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Ligand exchange processes at metal complexes underpin their reactivity and catalytic applications. While mechanisms of ligand exchange at single site complexes are well established, occurring through textbook associative, dissociative and interchange mechanisms, those involving heterometallic complexes are less well developed. Here we report the reactions of a well-defined Fe-Al dihydride complex with exogenous ligands (CO and CNR, R=Me, t Bu, Xyl=2,6-Me 2 C 6 H 3 ). Based on DFT calculations we suggest that these reactions occur through a dyotropic rearrangement, this involves initial coordination of the exogenous ligand at Al followed by migration to Fe, with simultaneous migration of a hydride ligand from Fe to Al. Such processes are rare for heterometallic complexes. We study the bonding and mechanism of the dyotropic rearrangement through in-depth computational analysis (NBO, IBOs, CLMO analysis, QTAIM, NCIplot, IGMH), shedding new light on how the electronic structure of the heterometallic core responds to the migration of ligands between metal sites. The dyotropic rearrangement fundamentally changes the nature of the hydride ligands, exposing new nucleophilic reactivity as evidenced by insertion reactions with CO 2 , isocyanates, as well as isocyanides., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Herein we present the first double deprotonation of acetonitrile (CH 3 CN) using two equivalents of a bimetallic iron-aluminium complex. The products of this reaction contain an exceeding simple yet rare [CHCN] 2- dianion moiety that bridges two metal fragments. DFT calculations suggest that the bonding to the metal centres occurs through heavily polarised covalent interactions. Mechanistic studies reveal the intermediacy of a monomeric [CH 2 CN] - complex, which has been characterised in situ. Our findings provide an important example in which a bimetallic metal complex achieves a new type of reactivity not previously encountered with monometallic counterparts. [1, 2] The isolation of a [CHCN] 2- dianion through simple deprotonation of CH 3 CN also offers the possibility of establishing a broader chemistry of this motif., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

An essential requirement for oral vaccines is the ability to survive the harsh environment of the stomach in an antigenically intact form. As bacteriophages are adapted to this environment we used epitope-displaying M13 bacteriophages as carriers for an experimental oral anti-IgE vaccine. The feasibility of this approach was tested in a simulated gastric fluid using two different mimotopes as well as an anti-idiotypic Fab of the non-anaphylactogenic monoclonal anti-IgE antibody BSW17. All phage clones remained infective after this treatment. However, only epitopes displayed on the pVIII protein were still recognized by BSW17 whereas pIII-expressed epitopes were rapidly inactivated. Surprisingly, when used for oral immunization of mice all phage clones induced anti-IgE antibodies. In contrast, oral immunization with the purified, pVIII protein displaying the mimotope induced anti-phage but no anti-IgE antibodies. After feeding a single dose of mimotope-displaying bacteriophage, phage DNA could be detected in mouse feces for 10 days. Our results show that epitope-displaying bacteriophages can be used to induce an epitope-specific antibody response via the oral route.

Antigen-specific human IgE is in short supply. Thus, we sought to determine the yet unknown specificity of a widely available human IgE, namely the myeloma cell line U266-derived IgE-ND. For this purpose highly specific peptides able to mimic the putative antigen recognized by IgE-ND were isolated from phage-display random peptide libraries. Interestingly, we found linear sequence homologies of the IgE-ND-binding peptides with self antigens and a xenoantigen from Thiobacillus ferrooxidans. However, none of these antigens was recognized by IgE-ND. Nevertheless, our approach may be applied to identify antigen specificities of myeloma antibodies. Importantly, the mimotopes were anaphylactogenic in a histamine release assay using human basophils sensitized with IgE-ND. Thus, our mimotopes represent functional albeit synthetic antigens and may be used to study human antigen-specific IgE responses.

