Immunostimulatory Properties Research Articles

Influence of loop permutation on immunostimulatory activities of CpG oligodeoxynucleotides forming monomeric guanine-quadruplex structures

Introduction: Existing research establishes the role of oligodeoxynucleotides (ODNs) containing cytosine-phosphate-guanine motifs (CpG ODNs) as adjuvants to vaccines against infectious diseases. However, the natural structure of ODN with phosphodiester (PD) linkages is prone to degradation by serum DNase, which limits the application of CpG ODNs. We recently engineered a monomeric guanine-quadruplex (G4)-structured CpG ODN (G4 CpG ODN) and determined that the monomeric G4 increased the DNase resistance, intracellular uptake, and cytokine induction of CpG ODN in vitro and in vivo. However, the effect of the primary sequence on G4 conformation and function of G4 CpG ODN is unclear. Methods: Therefore, in this study, we examine the role of loop permutation, a sequential order of three G4 loops, in G4 folding and immunostimulatory properties of G4 CpG ODNs. We explore the impact of loop permutation on the immunostimulatory activity of GD3_2L, a monomeric G4 CpG ODN that we reported previously. We exchanged the sequence of the second loop with the first and third loops of GD3_2L to obtain GD3_1L and GD3_3L, respectively. The structure of the obtained ODNs was determined by circular dichroism and polyacrylamide gel electrophoresis; the thermodynamic stability of ODN was evaluated through UV melting curve analysis. We assessed the immunostimulatory activity by examining the relative messenger RNA (mRNA) levels of cytokines produced in G4 CpG ODN-stimulated mouse macrophage-like RAW264 by reverse transcription-quantitative polymerase chain reaction method. Results: We found that changing the sequential order of CpG motifs from the second to the first loop resulted in a change in G4 conformation from hybrid to parallel and a decrease in the compactness and thermodynamic stability of GD3_2L. Moreover, swapping order between the second and third loop of GD3_2L led to a hybrid G4 structure that is as stable as GD3_2L but possesses a lower cytokine induction. Conclusion: In conclusion, the loop permutation does not always change the topology and structural compactness; instead, it changes the conformation of CpG motifs in the G4 structure, directly affecting the immunostimulatory properties. Our result demonstrates that the loop-transposition-mediated structural compactness decrease can, if exploited, increase the efficacy of immunostimulatory G4 CpG ODN.

Are Plants Capable of Pheomelanin Synthesis? Gas Chromatography/Tandem Mass Spectrometry Characterization of Thermally Degraded Melanin Isolated from Echinacea purpurea

Echinacea is a widely used plant medicine, valued especially for its well-documented ability to stimulate the immune system. It has been suggested that melanin could be one of the bioactive factors responsible for the immunostimulatory properties of the plant. The biological functions of melanin pigments are closely related to their chemical composition and structural features. The aim of this study was to characterize the melanin from Echinacea purpurea based on the analysis of thermal degradation products of the well-purified pigment extracted from the dried herb. The melanin was pyrolyzed, and the resulting products were separated by gas chromatography and identified using a triple quadrupole mass spectrometer operating in full scan and multiple reaction monitoring modes. Three groups of marker products were detected in the melanin pyrolysate: polyphenol derivatives, nitrogen-containing heterocycles devoid of sulfur, and benzothiazines/benzothiazoles. This suggests that E. purpurea produces three structurally different melanin pigments: allomelanin, eumelanin, and pheomelanin, which in turn may affect the biological activity of the herb. Our results provide the first-ever evidence that plants are capable of synthesizing pheomelanin, which until now, has only been described for representatives of the animal and fungal kingdoms.

Antigen Trafficking and Possibly Intrinsic Immunostimulatory Properties Shape High Immunogenicity of Factor VIII

Antigen Trafficking and Possibly Intrinsic Immunostimulatory Properties Shape High Immunogenicity of Factor VIII

The Effect of Pegbovigrastim Injection on Phagocytic and Oxidative Burst Activities of Peripheral Blood Granulocytes and Monocytes in Calves Challenged with Mycoplasma bovis.

