Toll-like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin-specific allergic airway disease: role of MyD88 adaptor molecule and interleukin-12/interferon-gamma axis

Background Epidemiological and experimental data suggest that bacteria] lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll-like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro-Type 1 T helper cells (Th 1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated. Objective We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways, we used TLR4-, MyD88-, TRIF-, or IL-12/IFN-gamma-deficient mice. Methods Mice were sensitized with subcutaneous injections of ovalbumin (OVA) with or without TLR4 agonists co-adsorbed onto alum and challenged with intranasally with OVA. The development of allergic lung disease was evaluated 24 h after last OVA challenge. Results Sensitization with OVA plus LPS co-adsorbed onto alum impaired in dose-dependent manner OVA-induced Th2-mediated allergic responses such as airway eosinophilia, type-2 cytokines secretion, airway hyper-reactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, Th1 -affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. Lipopolysaccharides impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules and via the IL-12/IFN-gamma axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. Conclusion Toll-like receptor 4 agonists co-adsorbed with allergen onto alum down-modulate allergic lung disease and prevent the development of polarized T cell-mediated airway inflammation.

Aluminium hydroxide stabilised MnFe2O4 and Fe3O4 nanoparticles as dual-modality contrasts agent for MRI and PET imaging

Magnetic nanoparticles (NPs) MnFe2O4 and Fe3O4 were stabilised by depositing an Al(OH)3 layer via a hydrolysis process. The particles displayed excellent colloidal stability in water and a high affinity to [18F]-fluoride and bisphosphonate groups. A high radiolabeling efficiency, 97% for 18F-fluoride and 100% for 64Cu-bisphosphonate conjugate, was achieved by simply incubating NPs with radioactivity solution at room temperature for 5min. The properties of particles were strongly dependant on the thickness and hardness of the Al(OH)3 layer which could in turn be controlled by the hydrolysis method. The application of these Al(OH)3 coated magnetic NPs in molecular imaging has been further explored. The results demonstrated that these NPs are potential candidates as dual modal probes for MR and PET. In vivo PET imaging showed a slow release of 18F from NPs, but no sign of efflux of 64Cu. © 2014 The Authors.

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund`s Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria. (C) 2010 Elsevier Ltd. All rights reserved.

Aluminum and Sulphate Removal by a Highly Al-Resistant Dissimilatory Sulphate-Reducing Bacteria Community

A highly Al-resistant dissimilatory sulphatereducing bacteria community was isolated from sludge of the wetland of Urgeiriça mine (community W). This community showed excellent sulphate removal at the presence of Al3+. After 27 days of incubation, 73,86 and 81% of sulphate was removed in the presence of 0.48, 0.90 and 1.30 mM of Al3+, respectively. Moreover,Al3+ was simultaneously removed: 55,85 and 78% of metal was removed in the presence of 0.48, 0.90 and 1.30 mM of Al3+, respectively. The dissociation of aluminiumlactate soluble complexes due to lactate consumption by dissimilatory sulphate-reducing bacteria can be responsible for aluminum removal, which probably precipitates as insoluble aluminium hydroxide. Phylogenetic analysis of 16S rRNA gene showed that this community was mainly composed by bacteria closely related to Desulfovibrio desulfuricans. However, bacteria affiliated to Proteus and Ralstonia were also present in the community.

Humoral immune responses induced by Kudoa sp. (Myxosporea: Multivalvulida) antigens in BALB/c mice

