Long-range, water-mediated interaction between a moderately active antifreeze protein molecule and the surface of ice.

Using molecular dynamics simulations, we show that a molecule of moderately active antifreeze protein (type III AFP, QAE HPLC-12 isoform) is able to interact with ice in an indirect manner. This interaction occurs between the ice binding site (IBS) of the AFP III molecule and the surface of ice, and it is mediated by liquid water, which separates these surfaces. As a result, the AFP III molecule positions itself at a specific orientation and distance relative to the surface of ice, which enables the effective binding (via hydrogen bonds) of the molecule with the nascent ice surface. Our results show that the final adsorption of the AFP III molecule on the surface of ice is not achieved by chaotic diffusion movements, but it is preceded by a remote, water-mediated interaction between the IBS and the surface of ice. The key factor that determines the existence of this interaction is the ability of water molecules to spontaneously form large, high-volume aggregates that can be anchored to both the IBS of the AFP molecule and the surface of ice. The results presented in this work for AFP III are in full agreement with the ones obtained by us previously for hyperactive CfAFP, which indicates that the mechanism of the remote interaction of these molecules with ice remains unchanged despite significant differences in the molecular structure of their ice binding sites. For that reason, we can expect that also other types of AFPs interact with the ice surface according to an analogous mechanism.

Solubility of carbon dioxide in water: Some useful results for hydrate nucleation.

In this paper, the solubility of carbon dioxide (CO2) in water along the isobar of 400 bar is determined by computer simulations using the well-known TIP4P/Ice force field for water and the TraPPE model for CO2. In particular, the solubility of CO2 in water when in contact with the CO2 liquid phase and the solubility of CO2 in water when in contact with the hydrate have been determined. The solubility of CO2 in a liquid-liquid system decreases as the temperature increases. The solubility of CO2 in a hydrate-liquid system increases with temperature. The two curves intersect at a certain temperature that determines the dissociation temperature of the hydrate at 400 bar (T3). We compare the predictions with T3 obtained using the direct coexistence technique in a previous work. The results of both methods agree, and we suggest 290(2) K as the value of T3 for this system using the same cutoff distance for dispersive interactions. We also propose a novel and alternative route to evaluate the change in chemical potential for the formation of hydrates along the isobar. The new approach is based on the use of the solubility curve of CO2 when the aqueous solution is in contact with the hydrate phase. It considers rigorously the non-ideality of the aqueous solution of CO2, providing reliable values for the driving force for nucleation of hydrates in good agreement with other thermodynamic routes used. It is shown that the driving force for hydrate nucleation at 400 bar is larger for the methane hydrate than for the carbon dioxide hydrate when compared at the same supercooling. We have also analyzed and discussed the effect of the cutoff distance of dispersive interactions and the occupancy of CO2 on the driving force for nucleation of the hydrate.

Selective tumor antigen vaccine delivery to human CD169+ antigen-presenting cells using ganglioside-liposomes.

Priming of CD8+ T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1+ antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14+ CD169+ monocytes and Axl+ CD169+ DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169+ moDCs and Axl+ CD169+ DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8+ T cells. Finally, Axl+ CD169+ DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169+ DCs to drive antitumor T cell responses.

Bone marrow-derived myeloid progenitors as driver mutation carriers in high- and low-risk Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a myeloid neoplasia, driven by sporadic activating mutations in the MAPK pathway. The misguided myeloid dendritic cell (DC) model proposes that high-risk, multisystem, risk-organ-positive (MS-RO+) LCH results from driver mutation in a bone marrow (BM)-resident multipotent hematopoietic progenitor, while low-risk, MS-RO- and single-system LCH would result from driver mutation in a circulating or tissue-resident, DC-committed precursor. We have examined the CD34+c-Kit+Flt3+ myeloid progenitor population as potential mutation carrier in all LCH disease manifestations. This population contains oligopotent progenitors of monocytes (Mo's)/macrophages (MΦs), osteoclasts (OCs), and DCs. CD34+c-Kit+Flt3+ cells from BM of MS-RO+ LCH patients produced Langerhans cell (LC)-like cells in vitro. Both LC-like and DC offspring from this progenitor carried the BRAF mutation, confirming their common origin. In both high- and low-risk LCH patients, CD34+c-Kit+Flt3+ progenitor frequency in blood was higher than in healthy donors. In one MS-RO+ LCH patient, CD34+c-Kit+Flt3+ cell frequency in blood and its BRAF-mutated offspring reported response to chemotherapy. CD34+c-Kit+Flt3+ progenitors from blood of both high- and low-risk LCH patients gave rise to DCs and LC-like cells in vitro, but the driver mutation was not easily detectable, likely due to low frequency of mutated progenitors. Mutant BRAF alleles were found in Mo's /MΦs, DCs, LC-like cells, and/or OC-like cells in lesions and/or Mo and DCs in blood of multiple low-risk patients. We therefore hypothesize that in both high- and low-risk LCH, the driver mutation is present in a BM-resident myeloid progenitor that can be mobilized to the blood.

