Regulatory T Cells Accumulate in the Lung Allergic Inflammation and Efficiently Suppress T-Cell Proliferation but Not Th2 Cytokine Production

Foxp3(+)CD25(+)CD4(+) regulatory T cells are vital for peripheral tolerance and control of tissue inflammation. In this study, we characterized the phenotype and monitored the migration and activity of regulatory T cells present in the airways of allergic or tolerant mice after allergen challenge. To induce lung allergic inflammation, mice were sensitized twice with ovalbumin/aluminum hydroxide gel and challenged twice with intranasal ovalbumin. Tolerance was induced by oral administration of ovalbumin for 5 consecutive days prior to OVA sensitization and challenge. We detected regulatory T cells (Foxp3(+)CD25(+)CD4(+) T cells) in the airways of allergic and tolerant mice; however, the number of regulatory T cells was more than 40-fold higher in allergic mice than in tolerant mice. Lung regulatory T cells expressed an effector/memory phenotype (CCR4(high)CD62L(low)CD44(high)CD54(high)CD69(+)) that distinguished them from naive regulatory T cells (CCR4(int)CD62L(high)CD44(int)CD54(int)CD69(-)). These regulatory T cells efficiently suppressed pulmonary T-cell proliferation but not Th2 cytokine production.

Photodynamic Effects of Curcumin Against Cariogenic Pathogens

Background data: The presence of Streptococcus mutans and Lactobacillus acidophilus in dental structure is an indicator of a cariogenic biofilm. Photodynamic therapy is a technique that involves the activation of photosensitizers by light in the presence of oxygen, resulting in the production of reactive radicals capable of inducing cell death. Reduction of bacteria levels can provide additional means of preventing dental caries. Objective: The present study evaluated the susceptibility of planktonic cultures of S. mutans (ATCC 25175) and L. acidophilus (ATCC-IAL-523) from the Adolfo Lutz Institute (IAL) to photodynamic therapy after sensitization with curcumin and exposure to blue light at 450 nm. Methods: Bacterial suspensions of S. mutans and L. acidophilus isolated (as single species) and combined (multspecies) were prepared and then evaluated. Four different groups were analyzed: L-D- (control group), L-D+ (drug group), L+D- (light group), and L+D+ (photodynamic therapy group). Two different concentrations of curcumin were tested (0.75 and 1.5 g/L) associated with a 5.7 J/cm(2) light emission diode. Results: Significant decreases (p < 0.05) in the viability of S. mutans were only observed when the bacterial suspensions were exposed to both curcumin and light. Then, reductions in viability of up to 99.99% were observed when using 1.5 g/L of the photosensitizer. The susceptibility of L. acidophilus was considerably lower (21% and 37.6%) for both curcumin concentrations. Conclusions: Photodynamic therapy was found to be effective in reducing S. mutans and L. acidophilus on planktonic cultures. No significant reduction was found for L-D+, proving the absence of dark toxicity of the drug.

Long-term amphetamine treatment exacerbates inflammatory lung reaction while decreases airway hyper-responsiveness after allergic stimulus in rats

Asthma is an allergic lung disease can be modulated by drugs that modify the activity of central nervous system (CNS) such as amphetamine (AMPH). AMPH is a highly abused drug that exerts potent effects on behavior and immunity. In this study we investigated the mechanism involved in the effects of long-term AMPH treatment on the increased magnitude of allergic lung response. We evaluated mast cells degranulation, cytokines release, airways responsiveness and, expression of adhesion molecules. Male Wistar rats were treated with AMPH or vehicle (PBS) for 21 days and sensitized with ovalbumin (OVA) one week after the first injection of vehicle or AMPH. Fourteen days after the sensitization, the rats were challenged with an OVA aerosol, and 24 h later their parameters were analyzed. In allergic rats, the treatment with AMPH exacerbated the lung cell recruitment due increased expression of ICAM-1, PECAM-1 and Mac-1 in granulocytes and macrophages recovered from bronchoalveolar lavage. Elevated levels of IL-4, but decreased levels of IL-10 were also found in samples of lung explants after AMPH treatment. Conversely, the ex-vivo tracheal hyper-responsiveness to methacholine (MCh) was reduced by AMPH treatment, whereas the force contraction of tracheal segments due to in vitro antigen challenge remained unaltered. Our findings suggest that lung inflammation and airway hyper-responsiveness due to OVA challenge are under the distinct control of AMPH during long-term treatment. Our data strongly indicate that AMPH positively modulates allergic lung inflammation via the increase of ICAM-1, PECAM-1, Mac-1 and IL-4. AMPH also abrogates the release of the anti-inflammatory cytokine IL-10. (c) 2012 Elsevier B.V. All rights reserved.

