p110γ/δ Double-Deficiency Induces Eosinophilia and IgE Production but Protects from OVA-Induced Airway Inflammation.

The catalytical isoforms p110γ and p110δ of phosphatidylinositide 3-kinase γ (PI3Kγ) and PI3Kδ play an important role in the pathogenesis of asthma. Two key elements in allergic asthma are increased levels of eosinophils and IgE. Dual pharmacological inhibition of p110γ and p110δ reduces asthma-associated eosinophilic lung infiltration and ameliorates disease symptoms, whereas the absence of enzymatic activity in p110γKOδD910A mice increases IgE and basal eosinophil counts. This suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. Here, we analysed mice genetically deficient for both catalytical subunits (p110γ/δ-/-) and determined basal IgE and eosinophil levels and the immune response to ovalbumin-induced asthma. Serum concentrations of IgE, IL-5 and eosinophil numbers were significantly increased in p110γ/δ-/- mice compared to single knock-out and wildtype mice. However, p110γ/δ-/- mice were protected against OVA-induced infiltration of eosinophils, neutrophils, T and B cells into lung tissue and bronchoalveolar space. Moreover, p110γ/δ-/- mice, but not single knock-out mice, showed a reduced bronchial hyperresponsiveness. We conclude that increased levels of eosinophils and IgE in p110γ/δ-/- mice do not abolish the protective effect of p110γ/δ-deficiency against OVA-induced allergic airway inflammation.

Comparison of the expression, activity, and fecal concentration of intestinal alkaline phosphatase between healthy dogs and dogs with chronic enteropathy.

OBJECTIVE To compare expression, activity, and fecal concentration of intestinal alkaline phosphatase (IAP) between healthy dogs and dogs with chronic enteropathy (CE). ANIMALS 9 healthy university-owned Beagles and 109 healthy client-owned dogs (controls) and 28 dogs with CE (cases). PROCEDURES Cases were defined as dogs with persistent (> 3 weeks) gastrointestinal signs that failed to respond to antimicrobials and anti-inflammatory doses of prednisolone or dietary trials, did not have mechanical gastrointestinal abnormalities as determined by abdominal radiography and ultrasonography, and had a diagnosis of lymphoplasmacytic enteritis or eosinophilic gastroenteritis on histologic examination of biopsy specimens. Duodenal and colonic mucosa biopsy specimens were obtained from the 9 university-owned Beagles and all cases for histologic examination and determination of IAP expression (by real-time quantitative PCR assay) and activity (by enzyme histochemical analysis). Fecal samples were obtained from all dogs for determination of fecal IAP concentration by a quantitative enzyme reaction assay. RESULTS For dogs evaluated, IAP expression and activity were localized at the luminal side of epithelial cells in the mucosa and intestinal crypts, although both were greater in the duodenum than in the colon. Active IAP was detected in the feces of all dogs. Intestinal alkaline phosphatase expression and activity were lower for cases than for controls, and fecal IAP concentration for dogs with moderate and severe CE was lower than that for dogs with mild CE. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CE had impaired IAP expression and activity. Additional research is necessary to elucidate the role of IAP in the pathogenesis of CE.

Serum levels of matrix metalloproteinase-13 in patients with eosinophilic fasciitis.

Matrix metalloproteinase-13 (MMP-13), a member of the collagenase family, has been implicated in the pathogenesis of connective tissue diseases characterized by extracellular matrix remodeling. Since serum MMP-13 levels reflect disease severity of systemic sclerosis and localized scleroderma, we evaluated the clinical significance of serum MMP-13 levels in eosinophilic fasciitis (EF). All the EF patients had serum MMP-13 levels lower than the mean - 2SD of healthy controls. Serum MMP-13 levels were also significantly decreased in EF patients compared with diffuse cutaneous systemic sclerosis, limited cutaneous systemic sclerosis, and generalized morphea patients. Although serum MMP-13 levels did not reflect any clinical and serological features of EF, these results indicate that MMP-13 may be involved in the development of this disease.

Feline eosinophilic dermatoses: a retrospective immunohistochemical and ultrastructural study of extracellular matrix remodelling.

