Aluminium adjuvants in vaccines - A way to modulate the immune response.

Aluminium salts have been used as adjuvants in vaccines for almost a century, but still no clear understanding of the mechanisms behind the immune stimulating properties of aluminium based adjuvants is recognized. Aluminium adjuvants consist of aggregates and upon administration of a vaccine, the aggregates will be recognized and phagocytosed by sentinel cells such as macrophages or dendritic cells. The adjuvant aggregates will persist intracellularly, maintaining a saturated intracellular concentration of aluminium ions over an extended time. Macrophages and dendritic cells are pivotal cells of the innate immune system, linking the innate and adaptive immune systems, and become inflammatory and antigen-presenting upon activation, thus mediating the initiation of the adaptive immune system. Both types of cell are highly adaptable, and this review will discuss and highlight how the occurrence of intracellular aluminium ions over an extended time may induce the polarization of macrophages into inflammatory and antigen presenting M1 macrophages by affecting the: endosomal pH; formation of reactive oxygen species (ROS); stability of the phagosomal membrane; release of damage associated molecular patterns (DAMPs); and metabolism (metabolic re-programming). This review emphasizes that a persistent intracellular presence of aluminium ions over an extended time has the potential to affect the functionality of sentinel cells of the innate immune system, inducing polarization and activation. The immune stimulating properties of aluminium adjuvants is presumably mediated by several discrete events, however, a persistent intracellular presence of aluminium ions appears to be a key factor regarding the immune stimulating properties of aluminium based adjuvants.

Metabolic Reprogramming of Macrophages upon In Vitro Incubation with Aluminum-Based Adjuvant.

Aluminum-based adjuvants have been extensively used in vaccines. Despite their widespread use, the mechanism behind the immune stimulation properties of these adjuvants is not fully understood. Needless to say, extending the knowledge of the immune-stimulating properties of aluminum-based adjuvants is of utmost importance in the development of new, safer, and efficient vaccines. To further our knowledge of the mode of action of aluminum-based adjuvants, the prospect of metabolic reprogramming of macrophages upon phagocytosis of aluminum-based adjuvants was investigated. Macrophages were differentiated and polarized in vitro from human peripheral monocytes and incubated with the aluminum-based adjuvant Alhydrogel®. Polarization was verified by the expression of CD markers and cytokine production. In order to recognize adjuvant-derived reprogramming, macrophages were incubated with Alhydrogel® or particles of polystyrene as control, and the cellular lactate content was analyzed using a bioluminescent assay. Quiescent M0 macrophages, as well as alternatively activated M2 macrophages, exhibited increased glycolytic metabolism upon exposure to aluminum-based adjuvants, indicating a metabolic reprogramming of the cells. Phagocytosis of aluminous adjuvants could result in an intracellular depot of aluminum ions, which may induce or support a metabolic reprogramming of the macrophages. The resulting increase in inflammatory macrophages could thus prove to be an important factor in the immune-stimulating properties of aluminum-based adjuvants.

Myeloperoxidase-immunoreactive cells are significantly increased in brain areas affected by neurodegeneration in Parkinson's and Alzheimer's disease.

Myeloperoxidase (MPO) is a key enzyme in inflammatory and degenerative processes, although conflicting reports have been presented concerning its expression in the brain. We studied the cellular localization of MPO and compared numbers of MPO cells in various brain regions between neurologically healthy individuals and patients with Parkinson's disease (PD) or Alzheimer's disease (AD; n = 10-25). We also investigated two rodent PD models. MPO immunoreactivity (ir) was detected in monocytes, perivascular macrophages and amoeboid microglia in the human brain parenchyma, whereas no co-localization with glial fibrillary acidic protein (GFAP) ir was observed. In the midbrain, caudate and putamen, we found a significant increase of MPO-immunoreactive cells in PD compared with control brains, whereas in the cerebellum, no difference was apparent. MPO ir was detected neither in neurons nor in occasional small beta-amyloid-immunoreactive plaques in PD or control cases. In the frontal cortex of AD patients, we found significantly more MPO-immunoreactive cells compared with control cases, together with intense MPO ir in extracellular plaques. In the hippocampus of several AD cases, MPO-like ir was observed in some pyramidal neurons. Neither rapid dopamine depletion in the rat PD model, nor slow degeneration of dopamine neurons in MitoPark mice induced the expression of MPO ir in any brain region. MPO mRNA was not detectable with radioactive in situ hybridization in any human or rodent brain area, although myeloid cells from bone marrow displayed clear MPO signals. Our results indicate significant increases of MPO-immunoreactive cells in brain regions affected by neurodegeneration in PD and AD, supporting investigations of MPO inhibitors in novel treatment strategies.