Mycoplasma bovis (M. bovis) is an important pathogen affecting cattle, causing various diseases including pneumonia which mainly occurring in calves. Control of M. bovis infections is difficult due to the lack of commercial vaccines in most parts of the world and increasing trends of antimicrobial resistance in field isolates of the pathogen; therefore, it seems reasonable to look for new solutions for the prevention of the infection. Pegbovigrastim is a pegylated form of naturally occurring circulating cytokine in cattle that affects bovine leukocytes and some cell functions. Most studies on pegbovigrastim have focused on reducing the occurrence of mastitis and other diseases occurring during the periparturient period in cows, while this study attempts to use pegbovigrastim in the prevention of respiratory diseases in calves, which are largely caused by M. bovis. Based on previous observations on the immunostimulatory properties of pegbovigrastim in cattle, for the first time, the effect of its injection on the number and phagocytic and oxidative burst activities of peripheral blood granulocytes and monocytes in calves experimentally infected with M. bovis was investigated. Pegbovigrastim administration in the calves significantly stimulated an increase in peripheral blood granulocyte and monocyte counts and phagocytic activity of the cells, especially granulocytes, which was also generally expressed in the course of M. bovis infection. In response to pegbovigrastim administration, a general increase in the oxygen burst activity of the cells was observed. This effect was also shown despite ongoing infection with M. bovis which, taken together, may indicate a beneficial effect of pegbovigrastim injection on the immunity of the affected animals.

Cell penetrating peptide: A potent delivery system in vaccine development.

One of the main obstacles to most medication administrations (such as the vaccine constructs) is the cellular membrane's inadequate permeability, which reduces their efficiency. Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) are well-known as potent biological nanocarriers to overcome this natural barrier, and to deliver membrane-impermeable substances into cells. The physicochemical properties of CPPs, the attached cargo, concentration, and cell type substantially influence the internalization mechanism. Although the exact mechanism of cellular uptake and the following processing of CPPs are still uncertain; but however, they can facilitate intracellular transfer through both endocytic and non-endocytic pathways. Improved endosomal escape efficiency, selective cell targeting, and improved uptake, processing, and presentation of antigen by antigen-presenting cells (APCs) have been reported by CPPs. Different in vitro and in vivo investigations using CPP conjugates show their potential as therapeutic agents in various medical areas such as infectious and non-infectious disorders. Effective treatments for a variety of diseases may be provided by vaccines that can cooperatively stimulate T cell-mediated immunity (T helper cell activity or cytotoxic T cell function), and immunologic memory. Delivery of antigen epitopes to APCs, and generation of a potent immune response is essential for an efficacious vaccine that can be facilitated by CPPs. The current review describes the delivery of numerous vaccine components by various CPPs and their immunostimulatory properties.

Application of Ozone Therapy in Paediatric Dentistry

The therapeutic use of ozone has been suggested for a long time in general dentistry and paediatric dentistry for its antimicrobial, virucidal, disinfectant, and biocompatible properties. Ozone has also anti-inflammatory, analgesic, and immunostimulant properties, and it promotes tissue regeneration. Dental treatment in children is often complex and ozone could support the clinician to enhance the approach and prognosis with young patients, non-cooperant children, or special needs patients. The efficiency of ozone will certainly increase if studies continue to show positive outcomes in a growing number of dental paediatric conditions. This review explores the recent literature of ozone therapy in paediatric dentistry and suggests fields of application for future randomized controlled trials (RCTs).

Characterization of immunomodulatory responses induced by manuka honey.

Manuka honey (MH) is known for its wound-healing, anti-microbial, anti-oxidant and anti-tumor properties. However, there is conflicting evidence regarding the role of MH in inflammatory responses, with some studies highlighting its pro-inflammatory capacity and others showing that it has a predominantly anti-inflammatory activity. The current study is aimed at characterizing the immunomodulatory capacity of MH using both in vitro and in vivo approaches, focusing on the underlying mechanisms. Treatment of RAW 264.7 macrophages with 1% MH (w/v) resulted in a significant increase in the gene expression (~26-fold) and secretion (~27-fold) of tumor necrosis factor-alpha (TNF-α). Similarly, an increase was observed in the gene expression of other inflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS), as well as the chemokines; (C-X-C motif) ligand 2 (CXCL2) and (C-C) motif ligand 2 (CCL2). Using an in vivo model, intraperitoneal (i.p.) administration of MH in C57BL/6 mice elicited a peritoneal response characterized by a significant expansion in the number of peritoneal exudate cells (PECs), which was mainly due to a 35-fold increase in the recruitment of neutrophils. Importantly, this response was evident in toll-like receptor 4 (TLR4)-defective C3H/HeJ mice, indicating that the observed stimulatory effect occurs independently of TLR4 and unlikely to be mediated by any lipopolysaccharide (LPS) contaminant. MH administration also led to changes in the phenotypic expression and functional maturation of peritoneal macrophages, as evidenced by a shift towards the CD11blo F4/80lo phenotype and an increase in the expression of major histocompatibility complex (MHC) class II proteins. In contrast, the MH-initiated peritoneal response was largely abrogated in mice deficient in myeloid differentiation primary response 88 (MyD88) protein, a critical adaptor of most TLR signaling pathways. Thus, the current findings help to characterize the immunostimulatory properties of MH and their dependence on TLR signaling, and highlight the potential utility of MH as an immunomodulatory agent in a variety of disorders.

