Eman Hamza

Equine insect bite hypersensitivity: what do we know?

Insect bite hypersensitivity (IBH) is an allergic dermatitis of the horse caused by bites of insects of the genus Culicoides and is currently the best characterized allergic disease of horses. This article reviews knowledge of the immunopathogenesis of IBH, with a particular focus on the causative allergens. Whereas so far hardly any research has been done on the role of antigen presenting cells in the pathogenesis of IBH, recent studies suggest that IBH is characterized by an imbalance between a T helper 2 (Th2) and regulatory T cell (T(reg)) immune response, as shown both locally in the skin and with stimulated peripheral blood mononuclear cells. Various studies have shown IBH to be associated with IgE-mediated reactions against salivary antigens from Culicoides spp. However, until recently, the causative allergens had not been characterized at the molecular level. A major advance has now been made, as 11 Culicoides salivary gland proteins have been identified as relevant allergens for IBH. Currently, there is no satisfactory treatment of IBH. Characterization of the main allergens for IBH and understanding what mechanisms induce a healthy or allergic immune response towards these allergens may help to develop new treatment strategies, such as immunotherapy.

The BCS Functional for General Pair Interactions

The Bardeen-Cooper-Schrieffer (BCS) functional has recently received renewed attention as a description of fermionic gases interacting with local pairwise interactions. We present here a rigorous analysis of the BCS functional for general pair interaction potentials. For both zero and positive temperature, we show that the existence of a non-trivial solution of the nonlinear BCS gap equation is equivalent to the existence of a negative eigenvalue of a certain linear operator. From this we conclude the existence of a critical temperature below which the BCS pairing wave function does not vanish identically. For attractive potentials, we prove that the critical temperature is non-zero and exponentially small in the strength of the potential.

Modulation of Allergy Incidence in Icelandic Horses Is Associated with a Change in IL-4-Producing T Cells

Background: Equine insect bite hypersensitivity (IBH) is an immediate-type hypersensitivity reaction provoked by insect-derived allergens. Icelandic horses living in Iceland do not have IBH due to absence of relevant insects, but acquire it at high frequency after being imported to mainland Europe. In contrast, their offspring born in mainland Europe has reduced IBH incidence. T helper 1 (Th1) and Th2 cells and cytokines were determined in Icelandic horses born in Iceland and on the continent and which either have IBH or are healthy. Methods: Peripheral blood mononuclear cells (PBMC) from these horses were stimulated for 18 h dur- ing summer and winter with polyclonal T cell stimuli, IBH allergen(s) or irrelevant allergen(s). Cells were analysed by flow cytometry for interferon-γ (IFN-γ) and interleukin-4 (IL-4); RNA was analysed for IFN-γ, IL-4, IL-5 and IL-13 mRNA. Results: During summer, but not during winter, IBH PBMC stimulated polyclonally showed reduced IFN-γ mRNA and IFN-γ-producing cells when compared with those of healthy horses, regardless of origin. PBMC stimulated polyclonally or with IBH allergen showed increased IL-4 mRNA levels and higher numbers of IL-4-producing cells when born in Iceland or showing IBH symptoms. IL-5 and IL-13 mRNA were modulated neither by disease nor by origin. Abrogation of IL-4 production in healthy horses born in mainland Europe may be due, at least in part, to IL-10. There was an increased level of IL-10 in supernatants from PBMC of healthy horses born in mainland Europe and stimulated polyclonally or with IBH allergen. Conclusions: Modulation of IBH incidence is governed by altered Th1/Th2 ratio, which might be influenced by IL-10.

Dynamical Localization in Disordered Quantum Spin Systems

We say that a quantum spin system is dynamically localized if the time-evolution of local observables satisfies a zero-velocity Lieb-Robinson bound. In terms of this definition we have the following main results: First, for general systems with short range interactions, dynamical localization implies exponential decay of ground state correlations, up to an explicit correction. Second, the dynamical localization of random xy spin chains can be reduced to dynamical localization of an effective one-particle Hamiltonian. In particular, the isotropic xy chain in random exterior magnetic field is dynamically localized.