BACKGROUND: Feline eosinophilic dermatoses (FEDs) are common diseases of cats with an unknown pathogenesis. They are histologically characterized by an eosinophilic infiltration and often by the presence of flame figures (FFs) and/or areas of loss of tissue architecture, here termed necrotic foci (NF). It has been postulated that an alteration in the degradation of the extracellular matrix could be responsible for these histological features. Matrix metalloproteinases (MMPs) are a group of proteases that are fundamental in extracellular matrix remodelling. HYPOTHESIS/OBJECTIVES: The aim of the study was to investigate retrospectively the expression of a subgroup of MMPs, in particular MMP-2 and MMP-9 gelatinases, in FEDs. The expression of one of their inhibitors, TIMP-2, was also investigated in order to establish the role of these molecules in the pathogenesis of FEDs. The ultrastructural characteristics of extracellular matrix in FFs and NF were subsequently assessed. METHODS: Fifty-one formalin-fixed, paraffin-embedded specimens from cutaneous and mucosal biopsies diagnosed as FEDs were investigated immunohistochemically. Two selected samples were processed for electron microscopy. RESULTS: This study revealed an increased expression of MMP-2 in NF and a decreased expression of this gelatinase in FFs. An imbalance between MMP-2 and TIMP-2 was evident using immunohistochemistry. No significative results were observed for MMP-9 expression. Electron microscopy confirmed the lack of normal collagen fibres in NF, whereas in FFs only occasional, amorphous material was observed among normal collagen fibres. CONCLUSIONS AND CLINICAL IMPORTANCE: Our study suggests that an imbalance in the expression of matrix metalloproteinases could be responsible for different morphological findings in FEDs. Further studies are needed to assess the role of matrix metalloproteinases in the pathogenesis of FEDs.

Ischaemic brain damage after cardiac arrest and induced hypothermia--a systematic description of selective eosinophilic neuronal death. A neuropathologic study of 23 patients.

PURPOSE: Although well characterized in animals, brain damage in humans treated with hypothermia after cardiac arrest has not been systematically explored. In this study we aimed to describe the characteristic trait of selective eosinophilic neuronal death (SEND), and its correlation with time to return of spontaneous circulation (ROSC) in cardiac arrest patients who died after hypothermia treatment and were referred for autopsy. METHODS: Brain autopsy microscopic slides and clinical data were gathered from 23 non-survivors of cardiac arrest who were treated with hypothermia. Based on the percentage of eosinophilic neurons, a damage score 0-4 was given in 6 brain regions, and a total damage score was calculated. The damage score was correlated with time to ROSC and with neuron-specific enolase (NSE) in peripheral blood at 48 h post arrest. RESULTS: Hippocampus had the highest damage score with a median of 3 (inter-quartile range 2-4) while the brainstem had the lowest median damage score of 0 (0-2). Total damage score showed the best correlation with time to ROSC (Spearman Rho=0.66). Serum NSE values >33 µg/L (n=6) was associated with significantly higher mean damage score than NSE <33 µg/L (n=9) (p=0.002). CONCLUSION: This is the first study to systematically describe regional SEND in patients treated with hypothermia after cardiac arrest. Hippocampus was the most vulnerable region whereas the brainstem was the most resistant. Although not directly compared here, the regional pattern of SEND seems not to be altered by hypothermia treatment, but maintains its profile distinctive for cardiac arrest pathogenesis.

Macro-AST: misleading finding in an adolescent with MCAD-deficiency.

BACKGROUND: MCAD-deficiency is the most common inborn error of fatty acid oxidation now included in many newborn screening programms using MS/MS. During prolonged catabolic episodes, patients may suffer from metabolic decompensation with dysfunction of liver, skeletal- and heart muscle as well as brain. In anabolism, neither clinical symptoms nor biochemical signs of organ dysfunction occur. CASE PRESENTATION: We report a female patient with MCAD-deficiency in whom at the age of 11 years isolated AST-elevation was found without any clinical or biochemical signs of organ dysfunction. We showed by polyethylene glycol precipitation that macro-AST formation was responsible for this biochemical finding. AST was probably complexed with immunoglobulins possibly related to an allergic disposition. Macro-AST formation is not a special feature of MCAD-deficiency but rather a non-specific, coincidental finding which also occurs in healthy individuals. The general practitioner consulted by the patient before coming to our outpatient clinic for inborn errors of metabolism was worried that isolated AST-elevation indicated cell damage in MCAD-deficiency. He ordered further diagnostic tests like ultrasound, ECG and echocardiography without any pathology. CONCLUSION: In isolated AST-elevation, macro-AST has to be considered in order to avoid unnecessary, costly and invasive evaluation. This is not only true for healthy persons but for patients with chronic diseases like MCAD as well.

Eosinophils preserve parasitic nematode larvae by regulating local immunity.

