mRNA-LNP COVID-19 Vaccine Lipids Induce Complement Activation and Production of Proinflammatory Cytokines: Mechanisms, Effects of Complement Inhibitors, and Relevance to Adverse Reactions.

A small fraction of people vaccinated with mRNA-lipid nanoparticle (mRNA-LNP)-based COVID-19 vaccines display acute or subacute inflammatory symptoms whose mechanism has not been clarified to date. To better understand the molecular mechanism of these adverse events (AEs), here, we analyzed in vitro the vaccine-induced induction and interrelations of the following two major inflammatory processes: complement (C) activation and release of proinflammatory cytokines. Incubation of Pfizer-BioNTech's Comirnaty and Moderna's Spikevax with 75% human serum led to significant increases in C5a, sC5b-9, and Bb but not C4d, indicating C activation mainly via the alternative pathway. Control PEGylated liposomes (Doxebo) also induced C activation, but, on a weight basis, it was ~5 times less effective than that of Comirnaty. Viral or synthetic naked mRNAs had no C-activating effects. In peripheral blood mononuclear cell (PBMC) cultures supplemented with 20% autologous serum, besides C activation, Comirnaty induced the secretion of proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1ß < TNF-α < IL-6 < IL-8. Heat-inactivation of C in serum prevented a rise in IL-1α, IL-1ß, and TNF-α, suggesting C-dependence of these cytokines' induction, although the C5 blocker Soliris and C1 inhibitor Berinert, which effectively inhibited C activation in both systems, did not suppress the release of any cytokines. These findings suggest that the inflammatory AEs of mRNA-LNP vaccines are due, at least in part, to stimulation of both arms of the innate immune system, whereupon C activation may be causally involved in the induction of some, but not all, inflammatory cytokines. Thus, the pharmacological attenuation of inflammatory AEs may not be achieved via monotherapy with the tested C inhibitors; efficacy may require combination therapy with different C inhibitors and/or other anti-inflammatory agents.

Flow Cytometry-Based Assay to Detect Alpha Galactosidase Enzymatic Activity at the Cellular Level.

BACKGROUND: Fabry disease is a progressive, X chromosome-linked lysosomal storage disorder with multiple organ dysfunction. Due to the absence or reduced activity of alpha-galactosidase A (AGAL), glycosphingolipids, primarily globotriaosyl-ceramide (Gb3), concentrate in cells. In heterozygous women, symptomatology is heterogenous and currently routinely used fluorometry-based assays measuring mean activity mostly fail to uncover AGAL dysfunction. The aim was the development of a flow cytometry assay to measure AGAL activity in individual cells. METHODS: Conventional and multispectral imaging flow cytometry was used to detect AGAL activity. Specificity was validated using the GLA knockout (KO) Jurkat cell line and AGAL inhibitor 1-deoxygalactonojirimycin. The GLA KO cell line was generated via CRISPR-Cas9-based transfection, validated with exome sequencing, gene expression and substrate accumulation. RESULTS: Flow cytometric detection of specific AGAL activity is feasible with fluorescently labelled Gb3. In the case of Jurkat cells, a substrate concentration of 2.83 nmol/mL and 6 h of incubation are required. Quenching of the aspecific exofacial binding of Gb3 with 20% trypan blue solution is necessary for the specific detection of lysosomal substrate accumulation. CONCLUSION: A flow cytometry-based assay was developed for the quantitative detection of AGAL activity at the single-cell level, which may contribute to the diagnosis of Fabry patients.

Eculizumab suppresses zymosan-induced release of inflammatory cytokines IL-1α, IL-1ß, IFN-γ and IL-2 in autologous serum-substituted PBMC cultures: Relevance to cytokine storm in Covid-19.