Seasonal influenza vaccination among cancer patients during the COVID-19 pandemic in Poland.

INTRODUCTION: In the era of the COVID-19 pandemic overlapping with the influenza season, the number of infections with the abovementioned viruses may result in overload in the healthcare system, difficulties in the diagnosis of respiratory diseases, poorer access to appropriate therapy, and increased mortality. AIM OF THE STUDY: The aim of this study was to analyze the influence of the COVID-19 pandemic on the decision to be vaccinated against seasonal influenza in cancer patients. MATERIAL AND METHODS: An anonymous survey prepared by the authors was made available to patients at the Chemotherapy Department at the Greater Poland Cancer Center. The survey covered 236 respondents, both female (67.4%, n = 159) and male (32.6%, n =77). A 0-10 point numerical scale was used to assess the fear of coronavirus infection and the influenza. Data were collected from June 8 to September 30, 2020. The survey included 25 questions. The patients were informed by physicians about the voluntary and anonymous nature of the survey, to which they gave their oral consent. IBM SPSS Statistics 26 was used for the analysis. RESULTS: The vast majority of patients (69.5%, n = 164) have never been vaccinated against influenza and 30.5% (n = 72) have been vaccinated at least once in the past. In the face of the COVID-19 pandemic, almost » of the patients (24.6%, n = 58) stated that they wanted to be vaccinated against influenza. Only 33.5% (n = 79) of the respondents believed that the influenza vaccine was effective. CONCLUSIONS: Action is needed to increase the percentage of cancer patients who will be regularly vaccinated against the influenza. The COVID-19 pandemic may raise the interest of cancer patients in influenza vaccination.

Why is the cubic structure preferred in newly formed ice?

Molecular dynamics was employed to explain the preference for the cubic structure in newly formed crystals of ice. The results showed that in supercooled liquid water the molecules connected by hydrogen bonds are more likely to adopt relative orientations similar to the ones characteristic for cubic ice. The observed preference for certain relative orientations of molecules in the hydrogen-bonded pairs results in the higher probability of the formation of ice with the cubic structure. On that basis, it was concluded that the main reason for the increased probability of the formation of cubic ice in solidifying water is the distinctive structure of liquid water.

Egg-specific IgE and basophil activation but not egg-specific T-cell counts correlate with phenotypes of clinical egg allergy.

BACKGROUND: Egg allergy is phenotypically heterogeneous. A subset of patients with egg allergy can tolerate egg in an extensively heated form. Inclusion of baked egg (BE) into the diet accelerates resolution of egg allergy. Conversely, BE reactivity is associated with persistent disease. The immune basis of this clinical heterogeneity is unknown. OBJECTIVES: We sought to study egg-specific antibody, basophil, and T-cell responses in children with reactivity or tolerance to BE. METHODS: All participants underwent double-blind, placebo-controlled challenges to BE, and those who tolerated BE were challenged with unheated egg white protein to confirm clinical egg reactivity. Laboratory studies included serum antibody measurements, basophil activation tests, and CD154-based detection of egg-responsive T cells by using flow cytometry. RESULTS: Of the 129 children studied, BE-reactive participants had significantly greater levels of egg-, ovalbumin-, and ovomucoid-specific IgE; lower ratios of egg-specific IgG4/IgE; and increased basophil activation in response to egg. Among all participants, CD154-based profiling revealed egg-responsive T cells producing IL-4 and IL-13 but little IL-10 or IFN-γ, as well as the presence of egg-responsive Foxp3+CD25+CD127low regulatory T cells. Egg-responsive T cells expressed CCR4, CCR6, and CXCR5, indicating capacity for homing to the skin, mucosa, and B-cell follicles. However, neither the frequency nor phenotype of egg-responsive T cells was different in those with tolerance or reactivity to BE. CONCLUSIONS: Egg-specific antibody and basophil responses, but not T-cell responses, are greater in those with reactivity versus tolerance to BE. Egg-specific antibody and T-cell responses were highly heterogeneous in this cohort. The clinical implications of this immune heterogeneity will need to be studied longitudinally.