The majority of Kudoa species infect the somatic muscle of fish establishing cysts. As there is no effective method to detect infected fish without destroying them these parasited fish reach the consumer. This work was developed to determine whether this parasite contains antigenic compounds capable of provoking an immune response in laboratory animals, in order to consider the possible immunopathological effects in man by the ingestion of Kudoa infected fish. BALB/c mice were injected by the subcutaneous route with the following extracts suspended in aluminium hydroxide: group 1 (black Kudoa sp. pseudocyst extract), group 2 (white Kudoa sp. pseudocyst extract), and group 3 (non-infected hake meat extract). Specific antibody levels were measured by ELISA against homologous and heterologous antigens. The highest responses were obtained from the black Kudoa sp. pseudocyst extract (group 1).The low optic density levels detected in group 3 proved that the results obtained in groups 1 and 2 were a consequence of the parasitic extract injection. The IgG1 was the predominant subclass. IgE detected in groups 1 and 2 showed the possible allergenic nature of some of the components of the parasitic extract. High IgA levels and medium IgG2a and IgG3 levels were obtained in groups 1 and 2. Low IgG2b responses were shown. No cross-reactions between Kudoa sp. pseudocyst extracts and the non-infected hake meat extract were observed.

Green propolis phenolic compounds act as vaccine adjuvants, improving humoral and cellular responses in mice inoculated with inactivated vaccines

Adjuvants play an important role in vaccine formulations by increasing their immunogenicity. In this study, the phenolic compound-rich J fraction (JFR) of a Brazilian green propolis methanolic extract stimulated cellular and humoral immune responses when co-administered with an inactivated vaccine against swine herpesvirus type 1 (SuHV-1). When compared to control vaccines that used aluminium hydroxide as an adjuvant, the use of 10 mg/dose of JFR significantly increased (p < 0.05) neutralizing antibody titres against SuHV-1, as well as the percentage of protected animals following SuHV-1 challenge (p < 0.01). Furthermore, addition of phenolic compounds potentiated the performance of the control vaccine, leading to increased cellular and humoral immune responses and enhanced protection of animals after SuHV-1 challenge (p < 0.05). Prenylated compounds such as Artepillin C that are found in large quantities in JFR are likely to be the substances that are responsible for the adjuvant activity.

Early Phase of Allergic Airway Inflammation in Diabetic Rats: Role of Insulin on the Signaling Pathways and Mediators

Background: Allergic lung inflammation is impaired in diabetic rats and is restored by insulin treatment. In the present study we investigated the effect of insulin on the signaling pathways triggered by allergic inflammation in the lung and the release of selected mediators. Methods: Diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 10 days) and matching controls were sensitized by s.c. injections of ovalbumin (OA) in aluminium hydroxide, 14 days before OA (1 mg/0.4 ml) or saline intratracheal challenge. A group of diabetic rats were treated with neutral protamine Hagedorn insulin (NPH, 4 IU, s.c.), 2 h before the OA challenge. Six hours after the challenge, bronchoalveolar lavage (BAL) was performed for mediator release and lung tissue was homogenized for Western blotting analysis of signaling pathways. Results: Relative to non-diabetic rats, the diabetic rats exhibited a significant reduction in OA-induced phosphorylation of the extracellular signal-regulated kinase (ERK, 59%), p38 (53%), protein kinase B (Akt, 46%), protein kinase C (PKC)-alpha (63%) and PKC-delta (38%) in lung homogenates following the antigen challenge. Activation of the NF-kappa B p65 subunit and phosphorylation of I kappa B alpha were almost suppressed in diabetic rats. Reduced expression of inducible nitric oxide synthase (iNOS, 32%) and cyclooxygenase-2 (COX-2, 46%) in the lung homogenates was also observed. The BAL concentration of prostaglandin (PG)-E(2), nitric oxide (NO) and interleukin (IL)-6 was reduced in diabetic rats (74%, 44% and 65%, respectively), whereas the cytokine-induced neutrophil chemoattractant (CINC)-2 concentration was not different from the control animals. Treatment of diabetic rats with insulin completely or partially restored all of these parameters. This protocol of insulin treatment only partially reduced the blood glucose levels. Conclusion: The data presented show that insulin regulates MAPK, PI3K, PKC and NF-kappa B pathways, the expression of the inducible enzymes iNOS and COX-2, and the levels of NO, PGE(2) and IL-6 in the early phase of allergic lung inflammation in diabetic rats. It is suggested that insulin is required for optimal transduction of the intracellular signals that follow allergic stimulation. Copyright (C) 2010 S. Karger AG, Basel

Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunised with human serum albumin

Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund`s adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated. (C) 2011 Elsevier Ltd. All rights reserved.

