Surface Modifications of Superparamagnetic Iron Oxide Nanoparticles with Chitosan, Polyethylene Glycol, Polyvinyl Alcohol, and Polyvinylpyrrolidone as Methylene Blue Adsorbent Beads.

Due to the negative impacts the dye may have on aquatic habitats and human health, it is often found in industrial effluent and poses a threat to public health. Hence, to solve this problem, this study developed magnetic adsorbents that can remove synthetic dyes like methylene blue. The adsorbent, in the form of beads, consists of a polymer blend of chitosan, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and superparamagnetic iron oxide nanoparticles (average size of 19.03 ± 4.25 nm). The adsorption and desorption of MB from beads were carried out at pH values of 7 and 3.85, respectively. At a concentration of 9 mg/L, the loading capacity and the loading amount of MB after 5 days peaked at 29.75 ± 1.53% and 297.48 ± 15.34 mg/g, respectively. Meanwhile, the entrapment efficiency of MB reached 29.42 ± 2.19% at a concentration of 8 mg/L. The cumulative desorption capacity of the adsorbent after 13 days was at its maximum at 7.72 ± 0.5%. The adsorption and desorption kinetics were evaluated.

Fusobacterium nucleatum infection modulates the transcriptome and epigenome of HCT116 colorectal cancer cells in an oxygen-dependent manner.

Fusobacterium nucleatum, a gram-negative oral bacterium, has been consistently validated as a strong contributor to the progression of several types of cancer, including colorectal (CRC) and pancreatic cancer. While previous in vitro studies have shown that intracellular F. nucleatum enhances malignant phenotypes such as cell migration, the dependence of this regulation on features of the tumor microenvironment (TME) such as oxygen levels are wholly uncharacterized. Here we examine the influence of hypoxia in facilitating F. nucleatum invasion and its effects on host responses focusing on changes in the global epigenome and transcriptome. Using a multiomic approach, we analyze epigenomic alterations of H3K27ac and global transcriptomic alterations sustained within a hypoxia and normoxia conditioned CRC cell line HCT116 at 24 h following initial infection with F. nucleatum. Our findings reveal that intracellular F. nucleatum activates signaling pathways and biological processes in host cells similar to those induced upon hypoxia conditioning in the absence of infection. Furthermore, we show that a hypoxic TME favors F. nucleatum invasion and persistence and therefore infection under hypoxia may amplify malignant transformation by exacerbating the effects induced by hypoxia alone. These results motivate future studies to investigate host-microbe interactions in tumor tissue relevant conditions that more accurately define parameters for targeted cancer therapies.

Type IV pili facilitated natural competence in Fusobacterium nucleatum.

OBJECTIVES: Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV pili facilitated natural competence in Fusobacterium nucleatum, which is a Gram-negative, anaerobic bacterium that participates in a range of infections and diseases including periodontitis, preterm birth, and cancer. METHODS: Here we used bioinformatics on multiple Fusobacterium species, as well as molecular genetics to characterize natural competence in strain F. nucleatum subsp. nucleatum ATCC 23726. RESULTS: We bioinformatically identified components of the Type IV conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type IV pili in natural competence in F. nucleatum ATCC 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. CONCLUSIONS: In summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.

Non-invasive intravenous administration of AAV9 transducing iduronate sulfatase leads to global metabolic correction and prevention of neurologic deficits in a mouse model of Hunter syndrome.