Potent reversible inhibition of myeloperoxidase by aromatic hydroxamates.

The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.

First and second generation γ-secretase modulators (GSMs) modulate amyloid-ß (Aß) peptide production through different mechanisms.

γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-ß (Aß) peptides. The Aß42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs), such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide, have been suggested to modulate the Alzheimer-related Aß production by targeting the APP. Here, we describe novel GSMs that are selective for Aß modulation and do not impair processing of Notch, EphB2, or EphA4. The GSMs modulate Aß both in cell and cell-free systems as well as lower amyloidogenic Aß42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM, E2012, but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a (3)H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined, these data provide compelling evidence of a growing class of in vivo active GSMs, which are selective for Aß modulation and have a different mechanism of action compared with the original class of GSMs described.

The immunobiology of aluminium adjuvants: how do they really work?

Aluminium adjuvants potentiate the immune response, thereby ensuring the potency and efficacy of typically sparingly available antigen. Their concomitant critical importance in mass vaccination programmes may have prompted recent intense interest in understanding how they work and their safety. Progress in these areas is stymied, however, by a lack of accessible knowledge pertaining to the bioinorganic chemistry of aluminium adjuvants, and, consequently, the inappropriate application and interpretation of experimental models of their mode of action. The objective herein is, therefore, to identify the many ways that aluminium chemistry contributes to the wide and versatile armoury of its adjuvants, such that future research might be guided towards a fuller understanding of their role in human vaccinations.

2-thioxanthines are mechanism-based inactivators of myeloperoxidase that block oxidative stress during inflammation.

Myeloperoxidase (MPO) is a prime candidate for promoting oxidative stress during inflammation. This abundant enzyme of neutrophils uses hydrogen peroxide to oxidize chloride to highly reactive and toxic chlorine bleach. We have identified 2-thioxanthines as potent mechanism-based inactivators of MPO. Mass spectrometry and x-ray crystal structures revealed that these inhibitors become covalently attached to the heme prosthetic groups of the enzyme. We propose a mechanism whereby 2-thioxanthines are oxidized, and their incipient free radicals react with the heme groups of the enzyme before they can exit the active site. 2-Thioxanthines inhibited MPO in plasma and decreased protein chlorination in a mouse model of peritonitis. They slowed but did not prevent neutrophils from killing bacteria and were poor inhibitors of thyroid peroxidase. Our study shows that MPO is susceptible to the free radicals it generates, and this Achilles' heel of the enzyme can be exploited to block oxidative stress during inflammation.

Intratumoral administration of the antisecretory peptide AF16 cures murine gliomas and modulates macrophage functions.

Glioblastoma has remained the deadliest primary brain tumor while its current therapy offers only modest survival prolongation. Immunotherapy has failed to record notable benefits in routine glioblastoma treatment. Conventionally, immunotherapy relies on T cells as tumor-killing agents; however, T cells are outnumbered by macrophages in glioblastoma microenvironment. In this study, we explore the effect of AF16, a peptide from the endogenous antisecretory factor protein, on the survival of glioma-bearing mice, the tumor size, and characteristics of the tumor microenvironment with specific focus on macrophages. We elucidate the effect of AF16 on the inflammation-related secretome of human and murine macrophages, as well as human glioblastoma cells. In our results, AF16 alone and in combination with temozolomide leads to cure in immunocompetent mice with orthotopic GL261 gliomas, as well as prolonged survival in immunocompromised mice. We recorded decreased tumor size and changes in infiltration of macrophages and T cells in the murine glioma microenvironment. Human and murine macrophages increased expression of proinflammatory markers in response to AF16 treatment and the same effect was seen in human primary glioblastoma cells. In summary, we present AF16 as an immunomodulatory factor stimulating pro-inflammatory macrophages with a potential to be implemented in glioblastoma treatment protocols.