BACKGROUND AND OBJECTIVE: Cytokine storm (CS) is a major contributor to the fatal outcome of severe infectious diseases, including Covid-19. Treatment with the complement (C) C5 inhibitor eculizumab was beneficial in end-stage Covid-19, however, the mechanism of this effect is unknown. To clarify this, we analyzed the relationship between C activation and production of pro-inflammatory cytokines in a PBMC model. METHODS: Human PBMC with or without 20 % autologous serum was incubated with C3a, C5a, zymosan or zymosan-pre-activated serum (ZAS) for 24 h with or without eculizumab or the C5a receptor antagonist, DF2593A. C activation (sC5b-9) and 9 inflammatory cytokines were measured by ELISA. RESULTS: In serum-free unstimulated PBMC only IL-8 release could be measured during incubation. Addition of C5a increased IL-8 secretion only, ZAS induced both IL-2 and IL-8, while zymosan led to significant production of all cytokines, most abundantly IL-8. In the presence of serum the above effects were greatly enhanced, and the zymosan-induced rises of IL-1α, IL-1ß IFN-γ and IL-2 were significantly attenuated by eculizumab but not by DF2593a. CONCLUSIONS: These data highlight the complexity of interrelationships between C activation and cytokine secretion under different experimental conditions. The clinically relevant findings include the abundant formation of the chemokine IL-8, which was stimulated by C5a, and the suppression of numerous inflammatory cytokines by eculizumab, which explains its therapeutic efficacy in severe Covid-19. These data strengthen the clinical relevance of the applied PBMC model for drug screening against CS, enabling the separation of complex innate immune cross-talks.

Role of anti-polyethylene glycol (PEG) antibodies in the allergic reactions to PEG-containing Covid-19 vaccines: Evidence for immunogenicity of PEG.

A small fraction of recipients who receive polyethylene-glycol (PEG)-containing COVID-19 mRNA-LNP vaccines (Comirnaty and Spikevax) develop hypersensitivity reactions (HSRs) or anaphylaxis. A causal role of anti-PEG antibodies (Abs) has been proposed, but not yet been proven in humans.We used ELISA for serial measurements of SARS-CoV-2 neutralizing Ab (anti-S) and anti-PEG IgG/IgM Ab levels before and after the first and subsequent booster vaccinations with mRNA-LNP vaccines in a total of 291 blood donors. The HSRs in 15 subjects were graded and correlated with anti-PEG IgG/IgM, just as the anti-S and anti-PEG Ab levels with each other. The impacts of gender, allergy, mastocytosis and use of cosmetics were also analyzed. Serial testing of two or more plasma samples showed substantial individual variation of anti-S Ab levels after repeated vaccinations, just as the levels of anti-PEG IgG and IgM, which were over baseline in 98-99 % of unvaccinated individuals. About 3-4 % of subjects in the strongly left-skewed distribution had 15-45-fold higher values than the median, referred to as anti-PEG Ab supercarriers. Both vaccines caused significant rises of anti-PEG IgG/IgM with >10-fold rises in about âˆ¼10 % of Comirnaty, and all Spikevax recipients. The anti-PEG IgG and/or IgM levels in the 15 vaccine reactors (3 anaphylaxis) were significantly higher compared to nonreactors. Serial testing of plasma showed significant correlation between the booster injection-induced rises of anti-S and anti-PEG IgGs, suggesting coupled anti-S and anti-PEG immunogenicity.Conclusions: The small percentage of people who have extremelevels of anti-PEG Ab in their blood may be at increased risk for HSRs/anaphylaxis to PEGylated vaccines and other PEGylated injectables. This risk might be further increased by the anti-PEG immunogenicity of these vaccines. Screening for anti-PEG Ab "supercarriers" may help predicting reactors and thus preventing these adverse phenomena.

Zymosan Particle-Induced Hemodynamic, Cytokine and Blood Cell Changes in Pigs: An Innate Immune Stimulation Model with Relevance to Cytokine Storm Syndrome and Severe COVID-19.

Hemodynamic disturbance, a rise in neutrophil-to-lymphocyte ratio (NLR) and release of inflammatory cytokines into blood, is a bad prognostic indicator in severe COVID-19 and other diseases involving cytokine storm syndrome (CSS). The purpose of this study was to explore if zymosan, a known stimulator of the innate immune system, could reproduce these changes in pigs. Pigs were instrumented for hemodynamic analysis and, after i.v. administration of zymosan, serial blood samples were taken to measure blood cell changes, cytokine gene transcription in PBMC and blood levels of inflammatory cytokines, using qPCR and ELISA. Zymosan bolus (0.1 mg/kg) elicited transient hemodynamic disturbance within minutes without detectable cytokine or blood cell changes. In contrast, infusion of 1 mg/kg zymosan triggered maximal pulmonary hypertension with tachycardia, lasting for 30 min. This was followed by a transient granulopenia and then, up to 6 h, major granulocytosis, resulting in a 3-4-fold increase in NLR. These changes were paralleled by massive transcription and/or rise in IL-6, TNF-alpha, CCL-2, CXCL-10, and IL-1RA in blood. There was significant correlation between lymphopenia and IL-6 gene expression. We conclude that the presented model may enable mechanistic studies on late-stage COVID-19 and CSS, as well as streamlined drug testing against these conditions.

