Eosinophil-Associated Gene Pathways but not Eosinophil Numbers are Differentially Regulated between Synchrotron Microbeam Radiation Treatment and Synchrotron Broad-Beam Treatment by 48 Hours Postirradiation.

Synchrotron microbeam radiation treatment (MRT) is a preclinical radiotherapy technique with considerable clinical promise, although some of the underlying radiobiology of MRT is still not well understood. In recently reported studies, it has been suggested that MRT elicits a different tumor immune profile compared to broad-beam treatment (BB). The aim of this study was to investigate the effects of synchrotron MRT and BB on eosinophil-associated gene pathways and eosinophil numbers within and around the tumor in the acute stage, 48 h postirradiation. Balb/C mice were inoculated with EMT6.5 mouse mammary tumors and irradiated with microbeam radiation (112 and 560 Gy) and broad-beam radiation (5 and 9 Gy) at equivalent doses determined from a previous in vitro study. After tumors were collected 24 and 48 h postirradiation, RNA was extracted and quantitative PCR performed to assess eosinophil-associated gene expression. Immunohistochemistry was performed to detect two known markers of eosinophils: eosinophil-associated ribonucleases (EARs) and eosinophil major basic protein (MBP). We identified five genes associated with eosinophil function and recruitment (Ear11, Ccl24, Ccl6, Ccl9 and Ccl11) and all of them, except Ccl11, were differentially regulated in synchrotron microbeam-irradiated tumors compared to broad-beam-irradiated tumors. However, immunohistochemical localization demonstrated no significant differences in the number of EAR- and MBP-positive eosinophils infiltrating the primary tumor after MRT compared to BB. In conclusion, our work demonstrates that the effects of MRT on eosinophil-related gene pathways are different from broad-beam radiation treatment at doses previously demonstrated to be equivalent in an in vitro study. However, a comparison of the microenvironments of tumors, which received MRT and BB, 48 h after exposure showed no difference between them with respect to eosinophil accumulation. These findings contribute to our understanding of the role of differential effects of MRT on the tumor immune response.

Single high-dose irradiation aggravates eosinophil-mediated fibrosis through IL-33 secreted from impaired vessels in the skin compared to fractionated irradiation.

We have revealed in a porcine skin injury model that eosinophil recruitment was dose-dependently enhanced by a single high-dose irradiation. In this study, we investigated the underlying mechanism of eosinophil-associated skin fibrosis and the effect of high-dose-per-fraction radiation. The dorsal skin of a mini-pig was divided into two sections containing 4-cm(2) fields that were irradiated with 30 Gy in a single fraction or 5 fractions and biopsied regularly over 14 weeks. Eosinophil-related Th2 cytokines such as interleukin (IL)-4, IL-5, and C-C motif chemokine-11 (CCL11/eotaxin) were evaluated by quantitative real-time PCR. RNA-sequencing using 30 Gy-irradiated mouse skin and functional assays in a co-culture system of THP-1 and irradiated-human umbilical vein endothelial cells (HUVECs) were performed to investigate the mechanism of eosinophil-mediated radiation fibrosis. Single high-dose-per-fraction irradiation caused pronounced eosinophil accumulation, increased profibrotic factors collagen and transforming growth factor-ß, enhanced production of eosinophil-related cytokines including IL-4, IL-5, CCL11, IL-13, and IL-33, and reduced vessels compared with 5-fraction irradiation. IL-33 notably increased in pig and mouse skin vessels after single high-dose irradiation of 30 Gy, as well as in irradiated HUVECs following 12 Gy. Blocking IL-33 suppressed the migration ability of THP-1 cells and cytokine secretion in a co-culture system of THP-1 cells and irradiated HUVECs. Hence, high-dose-per-fraction irradiation appears to enhance eosinophil-mediated fibrotic responses, and IL-33 may be a key molecule operating in eosinophil-mediated fibrosis in high-dose-per fraction irradiated skin.

