Novel nanoadjuvants balance immune activation with modest inflammation: implications for older adult vaccines.

BACKGROUND: Age-associated impairments of immune response and inflammaging likely contribute to poor vaccine efficacy. An appropriate balance between activation of immune memory and inflammatory response may be more effective in vaccines for older adults; attempts to overcome reduced efficacy have included the addition of adjuvants or increased antigenic dose. Next generation vaccine formulations may also use biomaterials to both deliver and adjuvant vaccine antigens. In the context of aging, it is important to determine the degree to which new biomaterials may enhance antigen-presenting cell (APC) functions without inducing potent inflammatory responses of APCs or other immune cell types (e.g., T cells). However, the effect of newer biomaterials on these cell types from young and older adults remains unknown. RESULTS: In this pilot study, cells from young and older adults were used to evaluate the effect of novel biomaterials such as polyanhydride nanoparticles (NP) and pentablock copolymer micelles (Mi) and cyclic dinucleotides (CDN; a STING agonist) on cytokine and chemokine secretion in comparison to standard immune activators such as lipopolysaccharide (LPS) and PMA/ionomycin. The NP treatment showed adjuvant-like activity with induction of inflammatory cytokines, growth factors, and select chemokines in peripheral blood mononuclear cells (PBMCs) of both young (n = 6) and older adults (n = 4), yet the degree of activation was generally less than LPS. Treatment with Mi or CDN resulted in minimal induction of cytokines and chemokine secretion with the exception of increased IFN-α and IL-12p70 by CDN. Age-related decreases were observed across multiple cytokines and chemokines, yet IFN-α, IL-12, and IL-7 production by NP or CDN stimulation was equal to or greater than in cells from younger adults. Consistent with these results in aged humans, a combination nanovaccine composed of NP, Mi, and CDN administered to aged mice resulted in a greater percentage of antigen-specific CD4+ T cells and greater effector memory cells in draining lymph nodes compared to an imiquimod-adjuvanted vaccine. CONCLUSIONS: Overall, our novel biomaterials demonstrated a modest induction of cytokine secretion with a minimal inflammatory profile. These findings suggest a unique role for biomaterial nanoadjuvants in the development of next generation vaccines for older adults.

"Just right" combinations of adjuvants with nanoscale carriers activate aged dendritic cells without overt inflammation.

BACKGROUND: The loss in age-related immunological markers, known as immunosenescence, is caused by a combination of factors, one of which is inflammaging. Inflammaging is associated with the continuous basal generation of proinflammatory cytokines. Studies have demonstrated that inflammaging reduces the effectiveness of vaccines. Strategies aimed at modifying baseline inflammation are being developed to improve vaccination responses in older adults. Dendritic cells have attracted attention as an age-specific target because of their significance in immunization as antigen presenting cells that stimulate T lymphocytes. RESULTS: In this study, bone marrow derived dendritic cells (BMDCs) were generated from aged mice and used to investigate the effects of combinations of adjuvants, including Toll-like receptor, NOD2, and STING agonists with polyanhydride nanoparticles and pentablock copolymer micelles under in vitro conditions. Cellular stimulation was characterized via expression of costimulatory molecules, T cell-activating cytokines, proinflammatory cytokines, and chemokines. Our results indicate that multiple TLR agonists substantially increase costimulatory molecule expression and cytokines associated with T cell activation and inflammation in culture. In contrast, NOD2 and STING agonists had only a moderate effect on BMDC activation, while nanoparticles and micelles had no effect by themselves. However, when nanoparticles and micelles were combined with a TLR9 agonist, a reduction in the production of proinflammatory cytokines was observed while maintaining increased production of T cell activating cytokines and enhancing cell surface marker expression. Additionally, combining nanoparticles and micelles with a STING agonist resulted in a synergistic impact on the upregulation of costimulatory molecules and an increase in cytokine secretion from BMDCs linked with T cell activation without excessive secretion of proinflammatory cytokines. CONCLUSIONS: These studies provide new insights into rational adjuvant selection for vaccines for older adults. Combining appropriate adjuvants with nanoparticles and micelles may lead to balanced immune activation characterized by low inflammation, setting the stage for designing next generation vaccines that can induce mucosal immunity in older adults.

