A new class of recombinant human albumin with multiple surface thiols exhibits stable conjugation and enhanced FcRn binding and blood circulation.

Human serum albumin is an endogenous ligand transport protein whose long circulatory half-life is facilitated by engagement with the human cellular recycling neonatal Fc receptor (hFcRn). The single free thiol located at Cys-34 in domain I of albumin has been exploited for monoconjugation of drugs. In this work, we increased the drug-to-albumin ratio potential by engineering recombinant human albumin (rHSA) variants with varying hFcRn affinity to contain three free, conjugation-competent cysteines. Structural analysis was used to identify positions for cysteine introduction to maximize rHSA stability and formation of the conjugated product without affecting hFcRn binding. The thiol rHSA variants exhibited up to 95% monomeric stability over 24 months and retained hFcRn engagement compared with a WT unconjugated control demonstrated by Biolayer Interferometry. The additional cysteines were further introduced into a panel of rHSA variants engineered with different affinities for hFcRn. After conjugation with three Alexa Fluor 680 (AF680) fluorophores, hFcRn binding was similar to that of the original triple-thiol nonconjugated rHSA variants (0.88 and 0.25 µm for WT albumin with or without 3xAF680 respectively, and 0.04 and 0.02 µm for a high hFcRn-binding variant with or without 3xAF680, respectively). We also observed a 1.3-fold increase in the blood circulatory half-life of a high hFcRn-binding triple-thiol variant conjugated with AF680 (t½ = 22.4 h) compared with its WT counterpart (t½ = 17.3 h) in mice. Potential high drug-to-albumin ratios combined with high hFcRn engagement are attractive features of this new class of albumins that offer a paradigm shift for albumin-based drug delivery.

Lipidoid-siRNA Nanoparticle-Mediated IL-1ß Gene Silencing for Systemic Arthritis Therapy in a Mouse Model.

Interleukin-1 beta (IL-1ß) plays a central role in the induction of rheumatoid arthritis (RA). In the present study, we demonstrated that lipidoid-polymer hybrid nanoparticle (FS14-NP) can efficiently deliver siRNA against IL-1ß (siIL-1ß) to macrophages and effectively suppress the pathogenesis of experimental arthritis induced by collagen antibody (CAIA mice). FS14-NP/siIL-1ß achieved approximately 70% and 90% gene-silencing efficiency in the RAW 264.7 cell line and intraperitoneal macrophages, respectively. Intravenous administration of FS14-NP/siRNA led to rapid accumulation of siRNA in macrophages within the arthritic joints. Furthermore, FS14-NP/siIL-1ß treatment lowered the expression of pro-inflammatory cytokines in arthritic joints and dramatically attenuated ankle swelling, bone erosion, and cartilage destruction. These results demonstrate that FS14-NP/siIL-1ß may represent an effective therapy for systemic arthritis and other inflammatory disorders.

Molecular, Macromolecular, and Supramolecular Glucuronide Prodrugs: Lead Identified for Anticancer Prodrug Monotherapy.

In this work, a tumor growth intervention by localized drug synthesis within the tumor volume, using the enzymatic repertoire of the tumor itself, is presented. Towards the overall success, molecular, macromolecular, and supramolecular glucuronide prodrugs were designed for a highly potent toxin, monomethyl auristatin E (MMAE). The lead candidate exhibited a fold difference in toxicity between the prodrug and the drug of 175, had an engineered mechanism to enhance the deliverable payload to tumours, and contained a highly potent toxin such that bioconversion of only a few prodrug molecules created a concentration of MMAE sufficient enough for efficient suppression of tumor growth. Each of these points is highly significant and together afford a safe, selective anticancer measure, making tumor-targeted glucuronides attractive for translational medicine.

cAIMP administration in humanized mice induces a chimerization-level-dependent STING response.