Penfigoide ampolloso inducido por crioterapia / Bullous pemphigoid induced by cryotherapy

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Acute exposure of rabbit eyes to artificial light in vivo: effect on corneal and third eyelid conjunctival histology and the gene expression of PAFR.

PURPOSE: To study the effect of acute exposure of rabbit eyes to artificial sunlight in vivo, on the integrity of corneal and conjunctival tissue as well as on the gene expression of the receptor for platelet activating factor (PAFR). METHODS: New Zealand albino rabbits were immobilized opposite a 300 W Osram Ultra-Vitalux® light bulb with an emission radiation spectrum similar to that of normal sunlight at noon, and exposed to ultraviolet B radiation in the range of the reported threshold for corneal damage. Corneal and third eyelid tissue samples were removed from exposed eyes at 2, 6 and 24 h following the end of the exposure to the bulb light and were subsequently processed for histochemical staining and RNA extraction. The gene expression of PAFR was detected with real time reverse transcription polymerase chain reaction. RESULTS: Some epithelial shedding was detected in the corneal tissue as a result of acute exposure to artificial sunlight. In the eyelid conjunctiva, a marked accumulation of eosinophils was noticed, as early as 2 h post-exposure, apparently directed toward the upper part of the epithelial layer. This effect appears to subside by hour 24. No statistically significant changes in gene expression were detected in the corneal tissue, whereas in the third eyelid, PAFR gene expression was significantly induced, most prominently at t = 2 and 6 h post-exposure. CONCLUSION: Acute exposure of rabbit eyes to artificial sunlight induced a marked infiltration of eosinophils into the epithelial layer of the conjunctiva but no gross alterations in the cornea or the third eyelid. The gene expression of PAFR was upregulated, as an effect of light exposure, in the third eyelid but not in the cornea.

Analysis of minocycline as a countermeasure against acute radiation syndrome.

BACKGROUND/AIM: To evaluate the impact of an antibiotic, minocycline, on several immune parameters in response to radiation in a mouse model. MATERIALS AND METHODS: C57BL/6 mice were treated with minocycline (i.p.) for 5 days, beginning immediately before radiation with 1-3 Gy (60)Co γ-rays. Spleen and blood were collected on day 4 post-irradiation. Cell populations were determined in the blood and spleen. Splenocytes were activated with anti-CD3 antibody for 48 h and cytokines were quantified. RESULTS: Minocycline increased the counts and/or percentages of splenic macrophages, granulocytes, natural killer, T- and CD8(+) T-cells (p<0.05 versus radiation alone). Minocycline significantly increased the expression of interleukin-1α and ß, which are radioprotective, as well as the ones of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor, which accelerate neutrophil recovery (p<0.05 versus radiation alone), while suppressing cytokines that could prevent hematopoiesis, e.g. macrophage inflammatory protein-1α, tumor necrosis factor-α and interferon-γ. CONCLUSION: These data indicate that minocycline should be further tested for use in restoration of the hematopoietic system after radiation exposure.

Selective depletion of eosinophils or neutrophils in mice impacts the efficiency of apoptotic cell clearance in the thymus.

Developing thymocytes undergo a rigorous selection process to ensure that the mature T cell population expresses a T cell receptor (TCR) repertoire that can functionally interact with major histocompatibility complexes (MHC). Over 90% of thymocytes fail this selection process and die. A small number of macrophages within the thymus are responsible for clearing the large number of dying thymocytes that must be continuously cleared. We studied the capacity of thymic macrophages to clear apoptotic cells under acute circumstances. This was done by synchronously inducing cell death in the thymus and then monitoring the clearance of apoptotic thymocytes. Interestingly, acute cell death was shown to recruit large numbers of CD11b(+) cells into the thymus. In the absence of a minor CSF-1 dependent population of macrophages, the recruitment of these CD11b(+) cells into the thymus was greatly reduced and the clearance of apoptotic cells was disrupted. To assess a possible role for the CD11b(+) cells in the clearance of apoptotic cells, we analyzed mice deficient for eosinophils and mice with defective trafficking of neutrophils. Failure to attract either eosinophils or neutrophils to the thymus resulted in the impaired clearance of apoptotic cells. These results suggested that there is crosstalk between cells of the innate immune system that is necessary for maximizing the efficiency of apoptotic cell removal.

