AllergoOncology: ultra-low IgE, a potential novel biomarker in cancer-a Position Paper of the European Academy of Allergy and Clinical Immunology (EAACI).

Elevated serum IgE levels are associated with allergic disorders, parasitosis and specific immunologic abnormalities. In addition, epidemiological and mechanistic evidence indicates an association between IgE-mediated immune surveillance and protection from tumour growth. Intriguingly, recent studies reveal a correlation between IgE deficiency and increased malignancy risk. This is the first review discussing IgE levels and links to pathological conditions, with special focus on the potential clinical significance of ultra-low serum IgE levels and risk of malignancy. In this Position Paper we discuss: (a) the utility of measuring total IgE levels in the management of allergies, parasitosis, and immunodeficiencies, (b) factors that may influence serum IgE levels, (c) IgE as a marker of different disorders, and d) the relationship between ultra-low IgE levels and malignancy susceptibility. While elevated serum IgE is generally associated with allergic/atopic conditions, very low or absent IgE may hamper anti-tumour surveillance, indicating the importance of a balanced IgE-mediated immune function. Ultra-low IgE may prove to be an unexpected biomarker for cancer risk. Nevertheless, given the early stage of investigations conducted mostly in patients with diseases that influence IgE levels, in-depth mechanistic studies and stratification of malignancy risk based on associated demographic, immunological and clinical co-factors are warranted.

An immunologically relevant rodent model demonstrates safety of therapy using a tumour-specific IgE.

BACKGROUND: Designing biologically informative models for assessing the safety of novel agents, especially for cancer immunotherapy, carries substantial challenges. The choice of an in vivo system for studies on IgE antibodies represents a major impediment to their clinical translation, especially with respect to class-specific immunological functions and safety. Fcε receptor expression and structure are different in humans and mice, so that the murine system is not informative when studying human IgE biology. By contrast, FcεRI expression and cellular distribution in rats mirror that of humans. METHODS: We are developing MOv18 IgE, a human chimeric antibody recognizing the tumour-associated antigen folate receptor alpha. We created an immunologically congruent surrogate rat model likely to recapitulate human IgE-FcεR interactions and engineered a surrogate rat IgE equivalent to MOv18. Employing this model, we examined in vivo safety and efficacy of antitumour IgE antibodies. RESULTS: In immunocompetent rats, rodent IgE restricted growth of syngeneic tumours in the absence of clinical, histopathological or metabolic signs associated with obvious toxicity. No physiological or immunological evidence of a "cytokine storm" or allergic response was seen, even at 50 mg/kg weekly doses. IgE treatment was associated with elevated serum concentrations of TNFα, a mediator previously linked with IgE-mediated antitumour and antiparasitic functions, alongside evidence of substantially elevated tumoural immune cell infiltration and immunological pathway activation in tumour-bearing lungs. CONCLUSION: Our findings indicate safety of MOv18 IgE, in conjunction with efficacy and immune activation, supporting the translation of this therapeutic approach to the clinical arena.

AllergoOncology: Opposite outcomes of immune tolerance in allergy and cancer.

While desired for the cure of allergy, regulatory immune cell subsets and nonclassical Th2-biased inflammatory mediators in the tumour microenvironment can contribute to immune suppression and escape of tumours from immunological detection and clearance. A key aim in the cancer field is therefore to design interventions that can break immunological tolerance and halt cancer progression, whereas on the contrary allergen immunotherapy exactly aims to induce tolerance. In this position paper, we review insights on immune tolerance derived from allergy and from cancer inflammation, focusing on what is known about the roles of key immune cells and mediators. We propose that research in the field of AllergoOncology that aims to delineate these immunological mechanisms with juxtaposed clinical consequences in allergy and cancer may point to novel avenues for therapeutic interventions that stand to benefit both disciplines.

Topical ivermectin improves allergic skin inflammation.

BACKGROUND: Ivermectin (IVM) is widely used in both human and veterinary medicine to treat parasitic infections. Recent reports have suggested that IVM could also have anti-inflammatory properties. METHODS: Here, we investigated the activity of IVM in a murine model of atopic dermatitis (AD) induced by repeated exposure to the allergen Dermatophagoides farinae, and in standard cellular immunological assays. RESULTS: Our results show that topical IVM improved allergic skin inflammation by reducing the priming and activation of allergen-specific T cells, as well as the production of inflammatory cytokines. While IVM had no major impact on the functions of dendritic cells in vivo and in vitro, IVM impaired T-cell activation, proliferation, and cytokine production following polyclonal and antigen-specific stimulation. CONCLUSION: Altogether, our results show that IVM is endowed with topical anti-inflammatory properties that could have important applications for the treatment of T-cell-mediated skin inflammatory diseases.

