The quest for down scale representativeness: how to exploit CFD to design a shear study for vaccines.

In this work, we exploit computational fluid dynamics (CFD) to evaluate stirred tank reactor (STR) process engineer parameters (PEP) and design a scale-down system (SDS) to be representative of the formulation and filling process steps for an Aluminum adjuvanted vaccine drug product (DP). To study the shear history in the SDS we used the concept of number of passages, combined with an appropriate stirring speed down scale strategy comprising of either (i) tip speed equivalence, widely used as a scale-up criterion for a shear-sensitive product, or (ii) rotating shear, a shear metric introduced by Metz and Otto in 1957 but never used as scaling criterion. The outcome of the CFD simulations shows that the tip equivalence generates a worst-case SDS in terms of shear, whereas the rotating shear scaling approach could be used to design a more representative SDS. We monitored the trend over time for "In Vitro Relative Potency" as DP Critical Quality Attribute for both scaling approaches, which highlighted the crucial role of choosing the appropriate scaling-down approach to be representative of the manufacturing scale during process characterization studies.

Maturation of Aluminium Adsorbed Antigens Contributes to the Creation of Homogeneous Vaccine Formulations.

Although aluminium-based vaccines have been used for almost over a century, their mechanism of action remains unclear. It is established that antigen adsorption to the adjuvant facilitates delivery of the antigen to immune cells at the injection site. To further increase our understanding of aluminium-based vaccines, it is important to gain additional insights on the interactions between the aluminium and antigens, including antigen distribution over the adjuvant particles. Immuno-assays can further help in this regard. In this paper, we evaluated how established formulation strategies (i.e., sequential, competitive, and separate antigen addition) applied to four different antigens and aluminium oxyhydroxide, lead to formulation changes over time. Results showed that all formulation samples were stable, and that no significant changes were observed in terms of physical-chemical properties. Antigen distribution across the bulk aluminium population, however, did show a maturation effect, with some initial dependence on the formulation approach and the antigen adsorption strength. Sequential and competitive approaches displayed similar results in terms of the homogeneity of antigen distribution across aluminium particles, while separately adsorbed antigens were initially more highly poly-dispersed. Nevertheless, the formulation sample prepared via separate adsorption also reached homogeneity according to each antigen adsorption strength. This study indicated that antigen distribution across aluminium particles is a dynamic feature that evolves over time, which is initially influenced by the formulation approach and the specific adsorption strength, but ultimately leads to homogeneous formulations.

Asn25 Deamidation as an Allosteric Tool to Increase IFNß-1a Biological Activity.

Interferon beta (IFNß) is a well-known cytokine, belonging to the type I family, that exerts antiviral, immunomodulatory, and antiproliferative activity. It has been reported that the artificially deamidated form of recombinant IFNß-1a at Asn25 position shows an increased biological activity. As a deepening of the previous study, the molecular mechanism underlying this biological effect was investigated in this work by combining experimental and computational techniques. Specifically, the binding to IFNAR1 and IFNAR2 receptors and the canonical pathway of artificially deamidated IFNß-1a molecule were analyzed in comparison to the native form. As a result, a change in receptor affinity of deamidated IFNß-1a with respect to the native form was observed, and to better explore this molecular interaction, molecular dynamics simulations were carried out. Results confirmed, as previously hypothesized, that the N25D mutation can locally change the interaction network of the mutated residue but also that this effect can be propagated throughout the molecule. In fact, many residues not involved in the interaction with IFNAR1 in the native form participate to the recognition in the deamidated molecule, enhancing the binding to IFNAR1 receptor and consequently an increase of signaling cascade activation. In particular, a higher STAT1 phosphorylation and interferon-stimulated gene expression was observed under deamidated IFNß-1a cell treatment. In conclusion, this study increases the scientific knowledge of deamidated IFNß-1a, deciphering its molecular mechanism, and opens new perspectives to novel therapeutic strategies.

Epitope Mapping and Binding Assessment by Solid-State NMR Provide a Way for the Development of Biologics under the Quality by Design Paradigm.

