Absolute Number Of Eosinophils Research Articles

Comprehensive analysis of immune, extracellular matrices and pathogens profile in lung granulomatosis of unexplained etiology

This study analyzed the type 1 and type 2T helper (Th1/Th2) cytokines (including interleukins), immune cellular, matrix profile, and pathogens in granulomas with unexplained etiology compared to those with infectious and noninfectious etiology. Surgical lung biopsies from 108 patients were retrospectively reviewed. Histochemistry, immunohistochemistry, immunofluorescence, morphometry and polymerase chain reaction were used, respectively, to evaluate total collagen and elastin fibers, collagen I and III, immune cells, cytokines, matrix metalloproteinase-9, myofibroblasts, and multiple usual and unusual pathogens. No relevant polymerase chain reaction expression was found in unexplained granulomas. A significant difference was found between the absolute number of eosinophils, macrophages, and lymphocytes within granulomas compared to uninvolved lung tissue. Granulomas with unexplained etiology (UEG) presented increased number of eosinophils and high expression of interleukins (ILs) IL-4/IL-5 and transforming growth factor-β. In sarcoidosis, CD4/CD8 cell number was significantly higher within and outside granulomas, respectively; the opposite was detected in hypersensitivity pneumonitis. Again, a significant difference was found between the high number of myofibroblasts and matrix metalloproteinase-9 in UEG, hypersensitivity pneumonitis, and sarcoidosis compared to granulomas of tuberculosis. Granulomas of paracoccidioisis exhibited increased type I collagen and elastic fibers. Th1 immune cellular profile was similar among granulomas with unexplained, infectious, and noninfectious etiology. In contrast, modulation of Th2 and matrix remodeling was associated with more fibroelastogenesis and scarring of lung tissue in UEG compared to infectious and noninfectious. We concluded that IL-4/IL-5 and transforming growth factor-β might be used as surrogate markers of early fibrosis, reducing the need for genotyping, and promise therapeutic target in unexplained granulomas.

Epizootical situation on herpes virus infections, leptospirosis and gelminthosis in horses in several regions of Ukraine

The distribution of herpes virus infections of the first and second type in tribal horses for the last five years has been established. During this period, a 1301 samples of blood serum samples of horses were investigated in the diffusion precipitation process. Out of these, 412 or 31.6% of the studied specimens were positive for the herpes virus infection of the first type, 473 or 36.4% for the second-generation herpes virus infection, and 345 or 26.5% of the animals were co-infected with herpes virus infections of the first and second type, respectively. The determination of the epizootic situation in the regions shows that the highest percentage of infections with herpes virus of the first and second types is established in Ternopil region, and the smallest in Chernivtsi. In breeding reproducers of the Chernivtsi region, the level of infection of EHV 1 and EHV 2 types does not exceed 10% of the population. We conducted a study on the coexistence of herpes virus infections, leptospirosis and helminthiasis in horses. The results of the coprological research provide an opportunity to state that in all regions the strongilidosis of horses are the most common. Also, frequent cases of infection with dictiocaulosis testify to the grazing of horses on compatible pastures with cattle. The study, based on the latent course of leptospirosis, depending on the severity of the degree of damage by strontium glands, suggests that with a consistent course of leptospirosis and strontium glands increases the number of leukocytes, eosinophils, neutrophils and total protein. In horses positive in the RMA for leptospirosis and free from strontium glands, a decrease in the number of erythrocytes, lymphocytes, eosinophils and an increase in the number of leukocytes, neutrophils and total protein is observed. In the horses positive in the RMA and affected by strontium, there is a tendency to increase the number of red blood cells, leukocytes, and eosinophils. However, it was found that with an increase in antibody titers in the RMA 1:100 and the intensity of invasion by strontials of 100 and more eggs in 1 gram of feces, a significant decrease (P < 0.001) of the absolute number of lymphocytes and a significant increase (P < 0.001) of the absolute number of eosinophils and neutrophils. A significant decrease in the absolute number of lymphocytes in horses with latent leptospirosis with antibody titres of 1:100 in the RMA and the presence of 100 and more eggs in 1 gram of feces may indicate the development of the immunodeficiency state in such animals and the desirability of immediate demyelination.

Possible Role of IL-25 in Eosinophilic Lung Inflammation in Patients with Chronic Eosinophilic Pneumonia.

Interleukin (IL)-25 and IL-33 induce IL-5 production by various types of cells, such as type 2 helper T (Th2) cells and type 2 innate lymphoid cells. The number of Th2 cells and concentration of IL-5 in the bronchoalveolar lavage fluid (BALF) are increased in patients with eosinophilic pneumonia (EP). To examine the contribution of IL-25 and IL-33 to eosinophilic inflammation of the lung in humans, we evaluated IL-5, IL-25 and IL-33 levels in the BALF of patients with EP. IL-5, IL-25, and IL-33 concentrations in the BALF were measured by enzyme-linked immunosorbent assay in patients with acute eosinophilic pneumonia (AEP), chronic eosinophilic pneumonia (CEP), idiopathic pulmonary fibrosis (IPF), and sarcoidosis. The absolute number of eosinophils, and IL-5 levels, but not IL-33 levels, in the BALF were significantly higher in patients with EP than in patients with IPF and sarcoidosis. IL-25 levels in the BALF were significantly higher in patients with CEP, but not in patients with AEP, than in patients with IPF and sarcoidosis. The absolute number of eosinophils was significantly correlated with the IL-5 concentration in the BALF of patients with EP. IL-5 concentrations were significantly correlated with IL-25 concentrations in the BALF of patients with CEP, but not in patients with AEP. IL-5 levels were not correlated with IL-33 levels in the BALF of patients with EP. Our findings suggest that IL-25 plays an important role via IL-5 in eosinophilic lung inflammation in patients with CEP.