Beta-Glucan as a Novel Functional Fiber: Functional Properties, Health Benefits and Food Applications

Recently the demand for functional food components such as prebiotics, probiotics, and phenolic compounds are increased. Nowadays, β-glucan, dietary fiber, and biologically active natural polysaccharide, exhibit properties like a prebiotic effect and immune system support. Furthermore, clinical studies have shown that daily intake of 250-500 mg β-glucan has several benefits on the immune system, showing low glycemic index, antimicrobial and anti-inflammatory properties. It is a polysaccharide composed of D-glucose monomers connected with β-glycoside bonds, found in natural sources like yeast (Saccharomyces cerevisiae), some edible mushrooms, algae, and cereal grains. β-Glucan is more resistant and preserves its stability during food processing technologies (drying, freezing, etc.). Especially, β-glucan originated from baker's yeast (S. cerevisiae) has many immunostimulatory properties, such as hypoglycemic, anti-inflammatory, immune-modulatory, antioxidant, and anticancer activities. Also, the utilization of β-glucan in the food industry has increased regarding to their techno-functional properties like gelation, viscosity, solubility, and encapsulating agent. Therefore, this review highlights recently published research results on the functional properties, health benefits and clinical studies, the effects on the interaction with the other food compounds, and the potential food applications of β-glucans.

Dynamics of morphological and biochemical indicators of blood of young pigs using Globigen® Pig Doser and Globigen ® Jump Start with different methods of their keeping

The article deals with the influence of Globigen® Pig Doser and Globigen® Jump Start products, which contain specific Ig Y, which is characterized by pronounced immunostimulating properties. A comprehensive analysis of the use and influence of these agents on the morphological and biochemical indicators of the blood of young pigs under conditions of intensive technologies under different methods of cultivation in different periods of their growth and development – 30, 60, and 120 days – was carried out. It was set that the use of the proposed means had a prolonged positive effect, which concerning hematological indicators, mainly appeared by an increase in the number of erythrocytes and the concentration of hemoglobin in the blood of experimental groups of animals of both farms of all age periods. It is worth noting that in the farm with single-phase breeding of pigs (FE PE “Glynyany Agro”) under the influence of Globigen®Pig Doser, the number of erythrocytes in the blood of animals was increased and, corresponding to the physiological norm, fluctuated between 7.09 and 7.43 T/l, and the concentration of hemoglobin, having reached the maximum value, in the blood of all experimental groups of animals was 108.62–112.77 g/l. In the farm, which practices three-phase pig breeding (“Meat Resources” Ltd.), under the influence of Globigen® Pig Doser, a pronounced effect has appeared in animals 60 and 120 days old; the number of erythrocytes in their blood was increased to 7.46–7.69 T/l, and under the influence of Globigen® Jump Start, a tendency to a gradual increase in hemoglobin concentration was observed, which stopped at 99.28 g/l in 120-day-old piglets. Evaluating the level of protein metabolism in animals under the influence of Globigen® Pig Doser and Globigen® Jump Start, a tendency to increase the total protein content, compared to the control, in the blood of animals in all age periods in both farms was set. It was noted that under the action of Globigen®Jump Start, the total protein content in the blood of 120-day-old piglets from a farm with single-phase farming (FE PE “Glynyany Agro”) was 76.38 g/l, and in a farm with three-phase farming (Ltd. “Meat resources”) 75.08 g/l. In addition, it was set that the normalization of the ratio of protein fractions, in particular, the use of Globigen® Pig Doser and Globigen® Jump Start, increased the synthesis of albumin and normalized gamma-globulin to the level of the physiological norm. The “Glynyany Agro” FE found a more pronounced effect with single-phase piglet rearing. It was concluded that Globigen® Pig Doser and Globigen® Jump Start positively influence maintaining body homeostasis.