Maternal immune activation in late gestation enhances locomotor response to acute but not chronic amphetamine treatment in male mice offspring: Role of the D1 receptor

Exposure to elevated levels of maternal cytokines can lead to functional abnormalities of the dopaminergic system in the adult offspring, including enhanced amphetamine (AMPH)-induced locomotion. Therefore, it seems reasonable to consider that offspring of challenged mothers would behave differently in models of addictive behavior, such as behavioral sensitization. Thus, we sought to evaluate the effects of prenatal exposure to lipopolysaccharide (LPS) on the locomotor response to acute and chronic AMPH treatment in male mice offspring. For this purpose, LPS (Escherichia coli 0127:B8; 120 mu g/kg) was administered intraperitoneally to pregnant Swiss mice on gestational day 17. At adulthood, male offspring were studied under one of the following conditions: (1) locomotor response to acute AMPH treatment (2.5 or 5.0 mg/kg) in an open field test; (2) behavioral sensitization paradigm, which consists of a daily injection of AMPH (1.0 mg/kg) for 10 days and observation of locomotion in the open field on days 1, 5, 10 (development phase), 15 and 17 (expression phase). The LPS stimulated offspring showed enhancement of the locomotor-stimulant effect after an acute AMPH challenge in comparison to baseline and saline pre-treated mice. They also showed development of behavioral sensitization earlier than the saline pre-treated group, although no changes between saline and LPS pre-treated groups were observed on development or expression of locomotor behavioral sensitization to AMPH. Furthermore, there was up-regulation of D1 receptor protein level within striatum in the LPS-stimulated offspring which was strongly correlated with increased grooming behavior. Taken together, our results indicate that motor and dopaminergic alterations caused by maternal immune activation are restricted to the acute AMPH challenge, mostly due to up-regulation of the D1 receptor within the mesolimbic and nigrostriatal pathways, but no locomotor differences were observed for behavioral sensitization to AMPH. (C) 2012 Elsevier B.V. All rights reserved.

Tb3+-> Eu3+ Energy Transfer in Mixed-Lanthanide-Organic Frameworks

In this work, we report a theoretical and experimental investigation of the energy transfer mechanism in two isotypical 2D coordination polymers, (infinity)[(Tb1-xEux)(DPA)(HDPA)], where H(2)DPA is pyridine 2,6-dicarboxylic acid and x = 0.05 or 0.50. Emission spectra of (infinity)[(Tb0.95Eu0.05)(DPA)(HDPA)] and (infinity)[(Tb0.5Eu0.5)(DPA)(HDPA)], (I) and (2), show that the high quenching effect on Tb3+ emission caused by Eu3+ ion indicates an efficient Tb3+-> Eu3+ energy transfer (ET). The k(ET) of Tb3+-> Eu3+ ET and rise rates (k(r)) of Eu3+ as a function of temperature for (1) are on the same order of magnitude, indicating that the sensitization of the Eu3+5D0 level is highly fed by ET from the D-5(4) level of Tb3+ ion. The eta(ET) and R-0 values vary in the 67-79% and 7.15 to 7.93 angstrom ranges. Hence, Tb3+ is enabled to transfer efficiently to Eu3+ that can occupy the possible sites at 6.32 and 6.75 angstrom. For (2), the ET processes occur on average with eta(ET) and R-0 of 97% and 31 angstrom, respectively. Consequently, Tb3+ ion is enabled to transfer energy to Eu3+ localized at different layers. The theoretical model developed by Malta was implemented aiming to insert more insights about the dominant mechanisms involved in the ET between lanthanides ions. Calculated single Tb3+-> Eu3+ ETs are three orders of magnitude inferior to those experimentally; however, it can be explained by the theoretical model that does not consider the role of phonon assistance in the Ln(3+)-> Ln(3+) ET processes. In addition, the Tb3+-> Eu3+ ET processes are predominantly governed by dipole-dipole (d-d) and dipole-quadrupole (d-q) mechanisms.