Hunter syndrome is a rare x-linked recessive genetic disorder that affects lysosomal metabolism due to deficiency of iduronate-2-sulfatase (IDS), with subsequent accumulation of glycosaminoglycans heparan and dermatan sulfates (GAG). Enzyme replacement therapy is the only FDA-approved remedy and is an expensive life-time treatment that alleviates some symptoms of the disease without neurocognitive benefit. We previously reported successful treatment in a mouse model of mucopolysaccharidosis type II (MPS II) using adeno-associated viral vector serotype 9 encoding human IDS (AAV9.hIDS) via intracerebroventricular injection. As a less invasive and more straightforward procedure, here we report intravenously administered AAV9.hIDS in a mouse model of MPS II. In animals administered 1.5 × 1012 vg of AAV9.hIDS at 2 months of age, we observed supraphysiological levels of IDS enzyme activity in the circulation (up to 9100-fold higher than wild-type), in the tested peripheral organs (up to 560-fold higher than wild-type), but only 4% to 50% of wild type levels in the CNS. GAG levels were normalized on both sides of the blood-brain-barrier (BBB) in most of tissues tested. Despite low levels of the IDS observed in the CNS, this treatment prevented neurocognitive decline as shown by testing in the Barnes maze and by fear conditioning. This study demonstrates that a single dose of IV-administered AAV9.hIDS may be an effective and non-invasive procedure to treat MPS II that benefits both sides of the BBB, with implications for potential use of IV-administered AAV9 for other neuronopathic lysosomal diseases.

Neurologic Recovery in MPS I and MPS II Mice by AAV9-Mediated Gene Transfer to the CNS After the Development of Cognitive Dysfunction.

The mucopolysaccharidoses (MPS) are a group of recessively inherited conditions caused by deficiency of lysosomal enzymes essential to the catabolism of glycosaminoglycans (GAG). MPS I is caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA), while MPS II is caused by a lack of iduronate-2-sulfatase (IDS). Lack of these enzymes leads to early mortality and morbidity, often including neurological deficits. Enzyme replacement therapy has markedly improved the quality of life for MPS I and MPS II affected individuals but is not effective in addressing neurologic manifestations. For MPS I, hematopoietic stem cell transplant has shown effectiveness in mitigating the progression of neurologic disease when carried out in early in life, but neurologic function is not restored in patients transplanted later in life. For both MPS I and II, gene therapy has been shown to prevent neurologic deficits in affected mice when administered early, but the effectiveness of treatment after the onset of neurologic disease manifestations has not been characterized. To test if neurocognitive function can be recovered in older animals, human IDUA or IDS-encoding AAV9 vector was administered by intracerebroventricular injection into MPS I and MPS II mice, respectively, after the development of neurologic deficit. Vector sequences were distributed throughout the brains of treated animals, associated with high levels of enzyme activity and normalized GAG storage. Two months after vector infusion, treated mice exhibited spatial navigation and learning skills that were normalized, that is, indistinguishable from those of normal unaffected mice, and significantly improved compared to untreated, affected animals. We conclude that cognitive function was restored by AAV9-mediated, central nervous system (CNS)-directed gene transfer in the murine models of MPS I and MPS II, suggesting that gene transfer may result in neurodevelopment improvements in severe MPS I and MPS II when carried out after the onset of cognitive decline.

Enhanced Fusobacterium nucleatum Genetics Using Host DNA Methyltransferases To Bypass Restriction-Modification Systems.

