Gergely Tibor Kozma

The development of asthma in children infected with Chlamydia pneumoniae is dependent on the modifying effect of mannose-binding lectin

BACKGROUND Although several studies found associations between infection with Chlamydia pneumoniae and asthma, these were mainly restricted to the exacerbation of the symptoms in adults with known asthma. Data about the role of C pneumoniae in the initiation and development of asthma in children are controversial. OBJECTIVE We investigated the role of C pneumoniae infection in 139 children with asthma, comparing them with 174 healthy control subjects. Furthermore, we studied the modifying effect of mannose-binding lectin (MBL) variant alleles on the susceptibility to asthma in children infected with C pneumoniae. METHODS C pneumoniae-specific antibodies were measured by means of ELISA, and MBL genotypes were determined by means of PCR-RFLP. RESULTS There were no significant differences in the percentage of children with positive results for C pneumoniae-specific antibodies between patients and control subjects. Among asthmatic children carrying variant MBL alleles, there were significantly more patients with positive results for C pneumoniae-specific IgG than among control children with variant MBL genotypes (63.7% vs 40.7% of asthmatic vs control children, respectively; odds ratio adjusted for age and sex, 2.21; 95% CI, 1.10-4.41; P =.02). Infected children with variant MBL alleles were found to have a higher risk of asthma development than infected children with normal MBL genotype. This risk was especially high in children with chronic or recurrent infection (positive results for both IgA and IgG; adjusted odds ratio, 5.38; 95% CI, 1.75-14.36; P =.01), but no increased risk was seen in children with current C pneumoniae infection (positive results for IgM). CONCLUSION This study indicates the important role of variant MBL alleles in the susceptibility to asthma in children infected with C pneumoniae.

Lack of association between atopic eczema/dermatitis syndrome and polymorphisms in the promoter region of RANTES and regulatory region of MCP-1

Background: Chemokines play an important role in the pathophysiology of atopic eczema/dermatitis syndrome (AEDS) and allergy. Recently polymorphisms in the promoter region of RANTES (regulated on activation normal T cell expressed and secreted) and in the gene regulatory region of MCP‐1 (monocyte chemoattractant protein‐1) have been found, which increase the expression of these chemokines. The − 403A allele of the RANTES promoter region was found associated with AEDS in German children. We investigated whether the presence of these polymorphisms was associated with AEDS or allergy in Hungarian children.

Galectin-9 in allergic airway inflammation and hyper-responsiveness in mice.

Background: Galectin-9 (Gal-9) is a member of the growing family of β-galactoside-binding lectins. Gal-9 is an eosinophil chemoattractant and inducer of Th1 cell apoptosis. These effects suggest its potential role in the pathogenesis of asthma. Our aim was to study the expression of Gal-9 in an ovalbumin (OVA)-induced mouse model of allergic asthma. Methods: To investigate the significance of Gal-9 in allergic inflammation and airway hyperresponsiveness (AHR), a group of BALB/c mice was sensitized and challenged with OVA (GOVA). Another group of animals was allergized with OVA and also treated with dexamethasone (DEX) (GOVA+DEX). The control group (GPBS) received phosphate-buffered saline instead of OVA as placebo. Airway reactivity to intravenous methacholine was assessed. Results: The percentage of Gal-9-positive cells and their intracellular Gal-9 content and Th1/Th2 cytokine levels in the bronchoalveolar lavage (BAL) were determined by flow cytometry. Gal-9 mRNA expression and protein level were measured in the lung tissue by real-time RT-PCR and Western blot. In GOVA mice, airway inflammation and mucus hypersecretion developed. DEX treatment inhibited the main features of experimental asthma. The number of Gal-9-positive lymphocytes, eosinophil and neutrophil granulocytes and the levels of Th2 cytokines were higher in the BAL of GOVA compared to GPBS or GOVA+DEX mice. Moreover, Gal-9 protein level was elevated in the lungs of GOVA mice. Conclusions: These results suggest that Gal-9 plays a role as a mediator contributing to the development of allergic airway inflammation. Gal-9 may serve as a recruiter of eosinophil granulocytes and promoter of Th2 dominance.

Lipopolysaccharide exposure makes allergic airway inflammation and hyper-responsiveness less responsive to dexamethasone and inhibition of iNOS.

