Janina Grzegorczyk

Effect of 7 mT static magnetic field and iron ions on rat lymphocytes: apoptosis, necrosis and free radical processes

Simultaneous exposure of rat lymphocytes to 7 mT static magnetic field (SMF) and iron ions caused an increase in the number of cells with DNA damage. The mechanism by which MF induces DNA damage and the possible cytotoxic consequences are not known. However, we suppose that free radicals are involved. Potentially, the deterioration of DNA molecules by simultaneous exposure to 7 mT SMF and iron ions may lead to cell death: apoptosis or necrosis. The possible prooxidative properties of these two agents may result in an induction of the lipid peroxidation process as a marker of free radical mechanism in the cells. Experiments were performed on rat blood lymphocytes incubated for 3 h in Helmholtz coils at SMF of flux density 7 mT. During SMF exposure, some samples were treated with ferrous chloride (10 microg/ml), the rest serving as controls. We used the dye exclusion method with the DNA-fluorochromes: ethidium bromide and acridine orange. No significant differences were observed between unexposed lymphocytes incubated with medium alone and lymphocytes exposed to 7 mT SMF. Three-hour incubation with FeCl(2) (10 microg/ml) did not affect cell viability. However, when lymphocytes were exposed to 7 mT SMF and simultaneously treated with FeCl(2), there was a significant increase in the percentage of apoptotic and necrotic cells accompanied by significant alterations in cell viability. As compared to lipid peroxidation, there is a significant increase in the amount of lipid peroxidation end products MDA+4 HNE in rat lymphocytes after simultaneous exposure to 7 mT SMF and FeCl(2) (vs. to the control samples and those exposed to SMF alone). This suggests that 7 mT static magnetic field in the presence of Fe(2+) ions can increase the concentration of oxygen free radicals and thus may lead to cell death.

A single weekly dose of imatinib is sufficient to induce and maintain remission of chronic eosinophilic leukaemia in FIP1L1-PDGFRA-expressing patients

Hypereosinophilic syndrome (HES) is defined as chronic, unexplained hypereosinophilia with organ involvement. A subset of HES patients presents an interstitial deletion in chromosome 4q12, which leads to the expression of an imatinib‐responsive fusion gene, FIP1L1‐PDGFRA. These patients are diagnosed as chronic eosinophilic leukaemia (CEL). We treated seven CEL and HES patients, six of which expressed FIP1L1‐PDGFRA, with imatinib using initial daily doses ranging from 100 to 400 mg. In a remission maintenance phase, the patients were treated with imatinib once weekly. All imatinib‐treated patients achieved a complete haematological remission (CHR), and five of the six patients with FIP1L1‐PDGFRA expression exhibited molecular remission. The decreased imatinib doses were as follows: 200 mg/week in three patients, 100 mg/week in two patients and 100 mg/d in the remaining two patients. For remission maintenance, imatinib doses were set at 100 mg/week in five patients and 200 mg/week in two patients. At a median follow‐up of 30 months all patients remained in CHR and FIP1L1‐PDGFRA expression was undetectable in five of the six FIP1L1‐PDGFRA‐expressing patients. These data suggest that a single weekly dose of imatinib is sufficient to maintain remission in FIP1L1‐PDGFRA‐ positive CEL patients.

RANTES and Chemotactic Activity in Synovial Fluids From Patients With Rheumatoid Arthritis and Osteoarthritis

A massive accumulation of inflammatory cells in synovial tissues is a major pathological feature of rheumatoid arthritis (RA). Neutrophiles dominate synovial fluid while rheumatoid synovium is infiltrated with mononuclear cells. Mechanisms regulating influx of particular subpopulations of leukocytes into articular cavity and synovium compartment are not completely defined. An increasing amount of data supports a crucial role of a C-C chemokine RANTES in the RA pathogenesis. Our objective is to evaluate chemotactic activity for neutrophils (NCA), lymphocytes (LCA), and monocytes (MoCA) in SFs obtained from patients with RA and osteoarthritis (OA). We also aimed to characterise the relation between chemotactic activity, RANTES, and percentage distribution of leukocytes in SF. SFs from 11 patients with RA and 6 with OA were included in the study. Modified microchamber Boyden method was employed to assess chemotactic activity. Cytological and biochemical analysis of SF was performed. RANTES was measured with ELISA. Rheumatoid SFs were rich in cells with predominance of neutrophiles while osteoarthritic fluids were lymphocytic. RA SFs were also characterised by increased lactoferrin level. Both NCA and LCA were higher in SF from patients with RA (62 ± 12 and 24 ± 6 cells/HPF, resp) as compared to patients with OA (23 ± 6; P < .05 and 6 ± 2 cells/HPF; P < 0.05). The chemoattractive effect of RA SF was more pronounced on neutrophiles than on lymphocytes. RA SF expressed high RANTES levels (145 ± 36 pg/mL), while OA SF was characterised by only trace amount of this chemokine (2 ± 1 pg/mL). We found positive correlation of RANTES with chemotactic activity for mononuclear cells (LCA+MoCA; R = 0.61; P < .05). Surprisingly, RANTES correlated also positively with neutrophiles number (R = 0.77; P < 0.001). Rheumatoid SF possesses strong chemotactic potency for leukocytes. RANTES is overexpressed in RA SF and is a potential mediator influencing intensity and composition of cellular infiltration in joints affected with inflammatory arthritis.