Alterations of hematological variations in rats exposed to extremely low frequency magnetic fields (50 Hz).

BACKGROUND AND AIMS: The aim of this study was to evaluate the possible effects of in vivo exposure to extremely low frequency electromagnetic fields (ELF-EMF) on whole blood parameters (hematological parameters) in rats. METHODS: Forty eight female Wistar rats, obtained from the Medical Science Application and Research Center, Dicle University, Turkey in 2004 were divided into four separate groups: two exposed groups (0.97 mT, 50 and 100 days, 3h/day) and two controls (sham). RESULTS: Eosinophil, hemoglobin and MPV levels significantly decreased in rats that were exposed to EMF for 50 days. When the data for rats exposed for 50 days and 100 days were compared, it was found that MPV levels in rats exposed for 100 days were significantly lower. There was no significant difference in total leukocyte, neutrofil, lymphocyte, monocyte, eosinophil and basophil counts, or in erythrocyte, Hct, MCH, MCHC, RDW, PLT and PDW levels between the exposed and sham-exposed groups. ELF-EMF exposure had no effect on body weight. Also, liver weight did not show any significant difference between groups. CONCLUSIONS: Our results indicate that the applied ELF-EMF exposure may induce slight but statistically significant alterations in some hematological parameters of rats, within the physiological range.

[Age related changes in rat blood leucocyte count and morpho-metrical parameters of large granular lymphocytes under different light regimens].

The effect of different light conditions on the age related level of blood leucocytes, leucocytes differential count and the morpho-metrical parameters of large granular lymphocytes in rats was investigated. It has been found that the senescent animals demonstrate disturbance in typical lymphocytes/ neutrophils ratio under condition with light and darkness changes natural and regular illumination. The total leucocytes number, the level of neutrophilic bands, eosynophils and large granular lymphocytes increased under continuous light illumination and natural photoperiod in Karelia. The comparatively lower level of lymphocytes and higher of neutrophils was formed earlier under light conditions without light and dark interchange, i.e. under long time light deprivation (DP) and under constant illumination (LL).

Circulating leukocyte and cytokine responses to measles and poliovirus vaccination in children after ultraviolet radiation exposures.

Suberythemal ultraviolet radiation (UVR) exposures of children are used routinely in Russia to prevent rickets and to strengthen general health. The aim of the present study was to re-evaluate the effects of such a regime on immune responses as UVR is now recognised to suppress cell-mediated immunity in many animal models. Seventeen infants were immunised with attenuated measles and recall polio vaccines of whom 10 had been given a course of prophylactic UV exposures before the vaccinations. All the infants in the study developed an acute infectious conjunctivitis one week prior to the vaccinations and were convalescent at the time of the vaccination. They were bled on the day of the vaccinations and at several times thereafter to assess leukocyte percentages and plasma cytokine levels. On the day of the vaccinations, an active immune response was apparent. The UV-exposed children differed from the unexposed children by having a smaller percentage of natural killer cells and a higher percentage of CD25-positive cells. In the days following the vaccinations, the UV-exposed infants had a lowered percentage of total lymphocytes with increased percentages of monocytes, eosinophils, neutrophils and HLA-DR-positive cells as well as higher concentrations of plasma IL-1beta and IL-10 compared with the unexposed infants. There were no local or systemic clinical reactions to the vaccines in the UV-group while a moderate rise in temperature of three children in the unexposed group occurred. Thus the UV irradiations modulated leukocyte percentages and plasma cytokine levels following the vaccinations, perhaps through the activation of a T helper 2-like response.

Rhinophototherapy: a new therapeutic tool for the management of allergic rhinitis.