Intravenous immunoglobulin G (IVIg) concurrently promotes Th2 and T regulatory cell development while abrogating Th17 cells

Abstract We aimed to further elucidate the mechanisms by which IVIg modulates the balance between Th17, Th2, and Treg. Using an OVA-driven murine model of allergic airway disease exhibiting a mixed Th17/Th2 response, we examined lung inflammation by H & E staining and histology, and performed cellular phenotyping of lung homogenates using flow cytometry. In addition, we assessed cytokine production by intracellular staining and ELISA, and performed co-culture studies. IVIg treatment reduced lung inflammation and diminished the frequency and absolute number of lung Th17 cells, IL-17A production, and neutrophilia. In contrast, the frequency and absolute number of eosinophils and basophils was increased, while IgE production was abrogated in the IVIg group. This was accompanied by a significant increase in IL-13+ and IL-5+ T cells and in IL-5 concentration in the BAL. The frequency of Foxp3+IL10+ + cells within CD4+ T cells (1.43 % ± .061 vs 2.57 % ± .30, p < .01) and IL-10 secreting dendritic cells (0.28 % ± 0.060 vs 1.20 % ± 0.27, p < .05) was augmented. We also observed an increase in the frequency of CD206 expressing macrophages, or M2 macrophages, in the IVIg group (10.81% ± 2.21 vs 19.80 % ± 1.81, p < .01). Using an in vitro DC: T cell co-culture, IVIg-treated DC inhibited IL-17A production by T cells and increased IL-13 production and Treg differentiation. In conclusion, IVIg modulates Th2/Th17/Treg balance by favoring Treg and Th2 cells while inhibiting Th17 cells. Furthermore, IVIg renders both dendritic cells and macrophages tolerogenic.

Allergic bronchopulmonary aspergillosis in patients with asthma: dynamics of immunological parameters during antimycotic therapy

Background. To determine the frequency of allergic bronchopulmonary aspergillosis in patients with asthma and to study the dynamics of immunological parameters in patients with allergic bronchopulmonary aspergillosis during antimycotic therapy. Methods. During investigation of 176 patients with asthma the group of patients with allergic bronchopulmonary aspergillosis was isolated. Allergological (skin tests with fungal allergens, serum total IgE, specific IgE to fungal allergens), immunological (IFN-γ, IL-10) and mycological (microscopy and culture of respiratory samples) examination was performed. Computer tomography of the chest was done when indicated. Results. In patients with asthma frequency of sensitization to Aspergillus spp. was 27%, with allergic bronchopulmonary aspergillosis - 4%. The increased activity of T-helper type 2 in patients with allergic bronchopulmonary aspergillosis (n=7) was revealed. After itraconazole treatment during 24 weeks serum total IgE reduced (p=0,04), spontaneous and induced production of IFN-γ ratio was normalized. The reduction of the absolute number of eosinophils in 4 (80%) patients, decreased production of sIgE to Aspergillus spp. in 3 (60%) patients were noted. Conclusion. All patients with severe asthma needed additional allergological and mycological examination for the detection of allergic bronchopulmonary aspergillosis. Itraconazole therapy was effective, reduced fungal burden, and resulted to restoring of Th2/Th1 imbalance in patients with allergic bronchopulmonary aspergillosis.

Volatile organic compounds discriminate between eosinophilic and neutrophilic inflammation in vitro

Inflammation associated oxidative stress leads to peroxidation of polyunsaturated fatty acids thereby generating volatile organic compounds (VOCs). The integrative analysis of the total amount of VOCs released by eosinophils and neutrophils in vitro enables the search for those compounds that discriminates between various inflammatory conditions.The approach comprises isolating eosinophils and neutrophils from 30 ml of blood of healthy non-smoking volunteers by gradient centrifugation, using lymphoprep. Eosinophils are separated from neutrophils by immunomagnetic cell separation using anti-CD16. Cells are activated with phorbol 12-myristate 13-acetate and VOCs from the headspace are collected at time 0′, 30′, 60′ and 90′ by introduction of ultra-pure nitrogen in the closed flasks at a flow rate of 200 ml min−1 during 10 min. The gases are trapped onto a sorption tube and analyzed by gas chromatography—time-of-flight—mass spectometry (GC-TOF-MS) in order to identify VOCs released in the headspace by activated neutrophils and eosinophils.Eosinophils and neutrophils were isolated from 26 healthy non-smoking volunteers. The average absolute number of eosinophils and neutrophils upon isolation was 3.5 × 106 and 19.4 × 106, respectively. The volatome in headspace consisted of 2116 compounds and those compounds present in at least 8% of the samples (1123 compounds) were used for further discriminant analysis. Discriminant analysis showed that two VOCs were able to distinguish between eosinophilic and neutrophilic cultures in the unactivated state with 100% correct classification of the entire data set and upon cross validation while five VOCs were able to discriminate between activated eosinophils and neutrophils with 96% correct classification in the original set and upon cross-validation.Analysis of VOCs seems to be a very promising approach in identifying eosinophilic and neutrophilic inflammation but it needs further development and in vivo confirmation.