Results of surgery for sporadic primary hyperparathyroidism in patients with preoperatively negative sestamibi scintigraphy and ultrasound.

PURPOSE: The purpose of this study is to investigate the results of first-time surgery for sporadic primary hyperparathyroidism (pHPT) in patients with preoperatively negative sestamibi scintigraphy and ultrasound. METHODS: Data were gathered prospectively in a multicenter database for quality control in parathyroid surgery. Between 2004 and 2008, 3,158 patients underwent first-time surgery for sporadic pHPT. A total of 984 patients were subjected to preoperative localization with ultrasound and sestamibi scintigraphy, and in 173 patients, both investigations were negative. Intraoperative findings and early outcome are reported. RESULTS: One hundred and fifty-five of 173 patients underwent bilateral neck exploration. The median weight of excised parathyroid tissue was 350 mg. In 23 patients (13.3%), the exploration was negative. A total of 112 patients (64.7%) had a histological diagnosis of parathyroid adenoma and 38 patients (22%) had multiglandular disease. Six weeks after operation, 164 patients were available for analysis, and 30 patients (18%) had persistent pHPT. The risk for persistent pHPT increased for patients with few intraoperatively identified (p = 0.001) and excised (p = 0.024) parathyroid glands. Patients operated with intraoperative parathyroid hormone (iOPTH) had lower risk for persistent pHPT 7/79 (9%) compared with 23/85 patients (27%) operated without iOPTH (p = 0.003). CONCLUSIONS: Negative localization with sestamibi and ultrasound in pHPT infers a highly selected patient population with small parathyroid adenomas, an alarmingly high rate of negative exploration, and an increased risk for persistent disease. The use of iOPTH influences cure rate favorably.

Tree and shrub expansion over the past 34 years at the tree-line near Abisko, Sweden.

Shrubs and trees are expected to expand in the sub-Arctic due to global warming. Our study was conducted in Abisko, sub-arctic Sweden. We recorded the change in coverage of shrub and tree species over a 32- to 34-year period, in three 50 x 50 m plots; in the alpine-tree-line ecotone. The cover of shrubs and trees (<3.5 cm diameter at breast height) were estimated during 2009-2010 and compared with historical documentation from 1976 to 1977. Similarly, all tree stems (> or =3.5 cm) were noted and positions determined. There has been a substantial increase of cover of shrubs and trees, particularly dwarf birch (Betula nana), and mountain birch (Betula pubescens ssp. czerepanovii), and an establishment of aspen (Populus tremula). The other species willows (Salix spp.), juniper (Juniperus communis), and rowan (Sorbus aucuparia) revealed inconsistent changes among the plots. Although this study was unable to identify the causes for the change in shrubs and small trees, they are consistent with anticipated changes due to climate change and reduced herbivory.

Multi-decadal changes in tundra environments and ecosystems: synthesis of the International Polar Year-Back to the Future project (IPY-BTF).

Understanding the responses of tundra systems to global change has global implications. Most tundra regions lack sustained environmental monitoring and one of the only ways to document multi-decadal change is to resample historic research sites. The International Polar Year (IPY) provided a unique opportunity for such research through the Back to the Future (BTF) project (IPY project #512). This article synthesizes the results from 13 papers within this Ambio Special Issue. Abiotic changes include glacial recession in the Altai Mountains, Russia; increased snow depth and hardness, permafrost warming, and increased growing season length in sub-arctic Sweden; drying of ponds in Greenland; increased nutrient availability in Alaskan tundra ponds, and warming at most locations studied. Biotic changes ranged from relatively minor plant community change at two sites in Greenland to moderate change in the Yukon, and to dramatic increases in shrub and tree density on Herschel Island, and in subarctic Sweden. The population of geese tripled at one site in northeast Greenland where biomass in non-grazed plots doubled. A model parameterized using results from a BTF study forecasts substantial declines in all snowbeds and increases in shrub tundra on Niwot Ridge, Colorado over the next century. In general, results support and provide improved capacities for validating experimental manipulation, remote sensing, and modeling studies.