Multispecific biologics are an emerging class of drugs, in which antibodies and/or proteins designed to bind pharmacological targets are covalently linked or expressed as fusion proteins to increase both therapeutic efficacy and safety. Epitope mapping on the target proteins provides key information to improve the affinity and also to monitor the manufacturing process and drug stability. Solid-state NMR has been here used to identify the pattern of the residues of the programmed cell death ligand 1 (PD-L1) ectodomain that are involved in the interaction with a new multispecific biological drug. This is possible because the large size and the intrinsic flexibility of the complexes are not limiting factors for solid-state NMR.

Impact of four inorganic impurities - iron, copper, nickel and zinc - on the quality attributes of a Fc-fusion protein upon incubation at different temperatures.

A key aspect that must be supervised during the development of a biotherapeutic is the presence of elemental impurities in the final drug product: they must be quantified as to ensure that their concentrations does not affect patients' safety. Regulatory guidelines such as ICH Q3D provides Permitted Daily Exposure (PDE) limits for those impurities considered having a higher potential safety risk. However, one of the limits of such PDE values is that they account for the safety risk, while alterations of certain Quality Attributes (QA) of a biologic may also take place. In order to understand how certain impurities could affect not only the safety of patients, but also the physicochemical properties of biotherapeutics, here we present a study in which we examined how four commonly observed elemental impurities could impact the QAs of a Fc-fusion protein, under normal storage conditions and after six weeks of incubation at +25 °C and +40 °C. The molecule was indeed treated with increasing concentrations of Ni2+, Cu2+, Zn2+ and Fe3+ and the potential changes in conformation, oxidation, aggregation, and fragmentation were monitored. Our data suggest that keeping the levels of these impurities under the safety threshold limits does not guarantee the product quality. While nickel and zinc slightly altered the physicochemical properties of our Fc-fusion protein, iron and copper appeared to be more harmful for the QAs stability. Indeed, these latter elements might cause significant alterations of the product quality such as to potentially alter its efficacy.

IgG1 conformational behavior: elucidation of the N-glycosylation role via molecular dynamics.

Currently, monoclonal antibodies (mAbs) are the most used biopharmaceuticals for human therapy. One of the key aspects in their development is the control of effector functions mediated by the interaction between fragment crystallizable (Fc) and Fcγ receptors, which is a secondary mechanism of the action of biotherapeutics. N-glycosylation at the Fc portion can regulate these mechanisms, and much experimental evidence suggests that modifications of glycosidic chains can affect antibody binding to FcγRIIIa, consequently impacting the immune response. In this work, we try to elucidate via in silico procedures the structural role exhibited by glycans, particularly fucose, in mAb conformational freedom that can potentially affect the receptor recognition. By using adalimumab, a marketed IgG1, as a general template, after rebuilding its three-dimensional (3D) structure through homology modeling approaches, we carried out molecular dynamics simulations of three differently glycosylated species: aglycosylated, afucosylated, and fucosylated antibody. Trajectory analysis showed different dynamical behaviors and pointed out that sugars can influence the overall 3D structure of the antibody. As a result, we propose a putative structural mechanism by which the presence of fucose introduces conformational constraints in the whole antibody and not only in the Fc domain, preventing a conformation suitable for the interaction with the receptor. As secondary evidence, we observed a high flexibility of the antibodies that is translated into an asymmetric behavior of Fab portions shown by all the simulated biopolymers, making the dynamical asymmetry a new, to our knowledge, molecular aspect that may be further investigated. In conclusion, these findings can help understand the contribution of sugars on the structural architecture of mAbs, paving the way to novel strategies of pharmaceutical development.

The indirubin derivative 6-bromoindirubin-3'-glycerol-oxime ether (6BIGOE) potently modulates inflammatory cytokine and prostaglandin release from human monocytes through GSK-3 interference.