Azithromycin for the treatment of eosinophilic nasal polyposis: Clinical and histologic analysis.

Introduction:Macrolides used as immunomodulators are a promising tool for chronic inflammatory airway diseases. Eosinophilic nasal polyposis (ENP) is still considered a disease that is difficult to control with the currently standardized treatments.Objectives:To evaluate prolonged treatment with low-dose azithromycin for ENP based on clinical and histopathologic variables.Methods:The present investigation was a self-paired case study of 33 patients with ENP. A comparison was performed between patients before and after treatment with azithromycin for 8 weeks. The patients were subjected to clinical examinations, staging (three-dimensional imaging by endoscopy), application of the questionnaire, and biopsy of nasal polyps at the beginning and at the end of the treatment.Results:The treatment yielded a clinical improvement regarding the two variables studied: polyposis staging (69.7%) and questionnaire (57.6%). We did not find significant differences in the inflammatory pattern and in the percentage or absolute number of eosinophils per field between samples obtained before and after the treatment (p > 0.05). There was no difference between the answers obtained from groups with and without asthma and/or aspirin intolerance (p > 0.3). The patients with advanced initial staging exhibited lower subjective improvement index and staging reduction (p = 0.031 and p = 0.012, respectively).Conclusion:Based on this study, azithromycin may be considered as another therapeutic option for ENP. However, further studies are necessary to define the real mechanism of action involved.

Prospective assessment of the diagnostic utility of esophageal brushings in adults with eosinophilic esophagitis.

Patients with eosinophilic esophagitis (EoE) undergo multiple endoscopies with biopsy for both diagnosis and assessment of treatment response, which is inconvenient and costly. Brush cytology has been examined in Barrett's esophagus to reduce the need for repeated endoscopic biopsies. The aim of this pilot study was to evaluate the ability of brush cytology to detect mucosal eosinophilia in patients with EoE. This prospective study included adults with untreated and treated esophageal eosinophilia undergoing endoscopy at a tertiary care center. Patients received paired brushings and biopsies at the proximal and distal esophagus. A blinded pathologist quantified the number of eosinophils and epithelial cells per high-power field (hpf) on the cytology slides. The ratio of eosinophils/epithelial cells was used to normalize the cytology specimens for density of cells collected. The main outcome measures were sensitivity and specificity of brush cytology, and correlation between cytology and histology. Twenty-eight patients enrolled. The average age of the cohort was 37.7 ± 10.4 years; 75% of subjects were male. The sensitivity of cytology was 67-69% at the proximal esophagus and 70-72% at the distal esophagus. The specificity was 61-67% proximally and 70-75% distally. Histology was not significantly correlated with the max ratio of eosinophils/epithelial cells per hpf or the absolute number of eosinophils on cytology slides. Cytology using esophageal brushing has limited sensitivity and specificity for the detection of esophageal mucosal eosinophilia. The presence of exudates on endoscopy increased the detection of eosinophilia, which could make cytology useful in pediatric EoE, which often has a more exudative presentation. Diagnostic yield may improve with alternative acquisition techniques or the incorporation of eosinophil degranulation proteins.

ITCH Deficiency Promotes an MPN Phenotype with Eosinophilia, Inflammation, Mislocalization and Overexpression of the Transcription Factor NF-E2