Natural antibodies (Ab) reacting with self antigens have been shown to be present in all individuals. These autoantibodies (auto-Ab) can be either pathogenic or non-pathogenic. Auto-Ab reacting with the alpha-subunit of the high-affinity receptor for IgE (FcepsilonRIalpha) have been implicated in the pathogenesis of a subset of patients with chronic idiopathic urticaria (CIU). Intravenous immunoglobulin (IVIg) preparations have been used with variable clinical benefit in the treatment of these patients. Here we show that anti-FcepsilonRIalpha auto-Ab are present in a therapeutic IVIg preparation as well as in atopic and chronic urticaria patients and healthy individuals. We affinity-purified the anti-FcepsilonRIalpha Ab from an IVIg preparation using recombinant FcepsilonRIalpha. Interestingly, these anti-FcepsilonRIalpha auto-Ab showed no evidence of histamine release but strongly cross-reacted with an external antigen, tetanus toxoid (TTd) with a higher affinity for TTd than for the FcepsilonRIalpha. Since the cross-reacting Ab are non-anaphylactogenic, there is no evidence that TTd immunization may contribute to the pathogenesis of CIU. However, our results may indicate that the anti-FcepsilonRIalpha auto-Ab belong to the natural Ab and serve as the parental Ab for some anti-TTd Ab.

Birch pollen and mugwort pollen allergies are often associated with hypersensitivity to plant foods. This clinical and serological cross-reactivity is mediated by IgE antibodies reacting with homologous proteins in pollen and food. Cross-reacting homologs of the important birch pollen allergen Bet v 2 (profilin) could be detected in other pollen, fruits, nuts, and vegetables, such as celery tuber. We purified IgG/IgE antibodies from the serum of an exclusively profilin-allergic patient using affinity columns either coupled with protein extracts from mugwort pollen, birch pollen, or celery tuber. Constrained and unconstrained random nonapeptide libraries were pooled and screened with the anti-profilin antibody preparations to define cross-reactive ligands. Specific ligands were enriched by successive panning rounds using the profilin-specific antibodies in series. After the last panning round enriched phage clones were screened with purified profilin-specific antibodies and IgE-binding clones were sequenced. Five out of eight positive clones (62.5 %) displayed the same circular peptide CAISGGYPVC. This peptide was synthesized and examined for its ability to inhibit IgE binding to blotted mugwort pollen, birch pollen, or celery tuber profilin. Inhibition studies showed reduction of IgE binding to profilins in all three protein extracts. As the sequence of the mimotope did not show any homology to the known birch profilin sequence this peptide is considered to mimic a common conformational IgE epitope for these examined profilins.

We used a selection of well-characterized murine monoclonal anti-IgE antibodies to investigate their effect on human in vitro IgE synthesis. We found anti-IgE antibodies that either inhibited or enhanced interleukin-4 plus anti-CD40-induced in vitro IgE synthesis in peripheral blood mononuclear cells (PBMC). This differential activity was isotype specific as neither IgM nor IgG synthesis were affected. Interestingly, only coding IgE mRNA was down-regulated, whereas germ-line epsilon RNA expression was not influenced by anti-IgE monoclonal antibody (mAb). On purified B cells all anti-IgE mAb inhibited interleukin-4 plus anti-CD40-induced IgE synthesis, implying a role of non-B cells for the enhancing activity observed in PBMC. Using Fab and F(ab')2 of an inhibitory anti-IgE mAb we could show that divalent recognition was required for inhibition of IgE synthesis.

We determined the effect of adrenocorticotropin hormone (ACTH) on the regulation of IgE synthesis. Depending on the concentration, ACTH enhanced or inhibited IgE synthesis in a culture system where IgE synthesis was induced with interleukin-4 (IL-4) and anti-CD40 monoclonal antibody in peripheral blood mononuclear cells. Similar effects on IgE synthesis were observed by adding ACTH-related peptides, e.g. corticotropin-releasing factor (CRF), the inducer of ACTH, or alpha-melanocyte stimulating hormone (alpha-MSH), a cleavage product of ACTH. However, ACTH had no effect on IgG or IgM synthesis in this culture system. ACTH did not act directly on either B or T cells as there was no influence on IgE synthesis in a system using purified B cells alone or co-cultured with T cells. The effect of ACTH on IgE synthesis was mediated by accessory cells. This was shown by priming purified CD14-positive monocytes with ACTH and reconstitution experiments. Therefore, these findings suggest that ACTH and the related peptides CRF and alpha-MSH can influence the microenvironment modulating an IL-4 and anti-CD40 monoclonal antibody driven class switching to IgE via accessory cells.