A naturally hypersensitive porcine model may help understand the mechanism of COVID-19 mRNA vaccine-induced rare (pseudo) allergic reactions: complement activation as a possible contributing factor.

A tiny fraction of people immunized with lipid nanoparticle (LNP)-enclosed mRNA (LNP-mRNA) vaccines develop allergic symptoms following their first or subsequent vaccinations, including anaphylaxis. These reactions resemble complement (C) activation-related pseudoallergy (CARPA) to i.v. administered liposomes, for which pigs provide a naturally oversensitive model. Using this model, we injected i.v. the human vaccination dose (HVD) of BNT162b2 (Comirnaty, CMT) or its 2-fold (2x) or 5-fold (5x) amounts and measured the hemodynamic changes and other parameters of CARPA. We observed in 6 of 14 pigs transient pulmonary hypertension along with thromboxane A2 release into the blood and other hemodynamic and blood cell changes, including hypertension, granulocytosis, lymphopenia, and thrombocytopenia. One pig injected with 5x CMT developed an anaphylactic shock requiring resuscitation, while a repeat dose failed to induce the reaction, implying tachyphylaxis. These typical CARPA symptoms could not be linked to animal age, sex, prior immune stimulation with zymosan, immunization of animals with Comirnaty i.v., or i.m. 2 weeks before the vaccine challenge, and anti-PEG IgM levels in Comirnaty-immunized pigs. Nevertheless, IgM binding to the whole vaccine, used as antigen in an ELISA, was significantly higher in reactive animals compared to non-reactive ones. Incubation of Comirnaty with pig serum in vitro showed significant elevations of C3a anaphylatoxin and sC5b-9, the C-terminal complex. These data raise the possibility that C activation plays a causal or contributing role in the rare HSRs to Comirnaty and other vaccines with similar side effects. Further studies are needed to uncover the factors controlling these vaccine reactions in pigs and to understand their translational value to humans.

Mini-Factor H Modulates Complement-Dependent IL-6 and IL-10 Release in an Immune Cell Culture (PBMC) Model: Potential Benefits Against Cytokine Storm.

Cytokine storm (CS), an excessive release of proinflammatory cytokines upon overactivation of the innate immune system, came recently to the focus of interest because of its role in the life-threatening consequences of certain immune therapies and viral diseases, including CAR-T cell therapy and Covid-19. Because complement activation with subsequent anaphylatoxin release is in the core of innate immune stimulation, studying the relationship between complement activation and cytokine release in an in vitro CS model holds promise to better understand CS and identify new therapies against it. We used peripheral blood mononuclear cells (PBMCs) cultured in the presence of autologous serum to test the impact of complement activation and inhibition on cytokine release, testing the effects of liposomal amphotericin B (AmBisome), zymosan and bacterial lipopolysaccharide (LPS) as immune activators and heat inactivation of serum, EDTA and mini-factor H (mfH) as complement inhibitors. These activators induced significant rises of complement activation markers C3a, C4a, C5a, Ba, Bb, and sC5b-9 at 45 min of incubation, with or without ~5- to ~2,000-fold rises of IL-1α, IL-1ß, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13 and TNFα at 6 and 18 h later. Inhibition of complement activation by the mentioned three methods had differential inhibition, or even stimulation of certain cytokines, among which effects a limited suppressive effect of mfH on IL-6 secretion and significant stimulation of IL-10 implies anti-CS and anti-inflammatory impacts. These findings suggest the utility of the model for in vitro studies on CS, and the potential clinical use of mfH against CS.

Anti-PEG antibodies: Properties, formation, testing and role in adverse immune reactions to PEGylated nano-biopharmaceuticals.