Indirubin is a natural bis-indole alkaloid contained as active ingredient in the traditional Chinese remedy Danggui Longhui Wan. Indirubin and its 3'-oxime derivatives exhibit anti-cancer and anti-inflammatory properties and they inhibit glycogen synthase kinase (GSK)-3 in cell-free assays where 6-bromoindirubin-3'-oxime (6BIO) is among the most potent analogs. Here, we reveal 6-bromoindirubin-3'-glycerol-oxime ether (6BIGOE) as highly potent derivative able to inhibit pro-inflammatory cytokine, chemokine and prostaglandin (PG) release in human primary monocytes while increasing anti-inflammatory interleukin (IL)-10 levels. 6BIGOE suppressed lipopolysaccharide (LPS)-induced IL-1ß and PGE2 release with IC50 of 0.008 and 0.02 µM, respectively, being ≥ 12-fold more potent than 6BIO. The effects of 6BIGOE are mediated via intracellular inhibition of GSK-3, where 6BIGOE again surpassed the effectiveness of 6BIO despite the higher potency of the latter in cell-free GSK-3 activity assays. Side-by-side comparison of 6BIGOE (0.1 µM) with the selective GSK-3 inhibitor SB216763 (5 µM) revealed congruent properties such as enrichment of ß-catenin and suppression of cyclooxygenase (COX)-2 protein levels due to GSK-3 inhibition. Metabololipidomics using ultra-performance liquid chromatography-tandem mass spectrometry showed that 6BIGOE selectively decreases pro-inflammatory COX-derived product formation without marked modulation of other lipid mediators. In summary, 6BIGOE is a highly potent indirubin derivative in the cellular context that favorably modulates pro- and anti-inflammatory cytokines as well as COX-2-derived PG via interference with GSK-3.

Mannosylation of LNP Results in Improved Potency for Self-Amplifying RNA (SAM) Vaccines.

Mannosylation of Lipid Nanoparticles (LNP) can potentially enhance uptake by Antigen Presenting Cells, which are highly abundant in dermal tissues, to improve the potency of Self Amplifying mRNA (SAM) vaccines in comparison to the established unmodified LNP delivery system. In the current studies, we evaluated mannosylated LNP (MLNP), which were obtained by incorporation of a stable Mannose-cholesterol amine conjugate, for the delivery of an influenza (hemagglutinin) encoded SAM vaccine in mice, by both intramuscular and intradermal routes of administration. SAM MLNP exhibited in vitro enhanced uptake in comparison to unglycosylated LNP from bone marrow-derived dendritic cells, and in vivo more rapid onset of the antibody response, independent of the route. The increased binding antibody levels also translated into higher functional hemagglutinin inhibition titers, particularly following intradermal administration. T cell assay on splenocytes from immunized mice also showed an increase in antigen specific CD8+ T responses, following intradermal administration of MLNP SAM vaccines. Induction of enhanced antigen specific CD4+ T cells, correlating with higher IgG2a antibody responses, was also observed. Hence, the present work illustrates the benefit of mannosylation of LNPs to achieve a faster immune response with SAM vaccines and these observations could contribute to the development of novel skin delivery systems for SAM vaccines.

Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males.

Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO-activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor-induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.

Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase.

Pharmacological interference with vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, is considered a potential strategy for cancer therapy. Macrophages are critically involved in tumor progression and may occur as pro-tumoral M2 phenotype, whereas classically-activated M1 can inhibit tumor development for example by releasing tumor-suppressing molecules, including tumor necrosis factor (TNF)α. Here, we show that targeting V-ATPase by selective inhibitors such as archazolid upregulates the expression and secretion of TNFα in lipopolysaccharide (LPS)- or LPS/interferon (INF)γ-activated M1-like macrophages derived from human blood monocytes. In contrast, archazolid failed to elevate TNFα production from uncommitted (M0) or interleukin (IL)-4-treated M2-like macrophages. Secretion of other relevant cytokines (i.e., IL-1ß, IL-6, IL-10) or chemokines (i.e. IL-8 and monocyte chemotactic protein-1) from M1 was not affected by archazolid. Though V-ATPase inhibitors elevated the lysosomal pH in M1 comparable to chloroquine or ammonium chloride, the latter agents suppressed TNFα secretion. Archazolid selectively increased TNFα mRNA levels, which was abolished by dexamethasone. Interestingly, archazolid enhanced the phosphorylation and nuclear translocation of the p65 subunit of NFκB and stimulated phosphorylation of SAPK/JNK. In a microfluidically-supported human tumor biochip model, archazolid-treated M1 significantly reduced tumor cell viability. Together, our data show that V-ATPase inhibition selectively upregulates TNFα production in classically-activated macrophages along with NFκB and SAPK/JNK activation. Such increased TNFα release caused by V-ATPase inhibitors may contribute to tumor suppression in addition to direct targeting cancer cells.