It is well understood that the STING signalling pathway is critical for generating a robust innate immune response to pathogens. Human and mouse STING signalling pathways are not identical, however. For example, mice lack IFI16, which has been proven important for the human STING pathway. Therefore, we investigated whether humanized mice are an appropriate experimental platform for exploring the human STING signalling cascade in vivo. We found that NOG mice reconstituted with human cord blood haematopoietic stem cells (humanized NOG mice) exhibit human STING signalling responses to an analogue of the cyclic di-nucleotide cGAMP. There was an increase in the proportions of monocytes in the lungs of mice receiving cGAMP analogue. The most robust levels of STING expression and STING-induced responses were observed in mice exhibiting the highest levels of human chimerization. Notably, differential levels of STING in lung versus spleen following cGAMP analogue treatment suggest that there are tissue-specific kinetics of STING activation and/or degradation in effector versus inductive sites. We also examined the mouse innate immune response to cGAMP analogue treatment. We detected that mouse cells in the immunodeficient NOG mice responded to the cGAMP analogue and they do so with distinct kinetics from the human response. In conclusion, humanized NOG mice represent a valuable experimental model for examining in vivo human STING responses.

Analysis of Factor D Isoforms in Malpuech-Michels-Mingarelli-Carnevale Patients Highlights the Role of MASP-3 as a Maturase in the Alternative Pathway of Complement.

Factor D (FD), which is also known as adipsin, is regarded as the first-acting protease of the alternative pathway (AP) of complement. It has been suggested that FD is secreted as a mature enzyme that does not require subsequent activation. This view was challenged when it was shown that mice lacking mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1) and MASP-3 contain zymogenic FD (pro-FD), and it is becoming evident that MASP-3 is implicated in pro-FD maturation. However, the necessity of MASP-3 for pro-FD cleavage has been questioned, because AP activity is still observed in sera from MASP-1/3-deficient Malpuech-Michels-Mingarelli-Carnevale (3MC) patients. The identification of a novel 3MC patient carrying a previously unidentified MASP-3 G665S mutation prompted us to develop an analytical isoelectric focusing technique that resolves endogenous FD variants in complex samples. This enabled us to show that although 3MC patients predominantly contain pro-FD, they also contain detectable levels of mature FD. Moreover, using isoelectric focusing analysis, we show that both pro-FD and FD are present in the circulation of healthy donors. We characterized the naturally occurring 3MC-associated MASP-3 mutants and found that they all yielded enzymatically inactive proteins. Using MASP-3-depleted human serum, serum from 3MC patients, and Masp1/3-/- mice, we found that lack of enzymatically active MASP-3, or complete MASP-3 deficiency, compromises the conversion of pro-FD to FD. In summary, our observations emphasize that MASP-3 acts as an important maturase in the AP of complement, while also highlighting that there exists MASP-3-independent pro-FD maturation in 3MC patients.

Endogenous Natural Complement Inhibitor Regulates Cardiac Development.

Congenital heart defects are a major cause of perinatal mortality and morbidity, affecting >1% of all live births in the Western world, yet a large fraction of such defects have an unknown etiology. Recent studies demonstrated surprising dual roles for immune-related molecules and their effector mechanisms during fetal development and adult homeostasis. In this article, we describe the function of an endogenous complement inhibitor, mannan-binding lectin (MBL)-associated protein (MAp)44, in regulating the composition of a serine protease-pattern recognition receptor complex, MBL-associated serine protease (MASP)-3/collectin-L1/K1 hetero-oligomer, which impacts cardiac neural crest cell migration. We used knockdown and rescue strategies in zebrafish, a model allowing visualization and assessment of heart function, even in the presence of severe functional defects. Knockdown of embryonic expression of MAp44 caused impaired cardiogenesis, lowered heart rate, and decreased cardiac output. These defects were associated with aberrant neural crest cell behavior. We found that MAp44 competed with MASP-3 for pattern recognition molecule interaction, and knockdown of endogenous MAp44 expression could be rescued by overexpression of wild-type MAp44. Our observations provide evidence that immune molecules are centrally involved in the orchestration of cardiac tissue development.

CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma.