Crystallization of nordstrandite in ethylene glycol water solutions: electron microscopic studies

The present work shows the growth of nordstrandile microcrystals observed by transmission and scanning electron microscopy. Nordstrandite was synthesised from non-crystalline aluminium hydroxide reacted in 20% ethylene glycol/water solution, at room temperature. This material was characterized by TEM, SEM, SAED, XRD and EDS/TEM, during six month and revealed the formation and growth of nordstrandite. Fibrillar pseudoboehmite is the only aluminium hydroxide which could be identified during the first two weeks. The nuclei grow, from complete dissolution/recrystallization of pseudoboehmite fibrils, into platy rectangular microscrystals of nordstrandite. Some tabular microcrystals recrystallise, forming after six months only the mufti-point nordstrandite stars. This electron-optical study suggest that the star shape results from the overlapping of rectangular plates, and pseudoboehmite fibrils act as the precursor of nordstrandite crystallisation in ethylene glycol/water solution.

Crystallization of nordstrandite in ethylene glycol water solutions: electron microscopic studies

The present work shows the growth of nordstrandile microcrystals observed by transmission and scanning electron microscopy. Nordstrandite was synthesised from non-crystalline aluminium hydroxide reacted in 20% ethylene glycol/water solution, at room temperature. This material was characterized by TEM, SEM, SAED, XRD and EDS/TEM, during six month and revealed the formation and growth of nordstrandite. Fibrillar pseudoboehmite is the only aluminium hydroxide which could be identified during the first two weeks. The nuclei grow, from complete dissolution/recrystallization of pseudoboehmite fibrils, into platy rectangular microscrystals of nordstrandite. Some tabular microcrystals recrystallise, forming after six months only the mufti-point nordstrandite stars. This electron-optical study suggest that the star shape results from the overlapping of rectangular plates, and pseudoboehmite fibrils act as the precursor of nordstrandite crystallisation in ethylene glycol/water solution.

Comportamento mecânico e de flamabilidade de compósito de polipropileno reclicado com fibra de coco e hidróxido de alumínio

A reciclagem de plásticos tem sido uma possibilidade interessante para minimizar o problema de destino dos resíduos plásticos. O polipropileno (PP) está entre os tipos de polímeros de maior consumo, portanto a reutilização deste material tem possibilitado o desenvolvimento de estudos de grande relevância científica e social. Este polímero apresenta excelente relação custo/benefício, além de ser facilmente conformável e exibir propriedades mecânicas que o torna útil em várias aplicações. Entretanto, esse material ao ser queimado gera produtos que agem como combustíveis de modo que, para alguns usos, boa resistência à chama é necessária. Isso pode ser obtido pela adição de retardante de chama, que tem o propósito de aumentar a resistência desse material à ignição e, ao mesmo tempo, reduzir a velocidade de propagação da chama. O hidróxido de alumínio, ou simplesmente hidrato de alumina, é o agente retardante de chama mais utilizado no mercado, pois, age também como supressor de fumaça e não libera gases tóxicos durante a queima. No entanto, para tais propriedades, altas concentrações de alumina hidratada são necessárias. Isto causa deterioração nas propriedades físicas dos materiais, por não ter caráter reforçante. As fibras naturais possuem boa capacidade de reforço quando combinadas adequadamente com polímeros. Apresentando também vantagens como baixo custo, baixa densidade, biodegradabilidade e na combustão não emana gases tóxicos. Neste trabalho, misturas contendo alumina hidratada e fibras de coco foram incorporadas ao polipropileno com o objetivo de se encontrar um balanço adequado de propriedades para utilização deste compósito com características de resistência à chama e desempenho mecânico. Os compósitos foram moldados por compressão a quente e caracterizados por IV, DRX, MEV, testes mecânicos e de inflamabilidade. Foi observado aumento no módulo de elasticidade dos compósitos em geral, bem como aumento na resistência a tenacidade do compósito PP/fibra de coco em relação ao PP puro. Os resultados indicaram a eficiência da alumina hidratada como antichama, em todos os compósitos, exceto PP/F, classificando os materiais como V-0 segundo a norma internacional UL 94V.