Evaluation of Dual 5-Lipoxygenase/Microsomal Prostaglandin E2 Synthase-1 Inhibitory Effect of Natural and Synthetic Acronychia-Type Isoprenylated Acetophenones.

Among the pathways responsible for the development of inflammatory responses, the cyclooxygenase and lipoxygenase pathways are among the most important ones. Two key enzymes, namely, 5-LO and mPGES-1, are involved in the biosynthesis of leukotrienes and prostaglandins, respectively, which are considered attractive therapeutic targets, so their dual inhibition might be an effective strategy to control inflammatory deregulation. Several natural products have been identified as 5-LO inhibitors, with some also being dual 5-LO/mPGES-1 inhibitors. Here, some prenylated acetophenone dimers from Acronychia pedunculata have been identified for their dual inhibitory potency toward 5-LO and mPGES-1. To gain insight into the SAR of this family of natural products, the synthesis and biological evaluation of analogues are presented. The results show the ability of the natural and synthetic molecules to potently inhibit 5-LO and mPEGS-1 in vitro. The potency of the most active compound (10) has been evaluated in vivo in an acute inflammatory mouse model and displayed potent anti-inflammatory activity comparable in potency to the drug zileuton used as a positive control.

Modulation of actin dynamics as potential macrophage subtype-targeting anti-tumour strategy.

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells.

Multiplex immunoassay for in vitro characterization of acellular pertussis antigens in combination vaccines.

Vaccines characterization is required to ensure physical, chemical, and biological integrity of antigens and adjuvants. Current analytical methods mostly require complete antigen desorption from aluminum-based adjuvants and are not always suitable to distinguish individual antigens in multivalent formulations. Here, Luminex technology is proposed to improve the analytics of vaccine characterization. As proof of concept, TdaP (tetanus, diphtheria and acellular pertussis) combination, adjuvanted with aluminum hydroxide, was chosen as model formulation to quantify and determine the level of adsorption of acellular pertussis (aP) antigens onto adjuvant surface at the same time. The assay used specific antibodies bound to magnetic microspheres presenting unique digital signatures for each pertussis antigen, allowing the simultaneous recognition of respective antigens in the whole vaccine, avoiding laborious procedures for adjuvant separation. Accurate and reproducible quantification of aP antigens in TdaP vaccine has been achieved in the range 0.78-50 ng/mL, providing simultaneously information on antigen identity, quantity, and degree of adsorption to aluminum hydroxide. The current study could further be considered as a model to set up in vitro potency assays thus supporting the replacement of animal tests accordingly to the 3Rs concept.

2,3-Dihydrobenzofuran privileged structures as new bioinspired lead compounds for the design of mPGES-1 inhibitors.

2,3-Dihydrobenzofurans are proposed as privileged structures and used as chemical platform to design small compound libraries. By combining molecular docking calculations and experimental verification of biochemical interference, we selected some potential inhibitors of microsomal prostaglandin E2 synthase (mPGES)-1. Starting from low affinity natural product 1, by our combined approach we identified the compounds 19 and 20 with biological activity in the low micromolar range. Our data suggest that the 2,3-dihydrobenzofuran derivatives might be suitable bioinspired lead compounds for development of new generation mPGES-1 inhibitors with increased affinity.

Can Small Chemical Modifications of Natural Pan-inhibitors Modulate the Biological Selectivity? The Case of Curcumin Prenylated Derivatives Acting as HDAC or mPGES-1 Inhibitors.

Curcumin, or diferuloylmethane, a polyphenolic molecule isolated from the rhizome of Curcuma longa, is reported to modulate multiple molecular targets involved in cancer and inflammatory processes. On the basis of its pan-inhibitory characteristics, here we show that simple chemical modifications of the curcumin scaffold can regulate its biological selectivity. In particular, the curcumin scaffold was modified with three types of substituents at positions C-1, C-8, and/or C-8' [C5 (isopentenyl, 5-8), C10 (geranyl, 9-12), and C15 (farnesyl, 13, 14)] in order to make these molecules more selective than the parent compound toward two specific targets: histone deacetylase (HDAC) and microsomal prostaglandin E2 synthase-1 (mPGES-1). From combined in silico and in vitro analyses, three selective inhibitors by proper substitution at position 8 were revealed. Compound 13 has improved HDAC inhibitory activity and selectivity with respect to the parent compound, while 5 and 9 block the mPGES-1 enzyme. We hypothesize about the covalent interaction of curcumin, 5, and 9 with the mPGES-1 binding site.