Monophosphoryl lipid A-adjuvanted nucleoprotein-neuraminidase nanoparticles improve immune protection against divergent influenza viruses

Universal influenza vaccines are urgently needed to prevent recurrent influenza epidemics and inevitable pandemics. We generated double-layered protein nanoparticles incorporating two conserved influenza antigens—nucleoprotein and neuraminidase—through a two-step desolvation-crosslinking method. These protein nanoparticles displayed immunostimulatory properties to antigen-presenting cells by promoting inflammatory cytokine (IL-6 and TNF-α) secretion from JAWS II dendric cells. The nanoparticle immunization induced significant antigen-specific humoral and cellular responses, including antigen-binding and neutralizing antibodies, antibody- and cytokine (IFN-γ and IL-4)-secreting cells, and NP147–155 tetramer-specific cytotoxic T lymphocyte (CTL) responses. Co-administration of monophosphoryl lipid A (MPLA, a toll-like receptor 4 agonist) with the protein nanoparticles further improved immune responses and conferred heterologous and heterosubtypic influenza protection. The MPLA-adjuvanted nanoparticles reduced lung inflammation post-infection. The results demonstrated that the combination of MPLA and conserved protein nanoparticles could be developed into an improved universal influenza vaccine strategy.

Localized Nano-mediated Interleukin-12 Gene Therapy: Promising Candidate for Cancer Immunotherapeutics.

Interleukin-12 (IL-12) has a pleiotropic nature that allows it to induce immune responses while reversing tumour-induced immunosuppression. Therefore, this paper discusses the application and potential of IL-12 as an antitumor immunotherapeutic agent, emphasizing its advantages and limitations and the need for and the development of localized IL-12 nano-delivery strategies in cancer immunotherapy. Several databases from the National Centre for Biotechnology Information, WorldCat.org and the National Library of Medicine were searched for peer-reviewed studies to assess the potential of localized nano-mediated interleukin-12 gene therapy for cancer treatment. The literature search showed that IL-12 is a promising cancer immunotherapeutic agent. However, the systemic delivery of IL-12 was compromised by severe dose-limiting side effects, prompting the need for localized gene therapy to express the interleukin within the tumour microenvironment while minimizing systematic exposure. Although viral and non-viral gene therapy have demonstrated some efficacy in preclinical trials, the era of nanomedicine has opened novel avenues to improve therapeutic indices with minimal side effects. IL-12 activity can be further potentiated with other anticancer molecules that display immunostimulatory, autoantigenic and cytotoxic properties. Combination therapy has gained significant interest in the last decade as it increases gene therapy's therapeutic properties by decreasing the threshold for IL-12 efficacy and preventing systematic toxicity. The findings of this article will provide researchers with the knowledge to create immunotherapeutic nanovectors which work synergistically with their therapeutic payload to enhance the therapeutic effect of the IL-12 gene to eliminate cancer cells.

Testing of Medical Sheep Fur with Antimicrobial Properties – Part 1

The medical treatment of patients with rheumatic, joint and muscle diseases can be improved by alternative methods. The essential oils extracted from different plants have certain analgesic, anti-inflammatory, antiseptic, antibacterial, immunostimulating properties, etc. Products have been made based on essential oils with therapeutic properties (mint, cajeput, eucalyptus), which can be used to treat the surface of finished sheep fur for medical purposes. The sheep furs were tanned (without metals) with syntans based on phenolsulfonic acids and aromatic oxysulfones and treated with products based on essential oils with therapeutic properties, to be used to make medical fur articles (lumbar and cervical belts, knee pads, elbow pads, etc.). They can improve rheumatic, muscular and circulatory conditions, complementing the medical treatment of patients suffering from these conditions. The work presents the chemical, physical-mechanical and microbiological characterization of natural furs for medical use.