Increased apoptosis of peripheral blood mononuclear cells in patients with perennial allergic asthma/rhinitis: relation to serum markers of apoptosis.

BACKGROUND: The goal of our study was to examine spontaneous and stimulated apoptosis of peripheral blood MNC from allergic patients, sensitized to Der p I antigen as compared to cells from non-atopic subjects. Furthermore we aimed to investigate which populations of mononuclear cells (lymphocytes, monocytes) undergo the apoptosis and to determine relations between apoptosis and serum levels of sFas/APO-1, ICE/caspase-1 or TNF-alpha. METHODS: The study included 17 patients with perennial, allergic asthma and/or allergic rhinitis [6 male and 11 female; mean age 29,5 years; (range 15-49)]. Apoptosis was assessed by fluorescence technique and confirmed by flow-cytometric method and DNA ladder. Serum levels of sFas, ICE/caspase-1 or TNF-alpha were determined by immunoassays (ELISA). RESULTS: Apoptotic index of unfractionated mononuclear cells (MNC) and lymphocytes (but not monocytes) were significantly higher in allergic patients as compared to non-allergic subjects after 48 and 72 hours of culture (p<0.05). Incubation of cells with ConA (10 microg/ml) resulted in a significant increase in the proportion of apoptotic cells in all populations once the apoptotic index for MNC and lymphocytes (but not monocytes) was again significantly higher in allergic as compared to non-allergic subjects after 24, 48 and 72 hour of culture. In allergic patients, mean serum sFas level, was significantly lower then in non-allergic group (mean value 624.8 pg/ml +/- 25.67 versus 802.0 pg/ml +/- 31.91; p = 0.003) and in both groups sFas level correlated inversely with apoptosis of MNC. The mean ICE/caspase-1 concentration was significantly higher in sera of allergic patients as compared to non-allergic group (mean value 27.71 pg/ml +/- 3.79 vs 23.54 pg/ml respectively; p<0.01). ICE/caspase-1 levels in allergic patients correlated with apoptotic index of mononuclear cells (r = 0.57; p<0.001). CONCLUSIONS: An increased spontaneous and mitogen-induced apoptosis of MNC from peripheral blood of atopic patients as well as different serum levels of sFas and ICE/caspase-1 correlating with apoptosis, suggest different regulation of apoptotic process in peripheral blood mononuclear cells of patients with allergic asthma and/or rhinitis.

Influence of a 7 mT Static Magnetic Field and Iron Ions on Apoptosis and Necrosis in Rat Blood Lymphocytes

In recent years several epidemiological and laboratory studies have suggested possible bioeffects of magnetic fields (MFs) and implied that those fields, especially at extremely low frequencies (50/60 Hz), could exert adverse effects on human health (various types of cancer and immunological disturbances). The mechanism of this effect remains unknown, but one of the most recognised hypotheses is the radical pairs mechanism in which an external magnetic field influences the kinetics of chemical reactions with radical pair intermediates and could increase the concentration of free radicals (oxygen free radicals) in the cells. This bioeffect may occur immediately after the MF exposure as acute or transient event in cells but can also give rise to more long-lasting events by affecting cell function or metabolism. We have previously shown that simultaneous exposure of rat lymphocytes to 7 mT MF (static or 50 Hz) and iron ions caused a significant increase in the number of cells with DNA damage, compared to the control samples or those incubated with iron ions or exposed to MF alone, and that melatonin, an established free radicals scavenger, provides protection against that DNA damage . We were therefore able to suggest that free radicals can be involved in 7 mT MF and iron ioninduced DNA damage in rat blood lymphocytes as a result of the MF effect due to the radical pair mechanism, but the possible cytotoxic consequences are not known. In the present study we investigated whether the oxidative DNA damage caused by simultaneous exposure of rat lymphocytes to a 7 mT static magnetic field (SMF) and iron ions may lead to cell death: apoptosis or necrosis. This communication provides some evidence of this effect.