Bacterial restriction-modification (R-M) systems are a first-line immune defense against foreign DNA from viruses and other bacteria. While R-M systems are critical in maintaining genome integrity, R-M nucleases unfortunately present significant barriers to targeted genetic modification. Bacteria of the genus Fusobacterium are oral, Gram-negative, anaerobic, opportunistic pathogens that are implicated in the progression and severity of multiple cancers and tissue infections, yet our understanding of their direct roles in disease have been severely hindered by their genetic recalcitrance. Here, we demonstrate a path to overcome these barriers in Fusobacterium by using native DNA methylation as a host mimicry strategy to bypass R-M system cleavage of transformed plasmid DNA. We report the identification, characterization, and successful use of Fusobacterium nucleatum type II and III DNA methyltransferase (MTase) enzymes to produce a multifold increase in gene knockout efficiency in the strain Fusobacterium nucleatum subsp. nucleatum 23726, as well as the first system for efficient gene knockouts and complementations in F. nucleatum subsp. nucleatum 25586. We show plasmid protection can be accomplished in vitro with purified enzymes, as well as in vivo in an Escherichia coli host that constitutively expresses F. nucleatum subsp. nucleatum MTase enzymes. In summary, this proof-of-concept study characterizes specific MTases that are critical for bypassing R-M systems and has enhanced our understanding of enzyme combinations that could be used to genetically modify clinical isolates of Fusobacterium that have thus far been inaccessible to molecular characterization. IMPORTANCE Fusobacterium nucleatum is an oral opportunistic pathogen associated with diseases that include cancer and preterm birth. Our understanding of how this bacterium modulates human disease has been hindered by a lack of genetic systems. Here, we show that F. nucleatum DNA methyltransferase-modified plasmid DNA overcomes the transformation barrier and has allowed the development of a genetic system in a previously inaccessible strain. We present a strategy that could potentially be expanded to enable the genetic modification of highly recalcitrant strains, thereby fostering investigational studies to uncover novel host-pathogen interactions in Fusobacterium.

Fusobacterium nucleatum induces proliferation and migration in pancreatic cancer cells through host autocrine and paracrine signaling.

The tumor microbiome is increasingly implicated in cancer progression and resistance to chemotherapy. In pancreatic ductal adenocarcinoma (PDAC), high intratumoral loads of Fusobacterium nucleatum correlate with shorter survival in patients. Here, we investigated the potential mechanisms underlying this association. We found that F. nucleatum infection induced both normal pancreatic epithelial cells and PDAC cells to secrete increased amounts of the cytokines GM-CSF, CXCL1, IL-8, and MIP-3α. These cytokines increased proliferation, migration, and invasive cell motility in both infected and noninfected PDAC cells but not in noncancerous pancreatic epithelial cells, suggesting autocrine and paracrine signaling to PDAC cells. This phenomenon occurred in response to Fusobacterium infection regardless of the strain and in the absence of immune and other stromal cells. Blocking GM-CSF signaling markedly limited proliferative gains after infection. Thus, F. nucleatum infection in the pancreas elicits cytokine secretion from both normal and cancerous cells that promotes phenotypes in PDAC cells associated with tumor progression. The findings support the importance of exploring host-microbe interactions in pancreatic cancer to guide future therapeutic interventions.

A Paradoxical Effect of Interleukin-32 Isoforms on Cancer.

IL-32 plays a contradictory role such as tumor proliferation or suppressor in cancer development depending on the cancer type. In most cancers, it was found that the high expression of IL-32 was associated with more proliferative and progression of cancer. However, studying the isoforms of IL-32 cytokine has placed its paradoxical role into a wide range of functions based on its dominant isoform and surrounding environment. IL-32ß, for example, was found mostly in different types of cancer and associated with cancer expansion. This observation is legitimate since cancer exhibits some hypoxic environment and IL-32ß was known to be induced under hypoxic conditions. However, IL-32θ interacts directly with protein kinase C-δ reducing NF-κB and STAT3 levels to inhibit epithelial-mesenchymal transition (EMT). This effect could explain the different functions of IL-32 isoforms in cancer. However, pro- or antitumor activity which is dependant on obesity, gender, and age as it relates to IL-32 has yet to be studied. Obesity-related IL-32 regulation indicated the role of IL-32 in cancer metabolism and inflammation. IL-32-specific direction in cancer therapy is difficult to conclude. In this review, we address that the paradoxical effect of IL-32 on cancer is attributed to the dominant isoform, cancer type, tumor microenvironment, and genetic background. IL-32 seems to have a contradictory role in cancer. However, investigating multiple IL-32 isoforms could explain this doubt and bring us closer to using them in therapy.

Comparison of the Seven Interleukin-32 Isoforms' Biological Activities: IL-32θ Possesses the Most Dominant Biological Activity.