AllergoOncology - the impact of allergy in oncology: EAACI position paper.

Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both antitumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE-mediated immune responses against cancer, to derive novel insights into more effective treatments. Coincident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state-of-the-art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intratumoral innate allergy effector cells and mediators, IgE-mediated tumour antigen cross-presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the crosstalk between allergic response and cancer is paving the way for new avenues of treatment.

Natural killer cells and mast cells from gp49B null mutant mice are functional.

Immune responses are controlled by a combination of positive and negative cellular signals. Effector cells in the immune system express inhibitory receptors that serve to limit effector cell expansion and to protect the host from autoreactivity. gp49B is a receptor of unknown function that is expressed on activated mast cells and natural killer (NK) cells and whose cytoplasmic tail endows it with inhibitory potential. To gain insight into the function of gp49B in mice, we disrupted the gp49B gene by homologous recombination. gp49B(0) mice were born at expected ratios, were healthy and fertile, and displayed normal long-term survival rates. gp49B(0) mice showed no defect in NK or mast cell development. Furthermore, NK and mast cells from the gp49B(0) mice showed activation properties in vitro similar to those of cells isolated from wild-type mice. Therefore, gp49B is not critical for the development, expansion, and maturation of mast cells and NK cells in vivo. The healthy status of gp49B(0) mice makes them suitable for testing the role of gp49B in immune responses to infectious agents.

Expression of a functional Fc epsilon RI on rat eosinophils and macrophages.

Besides its crucial role in type I hypersensitivity reactions, IgE is involved in anti-parasite immunity. This role has been clearly demonstrated in both human and rat schistosomiasis, but remains controversial in the mouse. Since the cellular distribution of the high affinity IgE receptor, Fc epsilon RI, differs in humans and mice, it might explain the differences in effector function of IgE between the two species. In humans, eosinophils and macrophages induce IgE-dependent cytotoxicity toward Schistosoma mansoni larvae, which involves Fc epsilon RI in the case of eosinophils. In the present study, we have investigated the expression and function of Fc epsilon RI in rat eosinophils and macrophages. We demonstrate, by flow cytometry, fluorescence microscopy, and western blot analysis, that in rats, as in humans, a functional alpha gamma 2 trimeric Fc epsilon RI is expressed on eosinophils and macrophages. We also show that these two cell types can induce IgE-mediated, Fc epsilon RI-dependent cellular cytotoxicity toward schistosomula. These results thus provide a molecular basis for the differences observed between rat and mouse regarding IgE-mediated anti-parasite immunity.

Expression of CD28 and CD86 by human eosinophils and role in the secretion of type 1 cytokines (interleukin 2 and interferon gamma): inhibition by immunoglobulin a complexes.

Eosinophils are the source of various immunoregulatory cytokines, but the membrane molecules involved in their secretion have not been clearly identified. Here we show that peripheral blood eosinophils from hypereosinophilic patients could express membrane CD86 but not CD80. The T cell costimulatory molecule CD28 is also detected on the eosinophil surface. CD28 ligation but not CD86 ligation resulted in interleukin (IL)-2 and interferon (IFN)-gamma secretion by eosinophils, whereas IL-4, IL-5, and IL-10 were not detected. In contrast to T cells requiring two signals for effective stimulation, CD28 ligation alone was sufficient for optimal eosinophil activation. Eosinophil-derived IL-2 and IFN-gamma were biologically active, as supernatants from anti-CD28-treated cells were able to induce CTLL-2 proliferation and major histocompatibility complex class II expression on the colon carcinoma cell line Colo 205, respectively. Addition of secretory immunoglobulin (Ig)A-anti-IgA complexes, which could induce the release of IL-10, very significantly inhibited both CD28-mediated IL-2 and IFN-gamma release. These results suggest that the release of type 1 (IFN-gamma and IL-2) versus type 2 cytokines by eosinophils is not only differential but also dependent on cross-regulatory signals. They confirm that through activation of costimulatory molecules, eosinophils could function as an immunoregulatory cell involved in the release of both type 1 and type 2 cytokines.

Allergy-associated FcRbeta is a molecular amplifier of IgE- and IgG-mediated in vivo responses.

