Effect of natural seasonal pollen exposure and repeated nasal allergen provocations on elevation of exhaled nitric oxide

BACKGROUND: Exhaled nitric oxide (FENO) is a marker for allergic airway inflammation. We wondered whether in patients with intermittent allergic rhinitis only (i) natural pollen exposure and (ii) artificial pollen exposure by repeated nasal allergen provocations may lead to an elevation of FENO. METHODS: In two prospective studies, we compared the FENO of nonatopic controls with the FENO of nonasthmatic individuals with mild intermittent rhinitis to tree and/or grass pollen. Study I: 13 atopic individuals and seven controls had measurements of FENO, blood eosinophils and eosinophilic cationic protein (ECP) before, during and after pollen season. Study II: 16 atopic individuals and 12 controls had nasal allergen provocations on four following days out of pollen season, with daily measurements of FENO before, 2 and 6 h after provocation, and determination of blood eosinophils, ECP and FEV1 at baseline, on days 5 and 10-12. RESULTS: Natural pollen exposure (study I) caused a significant elevation of FENO in allergic individuals. Nasal allergen provocations (study II) did not elicit a statistically significant rise neither of FENO nor of blood eosinophils between baseline and day 5. However, a subgroup of four individuals with a rise of blood eosinophils during nasal allergen provocations showed also a rise of FENO. CONCLUSIONS: We suppose that in allergic rhinitis a concomitant reaction of the bronchial system is dependent on a strong local inflammation leading to a generalized immune stimulation.

Anti-eosinophil activity and clinical efficacy of the CRTH2 antagonist OC000459 in eosinophilic esophagitis

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic, Th2-type inflammatory disease. Chemoattractant receptor-homologous molecule on Th2 cells (CRTH2) is a prostaglandin D(2) (PGD(2)) receptor, expressed by Th2 cells and other inflammatory cells, including eosinophils and basophils, that mediates chemotaxis and activation. OC000459 is a selective CRTH2 antagonist and would be expected to suppress eosinophilic tissue inflammation. The purpose of this study was to evaluate the efficacy and safety of an OC000459 monotherapy in adult patients with active, corticosteroid-dependent or corticosteroid-refractory EoE. METHODS: In this randomized, double-blind, placebo-controlled trial, 26 adult patients (m/f = 22/4; mean age 41 years, range 22-69 years) with active EoE, dependent or resistant to corticosteroids, were treated either with 100 mg OC000459 (n = 14) or placebo (n = 12) twice daily. Pre- and post-treatment disease activity was assessed clinically, endoscopically, histologically, and via biomarkers. The primary end point was the reduction in esophageal eosinophil infiltration. RESULTS: After an 8-week OC000459 treatment, the esophageal eosinophil load decreased significantly, from 114.83 to 73.26 eosinophils per high-power field [(eos/hpf), P = 0.0256], whereas no reduction was observed with placebo (102.80-99.47 eos/hpf, P = 0.870). With OC000459, the physician's global assessment of disease activity improved from 7.13 to 5.18 (P = 0.035). OC000459 likewise reduced extracellular deposits of eosinophil peroxidase and tenascin C, the effects not seen with placebo. No serious adverse events were observed. CONCLUSIONS: An 8-week treatment with the CRTH2-antagonist, OC000459, exerts modest, but significant, anti-eosinophil and beneficial clinical effects in adult patients with active, corticosteroid-dependent or corticosteroid-refractory EoE and is well tolerated.

Extracellular DNA traps in allergic, infectzious, and autoimmune diseases

Extracellular DNA traps are part of the innate immune response and are seen with many infectious, allergic, and autoimmune diseases. They can be generated by several different leukocytes, including neutrophils, eosinophils, and monocytes, as well as mast cells. Here, we review the composition of these extracellular DNA-containing structures as well as potential mechanisms for their production and function. In general, extracellular DNA traps have been described as binding to and killing pathogens, particularly bacteria, fungi, but also parasites. On the other hand, it is possible that DNA traps contribute to immunopathology in chronic inflammatory diseases, such as bronchial asthma. In addition, it has been demonstrated that they can initiate and/or potentiate autoimmune diseases. Extracellular DNA traps represent a frequently observed phenomenon in inflammatory diseases, and they appear to participate in the cross-talk between different immune cells. These new insights into the pathogenesis of inflammatory diseases may open new avenues for targeted therapies.

