Stimulation Of Immune Cells Research Articles

Expression kinetics of ISG15, IRF3, IFNγ, IL10, IL2 and IL4 genes vis-a-vis virus shedding, tissue tropism and antibody dynamics in PPRV vaccinated, challenged, infected sheep and goats

Here, we studied the in vivo expression of Th1 (IL2 and IFN gamma) and Th2 (IL4 and IL10) - cytokines and antiviral molecules - IRF3 and ISG15 in peripheral blood mononuclear cells in relation to antigen and antibody dynamics under Peste des petits ruminants virus (PPRV) vaccination, infection and challenge in both sheep and goats. Vaccinated goats were seropositive by 9 days post vaccination (dpv) while in sheep idiosyncratic response was observed between 9 and 14 dpv for different animals. Expression of PPRV N gene was not detected in PBMCs of vaccinated and vaccinated challenged groups of both species, but was detected in unvaccinated infected PBMCs at 9 and 14 days post infection. The higher viral load at 9 dpi coincided with the peak clinical signs of the disease. The peak in viral replication at 9 dpi correlated with significant expression of antiviral molecules IRF3, ISG15 and IFN gamma in both the species. With the progression of disease, the decrease in N gene expression also correlated with the decrease in expression of IRF3, ISG15 and IFN gamma. In the unvaccinated infected animals ISG15, IRF3, IFN gamma and IL10 expression was higher than vaccinated animals. The IFN gamma expression predominated over IL4 in both vaccinated and infected animals with the infected exhibiting a stronger Th1 response. The persistent upregulation of this antiviral molecular signature - ISG15 and IRF3 even after 2 weeks post vaccination, presumably reflects the ongoing stimulation of innate immune cells.

Synthetic Cancer-Targeting Innate Immune Stimulators Give Insights into Avidity Effects.

Multispecific and multivalent antibodies are seen as promising cancer therapeutics, and numerous antibody fragments and derivatives have been developed to exploit avidity effects that result in increased selectivity. Most of these multispecific and multivalent antibody strategies make use of recombinant expression of antigen-binding modules. In contrast, chemical synthesis and chemoselective ligations can be used to generate a variety of molecules with different numbers and combinations of binding moieties in a modular and homogeneous fashion. In this study we synthesized a series of targeted immune system engagers (ISErs) by using solid-phase peptide synthesis and chemoselective ligations. To explore avidity effects, we constructed molecules bearing different numbers and combinations of two "binder" peptides that target ephrin A2 and integrin α3 receptors and an "effector" peptide that binds to formyl peptide receptors and stimulates an immune response. We investigated various strategies for generating multivalent and multispecific targeted innate immune stimulators and studied their activities in terms of binding to cancer cells and stimulation of immune cells. This study gives insights into the influence that multivalency and receptor density have on avidity effects and is useful for the design of potential anticancer therapeutics.

Modelling Combined Virotherapy and Immunotherapy: Strengthening the Antitumour Immune Response Mediated by IL-12 and GM-CSF Expression

Combined virotherapy and immunotherapy has been emerging as a promising and effective cancer treatment for some time. Intratumoural injections of an oncolytic virus instigate an immune reaction in the host, resulting in an influx of immune cells to the tumour site. Through combining an oncolytic viral vector with immunostimulatory cytokines an additional antitumour immune response can be initiated, whereby immune cells induce apoptosis in both uninfected and virus infected tumour cells. We develop a mathematical model to reproduce the experimental results for tumour growth under treatment with an oncolytic adenovirus co-expressing the immunostimulatory cytokines interleukin 12 (IL-12) and granulocyte-monocyte colony stimulating factor (GM-CSF). By exploring heterogeneity in the immune cell stimulation by the treatment, we find a subset of the parameter space for the immune cell induced apoptosis rate, in which the treatment will be less effective in a short time period. Therefore, we believe the bivariate nature of treatment outcome, whereby tumours are either completely eradicated or grow unbounded, can be explained by heterogeneity in this immune characteristic. Furthermore, the model highlights the apparent presence of negative feedback in the helper T cell and APC stimulation dynamics, when IL-12 and GM-CSF are co-expressed as opposed to individually expressed by the viral vector.