Eosinophils play important roles in regulation of cellular responses under conditions of homeostasis or infection. Intestinal infection with the parasitic nematode, Trichinella spiralis, induces a pronounced eosinophilia that coincides with establishment of larval stages in skeletal muscle. We have shown previously that in mouse strains in which the eosinophil lineage is ablated, large numbers of T. spiralis larvae are killed by NO, implicating the eosinophil as an immune regulator. In this report, we show that parasite death in eosinophil-ablated mice correlates with reduced recruitment of IL-4(+) T cells and enhanced recruitment of inducible NO synthase (iNOS)-producing neutrophils to infected muscle, as well as increased iNOS in local F4/80(+)CD11b(+)Ly6C(+) macrophages. Actively growing T. spiralis larvae were susceptible to killing by NO in vitro, whereas mature larvae were highly resistant. Growth of larvae was impaired in eosinophil-ablated mice, potentially extending the period of susceptibility to the effects of NO and enhancing parasite clearance. Transfer of eosinophils into eosinophil-ablated ΔdblGATA mice restored larval growth and survival. Regulation of immunity was not dependent upon eosinophil peroxidase or major basic protein 1 and did not correlate with activity of the IDO pathway. Our results suggest that eosinophils support parasite growth and survival by promoting accumulation of Th2 cells and preventing induction of iNOS in macrophages and neutrophils. These findings begin to define the cellular interactions that occur at an extraintestinal site of nematode infection in which the eosinophil functions as a pivotal regulator of immunity.

Matrix metalloproteinase-12 leads to elastin degradation in BALB/c mice with eosinophilic meningitis caused by Angiostrongylus cantonensis.

The rat lugworm Angiostrongylus cantonensis can cause eosinophilic meningitis. The purpose of this study was to determine whether matrix metalloproteinase (MMP)-12 and its substrate elastin participate in this inflammatory response. We showed that the MMP-12/tissue inhibitor of metalloproteinase-1 ratio was significantly increased in the CSF of A. cantonensis-infected mice from day 10 p.i., and reached high levels on days 20 and 25 p.i. MMP-12 production was correlated with elastin degradation, eosinophil count, blood-CSF barrier permeability and pathological changes in the subarachnoid space. Also, MMP-12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. Simultaneous administration of albendazole and doxycycline significantly reduced the levels of MMP-12, elastin and Evans blue in mice with meningitis. These results imply that MMP-12 contributes to the elastin degradation that occurs in angiostrongyliasis meningitis, and doxycycline can reverse related inflammatory events by inhibition of MMP-12.

In vivo characterization of GSK256066, a high-affinity inhaled phosphodiesterase 4 inhibitor.

Oral phosphodiesterase (PDE) 4 inhibitors have demonstrated clinical efficacy in chronic obstructive pulmonary disease and asthma. Preclinical and clinical investigation of inhaled PDE4 inhibitors is ongoing. 6-({3-[(Dimethylamino)carbonyl]phenyl}sulfonyl)-8-methyl-4-{[3-methyloxy)phenyl]amino}-3-quinolinecarboxamide (GSK256066) is an exceptionally high-affinity and selective inhibitor of PDE4 designed for inhaled delivery. The aim of these studies was to investigate the potency, duration of action, and therapeutic index of GSK256066 in animal models of pulmonary inflammation. The effects of intratracheally administered GSK256066 were investigated in rat lipopolysaccharide (LPS)- and ovalbumin (OVA)-induced models of acute pulmonary inflammation. In some studies, fluticasone propionate (FP) was included as a comparator. The therapeutic index (anti-inflammatory effect versus emesis) of GSK256066 was studied in ferrets where acute pulmonary inflammation was induced with inhaled LPS. In rats, GSK256066 and FP caused significant (p < 0.05) inhibition of LPS-induced pulmonary neutrophilia. The duration of action of GSK256066 at 10 × ED(50) dose (10 µg/kg) was 12 h. GSK256066 and FP also inhibited LPS-induced increases in exhaled nitric oxide (ED(50) 35 and 92 µg/kg, respectively). In addition, GSK256066 inhibited pulmonary eosinophilia in rats exposed to OVA (ED(50) 0.4 µg/kg). In ferrets, inhaled GSK256066 inhibited LPS-induced pulmonary neutrophilia (ED(50) 18 µg/kg), and no emetic episodes were observed. Thus, GSK256066 may have an improved therapeutic index compared with oral PDE4 inhibitors, e.g., cilomilast and roflumilast. In summary, GSK256066 demonstrates potent and long-lasting anti-inflammatory effects in animal models of pulmonary inflammation and does not induce emetic episodes in ferrets. GSK256066 has potential as an inhaled therapeutic for the treatment of asthma and chronic obstructive pulmonary disease.

Marked deposition of eosinophil-derived neurotoxin in adult patients with eosinophilic esophagitis.