Pathology of sarcoptic mange in red foxes (Vulpes vulpes): macroscopic and histologic characterization of three disease stages

Sarcoptic mange is a highly contagious skin disease that can have a devastating impact on affected wild mammal populations. There are notable variations in the clinical and pathologic picture of sarcoptic mange among species and among conspecifics. However, the origin of these variations is unclear. We propose a classification scheme for skin lesions associated with Sarcoptes scabiei infestation to provide a basis for a subsequent risk factor analysis. We conducted a case-control study focused on macroscopic and histologic examination of the skin, using 279 red foxes (Vulpes vulpes) found dead or shot in Switzerland between November 2004 and February 2006. All animals were submitted to gross necropsy following a detailed protocol. Selection criteria for cases (n=147) vs. controls (n=111) were the presence or absence of mange-like lesions, mite detection by isolation or histologic examination, and serologic testing for S. scabiei antibodies. Characteristic features of mange lesions were scored macroscopically in all foxes and histologically in 67 cases and 15 controls. We classified skin lesions and associated necropsy findings into three types of mange: A) early stage (n=45): focal-extensive skin lesions, thin crusts, mild to moderate alopecia, few mites, numerous eosinophils, and mild lymph node enlargement; B) hyperkeratotic, fatal form (n=86): generalized skin lesions, thick crusts with or without alopecia, foul odor, abundance of mites, numerous bacteria and yeasts, numerous lymphocytes and mast cells, severe lymph node enlargement, and emaciation; C) alopecic, healing form (n=16): focal lesions, no crusts, severe alopecia, hyperpigmentation and lichenification, absence of mites, mixed cell infiltration, and rare mild lymph node enlargement. We hypothesize that after stage A, the animal either enters stage B and dies, or stage C and survives, depending on largely unknown extrinsic or intrinsic factors affecting the host ability to control mite infestation.

Th17 cells and tissue remodeling in atopic and contact dermatitis

BACKGROUND: Eczematous skin lesions of atopic dermatitis (AD) as well as allergic and irritant contact dermatitis (ACD, ICD) are characterized by the same typical clinical signs, although due to different causes. In both AD and ACD, the presence of T helper 17 cells which play an important role in host defense, has been reported. Furthermore, IL-17 is involved in tissue repair and remodeling. This study aimed to investigate IL-17 expression in acute eczematous skin lesions and correlate it with markers of remodeling in AD, ACD, and ICD. METHODS: Skin specimens were taken from positive patch test reactions to aeroallergens, contact allergens, and irritants at days 2, 3, and 4. Inflammatory cells as well as the expression of cytokines and extracellular matrix proteins were evaluated by immunofluorescence staining and confocal microscopy. RESULTS: Allergic contact dermatitis and ICD were characterized by IFN-γ expression, whereas in AD lesions, IL-13 expression and high numbers of eosinophils were the prominent phenotype. Expression of IL-17, but also IL-21 and IL-22, was observed in all eczema subtypes. The number of IL-22+ T cells correlated with the number of eosinophils. Markers of remodeling such as MMP-9, procollagen-3, and tenascin C were observed in all acute eczematous lesions, while a correlation of IL-17+ T cell numbers with tenascin C-expressing cells and MMP-9+ eosinophils was apparent. CONCLUSION: The expression of IL-17 and related cytokines, such as IL-22, was demonstrated in acute eczematous lesions independent of their pathogenesis. Our results suggest a potential role for IL-17 in remodeling of the skin.

CD8+ T cells and NK cells from blister fluid of an EEM/SJS overlap highly express Fas ligand and Granulysin