Background: We have described overexpression of the transcription factor NF-E2 in MPN patients and shown that elevated NF-E2 levels cause a MPN phenotype in transgenic mice. This includes thrombocytosis, leukocytosis, splenomegaly as well as an expansion of the stem- and progenitor cell compartments in the bone marrow.Recently, we have shown that, counterintuitively for a transcription factor, NF-E2 is located exclusively in the cytoplasm in the vast majority of erythroid cells in the bone marrow (85%). Patients with PMF show a statistically highly significant elevation in the proportion of cells displaying nuclear NF-E2 compared to either healthy controls or ET and PV patients. However, the molecular mechanisms regulating the subcellular localization of NF-E2 and its aberrant localization in PMF remain to be investigated.The E3 ubiquitin ligase ITCH has been postulated to stabilize and retain NF-E2 in the cytosol by protein-protein interaction and subsequent ubiquitinylation. The phenotype of ITCH deficient mice, however, has only been described briefly: animals display splenomegaly and an expansion of the stem cell compartment. The effect of ITCH deficiency on peripheral blood counts and on NF-E2 activity has not been determined.Aims: To characterize the phenotype of ITCH deficient mice and investigate the effect of ITCH deficiency on NF-E2 localization and activity.Methods: The peripheral blood and bone marrow of ITCH knock out mice as well as of heterozygous and wild-type control animals was analyzed: CBCs were determined every four weeks, stem- and progenitor populations in the bone marrow were assessed by 7-color FACS. Expression levels of NF-E2 and its targets genes were measured by quantitative PCR. Plasma cytokine concentrations were measured by Cytometric Bead Array. To determine the subcellular localization of NF-E2, immunohistochemical stainings of ITCH knock out BMs and wild-type controls were conducted.Results: At several consecutive time points ITCH knock out mice displayed a statistically significant elevation in WBC compared to heterozygous and wild-type littermates. Interestingly, both the percentage and the absolute number of eosinophils were significantly increased, some animals presenting with a drastic eosinophilia, the differential containing over 60% eosinophils. Furthermore, ITCH knock out mice display a significant decrease in platelet count, accompanied by an increase in platelet mass and volume, indicative of giant platelets. In the bone marrow ITCH deficient mice show a significant increase in the absolute number of Common Myeloid Progenitors (CMP).NF-E2 expression levels in the peripheral blood as well as in the bone marrow were highly statistically significantly increased compared to the levels measured in wild-type or heterozygous control mice. Consequently, the NF-E2 target gene Thromboxane Synthase A was statistically significantly overexpressed in peripheral blood of ITCH knock out mice.Plamsa concentrations of the inflammatory cytokines INF-γ and TNF were statistically significantly elevated, reaching two to threefold higher levels in ITCH knock out mice compared to wild-type littermates.Lastly, NF-E2 subcellular localization was altered in ITCH deficient mice, which display a significant increase in the proportion of megakaryocytes positive for nuclear NF-E2.Summary/Conclusions: Our data identify the E3 ubiquitin ligase ITCH as a regulator of NF-E2 activity. Impaired ITCH activity leads to both an NF-E2 overexpression and an increased nuclear NF-E2 localization that together drive overexpression of NF-E2 target genes. Furthermore, ITCH deficiency leads to higher inflammatory cytokine levels, comparable to those seen in PMF patients. All of these factors contribute to the resulting myeloproliferative phenotype with eosinophilia. Our data provide the first pathophysiological explanation of the pathognomonic symptom of ITCH deletion: pruritus in “itchy” mice. Moreover, given the aberrant NF-E2 localization in PMF patients, our data provide a possible mechanism and underscore the role of elevated NF-E2 activity in the pathophysiology of myeloproliferative neoplasms. DisclosuresNo relevant conflicts of interest to declare.

Decreased Levels of Lipoxin A4 and Annexin A1 in Wheezy Infants

Objective: Wheezing is a common and challenging health issue in infancy and early childhood. Asthma diagnosis is frequent in patients with a history of recurrent wheezing. A relationship has been reported between asthma and anti-inflammatory mediators such as lipoxin A4 and annexin A1. However, this remains uncertain in wheezy infants. The aim of the present study was to determine lipoxin A4 and annexin A1 levels in wheezy infants. Materials and Methods: Eighty-seven patients aged 6-36 months were included in this study. Demographic characteristics, clinical features, laboratory data, clinical diagnoses, and treatments, if present, were recorded. Patients were divided into 2 groups: patients with wheezing (n = 59) and healthy controls (n = 28). Blood samples were taken and lipoxin A4 and annexin A1 levels were evaluated by ELISA. Results: Lipoxin A4 and annexin A1 levels were significantly lower in the wheezing group than in the control group (p < 0.05). A significant correlation was found between the serum total immunoglobulin E (IgE) level and the percentage and absolute number of eosinophils (p < 0.05). No significant correlation was found in terms of lipoxin A4 and annexin A1 levels, the serum total IgE level, and the percentage and absolute number of eosinophils among groups (p > 0.05). Conclusion: This is the first study to assess lipoxin A4 and annexin A1 levels in wheezy infants. The levels of lipoxin A4 and annexin A1 were found to be low in wheezy infants. We hope that these results will lead to novel therapeutic options for asthma in cases where an optimal treatment modality is lacking.

White Blood Count Components as Risk Predictors for Coronary Heart Disease: The Role of Eosinophils