OBJECTIVES: Eosinophilic esophagitis (EoE) is characterized by infiltration of eosinophils into esophageal epithelium. Blood levels of an eosinophil granule protein, eosinophil-derived neurotoxin (EDN), have been proposed as a biomarker for EoE. However, information regarding localization of EDN in the diseased tissues has not been available. The goal of this study was to evaluate the magnitude and distribution of EDN deposition in tissue specimens from the esophagus of EoE patients. METHODS: We studied specimens from 10 adult EoE patients and eight histologically normal controls (three under age 17). Sections from mid-esophageal biopsy specimens were stained for EDN by immunofluorescence, using a polyclonal rabbit antibody to EDN. Cellular staining (i.e., infiltration of intact eosinophils) and extracellular staining (i.e., deposition of released EDN) were scored in a blinded manner on an established 7-point scale. RESULTS: Esophageal biopsy specimens from histologically normal controls showed no or few intact eosinophils and no or minimal extracellular EDN deposition. In contrast, EDN staining was clearly observed in specimens from all EoE patients. In some EoE patients, marked extracellular EDN deposition was observed despite relatively small numbers of intact eosinophils. Overall, there was no correlation between the eosinophil infiltration and the extracellular EDN staining scores. CONCLUSIONS: Marked tissue deposition of extracellular EDN is present in the esophagus of EoE patients. Tissue eosinophil counts may underestimate how extensively eosinophils are involved, particularly in individuals with marked eosinophil degranulation. Evaluation of EDN staining in esophageal biopsy specimens may be useful to diagnose and manage patients with EoE.

Ym1/2 promotes Th2 cytokine expression by inhibiting 12/15(S)-lipoxygenase: identification of a novel pathway for regulating allergic inflammation.

The Ym1/2 lectin is expressed abundantly in the allergic mouse lung in an IL-13-dependent manner. However, the role of Ym1/2 in the development of allergic airways disease is largely unknown. In this investigation, we show that treatment of mice with anti-Ym1/2 Ab during induction of allergic airways disease attenuated mediastinal lymph node production of IL-5 and IL-13. Ym1/2 was found to be expressed by dendritic cells (DCs) in an IL-13-dependent manner and supplementation of DC/CD4(+) T cell cocultures with Ym1/2 enhanced the ability of IL-13(-/-) DCs to stimulate the secretion of IL-5 and IL-13. Affinity chromatography identified 12/15(S)-lipoxygenase (12/15-LOX) as a Ym1/2-interacting protein and functional studies suggested that Ym1/2 promoted the ability of DCs to stimulate cytokine production by inhibiting 12/15-LOX-mediated catalysis of 12-hydroxyeicosatetraenoic acid (12(S)-HETE). Treatment of DC/CD4(+) T cell cultures with the 12/15-LOX inhibitor baicalein enhanced, whereas 12(S)-HETE inhibited the production of Th2 cytokines. Notably, delivery of 12(S)-HETE to the airways of mice significantly attenuated the development of allergic airways inflammation and the production of IL-5 and IL-13. In summary, our results suggest that production of Ym1/2 in response to IL-13 promotes Th2 cytokine production and allergic airways inflammation by inhibiting the production of 12(S)-HETE by 12/15-LOX.

Myeloproliferative disorder with eosinophilia and ETV6-ABL gene rearrangement: efficacy of second-generation tyrosine kinase inhibitors.

ETV6/ABL is a rare gene rearrangement that has rarely been detected in Philadelphia-negative chronic myeloproliferative disorders (C-MPD) and found to have tyrosine kinase activity similar to the BCR/ABL fusion protein. We describe a case of a 61-year-old female with a C-MPD associated with an ETV6/ABL gene rearrangement. She achieved complete cytogenetic remission on imatinib 400mg daily for 17 months, but then developed morphologic and cytogenetic relapse. After starting nilotinib 400mg orally twice daily, she achieved CCyR at 3, 6, and 11 months, suggesting that second-generation TKIs can result in favorable responses in patients with ETV6/ABL rearrangement who relapse after imatinib.

Gamma-secretase inhibitor reduces allergic pulmonary inflammation by modulating Th1 and Th2 responses.