Clinical Treatment of Dry Eye Using 0.03% Tacrolimus Eye Drops

Purpose: To report the clinical outcome of the treatment of dry eyes using 0.03% tacrolimus eye drops (olive oil + tacrolimus 0.03%) (Ophthalmos, Sao Paulo, Brazil). Methods: Sixteen eyes of 8 patients with Sjogren syndrome dry eyes (age, 51.13 +/- 9.45 years) were enrolled in this study (prospective noncontrolled interventional case series). Patients were instructed to use topical 0.03% tacrolimus eye drops twice a day (every 12 hours) in the lower conjunctival sac. Schirmer I test, break-up time, corneal fluorescein, and rose bengal staining score were performed in all patients 1 day before, and 14, 28, and 90 days after treatment with 0.03% tacrolimus eye drops. Results: The average fluorescein staining and rose bengal staining scores improved statistically significantly after 14 days of treatment and improved even more after 28 and 90 days. The average Schirmer I test did not improve statistically significantly after 28 days of treatment, although we did observe a significant improvement after 90 days of treatment with 0.03% tacrolimus eye drops. The average break-up time did not improve statistically after 14 days of treatment, although we observed a significant improvement after 28 and 90 days of treatment with 0.03% tacrolimus eye drops. Conclusions: Topical 0.03% tacrolimus eye drops successfully improved tear stability and ocular surface status in patients with dry eyes.

Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor FcγRIIB expression on B cells

Abstract Background Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period. Results Maternal immunization with OVA showed increased levels of FcγRIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-γ-secreting T cells and IL-4 and IL-12- secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced FcγRIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers. Conclusion Maternal immunization upregulates the inhibitory FcγRIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.

Formation and stability of oil-in-water nanoemulsions containing rice bran oil: in vitro and in vivo assessments

Abstract Background Nanoemulsions have practical application in a multitude of commercial areas, such as the chemical, pharmaceutical and cosmetic industries. Cosmetic industries use rice bran oil in sunscreen formulations, anti ageing products and in treatments for skin diseases. The aim of this study was to create rice bran oil nanoemulsions using low energy emulsification methods and to evaluate their physical stability, irritation potential and moisturising activity on volunteers with normal and diseased skin types. Results The nanoemulsion developed by this phase diagram method was composed of 10% rice bran oil, 10% surfactants sorbitan oleate/PEG-30 castor oil, 0.05% antioxidant and 0.50% preservatives formulated in distilled water. The nanoemulsion was stable over the time course of this study. In vitro assays showed that this formulation has a low irritation potential, and when applied to human skin during in vivo studies, the nanoemulsion improved the skin's moisture and maintained normal skin pH values. Conclusion The results of irritation potential studies and in vivo assessments indicate that this nanoemulsion has potential to be a useful tool to treat skin diseases, such as atopic dermatitis and psoriasis.

Brazilian blood donation eligibility criteria for dermatologic patients

A focused and commented review on the impact of dermatologic diseases and interventions in the solidary act of donating blood is presented to dermatologists to better advise their patients. This is a review of current Brazilian technical regulations on hemotherapeutic procedures as determined by Ministerial Directive #1353/2011 by the Ministry of Health and current internal regulations of the Hemotherapy Center of Ribeirão Preto, a regional reference center in hemotherapeutic procedures. Criteria for permanent inaptitude: autoimmune diseases (>1 organ involved), personal history of cancer other than basal cell carcinoma, severe atopic dermatitis or psoriasis, pemphigus foliaceus, porphyrias, filariasis, leprosy, extra pulmonary tuberculosis or paracoccidioidomycosis, and previous use of etretinate. Drugs that impose temporary ineligibility: other systemic retinoids, systemic corticosteroids, 5-alpha-reductase inhibitors, vaccines, methotrexate, beta-blockers, minoxidil, anti-epileptic, and anti-psychotic drugs. Other conditions that impose temporary ineligibility: occupational accident with biologic material, piercing, tattoo, sexually transmitted diseases, herpes, and bacterial infections, among others. Discussion: Thalidomide is currently missing in the teratogenic drugs list. Although finasteride was previously considered a drug that imposed permanent inaptitude, according to its short halflife current restriction of 1 month is still too long. Dermatologists should be able to advise their patients about proper timing to donate blood, and discuss the impact of drug withdrawal on treatment outcomes and to respect the designated washout periods.