Dynamical Localization for Unitary Anderson Models

This paper establishes dynamical localization properties of certain families of unitary random operators on the d-dimensional lattice in various regimes. These operators are generalizations of one-dimensional physical models of quantum transport and draw their name from the analogy with the discrete Anderson model of solid state physics. They consist in a product of a deterministic unitary operator and a random unitary operator. The deterministic operator has a band structure, is absolutely continuous and plays the role of the discrete Laplacian. The random operator is diagonal with elements given by i.i.d. random phases distributed according to some absolutely continuous measure and plays the role of the random potential. In dimension one, these operators belong to the family of CMV-matrices in the theory of orthogonal polynomials on the unit circle. We implement the method of Aizenman-Molchanov to prove exponential decay of the fractional moments of the Green function for the unitary Anderson model in the following three regimes: In any dimension, throughout the spectrum at large disorder and near the band edges at arbitrary disorder and, in dimension one, throughout the spectrum at arbitrary disorder. We also prove that exponential decay of fractional moments of the Green function implies dynamical localization, which in turn implies spectral localization. These results complete the analogy with the self-adjoint case where dynamical localization is known to be true in the same three regimes.

CD4+CD25+ T cells expressing FoxP3 in Icelandic horses affected with insect bite hypersensitivity.

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-β1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses.

Increased IL-4 and decreased regulatory cytokine production following relocation of Icelandic horses from a high to low endoparasite environment

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland where Culicoides are absent. However, following importation into continental Europe where Culicoides are present, >or=50% of Icelandic horses (1st generation) develop IBH but <or=10% of their offspring born in Europe (2nd generation) do so. Recently, we showed that PBMC from 1st generation horses produce more IL-4 than 2nd generation horses. Since helminths and allergens induce Th2 responses, we investigated whether horses domiciled in Iceland are Th2-biased, and whether this is determined by helminth infection. We compared the parasite burden and T cell responses between Icelandic horses living in Iceland or Switzerland. Horses in Iceland have higher faecal egg counts, higher tapeworm-specific IgG(T) levels and higher total serum IgE levels than horses in Switzerland. Nevertheless, horses in Iceland displayed a low proportion of IL-4-producing cells in PBMC cultures after polyclonal or parasite extracts stimulation. No IL-4-producing cells were found in PBMC from horses after stimulation by Culicoides extract. Addition of anti-IL-10 and anti-TGF-beta1 to PBMC cultures of horses in Iceland increased the proportion of IL-4-producing cells after polyclonal or parasite antigens stimulation but not stimulation with Culicoides extract. This paralleled the high levels of IL-10 and TGF-beta1 found in supernatants from PBMC cultures of horses in Iceland. Collectively, horses living in Iceland have a high parasite burden but low IL-4 production. This supports the hypothesis that heavy helminth infections have a suppressive effect on IL-4 production mediated by IL-10 and TGF-beta1.

Equine CD4(+) CD25(high) T cells exhibit regulatory activity by close contact and cytokine-dependent mechanisms in vitro.

Horses are particularly prone to allergic and autoimmune diseases, but little information about equine regulatory T cells (Treg) is currently available. The aim of this study therefore was to investigate the existence of CD4+ Treg cells in horses, determine their suppressive function as well as their mechanism of action. Freshly isolated peripheral blood mononuclear cells (PBMC) from healthy horses were examined for CD4, CD25 and forkhead box P3 (FoxP3) expression. We show that equine FoxP3 is expressed constitutively by a population of CD4+ CD25+ T cells, mainly in the CD4+ CD25high subpopulation. Proliferation of CD4+ CD25− sorted cells stimulated with irradiated allogenic PBMC was significantly suppressed in co‐culture with CD4+ CD25high sorted cells in a dose‐dependent manner. The mechanism of suppression by the CD4+ CD25high cell population is mediated by close contact as well as interleukin (IL)‐10 and transforming growth factor‐β1 (TGF‐β1) and probably other factors. In addition, we studied the in vitro induction of CD4+ Treg and their characteristics compared to those of freshly isolated CD4+ Treg cells. Upon stimulation with a combination of concanavalin A, TGF‐β1 and IL‐2, CD4+ CD25+ T cells which express FoxP3 and have suppressive capability were induced from CD4+ CD25− cells. The induced CD4+ CD25high express higher levels of IL‐10 and TGF‐β1 mRNA compared to the freshly isolated ones. Thus, in horses as in man, the circulating CD4+ CD25high subpopulation contains natural Treg cells and functional Treg can be induced in vitro upon appropriate stimulation. Our study provides the first evidence of the regulatory function of CD4+ CD25+ cells in horses and offers insights into ex vivo manipulation of Treg cells.