Direct and Intestinal Epithelial Cell-Mediated Effects of TLR8 Triggering on Human Dendritic Cells, CD14+CD16+ Monocytes and γδ T Lymphocytes.

Toll-like receptor (TLR)7/8 plays a crucial role in host recognition/response to viruses and its mucosal expression directly correlates with intestinal inflammation. The aim of this study was to investigate the role of TLR7/8 stimulation of intestinal epithelium in shaping the phenotype and functions of innate immunity cell subsets, and to define direct and/or epithelial cell-mediated mechanisms of the TLR7/8 agonist R848 immunomodulatory activity. We describe novel, TLR8-mediated, pro- and anti-inflammatory effects of R848 on ex vivo cultured human blood monocytes and γδ T lymphocytes, either induced by direct immune cell stimulation or mediated by intestinal epithelial cells (IEC). Apical stimulation with R848 led to its transport across normal polarized epithelial cell monolayer and resulted in the inhibition of monocyte differentiation toward immunostimulatory dendritic cells and Th1 type response. Furthermore, γδ T lymphocyte activation was promoted following direct exposure of these cells to the agonist. Conversely, a selective enrichment of the CD14+CD16+ monocyte subpopulation was observed, which required a CCL2-mediated inflammatory response of normal epithelial cells to R848. Of note, a TLR-mediated activation of control γδ T lymphocytes was promoted by inflamed intestinal epithelium from active Crohn’s disease patients. This study unravels a novel regulatory mechanism linking the activation of the TLR8 pathway in IEC to the monocyte-mediated inflammatory response, and highlights the capacity of the TLR7/8 agonist R848 to directly enhance the activation of γδ T lymphocytes. Overall these results expand the range of cell targets and immune responses controlled by TLR8 triggering that may contribute to the antiviral response, to chronic inflammation, as well as to the adjuvant activity of TLR8 agonists, highlighting the role of intestinal epithelium microenvironment in shaping TLR agonist-induced responses.

Gravity-Related Immunological Changes in Human Whole Blood Cultured Under Simulated Microgravity Using an In Vitro Cytokine Release Assay.

Although immune dysfunction by space conditions has been reported postflight, as well as during ground-based experiments, the cause(s) and nature of the immunological changes are not completely understood. Microgravity has been suggested as one of the factors responsible for the observed immune dysregulation. The goal of this study was to assess immune changes in simulated microgravity (s-μG) using an in vitro cytokine release assay. The effect of s-μG provided by the desktop random positioning machine on cell-mediated immunity was examined by analyzing interleukin 2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 10 (IL-10), in response to immune cell stimulation in whole blood samples (n = 10). Stimuli used were bacterial recall antigens, pokeweed mitogen (PWM), lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM). S-μG caused an overall inhibition of the IL-2 and IFN-γ responses to recall antigen and mitogen stimulation. More specifically, s-μG most strongly influenced the levels of all four cytokines elicited by bacterial recall antigen stimulation. In contrast, HKLM-induced TNF-α secretion was elevated. The average concentrations of TNF-α in response to PWM and LPS and IL-10 release stimulated by PWM, LPS, and HKLM were not significantly altered by s-μG. However, a variable response between individual subjects could be observed. In conclusion, our results demonstrate that the in vitro cytokine release assay can detect gravity-related immune alterations. Furthermore, the use of multiple stimuli and the associated changes in cytokine secretion has the potential to reveal information on the underlying mechanisms affected by s-μG.