Ovariectomized OVA-sensitized mice display increased frequency of CD4+Foxp3+ T regulatory cells in the periphery

It is well established that female sex hormones have a pivotal role in inflammation. For instance, our group has previously reported that estradiol has proinflammatory actions during allergic lung response in animal models. Based on these findings, we have decided to further investigate whether T regulatory cells are affected by female sex hormones absence after ovariectomy. We evaluated by flow cytometry the frequencies of CD4+Foxp3+ T regulatory cells (Tregs) in central and peripheral lymphoid organs, such as the thymus, spleen and lymph nodes. Moreover, we have also used the murine model of allergic lung inflammation a to evaluate how female sex hormones would affect the immune response in vivo. To address that, ovariectomized or sham operated female Balb/c mice were sensitized or not with ovalbumin 7 and 14 days later and subsequently challenged twice by aerosolized ovalbumin on day 21. Besides the frequency of CD4+Foxp3+ T regulatory cells, we also measured the cytokines IL-4, IL-5, IL-10, IL-13 and IL-17 in the bronchoalveolar lavage from lungs of ovalbumine challenged groups. Our results demonstrate that the absence of female sex hormones after ovariectomy is able to increase the frequency of Tregs in the periphery. As we did not observe differences in the thymus-derived natural occurring Tregs, our data may indicate expansion or conversion of peripheral adaptive Tregs. In accordance with Treg suppressive activity, ovariectomized and ovalbumine-sensitized and challenged animals had significantly reduced lung inflammation. This was observed after cytokine analysis of lung explants showing significant reduction of pro-inflammatory cytokines, such as IL-4, IL-5, IL-13 and IL-17, associated to increased amount of IL-10. In summary, our data clearly demonstrates that OVA sensitization 7 days after ovariectomy culminates in reduced lung inflammation, which may be directly correlated with the expansion of Tregs in the periphery and further higher IL-10 secretion in the lungs.

Measurement and model calculation of the temperature dependence of ligand-to-metal energy transfer rates in lanthanide complexes

Temperature dependent transient curves of excited levels of a model Eu3+ complex have been measured for the first time. A coincidence between the temperature dependent rise time of the 5D0 emitting level and decay time of the 5D1 excited level in the [Eu(tta)3(H2O)2] complex has been found, which unambiguously proves the T1→5D1→5D0 sensitization pathway. A theoretical approach for the temperature dependent energy transfer rates has been successfully applied to the rationalization of the experimental data.