Polymeric Nanoparticle-Based Vaccine Adjuvants and Delivery Vehicles.

As vaccine formulations have progressed from including live or attenuated strains of pathogenic components for enhanced safety, developing new adjuvants to more effectively generate adaptive immune responses has become necessary. In this context, polymeric nanoparticles have emerged as a promising platform with multiple advantages, including the dual capability of adjuvant and delivery vehicle, administration via multiple routes, induction of rapid and long-lived immunity, greater shelf-life at elevated temperatures, and enhanced patient compliance. This comprehensive review describes advances in nanoparticle-based vaccines (i.e., nanovaccines) with a particular focus on polymeric particles as adjuvants and delivery vehicles. Examples of the nanovaccine approach in respiratory infections, biodefense, and cancer are discussed.

Gut Organoid as a New Platform to Study Alginate and Chitosan Mediated PLGA Nanoparticles for Drug Delivery.

Intestinal organoids can be used as an ex vivo epithelial model to study different drug delivery effects on epithelial cells' luminal surface. In this study, the impact of surface charge on the delivery of 5-ASA loaded PLGA nanoparticles into the lumen of organoids was investigated. Alginate and chitosan were used to coat the nanoparticles and provide negative and positive charges on the particles, respectively. The organoid growth and viability were not affected by the presence of either alginate- or chitosan-coated nanoparticles. It was shown that nanoparticles could be transported from the serosal side of the organoids to the lumen as the dye gradually accumulated in the lumen by day 2-3 after adding the nanoparticles to the Matrigel. By day 5, the dye was eliminated from the lumen of the organoids. It was concluded that the positively charged nanoparticles were more readily transported across the epithelium into the lumen. It may be attributed to the affinity of epithelial cells to the positive charge. Thus, the organoid can be utilized as an appropriate model to mimic the functions of the intestinal epithelium and can be used as a model to evaluate the benefits of nanoparticle-based drug delivery.

Derivation of adult canine intestinal organoids for translational research in gastroenterology.

BACKGROUND: Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures. RESULTS: Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research. CONCLUSIONS: We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.

Intranasal delivery of influenza antigen by nanoparticles, but not NKT-cell adjuvant differentially induces the expression of B-cell activation factors in mice and swine.

Intranasal vaccination of pigs with poly lactic-co-glycolic acid and polyanhydride nanoparticles delivered inactivated influenza virus provides cross-reactive T-cell response, but not antibody response, resulting in incomplete protection and no reduction in nasal virus shedding. Expression of BAFF and Th2 transcription factor GATA-3 were downregulated in lungs of pigs vaccinated with influenza nanovaccine, but in mice it upregulated the expression of BAFF and cytokine TGFß in cervical lymph nodes. However, the intranasal iNKT cell adjuvant, α-Galctosylceramide upregulates the expression of BAFF in pig lungs. In conclusion, expression of BAFF is differentially regulated by intranasal nanovaccine and α-Galctosylceramide in pig respiratory tract.

Pulmonary biodistribution and cellular uptake of intranasally administered monodisperse particles.

PURPOSE: For the rational design of nanovaccines against respiratory pathogens, careful selection of optimal particle size and chemistry is paramount. This work investigates the impact of these properties on the deposition, biodistribution, and cellular interactions of nanoparticles within the lungs. METHOD: In this work, biodegradable poly(sebacic anhydride) (poly(SA)) nanoparticles of multiple sizes were synthesized with narrow particle size distributions. The lung deposition and retention as well as the internalization by phagocytic cells of these particles were compared to that of non-degradable monodisperse polystyrene nanoparticles of similar sizes. RESULTS: The initial deposition of intranasally administered particles in the lungs was dependent on primary particle size, with maximal deposition occurring for the 360-470 nm particles, regardless of chemistry. Over time, both particle size and chemistry affected the frequency of particle-positive cells and the specific cell types taking up particles. The biodegradable poly(SA) particles associated more closely with phagocytic cells and the dynamics of this association impacted the clearance of these particles from the lung. CONCLUSIONS: The findings reported herein indicate that both size and chemistry control the fate of intranasally administered particles and that the dynamics of particle association with phagocytic cells in the lungs provide important insights for the rational design of pulmonary vaccine delivery vehicles.