Anti-IgE antibodies have been detected in sera of patients with an allergic disease where they might play a role in the regulation of (Ig)E-mediated reactions. Using a recombinant phage surface display expression system a combinatorial library of antibody heavy and light chains was constructed from peripheral blood mononuclear cells from an atopic donor immunized with tetanus toxoid. Screening of the library allowed the identification of a large number of phages displaying human monovalent antigen-binding fragments (Phab) against tetanus toxoid and IgE. Surprisingly, we found a high frequency of Phab against particular IgE myelomas that was comparable to the frequency found for Phab against tetanus toxoid. However, most of these Phab were directed to different idiotypic determinants, depending on the IgE myeloma used for the panning procedure. Nevertheless, two clones were found to have anti-isotypic specificity and were shown to react specifically with the CH2 domain of the IgE heavy chain.

Expression of human IgE mRNA by peripheral blood lymphocytes (PBL) and an IgE-producing myeloma cell line, U-266, was examined by Northern blot hybridization and compared with IgE levels in culture supernatants. A 2.35-kb IgE mRNA was detected in unstimulated atopic PBL and U-266 cells but not in normal PBL, and its levels correlated with IgE protein levels in the supernatant. Upon stimulation with interleukin 4, a new 1.75-kb transcript was revealed in both atopic and normal PBL but not in U-266 cells. Its expression did not correlate with IgE levels in the supernatant. Pokeweed mitogen also induced the expression of the 1.75-kb transcript without concomitant induction of IgE synthesis by normal PBL and even suppressed the spontaneous expression of the 2.35-kb transcript and IgE protein synthesis by atopic PBL. Interferon-gamma, which suppressed both the 2.35-kb transcript and IgE protein production, had no inhibitory effect on the 1.75-kb transcript. Expression of the 1.75-kb transcript was already high after 2 days of stimulation and peaked around day 4. The length of the transcript is smaller than that of mRNA coding for secreted human IgG and IgA and contains all four C epsilon exon sequences, suggesting it might be a truncated transcript without v region and might be a human counterpart of the murine germ-line C epsilon transcript.

This study shows that both recombinant human interleukin (rhIL)3 and rhIL4 induced proliferation in bone marrow (BM) cells of myelogenous leukemia patients in a manner similar to that reported using normal BM cells. However, we additionally found that these cytokines also influenced expression of other cytokines. Namely, using a reproducible dot blot hybridization technique we observed on the one hand that BM cells were capable of constitutively expressing low levels of cytokine mRNA coding for IL3, IL4, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte (G)-CSF and IL 1 beta, and on the other hand that in normal peripheral blood mononuclear cells rhIL4 inhibited mRNA expression coding for GM-CSF, G-CSF, IL3 and IL 1 beta, while IL4 mRNA and 28S rRNA was not affected. In contrast, rhIL3 marginally enhanced mRNA coding for IL3, GM-CSF, G-CSF and IL 1 beta and counteracted the inhibitory effect of IL4. In long-term cultures rhIL3 and rhIL4 had no significant effect on spontaneous cytokine gene expression of myelogenous leukemia-derived peripheral blood or BM cells, but made these cells more sensitive for subsequent stimulation with different polyclonal stimuli. Thus, IL3 and IL4 already modulate cytokine gene expression during the initiation of cell culture and differentiate BM cells into populations of cells which are capable of responding with an enhanced cytokine gene expression after polyclonal stimulation.

Mitogens such as pokeweed mitogen, Staphylococcus aureus Cowan I, concanavalin A and phytohemagglutinin and monoclonal anti-CD antibodies were examined for their capacity to induce IgE synthesis by peripheral blood leukocytes obtained from both atopic and nonatopic individuals. While lectins failed to induce IgE synthesis, monoclonal anti-CD3 antibodies were very potent stimuli for the induction of human in vitro IgE synthesis, possibly due to the activation of T cells. Activation via CD4 or CD8 molecules by OKT4 or OKT8 antibodies did not lead to a T cell-dependent modulation of IgE synthesis. PWM acted in synergy with the enhancing effect of anti-CD3 antibodies for IgE synthesis. In vitro IgG production was less affected. These results indicate that activation of T cell via CD3 molecules may be important in the regulation of IgE immune responses in man. Furthermore, the successful induction of IgE synthesis by anti-CD3 antibody in unfractionated peripheral blood leukocytes culture provides a simple model for investigation of human IgE regulatory mechanism in vitro.