Bronchipret® syrup containing thyme and ivy extracts suppresses bronchoalveolar inflammation and goblet cell hyperplasia in experimental bronchoalveolitis.

BACKGROUND/PURPOSE: Acute bronchitis (AB) is a common lung condition characterized by inflammation of the large bronchi in response to infection. Bronchipret(®) syrup (BRO), a fixed combination of thyme and ivy extracts has been effectively used for the treatment of AB. Combining in vivo and mechanistic in vitro studies we aimed to provide a better understanding of the therapeutic potential of BRO on key aspects of AB and to identify potential mechanisms of action. METHODS: Bronchoalveolitis in rats was induced by intratracheal LPS instillation. BRO was administered p.o. once daily at 1- to 10-fold equivalents of the human daily dose. Animals were sacrificed 24-72 h post LPS challenge to analyze leukocyte numbers in lung tissue, bronchoalveolar lavage fluid (BALF) and blood as well as goblet cells in bronchial epithelium. Inhibitory effects of BRO analogue on leukotriene (LT) production were determined in human neutrophils and monocytes as well as on isolated 5-lipoxygenase (5-LO). RESULTS: BRO significantly reversed the LPS-induced increase in leukocyte numbers in lung tissue, BALF and blood as well as goblet cell numbers in bronchial epithelium. In vitro, BRO analogue suppressed cellular release of LTB4 (IC50 = 36 µg⋅ml(-1)) and cysLT (IC50 = 10 µg⋅ml(-1)) and inhibited the activity of isolated 5-LO (IC50 = 19 µg⋅ml(-1)). CONCLUSION: BRO exerts significant anti-inflammatory effects and attenuates goblet cell metaplasia in LPS-induced bronchoalveolitis in vivo potentially via interference with 5-LO/LT signaling. These effects may contribute to its observed clinical efficacy in AB.

Role of p38 mitogen-activated protein kinase in linking stearoyl-CoA desaturase-1 activity with endoplasmic reticulum homeostasis.

Endoplasmic reticulum (ER) homeostasis is regulated by a network of signaling pathways to which stearoyl-CoA desaturase (SCD)-1, p38 mitogen-activated protein kinase (MAPK) and the unfolded protein response (UPR) belong. Because all these pathways are located at the interface of cell cycle control and cell stress, we hypothesized a cross-regulation. Interference with SCD-1, either by small interfering (si)RNA or the specific SCD-1 inhibitor CAY10566 (EC50 1 µM; ≥ 24 h), specifically induced phosphorylation and thus activation of p38 MAPK in NIH-3T3 mouse fibroblasts (1.5- to 2-fold; 48 hours). During lipotoxic and cell cycle stress, prolonged activation of p38 MAPK due to SCD-1 inhibition induced ER stress, the UPR, and ER/Golgi remodeling as shown by Western blot and immunofluorescence microscopy (1.2- to 3.5-fold). Specific inhibition of p38 MAPK by Skepinone-L [half maximal inhibitory concentration (IC50) 25-50 nM] reversed these effects (at 1 µM; 48 hours). The specificity by which SCD-1 modulates the phospholipid composition and inhibits p38 MAPK signaling (among survival/stress pathways), thereby preventing ER stress (but not other SCD-1-dependent responses), suggests selective protein-lipid interactions. Palmitoleoyl/oleoyl-phosphatidylinositol (PI) was accordingly identified as potential lipid mediator using chromatography-coupled ESI tandem mass spectrometry. We conclude that the negative regulation of p38 MAPK mediates the protective effects of SCD-1 on ER homeostasis under distinct stress conditions.

Progesterone rapidly down-regulates the biosynthesis of 5-lipoxygenase products in human primary monocytes.

