactivity. Ni-allergic subjects were patch tested three times with three month intervals and PBMC obtained at the same time points were assessed for in vitro reactivity to Ni. The overall reactivity in the patch test and the in vitro test was well correlated confirming that both methods provide a good and comparable estimate of the systemic reactivity to Ni. However, fluctuations in the patch test reactivity over time were not well correlated with variations in the cytokine response elicited in vitro suggesting that other parameters besides changes in the systemic reactivity could significantly contribute to the variation in patch test reaction over time.
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| 7. |
- Masjedi, Khosro, et al.
(författare)
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T-Helper 22 Cell Type Responses to Nickel in Contact Allergic Subjects Are Associated with T-Helper 1, T-Helper 2, and T-Helper 17 Cell Cytokine Profile Responses and Patch Test Reactivity
- 2023
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Ingår i: International Archives of Allergy and Immunology. - 1018-2438 .- 1423-0097. ; 184:8, s. 832-840
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Contact allergy to nickel (Ni) is a delayed-type hypersensitivity reaction mediated by Ni-reactive T cells producing the hallmark cytokines of several T-helper cell (Th) populations including IFN-γ(Th1), IL-4, IL-5 and IL-13 (Th2), and IL-17A (Th17). IL-22-expressing CD4+ cells, which could be either Th17 co-expressing IL-22 or Th22, expressing IL-22 in the absence of IL-17A, have also been found in Ni-provoked skin of allergic subjects. It has been unclear if Ni-reactive T cells consist of distinct Th cell type populations or if they secrete a mix of Th cell hallmark cytokines. The aim herein was to assess if cellular cytokine responses to Ni, in ex vivo-stimulated peripheral blood mononuclear cells (PBMCs) from Ni-allergic subjects, include not only Th1, Th2, and Th17 but also Th22 hallmark cytokines and to define if the cytokines are produced by distinct cell populations representing different Th profiles. Methods: PBMC from Ni-allergic subjects (n = 15) with different degrees of patch test reactivity and non-allergic controls (n = 5) were in vitro stimulated with Ni. Cytokine levels in PBMC supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) (IFN-γ, IL-2, IL-3, IL-5, IL-6, IL-13, IL-17A, IL-22, and IL-31). FluoroSpot was used to assess if individual Ni-reactive cells produced single, or combinations of, cytokines representing different Th profiles. Cytokine combinations analyzed were IL-17A/IL-22/IFN-γ, IL-5/IL-17A/IFN-γ, IL-13/IL-22/IFN-γ, and IL-5/IL-13. Results: IL-22 as well as all other cytokines measured by ELISA were induced by Ni at higher levels in PBMC from allergic versus non-allergic subjects, with higher levels being associated with stronger patch test reactivity. The levels of most Ni-induced cytokines were positively correlated with each other; IL-2 displayed the highest correlation with other cytokines and IL-6 the lowest. FluoroSpot analysis showed that Th signature cytokines, IFN-γ(Th1), IL-5 and IL-13 (Th2), IL-17A (Th17), and IL-22 (Th22), were almost exclusively produced by distinct cell populations. Conclusion: Distinct Th cell populations, including Ni-reactive cells displaying Th1, Th2, Th17, and Th22 cytokine profiles, are all increased in PBMC from Ni-allergic subjects and positively associated with patch test reactivity. The relevance of these different Th profile populations for the up- or down-regulation of inflammatory reactions in the skin of Ni-allergic subjects remains to be clarified.
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