In the present study, interleukin 1 (IL 1)-containing media from different sources, namely a murine macrophage cell line (P388D1), rabbit peritoneal macrophages, and human peripheral blood mononuclear cells, were compared for their effect on thymocyte proliferation and on collagenase and PGE2 secretion by chondrocytes. A high correlation was found between the enhancement of thymocyte proliferation and the induction of collagenase and PGE2 secretion by chondrocytes. Furthermore, a highly purified IL 1-like factor, namely mononuclear cell factor (MCF) was also active on chondrocytes. The addition of highly purified IL 2 to rabbit chondrocytes had no effect on collagenase and PGE2 secretion induced by IL 1-containing media. Our findings suggest that the factor which induced collagenase and PGE2 secretion by rabbit chondrocytes was an IL 1-like factor. Thus, collagenase secretion by chondrocytes may be used as an IL 2-insensitive assay for the detection of IL 1-like factors.

Recombinant human interleukin 4 (IL 4) alone enhanced the spontaneous IgE synthesis in cultures of peripheral blood leukocytes (PBL) from atopic patients as well as from nonatopic individuals, suggesting the existence of preactivated PBL sensitive for IL 4. Preactivated cells were also obtained by stimulation with Staphylococcus aureus strain Cowan I (SAC). However, co-stimulation of PBL by IL 4 with SAC or anti-IgM antibody and pokeweed mitogen did not result in an enhanced IgE synthesis. Optimal IL 4 concentrations for the induction of IgE synthesis coincided with optimal proliferative responses in PBL. The effect of IL 4 was not isotype specific, and in terms of protein even more IgG and IgM antibodies were formed. The effect of IL 4 on IgE synthesis was counteracted by very low concentrations of interferon-gamma (IFN-gamma), suggesting that both IL 4 and IFN-gamma might be decisive cytokines for the human in vitro IgE synthesis.

The relative RNA content of single macrophages was measured by cytofluorometry after differential staining of cellular DNA and RNA with the metachromatic fluorescent dye acridine orange. This method allowed the differentiation of two major groups in the adherent macrophage population differing in their RNA content. After in vivo stimulation of mice (by injection of thioglycollate medium) or guinea pigs (by injection of oil), an increasing percentage of macrophages possessing a high RNA content is recovered. In vitro stimulation of macrophage cultures with lipopolysaccharide had the same enhancing effect on cellular RNA content. Cytofluorometric measurement of RNA content may possibly become an efficient and rapid method of measuring macrophage activation.

Levels of cytokine mRNA coding for granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), interleukin (IL) 2, IL 1 beta, IL 4 or IL 6 have been measured by Northern blot analysis after antigen stimulation. As source for RNA we used peripheral blood mononuclear cells (PBMC) from donors which showed a proliferative response after tetanus toxoid or Candida albicans stimulation. For comparison PBMC were also stimulated with lectins and anti-CD3 antibody. With some variations among donors, antigens clearly induced measurable levels of IFN-gamma, GM-CSF and IL 2 mRNA. Increased levels for IL 6 were also detected after antigen stimulation. In contrast to polyclonal T cell stimuli, antigens showed delayed kinetics of mRNA steady-state levels and resembled in this respect more closely the stimulation with pokeweed mitogen. Thus, cytokine mRNA levels may be assessed in unfractionated PBMC after antigen stimulation. The two tested antigens also clearly show a cytokine pattern distinct from that induced in polyclonal stimulations such as anti-CD3.

Steady-state mRNA levels for lymphokines were studied in human peripheral blood mononuclear cells stimulated by various agents. The pattern of mRNA levels differed according to the way in which cells were stimulated or activated. Stimulation by anti-CD3 antibodies led to increased mRNA levels for interferon-gamma but only extremely low levels of interleukin 2 mRNA were detected. In order to observe levels of interleukin 2 mRNA, it was necessary to use phorbol myristate acetate (PMA) in addition to either lectins or anti-CD3 antibodies. PMA by itself was already able to induce high levels of mRNA for granulocyte macrophage colony-stimulating factor. Most interestingly, different patterns of lymphokine production were obtained with stimuli yielding the same number of activated cells and the same ratio of activated CD4 and CD8 cells. Thus, different T cell stimuli induced distinct patterns of lymphokine mRNA levels.