Stability of pro- and anti-inflammatory immune biomarkers for human cohort studies.

BACKGROUND: Although discovery research has identified the importance of dozens of pro- and anti-inflammatory immune mediators in the pathogenesis, maintenance, exacerbation and resolution of inflammatory diseases, most human cohort studies have incorporated few or no immunological intermediate phenotypes in their analyses. Significant hindrances have been (1) the limited panel of biomarkers known to be readily detected in healthy human populations and (2) the stability, hence utility, of such biomarkers to repeated analysis. METHODS: The frequency and stability of 14 plasma biomarkers linked to in vivo immune regulation of allergic and autoimmune inflammatory disorders was determined in 140 healthy pediatric and adult participants. The impact of initial and multiple subsequent freeze/thaw cycles on pro-inflammatory (CCL2, CXCL10, IL-18, TNFα, IL-6), anti-inflammatory (IL-10, sTNF-RII, IL-1Ra), acute phase proteins (CRP, PTX3) and other biomarkers (sST2, IL-1RAcP) was subsequently quantified. RESULTS: Multiple biomarkers capable of providing an innate immune signature of inflammation were readily detected directly ex vivo in healthy individuals. These biomarker levels were unaffected when comparing paired data sets from freshly obtained, never frozen plasma or serum and matched aliquots despite extensive freeze/thaw cycles. Neither age nor sex affected stability. Similarly, no quantitative differences were found following repetitive analysis of inflammatory biomarkers in culture samples obtained following in vitro stimulation with TLR and RLR ligands. CONCLUSIONS: A broad panel of in vivo and ex vivo cytokine, chemokine and acute phase protein biomarkers that have been linked to human chronic inflammatory disorders are readily detected in vivo and remain stable for analysis despite multiple freeze thaw cycles. These data provide the foundation and confidence for large scale analyses of panels of inflammatory biomarkers to provide better understanding of immunological mechanisms underlying health versus disease.

Limiting dilution analysis of antigen stimulated IL-4 and IFN-gamma production in human mononuclear cell populations.

Limiting dilution analysis (LDA) of fresh human mononuclear cell populations has previously been used to estimate the frequency of specific B cells, CTL, proliferative T cells, or cells capable of IL-2 production in various clinical situations. Such approaches evaluate the intensity of the response but provide little information concerning the balance between Th1- vs. Th2-like patterns of cytokine gene expression. Here, we describe development of an LDA method to obtain quantitative estimates of the frequency of antigen-specific IFN-gamma or IL-4 producing cells in human peripheral blood. The approach utilizes 3-4 day antigen-mediated restimulation of mononuclear cell populations freshly derived from grass pollen sensitive allergic rhinitis subjects. IFN-gamma and IL-4 production in culture supernatants are determined by ELISA and CT.h4S bioassay. Cytokine production elicited in this assay is CD4 dependent and antigen specific. As such, it provides a useful non-invasive approach for rapid evaluation of low frequency, antigen-induced cytokine production in the circulating repertoire. This method can readily be extended to analysis of other cytokines in other immunologic disorders or in infectious disease states, allowing longitudinal analysis of individuals and facilitating efforts to establish clear correlations between in vivo patterns of cytokine gene expression and disease exacerbation and remission.

Antigen-specific inhibition of ongoing murine IgE responses. II. Inhibition of IgE responses induced by treatment with glutaraldehyde-modified allergens is paralleled by reciprocal increases in IgG2a synthesis.

Administration of high m.w. glutaraldehyde-polymerized OVA (termed OVA-POL) before OVA-[A1(OH)3] immunization of C57BL/6 mice markedly impairs their capacity to generate OVA-specific IgE responses, while simultaneously resulting in striking enhancement of Ag-specific IgG2a responses. We demonstrate here that treatment with this class of chemically modified allergen also results in pronounced inhibition of ongoing IgE responses in vivo. The abrogation of well established murine IgE responses that is elicited after treatment with OVA-POL (i) is potent (97%), (ii) is long lived, and (iii) reflects reciprocal regulation of Ag-specific IgE and IgG2a responses in vivo. Moreover, the capacity of OVA-POL-treated mice to generate secondary IgE responses remains strongly decreased for at least 260 days and six subsequent immunizations with native allergen, despite there being no further treatment with modified allergen. These changes in IgE and IgG2a responsiveness are Ag specific and T cell dependent.