Insulin modulates cytokine release and selectin expression in the early phase of allergic airway inflammation in diabetic rats

Abstract Background Clinical and experimental data suggest that the inflammatory response is impaired in diabetics and can be modulated by insulin. The present study was undertaken to investigate the role of insulin on the early phase of allergic airway inflammation. Methods Diabetic male Wistar rats (alloxan, 42 mg/Kg, i.v., 10 days) and controls were sensitized by s.c. injection of ovalbumin (OA) in aluminium hydroxide 14 days before OA (1 mg/0.4 mL) or saline intratracheal challenge. The following analyses were performed 6 hours thereafter: a) quantification of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and cytokine-induced neutrophil chemoattractant (CINC)-1 in the bronchoalveolar lavage fluid (BALF) by Enzyme-Linked Immunosorbent Assay, b) expression of E- and P- selectins on lung vessels by immunohistochemistry, and c) inflammatory cell infiltration into the airways and lung parenchyma. NPH insulin (4 IU, s.c.) was given i.v. 2 hours before antigen challenge. Results Diabetic rats exhibited significant reduction in the BALF concentrations of IL-1β (30%) and TNF-α (45%), and in the lung expression of P-selectin (30%) compared to non-diabetic animals. This was accompanied by reduced number of neutrophils into the airways and around bronchi and blood vessels. There were no differences in the CINC-1 levels in BALF, and E-selectin expression. Treatment of diabetic rats with NPH insulin, 2 hours before antigen challenge, restored the reduced levels of IL-1β, TNF-α and P-selectin, and neutrophil migration. Conclusion Data presented suggest that insulin modulates the production/release of TNF-α and IL-1β, the expression of P- and E-selectin, and the associated neutrophil migration into the lungs during the early phase of the allergic inflammatory reaction.

The influence of heat treatment on the phase relations in mineral growth systems

ABSTRACT Corundum is one of the most famous gems materials. Different heat treatment methods for enhancement purposes are commonly applied and accepted in the gem market. With this reason, the identification of the natural, unheated corundum is intensively investigated. In this study, aluminium hydroxide minerals and zircon are focused to observe the crystallization and phase change of these minerals during heat treatment procedures. Aluminium hydroxide minerals can be transformed to alumina with the corundum structure by heating. The reaction history of aluminium hydroxide minerals containing corundum was investigated comparing it with diaspore, boehmite, gibbsite and bayerite by TG and DTA methods. These hydroxide minerals were entirely transformed to corundum after heating at 600°C. Zircon inclusions in corundums from Ilakaka, Madagascar, were investigated for the influence of different heat-treatment temperatures on the recovery of their crystalline structure and on possible reactions within and with the host crystals. The host corundum was heated at 500, 800, 1000, 1200, 1400, 1600 and 1800°C. The crystallinity, the trapped pressure, and the decomposition of the zircon inclusions within the host corundum have been investigated by Raman spectroscopy. Radiation-damaged zircon inclusions may be used as an indicator for unheated Ilakaka corundum crystals. They are fully recrystallized after heating at 1000°C influencing the lowering of the 3 Raman band shift, the decreasing of FWHM of the 3 Raman band and the decreasing of the trapped pressure between the inclusion and the host corundum. Under microscopic observation, surface alterations of the inclusions can be firstly seen from transparent into frosted-like appearance at 1400°C. Then, between 1600°C and 1800 °C, the inclusion becomes partly or even completely molten. The decomposition of the zircon inclusion to m-ZrO2 and SiO2-glass phases begins at the rim of the inclusion after heating from 1200°C to 1600°C which can be detected by the surface change, the increase of the 3 Raman band position and the trapped pressure. At 1800°C, the zircon inclusions entirely melt transforming to solid phases during cooling like m-ZrO2 and SiO2-glass accompanied by an increase of pressure between the transformed inclusion and its host.