Conjugation of polyethylene glycols (PEGs) to proteins or drug delivery nanosystems is a widely accepted method to increase the therapeutic index of complex nano-biopharmaceuticals. Nevertheless, these drugs and agents are often immunogenic, triggering the rise of anti-drug antibodies (ADAs). Among these ADAs, anti-PEG IgG and IgM were shown to account for efficacy loss due to accelerated blood clearance of the drug (ABC phenomenon) and hypersensitivity reactions (HSRs) entailing severe allergic symptoms with occasionally fatal anaphylaxis. In addition to recapitulating the basic information on PEG and its applications, this review expands on the physicochemical factors influencing its immunogenicity, the prevalence, features, mechanism of formation and detection of anti-PEG IgG and IgM and the mechanisms by which these antibodies (Abs) induce ABC and HSRs. In particular, we highlight the in vitro, animal and human data attesting to anti-PEG Ab-induced complement (C) activation as common underlying cause of both adverse effects. A main message is that correct measurement of anti-PEG Abs and individual proneness for C activation might predict the rise of adverse immune reactions to PEGylated drugs and thereby increase their efficacy and safety.

Liposome-induced hypersensitivity reactions: Risk reduction by design of safe infusion protocols in pigs.

Intravenous administration of liposomal drugs can entail infusion reactions, also known as hypersensitivity reactions (HSRs), that can be severe and sometimes life-threatening in a small portion of patients. One empirical approach to prevent these reactions consists of lowering the infusion speed and extending the infusion time of the drug. However, different liposomal drugs have different levels of reactogenicity, which means that the optimal protocol for each liposomal drug may differ and should be identified and evaluated to make the treatment as safe and convenient as possible. The goal of the present study was to explore the use of pigs for the above purpose, using PEGylated liposomal prednisolone (PLP) as a model drug. We compared the reactogenicities of bolus versus infusion protocols involving 2-, 3- and 4-step dose escalations for a clinically relevant total dose, also varying the duration of infusions. The strength of HSRs was measured via continuous recording of hemodynamic parameters and blood thromboxane B2 levels. We showed that bolus administration or rapid infusion of PLP caused transient changes in systemic and pulmonary blood pressure and heart rate, most notably pulmonary hypertension with paralleling rises in plasma thromboxane B2. These adverse responses could be significantly reduced or eliminated by slow infusion of PLP, with the 3-h 3-step dose escalation protocol being the least reactogenic. These data suggest that the pig model enables the development of safe infusion protocols for reactogenic nanomedicines.

Pseudo-anaphylaxis to Polyethylene Glycol (PEG)-Coated Liposomes: Roles of Anti-PEG IgM and Complement Activation in a Porcine Model of Human Infusion Reactions.

Polyethylene glycol (PEG)-coated nanopharmaceuticals can cause mild to severe hypersensitivity reactions (HSRs), which can occasionally be life threatening or even lethal. The phenomenon represents an unsolved immune barrier to the use of these drugs, yet its mechanism is poorly understood. This study showed that a single i.v. injection in pigs of a low dose of PEGylated liposomes (Doxebo) induced a massive rise of anti-PEG IgM in blood, peaking at days 7-9 and declining over 6 weeks. Bolus injections of PEG-liposomes during seroconversion resulted in anaphylactoid shock (pseudo-anaphylaxis) within 2-3 min, although similar treatments of naïve animals led to only mild hemodynamic disturbance. Parallel measurement of pulmonary arterial pressure (PAP) and sC5b-9 in blood, taken as measures of HSR and complement activation, respectively, showed a concordant rise of the two variables within 3 min and a decline within 15 min, suggesting a causal relationship between complement activation and pulmonary hypertension. We also observed a rapid decline of anti-PEG IgM in the blood within minutes, increased binding of PEGylated liposomes to IgM+ B cells in the spleen of immunized animals compared to control, and increased C3 conversion by PEGylated liposomes in the serum of immunized pigs. These observations taken together suggest rapid binding of anti-PEG IgM to PEGylated liposomes, leading to complement activation via the classical pathway, entailing anaphylactoid shock and accelerated blood clearance of liposome-IgM complexes. These data suggest that complement activation plays a causal role in severe HSRs to PEGylated nanomedicines and that pigs can be used as a hazard identification model to assess the risk of HSRs in preclinical safety studies.

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.

From genomes to diaries: a 3-year prospective, real-life study of ragweed-specific sublingual immunotherapy.