INTRODUCTION Erythema exsudativum multiforme majus (EEMM) and Stevens-Johnson Syndrome (SJS) are severe cutaneous reaction patterns caused by infections or drug hypersensitivity. The mechanism by which widespread keratinocyte death is mediated by the immune system in EEMM/SJS are still to be elucidated. Here, we characterized the blister cells isolated from a patient with EEMM/SJS overlap and investigated its cause. METHODS Clinical classification of the cutaneous eruption was done according to the consensus definition of severe blistering skin reactions and histological analysis. Common infectious causes of EEMM were investigated using standard clinical techniques. T cell reactivity for potentially causative drugs was assessed by lymphocyte transformation tests (LTT). Lymphocytes isolated from blister fluid were analyzed for their expression of activation markers and cytotoxic molecules using flow cytometry. RESULTS The healthy 58 year-old woman suffered from mild respiratory tract infection and therefore started treatment with the secretolytic drug Ambroxol. One week later, she presented with large palmar and plantar blisters, painful mucosal erosions, and flat atypical target lesions and maculae on the trunc, thus showing the clinical picture of an EEMM/SJS overlap (Fig. 1). This diagnosis was supported by histology, where also eosinophils were found to infiltrate the upper dermis, thus pointing towards a cutaneous adverse drug reaction (cADR). Analysis of blister cells showed that they mainly consisted of CD8+ and CD4+ T cells and a smaller population of NK cells. Both the CD8+ T cells and the NK cells were highly activated and expressed Fas ligand and the cytotoxic molecule granulysin (Fig. 2). In addition, in comparison to NK cells from PBMC, NK cells in blister fluids strongly upregulated the expression of the skin-homing chemokine receptor CCR4 (Fig 4). Surprisingly, the LTT performed on PBMCs in the acute phase was positive for Ambroxol (SI=2.9) whereas a LTT from a healthy but exposed individual did not show unspecific proliferation. Laboratory tests for common infectious causes of EEMM were negative (HSV-1/-2, M. pneumoniae, Parvovirus B19). However, 6 weeks later, specific proliferation to Ambroxol could no longer be observed in the LTT (Fig 4.).

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

GENE EXPRESSION IN CHRONIC MYELOGENOUS LEUKEMIA

The goal of the present work was to identify and characterize gene sequences that are preferentially expressed in CML in an effort to better understand the molecular basis of the disease. As high abundance mRNAs generally encode proteins that are phenotypically characteristic of cells, positive-negative screening of a CML cDNA library was used to identify cDNA clones containing sequences preferentially transcribed in CML. One cDNA sequence that fulfilled this criterion, C-A3, has been characterized in some detail. It represents a small mRNA ((TURN)496 nucleotides) that is highly abundant ((TURN)2% of the poly(A('+))RNA) in cells from the chronic phase of CML. In situ hybridization to whole cells indicates the principal leukocytes that express C-A3 sequences are eosinophils, basophils and immature myelocytes. Surprisingly, CML patients with high numbers of myeloblasts do not have an abundance of C-A3 transcripts, although transcript levels remain elevated in patients with lymphoblasts. In AML, high transcript levels are only found sporadically and occasionally different sized transcripts can be detected. Sequences from the 3' end of the C-A3 message are present in 2-5 copies per haploid genome. The 3' end of C-A3 localizes to bands 8q21.1 and 8q23 by in situ chromosomal hybridization. This is a region that is often involved in hematopoietic malignancies. Restriction digests of human genomic DNA show a correlation between the presence of a 2.3 kb Hind III fragment and certain types of leukemia. All of the leukemic DNAs tested had this fragment. In comparison, only one of five normal DNAs had a band this size. Analysis of the nucleotide sequence indicates that C-A3 probably encodes a small, hydrophobic peptide which may be part of a larger protein. ^

Characterization of the inflammatory infiltrate and cytokine expression in the skin of horses with recurrent urticaria.

