Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis.

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (CH 1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between VH -CH 1 and VL -CL was improved by the CH 1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91-62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.

A role for inflammatory mediators in heterologous desensitization of CysLT1 receptor in human monocytes.

Cysteinyl-leukotrienes (cysteinyl-LT) are rapidly generated at sites of inflammation and, in addition to their role in asthma, rhinitis, and other immune disorders, are increasingly regarded as significant inflammatory factors in cancer, gastrointestinal, cardiovascular diseases. We recently demonstrated that in monocyte/macrophage-like U937 cells, extracellular nucleotides heterologously desensitize CysLT(1) receptor (CysLT(1)R)-induced Ca(2+) transients. Given that monocytes express a number of other inflammatory and chemoattractant receptors, this study was aimed at characterizing transregulation between these different stimuli. We demonstrate that in U937 cells and in primary human monocytes, a series of inflammatory mediators activating G(i)-coupled receptor (FPR1, BLT(1)) desensitize CysLT(1)R-induced Ca(2+) response unidirectionally through activation of PKC. Conversely, PAF-R, exclusively coupled to G(q), cross-desensitizes CysLT(1)R without the apparent involvement of any kinase. Interestingly, G(s)-coupled receptors (beta(2)AR, H(1/2)R, EP(2/4)R) are also able to desensitize CysLT(1)R response through activation of PKA. Heterologous desensitization seems to affect mostly the G(i)-mediated signaling of the CysLT(1)R. The hierarchy of desensitization among agonists may be important for leukocyte signal processing at the site of inflammation. Considering that monocytes/macrophages are likely to be the major source of cysteinyl-LT in many immunological and inflammatory processes, shedding light on how their receptors are regulated will certainly help to better understand the role of these cells in orchestrating this complex network of integrated signals.

Bacterial lipopolysaccharide-stimulated release of tumor necrosis factor-alpha from the isolated rat heart: the effect of aprotinin and forskolin.

Aprotinin has been reported to reduce plasma levels of inflammatory cytokines associated with cardiopulmonary bypass (CPB). Because CPB is also associated with elevated levels of bacterial lipopolysaccharide (LPS) and LPS stimulates release of inflammatory cytokines from the heart we tested the hypothesis that aprotinin would inhibit cardiac release of tumor necrosis factor-alpha (TNF) provoked by LPS. Isolated rat hearts were perfused Langendorf style. After 30 minutes of equilibration LPS (100 ng/mL) was infused for 60 minutes. Timed samples of coronary effluent were collected at 0, 30, 60, 90, 120, and 150 minutes after the initiation of LPS for the measurement of coronary flow and the determination of TNF and cyclic AMP. Other hearts were perfused with buffer containing aprotinin [137 kallikrein-inhibiting units (KIU)/mL or 250 KIU/mL] and then infused with LPS. An additional group received forskolin (10 microM) and LPS. In hearts perfused as controls with buffer alone no TNF was detected in the coronary effluent. In hearts perfused with LPS TNF was reliably detected in the coronary effluent at 60 minutes (606 +/- 450 pg/min) and increased with time to a level of 1792 +/- 650 pg/min at 150 minutes. The addition of aprotinin had no significant effect on LPS-stimulated TNF release. For instance in hearts perfused with 137 KIU/mL aprotinin LPS-stimulated release at 150 minutes was 2141 +/- 732 pg/min and in hearts perfused with 250 KIU/mL LPS-stimulated TNF release was 2049 +/- 789 pg/min. Forskolin administration was associated with release of cyclic AMP from the heart and completely inhibited LPS-stimulated TNF release. We conclude that LPS stimulated release of TNF from the heart. Adding aprotinin to the perfusion buffer in either high or low concentrations did not attenuate LPS-stimulated cytokine release. Elevating myocardial cyclic AMP with forskolin completely attenuated LPS-stimulated TNF release.

The induction of systemic and mucosal immune responses to antigen-adjuvant compositions administered into the skin: alterations in the migratory properties of dendritic cells appears to be important for stimulating mucosal immunity.