During the last decades, the prevalence of allergy has dramatically increased. Allergen-specific immunotherapy is the only currently available medical intervention that has the potential to affect the natural course of the disease, but there are still many questions and unmet needs hindering its widespread use to fulfill its treatment potential and maximize its benefits for the society. To provide a comprehensive phenome-wide overview in sublingual immunotherapy, using ragweed allergy as a target, we planned and carried out a longitudinal, prospective, observational, open-label study (DesensIT). In this paper we present challenges of using deep and comprehensive phenotypes embracing biological, clinical and patient-reported outcomes in allergen-specific immunotherapy and show how we designed the DesensIT project to optimize data collection, processing and evaluation.

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.

Increased synthesis of vascular endothelial growth factor in allergic airway inflammation in histidine decarboxylase knockout (HDC(-/-)) mice.

Histamine and vascular endothelial growth factor (VEGF) have been implicated in the pathogenesis of allergic asthma; they enhance inflammation, vascular permeability, and mucus secretion. Histamine was suggested to alter the level of VEGF via the H2 receptors. Here the authors have applied histidine decarboxylase gene-targeted (HDC(-/-)) mice, lacking histamine, to investigate the effect of histamine deficiency on VEGF expression in an animal model of asthma. HDC(-/-) and wild-type (WT) mice were sensitized and challenged with ovalbumin (OVA). VEGF mRNA expression and protein level were determined in the lung. Number of VEGF-positive immune cells of bronchoalveolar lavage (BAL) and their intracellular VEGF content were measured by flow cytometry. VEGF protein level in the lung and in the BAL cells was increased in OVA treated (HDC(-/-)(ova) as well as in WT(ova)) animals compared to their controls. However, there was no difference in the VEGF levels between HDC(-/-) or WT animals, either in the lung or in the BAL cells. In conclusion, increased VEGF production of the lung or BAL immune cells can be induced by allergen provocation independently from the genetic background of the animals. These data suggest that VEGF-mediated allergic processes can persist in the absence of histamine.

Dynamic regulation of Gata1 expression during the maturation of conventional dendritic cells.

OBJECTIVES: To identify the regulatory sequences driving Gata1 expression in conventional dendritic cells (cDC). MATERIALS AND METHODS: The number and expression levels of Gata1, Gata1-target genes and hypersensitive site (HS) 2 (the eosinophil-specific enhancer)-driven green fluorescent protein (GFP) reporter of cDCs from mice lacking HS1 (the erythroid/megakaryocytic-specific enhancer, Gata1(low) mutation) and wild-type littermates, as well as the response to lipopolysaccharide of ex vivo-generated wild-type and Gata1(low) DCs were investigated. RESULTS: cDC maturation was associated with bell-shaped changes in Gata1 expression that peaked in cDCs precursors from blood. The Gata1(low) mutation did not affect Gata1 expression in cDC precursors and these cells expressed the HS2-driven reporter, indicating that Gata1 expression is HS2-driven in these cells. By contrast, the Gata1(low) mutation reduced Gata1 expression in mature cDCs and these cells did not express GFP, indicating that mature cDCs express Gata1 driven by HS1. In blood, the number of cDC precursors expressing CD40/CD80 was reduced in Gata1(low) mice, while CD40(pos)/CD80(pos) cDC precursors from wild-type mice expressed the HS2-GFP reporter, suggesting that Gata1 expression in these cells is both HS1- and HS2-driven. In addition, the antigen and accessory molecules presentation process induced by lipopolysaccharide in ex vivo-generated wild-type DC was associated with increased acetylated histone 4 occupancy of HS1, while ex vivo-generated Gata1(low) cDCs failed to respond to lipopolysaccharide, suggesting that HS1 activation is required for cDC maturation. CONCLUSION: These results identify a dynamic pattern of Gata1 regulation that switches from an HS1 to an HS2-dependent phase during the maturation of cDCs associated with the antigen-presentation process in the blood.

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

BACKGROUND: Galectin-9 (Gal-9) is a member of the growing family of beta-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 (G(OVA)). Another group of animals was allergized with OVA and also treated with dexamethasone (DEX) (G(OVA+DEX)). The control group (G(PBS)) 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 G(OVA )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 G(OVA) compared to G(PBS) or G(OVA+DEX )mice. Moreover, Gal-9 protein level was elevated in the lungs of G(OVA) 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.

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.

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.