A role for the Fc receptor beta chain (FcRbeta) in the pathogenesis of allergy has been suggested by genetic studies. FcRbeta is a subunit common to the high-affinity IgE (FcepsilonRI) and low-affinity IgG (FcgammaRIII) receptors, both of which contribute to the initiation of allergic reactions. Current in vitro data suggest that FcRbeta can function as either a positive or negative regulator, leaving a mechanistic explanation for its association with the development of atopy unclear. To address this controversy, we have generated novel mouse models relevant to human Fc receptor function. Analysis of FcepsilonRI- and FcgammaRIII-dependent responses in these mice provides unequivocal genetic evidence that FcRbeta functions as an amplifier of early and late mast cell responses and, remarkably, in vivo anaphylactic responses.

Fc epsilonRI-deficient mice infected with Schistosoma mansoni mount normal Th2-type responses while displaying enhanced liver pathology.

The IgE/Fc epsilonRI interaction is postulated to play an important role in resistance to helminths both at the level of anti-parasitic effector cell function and in the initiation of Th2 responses through IL-4 produced by Fc epsilonRI+ non-B, non-T (NBNT) cells. To formally evaluate the role of IgE/Fc epsilonRI signaling in the host response to helminths we studied Schistosoma mansoni infection in Fc epsilonRI knockout (KO) mice. Infected wild-type (wt) and KO animals showed comparable adult worm and tissue egg burdens, arguing against a role for Fc epsilonRI interactions in host resistance. Significantly, NBNT cells from infected KO, in contrast to wt animals, did not secrete IL-4 when stimulated with anti-IgE Ab or soluble parasite Ag. Nevertheless, serum IgE levels and Th2 cytokine production profiles were comparable in both strains of mice, demonstrating that the Ag-dependent stimulation of IL-4 secretion by NBNT cells is not essential for helminth-induced Th2 differentiation. However, when stimulated with low Ag doses, splenocytes from infected Fc epsilonRI-deficient mice produced less IL-4 in vitro than similar cultures from infected wt animals, an effect attributable to their defective NBNT cell function. Moreover, infected KO mice showed enhanced egg granuloma formation and hepatic fibrosis, revealing that the IgE/Fc epsilonRI interaction, while not essential for Th2 response development or resistance to primary infection, plays a significant role in down-regulating host pathology.

Absence of Fc epsilonRI alpha chain results in upregulation of Fc gammaRIII-dependent mast cell degranulation and anaphylaxis. Evidence of competition between Fc epsilonRI and Fc gammaRIII for limiting amounts of FcR beta and gamma chains.

In mouse mast cells, both Fc epsilonRI and Fc gammaRIII are alpha beta gamma2 tetrameric complexes in which different alpha chains confer IgE or IgG ligand recognition while the signaling FcR beta and gamma chains are identical. We used primarily noninvasive techniques (changes in body temperature, dye extravasation) to assess systemic anaphylactic responses in nonanesthetized wild-type, Fc epsilonRI alpha chain -/- and FcR gamma chain -/- mice. We confirm that systemic anaphylaxis in mice can be mediated largely through IgG1 and Fc gammaRIII and we provide direct evidence that these responses reflect activation of Fc gammaRIII rather than Fc gammaRI. Furthermore, we show that Fc gammaRIII-dependent responses are more intense in normal than in congenic mast cell-deficient KitW/KitW-v mice, indicating that Fc gammaRIII responses have mast cell-dependent and -independent components. Finally, we demonstrate that the upregulation of cell surface expression of Fc gammaRIII seen in Fc epsilonRI alpha chain -/- mice corresponds to an increased association of Fc gammaRIII alpha chains with FcR beta and gamma chains and is associated with enhanced Fc gammaRIII-dependent mast cell degranulation and systemic anaphylactic responses. Therefore, the phenotype of the Fc epsilonRI alpha chain -/- mice suggests that expression of Fc epsilonRI and Fc gammaRIII is limited by availability of the FcR beta and gamma chains and that, in normal mice, changes in the expression of one receptor (Fc epsilonRI) may influence the expression of functional responses dependent on the other (Fc gammaRIII).

Pituitary control of proliferation and differentiation of Leydig cells and their putative precursors in immature hypophysectomized rat testis.