Cytokines are significantly associated with the homeostasis of immune responses in health and disease. Interleukin-32 (IL-32) is a cytokine originally discovered in natural killer cell transcript 4. IL-32 with different disorders has been described in terms of pathogenesis and the progression of diseases. Clinical studies have investigated IL-32 under various conditions, such as viral infection, autoimmune diseases, inflammatory diseases, certain types of cancer, vascular disease, and pulmonary diseases. The high expression of IL-32 was identified in different tissues with various diseases and found to have multiple transcripts of up to seven isoforms. However, the purification and biological activities of these isoforms have not been investigated yet. Therefore, in this study, we purified and compared the biological activity of recombinant IL-32 (rIL-32) isoforms. This is the first time for seven rIL-32 isoforms (α, ß, δ, γ, ϵ, ζ, and θ) to be cloned and purified using an Escherichia coli expression system. Next, we evaluate the biological activities of these seven rIL-32 isoforms, which were used to treat different types of cells by assessing the levels of inflammatory cytokine production. The results revealed that rIL-32θ possessed the most dominant biological activity in both immune and non-immune cells.

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Comparative Analysis of Colon Cancer-Derived Fusobacterium nucleatum Subspecies: Inflammation and Colon Tumorigenesis in Murine Models.

Fusobacteria are commonly associated with human colorectal cancer (CRC), but investigations are hampered by the absence of a stably colonized murine model. Further, Fusobacterium nucleatum subspecies isolated from human CRC have not been investigated. While F. nucleatum subspecies are commonly associated with CRC, their ability to induce tumorigenesis and contributions to human CRC pathogenesis are uncertain. We sought to establish a stably colonized murine model and to understand the inflammatory potential and virulence genes of human CRC F. nucleatum, representing the 4 subspecies, animalis, nucleatum, polymorphum, and vincentii. Five human CRC-derived and two non-CRC derived F. nucleatum strains were tested for colonization, tumorigenesis, and cytokine induction in specific-pathogen-free (SPF) and/or germfree (GF) wild-type and ApcMin/+ mice, as well as in vitro assays and whole-genome sequencing (WGS). SPF wild-type and ApcMin/+ mice did not achieve stable colonization with F. nucleatum, whereas certain subspecies stably colonized some GF mice but without inducing colon tumorigenesis. F. nucleatum subspecies did not form in vivo biofilms or associate with the mucosa in mice. In vivo inflammation was inconsistent across subspecies, whereas F. nucleatum induced greater cytokine responses in a human colorectal cell line, HCT116. While F. nucleatum subspecies displayed genomic variability, no distinct virulence genes associated with human CRC strains were identified that could reliably distinguish these strains from non-CRC clinical isolates. We hypothesize that the lack of F. nucleatum-induced tumorigenesis in our model reflects differences in human and murine biology and/or a synergistic role for F. nucleatum in concert with other bacteria to promote carcinogenesis. IMPORTANCE Colon cancer is a leading cause of cancer morbidity and mortality, and it is hypothesized that dysbiosis in the gut microbiota contributes to colon tumorigenesis. Fusobacterium nucleatum, a member of the oropharyngeal microbiome, is enriched in a subset of human colon tumors. However, it is unclear whether this genetically varied species directly promotes tumor formation, modulates mucosal immune responses, or merely colonizes the tumor microenvironment. Mechanistic studies to address these questions have been stymied by the lack of an animal model that does not rely on daily orogastric gavage. Using multiple murine models, in vitro assays with a human colon cancer cell line, and whole-genome sequencing analysis, we investigated the proinflammatory and tumorigenic potential of several F. nucleatum clinical isolates. The significance of this research is development of a stable colonization model of F. nucleatum that does not require daily oral gavages in which we demonstrate that a diverse library of clinical isolates do not promote tumorigenesis.

Harnessing Tissue Engineering Tools to Interrogate Host-Microbiota Crosstalk in Cancer.