Autotolerant ceruloplasmin based biocathodes for implanted biological power sources.

High-performance autotolerant bioelectrodes should be ideally suited to design implantable bioelectronic devices. Because of its high redox potential and ability to reduce oxygen directly to water, human ceruloplasmin, HCp, the only blue multicopper oxidase present in human plasma, appears to be the ultimate biocatalyst for oxygen biosensors and also biocathodes in biological power sources. In comparison to fungal and plant blue multicopper oxidases, e.g. Myrothecium verrucaria bilirubin oxidase and Rhus vernicifera laccase, respectively, the inflammatory response to HCp in human blood is significantly reduced. Partial purification of HCp allowed to preserve the native conformation of the enzyme and its biocatalytic activity. Therefore, electrochemical studies were carried out with the partially purified enzyme immobilised on nanostructured graphite electrodes at physiological pH and temperature. Amperometric investigations revealed low reductive current densities, i.e. about 1.65 µA cm-2 in oxygenated electrolyte and in the absence of any mediator, demonstrating nevertheless direct electron transfer based O2 bioelectroreduction by HCp for the first time. The reductive current density obtained in the mediated system was about 12 µA cm-2. Even though the inflammatory response of HCp is diminished in human blood, inadequate bioelectrocatalytic performance hinders its use as a cathodic bioelement in a biofuel cell.

Ligand surface density is important for efficient capture of immunoglobulin and phosphatidylcholine coated particles by human peripheral dendritic cells.

A unique property of dealuminated zeolite particles is the exceptional ability to bind both hydrophilic and hydrophobic biomolecules without any covalent linkages. By adsorbing phospholipids onto the particle surface, capture of particles by human peripheral myeloid dendritic cells (mDCs) was observed. Capture of zeolite particles was only seen when a low density of phosphatidylcholine was present on the particles, indicating a specific recognition of the structural features realised by phosphatidylcholine after adsorption on the particle. Adsorbing IgG on the particles revealed capture by mDCs that was dependent upon the density of the IgG molecules. To obtain a smaller particle exposing a high density of IgG molecules, immune complexes (ICs) were formed and both mDCs and pDCs (peripheral plasmacytoid DCs) captured immune complexes, although the mDCs showed a more efficient capture of ICs. As expected, mDCs captured and internalized ICs, whereas pDCs captured ICs but showed no internalization of ICs.

Impact of modern techniques on short-term outcome after surgery for primary hyperparathyroidism: a multicenter study comprising 2,708 patients.

PURPOSE: Preoperative localization procedures and the use of intraoperative parathyroidism (iOPTH) have led to a shift of paradigm from bilateral neck exploration to focused parathyroidectomy in primary hyperparathyroidism (pHPT). However, only a small number of randomized trials from specialized centers have been published. The main purpose of the study was to analyze the impact of localization procedures and iOPTH on short-term outcome after pHPT surgery in a multi-institutional setting. METHODS: An audit for quality assurance in pHPT surgery was performed in 23 Scandinavian departments in 2004-2008. Data were gathered prospectively in a database. Two thousand seven hundred and eight patients were registered and 78% were females. The median serum calcium level was 2.79 mmol/l. RESULTS: Localization procedures were performed in 1,831 patients (68%), (sestamibi in 54% and ultrasound in 41%) and iOPTH in 792 operations (29%). Bilateral exploration was performed in 61%, focused parathyroidectomy in 17%, and unilateral exploration in 22%. Histology showed parathyroid adenoma in 82%, with the median weight of 0.6 g. The alleviation of hypercalcemia at the first follow-up was 93% (94% for primary operation). In the multivariate logistic regression analysis, iOPTH increased cure rate (OR 1.70, 95% CI 1.14-2.53, p = 0.0092). The risk for postoperative medically treated hypocalcemia decreased with the use of localization procedures (OR 0.56, 95% CI 0.43-0.78, p = 0.0004) and iOPTH (OR 0.56, 95% CI 0.39-0.90, p = 0.0015). CONCLUSIONS: Localization procedures and iOPTH decreased the risk for hypocalcemia after pHPT surgery. Additionally, iOPTH influenced short-term cure rate favorably.