Long-lived reciprocal regulation of antigen-specific IgE and IgG2a responses in mice treated with glutaraldehyde-polymerized ovalbumin.

Previously, we discovered that administration of high Mr glutaraldehyde-polymerized ovalbumin (OA) to C57BL/6 mice prior to immunization with OA in A1(OH)3 adjuvant resulted in induction of an interferon-gamma (IFN-gamma) dependent, split tolerance in which maximal OA-specific IgE responses were 1-3% of those observed in saline-treated OA-[A1(OH)3] immunized controls. Concomitantly, these mice exhibited up to 10(3)-fold increases in OA-specific IgG2a synthesis. In this report we examine the longevity and resilience of these reciprocal effects on IgE inhibition/IgG2a enhancement over extended periods of time and following multiple re-exposures to the sensitizing allergen. The data indicate that the T-cell mediated changes in responsiveness which are induced upon exposure to glutaraldehyde-modified protein allergen, but not unmodified allergen, are (i) extremely long-lived (greater than 350 days); (ii) resistant to at least five re-immunizations with OA in A1(OH)3 adjuvant; and (iii) antigen-specific. The results are consistent with a virtually permanent shift in the OA-specific T-cell repertoire in vivo from one dominated by Th2-like patterns of cytokine synthesis (IL-4) to one dominated by Th1-like (IFN-gamma) cytokine production.

Immunochemical properties of some monoclonal IgE antibodies to 4-hydroxy-3-nitrophenylacetyl (NP).

Several hybridoma cell lines secreting NP-specific, murine IgE antibodies were generated by fusion of P3-X20 (gamma, kappa) tumour cells with spleen cells from (BALB/c X C57B1/6)F1 (CB6F1) mice previously immunized with NP-ovalbumin. Four subclones (designated NP-epsilon-3.57, NP-epsilon-15.88, NP-epsilon-91.58 and NP-epsilon-95.31) were propagated in vivo and milligram quantities of the corresponding IgE antibodies were purified from ascitic fluid by gel filtration, ion exchange chromatography and affinity chromatography. Immunological analyses and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that NP-epsilon-15.88, NP-epsilon-91.58 and NP-epsilon-95.31 all possessed lambda 1 (or possibly lambda 3) light chains; and that NP-epsilon-3.57 possessed lambda 2 light chains; NP-epsilon-95.31 also expressed the P3-X20 derived, MOPC-21 kappa light chain. Radioallergosorbent test (RAST) titration curves, generated from the interaction of the four monoclonal IgE antibodies with NP-BSA attached to paper discs (NP-BSA-P) were found to be non-overlapping. Measurements of the relative amounts of NP-epsilon-aminocaproic acid (NP-CAP) and 4-hydro-3-iodo-5-nitrophenylacetyl-epsilon-aminocaproic acid (NIP-CAP) that were required to inhibit by 50% the binding of the 4 IgE antibodies to NP-BSA-P indicated that these antibodies were all heteroclitic, since their affinity for NIP appeared to be higher than their affinity for NP. These results, in conjunction with other findings reported in the literature, suggested that the V regions of NP-specific IgE antibodies are similar to the V regions of NP-specific IgM and IgG antibodies, produced by the same mouse strains. Finally, in vitro histamine release measurements demonstrated that two of these monoclonal IgE antibodies could mediate antigen induced histamine release from passively sensitized rat peritoneal mast cells.

The suppression of insulin-specific immune responses by the administration of insulin-derivatized syngeneic cells and soluble lysates from these cells.

The results of this study demonstrated that the i.v. administration of insulin, in the form of a conjugate with either syngeneic spleen cells (SC) or peritoneal exudate cells (PEC), markedly reduced the capacity of recipient mice to develop insulin-specific immune responses, as manifested by diminished in vivo IgE antibody production and by depressed in vitro, lymph node cell proliferation responses, respectively. Furthermore, it was shown that i.v. injection of insulin-PEC conjugates induced the activation of suppressor cells that had the capacity to downregulate insulin-specific IgG plaque-forming cell (PFC) responses. Finally, it was also determined that freezing and thawing of the insulin-PEC conjugates resulted in the release of a soluble tolerogenic molecule and/or membrane preparation that could also markedly depress insulin-specific IgG antibody production.