Developing a preventive immunization approach against insect bite hypersensitivity using recombinant allergens: A pilot study.

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of midges (Culicoides spp.). IgE-mediated reactions are often involved in the pathogenesis of this disease. IBH does not occur in Iceland due to the absence of Culicoides, but it occurs with a high frequency in Icelandic horses exported to mainland Europe, where Culicoides are present. We hypothesize that immunization with the Culicoides allergens before export could reduce the incidence of IBH in exported Icelandic horses. The aim of the present study was therefore to compare intradermal and intralymphatic vaccination using four purified recombinant allergens, in combination with a Th1 focusing adjuvant. Twelve horses were vaccinated three times with 10μg of each of the four recombinant Culicoides nubeculosus allergens. Six horses were injected intralymphatically, three with and three without IC31(®), and six were injected intradermally, in the presence or absence of IC31(®). Antibody responses were measured by immunoblots and ELISA, potential sensitization in a sulfidoleukotriene release test and an intradermal test, cytokine and FoxP3 expression with real time PCR following in vitro stimulation of PBMC. Immunization with the r-allergens induced a significant increase in levels of r-allergen-specific IgG1, IgG1/3, IgG4/7, IgG5 and IgG(T). Application of the r-allergens in IC31(®) adjuvant resulted in a significantly higher IgG1, IgG1/3, IgG4/7 allergen-specific response. Intralymphatic injection was slightly more efficient than intradermal injection, but the difference did not reach significance. Testing of the blocking activity of the sera from the horses immunized intralymphatically with IC31(®) showed that the generated IgG antibodies were able to partly block binding of serum IgE from an IBH-affected horse to these r-allergens. Furthermore, IgG antibodies bound to protein bands on blots of C. nubeculosus salivary gland extract. No allergen-specific IgE was induced and there was no indication of induction of IgE-mediated reactions, as horses neither responded to Culicoides extract stimulation in a sulfidoleukotriene release test, nor developed a relevant immediate hypersensitivity reaction to the recombinant allergens in skin test. IL-4 expression was significantly higher in horses vaccinated intralymphatically without IC31(®), as compared to horses intradermally vaccinated with IC31(®). Both routes gave higher IL-10 expression with IC31(®). Both intralymphatic and intradermal vaccination of horses with recombinant allergens in IC31(®) adjuvant induced an immune response without adverse effects and without IgE production. The horses were not sensitized and produced IgG that could inhibit allergen-specific IgE binding. We therefore conclude that both the injection routes and the IC31(®) adjuvant are strong candidates for further development of immunoprophylaxis and therapy in horses.

The Transcriptome of Equine Peripheral Blood Mononuclear Cells

Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To improve the horse genome annotation we performed RNA-seq on 561 samples of peripheral blood mononuclear cells (PBMCs) derived from 85 Warmblood horses. The mapped sequencing reads were used to build a new transcriptome assembly. The new assembly revealed many alternative isoforms associated to known genes or to those predicted by the Ensembl and/or Gnomon pipelines. We also identified 7,531 transcripts not associated with any horse gene annotated in public databases. Of these, 3,280 transcripts did not have a homologous match to any sequence deposited in the NCBI EST database suggesting horse specificity. The unknown transcripts were categorized as coding and noncoding based on predicted coding potential scores. Among them 230 transcripts had high coding potential score, at least 2 exons, and an open reading frame of at least 300 nt. We experimentally validated 9 new equine coding transcripts using RT-PCR and Sanger sequencing. Our results provide valuable detailed information on many transcripts yet to be annotated in the horse genome.