The objective of this study was to determine the effects of pituitary hormones (luteinizing hormone [LH], follicle-stimulating hormone [FSH], growth hormone [GH], and prolactin [PRL]) on interstitial cell proliferation and differentiation in the testis of immature hypophysectomized rats. Macrophages, Leydig cells, precursor mesenchymal cells, endothelial lymphatic cells, and myoid cells were studied. Our experimental approach was aimed at determining whether changes in a cellular subpopulation observed after pituitary hormone treatments were the result of division of existing cells in the population, of differentiation of interstitial precursor cells, or both. In this context, it must be stressed that our data reflected the effects of hormones to prevent the decline of cells due to hypophysectomy rather than their recovery. Macrophage proliferation was taken into account because macrophages closely resemble Leydig cells and are known to proliferate after hormonal treatment. A double-labeling procedure (acid phosphatase and anti-bromodeoxyuridine [anti-BUdR]) revealed that LH, FSH, and PRL increased the number of testicular macrophages 105-, 104-, and 103-fold, respectively, in hypophysectomized rats compared to hypophysectomized control animals. BUdR incorporation in testicular macrophages was greater after PRL treatment than after LH and FSH supplementation. In contrast, we were unable to demonstrate any effect of rat GH on the macrophage population. Light microscopic analysis of plastic embedded sections of treated rat testis revealed that LH increased the numbers of Leydig, precursor mesenchymal, and myoid cells 6-, 4-, and 1.3-fold, respectively. LH also stimulated BUdR incorporation into all interstitial cell types. PRL administration increased both the number of Leydig and precursor mesenchymal cells (each 3-fold) but decreased the number of endothelial lymphatic cells (1.5-fold) when compared to the control animals. In contrast, FSH did not increase the number and proliferation of Leydig cells but exerted a slight proliferative effect on the other interstitial cell populations. In GH-treated rats, the number of precursor mesenchymal cells increased two fold above the control rats. GH also exerted slight proliferative effects on both precursor mesenchymal and myoid cells. Immunohistochemical studies of steroidogenic enzymes in the testicular interstitium of treated rats demonstrated the presence of steroidogenic enzymes, not only in Leydig and precursor mesenchymal cells, but also in some (1%-2%) endothelial lymphatic cells and myoid cells. This may indicate that both of these cell types are also constitutively equipped to perform steroidogenesis or that they are precursor cells undergoing differentiation. Taken together, changes in the number of Leydig cells in our animal model appeared more likely to be dependent on the transformation of precursor cells than on division of preexisting mature Leydig cells.

Benign prostatic hyperplasia and normal prostate aging: differences in types I and II 5 alpha-reductase and steroid hormone receptor messenger ribonucleic acid (mRNA) levels, but not in insulin-like growth factor mRNA levels.

Benign prostatic hyperplasia (BPH) is so common in elderly men that the development of adenomatous nodules in this organ can be seen as a normal age-dependent process. In this work, we used Northern blotting to compare the levels of androgen, estrogen, and insulin-like growth factor-I (IGF-I) receptor in young (age range, 23-33; n = 3), old normal (age range, 52-80; n = 3), and BPH-affected subjects (age range, 66-87; n = 15). We have also investigated in these groups the expression of genes coding for the two 5 alpha-reductases (types I and II), aromatase, IGF-I, and IGF-II. Our results show significantly increased levels of IGF mRNA in old healthy and BPH-affected subjects; the respective rises for IGF-I, IGF-II, and IGF-I receptor mRNAs were 3.0-, 2.9-, and 1.5-fold (BPH) and 2.7-, 2.4-, and 1.8-fold (old normal controls). For estrogen receptor, androgen receptor, and type I and II 5 alpha-reductase mRNAs, a marked but opposite effect was observed in adenomatous tissues only; the respective levels were 2.2-, 1.8-, 3.9-, and 1.7-fold lower than those in young adult subjects, whereas no significant differences were recorded between the two normal groups. Morphometric analysis of each tissue specimen confirmed the significantly lower epithelium/stroma ratio in adenomas compared to young or old healthy tissues. Together, these observations suggest that prostatic adenomas may result from at least two conjugate processes: one characterized by a drop in the mRNA levels of steroid hormone receptors, which might be associated with a lower epithelium/stroma ratio, and another characterized by normal aging phenomena, of which the increased production of IGFs and IGF-I receptor transcripts could be biochemical markers.

Dose-dependent effects of human prolactin on the immature hypophysectomized rat testis.

