IL-3 but not monomeric IgE regulates FcεRI levels and cell survival in primary human basophils.

Binding of allergen-specific IgE to its primary receptor FcεRI on basophils and mast cells represents a central event in the development of allergic diseases. The high-affinity interaction between IgE and FcεRI results in permanent sensitization of these allergic effector cells and critically regulates their release of pro-inflammatory mediators upon IgE cross-linking by allergens. In addition, binding of monomeric IgE has been reported to actively regulate FcεRI surface levels and promote survival of mast cells in the absence of allergen through the induction of autocrine cytokine secretion including interleukin-3 (IL-3). As basophils and mast cells share many biological commonalities we sought to assess the role of monomeric IgE binding and IL-3 signaling in FcεRI regulation and cell survival of primary human basophils. FcεRI cell surface levels and survival of isolated blood basophils were assessed upon addition of monomeric IgE or physiologic removal of endogenous cell-bound IgE with a disruptive IgE inhibitor by flow cytometry. We further determined basophil cell numbers in both low and high serum IgE blood donors and mice that are either sufficient or deficient for FcεRI. Ultimately, we investigated the effect of IL-3 on basophil surface FcεRI levels by protein and gene expression analysis. Surface levels of FcεRI were passively stabilized but not actively upregulated in the presence of monomeric IgE. In contrast to previous observations with mast cells, monomeric IgE binding did not enhance basophil survival. Interestingly, we found that IL-3 transcriptionally regulates surface levels of FcεRI in human primary basophils. Our data suggest that IL-3 but not monomeric IgE regulates FcεRI expression and cell survival in primary human basophils. Thus, blocking of IL-3 signaling in allergic effector cells might represent an interesting approach to diminish surface FcεRI levels and to prevent prolonged cell survival in allergic inflammation.

A revival of parabiosis in biomedical research.

Modern medicine wields the power to treat large numbers of diseases and injuries most of us would have died from just a hundred years ago, yet many of the most devastating diseases of our time are still untreatable. Chronic conditions of age such as cardiovascular disease, diabetes, osteoarthritis or Alzheimer's disease turn out to be of a complexity that may require transformative ideas and paradigms to understand and treat them. Parabiosis, which is characterised by a shared blood supply between two surgically connected animals, may just provide such a transformative experimental paradigm. Although forgotten and shunned now in many countries, it has contributed to major breakthroughs in tumour biology, endocrinology and transplantation research in the past century. Interestingly, recent studies from the United States and Britain are reporting stunning advances in stem cell biology and tissue regeneration using parabiosis between young and old mice, indicating a possible revival of this paradigm. We review here briefly the history of parabiosis and discuss its utility to study physiological and pathophysiological processes. We argue that parabiosis is a technique that should enjoy wider acceptance and application, and that policies should be revisited to allow its use in biomedical research.

DARPins against a functional IgE epitope.

The monoclonal anti-IgE antibody omalizumab (Xolair is mostly used for the treatment of severe allergic asthma. However, the requirement of high doses and suboptimal cost-effectiveness limits the use of the treatment. Here we propose to use a new drug format based on non-immunoglobulin structures, potentially offering increased clinical efficacy while being more cost-effective. For this purpose, DARPins™ (designed ankyrin repeat proteins) against the constant heavy chain region of IgE have been isolated. DARPins were binding to IgE with high specificity and affinities in the low nanomolar range. Selected DARPins antagonized the interaction between IgE and its high-affinity receptor in inhibition assays. Furthermore, anti-IgE DARPins were shown to inhibit proinflammatory mediator release from rat basophilic leukemia cells expressing human high-affinity IgE receptors with higher efficacy than the monoclonal anti-IgE antibody omalizumab. DARPins may thus represent promising future drug candidates for the treatment of allergy.

DARPins as bispecific receptor antagonists analyzed for immunoglobulin E receptor blockage.

The concept of multispecific antibodies is of high therapeutic interest but has failed to produce pharmaceutical products due to the poor biophysical properties of such molecules. Here, we propose an alternative and simple way to generate bispecific binding molecules using designed ankyrin repeat proteins (DARPins). For this purpose, monovalent DARPins with different epitope specificities were selected against the alpha chain of the high-affinity receptor for human immunoglobulin E (IgE) (FcepsilonRIalpha). Two of the isolated binders interfering with IgE binding to the receptor were joined to each other or to themselves via a flexible protein linker. The resulting bivalent and bispecific DARPins were tested for their ability to prevent allergen-induced cell degranulation using rat basophilic leukemia cells stably transfected with human FcepsilonRIalpha. The bispecific DARPin construct was the most potent one, efficiently blocking the IgE-FcepsilonRI interaction and preventing the release of proinflammatory mediators. Noteworthy, the multivalent and multispecific DARPin construct did not show any alteration of the beneficial biophysical properties of the monovalent parental DARPins. Hence, bispecific DARPins may be used to generate receptor antagonists simultaneously targeting different epitopes on the same molecule. Moreover, they easily overcome the limiting immunoglobulin binding paradigm (one binding molecule=one epitope) and thereby represent an alternative to monoclonal antibodies in cases where the immunoglobulin scaffold is unsuitable.