BACKGROUND & AIMS: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development. METHODS: We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8-week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1-Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. RESULTS: Livers from 12 of the 15 mice given the vectors to induce the Dnajb1-Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1-Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1-Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC. CONCLUSIONS: Using CRISPR/Cas9 technology, we found generation of the Dnajb1-Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1-PRKACA might be developed as therapeutics for this form of liver cancer.

Fatty Acid-Modified Gapmer Antisense Oligonucleotide and Serum Albumin Constructs for Pharmacokinetic Modulation.

Delivery technologies are required for realizing the clinical potential of molecular medicines. This work presents an alternative technology to preformulated delivery systems by harnessing the natural transport properties of serum albumin using endogenous binding of gapmer antisense oligonucleotides (ASOs)/albumin constructs. We show by an electrophoretic mobility assay that fatty acid-modified gapmer and human serum albumin (HSA) can self-assemble into constructs that offer favorable pharmacokinetics. The interaction was dependent on fatty acid type (either palmitic or myristic acid), number, and position within the gapmer ASO sequence, as well as phosphorothioate (PS) backbone modifications. Binding correlated with increased blood circulation in mice (t1/2 increased from 23 to 49 min for phosphodiester [PO] gapmer ASOs and from 28 to 66 min for PS gapmer ASOs with 2× palmitic acid modification). Furthermore, a shift toward a broader biodistribution was detected for PS compared with PO gapmer ASOs. Inclusion of 2× palmitoyl to the ASOs shifted the biodistribution to resemble that of natural albumin. This work, therefore, presents a novel strategy based on the proposed endogenous assembly of gapmer ASOs/albumin constructs for increased circulatory half-life and modulation of the biodistribution of gapmer ASOs that offers tunable pharmacokinetics based on the gapmer modification design.

Lack of immunological DNA sensing in hepatocytes facilitates hepatitis B virus infection.

UNLABELLED: Hepatitis B virus (HBV) is a major human pathogen, and about one third of the global population will be exposed to the virus in their lifetime. HBV infects hepatocytes, where it replicates its DNA and infection can lead to acute and chronic hepatitis with a high risk of liver cirrhosis and hepatocellular carcinoma. Despite this, there is limited understanding of how HBV establishes chronic infections. In recent years it has emerged that foreign DNA potently stimulates the innate immune response, particularly type 1 interferon (IFN) production; and this occurs through a pathway dependent on the DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase and the downstream adaptor protein stimulator of IFN genes (STING). In this work we describe that human and murine hepatocytes do not express STING. Consequently, hepatocytes do not produce type 1 IFN in response to foreign DNA or HBV infection and mice lacking STING or cyclic guanosine monophosphate-adenosine monophosphate synthase exhibit unaltered ability to control infection in an adenovirus-HBV model. Stimulation of IFN production in the murine liver by administration of synthetic RNA decreases virus infection, thus demonstrating that IFN possesses anti-HBV activity in the liver. Importantly, introduction of STING expression specifically in hepatocytes reconstitutes the DNA sensing pathway, which leads to improved control of HBV in vivo. CONCLUSION: The lack of a functional innate DNA-sensing pathway in hepatocytes hampers efficient innate control of HBV infection; this may explain why HBV has adapted to specifically replicate in hepatocytes and could contribute to the weak capacity of this cell type to clear HBV infection. (Hepatology 2016;64:746-759).

Cholinergic PET imaging in infections and inflammation using 11C-donepezil and 18F-FEOBV.