Attenuation of Colitis by Serum-Derived Bovine Immunoglobulin/Protein Isolate in a Defined Microbiota Mouse Model.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) is complex and multifaceted including genetic predisposition, environmental components, microbial dysbiosis, and inappropriate immune activation to microbial components. Pathogenic bacterial provocateurs like adherent and invasive E. coli have been reported to increase susceptibility to Crohn's disease. Serum-derived bovine immunoglobulin/protein isolate (SBI) is comprised primarily of immunoglobulins (Igs) that bind to conserved microbial components and neutralize exotoxins. AIM: To demonstrate that oral administration of SBI may modulate mucosal inflammation following colonization with E. coli, LF82, and exposure to dextran sodium sulfate (DSS). METHODS: Defined microbiota mice harboring the altered Schaedler flora (ASF) were administered SBI or hydrolyzed collagen twice daily starting 7 days prior to challenge with E. coli LF82 and continuing for the remainder of the experiment. Mice were treated with DSS for 7 days and then evaluated for evidence of local and peripheral inflammation. RESULTS: Igs within SBI bound multiple antigens from all eight members of the ASF and E. coli LF82 by western blot analysis. Multiple parameters of LF82/DSS-induced colitis were reduced following administration of SBI, including histological lesion scores, secretion of cytokines and chemokines from cecal biopsies, intestinal fatty acid binding protein (I-FABP) and serum amyloid A from plasma. CONCLUSIONS: Oral administration of SBI attenuated clinical signs of LF82/DSS-induced colitis in mice. The data are consistent with the hypothesis that SBI immunoglobulin binding of bacterial antigens in the intestinal lumen may inhibit the inflammatory cascades that contribute to IBD, thus attenuating DSS-induced colitis.

Inhibiting the cGAS-STING Pathway in Ulcerative Colitis with Programmable Micelles.

Ulcerative colitis is a chronic condition in which a dysregulated immune response contributes to the acute intestinal inflammation of the colon. Current clinical therapies often exhibit limited efficacy and undesirable side effects. Here, programmable nanomicelles were designed for colitis treatment and loaded with RU.521, an inhibitor of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. STING-inhibiting micelles (SIMs) comprise hyaluronic acid-stearic acid conjugates and include a reactive oxygen species (ROS)-responsive thioketal linker. SIMs were designed to selectively accumulate at the site of inflammation and trigger drug release in the presence of ROS. Our in vitro studies in macrophages and in vivo studies in a murine model of colitis demonstrated that SIMs leverage HA-CD44 binding to target sites of inflammation. Oral delivery of SIMs to mice in both preventive and delayed therapeutic models ameliorated colitis's severity by reducing STING expression, suppressing the secretion of proinflammatory cytokines, enabling bodyweight recovery, protecting mice from colon shortening, and restoring colonic epithelium. In vivo end points combined with metabolomics identified key metabolites with a therapeutic role in reducing intestinal and mucosal inflammation. Our findings highlight the significance of programmable delivery platforms that downregulate inflammatory pathways at the intestinal mucosa for managing inflammatory bowel diseases.

Next-generation nanovaccine induces durable immunity and protects against SARS-CoV-2.