Books: In Pursuit of Memory: the Fight Against Alzheimer’s

Joseph Jebelli John Murray, 2017 , HB, 320 pp, £18.99, 978-1473635739 Joseph Jebelli is a researcher at the Institute of Neurology at University College London, working on rats genetically programmed to develop Alzheimer’s disease — specifically, on immune stimulation of microglial cells in the hope that this will make some contribution to an eventual cure. He begins with an account of the first formal description of the disease by Alois Alzheimer in 1906, whose presentation to a psychiatric convention was met with a distinctly uninterested response from his psychoanalytically inclined audience. His work was rewarded in 1910 when Kraepelin, in his Handbook of Psychiatry , used the term ‘Alzheimer’s disease’ for the first time. Moving on to describe the major research findings to …

Therapeutic Tumor Control of HER2 DNA Vaccines Is Achieved by an Alteration of Tumor Cells and Tumor Microenvironment by Gemcitabine and Anti-Gr-1 Ab Treatment in a HER2-Expressing Tumor Model.

Therapeutic control of tumors is challenging as they tend to alter their biological functions and microenvironment. In a CT26/HER2 tumor model, HER2 DNA vaccines and even anti-PD-L1 Abs failed to display antitumor therapeutic activity while inducing Ag-specific cytotoxic T lymphocyte (CTL) activity. To clarify this contradictory finding, we selected tumor cells (CT26/HER2-1) from one tumor-bearing animal in the therapeutic model. CT26/HER2-1 cells behaved similar to wild-type CT26/HER2 cells in their HER2 expression, immune cell stimulation for IFN-γ production, and antitumor immune sensitivity. A similar finding was obtained with additional CT26/HER2-2, -3, -4, -5, and -6 cells from the therapeutic model, suggesting that a lack of antitumor therapeutic activity of HER2 DNA vaccines might be ascribed to a factor in the tumor microenvironment, but not to an alteration in tumor cell functions. When tumor-bearing mice were depleted of myeloid-derived suppressor cells (MDSCs) by anti-Gr-1 Ab treatment, they displayed HER2 vaccine-mediated antitumor activity, suggesting a role of MDSCs in blocking antitumor activity. Moreover, when tumor-bearing mice were treated with gemcitabine, they displayed HER2 vaccine-mediated antitumor activity, suggesting that cytotoxic drug treatment makes tumor cells susceptible to lysis by CTLs. Thus, these studies show that therapeutic control of HER2 DNA vaccines can be achieved by anti-Gr-1 Ab treatment through MDSC depletion and by gemcitabine treatment through sensitization of tumor cells to CTL-mediated killing in this model. These findings may have implications for achieving therapeutic control of CTL-resistant tumors in cancer therapy.

Abstract 3677: Early effects of CSF-470 melanoma vaccine plus adjuvants in immune cell stimulation

Abstract Background: In a Phase II/III clinical trial (CASVAC-0401, NCT01729663), cutaneous melanoma (CM) patients (pts) stages II/III were randomized to receive CSF-470 vaccine or IFN-α (2:1). Vaccine CSF-470 is composed of four lethally-irradiated allogeneic CM cell lines; BCG (Bacillus Calmette-Guérin) and rhGM-CSF were used as adjuvants. At the end of the Phase II part of the study (30 pts), a higher distant-metastases-free-survival was observed in vaccinated pts. All vaccinated pts developed inflammation at the vaccination site. Therefore, in an attempt to understand these first steps of immune reaction, leukocytes migration, cytokines synthesis and monocyte phagocytosis promoted by the vaccine components were studied in vitro. Methods: Cell migration of healthy donors’ leukocytes or mononuclear cells (MN) towards vaccine components was assayed in 0,5 μm transwells. Cytokines were measured by ELISA in supernatants of CSF-470 vaccine and MN (1:1, 0,5x106 each) cocultures. For phagocytosis assays, CM cell lines were labeled with PKH67 lipophilic dye, irradiated, and cocultured with purified monocytes. After 24 hs, cultures were stained with CD14 and double staining (CD14+PKH67+) was analyzed by flow cytometry. Results: In migration assays, CSF-470 vaccine induced higher attraction of leukocytes as compared to culture medium (p=0.003, unpaired-T-test). GM-CSF exerted little attraction, although combined with BCG it increased the migration triggered by CSF-470 (vaccine vs vaccine plus adjuvants, p=0.034, unpaired-T-test). When using MN, CSF-470 alone or plus adjuvants, highly attracted monocytes, which represented 10%-65% of the migrating CD45+ population (before migration 6-15%) (p<0.0001, paired-T-test). In the lymphocyte population, natural killer cells were mainly attracted towards the vaccine either with or without adjuvants, representing 12%-33% of total migrated lymphocyte population (before migration 4-25% ) (p=0.002, paired-T-test). Regarding cytokine production, 6 hs cocultures of CSF-470 vaccine plus adjuvants and MN, promoted TNFα production (385±217 pg/ml). IFNγ release was detected in CSF-470:MN cocultures after 48 hs (109,2 pg/ml). BCG appears to be the most important adjuvant for production of these proinflammatory cytokines. IL-10, but not IL-12p70, was also detected. Interestingly, approximately 60% of monocytes phagocytosed CSF-470 cells after 24 hs of coculture, either in the presence or absence of adjuvants. Conclusions: Our in vitro results suggest that recruitment of leukocytes, mostly from the innate immune system, is an early event after CSF-470 vaccine + BCG + GM-CSF interaction with immune cells. Also, early release of TNFα and IFNγ proinflammatory cytokines and efficient vaccine phagocytosis by monocytes would favor subsequent antigen processing and presentation. Further experiments will be performed to address the initial steps triggered after CSF-470 vaccination. Citation Format: Maria B. Pampena, Estefanía P. Juliá, Yamila S. Rocca, Paula A. Blanco, María M. Barrio, José Mordoh, Estrella M. Levy. Early effects of CSF-470 melanoma vaccine plus adjuvants in immune cell stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3677. doi:10.1158/1538-7445.AM2017-3677

Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses.The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4+ and CD8+ T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4+ and CD8+ lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized.DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30–40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll-like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4+ and CD8+ T cells, in vivo.Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects.

Reactive oxygen species modulate immune cell effector function

Abstract During an infection or carcinogenesis, the stimulation of immune cells may lead to elevated intracellular levels of reactive oxygen species (ROS), and this oxidative stress can cause damage to the cell. Interestingly, previous reports have shown that ROS produced through cell activation play a critical role as signaling messengers in immune responses. Yet, little is known in regard to the role of ROS in natural killer (NK) cell effector functions. Our data suggest that the levels of ROS increase in NK cells upon stimulation. Therefore, we hypothesize that, upon stimulation of NK cells, ROS accumulate and provide positive signaling that leads to the upregulation of NK cell effector functions. To test this hypothesis, we examined NK cell function and proliferation in Glrx2-deficient mice in which the ROS levels are elevated in the mitochondrial matrix due to a defect in the redox system. We found that lack of Glrx2 in NK and T cells does not improve their cytotoxic potential or their ability to produce IFNg. However, Glrx2-deficient NK cells showed enhanced proliferation when stimulated with IL-2, indicating that during prolonged stress, the elevated levels of ROS drive NK cells to undergo robust cell division. This finding will elucidate a link between the levels of ROS and immune cell proliferation during viral infection and carcinogenesis.

Alternative splicing switches: Important players in cell differentiation.

Alternative splicing (AS) greatly expands the coding capacities of genomes by allowing the generation of multiple mature mRNAs from a limited number of genes. Although the massive switch in AS profiles that often accompanies variations in gene expression patterns occurring during cell differentiation has been characterized for a variety of models, their causes and mechanisms remain largely unknown. Here, we integrate foundational and recent studies indicating the AS switches that govern the processes of cell fate determination. We include some distinct AS events in pluripotent cells and somatic reprogramming and discuss new progresses on alternative isoform expression in adipogenesis, myogenic differentiation and stimulation of immune cells. Finally, we cover novel insights on AS mechanisms during neuronal differentiation, paying special attention to the role of chromatin structure.

Engineered Hydrogen-Bonded Glycopolymer Capsules and Their Interactions with Antigen Presenting Cells.