INTRODUCTION: Immune cells utilize acetylcholine as a paracrine-signaling molecule. Many white blood cells express components of the cholinergic signaling pathway, and these are up-regulated when immune cells are activated. However, in vivo molecular imaging of cholinergic signaling in the context of inflammation has not previously been investigated. METHODS: We performed positron emission tomography (PET) using the glucose analogue 18F-FDG, and 11C-donepezil and 18F-FEOBV, markers of acetylcholinesterase and the vesicular acetylcholine transporter, respectively. Mice were inoculated subcutaneously with Staphylococcus aureus, and PET scanned at 24, 72, 120, and 144 h post-inoculation. Four pigs with post-operative abscesses were also imaged. Finally, we present initial data from human patients with infections, inflammation, and renal and lung cancer. RESULTS: In mice, the FDG uptake in abscesses peaked at 24 h and remained stable. The 11C-donepezil and 18F-FEOBV uptake displayed progressive increase, and at 120-144 h was nearly at the FDG level. Moderate 11C-donepezil and slightly lower 18F-FEOBV uptake were seen in pig abscesses. PCR analyses suggested that the 11C-donepezil signal in inflammatory cells is derived from both acetylcholinesterase and sigma-1 receptors. In humans, very high 11C-donepezil uptake was seen in a lobar pneumonia and in peri-tumoral inflammation surrounding a non-small cell lung carcinoma, markedly superseding the 18F-FDG uptake in the inflammation. In a renal clear cell carcinoma no 11C-donepezil uptake was seen. DISCUSSION: The time course of cholinergic tracer accumulation in murine abscesses was considerably different from 18F-FDG, demonstrating in the 11C-donepezil and 18F-FEOBV image distinct aspects of immune modulation. Preliminary data in humans strongly suggest that 11C-donepezil can exhibit more intense accumulation than 18F-FDG at sites of chronic inflammation. Cholinergic PET imaging may therefore have potential applications for basic research into cholinergic mechanisms of immune modulation, but also clinical applications for diagnosing infections, inflammatory disorders, and cancer inflammation.

Pancreas specific expression of oncogenes in a porcine model.

Pancreatic cancer is the fourth leading course of cancer death and early detection of the disease is crucial for successful treatment. However, pancreatic cancer is difficult to detect in its earliest stages and once symptoms appear, the cancer has often progressed beyond possibility for curing. Research into the disease has been hampered by the lack of good models. We have generated a porcine model of pancreatic cancer with use of transgenic overexpression of an oncogene cassette containing MYC, KRAS G12D and SV40 LT. The expression was initiated from a modified Pdx-1 promoter during embryogenesis in a subset of pancreatic epithelial cells. Furthermore, cells expressing the oncogenes also expressed a yellow fluorescent protein (mVenus) and an inducible negative regulator protein (rtTR-KRAB). Cells where the Pdx-1 promoter had not been activated, expressed a red fluorescent protein (Katushka). In vitro analyses of cells obtained from the transgenic pigs showed increased proliferation and expression of the transgenes when activated. Induction of the repressor protein eliminated the oncogene expression and decreased cell proliferation. In vivo analysis identified foci of pancreatic cells expressing the oncogenes at day zero post farrowing. These populations expanded and formed hyperplastic foci, with beginning abnormality at day 45. Cells in the foci expressed the oncogenic proteins and the majority of the cells were positive for the proliferation marker, Ki67. We predict that this model could be used for advanced studies in pancreatic cancer in a large animal model with focus on early detection, treatment, and identification of new biomarkers.

Cell therapy centered on IL-1Ra is neuroprotective in experimental stroke.

Cell-based therapies are emerging as new promising treatments in stroke. However, their functional mechanism and therapeutic potential during early infarct maturation has so far received little attention. Here, we asked if cell-based delivery of the interleukin-1 receptor antagonist (IL-1Ra), a known neuroprotectant in stroke, can promote neuroprotection, by modulating the detrimental inflammatory response in the tissue at risk. We show by the use of IL-1Ra-overexpressing and IL-1Ra-deficient mice that IL-1Ra is neuroprotective in stroke. Characterization of the cellular and spatiotemporal production of IL-1Ra and IL-1α/ß identifies microglia, not infiltrating leukocytes, as the major sources of IL-1Ra after experimental stroke, and shows IL-1Ra and IL-1ß to be produced by segregated subsets of microglia with a small proportion of these cells co-expressing IL-1α. Reconstitution of whole body irradiated mice with IL-1Ra-producing bone marrow cells is associated with neuroprotection and recruitment of IL-1Ra-producing leukocytes after stroke. Neuroprotection is also achieved by therapeutic injection of IL-1Ra-producing bone marrow cells 30 min after stroke onset, additionally improving the functional outcome in two different stroke models. The IL-1Ra-producing bone marrow cells increase the number of IL-1Ra-producing microglia, reduce the availability of IL-1ß, and modulate mitogen-activated protein kinase (MAPK) signaling in the ischemic cortex. The importance of these results is underlined by demonstration of IL-1Ra-producing cells in the human cortex early after ischemic stroke. Taken together, our results attribute distinct neuroprotective or neurotoxic functions to segregated subsets of microglia and suggest that treatment strategies increasing the production of IL-1Ra by infiltrating leukocytes or microglia may also be neuroprotective if applied early after stroke onset in patients.