Recent studies have begun to highlight the diverse and tumor-specific microbiomes across multiple cancer types. We believe this work raises the important question of whether the classical "Hallmarks of Cancer" should be expanded to include tumor microbiomes. To answer this question, the causal relationships and co-evolution of these microbiotic tumor ecosystems must be better understood. Because host-microbe interactions should be studied in a physiologically relevant context, animal models have been preferred. Yet these models are often poor mimics of human tumors and are difficult to interrogate at high spatiotemporal resolution. We believe that in vitro tissue engineered platforms could provide a powerful alternative approach that combines the high-resolution of in vitro studies with a high degree of physiological relevance. This review will focus on tissue engineered approaches to study host-microbe interactions and to establish their role as an emerging hallmark of cancer with potential as a therapeutic target.

Plastic Surgery and Cosmetic Procedures: Facial Injection Procedures.

Nonsurgical cosmetic procedures, including injection procedures, are among the fastest growing medical procedures. In 2018, botulinum toxin and hyaluronic acid injections were the two most common nonsurgical cosmetic procedures performed in the United States. Botulinum toxin is a neuromodulator with seven serotypes, but only serotypes A and B are in clinical use. The facial areas in which botulinum toxin is used most commonly include the forehead, glabella, and lateral canthal lines (ie, crow's feet). Adverse effects are transient. Several types of injectable dermal fillers are available. Hyaluronic acid is the most commonly used filler and is hypoallergenic. Others include calcium hydroxylapatite, poly L-lactic acid, and polymethylmethacrylate. The characteristics of fillers, including their elasticity and viscosity, are used to determine which should be used for specific applications. Potential serious complications include vascular occlusion leading to tissue necrosis or blindness. Immediate recognition and management of complications are needed to prevent long-term sequelae. With appropriate training, physicians and other clinicians can perform these injections safely in the office setting.

Plastic Surgery and Cosmetic Procedures: Facial Plastic Surgery.

Facial plastic surgery deals with improving the form, function, and appearance of the face. Blepharoplasty (ie, eyelid surgery) is the most commonly performed surgical procedure in men and women 65 years and older. It has been shown to be effective for improving attractiveness and eyelid function (eg, improved visual field). Indications for upper and lower eyelid surgery include aesthetic concerns, ptosis, and peripheral field vision impairment. Facelift procedures resuspend, reposition, and release underlying facial structures and excise excess skin to improve the appearance of the face. Facelifts mainly involve manipulation of the superficial musculo-aponeurotic system (SMAS), a group of muscles in the lateral face. Types of facelifts include subcutaneous facelift with SMAS manipulation to allow for facial shaping of the deep soft tissue. Such manipulation can include SMAS-ectomy, SMAS plication, and SMAS flap creation. Deep plane facelift involves the SMAS and release of the facial and cervical retaining ligaments. Hematoma is a common serious complication of facelifts. Less common complications include skin flap necrosis, nerve damage, and scarring. Rhinoplasty is another commonly performed facial surgery. The two main forms are open rhinoplasty and closed rhinoplasty.

Plastic Surgery and Cosmetic Procedures: Liposuction and Nonsurgical Fat Reduction Procedures.

Liposuction and nonsurgical fat reduction modalities (ie, body contouring) are among the most commonly performed cosmetic procedures in the United States. Liposuction is an invasive surgical procedure in which adipose tissue is dislodged and suction is used to remove it. The procedure originally was performed with a regional nerve block or general anesthesia; a tumescent technique for local anesthesia now is used. There are several types of liposuction, each uses application of energy to allow for removal of fat. These methods include laser-assisted, power-assisted, and ultrasound-assisted liposuction. Abdominoplasty can be performed in combination with liposuction for management of loose skin. Nonsurgical fat reduction modalities include cryolipolysis, use of radiofrequency lipolysis devices, and ultrasonographic modalities. Cryolipolysis uses cold temperatures to achieve lipolysis, whereas radiofrequency lipolysis devices use heat to destroy adipose tissue. Nonthermal low-intensity, low-frequency ultrasonography uses mechanical methods to destroy fat cells. High-intensity focused ultrasound uses thermal methods. Body contouring procedures can be performed alone or in combination with other procedures.