Hollow glycopolymer microcapsules were fabricated by hydrogen-bonded layer-by-layer (LbL) assembly, and their interactions with a set of antigen presenting cells (APCs), including dendritic cells (DCs), macrophages (MACs), and myeloid derived suppressor cells (MDSCs), were investigated. The glycopolymers were obtained by cascade postpolymerization modifications of poly(oligo(2-ethyl-2-oxazoline methacrylate)-stat-glycidyl methacrylate) involving the modification of the glycidyl groups with propargylamine and the subsequent attachment of mannose azide by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Multilayer assembly of the hydrogen-bonding pair (glycopolymer/poly(methacrylic acid) (PMA)) onto planar and particulate supports (SiO2 particles, d = 1.16 μm) yielded stable glycopolymer films upon cross-linking by CuAAC. The silica (SiO2) particle templates were removed yielding hollow monodisperse capsules, as demonstrated by fluorescence and scanning electron microscopy. Cellular uptake studies using flow cytometry revealed the preferential uptake of the capsules by DCs when compared to MACs or MDSCs. Mannosylated capsules showed a cytokine independent cis-upregulation of CD80 specifically on DCs and a trans-downregulation of PDL-1 on MDSCs. Thus, the glycopolymer capsules may have potential as vaccine carriers, as they are able to upregulate costimulatory molecules for immune cell stimulation on DCs and at the same time downregulate immune inhibitory receptors on suppressor APC such as MDSCs.

Harnessing RNA sequencing for global, unbiased evaluation of two new adjuvants for dendritic-cell immunotherapy

Effective stimulation of immune cells is crucial for the success of cancer immunotherapies. Current approaches to evaluate the efficiency of stimuli are mainly defined by known flow cytometry-based cell activation or cell maturation markers. This method however does not give a complete overview of the achieved activation state and may leave important side effects unnoticed. Here, we used an unbiased RNA sequencing (RNA-seq)-based approach to compare the capacity of four clinical-grade dendritic cell (DC) activation stimuli used to prepare DC-vaccines composed of various types of DC subsets; the already clinically applied GM-CSF and Frühsommer meningoencephalitis (FSME) prophylactic vaccine and the novel clinical grade adjuvants protamine-RNA complexes (pRNA) and CpG-P. We found that GM-CSF and pRNA had similar effects on their target cells, whereas pRNA and CpG-P induced stronger type I interferon (IFN) expression than FSME. In general, the pathways most affected by all stimuli were related to immune activity and cell migration. GM-CSF stimulation, however, also induced a significant increase of genes related to nonsense-mediated decay, indicating a possible deleterious effect of this stimulus. Taken together, the two novel stimuli appear to be promising alternatives. Our study demonstrates how RNA-seq based investigation of changes in a large number of genes and gene groups can be exploited for fast and unbiased, global evaluation of clinical-grade stimuli, as opposed to the general limited evaluation of a pre-specified set of genes, by which one might miss important biological effects that are detrimental for vaccine efficacy.

Food-grade TiO2 impairs intestinal and systemic immune homeostasis, initiates preneoplastic lesions and promotes aberrant crypt development in the rat colon

Food-grade titanium dioxide (TiO2) containing a nanoscale particle fraction (TiO2-NPs) is approved as a white pigment (E171 in Europe) in common foodstuffs, including confectionary. There are growing concerns that daily oral TiO2-NP intake is associated with an increased risk of chronic intestinal inflammation and carcinogenesis. In rats orally exposed for one week to E171 at human relevant levels, titanium was detected in the immune cells of Peyer’s patches (PP) as observed with the TiO2-NP model NM-105. Dendritic cell frequency increased in PP regardless of the TiO2 treatment, while regulatory T cells involved in dampening inflammatory responses decreased with E171 only, an effect still observed after 100 days of treatment. In all TiO2-treated rats, stimulation of immune cells isolated from PP showed a decrease in Thelper (Th)-1 IFN-γ secretion, while splenic Th1/Th17 inflammatory responses sharply increased. E171 or NM-105 for one week did not initiate intestinal inflammation, while a 100-day E171 treatment promoted colon microinflammation and initiated preneoplastic lesions while also fostering the growth of aberrant crypt foci in a chemically induced carcinogenesis model. These data should be considered for risk assessments of the susceptibility to Th17-driven autoimmune diseases and to colorectal cancer in humans exposed to TiO2 from dietary sources.