Induction of atherosclerosis in mice and hamsters without germline genetic engineering.

RATIONALE: Atherosclerosis can be achieved in animals by germline genetic engineering, leading to hypercholesterolemia, but such models are constrained to few species and strains, and they are difficult to combine with other powerful techniques involving genetic manipulation or variation. OBJECTIVE: To develop a method for induction of atherosclerosis without germline genetic engineering. METHODS AND RESULTS: Recombinant adeno-associated viral vectors were engineered to encode gain-of-function proprotein convertase subtilisin/kexin type 9 mutants, and mice were given a single intravenous vector injection followed by high-fat diet feeding. Plasma proprotein convertase subtilisin/kexin type 9 and total cholesterol increased rapidly and were maintained at high levels, and after 12 weeks, mice had atherosclerotic lesions in the aorta. Histology of the aortic root showed progression of lesions to the fibroatheromatous stage. To demonstrate the applicability of this method for rapid analysis of the atherosclerosis susceptibility of a mouse strain and for providing temporal control over disease induction, we demonstrated the accelerated atherosclerosis of mature diabetic Akita mice. Furthermore, the versatility of this approach for creating atherosclerosis models also in nonmurine species was demonstrated by inducing hypercholesterolemia and early atherosclerosis in Golden Syrian hamsters. CONCLUSIONS: Single injections of proprotein convertase subtilisin/kexin type 9-encoding recombinant adeno-associated viral vectors are a rapid and versatile method to induce atherosclerosis in animals. This method should prove useful for experiments that are high-throughput or involve genetic techniques, strains, or species that do not combine well with current genetically engineered models.

Macrophage-mediated nanoparticle delivery to the periodontal lesions in established murine model via Pg-LPS induction.

We established a murine periodontitis model by local injection of lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS) into the gingival sulcus of mandibular left incisor four times with 48-h interval. The histological examination of the periodontal tissues demonstrated that significant loss of periodontal bone and ligaments was observed in the lesion side with abundant inflammatory cell infiltration. Two days after the last injection, Cy5-labelled siRNA/chitosan particles were injected intraperitoneally (ip). The chitosan/siRNA particles were taken up by peritoneal macrophages, which subsequently migrated to the inflamed gingival area evaluated by in vivo imaging. The localization of macrophages in the inflamed region was further confirmed by immunofluorescent staining. The present report demonstrates that intragingival injection of Pg-LPS can be used to create an experimental model of periodontal inflammation in mice and that recruitment of macrophages with chitosan/siRNA nanoparticles to the inflamed area opens the possibility of an RNAi-based therapeutic approach using chitosan as a carrier in periodontitis.

Mitochondria and the lectin pathway of complement.