A review on Immunostimulatory and antioxidant potential of herbs, Curcuma longa L., Camellia sinensis L. Zingiber officinale and Allium sativum Linn. in fish health: a sustainable approach for a healthy aquaculture

The usage of antibiotics and chemotherapeutics agents in aquaculture and their undesired consequencesleads to the search for other alternatives. Plants are rich in active compounds includes alkaloids, flavonoids,terpenoids, phenols and polypeptides which proved to maintain a good health status in fish by acting asgrowth promoters, stress resistance and preventions of infections. Phytomedicine provides a cheaper sourcefor the treatment and greater accuracy than chemotherapeutic agents without causing toxicity. There arediverse medicinal plants in India which are being used extensively in aquaculture for several purposes.Present review focuses only on the immunostimulatory and antioxidant property of some common herbs;turmeric, green tea, ginger and garlic owing to their effectiveness and availability. The studied herbs possesspotent immunostimulant and antioxidant compounds which conferring early activation to the non-specificdefence mechanisms of fish and boosted the immune responses like phagocytic and respiratory burst activity,lysozyme activity, complement proteins, immunoglobulins (Igs), protease, anti-protease and alkalineprotease (ALP) and leucocytes promotion etc. It also inhibits the generation of oxygen anions and scavengesfree radical by increasing the antioxidant enzymes activity like catalase (CAT), glutathione (GSH), glutathioneperoxidase (GPx) and superoxide dismutase (SOD) during emergence of physiological disturbances. Theseherbs will promote the eco-friend health management of highly commercial fish species withoutcompromising the aquatic environment and ensure sustainable aquaculture with higher yield and nutrientcontent.

LACpG10-HL Functions Effectively in Antibiotic-Free and Healthy Husbandry by Improving the Innate Immunity.

Antibiotics are broadly restricted in modern husbandry farming, necessitating the need for efficient and low-cost immunomodulatory preparations in antibiotic-free and healthful farming. As is known to all, CpG oligonucleotides (CpG-ODNs, an effective innate immunostimulatory agent) recognized by TLR9 in mammals (while TLR21 in avians) could collaborate with some united agent to induce stronger immune responses, but the cost is prohibitively expensive for farmers. Here, considering the coordination between TLR2 and TLR9/TLR21, we firstly proposed the idea that the well-fermented Lactococcus lactis could be utilized as a CpG-plasmid carrier (LACpG10) to enhance the host’s innate immunity against pathogenic invasion. In the present study, after obtaining LACpG10-HL from homogenized and lyophilized recombinant strain LACpG10, we treated primary chicken lymphocytes, two cell lines (HD11 and IPEC-J2), and chickens with LACpG10-HL, CpG plasmids (pNZ8148-CpG10), and other stimulants, and respectively confirmed the effects by conducting qRT-PCR, bacterial infection assays, and a zoological experiment. Our data showed that LACpG10-HL could induce excellent innate immunity by regulating autophagy reactions, cytokine expression, and motivating PRRs. Interestingly, despite having no direct antiseptic effect, LACpG10-HL improved the antibacterial capacities of lymphocytes and enterocytes at the first line of defense. Most importantly, water-supplied LACpG10-HL treatment reduced the average adverse event rates, demonstrating that LACpG10-HL maintained its excellent immunostimulatory and protective properties under farming conditions. Our research not only contributes to revealing the satisfactory effects of LACpG10-HL but also sheds new light on a cost-effective solution with optimal immune effects in green, antibiotic-free, and healthful husbandry farming.

C3H Expression Is Crucial for Methyl Jasmonate Induction of Chicoric Acid Production by Echinacea purpurea (L.) Moench Cell Suspension Cultures.

Echinacea purpurea (L.) Moench is one of the most economically important medicinal plants, cultivated worldwide for its high medicinal value and with several industrial applications in both pharmaceutical and food industries. Thanks to its various phytochemical contents, including caffeic acid derivatives (CADs), E. purpurea extracts have antioxidant, anti-inflammatory, and immuno-stimulating properties. Among CADs, chicoric acid is one of the most important compounds which have shown important pharmacological properties. The present research was aimed at optimizing the production of chicoric acid in E. purpurea cell culture. Methyl jasmonate (MeJa) at different concentrations and for different duration of treatments was utilized as elicitor, and the content of total polyphenols and chicoric acid was measured. Several genes involved in the chicoric acid biosynthetic pathway were selected, and their expression evaluated at different time points of cell culture growth. This was performed with the aim of identifying the most suitable putative molecular markers to be used as a proxy for the early prediction of chicoric acid contents, without the need of expensive quantification methods. A correlation between the production of chicoric acid in response to MeJa and an increased response to oxidative stress was also proposed.