To the Editor: We read with great interest the paper by Madjid and Fatemi1 about the components of the complete blood count as indicators of inflammation and risk predictors of coronary heart disease. Although all subtypes of the white blood count have been associated with an increased risk of coronary heart disease, the role of eosinophils has been underestimated. The authors1 correctly refer to genetic analysis of the inflammatory role of eosinophils, because a single nucleotide polymorphism at 12q24, in SH2B3, was found to have a significant association with myocardial infarction (MI). Eosinophils play an important role in inflammation, and their existence denotes hypersensitivity inflammation. Hypersensitivity inflammation can induce acute MI, and the Kounis hypersensitivity acute coronary syndrome is well described.2 In patients who had vasospastic angina pectoris, eosinophil counts were significantly increased and could predict the severity of the disease. After these patients underwent medical therapy and attained relief from chest symptoms, their eosinophil counts decreased significantly, to the same level as those of the control group.3 Clinical reports have shown that the absolute number of eosinophils and the eosinophil-to-leukocyte ratio are significantly elevated in patients who have coronary artery disease.4 Coronary vasospasm associated with eosinophilia responds poorly to conventional vasodilator therapy, and although the risk of recurrent coronary events is high, most patients respond to therapy that suppresses eosinophilia, such as corticosteroids.5 Eosinophils are pleiotropic multifunctional leukocytes involved in the initiation and propagation of inflammatory responses, and they express H4 histamine receptors on their surface. These receptors facilitate eosinophil chemotaxis toward mast cells, which are a source of inflammatory soluble mediators. These mediators can induce the coronary hypersensitivity associated with Kounis syndrome that manifests itself as vasospastic angina, acute MI, or both.2 Soluble mediators secreted by mast cells and eosinophils also modulate reciprocal interactions between these 2 cells in the so-called “allergic effector unit.” Major basic cationic protein released from eosinophils can activate mast cells.6 This activation elicits exocytosis and new eicosanoid and cytokine production, both of which are prominent responses after Fc∊RI-dependent activation of mast cells. Mediators similar to those found in Kounis syndrome are found in cases with nonallergic causes, which suggests that this is a more general problem. A common pathway between allergic and nonallergic coronary syndromes seems to exist.2 Eosinophil count has emerged as a novel biomarker of risk stratification in patients who have coronary artery disease. This reality is essential for elucidating the cause of inflammation.

Hydroxycarbamide reduces eosinophil adhesion and degranulation in sickle cell anaemia patients

Inflammation, leucocyte and red cell adhesion to the endothelium contribute to the pathogenesis of sickle cell anaemia. Neutrophils appear to be important for vaso-occlusion, however, eosinophils may also participate in this phenomenon. The role of eosinophils in the pathophysiology of sickle cell anaemia (SCA) and the effect of hydroxycarbamide (HC) therapy on the functional properties of these cells are not understood. Patients with SCA and those on HC therapy (SCAHC) were included in the study. SCAHC individuals presented significantly lower absolute numbers of eosinophils than SCA. Furthermore, SCAHC eosinophils demonstrated significantly lower adhesive properties, compared to SCA eosinophils. SCA and SCAHC eosinophils presented greater spontaneous migration when compared with control eosinophils. Baseline eosinophil peroxidase and reactive oxygen species release was higher for SCA individuals than for control individuals, as were plasma levels of eosinophil derived neurotoxin. SCAHC eosinophil degranulation was lower than that of SCA eosinophil degranulation. Eotaxin-1 and RANTES levels were higher in the plasma of SCA and SCAHC individuals, when compared with controls. These data suggest that eosinophils exist in an activated state in SCA and indicate that these cells play a role in the vaso-occlusive process. The exact mechanism by which HC may alter SCA eosinophil properties is not clear.

Laboratorial characteristics of patients with diarrhoea suffering from egg white allergy

BackgroundEgg allergy is associated with diarrhoeal symptoms. However, the mechanism underlying allergic diarrhoea remains unclear. ObjectiveTo determine whether egg white-specific IgE antibodies coexist with egg white-specific IgG antibodies in patients with egg allergy featuring diarrhoeal symptoms, and whether there is any relationship between these two antibody types. MethodsA total of 89 patients with egg allergy featuring diarrhoeal symptoms (average age, 23.2 years; range, 1–78 years), all of whom tested positive for egg white-specific IgG, were enrolled in this study. The concentration of total IgE, egg white-specific IgE and number of eosinophils in the serum were determined. ResultsAmong the 89 egg white allergic patients tested, 49 (55.1%) patients showed high reactivity to egg white-specific IgG, 48 (53.9%) patients had elevated serum total IgE levels, and 25 (28.1%) patients had elevated absolute eosinophil numbers. Out of the 89 egg white allergic patients, 25 showed elevated egg white-specific IgE antibody levels. Of the 25 patients who were positive for egg white-specific IgE antibody, 21 presented high sensitive reaction to egg white-specific IgG, three presented moderate sensitive reaction to egg white-specific IgG, and one presented mild sensitive reaction to egg white-specific IgG. A moderate correlation between egg white-specific IgG and egg white-specific IgE, egg white-specific IgG and absolute eosinophil number was found in the egg white allergic patients (r=0.438, P=0.000; r=0.322, P=0.002). Egg white-specific IgE levels varied in different age groups; the egg white-specific IgE concentration of younger patients (age≤18 years, mean rank 54.29) was significantly higher than that of the adult patients (age>18 years, mean rank 34.61) (Z=−3.629, P=0.000). ConclusionEgg white-specific IgE antibody could coexist with egg white-specific IgG antibody in patients suffering from egg white allergy. Aberrant changes in the concentration of egg white-specific IgE antibody were associated with the presence of egg white-specific IgG antibody.