Design and Characterization of New Luminescent Sensors and Labels

The aim of this Ph.D. project has been the design and characterization of new and more efficient luminescent tools, in particular sensors and labels, for analytical chemistry, medical diagnostics and imaging. Actually both the increasing temporal and spatial resolutions that are demanded by those branches, coupled to a sensitivity that is required to reach the single molecule resolution, can be provided by the wide range of techniques based on luminescence spectroscopy. As far as the development of new chemical sensors is concerned, as chemists we were interested in the preparation of new, efficient, sensing materials. In this context, we kept developing new molecular chemosensors, by exploiting the supramolecular approach, for different classes of analytes. In particular we studied a family of luminescent tetrapodal-hosts based on aminopyridinium units with pyrenyl groups for the detection of anions. These systems exhibited noticeable changes in the photophysical properties, depending on the nature of the anion; in particular, addition of chloride resulted in a conformational change, giving an initial increase in excimeric emission. A good selectivity for dicarboxylic acid was also found. In the search for higher sensitivities, we moved our attention also to systems able to perform amplification effects. In this context we described the metal ion binding properties of three photoactive poly-(arylene ethynylene) co-polymers with different complexing units and we highlighted, for one of them, a ten-fold amplification of the response in case of addition of Zn2+, Cu2+ and Hg2+ ions. In addition, we were able to demonstrate the formation of complexes with Yb3+ an Er3+ and an efficient sensitization of their typical metal centered NIR emission upon excitation of the polymer structure, this feature being of particular interest for their possible applications in optical imaging and in optical amplification for telecommunication purposes. An amplification effect was also observed during this research in silica nanoparticles derivatized with a suitable zinc probe. In this case we were able to prove, for the first time, that nanoparticles can work as “off-on” chemosensors with signal amplification. Fluorescent silica nanoparticles can be thus seen as innovative multicomponent systems in which the organization of photophysically active units gives rise to fruitful collective effects. These precious effects can be exploited for biological imaging, medical diagnostic and therapeutics, as evidenced also by some results reported in this thesis. In particular, the observed amplification effect has been obtained thanks to a suitable organization of molecular probe units onto the surface of the nanoparticles. In the effort of reaching a deeper inside in the mechanisms which lead to the final amplification effects, we also attempted to find a correlation between the synthetic route and the final organization of the active molecules in the silica network, and thus with those mutual interactions between one another which result in the emerging, collective behavior, responsible for the desired signal amplification. In this context, we firstly investigated the process of formation of silica nanoparticles doped with pyrene derivative and we showed that the dyes are not uniformly dispersed inside the silica matrix; thus, core-shell structures can be formed spontaneously in a one step synthesis. Moreover, as far as the design of new labels is concerned, we reported a new synthetic approach to obtain a class of robust, biocompatible silica core-shell nanoparticles able to show a long-term stability. Taking advantage of this new approach we also showed the synthesis and photophysical properties of core-shell NIR absorbing and emitting materials that proved to be very valuable for in-vivo imaging. In general, the dye doped silica nanoparticles prepared in the framework of this project can conjugate unique properties, such as a very high brightness, due to the possibility to include many fluorophores per nanoparticle, high stability, because of the shielding effect of the silica matrix, and, to date, no toxicity, with a simple and low-cost preparation. All these features make these nanostructures suitable to reach the low detection limits that are nowadays required for effective clinical and environmental applications, fulfilling in this way the initial expectations of this research project.

Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential. I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here. First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall. Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication). I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface. Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy. In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants.

Genomic and transcriptional analysis of allergen genes in apple (Malus x domestica)