5-Lipoxygenase (5-LO), the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) from arachidonic acid, is regulated by androgens in human neutrophils and monocytes accounting for sex differences in LT formation. Here we show that progesterone suppresses the synthesis of 5-LO metabolites in human primary monocytes. 5-LO product formation in monocytes stimulated with Ca(2+)-ionophore A23187 or with lipopolysaccharide/formyl peptide was suppressed by progesterone at concentrations of 10-100 nM in cells from females and at 1 µM in cells from males. Progesterone down-regulated 5-LO product formation in a rapid and reversible manner, but did not significantly inhibit 5-LO activity in cell-free assays using monocyte homogenates. Also, arachidonic acid release and its metabolism to other eicosanoids in monocytes were not significantly reduced by progesterone. The inhibitory effect of progesterone on LTs was still observed when mitogen-activated protein kinases were pharmacologically blocked, stimulatory 1-oleoyl-2-acetyl-sn-glycerol was exogenously supplied, or extracellular Ca(2+) was removed by chelation. Instead, suppression of PKA by means of two different pharmacological approaches (i.e. H89 and a cell-permeable PKA inhibitor peptide) prevented inhibition of 5-LO product generation by progesterone, to a similar extent as observed for the PKA activators prostaglandin E2 and 8-Br-cAMP, suggesting the involvement of PKA. In summary, progesterone affects the capacity of human primary monocytes to generate 5-LO products and, in addition to androgens, may account for sex-specific effects on pro-inflammatory LTs.

Targeting V-ATPase in primary human monocytes by archazolid potently represses the classical secretion of cytokines due to accumulation at the endoplasmic reticulum.

The macrolide archazolid inhibits vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, and potently suppresses cancer cell growth at low nanomolar concentrations. In view of the growing link between inflammation and cancer, we investigated whether inhibition of V-ATPase by archazolid may affect primary human monocytes that can promote cancer by sustaining inflammation through the release of tumor-promoting cytokines. Human primary monocytes express V-ATPase, and archazolid (10-100nM) increases the vesicular pH in these cells. Archazolid (10nM) markedly reduced the release of pro-inflammatory (TNF-α, interleukin-6 and -8) but also of anti-inflammatory (interleukin-10) cytokines in monocytes stimulated with LPS, without affecting cell viability up to 1000nM. Of interest, secretion of interleukin-1ß was increased by archazolid. Comparable effects were obtained by the V-ATPase inhibitors bafilomycin and apicularen. The phosphorylation of p38 MAPK and ERK-1/2, Akt, SAPK/JNK or of the inhibitor of NFκB (IκBα) as well as mRNA expression of IL-8 were not altered by archazolid in LPS-stimulated monocytes. Instead, archazolid caused endoplasmic reticulum (ER) stress response visualized by increased BiP expression and accumulation of IL-8 (and TNF-α) at the ER, indicating a perturbation of protein secretion. In conclusion, by interference with V-ATPase, archazolid significantly affects the secretion of cytokines due to accumulation at the ER which might be of relevance when using these agents for cancer therapy.

In vivo sex differences in leukotriene biosynthesis in zymosan-induced peritonitis.

Leukotrienes (LTs) are 5-lipoxygenase (5-LO) metabolites which are implicated in sex-dependent inflammatory diseases (asthma, autoimmune diseases, etc.). We have recently reported sex differences in LT biosynthesis in in vitro models such as human whole blood, neutrophils and monocytes, due to down-regulation of 5-LO product formation by androgens. Here we present evidences for sex differences in LT synthesis and related inflammatory reactions in an in vivo model of inflammation (mouse zymosan-induced peritonitis). On the cellular level, differential 5-LO subcellular compartmentalization in peritoneal macrophages (PM) from male and female mice might be the basis for these differences. Sex differences in vascular permeability and neutrophil recruitment (cell number and myeloperoxidase activity) into peritoneal cavity were evident upon intraperitoneal zymosan injection, with more prominent responses in female mice. This was accompanied by higher levels of LTC4 and LTB4 in peritoneal exudates of female compared to male mice. Interestingly, LT peritoneal levels in orchidectomized mice were higher than in sham male mice. In accordance with the in vivo results, LT formation in stimulated PM from female mice was higher than in male PM, accompanied by alterations in 5-LO subcellular localization. The increased formation of LTC4 in incubations of PM from orchidectomized mice confirms a role of sex hormones. In conclusion, sex differences observed in LT biosynthesis during peritonitis in vivo may be related, at least in part, to a variant 5-LO localization in PM from male and female mice.