BACKGROUND Recurrent urticaria (RU) is a common skin disease of horses, but little is known about its pathogenesis. HYPOTHESIS/OBJECTIVE The aim of this study was to characterize the inflammatory cell infiltrate and cytokine expression pattern in the skin of horses with RU. ANIMALS   Biopsies of lesional and nonlesional skin of horses with RU (n = 8) and of skin from healthy control horses (n = 8) were evaluated. METHODS The inflammatory cell infiltrate was analysed by routine histology. Immunohistochemistry was used to identify T cells (CD3), B  ells (CD79), macrophages (MAC387) and mast cells (tryptase). Expression of T-helper 2 cytokines (interleukins IL-4, IL-5 and IL-13), a T-helper 1 cytokine (interferon-γ), IL-4 receptor α and thymic stromal lymphopoietin was assessed by quantitative RT-PCR. Results -  In subepidermal lesional skin of RU-affected horses, increased numbers of eosinophils (P ≤ 0.01), CD79-positive (P ≤ 0.01), MAC387-positive (P ≤ 0.01) and tryptase-positive cells (P ≤ 0.05) were found compared with healthy horses. Subepidermal lesional skin of RU-affected horses contained more eosinophils (P ≤ 0.05) and tryptase-positive cells (P ≤ 0.05) compared with nonlesional skin. There was no significant difference in infiltrating cells between nonlesional skin and skin of healthy horses. Expression of IL-4 (P ≤ 0.01), IL-13 (P ≤ 0.05), thymic stromal lymphopoietin (P ≤ 0.05) and IL-4 receptor α (P ≤ 0.05) was increased in lesional skin of RU-affected horses compared with control horses. Expression of IL-4 was higher (P ≤ 0.05) in lesional compared with nonlesional RU skin. CONCLUSIONS AND CLINICAL IMPORTANCE Analysis of cytokine expression and inflammatory infiltrate suggests that T-helper 2 cytokines, eosinophils, mast cells and presumptive macrophages play a role in the pathogenesis of equine RU.

Eosinophilic oesophagitis and proton pump inhibitor-responsive oesophageal eosinophilia have similar clinical, endoscopic and histological findings.

BACKGROUND Some patients with a phenotypic appearance of eosinophilic oesophagitis (EoE) respond histologically to PPI, and are described as having PPI-responsive oesophageal eosinophilia (PPI-REE). It is unclear if PPI-REE is a GERD-related phenomenon, a subtype of EoE, or a completely unique entity. AIM To compare demographic, clinical and histological features of EoE and PPI-REE. METHODS Two databases were reviewed from the Walter Reed and Swiss EoE databases. Patients were stratified into two groups, EoE and PPI-REE, based on recent EoE consensus guidelines. Response to PPI was defined as achieving less than 15 eos/hpf and a 50% decrease from baseline following at least a 6-week course of treatment. RESULTS One hundred and three patients were identified (63 EoE and 40 PPI-REE; mean age 40.2 years, 75% male and 89% Caucasian). The two cohorts had similar dysphagia (97% vs. 100%, P = 0.520), food impaction (43% vs. 35%, P = 0.536), and heartburn (33% vs. 32%, P = 1.000) and a similar duration of symptoms (6.0 years vs. 5.8 years, P = 0.850). Endoscopic features were also similar between EoE and PPI-REE; rings (68% vs. 68%, P = 1.000), furrows (70% vs. 70%, P = 1.000), plaques (19% vs. 10%, P = 0.272), strictures (49% vs. 30%, P = 0.066). EoE and PPI-REE were similar in the number of proximal (39 eos/hpf vs. 38 eos/hpf, P = 0.919) and distal eosinophils (50 vs. 43 eos/hpf, P = 0.285). CONCLUSIONS EoE and PPI-responsive oesophageal eosinophilia are similar in clinical, histological and endoscopic features and therefore are indistinguishable without a PPI trial. Further studies are needed to determine why a subset of patients with oesophageal eosinophilia respond to PPI.

Stability of 3-bromotyrosine in serum and serum 3-bromotyrosine concentrations in dogs with gastrointestinal diseases

Abstract BACKGROUND: 3-Bromotyrosine (3-BrY) is a stable product of eosinophil peroxidase and may serve as a marker of eosinophil activation. A gas chromatography/mass spectrometry method to measure 3-BrY concentrations in serum from dogs has recently been established and analytically validated. The aims of this study were to determine the stability of 3-BrY in serum, to determine the association between peripheral eosinophil counts and the presence of an eosinophilic infiltrate in the gastrointestinal tract, and to compare serum 3-BrY concentrations in healthy dogs (n = 52) and dogs with eosinophilic gastroenteritis (EGE; n = 27), lymphocytic-plasmacytic enteritis (LPE; n = 25), exocrine pancreatic insufficiency (EPI; n = 26), or pancreatitis (n = 27). RESULTS: Serum 3-BrY concentrations were stable for up to 8, 30, and 180 days at 4°C, -20°C, and -80°C, respectively. There was no significant association between peripheral eosinophil count and the presence of eosinophils in the GI tissues (P = 0.1733). Serum 3-BrY concentrations were significantly higher in dogs with EGE (median [range] = 5.04 [≤0.63-26.26] μmol/L), LPE (median [range] = 3.60 [≤0.63-15.67] μmol/L), and pancreatitis (median [range] = 1.49 [≤0.63-4.46] μmol/L) than in healthy control dogs (median [range] = ≤0.63 [≤0.63-1.79] μmol/L; P < 0.0001), whereas concentrations in dogs with EPI (median [range] = 0.73 [≤0.63-4.59] μmol/L) were not different compared to healthy control dogs. CONCLUSIONS: The present study revealed that 3-BrY concentrations were stable in serum when refrigerated and frozen. No relationship between peripheral eosinophil count and the presence of eosinophils infiltration in the GI tissues was found in this study. In addition, serum 3-BrY concentrations were increased in dogs with EGE, but also in dogs with LPE and pancreatitis. Further studies are needed to determine whether measurement of 3-BrY concentrations in serum may be useful to assess patients with suspected or confirmed EGE or LPE.