Utility of serum IgG, IgG4 and carbonic anhydrase II antibodies in distinguishing autoimmune pancreatitis from pancreatic cancer and chronic pancreatitis.

PURPOSE Autoimmune pancreatitis (AIP) can mimic pancreatic cancer in its clinical presentation, imaging features and laboratory parameters. The aim of our study was to compare IgG, IgG4 and anti-CAIIAb serum levels in patients with AIP, pancreatic adenocarcinoma (PA) and chronic pancreatitis (CP) and to assess their clinical significance and utility in differential diagnosis of pancreatic diseases. PATIENT/METHODS The study included 124 patients: 45 with PA, 24 with AIP and 55 with CP. Peripheral venous blood samples were obtained from all analyzed patients at the time of hospital admission and total IgG, IgG4 and anti-CAIIAB serum levels were measured using ELISA tests. RESULTS Serum levels of IgG, IgG4 and anti-CAIIAb were significantly higher in patients with AIP compared to PA and CP patients (p<0.001). In AIP patients the median IgG levels were 19.7 g/l, IgG4 levels - 301.9 mg/dl and anti-CAIIAb - 81.82 ng/ml, compared to 10.61 g/l, 123.2mg/dl and 28.6 ng/ml, respectively, in PA patients. IgG4 for the cut-off 210 mg/dl showed the best sensitivity and specificity (83.8% and 89.5%) in AIP diagnosis compared to IgG (69.3% and 87.3%, respectively) and anti-CAIIAb (45.3% and 74.3%). However, 16 (35.5%) patients with PA and 14 (25.4%) patients with CP had IgG4 levels greater than 140 mg/dl. Moreover, in 3 (6.67%) patients with pancreatic cancer those values were greater than 280 mg/dl. No patients with CP had IgG4 more than 280 mg/dl. CONCLUSIONS IgG4 at cut-off 210 mg/dl showed the best sensitivity and specificity in AIP diagnosis compared to IgG and anti-CAIIAb, however elevations of serum IgG4 may be seen in subjects without AIP, including pancreatic cancer.

Recruitment of CD34+ progenitor cells into peripheral blood and asthma severity.

BACKGROUND Previous studies have suggested that the number of progenitor cells is elevated in the peripheral blood of asthmatic patients and that the number of progenitors correlate with the severity of the disease. OBJECTIVE To evaluate the number of leukocyte progenitor and eosinophil progenitor cells in the peripheral blood of patients with bronchial asthma in relation to disease severity. METHODS The study involved 51 patients with asthma (25 patients with a mild form and 26 with a severe form of the disease) and a group of 12 healthy controls. Using the flow cytometric method, leukocyte (CD34+ leukocytes) and eosinophil progenitors (CD34+CD125+) were detected in the peripheral blood of both asthmatic patients and healthy controls. RESULTS Patients with asthma had significantly more leukocyte progenitor cells (median, 0.06% vs 0.016%) and eosinophil progenitor cells (median, 0.046% vs 0.004%) compared with the controls. Patients with severe asthma had more leukocyte progenitor cells (0.12% vs 0.035%) and more eosinophil progenitor cells (0.102% vs 0.019%) than patients with mild asthma. The number of circulating leukocyte and eosinophil progenitor cells inversely correlated with the forced expiratory volume in 1 second percentage of predicted value (r = -0.4 and r = -0.35, respectively) and positively correlated (r = 0.63 and r = 0.65, respectively) with the dose of inhaled steroids used to control asthma. CONCLUSION These data suggest that the presence of leukocyte precursors and eosinophil progenitor cells in the peripheral blood of asthmatic patients may reflect ongoing airway inflammation.