Sensitive detection of hydrophobic antigens using a novel lipid-aggregate based ELISA.

Antibodies against hydrophobic antigens are common in several autoimmune diseases. However, detection of such antibodies by standard immune-assays, such as ELISA, is problematic, in part because of the problems with coating hydrophobic molecules onto polystyrene multi-well plates. We describe a novel method of stably associating hydrophobic antigens to ELISA plates. By mixing the antigen with a hydrophobic molecule containing a hydrophilic anchor, we generate mixed lipid aggregates that can attach to ELISA plates, and are resistant to detergent wash. Using the ganglioside GM-1 and phosphatidylethanolamine conjugated to the hapten DNP (dinitrophenyl) as model antigens, we show that hydrophobic antigens incorporated into mixed lipid aggregates expose their antigenic determinants in a correct configuration. The detection limit of both GM-1 and DNP-PE was considerably improved compared to when these antigens were coated on ELISA plates using organic solvents. Furthermore GM-1 incorporated into mixed lipid aggregates can be detected by specific antibodies in patient serum. The method of incorporating hydrophobic antigens into mixed lipid aggregates for stable association to ELISA plates can presumably be applied to a vast array of hydrophobic antigens, and may well be developed into a large scale screening system for serum reactivity towards different hydrophobic antigens.

Receptor-mediated endocytosis of particles by peripheral dendritic cells.

Human peripheral dendritic cells (DCs) are antigen-presenting cells with the ability to internalize antigen and present antigen-derived peptides to T cells. Human DCs express several receptors on the surface for endocytosis and other recognition receptors that bind to microbes or microbial products, which are internalized and processed. Here, we report the use of nanometer-size zeolite particles as a tool to study receptor-mediated endocytosis by the two subsets of immature DCs, myeloid (mDC) and plasmacytoid (pDC) dendritic cells. A major difference in receptor-mediated endocytosis was observed between the two populations of peripheral DCs. The pDC population demonstrated an almost complete lack of receptor-mediated endocytosis of zeolite particles, whereas the mDC population demonstrated a clear receptor-mediated endocytosis. Fc receptors are expressed by both peripheral DC populations and lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are known ligands of the Toll-like receptor (TLR)-2 and TLR4, respectively, both TLRs expressed by human mDCs. An efficient receptor-mediated endocytosis of immunoglobulin G-, LTA-, and LPS-coated zeolite particles was observed by the mDC population and their endocytosing capacity depended strongly on the density of the ligand adsorbed onto the zeolite particles. In conclusion, an efficient receptor-mediated endocytosis was observed from the mDC population, whereas the pDCs demonstrated an almost complete lack of receptor-mediated endocytosis and nanometer-size dealuminated zeolite particles were a useful tool for studying receptor-mediated endocytosis in human peripheral DCs.

Controlled release of preservatives using dealuminated zeolite Y.

This study demonstrates that dealuminated zeolite Y can act as a depot after adsorption of phenol derived preservatives. Upon suspension of zeolite loaded with the preservative m-cresol, equilibrium was quickly reached between free and adsorbed m-cresol. The equilibrium concentration of m-cresol was below 1 mM; however, it was enough to kill bacteria such as Escherichia coli and Staphylococcus aureus under metabolically active conditions. Killing of bacteria was not obtained under non-proliferating conditions and m-cresol was only released from the zeolite upon bacterial activity. Together, these results demonstrate an interesting potential use of dealuminated zeolite Y containing adsorbed preservatives for preventing microbial growth in numerous applications in industry and clinical setting.

Early activation markers of human peripheral dendritic cells.