Toxicity of eosinophil MBP is repressed by intracellular crystallization and promoted by extracellular aggregation.

Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly.

Active eosinophilic esophagitis is characterized by epithelial barrier defects and eosinophil extracellular trap formation.

BACKGROUND Eosinophilic esophagitis (EoE) exhibits esophageal dysfunction owing to an eosinophil-predominant inflammation. Activated eosinophils generate eosinophil extracellular traps (EETs) able to kill bacteria. There is evidence of an impaired barrier function in EoE that might allow pathogens to invade the esophagus. This study aimed to investigate the presence and distribution of EETs in esophageal tissues from EoE patients and their association with possible epithelial barrier defects. METHODS Anonymized tissue samples from 18 patients with active EoE were analyzed. The presence of DNA nets associated with eosinophil granule proteins forming EETs and the expression of filaggrin, the protease inhibitor lympho-epithelial Kazal-type-related inhibitor (LEKTI), antimicrobial peptides, and cytokines were evaluated by confocal microscopy following immune fluorescence staining techniques. RESULTS Eosinophil extracellular trap formation occurred frequently and was detected in all EoE samples correlating with the numbers of infiltrating eosinophils. While the expression of both filaggrin and LEKTI was reduced, epithelial antimicrobial peptides (human beta-defensin-2, human beta-defensin-3, cathelicidin LL-37, psoriasin) and cytokines (TSLP, IL-25, IL-32, IL-33) were elevated in EoE as compared to normal esophageal tissues. There was a significant correlation between EET formation and TSLP expression (P = 0.02) as well as psoriasin expression (P = 0.016). On the other hand, a significant negative correlation was found between EET formation and LEKTI expression (P = 0.016). CONCLUSION Active EoE exhibits the presence of EETs. Indications of epithelial barrier defects in association with epithelial cytokines are also present which may have contributed to the activation of eosinophils. The formation of EETs could serve as a firewall against the invasion of pathogens.

A novel regulatory macrophage induced by a helminth molecule instructs IL-10 in CD4+ T cells and protects against mucosal inflammation

Immunomodulation is a common feature of chronic helminth infections and mainly attributed to the secretion of bioactive molecules, which target and modify host immune cells. In this study, we show that the helminth immunomodulator AvCystatin, a cysteine protease inhibitor, induces a novel regulatory macrophage (Mreg; AvCystatin-Mreg), which is sufficient to mitigate major parameters of allergic airway inflammation and colitis in mice. A single adoptive transfer of AvCystatin-Mreg before allergen challenge suppressed allergen-specific IgE levels, the influx of eosinophils into the airways, local and systemic Th2 cytokine levels, and mucus production in lung bronchioles of mice, whereas increasing local and systemic IL-10 production by CD4(+) T cells. Moreover, a single administration of AvCystatin-Mreg during experimentally induced colitis strikingly reduced intestinal pathology. Phenotyping of AvCystatin-Mreg revealed increased expression of a distinct group of genes including LIGHT, sphingosine kinase 1, CCL1, arginase-1, and costimulatory molecules, CD16/32, ICAM-1, as well as PD-L1 and PD-L2. In cocultures with dendritic cells and CD4(+) T cells, AvCystatin-Mreg strongly induced the production of IL-10 in a cell-contact-independent manner. Collectively, our data identify a specific suppressive macrophage population induced by a single parasite immunomodulator, which protects against mucosal inflammation.