RATIONALE: Gamma-secretase inhibitor (GSI) has been used to effectively block Notch signaling, which is implicated in the differentiation and functional regulation of T helper (Th) effector cells. In asthma, a subset of CD4(+) T cells is believed to initiate and perpetuate the disease. OBJECTIVES: The aim of this study was to evaluate the therapeutic potential of GSI against allergic asthma. METHODS: GSI was administered to an ovalbumin-sensitized mouse via an intranasal route at the time of ovalbumin challenge. MEASUREMENTS AND MAIN RESULTS: The administration of GSI inhibits asthma phenotypes, including eosinophilic airway inflammation, goblet cell metaplasia, methacholine-induced airway hyperresponsiveness, and serum IgE production. GSI treatment of bronchoalveolar lavage cells stimulated via TCR or non-TCR pathways led to a decrease in Th2 cytokine production with a concomitant increase in Th1 cytokine secretion. Expression of Hes-1, a target of Notch signaling, was down-regulated in conjunction with a reduction of Notch intracellular domain and GATA-3 levels after GSI treatment of bronchoalveolar lavage cells. GSI treatment resulted in an inhibition of NF-kappaB activation, and combined treatment with GSI and an NF-kappaB inhibitor augmented IFN-gamma production in a synergistic manner. CONCLUSIONS: These data suggest that GSI directly regulates Th1 and Th2 responses in allergic pulmonary inflammation through a Notch signaling-dependent pathway and that GSI is of high therapeutic value for treating asthma by inhibiting airway inflammatory responses.

Tear fluid concentration of mmp-8 is elevated in non-allergic eosinophilic conjunctivitis and correlates with conjunctival inflammatory cell infiltration.

BACKGROUND: To investigate tear fluid concentration of matrix metalloproteinase 8 (MMP-8) and its relation to conjunctival inflammatory cell infiltration in persistent non-allergic eosinophilic conjunctivitis (NAEC). METHODS: Two groups were included: 26 consecutive adult patients with NAEC (conjunctival eosinophils at least 1+ [1-10 eosinophils/slide], skin prick test [SPT] to common allergens negative), and 26 asymptomatic adult persons (no conjunctival eosinophils, SPT negative). MMP-8 tear fluid concentrations were determined by immunofluorometric assay, and conjunctival brush cytology samples from NAEC patients were used for MMP-8 immunocytochemistry. Gelatin zymography was used to illustrate proteolytic activity within the tear fluid samples. RESULTS: The mean MMP-8 concentration was significantly higher among NAEC patients (214.3 +/- 327.7 microg/l) than among healthy persons (50.4 +/- 62.3 microg/l, P < 0.0001). In the NAEC patients, tear fluid MMP-8 correlated with the numbers of conjunctival neutrophils (r = 0.66, P = 0.0002) as well as with goblet cells and columnar epithelial cells (r = 0.54 for both, P = 0.045), but not with the lymphocyte numbers (r = -0.36, P = 0.0741). By immunocytology, MMP-8 protein could also be detected in vivo in the inflammatory cell population within the conjunctiva. Zymography revealed that proteolysis was significantly higher in the NAEC group, and activated enzymes were practically found only in the NAEC group. CONCLUSIONS: The results showed that NAEC is an inflammatory condition characterized by increased tear fluid MMP-8 levels, probably derived from both inflammatory and structural conjunctival cells. The increased proteolytic activity in NAEC patients may indicate risk of conjunctival structural changes (remodeling).

Reciprocal regulation of iNOS and PARP-1 during allergen-induced eosinophilia.

Inducible nitric oxide synthase (iNOS) inhibition was recently shown to exert no effect on allergen challenge in human asthma, raising serious concerns about the role of the protein in the disease. The present study investigated the role of iNOS in ovalbumin-induced eosinophilia from the perspective of its relationship with poly(ADP-ribose) polymerase-1 (PARP-1) and oxidative DNA damage. A mouse model of ovalbumin-induced eosinophilia was used to conduct the studies. iNOS-associated protein nitration and tissue damage were partially responsible for allergen-induced eosinophilia. iNOS expression was required for oxidative DNA damage and PARP-1 activation upon allergen challenge. PARP-1 was required for iNOS expression and protein nitration, and this requirement was connected to nuclear factor-kappaB. PARP-1 was an important substrate for iNOS-associated by-products after ovalbumin-challenge. PARP-1 nitration blocked its poly(ADP-ribosyl)ation activity. Interleukin-5 re-establishment in ovalbumin-exposed PARP-1(-/-) mice reversed eosinophilia and partial mucus production without a reversal of iNOS expression, concomitant protein nitration or associated DNA damage. The present results demonstrate a reciprocal relationship between inducible nitric oxide synthase and poly(ADP-ribose) polymerase-1 and suggest that expression of inducible nitric oxide synthase may be dispensable for eosinophilia after interleukin-5 production. Inducible nitric oxide synthase may be required for oxidative DNA damage and full manifestation of mucus production. Such dispensability may explain, in part, the reported ineffectiveness of inducible nitric oxide synthase inhibition in preventing allergen-induced inflammation in humans.