BACKGROUND: Phototherapy has a profound immunosuppressive effect and is able to inhibit hypersensibility reactions in the skin. OBJECTIVE: We evaluated whether phototherapy using a combination of UV-B (5%), UV-A (25%), and visible light (70%), referred to as mUV/VIS, is effective in treating allergic rhinitis. METHODS: We conducted a randomized, double-blind study, in 49 patients with hay fever. The study was performed during the ragweed season. Each intranasal cavity was illuminated 3 times a week for 3 weeks with mUV/VIS or with low-intensity visible light. Symptom scores, inflammatory cells, and their mediators were assessed in nasal lavages. In vitro effects of mUV/VIS irradiation on T-cell and eosinophil apoptosis and its inhibitory effect on mediator release from basophils were examined. RESULTS: Rhinophototherapy was tolerated well and resulted in a significant improvement of clinical symptoms for sneezing (P < .016), rhinorrhea (P < .007), nasal itching (P < .014), and total nasal score (P < .004). None of the scores improved significantly in the control group. Scores for nasal obstruction slightly improved after mUV/VIS treatment and significantly increased in the control group (P < .017). In the nasal lavage, phototherapy significantly reduced the number of eosinophils and the level of eosinophil cationic protein and IL-5. In vitro irradiation of T cells and eosinophils with mUV/VIS light dose-dependently induced apoptosis. Furthermore, mUV/VIS irradiation inhibited the mediator release from RBL-2H3 basophils. CONCLUSION: These results suggest that phototherapy is an effective modality to treat allergic rhinitis and offer new options for the treatment of immune-mediated mucosal diseases.

Increased radiation sensitivity of an eosinophilic cell line following treatment with epigallocatechin-gallate, resveratrol and curcuma.

Ionizing radiation is widely used in radiotherapy, in order to promote an apoptotic response in cancerous cells. Since the need to find new substances that would enhance the radiation-induced apoptosis in cancerous cells is great, we studied the effect of epigallocatechin-gallate (EGCG, a tea component), resveratrol (a wine component) and curcuma on cell proliferation and radiation-induced apoptosis in the human leukaemic cell line, EOL-1, derived from a patient with eosinophilic leukaemia. Cells were X-irradiated with 0, 2, 4, 6 or 8 Gy and cultured in the presence of EGCG, resveratrol or curcuma (concentrations ranging from 0 to 200 microM) for 1, 2 or 3 days of culture. Cell proliferation was measured using trypan blue exclusion. Apoptosis was evaluated using light microscopy (morphology study after May-Grunwald Giemsa staining) and flow cytometry (annexin-V staining). Irradiation alone induced a dose-related reduction in cell proliferation and the appearance of polyploid cells in EOL-1 cells. Additionally, EOL-1 cells underwent a dose-related increase of apoptosis which, from the second day on, was accompanied by a dose-related increase of necrosis. When cells were exposed to EGCG, resveratrol or curcuma alone, a decrease in cell proliferation was observed, beginning from 25 microM EGCG and 50 microM resveratrol and curcuma, while an increase in the percentage of apoptotic cells was noted from 50 microM EGCG, 100 microM resveratrol and curcuma in EOL-1 cells, after only one day of culture. Simultaneous exposure to X-irradiation and, EGCG, resveratrol or curcuma resulted in a synergistic decrease of cell proliferation as well as in a synergistic increase of apoptosis and necrosis. These results suggest that, depending on the concentration, EGCG, resveratrol and curcuma enhance radiation-induced apoptosis in the leukaemic cell line, EOL-1 (EGCG >resveratrol >curcuma). In order to further characterise the radiation-induced apoptosis of this leukaemic cell line, other flow cytometrical analyses are in progress.

Effect of low-intensity laser radiation on peripheral blood eosinophils.

The study was performed on peripheral blood eosinophils isolated on a Ficoll-Verografin continuous-density gradient. Low-intensity laser radiation (lambda=0.89 mu, 25-30 mW power, 8 min exposure) induced degranulation of peripheral blood eosinophils. The effect of low-intensity laser radiation was realized via activation of calcium channels.