Glucocorticoid-induced immunoglobulin E synthesis by peripheral blood mononuclear cells from allergic and nonallergic subjects

BACKGROUND Glucocorticoids (GCS) have been shown to induce IgE synthesis in human peripheral blood mononuclear cells (PBMCs) and purified B cells in vitro. However, the differences in immunoglobulin E (IgE) response to GCS between allergic and non-allergic individuals and the mechanism this interaction have not been elucidated. OBJECTIVE We aimed to compare the effect of GCS (budesonide) on interleukin (IL)-4-driven IgE production in vitro in allergic and non allergic subjects and assess the engagement of intracellular mechanisms. METHODS The study included 22 patients with allergic asthma and/or allergic rhinitis and 24 healthy volunteers. PBMCs were cultured for 11 days with IL-4 and budesonide and IgE concentrations in supernatants were assessed by immunoassays. T and B cell markers were assessed by flow cytometry. RESULTS Budesonide enhanced IgE synthesis to higher extent in healthy donors than in allergic patients (mean increase of 16.5 vs 6.3 kU/L, P< .05 respectively) acting through glucocorticoid receptor. Budesonide significantly increased lymhoplasmocytoid cells percentage in both media-controlled (2.5-fold increase) and IL-4-stimulated PBMCs (2-fold increase). Added to IL-4 budesonide decreased the percentage of both T cells and CD40L(+) T cells, but strongly increased the percentage of B cells. Protein tyrosine kinase (PTK) inhibitor decreased, but NF-κB and protein kinase A (PKA) inhibitors expressed modulatory effects on budesonide-induced IgE synthesis. CONCLUSIONS Budesonide-induced IgE generation in PBMCs differs in magnitude and seems to involve different mechanisms in atopic and non-atopic subjects.

Prostaglandin E2 and lipoxin A4 in PBMCs are associated with immune tolerance during venom immunotherapy

To the Editor: The mechanisms of induction and sustaining of immune tolerance during allergen immunotherapy have not been fully elucidated. Successful venom immunotherapy (VIT) is associated with generation of IL-10–producing regulatory T and B cells, which induce tolerance to the relevant allergen in peripheral T cells and decrease IgE/IgG4 ratio and IL-4, IL-5, and IL-13 levels. Very little is known on the potential role of arachidonic acid metabolites (eicosanoids) in the development of immune tolerance and allergen-specific desensitization. Therefore, we assessed the effect of ultrarush VIT and maintenance dosing of venom allergen on the generation of eicosanoids and immunoregulatory cytokines by PBMCs in patients with venom allergy. We recruited 10 subjects with a history of severe allergic reaction to Hymenoptera stings and sensitization to wasp venom confirmed by the presence of specific serum IgE. Patients received ultrarush updosing of wasp venom extract and then completed 6-month immunotherapy. The blood was collected before ultrarush immunotherapy, 1 hour (T1) after the induction of desensitization to 100 mg/mL of venom, and after 10 days (T2), 3 months (T3), and 6 months (T4). Patients’ characteristics and methods used are presented in the article’s Online Repository at www.jacionline.org. The spontaneous release of prostaglandin E2 (PGE2) by PBMCs significantly decreased at 3 and 6 months as compared with before VIT (Fig 1, A, left panel). The decrease in spontaneous generation of PGE2 at the sixth month of VIT was paralleled by the decrease in cyclooxgenase-1 and cyclooxgenase-2 mRNA expression (Fig1, B, left and middle panels). Cyclooxgenase-1 was also significantly inhibited immediately after completion of the induction phase of immunotherapy. The spontaneous release of lipoxin A4 (LXA4) by PBMCs tended to increase immediately after the induction phase, and 10 days later more than doubled the initial value (Fig1, A,middle panel). After 3 months of VIT, spontaneous LXA4 release tended to decrease and at 6 months was significantly lower as compared with the 10th day of immunotherapy and did not differ from the release before at T0. Spontaneous 15-hydroxyicosatetraenoic acid (15-HETE) release by PBMCs tended to increase in some patients after 3 and 6 months, but the median values were not significantly different before and during VIT (Fig 1, A, right panel). The expression of 15-lipooxygenase mRNA in PBMCs changed during immunotherapy in parallel to LXA4 release into supernatants: it increased after 10 days of immunotherapy and then significantly decreased after 6 months as compared with T2 time point (Fig 1, B, right panel). The spontaneous IL-10 synthesis, which was not changed immediately or 10 days after the induction phase, significantly increased 3 and 6 months later (Fig 1, C, left panel). The spontaneous TGF-b synthesis by PBMCs significantly increased on day 10 and the third month after the induction phase as compared with that at T0 (Fig 1, C, right panel). After 6 months, TGF-b synthesis by PBMCs significantly decreased and did not differ from that at T0. During venom-specific immunotherapy, a negative longitudinal correlation between PGE2 and spontaneous release of IL-10 (R 5 20.37; P < .05) (Fig 1, D, left panel) was noticed, and a positive longitudinal correlation between spontaneous generation of LXA4 and TGF-b by PBMCs (R 5 0.36; P < .05) was observed (Fig 1, D, right panel). In vitro cell stimulation with specific venom allergen increased the generation of PGE2 and LXA4 at 48 hours as compared with unstimulated controls at all time points of VIT. However, there was no significant difference in the reactivity of PBMCs to allergen with regard to the generation of both PGE2 and LXA4 at any time point of VIT as compared with that at T0 (Table I). Only at third month of VIT, wasp allergen significantly increased mean 15-HETE generation as compared with unstimulated controls. Incubation of PBMCs with specific allergen did not affect IL-10 or TGF-b generation neither before the induction phase nor in the following time points after rush immunotherapy as compared with unstimulated controls. Treatment of PBMCs with anti-CD3 and anti-CD28 antibodies did not influence the production of eicosanoid at early stage of VIT; however, it stimulated the release of PGE2 and LXA4 (but not 15-HETE) at the late phase of VIT (at third and sixth months for PGE2 and at sixth month for LXA4) (Table I). In contrast, anti-CD3 and anti-CD28 antibodies induced dramatic increases in IL-10 generation at all time points of VIT. TGF-b generation was significantly increased only at the third month after the induction phase. This study demonstrated for the first time that venom allergenspecific immunotherapy is associated with modulation of the spontaneous generation of PGE2 and LXA4, in parallel to changes in IL-10 and TGF-b release by PBMCs, thus suggesting the engagement of arachidonic acid metabolites in induction and maintenance of allergen-specific tolerance during VIT. Development of immune tolerance during VITwas accompanied by progressive decrease in spontaneous PGE2 release and the decrease in cyclooxgenase-1 and cyclooxgenase-2 mRNA expression, which is in line with potential suppression of PGE2 capacity to drive TH2-type immune responses and mast cell–driven inflammation and to lead to the synthesis of IgE and IgG1. 5,6 Early increase in the release of LXA4, which is a potent inhibitor of TH2-mediated eosinophilic inflammation, 7 may reflect immune counteraction to the release of the inflammatory mediators following high cumulative dose of allergen during the induction phase of VIT. Changes in the generation of LXA4 during VIT were to some extent paralleled by changes in the expression of 15-lipoxygenase in PBMCs, suggesting that the modulatory effect of immunotherapy on LXA4 is exerted at the enzymatic level. The longitudinal negative correlation between the spontaneous generation of PGE2 and IL-10 by PBMCs and the positive correlation between LXA4 and TGF-b1 across the whole period of treatment suggest the possible engagement of arachidonic acid metabolites and enzymes of arachidonic acid pathway in the regulation of the development of immune tolerance to allergens during VIT.