While first generation SARS-CoV-2 vaccines were effective in slowing the spread and severity of disease during the COVID-19 pandemic, there is a need for vaccines capable of inducing durable and broad immunity against emerging variants of concern. Nanoparticle-based vaccines (i.e., "nanovaccines") composed of polyanhydride nanoparticles and pentablock copolymer micelles have previously been shown to protect against respiratory pathogens, including influenza A virus, respiratory syncytial virus, and Yersinia pestis. In this work, a nanovaccine containing SARS-CoV-2 spike and nucleocapsid antigens was designed and optimized. The optimized nanovaccine induced long-lived systemic IgG antibody responses against wild-type SARS-CoV-2 virus. In addition, the nanovaccine induced antibody responses capable of neutralization and cross-reactivity to multiple SARS-CoV-2 variants (including B.1.1.529) and antigen-specific CD4+ and CD8+ T cell responses. Finally, the nanovaccine protected mice against a lethal SARS-CoV-2 challenge, setting the stage for advancing particle-based SARS-CoV-2 nanovaccines. STATEMENT OF SIGNIFICANCE: First-generation SARS-CoV-2 vaccines were effective in slowing the spread and limiting the severity of COVID-19. However, current vaccines target only one antigen of the virus (i.e., spike protein) and focus on the generation of neutralizing antibodies, which may be less effective against new, circulating strains. In this work, we demonstrated the ability of a novel nanovaccine platform, based on polyanhydride nanoparticles and pentablock copolymer micelles, to generate durable and broad immunity against SARS-CoV-2. These nanovaccines induced long-lasting (> 62 weeks) serum antibody responses which neutralized binding to ACE2 receptors and were cross-reactive to multiple SARS-CoV-2 variants. Additionally, mice immunized with the SARS-CoV-2 nanovaccine showed a significant increase of antigen-specific T cell responses in the draining lymph nodes and spleens. Together, these nanovaccine-induced immune responses contributed to the protection of mice against a lethal challenge of live SARS-CoV-2 virus, indicating that this nanovaccine platform is a promising next-generation SARS-CoV-2 vaccine.

The E. coli pathobiont LF82 encodes a unique variant of σ70 that results in specific gene expression changes and altered phenotypes.

LF82, an adherent invasive Escherichia coli pathobiont, is associated with ileal Crohn's disease, an inflammatory bowel disease of unknown etiology. Although LF82 contains no virulence genes, it carries several genetic differences, including single nucleotide polymorphisms (SNPs), that distinguish it from nonpathogenic E. coli. We have identified and investigated an extremely rare SNP that is within the highly conserved rpoD gene, encoding σ70, the primary sigma factor for RNA polymerase. We demonstrate that this single residue change (D445V) results in specific transcriptome and phenotypic changes that are consistent with multiple phenotypes observed in LF82, including increased antibiotic resistance and biofilm formation, modulation of motility, and increased capacity for methionine biosynthesis. Our work demonstrates that a single residue change within the bacterial primary sigma factor can lead to multiple alterations in gene expression and phenotypic changes, suggesting an underrecognized mechanism by which pathobionts and other strain variants with new phenotypes can emerge.

Study Transportation of Drugs within Newly Established Murine Colon Organoid Systems.

The development of 3D organoids of the small intestine is a tremendous breakthrough in drug development and biological research. However, the development of colonic organoids (i.e., colonoids) is particularly challenging due to a lack of simple, cost-effective protocols for colonoid cultivation. Here, intestinal homogenates are described as a supplement to the culture medium for maintaining and replicating colonic stem cells. Colonoids generated by this cultivation protocol demonstrate substantial proliferation and differentiation (3 months). There is a similarity between cultured colonoids and primary colon tissue regarding structure and functionality. To evaluate the functionality of colonoids, permeability testing is performed with suspensions of 4 and 40 kDa fluorescein isothiocyanate-dextran (FITC-DEX). It is observed that neither can permeate the healthy epithelial barrier. The P-glycoprotein receptor, a vital drug efflux pump mitigating potential drug toxicity, is functionally manipulated, as evidenced by its inhibition function by verapamil and monitoring uptake of Rhodamin 123. In addition, Forskolin treatment which affects chloride transport results in organoid swelling; this confirms the functional expression of the CFTR transporter in the colonoids. This protocol to generate colonoids is promising for high-throughput drug screening, toxicity testing, and oral drug development.