Allergic airway disease can be refractory to anti‐inflammatory treatment, whose cause is unclarified. Therefore, in the present experiment, we have tested the hypothesis that co‐exposure to lipopolysacharide (Lps) and allergen results in glucocorticoid‐resistant eosinophil airway inflammation and hyper‐responsiveness (AHR). Ovalbumin (Ova)‐sensitized BALB/c mice were primed with 10 μg intranasal Lps 24 h before the start of Ova challenges (20 min on 3 consecutive days). Dexamethasone (5 mg/kg/day) was given on the last 2 days of Ova challenges. AHR, cellular build‐up, cytokine and nitrite concentrations of bronchoalveolar lavage fluid (BALF) and lung histology were examined. To assess the role of iNOS‐derived NO in airway responsiveness, mice were treated with a selective inhibitor of this enzyme (1400W) 2 h before AHR measurements. More severe eosinophil inflammation and higher nitrite formation were found in Lps‐primed than in non‐primed allergized mice. After Lps priming, AHR and concentrations of T‐helper type 2 cytokines in BALF were decreased, but still remained significantly higher than in controls. Eosinophil inflammation was partially, while nitrite production and AHR were observed to be largely dexamethasone resistant in Lps‐primed allergized animals. 1400W effectively and rapidly diminished the AHR in Ova‐sensitized and challenged mice, but failed to affect it after Lps priming plus allergization. In conclusion, Lps inhalation may exaggerate eosinophil inflammation and reduce responsiveness to anti‐inflammatory treatment in allergic airway disease.

Flow cytometric analysis of supravesicular structures in doxorubicin-containing pegylated liposomes.

In an attempt to develop a quantitative assay for supravesicular structures (SVS) - such as aggregates, fused liposomes or solid lipid particles - in liposome preparations, forward vs. side scattering of liposomal doxorubicin (Doxil/Caelyx) was analyzed by flow cytometry. Based on calibration with fluorescent latex beads, the size resolution was between about 500 and 1000 nm. Caelyx, just as structurally matched empty liposomes (Doxebo) produced dot plots clearly distinguishable from background, suggesting the presence of SVS in the above size region. A comparison of gated areas on the scattergrams obtained for different Caelyx preparations showed differences between current and expired samples, implying that SVS formation may be storage-time-dependent. Incubation of doxorubicin with Doxebo in a free drug and lipid concentration range that corresponds to that in Caelyx also led to varying SVS patterns, raising the possibility that free doxorubicin in Caelyx might contribute to SVS formation. Dynamic light scattering and transmission electron microscopic analysis of liposomes following gaiting and sorting of >500 nm particles from Caelyx confirmed the presence of SVS, providing independent evidence for their stable existence. Based on a rough estimation, the amount of SVS in Caelyx is some 60 billionth part of all liposomes. These observations raise the possibility that the presence of an exceedingly small fraction of >500 nm particles may be an intrinsic property of PEGylated small unilamellar liposomes, and that the described FACS analysis may be developed further as a quality assay for liposomal homogeneity.

Pediatric Asthmatic Patients Have Low Serum Levels of Monocyte Chemoattractant Protein-1

Serum levels of MCP-1 were measured in children with and without asthma in order to determine a possible correlation between the MCP-1-2518A/G polymorphism, serum levels of MCP-1 and asthma. Two groups of subjects −160 children with asthma and 158 healthy children were screened with a PCR-based genotyping assay. Serum MCP-1 level was measured by ELISA. The -2518G allele occurred at a significantly higher frequency in asthmatic children than in controls. The mean serum MCP-1 level was significantly lower in the asthmatic than in the control children. There was no significant association between the MCP-1 genotypes and the serum MCP-1 levels.

Variable association of complement activation by rituximab and paclitaxel in cancer patients in vivo and in their screening serum in vitro with clinical manifestations of hypersensitivity: A pilot study

Abstract To explore the role of complement (C) activation in the hypersensitivity reactions (HSRs) to some anticancer drugs, as well as the use of the C activation biomarkers (Cbiom) C3a, C5a and SC5b-9 in the prediction of HSRs, we measured these Cbiom in plasma samples of cancer patients during infusion therapy, and in their pretreatment (screening) serum incubated with these drugs in vitro. Rituximab and paclitaxel caused mild to severe HSRs in 8/20 and 4/4 patients, respectively, which were associated with rises or falls of plasma and/or serum Cbioms. Among these changes, a rise of C3a in the plasma of 8/8 rituximab reactors and strong rises of Cbioms in the screening sera of all paclitaxel patents were most prominent. However, in the case of rituximab, significant Cbiom changes were also seen in nonreactors, while Cbiom changes were absent in the screening serum. Thus, C activation may be causally involved, but it is not rate limiting factor to HSRs to rituximab. Additional initial data in this study suggest that a whole blood assay using hirudin is more sensitive to C activation by rituximab than the serum test; that trastuzumab and docetaxel also cause HSRs with changes of Cbioms, and that an anti-paclitaxel antibody (ADA) ELISA may be useful as a predictor test for HSRs to Paclitaxel.