Myxoma virus lacking the host range determinant M062 stimulates cGAS-dependent type 1 interferon response and unique transcriptomic changes in human monocytes/macrophages.

The evolutionarily successful poxviruses possess effective and diverse strategies to circumvent or overcome host defense mechanisms. Poxviruses encode many immunoregulatory proteins to evade host immunity to establish a productive infection and have unique means of inhibiting DNA sensing-dependent type 1 interferon (IFN-I) responses, a necessity given their dsDNA genome and exclusively cytoplasmic life cycle. We found that the key DNA sensing inhibition by poxvirus infection was dominant during the early stage of poxvirus infection before DNA replication. In an effort to identify the poxvirus gene products which subdue the antiviral proinflammatory responses (e.g., IFN-I response), we investigated the function of one early gene that is the known host range determinant from the highly conserved poxvirus host range C7L superfamily, myxoma virus (MYXV) M062. Host range factors are unique features of poxviruses that determine the species and cell type tropism. Almost all sequenced mammalian poxviruses retain at least one homologue of the poxvirus host range C7L superfamily. In MYXV, a rabbit-specific poxvirus, the dominant and broad-spectrum host range determinant of the C7L superfamily is the M062R gene. The M062R gene product is essential for MYXV infection in almost all cells tested from different mammalian species and specifically inhibits the function of host Sterile α Motif Domain-containing 9 (SAMD9), as M062R-null (ΔM062R) MYXV causes abortive infection in a SAMD9-dependent manner. In this study we investigated the immunostimulatory property of the ΔM062R. We found that the replication-defective ΔM062R activated host DNA sensing pathway during infection in a cGAS-dependent fashion and that knocking down SAMD9 expression attenuated proinflammatory responses. Moreover, transcriptomic analyses showed a unique feature of the host gene expression landscape that is different from the dsDNA alone-stimulated inflammatory state. This study establishes a link between the anti-neoplastic function of SAMD9 and the regulation of innate immune responses.

The Presence of Molds and Their Secondary Metabolites in Purple Coneflower-Based Dietary Supplements (Echinacea purpurea (L.) Moench).

Purple coneflower (Echinacea purpurea (L.) Moench) is a plant in the family Asteraceae, mainly grown in North America. Echinacea purpurea has been used in conventional medicine. The plant has immuno-stimulating and antibacterial properties, but neither mold contamination nor a mycotoxin presence have been evaluated. Our goal is to determine the degree to which molds and mycotoxins contaminate dietary supplements based on purple coneflower distributed on the Polish market. We analyzed 21 samples divided into four groups: sachets (n = 5), dry raw material (n = 3), capsules (n = 9), and tablets (n = 4). The mycological analysis of dietary supplements shows that the average number of molds is 1012 cfu/g, and the most common molds are Aspergillus spp., Phoma spp. and Eurotium spp. The mycotoxins most common in the samples are ZEN (18/21), DON (5/21) and T-2 toxin (3/21).

FLOT and CROSS chemotherapy regimens alter the frequency of CD27+ and CD69+ T cells in oesophagogastric adenocarcinomas: implications for combination with immunotherapy