Impacts of Gut Microbiota on the Immune System and Fecal Microbiota Transplantation as a Re-Emerging Therapy for Autoimmune Diseases.

The enormous and diverse population of microorganisms residing in the digestive tracts of humans and animals influence the development, regulation, and function of the immune system. Recently, the understanding of the association between autoimmune diseases and gut microbiota has been improved due to the innovation of high-throughput sequencing technologies with high resolutions. Several studies have reported perturbation of gut microbiota as one of the factors playing a role in the pathogenesis of many diseases, such as inflammatory bowel disease, recurrent diarrhea due to Clostridioides difficile infections. Restoration of healthy gut microbiota by transferring fecal material from a healthy donor to a sick recipient, called fecal microbiota transplantation (FMT), has resolved or improved symptoms of autoimmune diseases. This (re)emerging therapy was approved for the treatment of drug-resistant recurrent C. difficile infections in 2013 by the U.S. Food and Drug Administration. Numerous human and animal studies have demonstrated FMT has the potential as the next generation therapy to control autoimmune and other health problems. Alas, this new therapeutic method has limitations, including the risk of transferring antibiotic-resistant pathogens or transmission of genes from donors to recipients and/or exacerbating the conditions in some patients. Therefore, continued research is needed to elucidate the mechanisms by which gut microbiota is involved in the pathogenesis of autoimmune diseases and to improve the efficacy and optimize the preparation of FMT for different disease conditions, and to tailor FMT to meet the needs in both humans and animals. The prospect of FMT therapy includes shifting from the current practice of using the whole fecal materials to the more aesthetic transfer of selective microbial consortia assembled in vitro or using their metabolic products.

Structural Stability and Antigenicity of Universal Equine H3N8 Hemagglutinin Trimer upon Release from Polyanhydride Nanoparticles and Pentablock Copolymer Hydrogels.

Seasonal influenza A virus infections present substantial costs to both health and economic resources each year. Current seasonal influenza vaccines provide suboptimal protection and require annual reformulation to match circulating strains. In this work, a recombinant equine H3N8 hemagglutinin trimer (rH33) known to generate cross-protective antibodies and protect animals against sublethal, heterologous virus challenge was used as a candidate vaccine antigen. Nanoadjuvants such as polyanhydride nanoparticles and pentablock copolymer hydrogels have been shown to be effective adjuvants, inducing both rapid and long-lived protective immunity against influenza A virus. In this work, polyanhydride nanoparticles and pentablock copolymer hydrogels were used to provide sustained release of the novel rH33 while also facilitating the retention of its structure and antigenicity. These studies lay the groundwork for the development of a novel universal influenza A virus nanovaccine by combining the equine H3N8 rH33 and polymeric nanoadjuvant platforms.

Pathway Tools Management of Pathway/Genome Data for Microbial Communities.

The Pathway Tools (PTools) software provides a suite of capabilities for storing and analyzing integrated collections of genomic and metabolic information in the form of organism-specific Pathway/Genome Databases (PGDBs). A microbial community is represented in PTools by generating a PGDB from each metagenome-assembled genome (MAG). PTools computes a metabolic reconstruction for each organism, and predicts its operons. The properties of individual MAGs can be investigated using the many search and visualization operations within PTools. PTools also enables the user to investigate the properties of the microbial community by issuing searches across the full community, and by performing comparative operations across genome and pathway information. The software can generate a metabolic network diagram for the community, and it can overlay community omics datasets on that network diagram. PTools also provides a tool for searching for metabolic transformation routes across an organism community.

Vertical transmission of attaching and invasive E. coli from the dam to neonatal mice predisposes to more severe colitis following exposure to a colitic insult later in life.