Plastic Surgery and Cosmetic Procedures: Augmentation and Reconstruction Procedures.

In 2018, breast augmentation was the most common cosmetic surgical procedure performed in the United States. It was the most commonly performed cosmetic surgical procedure in women ages 18 to 34 years. Silicone gel-filled implants are the most commonly used in the United States, followed by saline-filled implants. The most common approach to placement of implants is through an incision in the inframammary fold. The majority of implants are placed subglandularly, under the breast tissue but on top of the pectoralis muscle. Postoperative complications include lumps, asymmetry, leakage or deflation, capsular contracture, changes or loss of nipple and areolar sensation, seroma, hematoma, changes in breast shape, and infection. Long-term complications include infection, implant rupture, capsular contracture, and breast implant-associated anaplastic large cell lymphoma. Any patient who has undergone breast augmentation who presents with a breast lump or mass should be referred to a breast unit for evaluation. Lumps can indicate implant rupture, capsular contracture, seroma, hematoma, breast cancer, or infection. Studies have shown no association between silicone-filled breast implants and connective tissue disorders. Breast reconstruction after mastectomy frequently involves placement of implants. Autologous reconstruction remains another option. Various implants are approved by the Food and Drug Administration for buttock and calf augmentation.

Molecular Basis of the Mechanisms Controlling MASTL.

The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases.

Structural Characteristics of Seven IL-32 Variants.

IL-32 exists as seven mRNA transcripts that can translate into distinct individual IL-32 variants with specific protein domains. These translated protein domains of IL-32 variants code for specific functions that allow for interaction with different molecules intracellularly or extracellularly. The longest variant is IL-32γ possessing 234 amino acid residues with all 11 protein domains, while the shortest variant is IL-32α possessing 131 amino acid residues with three of the protein domains. The first domain exists in 6 variants except IL-32δ variant, which has a distinct translation initiation codon due to mRNA splicing. The last eleventh domain is common domain for all seven IL-32 variants. Numerous studies in different fields, such as inflammation, autoimmunity, pathogen infection, and cancer biology, have claimed the specific biological activity of individual IL-32 variant despite the absence of sufficient data. There are 4 additional IL-32 variants without proper transcripts. In this review, the structural characteristics of seven IL-32 transcripts are described based on the specific protein domains.

Temporal Regulation of Natural Killer T Cell Interferon Gamma Responses by ß-Catenin-Dependent and -Independent Wnt Signaling.

Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator ß-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. ß-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and ß-catenin activity shape liver NKT cell functions in vivo in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of ß-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls), to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of ß-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/ß-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of ß-catenin activity. Our analyses in ICG001-treated mice confirmed a role for ß-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and ß-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of ß-catenin to NKT cell functions.

Expression, Purification and Characterization of GMZ2'.10C, a Complex Disulphide-Bonded Fusion Protein Vaccine Candidate against the Asexual and Sexual Life-Stages of the Malaria-Causing Plasmodium falciparum Parasite.

PURPOSE: Production and characterization of a chimeric fusion protein (GMZ2'.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2'.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite. METHODS: GMZ2'.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS. RESULTS: CP-GMZ2'.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2'.10C. CP-GMZ2'.10C and IP-GMZ2'.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus Pfs48/45 was analysed by tandem mass spectrometry and was established for GMZ2'.10C and two reference fusion proteins encompassing similar parts of Pfs48/45. CONCLUSION: GMZ2'.10C, combining GMZ2' and correctly-folded Pfs48/45 can be produced by the Lactoccus lactis P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of Pfs48/45 was revealed experimentally, providing an important guideline for employing the Pfs48/45 antigen in vaccine design.