Clinical and Immunological Correction of DOCK8 Deficiency by Allogeneic Hematopoietic Stem Cell Transplantation Following a Reduced Toxicity Conditioning Regimen

Dedicator of cytokinesis 8 protein (DOCK8) deficiency is a combined immunodeficiency disorder characterized by an expanding clinical picture with typical features of recurrent respiratory or gastrointestinal tract infections, atopic eczema, food allergies, chronic viral infections of the skin, and blood eosinophilia often accompanied by elevated serum IgE levels. The only definitive treatment option is allogeneic hematopoietic stem cell transplantation (HSCT). We report a patient with early severe manifestation of DOCK8 deficiency, who underwent unrelated allogeneic HSCT at the age of 3 years following a reduced toxicity conditioning regimen. The transplant course was complicated by pulmonary aspergilloma pretransplantation, adenovirus (ADV) reactivation, and cytomegalovirus (CMV) pneumonitis 4 weeks after transplantation. With antifungal and antiviral treatment the patient recovered. Seven months after transplantation the patient is in excellent clinical condition. Eczematous rash, chronic viral skin infections, and food allergies have subsided, associated with normalization of IgE levels and absolute numbers of eosinophils. Chimerism analysis shows stable full donor chimerism. DOCK8 deficiency can be successfully cured by allogeneic HSCT. This treatment option should be considered early after diagnosis, as opportunistic infections and malignancies that occur more frequently during the natural course of the disease are associated with higher morbidity and mortality.

Associations Between Mercury and Hepatic, Renal, Endocrine, and Hematological Parameters in Atlantic Bottlenose Dolphins (Tursiops truncatus) Along the Eastern Coast of Florida and South Carolina

We evaluated associations between total mercury (Hg) concentrations in blood and skin and endocrine, hepatic, renal, and hematological parameters in free-ranging bottlenose dolphins (Tursiops truncatus). Dolphins in Indian River Lagoon, FL had higher concentrations of Hg in blood (0.67 μg/l wet wt) and skin (7.24 μg/g dry wt) compared with those from Charleston Harbor, SC (0.15 μg/l wet wt, 1.68 μg/g dry wt). An inverse relationship was observed between blood and skin Hg concentrations and total thyroxine, triiodothyronine, absolute numbers of lymphocytes, eosinophils, and platelets. Adrenocorticotropic hormone (ACTH), blood urea nitrogen, and gamma-glutamyl transferase increased with increasing concentrations of Hg in blood and skin; lactate dehydrogenase and neutrophils increased with concentrations in skin only. Hemoglobin and mean corpuscular hemoglobin increased with increasing concentrations of Hg in blood. Selenium was negatively associated with free T4, progesterone, and absolute numbers of monocytes, and positively correlated with absolute numbers of eosinophils and lymphocytes, and mean corpuscular volume. The results suggest the potential for a deleterious effect of Hg in highly exposed dolphins.

Altered Functional Properties of Eosinophils In Sickle Cell Anemia and Effects of Hydroxyurea Therapy

AbstractAbstract 2656The pathophysiology of sickle cell anemia (SCA) is now recognized to include a chronic inflammatory condition that contributes to the recurrent episodes of vaso-occlusion that characterize this disease. The vaso-occlusive process is a multi-step process that involves different cell types, such as red blood cells, reticulocytes, activated endothelial cells, platelets and leukocytes. In vivo transgenic animal studies indicated that the neutrophil appears to be the most important leukocyte in vaso-occlusion in previous transgenic animal in vivo studies; however, eosinophils (EOs) may also participate in this phenomenon in humans. EOs are found in significantly elevated numbers in sickle cell disease individuals and, in static adhesion assays, demonstrate augmented adhesive properties suggesting an activated state of these cells. However, the role of EOs in the pathophysiology of SCD and the effect of hydroxyurea (HU) therapy on the functional properties of these cells are not well understood. The aim of this study was to evaluate EO adhesion, migration and degranulation in SCA patients and the effect of HU therapy on these cells. EOs were purified from the peripheral blood of healthy controls (CON) (n≥10), steady-state SCA patients (n≥12) and SCA patients on HU therapy (n≥15), using Percoll gradient separation, followed by immunomagnetic sorting. EO adhesion was determined by static adhesion assays and degranulation was evaluated by measurement of eosinophil peroxidase activity. Migration of EOs was determined in 96-multiwell chemotaxis chamber assays. Plasma levels of Eotaxin-1, 2 and RANTES, important eosinophil attracting chemokines, were determined by ELISA. The absolute number of EOs in the peripheral blood of SCD patients not treated with HU was significantly higher compared to control individuals (0.504±0.09 vs 0.188±0.04; P=0.01, respectively). SCA patients taking HU presented significantly lower numbers of EOs (0.200±0.05, P = 0.01). Basal adhesion of EOs from SCA patients was significantly higher than in healthy controls (22.8±2.6 vs 13.7±1.7%, respectively, P = 0.004). Furthermore, the EOs from SCA patients on HU therapy demonstrated a significantly lower adhesion to fibronectin (12.5±2.0%, P=0.002). Spontaneous EO chemotaxis was significantly increased in SCA patients, compared to healthy controls (12.9±1.56 vs 6.97±0.8 × 105/ml, respectively, P=0.009). Interestingly, the chemotatic response of EOs isolated from SCA patients taking HU was also higher than that of control EOs (12.1± 1.9 × 105/ml). EO chemotaxis in response to RANTES, Eotaxin and IL5 was significantly augmented in all groups compared to spontaneous chemotaxis (RANTES: CON: 15.3± 2.3; SCA: 19.9±3.0; SCAHU: 25.0±4.7; Eotaxin: CON: 18.7±3.0; SCA: 24.5±4.2; SCAHU: 24.0±5.2; IL5: CON: 13.4±2.4; SCA: 18.8±3.0, SCAHU: 16.6±4.5, P<0.05). Baseline eosinophil peroxidase release was higher in SCA EOs, compared to CON EOs (0.4±0.04 vs 0.2±0.02, respectively, P=0.001), but patients on HU presented a lower EOs degranulation than SCA not on HU (0.19±0.03, P=0.003). Plasma levels of Eotaxin-1 and RANTES were significantly higher in SCA individuals, compared to CON individuals (124±7; 1057±125 vs 78±7; 540±50.2 pg/ml, P=0.0002; P=0.0009, respectively). HU therapy was not associated with any change in Eotaxin-1 and RANTES plasma levels (117.9±9.7; 1175±126.2 pg/ml, respectively). Eotaxin-2 plasma levels did not vary among groups (485±80.8, 450±34, 398±47.8 pg/ml for CON, SCA and SCAHU, respectively). EOs of patients with SCA demonstrate a higher capacity for spontaneous migration, stimulated migration and degranulation. Therapy with HU is associated with reduced adhesion and degranulation of EOs in these patients, but had no effect on the chemotactic ability of these cells or chemokine levels. The presence of relative eosinophilia along with altered functional properties in SCA patients warrants further investigation of the role of EOs in the vaso-occlusive process. In particular, complications associated with allergic reactions, such as acute thoracic syndrome and asthma in SCA children, in which EO adhesion and degranulation may be more important, should be studied. The characteristics of the effect of HU on EO also suggest that SCA events in which EO chemotaxis plays a role are likely to respond poorly to HU therapy, and should lead to the development of alternative therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Effects of Exogenous Corticosterone on Circulating Leukocytes of a Salamander (Ambystoma talpoideum) with Unusually Abundant Eosinophils