The gastrointestinal microbiota begins to be acquired at birth and continually matures through early adolescence. Despite the relevance for gut health, few studies have evaluated the impact of pathobiont colonization of neonates on the severity of colitis later in life. LF82 is an adherent invasive E. coli strain associated with ileal Crohn's disease. The aim of this study was to evaluate the severity of dextran sodium sulfate (DSS)-induced colitis in mice following E. coli LF82 colonization. Gnotobiotic mice harboring the altered Schaedler flora (ASF) were used as the model. While E. coli LF82 is neither adherent nor invasive, it was been demonstrated that adult ASF mice colonized with E. coli LF82 develop more severe DSS-induced colitis compared to control ASF mice treated with DSS. Therefore, we hypothesized that E. coli LF82 colonization of neonatal ASF mice would reduce the severity of DSS-induced inflammation compared to adult ASF mice colonized with E. coli LF82. To test this hypothesis, adult ASF mice were colonized with E. coli LF82 and bred to produce offspring (LF82N) that were vertically colonized with LF82. LF82N and adult-colonized (LF82A) mice were given 2.0% DSS in drinking water for seven days to trigger colitis. More severe inflammatory lesions were observed in the LF82N + DSS mice when compared to LF82A + DSS mice, and were characterized as transmural in most of the LF82N + DSS mice. Colitis was accompanied by secretion of proinflammatory cytokines (IFNγ, IL-17) and specific mRNA transcripts within the colonic mucosa. Using 16S rRNA gene amplicon sequencing, LF82 colonization did not induce significant changes in the ASF community; however, minimal changes in spatial redistribution by fluorescent in situ hybridization were observed. These results suggest that the age at which mice were colonized with E. coli LF82 pathobiont differentially impacted severity of subsequent colitic events.

Resources to Facilitate Use of the Altered Schaedler Flora (ASF) Mouse Model to Study Microbiome Function.

Animals colonized with a defined microbiota represent useful experimental systems to investigate microbiome function. The altered Schaedler flora (ASF) represents a consortium of eight murine bacterial species that have been used for more than 4 decades where the study of mice with a reduced microbiota is desired. In contrast to germ-free mice, or mice colonized with only one or two species, ASF mice show the normal gut structure and immune system development. To further expand the utility of the ASF, we have developed technical and bioinformatic resources to enable a systems-based analysis of microbiome function using this model. Here, we highlighted four distinct applications of these resources that enable and improve (i) measurements of the abundance of each ASF member by quantitative PCR; (ii) exploration and comparative analysis of ASF genomes and the metabolic pathways they encode that comprise the entire gut microbiome; (iii) global transcriptional profiling to identify genes whose expression responds to environmental changes within the gut; and (iv) discovery of genetic changes resulting from the evolutionary adaptation of the microbiota. These resources were designed to be accessible to a broad community of researchers that, in combination with conventionally-reared mice (i.e., with complex microbiome), should contribute to our understanding of microbiome structure and function. IMPORTANCE Improved experimental systems are needed to advance our understanding of how the gut microbiome influences processes of the mammalian host as well as microbial community structure and function. An approach that is receiving considerable attention is the use of animal models that harbor a stable microbiota of known composition, i.e., defined microbiota, which enables control over an otherwise highly complex and variable feature of mammalian biology. The altered Schaedler flora (ASF) consortium is a well-established defined microbiota model, where mice are stably colonized with 8 distinct murine bacterial species. To take better advantage of the ASF, we established new experimental and bioinformatics resources for researchers to make better use of this model as an experimental system to study microbiome function.

Nanocarriers for pancreatic cancer imaging, treatments, and immunotherapies.