A photoreactive fluorescent marker for identifying eosinophils and their cytoplasmic granules in tissues.

Here we describe a simple histochemical technique that provides an improved approach to identifying eosinophil components in tissues through the formation of photoreactive complexes that produce stable fluorescent emissions. This method worked readily with histological tissue sections 6-60 microm thick, which were fixed in neutral buffered formalin (NBF), and with cell suspensions similarly fixed and unfixed. Deep red (>605 nm) fluorescent emissions were produced by eosinophil-specific granules when exposed to broadband excitation spectra from a 100-W mercury lamp source (510-590 nm), as well as single-wavelength excitations from both an argon laser (488 nm) and a UV-visible laser (514 nm). The fluorophore-granule complex emissions increased in intensity during the first minute of continuous photoexcitation, then remained stable (>10 min). All nonspecific autofluorescence phenomena associated with these tissues were photobleached in the first minute, including areas of background Biebrich scarlet binding where photoreactive complexes were not formed (i.e., collagen), indicating environmental influences on the fluorophore. This technique allows the visualization of eosinophil granules over a greater period of time than is usually permissible with standard fluorescent markers. Therefore, techniques such as confocal microscopy can be utilized to their fullest extent, providing much more detailed information on the location and distribution of the cytoplasmic contents of eosinophils.

Mononuclear cells in atopic dermatitis in vivo: immunomodulation of the cutaneous infiltrate by medium-dose UVA1 phototherapy.

INTRODUCTION: Recently, medium-dose UVA1 phototherapy (50 J/cm2) has achieved great therapeutic success within the treatment of severe atopic dermatitis (AD). Histologically, AD is recognised by a pathological perivascular dermal infiltrate including T lymphocytes, eosinophils and Langerhans cells. The purpose of our study was to investigate the extent to which UVA1 irradiation is able to modulate the mononuclear dermal inflammatory infiltrate using different monoclonal antibodies. PATIENTS AND METHODS: Biopsy specimens before and after treatment with medium-dose UVA1 irradiation (cumulative dose: 750 J/cm2) from 15 patients suffering from severe AD were analysed immunohistochemically concerning the presence of CD4+ and CD8+ T lymphocytes, CD1a+ Langerhans cells and EG2+ activated eosinophils. RESULTS: Compared to lesional skin of patients with AD before UVA1 irradiation, the relative number of CD4+ cells, CD1a+ dendritic cells and activated EG2+ eosinophils within the dermal infiltrate could be decreased significantly after treatment. In contrast, medium-dose UVA1 phototherapy led to a significant increase of the percentage of dermal CD8+ cells. These alterations were closely linked to a decrease of the absolute skin-infiltrating cells and a substantial clinical improvement of the skin. CONCLUSIONS: In summary, our findings demonstrate that medium-dose UVA1 irradiation leads to a remarkable modulation of the dermal mononuclear infiltrate in patients with severe atopic dermatitis referring to a decrease of dermal Langerhans cells, activated eosinophils and CD4 cell count as well as to a relative increase of CD8+ lymphocytes. The immunomodulation of the cutaneous infiltrate is associated with a depletion of cytotoxic agents, the defective IgE overproduction and the aberrant presence of T lymphocytes combined with the pathological cytokine pattern.

Intracellular effect of ultrashort electrical pulses.

A simple electrical model for biological cells predicts an increasing probability for electric field interactions with cell substructures of prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses with electric field intensities of up to 5.3 MV/m to human eosinophils in vitro. When 3-5 pulses of 60 ns duration were applied to human eosinophils, intracellular granules were modified without permanent disruption of the plasma membrane. In spite of the extreme electrical power levels applied to the cells thermal effects could be neglected because of the ultrashort pulse duration. The intracellular effect extends conventional electroporation to cellular substructures and opens the potential for new applications in apoptosis induction, gene delivery to the nucleus, or altered cell functions, depending on the electrical pulse conditions.

Dose-response studies of depletion and repopulation of rat intestinal mucosal mast cells after irradiation.