Entwicklung eines Tests zur Ablösung von Tierversuchen beim Nachweis von Pertussis-Toxin

Weltweit existiert keine zum Tierversuch alternative Methode, um adsorbierte Pertussis-Impfstoffe auf restliche Toxin-Aktivität hin zu untersuchen. Der im Europäischen Arzneibuch vorgeschriebene Tierversuch besitzt nach Erfahrungen der Industrie, internationaler Prüfbehörden sowie des Paul-Ehrlich-Institutes eine schlechte Aussagekraft. Er ist wenig standardisierbar und weist häufig ein zweifelhaftes Ergebnis auf, so dass Wiederholungen und damit einhergehend ein hoher Verbrauch an Versuchstieren unumgänglich sind. Enthält der Impfstoff Reste von nicht-inaktiviertem Pertussis-Toxin (PTx), muss mit schweren und schwersten Nebenwirkungen bei den Impflingen gerechnet werden. In dieser Arbeit wurde ein In vitro-Nachweis für aktives PTx entwickelt. rnAngeregt durch Publikationen, wonach Pertussis-Toxin humane Monozyten aktiviert, wurde zunächst versucht, diesen Effekt zum Toxin-Nachweis auszunutzen. Die vorliegende Arbeit zeigt jedoch eindeutig, dass Pertussis-Toxin selbst nicht zur Stimulation humaner Monozyten führt. Vielmehr konnte nachgewiesen werden, dass die Aktivierung dieser Immunzellen auf Kontaminationen durch Lipopolysaccharide zurückzuführen ist. Damit wurden die Aussagen in den oben erwähnten Veröffentlichungen widerlegt. Dieses Ergebnis wurde bereits zur Publikation eingereicht.rnNunmehr wurden verschiedene Ansätze zum Nachweis von Pertussis-Toxin entwickelt, welche seine enzymatischen Aktivitäten als NAD-Glycohydrolase und ADP-Ribosyltransferase ausnutzen. Zunächst wurde versucht, die Hydrolyse von NAD zu ADP-Ribose und Nicotinamid photometrisch nachzuweisen. Wegen unbefriedigender Sensitivität wurde dieses Verfahren zu einem fluorometrischen Nachweis weiterentwickelt. Verwendet wurde hier fluorogenes etheno-NAD, welches von Pertussis-Toxin als Substrat akzeptiert wird. Letzteres Prinzip ist zum In vitro-Nachweis von Pertussis-Toxin geeignet, wird jedoch durch das in Impfstoffen häufig verwendete Adsorbens Aluminiumhydroxid gestört. Deshalb wurde dieser Ansatz aufgegeben und ein neuer Weg verfolgt, welcher am Energiestoffwechsel von humanen Zellen ansetzt. Eine Konsequenz des Angriffs von Pertussis-Toxin auf seine Zielzellen im Respirationstrakt besteht – nach komplexen Reaktionen des Signaltransduktionsweges – im Absenken des ATP-Gehaltes. Als menschliche Surrogat-Zellen wurden frisch isolierte periphere mononukleäre Zellen (PBMCs) sowie die permanente Lymphozyten-Zelllinie Jurkat eingesetzt und deren ATP-Gehalt mittels Luziferin-Luziferase-Lumineszenz gemessen. Der Test wird nicht durch Lipopolysaccharid gestört und auch Aluminiumhydroxid übt erst nach mehreren Stunden Inkubation einen interferierenden Einfluss aus. Ebenso konnte aktives Pertussis-Toxin mit Hilfe kryokonservierter PBMCs detektiert werden, auch in orientierenden Versuchen mit komplexen Impfstoffen. Der Pertussis-ATP-Test kommt der In vivo-Situation in der Zelle sehr nahe, weil beide Untereinheiten des Toxins in einem Test überprüft werden. Demnach soll er Bestandteil einer geplanten internationalen Studie zu alternativen Pertussis-Toxin-Testungen sein.

Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.