Pancreatic tumors are highly desmoplastic and immunosuppressive. Delivery and distribution of drugs within pancreatic tumors are compromised due to intrinsic physical and biochemical stresses that lead to increased interstitial fluid pressure, vascular compression, and hypoxia. Immunotherapy-based approaches, including therapeutic vaccines, immune checkpoint inhibition, CAR-T cell therapy, and adoptive T cell therapies, are challenged by an immunosuppressive tumor microenvironment. Together, extensive fibrosis and immunosuppression present major challenges to developing treatments for pancreatic cancer. In this context, nanoparticles have been extensively studied as delivery platforms and adjuvants for cancer and other disease therapies. Recent advances in nanotechnology have led to the development of multiple nanocarrier-based formulations that not only improve drug delivery but also enhance immunotherapy-based approaches for pancreatic cancer. This review discusses and critically analyzes the novel nanoscale strategies that have been used for drug delivery and immunomodulation to improve treatment efficacy, including newly emerging immunotherapy-based approaches. This review also presents important perspectives on future research directions that will guide the rational design of novel and robust nanoscale platforms to treat pancreatic tumors, particularly with respect to targeted therapies and immunotherapies. These insights will inform the next generation of clinical treatments to help patients manage this debilitating disease and enhance survival rates.

Plasma caffeic acid is associated with statistical clustering of the anticolitic efficacy of caffeic acid in dextran sulfate sodium-treated mice.

We hypothesized that interindividual variability in the bioavailability of caffeic acid (CA) would influence its anticolitic efficacy and that mice may be appropriate for modeling human gut microbial metabolism of CA, which is thought to influence CA bioavailability. Anaerobic human fecal and mouse cecal sample mixtures were incubated with CA derivatives from Echinacea purpurea and compound disappearance rates were measured, which were similar in both sample types. CA metabolism, including formation of its main metabolite, m-hydroxyphenylpropionate, in the mouse cecum may usefully model human gut metabolism of this compound. Ten-week-old CD-1/IGS female mice were fed 120 mg CA/kg (n = 36) or control diet for 7 d (n = 12); one-half of each group then drank 1.25% dextran sulfate sodium (DSS) in water for 5 d. DSS-treated mice fed CA showed lessened colitic damage than did mice given DSS alone, with longer colons, greater body weight, and colonic Cyp4b1 expression. Cluster analysis of the cecal histopathological score showed that mice with severe cecal damage (mean cecal score = 8.5; n = 11) also had greater myeloperoxidase (MPO) activity and lower plasma CA compared with mice showing mild cecal damage (mean cecal score = 4.5; n = 4) (P < 0.05). Cecal score was positively correlated with colonic MPO activity (r = 0.72; P < 0.05) and negatively correlated with plasma CA (r = -0.57; P < 0.05). These studies indicated that the anticolitic efficacy of CA was related to variability in CA bioavailability, which may be influenced by gut microbial metabolism of this compound.

Mannose-functionalized "pathogen-like" polyanhydride nanoparticles target C-type lectin receptors on dendritic cells.

Targeting pathogen recognition receptors on dendritic cells (DCs) offers the advantage of triggering specific signaling pathways to induce a tailored and robust immune response. In this work, we describe a novel approach to targeted antigen delivery by decorating the surface of polyanhydride nanoparticles with specific carbohydrates to provide "pathogen-like" properties that ensure nanoparticles engage C-type lectin receptors on DCs. The surface of polyanhydride nanoparticles was functionalized by covalent linkage of dimannose and lactose residues using an amine-carboxylic acid coupling reaction. Coculture of functionalized nanoparticles with bone marrow-derived DCs significantly increased cell surface expression of MHC II, the T cell costimulatory molecules CD86 and CD40, the C-type lectin receptor CIRE and the mannose receptor CD206 over the nonfunctionalized nanoparticles. Both nonfunctionalized and functionalized nanoparticles were efficiently internalized by DCs, indicating that internalization of functionalized nanoparticles was necessary but not sufficient to activate DCs. Blocking the mannose and CIRE receptors prior to the addition of functionalized nanoparticles to the culture inhibited the increased surface expression of MHC II, CD40 and CD86. Together, these data indicate that engagement of CIRE and the mannose receptor is a key mechanism by which functionalized nanoparticles activate DCs. These studies provide valuable insights into the rational design of targeted nanovaccine platforms to induce robust immune responses and improve vaccine efficacy.