Apple consumption is highly recomended for a healthy diet and is the most important fruit produced in temperate climate regions. Unfortunately, it is also one of the fruit that most ofthen provoks allergy in atopic patients and the only treatment available up to date for these apple allergic patients is the avoidance. Apple allergy is due to the presence of four major classes of allergens: Mal d 1 (PR-10/Bet v 1-like proteins), Mal d 2 (Thaumatine-like proteins), Mal d 3 (Lipid transfer protein) and Mal d 4 (profilin). In this work new advances in the characterization of apple allergen gene families have been reached using a multidisciplinary approach. First of all, a genomic approach was used for the characterization of the allergen gene families of Mal d 1 (task of Chapter 1), Mal d 2 and Mal d 4 (task of Chapter 5). In particular, in Chapter 1 the study of two large contiguos blocks of DNA sequences containing the Mal d 1 gene cluster on LG16 allowed to acquire many new findings on number and orientation of genes in the cluster, their physical distances, their regulatory sequences and the presence of other genes or pseudogenes in this genomic region. Three new members were discovered co-localizing with the other Mal d 1 genes of LG16 suggesting that the complexity of the genetic base of allergenicity will increase with new advances. Many retrotranspon elements were also retrieved in this cluster. Due to the developement of molecular markers on the two sequences, the anchoring of the physical and the genetic map of the region has been successfully achieved. Moreover, in Chapter 5 the existence of other loci for the Thaumatine-like protein family in apple (Mal d 2.03 on LG4 and Mal d 2.02 on LG17) respect the one reported up to now was demonstred for the first time. Also one new locus for profilins (Mal d 4.04) was mapped on LG2, close to the Mal d 4.02 locus, suggesting a cluster organization for this gene family, as is well reported for Mal d 1 family. Secondly, a methodological approach was used to set up an highly specific tool to discriminate and quantify the expression of each Mal d 1 allergen gene (task of Chapter 2). In aprticular, a set of 20 Mal d 1 gene specific primer pairs for the quantitative Real time PCR technique was validated and optimized. As a first application, this tool was used on leaves and fruit tissues of the cultivar Florina in order to identify the Mal d 1 allergen genes that are expressed in different tissues. The differential expression retrieved in this study revealed a tissue-specificity for some Mal d 1 genes: 10/20 Mal d 1 genes were expressed in fruits and, indeed, probably more involved in the allergic reactions; while 17/20 Mal d 1 genes were expressed in leaves challenged with the fungus Venturia inaequalis and therefore probably interesting in the study of the plant defense mechanism. In Chapter 3 the specific expression levels of the 10 Mal d 1 isoallergen genes, found to be expressed in fruits, were studied for the first time in skin and flesh of apples of different genotypes. A complex gene expression profile was obtained due to the high gene-, tissue- and genotype-variability. Despite this, Mal d 1.06A and Mal d 1.07 expression patterns resulted particularly associated with the degree of allergenicity of the different cultivars. They were not the most expressed Mal d 1 genes in apple but here it was hypotized a relevant importance in the determination of allergenicity for both qualitative and quantitative aspects of the Mal d 1 gene expression levels. In Chapter 4 a clear modulation for all the 17 PR-10 genes tested in young leaves of Florina after challenging with the fungus V. inaequalis have been reported but with a peculiar expression profile for each gene. Interestingly, all the Mal d 1 genes resulted up-regulated except Mal d 1.10 that was down-regulated after the challenging with the fungus. The differences in direction, timing and magnitude of induction seem to confirm the hypothesis of a subfunctionalization inside the gene family despite an high sequencce and structure similarity. Moreover, a modulation of PR-10 genes was showed both in compatible (Gala-V. inaequalis) and incompatible (Florina-V. inaequalis) interactions contribute to validate the hypothesis of an indirect role for at least some of these proteins in the induced defense responses. Finally, a certain modulation of PR-10 transcripts retrieved also in leaves treated with water confirm their abilty to respond also to abiotic stress. To conclude, the genomic approach used here allowed to create a comprehensive inventory of all the genes of allergen families, especially in the case of extended gene families like Mal d 1. This knowledge can be considered a basal prerequisite for many further studies. On the other hand, the specific transcriptional approach make it possible to evaluate the Mal d 1 genes behavior on different samples and conditions and therefore, to speculate on their involvement on apple allergenicity process. Considering the double nature of Mal d 1 proteins, as apple allergens and as PR-10 proteins, the gene expression analysis upon the attack of the fungus created the base for unravel the Mal d 1 biological functions. In particular, the knowledge acquired in this work about the PR-10 genes putatively more involved in the specific Malus-V. inaequalis interaction will be helpful, in the future, to drive the apple breeding for hypo-allergenicity genotype without compromise the mechanism of response of the plants to stress conditions. For the future, the survey of the differences in allergenicity among cultivars has to be be thorough including other genotypes and allergic patients in the tests. After this, the allelic diversity analysis with the high and low allergenic cultivars on all the allergen genes, in particular on the ones with transcription levels correlated to allergencity, will provide the genetic background of the low ones. This step from genes to alleles will allow the develop of molecular markers for them that might be used to effectively addressed the apple breeding for hypo-allergenicity. Another important step forward for the study of apple allergens will be the use of a specific proteomic approach since apple allergy is a multifactor-determined disease and only an interdisciplinary and integrated approach can be effective for its prevention and treatment.

Beeinflussung der menschlichen allergischen Immunantwort durch Allergen-DNA-transfizierte dendritische Zellen

Immunreaktionen gegen Antigene können eine zelluläre Immunantwort, eingeleitet durch TH1-Zellen induzieren oder aber eine antikörpervermittelte humorale Immunantwort induzieren, die von Zellen des TH2-Typs bestimmt sind. Diese unterschiedlichen Immunreaktionen regulieren sich gegenseitig, indem sie sich wechselseitig unterdrücken. Dadurch stellt sich ein Gleichgewicht zwischen den TH1- und TH2-Immunantworten ein. Die Allergie vom Soforttyp ist TH2-dominiert und wird ausgelöst durch die Produktion spezifischer IgE-Antikörper gegen eigentlich harmlose Antigene. Durch die frühe Ausschüttung des Zytokins IL-4 von T-Zellen differenzieren naive allergenspezifische T-Zellen zu TH2-Zellen aus. Diese sezernieren dann ebenfalls IL-4 und auch IL-13, was die B-Zellen zu einer vermehrten Produktion von IgE-Molekülen stimuliert. IgE bindet an den hochaffinen IgE-Rezeptor auf Mastzellen, Basophilen und Eosinophilen. Bei weiterem Allergenkontakt kommt es durch Kreuzvernetzung der Fc-Rezeptoren zur Ausschüttung vo