Molecular dynamics study on the role of solvation water in the adsorption of hyperactive AFP to the ice surface.

Using computer simulations, the early stages of the adsorption of the CfAFP molecule to the ice surface were analyzed. We found that the ice and the protein interact at least as early as when the protein is about 1 nm away from the ice surface. These interactions are mediated by interfacial solvation water and are possible thanks to the structural ordering of the solvent. This ordering leads to positional preference of the protein relative to the ice crystal before the final attachment to the ice surface takes place, accompanied by the solidification of the interfacial water. It is possible because the solvation water of the ice-binding plane of CfAFP is susceptible to the overlapping with the solvation water of ice and is mostly changeable into ice itself. These remote interactions significantly increase efficacy of the adsorption process by facilitating the geometric adjustment of the active region of the CfAFP molecule to the ice surface. Because of the ordered nature of the water molecules at the ice-binding plane, the energy of their interactions with the ice-binding surface of the protein does not change upon the ongoing solidification of solvation water. However, the structure of the solvation water is not strictly ice-like and the growth of ice in the interfacial water is not initiated at the side of the protein. On the contrary, we find that solvation water of CfAFP solidifies slower than solvation water of ice - the solidification of interfacial water starts at the surface of ice. Moreover, in the presence of the CfAFP molecule, also solvation water of ice solidifies slower compared to the situation when the protein is not present next to the ice surface. Additionally, the presence of the protein molecule shifts the ratio of cubic to hexagonal ice that spontaneously forms at the ice surface, by introducing another layer of ordered water molecules - opposite to the ice lattice, at the other side of the crystallizing layer of water.

The accretion of the new ice layer on the surface of hexagonal ice crystal and the influence of the local electric field on this process.

The process of creation of a new layer of ice on the basal plane and on the prism plane of a hexagonal ice crystal is analyzed. It is demonstrated that the ordering of water molecules in the already existing crystal affects the freezing. On the basal plane, when the orientations of water molecules in the ice block are random, the arrangement of the new layer in a cubic manner is observed more frequently-approximately 1.7 times more often than in a hexagonal manner. When the water molecules in the ice block are more ordered, it results in the predominance of the oxygen atoms or the hydrogen atoms on the most outer part of the surface of the ice block. In this case, the hexagonal structure is formed more frequently when the supercooling of water exceeds 10 K. This phenomenon is explained by the influence of the oriented electric field, present as a consequence of the ordering of the dipoles of water molecules in the ice block. This field modifies the structure of solvation water (i.e., the layer of water in the immediate vicinity of the ice surface). We showed that the structure of solvation water predetermines the kind of the newly created layer of ice. This effect is temperature-dependent: when the temperature draws nearer to the melting point, the cubic structure becomes the prevailing form. The temperature at which the cubic and the hexagonal structures are formed with the same probabilities is equal to about 260 K. In the case of the prism plane, the new layer that is formed is always the hexagonal one, which is independent of the arrangement of water molecules in the ice block and is in agreement with previous literature data. For the basal plane, as well as for the prism plane, no evident dependence on the ordering of water molecules that constitute the ice block on the rate of crystallization can be observed.

Structure of solvation water around the active and inactive regions of a type III antifreeze protein and its mutants of lowered activity.

Water molecules from the solvation shell of the ice-binding surface are considered important for the antifreeze proteins to perform their function properly. Herein, we discuss the problem whether the extent of changes of the mean properties of solvation water can be connected with the antifreeze activity of the protein. To this aim, the structure of solvation water of a type III antifreeze protein from Macrozoarces americanus (eel pout) is investigated. A wild type of the protein is used, along with its three mutants, with antifreeze activities equal to 54% or 10% of the activity of the native form. The solvation water of the ice-binding surface and the rest of the protein are analyzed separately. To characterize the structure of solvation shell, parameters describing radial and angular characteristics of the mutual arrangement of the molecules were employed. They take into account short-distance (first hydration shell) or long-distance (two solvation shells) effects. The obtained results and the comparison with the results obtained previously for a hyperactive antifreeze protein from Choristoneura fumiferana lead to the conclusion that the structure and amino acid composition of the active region of the protein evolved to achieve two goals. The first one is the modification of the properties of the solvation water. The second one is the geometrical adjustment of the protein surface to the specific crystallographic plane of ice. Both of these goals have to be achieved simultaneously in order for the protein to perform its function properly. However, they seem to be independent from one another in a sense that very small antifreeze activity does not imply that properties of water become different from the ones observed for the wild type. The proteins with significantly lower activity still modify the mean properties of solvation water in a right direction, in spite of the fact that the accuracy of the geometrical match with the ice lattice is lost because of the mutations. Therefore, we do not observe any correlation between the antifreeze activity and the extent of modification of the properties of solvation water.