The properties of various vaccine-adjuvant formulations that are capable of inducing both systemic and common mucosal immunity subsequent to their intradermal administration are described. Effective mucosal adjuvants, including bacterial toxins, chemical enhancers of cyclic AMP, and the active form of vitamin D3, all shared the ability to promote dendritic cell migration from the skin to Peyer's patches subsequent to antigen induced maturation. Our data suggests that skin dendritic cells may function as effective antigen presenting cells for the induction of mucosal immune responses, if microenvironmental conditions are appropriately manipulated subsequent to their stimulation by antigen.

Requirements for the induction of the interleukin-2 receptor complex in a human pre-T-cell line.

Cell line PER-117 is a T-cell receptor negative human T-cell line that can be induced to express a functional interleukin-2 receptor (IL-2R). Recombinant interleukin-1 (IL-1) as well as certain combinations of inducer substances could be shown to stimulate the expression of the p55 (alpha)-chain of the IL-2R in PER-117 cells. The synergistic increases in IL-2R alpha expression were demonstrated at the cell surface as well as at the mRNA level. The results suggested that in PER-117 cells IL-1 appears to induce expression of the alpha-chain by pathways that are different to activation via protein kinase C (PKC), and that drug-induced cyclic AMP (cAMP) activation did not substitute for IL-1. We found that the regulation of mRNA for IL-2R beta (p75) differed significantly from that seen for IL-2R alpha. Moreover, the requirements for IL-2R alpha induction determined for this cell line differ from other human cell lines, which may reflect that there are distinct requirements for activation depending on the stage of differentiation and/or lineage of the cells. The PER-117 cell line provides a unique model to examine further the mechanism leading to induction of a functional IL-2R at an early stage of human T-cell differentiation.

Phorbol ester, prostaglandin E2, forskolin and okadaic acid differentially modulate interleukin-4-versus interleukin-2-dependent immunoglobulin induction in human cellular models, in contrast to other selected modifiers of cellular activation.

Interleukin 2 (IL2) and 4 (IL4) are the most important mediators for immunoglobulin (Ig) synthesis of human B lymphocytes. There is no obvious difference with regard to Ig isotypes induced by either lymphokine except for IgE: only IL4 induces this allergic antibody type. Monoclonal anti-CD40 antibodies enhance both IL2- and IL4-dependent Ig induction. Searching for drugs which may inhibit induction of IgE but not of rather non-pathogenic immunoglobulins, we selected commercial compounds which are commonly used as probes for transmembrane signalling pathways in other cellular systems. They included modulators of protein kinase C and intracellular calcium, inducers of cAMP, and inhibitors of protein tyrosine kinase, protein serine/threonine phosphatases and phosphodiesterases. The data presented suggest that IL2- and IL4-mediated B cell activation can be differentially modulated. Phorbol ester at non-cell-toxic doses inhibited IL4- but not IL2-dependent Ig induction. Prostaglandin E2 potently enhanced IgE production stimulated with IL4 alone but was inhibitory in the presence of anti-CD40 as a co-stimulatory signal. IgG1 responses elicited with IL2 plus anti-CD40, in contrast, were not affected. All other compounds did not discriminate between IL2- versus IL4-mediated Ig induction.

Production and assay of antibodies to an activator of adenylate cyclase, forskolin.

Forskolin is a unique diterpene activator of adenylate cyclase which has been extensively used in the study of cAMP generating systems. This report describes the production of antibodies to forskolin and the optimization of two sensitive assay methods for such antibodies. 7-0-Hemisuccinyl 7-deacetyl forskolin, coupled to either human serum albumin or goat IgG, was injected into goats to elicit antibodies to the forskolin hapten. Two assay methods, a radioimmunoassay with [12-3H]forskolin as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) with horse radish peroxidase-labelled rabbit anti-goat IgG as an indicator, were optimized to test for the presence of forskolin antibodies in antisera and isolated IgG fractions. The titers for forskolin antisera were 4000-10000. Both assay methods can be adapted to quantify forskolin and its protein conjugates. The availability of antibodies to this diterpene will be useful in accelerating the understanding of the mechanism of adenylate cyclase activation by forskolin.