Vaccine antigens are partly adsorbed onto aluminium-based adjuvant particles, forming an unstable corona. At the inoculation site, the corona will be restructured, and the adsorbed antigens will be released through replacement with biomolecules from the interstitial fluid of the recipient. Aluminium-based adjuvants (ABAs) carrying a corona of serum proteins as a model of particles with a pre-formed antigen corona were shown to adsorb several categories of cytokines and growth factors, as assessed from a protein array covering 18 different analytes. Out of the 18 analytes, 12 were shown to be adsorbed by the aluminium-based adjuvant Alhydrogel (R), which had a pre-formed protein corona. The adsorption of TNF-alpha, IL-2, IL-4, IL-10, and IFN-gamma was studied in detail. Among the studied cytokines, IL-2, IL-4, and IFN-gamma, were adsorbed by Alhydrogel (R). Adsorbed IFN-gamma was further studied to show that the adsorption of IFN-gamma did not denature the cytokine, and the cytokine could be desorbed from adjuvant particles in a biologically active form and in relevant amounts. The adsorption of immune-stimulating molecules onto ABAs at the administration site of a vaccine is a neglected event in the mode of action of aluminium-based adjuvants. This process may modulate the immune response with a profound impact on initiating the innate immune response and consequently the adaptive immune response.

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((R)). Polarization was verified by the expression of CD markers and cytokine production. In order to recognize adjuvant-derived reprogramming, macrophages were incubated with Alhydrogel((R)) 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.

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.

Molecularly imprinted polymers (MIPs) against sialic acid (SA) have been developed as a detection tool to target cancer cells. Before proceeding to in vivo studies, a better knowledge of the overall effects of MIPs on the innate immune system is needed. The aim of this study thus was to exemplarily assess whether SA-MIPs lead to inflammatory and/or cytotoxic responses when administered to phagocytosing cells in the innate immune system. The response of monocytic/macrophage cell lines to two different reference particles, Alhydrogel and PLGA, was compared to their response to SA-MIPs. In vitro culture showed a cellular association of SA-MIPs and Alhydrogel, as analyzed by flow cytometry. The reference particle Alhydrogel induced secretion of IL-1β from the monocytic cell line THP-1, whereas almost no secretion was provoked for SA-MIPs. A reduced number of both THP-1 and RAW 264.7 cells were observed after incubation with SA-MIPs and this was not caused by cytotoxicity. Digital holographic cytometry showed that SA-MIP treatment affected cell division, with much fewer cells dividing. Thus, the reduced number of cells after SA-MIP treatment was not linked to SA-MIPs cytotoxicity. In conclusion, SA-MIPs have a low degree of inflammatory properties, are not cytotoxic, and can be applicable for future in vivo studies.

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.

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.

Background: Aluminium salts have been used for decades in vaccines as adjuvants to facilitate the adaptive immune response against co-administered antigens. Two types of aluminium adjuvant are mostly used, aluminium oxyhydroxide and aluminium hydroxyphosphate. Both types of aluminium adjuvant consist of nanoparticles that form loose, micrometre sized aggregates at circumneutral pH. Aluminium adjuvants constitute a well-documented example of administration of nanomaterials to humanswith infrequent side effects and a safety record generally regarded as excellent. However, despite its prolonged use in human and veterinary medicine, the mechanisms behind the enhanced response and the immune stimulatory effect are still by and large unknown. Methods: The present paper reviews existing ideas regarding the immunostimulatory effects of aluminium adjuvants, with a focus on the induction of an inflammatory response by cellular stress. Reviewed information was obtained from peer-reviewed scientific papers published in 1988 to date with one exception, a paper published 1931. Results: Cellular stress causes extra cellular signalling of danger associated molecular patterns (DAMPs) and upon phagocytosis of aluminium adjuvants the cells need to manage the ingested particles. Conclusion: A persistent intracellular accumulation of aluminium adjuvants will be a solid depository of sparingly soluble aluminium salts maintaining a constant concentration of Al3+ ions in the cytoplasm and this will affect multiple biochemical processes. The cell will be under constant stress and DAMP signalling will occur and we would like to suggest the maintenance of a constant concentration Al3+ ions in the cytoplasm as a general underlying feature of the immune stimulation properties of aluminium adjuvants.

Background: Studies revealing the immune stimulatory properties of aluminium-based adjuvants (ABAs) have been impaired by the absence of simple and reliable methods of tracing the adjuvants and their effect on biochemical processes upon endocytosis. Objective: To verify that labelling of ABAs with lumogallion doesn’t affect the physicochemical properties of the adjuvant; tracing cellular interaction with aluminium adjuvants; explore their effect on metabolic activity upon endocytosis. Methods: Physicochemical characterization by Z-potential and size distribution of ABAs labelled with lumogallion. Cellular interactions with ABAs by flow cytometry and confocal microscopy. Metabolic activity explored by measuring transformation of tetrazolium into formazan. Results: No or minor change of zeta potential and average particle size of lumogallion labelled aluminium oxyhydroxide, AlO(OH) and aluminium hydroxyphosphate, Al(OH)x(PO4 )y. Both phagocytosing and non-phagocytosing leukocytes became associated with ABAs at concentrations expected after in vivo administration of a vaccine. The ABAs were relatively toxic, affecting both lymphocytes and monocytes, and Al(OH)x(PO4 )y was more toxic than AlO(OH). Endocytosed aluminium adjuvant particles were not secreted from the cells and remained intracellular throughout several cell divisions. The presence of ABAs increased the mitochondrial activity of the monocytic cell line THP-1 and peripheral monocytes, as based on the transformation of tetrazolium into formazan. Conclusion: Lumogallion labelled ABAs is a valuable tool tracing interactions between ABAs and cells. Labelled ABAs can be traced intracellularly and ABAs are likely to remain intracellular for a long period of time. Intracellular ABAs increase the mitochondrial activity and the presence of intracellular Al ions is suggested to cause an increased mitochondrial activity. Keywords: Aluminium based adjuvant, Lumogallion, Mitochondrial activity, MTT assay, Phagocytosis, ABAS, Zeolites.

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-1, 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, is hypothesised how induction of DAMP expression by ABAs and their concomitant adsorption by extra cellular adjuvants may affect the inflammatory properties of ABAs.