The effects of radiation on gut mucosal mast cells (MMC) and tissue eosinophils were examined. Groups of rat were given single doses of whole-body irradiation from 0.5 to 5 Gy. Serum rat mast cell protease II (RMCPII) concentration showed a significant dose-dependent fall after 1 Gy on day 3 and 1.5 Gy on day 7. MMC counts and tissue RMCPII values on day 7 decreased significantly by 70% after 1 Gy and were undetectable with larger doses. Rat with normal and expanded MMC populations were irradiated or given anaphylaxis. Serum RMCPII concentrations did not change after irradiation, but there was a 10-fold increase in RMCPII after anaphylaxis. Tissue eosinophils in jejunum were 50% of control at 7 days after 2 Gy, and this effect was progressively more marked with higher doses. Similar effects on MMC and eosinophils were demonstrated in ileum, ascending colon and rectum. After 4.5 Gy, repopulation of the gut with MMC did not occur until week 3-4 postirradiation and MMC counts were still 50% below those of controls at 5 weeks postirradiation. Counts of tissue eosinophils 5 weeks after 4.5 Gy irradiation had returned to control levels in jejunum but were still significantly depleted in colon. These experiments show that the high radiosensitivity of rat intestinal MMC is dose dependent, similar at four different levels in the gastrointestinal tract and does not lead to immediate release of granule protease; repopulation with MMC does not begin until at 3 weeks postirradiation.

Eosinophil chemiluminescence response to cytokines and opsonized zymosans in atopic dermatitis.

Chemiluminescence (CL) responsiveness of eosinophils (Eos) to zymosan particles coated with either IgG, C3 or both and eosinophilopoiesis-modulating cytokines was investigated in patients with atopic dermatitis (AD), and an attempt was made to correlate these CL responses with serum levels of eosinophil cationic protein (ECP) which increased in response to the extent of AD severity, but not blood eosinophil counts. A high degree of positive correlation existed between serum levels of ECP and either IgG containing opsonized zymosan- or interleukin-5 (IL-5)-induced CLs. The CL values induced by these two stimuli appeared to be directly correlated with the intensity of each ligand-linked receptor expression. These results suggested that Fc gamma RII-and/or IL-5R-mediated eosinophil activation at the site of lesional skins might implicate in deterioration of cutaneous inflammation, and consequently cause an elevation of serum levels of ECP in AD.

[Ultrastructural and radioautographic study of bronchoalveolar lavage cells in pulmonary tuberculosis in endobronchial laser therapy]. / Ultrastrukturnoe i radioavtograficheskoe issledovanie kletok bronkhoalveoliarnogo lavazha pri tuberkulëze lëgkikh v usloviiakh éndobronkhialnoi lazernoi terapii.

Seventy specimens of bronchoalveolar lavage were obtained from 30 patients with pulmonary tuberculosis during treatment with endobronchial laser. The lavage is shown to undergo marked qualitative and quantitative changes in response to He-Ne laser treatment: the proportion of macrophages goes up while that of neutrophils tends to a decrease. The laser is capable to induce metabolic and proliferative activity of alveolar macrophages indicated by RNA and DNA synthesis. Morphological alterations in the cells of the respiratory compartment suggest weakening of exudative-necrotic component of the inflammation as an underlying cause of laser efficacy.

[Spontaneous adhesion of blood leukocytes in cancer patients and healthy subjects]. / Izuchenie spontannoi adgezii leikotsitov krovi u onkologicheskikh bol'nykh i zdorovykh lits.

The adherence of peripheral blood leukocyte to a hydrophilic substrate (cotton wool) was studied in healthy donors and patients with Hodgkin's disease and solid tumors. It was shown that neutrophils, eosinophils and lymphocytes contribute to the adhesive ability. Patients with primary solid neoplasms revealed an increased leukocyte adherence. Following a single therapeutic irradiation, this index appeared significantly enhanced in those with Hodgkin's disease. Possible causes of the disturbances are discussed.