We have studied dose-dependent effects of highly purified human PRL (39 IU/mg) on the testis of immature (22-day-old) hypophysectomized rats daily supplemented for 7 days with 2, 10, or 30 micrograms hormone. Dose-dependent stimulation was observed for all parameters: testis weight (1.6- and 2-fold above control for 10 and 30 micrograms PRL), basal and hCG-stimulated testosterone (14- and 21-fold), intratesticular testosterone (7- and 21-fold) and estradiol (1.2- and 1.5-fold), LH receptor concentration (1.8- and 2.5-fold), in vitro pregnenolone production by cholesterol side-chain cleavage enzyme (3-, 5- and 7-fold), and aromatase activity (2.1- and 2.4-fold). The number of Leydig cells exhibiting immunoreactivity toward anti-P450scc, anti-P450(17 alpha), and anti-3 beta-hydroxysteroid dehydrogenase antibodies also underwent a dose-dependent increase (under conditions revealing many immunopositive cells in hypox control animals). The respective increases were 8- to 14-fold for anti-P450scc and P450(17 alpha) and 1.5- to 2-fold for anti-3 beta-hydroxysteroid dehydrogenase. The number of germ cells and the percentage of tubular sections containing late pachytene spermatocytes as most advanced stages of spermatogenesis were subject to similar dose-dependent effects. These results suggest that during puberty PRL stimulates testicular function by promoting multiplication and differentiation of Leydig cells (acting at various steps of steroidogenesis and on tissue responsiveness to LH) and germ cells.

A non-radioactive method to detect RNA or DNA using an oligonucleotide probe with bromodeoxyuridine free ends, a monoclonal antibody against bromodeoxyuridine and immunogold silver staining.

A non radioactive method for probing RNA or DNA on dot and Northern blots using a synthetic oligonucleotide with bromodeoxyuridine free ends is described. The present experiment was carried out with human testis and placental RNA's. The probe was the 21 base long sequence coding for the amino acids 18 to 24 of the insulin-like growth factor I (IGF-I) with two bromodeoxyuridine dinucleotides added at the 5' and 3' ends. The probe was detected with a monoclonal antibody against bromodeoxyuridine and immunogold silver staining (IGSS). Our method was compared to the peroxydase (HRP) revelation of the same probe. The results obtained show a lower background with IGSS than with HRP revelation. A sensitivity similar to that of 32P labelling was found with the advantages of an increase in the rapidity of the procedure (24 hours instead of 9 days exposure) and the absence of handling radioactive substances. Moreover, as the monoclonal antibody against BrdU detects single stranded DNA only, the use of BrdU free ends-labelled oligonucleotide allows the development of the revelation procedure without any previous denaturation of the hybrid. This particular point is an indisputable advantage for detecting hybridization in situ.

[In-vitro incorporation of BUDR by the prostate and by human urothelium and immunohistological detection in plastic-encased sections]. / Incorporation in vitro du BUDR par la prostate et l'urothélium humains et révélation immunohistologique sur coupes inclues dans le plastic.

The measurement of the S phase fraction, essential parameter of cell proliferation, may be done through the immunohistochemical revelation of BUDR incorporated in the cells. The technique is illustrated. The advantages of in vitro incorporation as compared with in vivo injection as discussed. Easier than autoradiography, this method may be useful, in clinical practice, to study the aggressiveness of cancer tissues.

Detection of cell proliferation in pig testis and intestine sections using monoclonal anti-bromodeoxyuridine antibody and immunogold silver staining.

For the first time a monoclonal antibody against 5-bromodeoxyuridine was used to detect cell proliferation in pig testis and intestine sections. The influence of several parameters such as mode of injection, addition of thymidine biosynthesis inhibitor, tissue fixation, hydrolysis and revelation was examined. The technique of choice consisted in intravenously injecting the animals with 50 mg/kg BUdR added to 10 mg/kg FUdR 2 h before tissue collection and Bouin fixation; hydrolysis of sections was performed by HC1 4N: Ethanol 70 degrees (1:1 v/v); revelation of BUdR was made by a secondary antibody linked to colloidal gold particles, followed by a silver enhancement step. The data were superior when compared to those obtained by direct immunofluorescence and by the PAP technique. The described method is convenient and sensitive, provides an intense nuclear labelling without background and allows simultaneous examination of histology. The advantages over the technique using tritiated thymidine are particularly obvious when fast screening of numerous samples is required or when new experimental protocols are developing.

Anti-bromodeoxyuridine monoclonal antibody: an alternative tool for the identification of replicated DNA at the electron microscope level.

A new method for identifying the replicated DNA at the electron microscope level is described. Cells were first exposed in vitro to 5-bromodeoxyuridine (BUdR) in conjunction with 5-fluorodeoxyuridine (FUdR) and BUdR incorporated into DNA was then detected on Lowicryl-embedded sections by immunogold technique using a monoclonal anti-BUdR antibody. After using this method, chromatin and chromosomes are strongly labelled.