Seasonal changes of proapoptotic soluble Fas ligand level in allergic rhinitis combined with asthma

The function of apoptosis is to eliminate unnecessary or dangerous cells. The balance between production and death is important in the control of cell numbers within physiological ranges. Cells involved in allergic reactions may have altered apoptosis. The aim of this study was to examine the seasonal changes of programmed cell death in children with pollen allergy. We measured serum levels of soluble Fas (sFas) and soluble Fas ligand (sFasL), and examined whether there was any correlation between soluble apoptosis markers and development of asthma and or rhinitis in children with pollen allergy. We examined two groups of patients with ragweed pollen allergy. The first group consisted of 17 children with ‘rhinitis only’. The second group consisted of 16 children with ‘asthma + rhinitis’. For seasonal analysis we pooled the two groups and termed this the ‘ragweed sensitive’ group (n = 33, 5–18 yr, 25 boys, eight girls). Measurements (sFas and sFasL) were taken during the ragweed pollen allergy season, while control measurements were performed during the symptom‐free period. There was no difference in sFas levels measured during and after [1941 ± 68, 1963 ± 83 pg/ml (mean±s.e.m, respectively)] the pollen season in the ‘ragweed sensitive’ group. The sFasL level showed seasonal change, which was significantly higher (p = 0.0086) in the symptomatic period compared to the symptom‐free state (99 ± 13 and 53 ± 16 pg/ml, respectively). There was a difference between the ‘rhinitis only’ and the ‘asthma + rhinitis’ groups in the measured parameters of apoptosis. Children having allergic rhinitis combined with asthma had a significantly (p = 0.03) higher sFas level in the symptom‐free state than the ‘rhinitis only’ group did (2115 ± 156 and 1820 ± 52 pg/ml, respectively). During the allergic symptom state the sFasL level of the ‘asthma + rhinitis’ group was significantly higher (p = 0.025) than that of the ‘rhinitis only’ group (125 ± 20 and 75 ± 14 pg/ml, respectively). In conclusion, the increased level of sFasL during the pollen season may signal its role in the pathogenesis of allergic airway diseases. There was no seasonal change in sFas levels in the examined ragweed allergic group, however in the symptomatic period we observed a diminished level of antiapoptotic factor (sFas) and an elevated level of proapoptotic factor (sFasL) if there was a combined disease with pollen allergic asthma. We suggest that there is a deviation in the apoptotic reaction in children that may increase the seasonal allergic inflammation.

Involvement of complement activation in the pulmonary vasoactivity of polystyrene nanoparticles in pigs: unique surface properties underlying alternative pathway activation and instant opsonization

Background It has been proposed that many hypersensitivity reactions to nanopharmaceuticals represent complement (C)-activation-related pseudoallergy (CARPA), and that pigs provide a sensitive animal model to study the phenomenon. However, a recent study suggested that pulmonary hypertension, the pivotal symptom of porcine CARPA, is not mediated by C in cases of polystyrene nanoparticle (PS-NP)-induced reactions. Goals To characterize PS-NPs and reexamine the contribution of CARPA to their pulmonary reactivity in pigs. Study design C activation by 200, 500, and 750 nm (diameter) PS-NPs and their opsonization were measured in human and pig sera, respectively, and correlated with hemodynamic effects of the same NPs in pigs in vivo. Methods Physicochemical characterization of PS-NPs included size, ζ-potential, cryo-transmission electron microscopy, and hydrophobicity analyses. C activation in human serum was measured by ELISA and opsonization of PS-NPs in pig serum by Western blot and flow cytometry. Pulmonary vasoactivity of PS-NPs was quantified in the porcine CARPA model. Results PS-NPs are monodisperse, highly hydrophobic spheres with strong negative surface charge. In human serum, they caused size-dependent, significant rises in C3a, Bb, and sC5b-9, but not C4d. Exposure to pig serum led within minutes to deposition of C5b-9 and opsonic iC3b on the NPs, and opsonic iC3b fragments (C3dg, C3d) also appeared in serum. PS-NPs caused major hemodynamic changes in pigs, primarily pulmonary hypertension, on the same time scale (minutes) as iC3b fragmentation and opsonization proceeded. There was significant correlation between C activation by different PS-NPs in human serum and pulmonary hypertension in pigs. Conclusion PS-NPs have extreme surface properties with no relevance to clinically used nanomedicines. They can activate C via the alternative pathway, entailing instantaneous opsonization of NPs in pig serum. Therefore, rather than being solely C-independent reactivity, the mechanism of PS-NP-induced hypersensitivity in pigs may involve C activation. These data are consistent with the “double-hit” concept of nanoparticle-induced hypersensitivity reactions involving both CARPA and C-independent pseudoallergy.

Infusion Reactions Associated with the Medical Application of Monoclonal Antibodies: The Role of Complement Activation and Possibility of Inhibition by Factor H

Human application of monoclonal antibodies (mAbs), enzymes, as well as contrast media and many other particulate drugs and agents referred to as “nanomedicines”, can initiate pseudoallergic hypersensitivity reactions, also known as infusion reactions. These may in part be mediated by the activation of the complement system, a major humoral defense system of innate immunity. In this review, we provide a brief outline of complement activation-related pseudoallergy (CARPA) in general, and then focus on the reactions caused by mAb therapy. Because the alternative pathway of complement activation may amplify such adverse reactions, we highlight the potential use of complement factor H as an inhibitor of CARPA.