When animals become stressed, their levels of glucocorticoid hormones increase, causing white blood cells to move from tissues to circulation or vice versa. The primary alteration is an increase in the abundance of circulating neutrophils and a decrease in lymphocytes in circulation. A lesser-known effect is a decrease in the number of circulating eosinophils. Salamanders in the genusAmbystomahave unusually high numbers of circulating eosinophils, and as such, any effect of stress hormones on circulating leukocytes (especially eosinophils) of these species should be especially pronounced. We conducted an experiment to determine the effect of corticosterone administration on leukocyte counts (from blood smears) ofA. talpoideumsalamanders. Salamanders were captured and sampled as reference animals(n=11), given a sham injection(n=8), or injected with 0.1cc of a 100 μg/mL corticosterone solution(n=28). After 24 hours, relative neutrophil counts were higher and relative lymphocyte counts lower, in the corticosterone group than the sham and control groups. Absolute counts showed that this effect was driven by a reduction in lymphocytes, since neutrophil counts were statistically similar across treatments. Importantly, relative and absolute numbers of eosinophils decreased in the sham and corticosterone groups, confirming the sensitivity of this cell to stress in amphibians.

“Clinical and Molecular Heterogeneity of Eosinophilia-Associated Myeloproliferative Neoplasms with a Translocation t(5;12)”.

Abstract 4983Chromosomal or molecular aberrations in eosinophilia-associated myeloproliferative neoplasms (Eos-MPN) often involve receptor (e.g. PDGFRA at 4q12, PDGFRB at 5q31, FGFR1 at 8p11) or intracellular (e.g. ABL1 at 9q34, JAK2 at 9p24) tyrosine kinases (TK). Fusion genes with rearrangement of PDGFRA or PDGFRB are known to be associated with rapid and durable complete hematological and clinical remission on treatment with imatinib; many of these cases also achieve complete molecular remission (undetectable fusion gene transcripts by nested reverse transcriptase polymerase chain reaction, RT-PCR). The most common fusion genes in Eos-MPN are FIP1L1-PDGFRA caused by a cytogenetically invisible interstitial deletion on 4q12, and ETV6-PDGFRB as consequence of a reciprocal translocation t(5;12)(q31-33;p13). Due to restricted availability of molecular diagnostic tools, a substantial proportion of Eos-MPN patients with t(5;12) are treated with imatinib based on karyotype but without confirmation of the underlying fusion gene by fluorescence-in-situ-hybridization (FISH) or RT-PCR. We report here on three patients (male, n=2; female, n=1) (median age 58 years, range 38-71) with Eos-MPN (median absolute eosinophil numbers in peripheral blood 8,100/μL, range 2,300-62,000) in chronic (n=2) or secondary acute myeloid leukemia (AML)/blast phase (n=1). Cytogenetic analysis revealed a t(5;12) (n=2) or a complex karyotype with involvement of chromosome arms 5q and 12p (n=1). Despite the fact that the clinical and hematological phenotype was resembling ETV6-PDGFRB positive disease (e.g. leukocytosis, eosinophilia, hypercellular marrow, splenomegaly), all three cases tested negative for ETV6-PDGFRB. A fourth patient with t(5;12)(q31;p13) but negative for ETV6-PDGFRB had identical clinical characteristics except basophilia of 7% without eosinophilia. Further molecular analyses revealed an ETV6-ACSL6 fusion gene in all four patients. Two patients received imatinib (400 mg/d) without knowledge of the molecular status and two patients were treated with sorafenib (400-800 mg/d) due to its multitargeted activity towards signal transduction molecules. No responses were observed to imatinib or sorafenib. Two male patients received an allogeneic stem cell transplantation (SCT) from a related or a matched unrelated donor, respectively. The first patient died on day +67 due to relapse of secondary AML/blast phase while the second patient died on day +64 due to transplant related complications. After failure to imatinib, the female patient with secondary AML/blast crisis only received supportive care because of comorbidity and died 7 months after diagnosis due to cytopenia-related complications. The remaining chronic phase male patient is alive 5 months after diagnosis. In addition to patients known from the literature (n=6), primary AML or rapid progression to secondary AML/blast phase has been observed in 7 of overall 10 patients with t(5;12) and an ETV6-ACSL6 fusion gene indicating a potentially aggressive clinical course. Consistent with the lack of involvement of a TK, the disease is primarily resistant to currently available TK-inhibitors and allogeneic SCT should be considered in eligible patients. In conclusion, treatment with TK-inhibitors in patients with myeloproliferative neoplasms and a t(5;12) should only be initiated if involvement of PDGFRB is confirmed by FISH or PCR analysis. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Influência do período pós-parto sobre o leucograma de fêmeas bovinas da raça holandesa