Mitochondria, the powerhouses of our cells, are remnants of a eubacterial endosymbiont. Notwithstanding the evolutionary time that has passed since the initial endosymbiotic event, mitochondria have retained many hallmarks of their eubacterial origin. Recent studies have indicated that during perturbations of normal homeostasis, such as following acute trauma leading to massive necrosis and release of mitochondria, the immune system might mistake symbiont for enemy and initiate an inappropriate immune response. The innate immune system is the first line of defense against invading microbial pathogens, and as such is the primary suspect in the recognition of mitochondria-derived danger-associated molecular patterns and initiation of an aberrant response. Conversely, innate immune mechanisms are also central to noninflammatory clearance of innocuous agents. Here we investigated the role of a central humoral component of innate immunity, the lectin pathway of complement, in recognition of mitochondria in vitro and in vivo. We found that the soluble pattern recognition molecules, mannan-binding lectin (MBL), L-ficolin, and M-ficolin, were able to recognize mitochondria. Furthermore, MBL in complex with MBL-associated serine protease 2 (MASP-2) was able to activate the lectin pathway and deposit C4 onto mitochondria, suggesting that these molecules are involved either in homeostatic clearance of mitochondria or in induction of untoward inflammatory reactions. We found that following mitochondrial challenge, C3 was consumed in vivo in the absence of overt inflammation, indicating a potential role of complement in noninflammatory clearance of mitochondria. Thus, we report here the first indication of involvement of the lectin pathway in mitochondrial immune handling.

Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT).

Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange (RMCE) in cells from a Göttingen minipig with four RMCE acceptor loci, each containing a green fluorescence protein (GFP) marker gene driven by a human UbiC promoter. The four RMCE acceptor loci segregated independent of each other, and expression profiles could be determined in various tissues. Using minicircles in RMCE in fibroblasts with all four acceptor loci and followed by SCNT, we produced piglets with a single copy of a transgene incorporated into one of the transcriptionally active acceptor loci. The transgene, consisting of a cDNA of the Alzheimer's disease-causing gene PSEN1M146I driven by an enhanced human UbiC promoter, had an expression profile in various tissues similar to that of the GFP marker gene. The results show that RMCE can be done in a pre-selected transcriptionally active acceptor locus for targeted transgenesis in pigs.

Peritoneal macrophages mediated delivery of chitosan/siRNA nanoparticle to the lesion site in a murine radiation-induced fibrosis model.

BACKGROUND: Radiation-induced fibrosis (RIF) is a dose-limiting complication of cancer radiotherapy and causes serious problems, i.e. restricted tissue flexibility, pain, ulceration or necrosis. Recently, we have successfully treated RIF in a mouse model by intraperitoneal administration of chitosan/siRNA nanoparticles directed towards silencing TNF alpha in local macrophage populations, but the mechanism for the therapeutic effect at the lesion site remains unclear. METHODS: Using the same murine RIF model we utilized an optical imaging technique and fluorescence microscopy to investigate the uptake of chitosan/fluorescently labeled siRNA nanoparticles by peritoneal macrophages and their subsequent migration to the inflamed tissue in the RIF model. RESULTS: We observed strong accumulation of the fluorescent signal in the lesion site of the irradiated leg up to 24 hours using the optical imaging system. We further confirm by immunohistochemical staining that Cy3 labeled siRNA resides in macrophages of the irradiated leg. CONCLUSION: We provide a proof-of-concept for host macrophage trafficking towards the inflamed region in a murine RIF model, which thereby suggests that the chitosan/siRNA nanoparticle may constitute a general treatment for inflammatory diseases using the natural homing potential of macrophages to inflammatory sites.

Targeting the hemoglobin scavenger receptor CD163 in macrophages highly increases the anti-inflammatory potency of dexamethasone.

Synthetic glucocorticoids are potent anti-inflammatory drugs but serious side effects such as bone mobilization, muscle mass loss, immunosuppression, and metabolic alterations make glucocorticoid therapy a difficult balance. The therapeutic anti-inflammatory effect of glucocorticoids relies largely on the suppressed release of tumor-necrosis factor-α and other cytokines by macrophages at the sites of inflammation. We have now developed a new biodegradable anti-CD163 antibody-drug conjugate that specifically targets the glucocorticoid, dexamethasone to the hemoglobin scavenger receptor CD163 in macrophages. The conjugate, that in average contains four dexamethasone molecules per antibody, exhibits retained high functional affinity for CD163. In vitro studies in rat macrophages and in vivo studies of Lewis rats showed a strong anti-inflammatory effect of the conjugate measured as reduced lipopolysaccharide-induced secretion of tumor-necrosis factor-α. The in vivo potency of conjugated dexamethasone was about 50-fold that of nonconjugated dexamethasone. In contrast to a strong systemic effect of nonconjugated dexamethasone, the equipotent dose of the conjugate had no such effect, measured as thymus lymphocytes apoptosis, body weight loss, and suppression of endogenous cortisol levels. In conclusion, the study shows antibody-drug conjugates as a future approach in anti-inflammatory macrophage-directed therapy. Furthermore, the data demonstrate CD163 as an excellent macrophage target for anti-inflammatory drug delivery.