A PTK7-targeted antibody-drug conjugate reduces tumor-initiating cells and induces sustained tumor regressions.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

Intracellular accumulation and immunological responses of lipid modified magnetic iron nanoparticles in mouse antigen processing cells.

Understanding the effects of magnetic iron nanoparticles (MINPs) on the immune response is vitally important for biomedical applications such as cancer therapy, disease diagnosis and novel cancer imaging. In this study, lipid modified MINPs were designed and prepared by introducing the neutral lipid DSPE-PEG or the zwitterionic lipid DSPE-PCB into hydrophobic MINPs through hydrophobic interaction (L-MINPs and ZL-MINPs, respectively). The effect of L-MINPs and ZL-MINPs on the intracellular accumulation and immune responses of three kinds of antigen processing cells was examined. The results indicated that the high cellular uptake efficiency of surface coated MINPs was strongly related to the nature of the coating lipid, with the zwitterionic lipid being more effective than PEGylated ones. Besides, the results from flow cytometry (FCM), confocal laser scanning microscopy (CLSM) and Prussian blue staining demonstrated a time- and concentration-dependent MINP internalization. The uptake of zwitterionic lipid modified MINPs (ZL-MINPs) induced very low cytotoxicity and a strong mixed Th1/Th2 type immune response. L-MINPs could induce a strong increase in pro-inflammatory cytokines with a slight secretion of Th2 cytokines. Besides, no IL-10 was observed in both groups, indicating that MINPs with lipid modification were absence of immunosuppression. In conclusion, this study addresses an important implication of the lipid type and Fe concentration on the immune stimulation of cells and supports the potential for further development of biomedical applications.

Chemical Synthesis, Versatile Structures and Functions of Tailorable Adjuvants for Optimizing Oral Vaccination.

Oral vaccines have become a recent focus because of their potential significance in disease prevention and therapy. In the development of oral vaccine-based therapeutics, synthetic materials with tailorable structures and versatile functions can act as antigen conveyers with adjuvant effects, reduce the time cost for vaccine optimization, and provide high security and enhanced immunity. This review presents an overview of the current status of tailoring synthetic adjuvants for oral vaccination, modification strategies for producing effectors with specific structures and functions, enhancement of immune-associated efficiencies, including the barrier-crossing capability to protect antigens in the gastrointestinal tract, coordination of the antigens penetrating mucosa and cell barriers, targeting of concentrated antigens to immune-associated cells, and direct stimulation of immune cells. Finally, we focus on prospective synthetic adjuvants that facilitate the use of oral vaccines via two approaches, namely, in vivo antigen expression and cancer immunotherapy.

Sensitization of immune cells following hexylaminolevulinate photodynamic therapy of cervical intraepithelial neoplasia

BackgroundEffects of photodynamic therapy (PDT) were tested with respect to immune cell stimulation in cervical intraepithelial neoplasia (CIN). MethodsA patient with CIN received hexaminolevulinate (HAL) and subsequent PDT. These data were compared to a placebo PDT patient and a healthy HPV16-vaccinated donor. Isolation of peripheral blood mononuclear cells (PBMC) was performed before PDT and at 4 different time points after PDT. The proliferation of these PBMC was tested by [3H]thymidine incorporation following stimulation with control or HPV16-L1 peptides. Potential effects on the CD4+ and CD8+ cell subsets were analysed. ResultsThe data revealed an unchanged or decreased proliferation of HPV16-L1 peptide-stimulated PBMC as compared to placebo patient. HPV16-L1 peptide incubation of PBMC from the HAL patient demonstrated significant proliferative capacity after PDT. The CD4+ and CD8+ T cell populations in placebo patient were slightly increased after HPV16-L1 peptide administration at each time point. Mixed results were obtained for CD4+ cells as compared to controls and nearly unchanged amounts of CD8+ cells were detectable following HPV16-L1 peptide exposure of PBMC from the HAL/PDT-treated patient. ConclusionsThese findings suggest a T cell reaction from CIN patients during repeated HAL/PDT treatment. However, further immune cell populations might be involved during PDT.

Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide.

Neisseria meningitidis contains a very potent hexa-acylated LPS that is too toxic for therapeutic applications. We used systematic molecular bioengineering of meningococcal LPS through deletion of biosynthetic enzymes in combination with induction of LPS modifying enzymes to yield a variety of novel LPS mutants with changes in both lipid A acylation and phosphorylation. Mass spectrometry was used for detailed compositional determination of the LPS molecular species, and stimulation of immune cells was done to correlate this with endotoxic activity. Removal of phosphethanolamine in lipid A by deletion of lptA slightly reduces activity of hexa-acylated LPS, but this reduction is even more evident in penta-acylated LPS. Surprisingly, expression of PagL deacylase in a penta-acylated lpxL1 mutant increased LPS activity, contradicting the general rule that tetra-acylated LPS is less active than penta-acylated LPS. Further modification included expression of lpxP, an enzyme known to add a secondary 9-hexadecenoic acid to the 2’ acyl chain. The LpxP enzyme is temperature-sensitive, enabling control over the ratio of expressed modified hexa- and penta-acylated LPS by simply changing the growth temperature. These LPS derivatives display a broad range of TLR4 activity and differential cytokine induction, which can be exploited for use as vaccine adjuvant or other TLR4-based therapeutics.

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We thank the editors for giving us an opportunity to comment on Drs Kalman and Goldberg’s excellent summary of our paper on the use of obeticholic acid (OCA) in an experimental model of cirrhosis. Their commentary raises several issues regarding the relevance of the inflamed gut as a target for the management of portal hypertension and related complications in cirrhosis. Our main finding was that OCA reduces gut bacterial translocation (GBT) by mitigating intestinal inflammation in a model of noncholestatic cirrhosis. Intestinal inflammation was measured as increased activated immune system cells and inflammation-related gene expression in the gut wall of rats with cirrhosis, especially in those with GBT. In this last setting, inflammation products damage the intestinal barrier and are major contributors to increased intestinal permeability and GBT. In turn, gut dysbiosis seems to be the driver of intestinal inflammation in cirrhosis, because the latter is ameliorated after lowering the intestinal bacterial burden with antibiotics (Hepatology 2012;56:1861–1869). FXR stimulation by OCA normalized increased nuclear factor-κB activation, the expression of inflammation-related cytokine genes and innate defense mechanisms in the bowel wall of cirrhotic rats, as the main mechanism leading to improved intestinal barrier function and reduced GBT. Bacterial infections such as spontaneous peritonitis are an increasing cause of mortality in cirrhosis, and their prophylaxis is circumscribed to selective bowel decontamination with quinolones, a risk factor for infection with MDR strains (Hepatology 2012;55:1551–1561). Targeting gut inflammation to halt GBT is a novel alternative to antibiotics as primary or secondary prophylaxis for bacterial infection that warrants further clinical investigation. As indicated by Kalman and Goldberg, a remarkable finding of our study was the improvement of hepatic fibrogenesis, liver function, and ascites in cirrhotic rats on OCA. Although we did not address the mechanisms involved, it is tempting to speculate that the beneficial effects of OCA at the hepatic level could result from reduced stimulation of liver immune cells owing to lowered endotoxinemia and passage of bacterial products from the gut, and direct inhibition of activated hepatic stellate cells. Additionally, OCA can lower portal pressure by improving the vascular tone of the cirrhotic liver through (1) increased intrahepatic nitric oxide bioavailability (Hepatology 2014;59:2286–2298) and (2) decreased hepatic macrophage activation due to reduced passage of bacterial products from the gut to the portal circulation. Our preclinical study in a noncholestatic model of cirrhosis opens the window to managing portal hypertension and related complications with drugs mechanistically targeting the gut. The Role of Obeticholic Acid in Gut Bacterial Translocation and InflammationGastroenterologyVol. 151Issue 4PreviewUbeda M, Lario M, and Munoz L, et al. Obetocholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats. J Hepatol 2016;64:1049–1057. Full-Text PDF