Para avaliar a influência do período pós-parto sobre os constituintes do leucograma de fêmeas bovinas da raça Holandesa, foram colhidas 142 amostras de sangue nos primeiros 90 dias pós-parto. As amostras foram colhidas de vacas clinicamente sadias e não reagentes ao antígeno (GP 51) do vírus da Leucose dos Bovinos, divididas em 9 grupos experimentais, de acordo com o momento da colheita. Nas amostras de sangue foi realizada a contagem do número total de leucócitos e contagem diferencial de leucócitos. Durante todo o período estudado o quadro leucocitário foi predominantemente linfocitário, sendo nas primeiras 24 horas após aparição observado uma leucocitose fisiológica por neutrofilia com desvio à esquerda regenerativo, linfocitose e eosinopenia. Na primeira semana após o parto ocorreu a diminuição gradual do número de leucócitos, devido à redução do número absoluto de neutrófilos e linfócitos, sendo que exceção feita ao número absoluto de eosinófilos, nas amostras colhidas entre 6 - 8 dias após o parto os valores já estavam nos patamares observados nos animais fora do puerpério. Os valores de referência na primeira semana de puerpério variaram: entre 18.977 ± 5.644 e 12.247 ± 5.058 leucócitos/mm³; 8.453 ± 2.956 e 4.376 ± 4.139 neutrófilos /mm³ (532 ± 547 e 88 ± 174 neutófilos bastonete /mm³; 7.920 ± 2.689 e 4.289 ± 4.039 neutrófilos segmentado/mm³); 334 ± 387 e 225 ± 298 eosinófilos/mm³; 50 ± 128 e 23 ± 45 basófilos /mm³; 10.004 ± 4.901 e 7.595 ± 3.127 linfócitos/mm³; 121 ± 178 e 21 ± 85 monócitos/mm³.

Comparison of cough reflex sensitivity after an inhaled antigen challenge between actively and passively sensitized guinea pigs

BackgroundLate asthmatic response is observed following antigen challenge in actively, but not passively, sensitized guinea pigs. Although cough reflex sensitivity is increased after antigen challenge in actively sensitized guinea pigs, it is unknown whether the antigen-induced increase in cough reflex sensitivity develops in passively sensitized animals. The aim of this study was to compare the cough reflex sensitivity to inhaled capsaicin after an inhaled antigen challenge between actively and passively sensitized guinea pigs.MethodsMeasurement of number of coughs elicited by increasing concentrations of capsaicin (10-6 and 10-4 M) and bronchial responsiveness to ascending concentrations of methacholine, and analysis of bronchoalveolar lavage fluid (BALF) were separately performed 24 h after an antigen challenge in actively and passively sensitized guinea pigs.ResultsPercentage of eosinophils in BALF and bronchial responsiveness to methacholine were increased 24 h after the antigen challenge in both actively and passively sensitized animals compared with saline-challenged actively and passively sensitized animals, respectively. Absolute number of eosinophils in BALF from actively sensitized and antigen-challenged guinea pigs was significantly greater than that from passively sensitized and antigen-challenged animals. Cough response to capsaicin and concentration of substance P in BALF were increased 24 h after the antigen challenge in actively sensitized guinea pigs, but not in passively sensitized guinea pigs. Bronchial responsiveness, cough reflex sensitivity and substance P concentration and total cells in BALF were increased in actively sensitized and saline challenged guinea pigs compared with passively sensitized and saline challenged animals.ConclusionThe results suggest that active sensitization per se increases cough reflex sensitivity accompanied by increased inflammatory cells and substance P level in BALF, and antigen challenge further increases them, while simple IgE- and/or IgG-mediated allergic reaction per se or the low intensity of eosinophil infiltration in the airway itself may not affect cough reflex sensitivity in guinea pigs.