Two major populations of dendritic cells (DCs), myeloid and plasmacytoid, can be isolated from human peripheral blood, and are distinguished by differential expression of the cell surface markers CD11c and CD123. These two populations of DCs also are different in their expression of Toll-like receptor (TLRs), which are involved in their activation. To investigate the early events during activation of peripheral DCs, the cells were stimulated in vitro with ligands for TLR-4 (as in lipopolysaccharides [LPS]) or TLR-9 (CpG-containing oligonucleotide [CpG]). The earliest change in protein expression detected after stimulating peripheral DCs with lipopolysaccharide (LPS) or CpG was increased production of the chemokine interleukin (IL)-8. Enhanced production of IL-8 occurred already within 2 hours of stimulation in both myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs), and preceded expression of the well established activation marker CD40. Although both populations of DCs secreted IL-8 upon activation, the levels of IL-8 produced was several times higher within the M-DCs compared with the P-DCs population. Before activation, both subsets of DCs expressed the IL-8 receptor type B (CD128b); but after stimulation the IL-8 receptor was down-regulated in both populations of DCs. Increased expression of MHC class II molecules is generally regarded as an early activation marker of DCs. However, only the P-DCs showed a significant up-regulation of MHC class II after stimulation. The M-DC population up-regulated MHC class II without any prior activation; thus care should be taken using increased expression of MHC class II molecules as an early activation marker of peripheral M-DCs after activation in vitro. In conclusion, we propose that during activation of human DCs the production of IL-8 and loss of CD128b are the earliest signs of activation preceding both MHC class II, CD40, CD80, and CD86 expression.

Studies on estrogen receptor (ER) alpha and beta responses on gene regulation in peripheral blood leukocytes in vivo using selective ER agonists.

Major reproductive events such as menstruation, ovulation, implantation, and cervical ripening are characterized by an increased number of invading leukocytes in the tissues. Sex steroid hormones, particularly estrogens, play an important role in these dynamic changes in the female reproductive tract. Estrogens have also been implicated in the pathogenesis of many common pathological conditions associated with leukocyte infiltration and immunological dysfunction, such as auto-immune diseases and atherosclerosis. Although the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, have been found in different leukocyte populations in tissues and in peripheral blood, there is still very little known about functional activity and importance of ERs in blood cells. To elucidate the different roles for ERalpha and ERbeta in peripheral blood leukocytes, we used microarray gene expression profiling of rat peripheral blood leukocytes subjected to in vivo treatment with estradiol (E2), the selective ERalpha agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), and the selective ERbeta agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN). We report the identification of genes that were commonly regulated by E2, PPT, and DPN, and genes that were regulated either by the ERalpha or ERbeta agonist. Further confirmatory analyses of the selected regulated genes 12-lipoxygenase, fibulin-1, furin, and calgranulin B are also presented. These results were then compared with those from the uterine tissue of the same animals. Our study demonstrates that peripheral blood leukocytes are responsive to estrogens. E2 and selective ERalpha and ERbeta agonists regulate a number of genes that may contribute to inflammation and remodeling of the extracellular matrix.

Sequestering of damage-associated molecular patterns (DAMPs): a possible mechanism affecting the immune-stimulating properties of aluminium adjuvants.

Aluminium-based adjuvants (ABAs) have been used in human and veterinary vaccines for decades, and for a long time, the adjuvant properties were believed to be mediated by an antigen depot at the injection site, prolonging antigen exposure to the immune system. The depot hypothesis is today more or less abandoned, and instead replaced by the assumption that ABAs induce an inflammation at the injection site. Induction of an inflammatory response is consistent with immune activation initiated by recognition of molecular patterns associated with danger or damage (DAMPs), and the latter are derived from endogenous molecules that normally reside intracellularly. When extracellularly expressed, because of damage, stress or cell death, a sterile inflammation is induced. In this paper, we report the induction of DAMP release by viable cells after phagocytosis of aluminium-based adjuvants. Two of the most commonly used ABAs in pharmaceutical vaccine formulations, aluminium oxyhydroxide and aluminium hydroxyphosphate, induced a vigorous extracellular expression of the two DAMP molecules calreticulin and HMGB1. Concomitantly, extracellular adjuvant particles adsorbed the DAMP molecules released by the cells whereas IL-1ß, a previously reported inflammatory mediator induced by ABAs, was not absorbed by the adjuvants. Induction of extracellular expression of the two DAMP molecules was more prominent using aluminium hydroxyphosphate compared to aluminium oxyhydroxide, whereas the extracellular adsorption of the DAMP molecules was more pronounced with the latter. Furthermore, it is hypothesised how induction of DAMP expression by ABAs and their concomitant adsorption by extracellular adjuvants may affect the inflammatory properties of ABAs.