SKG arthritis as a model for evaluating therapies in rheumatoid arthritis with special focus on bone changes.

The aim was to further characterize the SKG model of rheumatoid arthritis (RA) and its potential for studying intervention treatments, with special focus on bone targeting therapies. Three individual studies were conducted, using a total of 71 SKG mice, comparing arthritis induction with mannan versus zymosan A, female versus male mice, and the effect of dexamethasone intervention treatment initiated at different time points after arthritis induction. Hind paws were embedded undecalcified in methyl methacrylate, and sections were stained with Masson-Goldner trichrome. Areal Bone Mineral Density (aBMD) of the femora was determined with pDXA. RNA was extracted from the hind paws followed by the quantification by reverse transcriptase PCR. SKG mice stimulated with mannan presented a higher arthritis score than mice stimulated with zymosan A. Female SKG mice developed a more severe arthritis than male SKG mice. Dexamethasone inhibited arthritis clinically as well as histologically when the treatment was initiated prophylactically or within the first week of arthritis. Femoral aBMD was lower in animals with arthritis than in control animals. The RANKL RNA expression was elevated in arthritic mice, whereas OPG RNA expression was unchanged. The results suggest mannan as arthritis inductor and female instead of male mice in experiments as well as an optimal time window for the initiation of treatment. Systemic bone loss as well as local up regulation of RANKL was present early in SKG arthritis. These results demonstrate that SKG arthritis is a suitable new model for evaluating therapies in RA.

Timing of Toll-like receptor 9 agonist administration in pneumococcal vaccination impacts both humoral and cellular immune responses as well as nasopharyngeal colonization in mice.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs, CpG ODN, are Toll-like receptor 9 agonists (TLR9a), which have been used as adjuvants in pneumococcal vaccines to improve antibody responses in immunodeficient patients. Here, we examined whether the coadministration of TLR9a with pneumococcal CRM(197)-conjugate vaccine enhances protection against pneumococcal colonization, the levels of antipolysaccharide antibodies, and the CD4(+) T-cell responses. Wild-type BALB/c mice and B-cell-deficient BALB/c Igh-J(tm1Dhu) mice were immunized twice with the following: (i) PCV alone; (ii) simultaneous PCV and TLR9a; (iii) PCV and then TLR9a, after a 48-h delay; (iv) TLR9a alone; and (v) phosphate-buffered saline. Nasopharyngeal protection, serum antibodies, CD4(+) T-cell responses, and clearance of bacteremia after intraperitoneal challenge with Streptococcus pneumoniae 6B were evaluated. We found decreased nasopharyngeal protection against S. pneumoniae 6B colonization after simultaneous immunization with PCV and TLR9a compared to immunization with PCV alone in wild-type BALB/c mice (P = 0.037). A similar trend was observed in B-cell-deficient BALB/c Igh-J(tm1Dhu) mice. Simultaneous administration did not enhance antibody levels and lowered the CRM(197)-specific cytokine release of gamma interferon, interleukin-2 (IL-2), IL-5 and IL-13. Immunization with PCV and then TLR9a, after a 48-h delay, significantly improved nasopharyngeal protection compared to simultaneous administration (P = 0.011). Furthermore, delaying TLR9a delivery increased antibody titers compared to both simultaneous administration (P = 0.001) and PCV immunization alone (P = 0.026). In conclusion, the immunological and clinical impact of adjuvanting a pneumococcal conjugate vaccine (Prevnar; Pfizer) with a TLR9a is highly depended on timing of the adjuvant administration. Thus, careful timing of adjuvant administration may improve novel vaccine formulations.