Combining immunostimulatory chemotherapies with immunotherapy is an attractive strategy to enhance treatment responses in oesophagogastric junctional adenocarcinoma (OGJ). This study investigates the immunostimulatory properties of FLOT, CROSS and MAGIC chemotherapy regimens in the context of OGJ using in vitro and ex vivo models of the treatment-naïve and post-chemotherapy treated tumour microenvironment. FLOT and CROSS chemotherapy regimens increased surrogate markers of immunogenic cell death (HMGB1 and HLA-DR), whereas the MAGIC treatment regimen decreased HMGB1 and HLA-DR on OGJ cells (markedly for epirubicin). Tumour-infiltrating and circulating T cells had significantly lower CD27 expression and significantly higher CD69 expression post-FLOT and post-CROSS treatment. Similarly, the supernatant from FLOT- and CROSS-treated OGJ cell lines and from FLOT- and CROSS-treated OGJ biopsies cultured ex vivo also decreased CD27 and increased CD69 expression on T cells. Following 48 h treatment with post-FLOT and post-CROSS tumour conditioned media the frequency of CD69+ T cells in culture negatively correlated with the levels of soluble immunosuppressive pro-angiogenic factors in the conditioned media from ex vivo explants. Supernatant from FLOT- and CROSS-treated OGJ cell lines also increased the cytotoxic potential of healthy donor T cells ex vivo and enhanced OGJ patient-derived lymphocyte mediated-killing of OE33 cells ex vivo. Collectively, this data demonstrate that FLOT and CROSS chemotherapy regimens possess immunostimulatory properties, identifying these chemotherapy regimens as rational synergistic partners to test in combination with immunotherapy and determine if this combinatorial approach could boost anti-tumour immunity in OGJ patients and improve clinical outcomes.

Association of Indocyanine Green with Chitosan Oleate Coated PLGA Nanoparticles for Photodynamic Therapy.

Indocyanine green (ICG) is a safe dye widely used in the biomedical field. Its photodynamic effect (PDT), originating from laser irradiation at 803 nm, opens interesting perspectives in theranostic applications. To overcome its low water stability, ICG can be shielded with nanoparticles (NPs). In this work, previously developed NPs based on poly lactic-co-glycolic acid (PLGA) coated with chitosan oleate (CS-OA) and loaded with resveratrol as a hydrophobic model drug have been proposed as an ICG carrier. These systems have been selected for their observed immunostimulatory properties. The possible loading of the dye by adsorption onto NP surface by electrostatic interaction was studied here in comparison with the encapsulation into the PLGA core. The ICG-chitosan (CS) interaction has been characterized by spectrophotometry, spectroscopy and in-cell in vitro assays. Fluorescence quenching was observed due to the ionic interaction between ICG and CS and was studied considering the dye:polymer stoichiometry and the effect of the NP dilution in cell culture medium (DMEM). The NP systems have been compared in vitro, assessing their behaviour in Caco-2 cell lines. A reduction in cell viability was observed after irradiation of ICG associated with NPs, evident also for the samples loaded by adsorption. These findings open the opportunity to exploit the association of PDT’s effect on ICG with the properties of CS-OA coated NPs, whose immunostimulatory effect can be associated with PDT mechanism in cancer therapy.

Self-Assembled Immunostimulatory Tetrahedral Framework Nucleic Acid Vehicles for Tumor Chemo-immunotherapy.

Some chemotherapeutic agents, such as anthracyclines and oxaliplatin, can induce immunogenic cell death (ICD) with additional immune responses against cancer. However, ICD-based immunotherapy is limited by the nonspecific distribution of drugs and various side effects. Here, an immunostimulatory self-assembled tetrahedral framework nucleic acid (tFNA) vehicle was constructed to potentiate the chemo-immunotherapy, in which doxorubicin (DOX) acted as a chemotherapeutic agent and an ICD-inducer. Meanwhile, the immunostimulatory CpG-tFNA was employed as a nanocarrier to deliver DOX and an adjuvant to enhance the immunotherapy. Damage-associated molecular patterns (DAMPs) generated by DOX from dying tumor cells, such as calreticulin (CRT), high mobility group protein 1(HMGB1), and adenosine triphosphate (ATP), can activate dendritic cells (DCs) and trigger an immunological response. Afterward, CpG-tFNA with immunostimulatory properties works to boost the DOX-induced immunotherapy. Consequently, CpG-tFNA/DOX showed excellent antitumor effects and immunological activation, including CD8+ T cell proliferation and antitumor cytokine TNF-α and IFN-γ secretion. Moreover, chemo-immunotherapy can also be enhanced synergistically when coadministered with PD-L1. In conclusion, CpG-tFNA/DOX promotes the ICD-associated chemo-immunotherapy and strengthens the connection between traditional chemotherapy and immunotherapy, representing a novel strategy for clinical application. Moreover, the concept of ICD-related immunotherapy can also be extended to other treatments such as radiotherapy which can induce immunogenic cell death as well.