IgE-mediated 15-hydroxyeicosatetraenoic acid (15-HETE) generation by peripheral blood leukocytes: its association with basophil activation.

Introduction Allergen-induced basophil activation has been associated with the release of several mediators and with an increased expression of CD203c molecules on basophils. Aim To assess the influence of specific allergens on the generation of 15-hydroxyeicosatetraenoic (15-HETE) from peripheral blood leukocytes in relation to basophil activation, on the basis of CD203c molecule expression and histamine release. Material and methods The study included 15 patients with clinical symptoms of birch pollen allergy confirmed by a positive skin prick test with the birch allergen, and 6 healthy controls. Leukocytes isolated from peripheral blood were incubated with 3 concentrations of the birch pollen allergen (Bet v 1), anti-IgE or with ionophore A23187. Results In vitro challenge of leukocytes from allergic patients with 1 ng/ml of allergen induced a significant increase in 15-HETE generation. An increase above 30% was observed in almost half the allergic patients, with mean values ranging from 40% to 46%, but not in healthy controls. Anti-IgE antibodies increased 15-HETE generation in 5 patients (termed IgE+), and the allergen induced a significant increase in 15-HETE in all patients who reacted to anti-IgE. The mean CD203c expression on basophils of the allergic patients increased after allergen challenge, but a significant increase (> 30%) was observed only in patients who demonstrated an increased expression after anti-IgE exposure. A significant correlation was seen between 15-HETE generation and histamine release induced by the highest concentration of the allergen (r = 0.95; p < 0.01). Conclusions Allergen-induced, IgE-mediated activation of basophils is associated with a significant increase in 15-HETE generation.