Parasitization of invasive gobiids in the eastern part of the Central trans-European corridor of invasion of Ponto-Caspian hydrobionts.

Four gobiid species, Babka gymnotrachelus, Neogobius melanostomus, Neogobius fluviatilis, and Proterorhinus semilunaris, were parasitologically studied in different localities of the Dnieper and Vistula river basins. The highest number of parasitic species was found in N. fluviatilis (35 taxa). The parasite fauna of N. melanostomus, B. gymnotrachelus, and P. semilunaris consists of 23, 22, and 15 taxa, respectively. The species accumulation curves show stable accumulation of parasite species by all four fish hosts along the studied part of the corridor, from the Dnieper Estuary to the Vistula River delta. The plot reveals also that the studied gobies lose the parasites common in the host native range and accept new parasites from the colonized area. In the case of N. melanostomus, it complies with the enemy release hypothesis, as the parasite load was low in the invaded area if compared to the native range. The three other alien gobies are vector for Gyrodactylus proterorhini in the Baltic basin. Moreover, populations of this alien monogenean tend to be more abundant in their new range in comparison with the Black Sea basin. In general, the number of parasite species in the colonized area was of the same rank as in the native one for N. fluviatilis, and even higher for B. gymnotrachelus. This results from accumulating new parasite species along the gobiid invasion route. In particular, the N. fluviatilis, B. gymnotrachelus, and P. semilunaris lost some of their native parasites and gained the local ones after entering the post-dam part of the Vistula River; it can be interpreted as a partial escape from parasites.

Effects of Pilates Training on Cardiorespiratory Functions in Medical Conditions - Comprehensive Approach: A Narrative Review.

Cardiorespiratory fitness (CRF) is established as a clinical vital sign in therapeutic strategy to restoring health of patients in medical conditions inclusive of age-related diseases. The beneficial effects of Pilates training (PT) are recognized for various aspects of health and fitness, but limited data present an impact on cardiorespiratory fitness. Thus, the current narrative review discusses the impact of the PT interventions on indicators of cardiorespiratory function among different patient groups to identify the mechanisms linking CRF with PT. The authors searched systematically databases: PubMed, Web of Science from inception to March 2023 and analyzed available data including finally 20 papers. In description of the findings PEDro Scale and final score was used. Analyzed data indicated: a) pleiotropic input of PT on improving physical performance in medical conditions; b) specific parameters characterizing effectiveness of PT in each group of patients according of disease; c) different range of static significance and effect size especially for such following indicators as: VO2 at VT (ml•kg-1•min-1), VO2 peak/max (ml•kg-1•min-1), HR at VT (beats•min-1), HRmax (beats•min-1), VE (L•min-1). We also formulate and discuss potential physiological mechanisms of PT affecting CRF. This paper showed PT: a) has positive impact on broad spectrum of indicators of cardiorespiratory function by pleiotropic action among different patients' groups; b) significant ameliorates quality of life that may contribute to long-standing behavior change of patients related with overall physical activity.

Platelets interact with CD169+ macrophages and cDC1 and enhance liposome-induced CD8+ T cell responses.

Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169+ macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8+ T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro. In addition, platelets interacted with splenic CD169+ macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8+ T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8+ T cell activation during bacterial infection. However, after liposomal vaccination CD8+ T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169+ macrophages and cDC1 and emphasize the importance of platelets in induction of CD8+ T cell responses in the context of liposomal vaccination.

Chronic hyper-reactivity of platelets resulting in enhanced monocyte recruitment in patients after ischaemic stroke.

Although the platelet activation profile after stroke is a well-known issue, the platelet reactivity assessed prospectively after ischaemic stroke still remains equivocal. The aim of this study was to evaluate the reactivity of platelets in response to stimulation with thrombin receptor-activating peptide (TRAP) at 1, 10 and 90 days after ischaemic stroke and to compare it with results obtained in control groups. We determined the increment in surface expression of CD62P, CD40L and monocyte- and granulocyte-platelet aggregate formation using five-colour flow cytometry in 86 subjects after an ischaemic event, in 62 disease controls, and in 38 healthy volunteers. We assessed the plasma levels of CD62P and CD40L soluble forms. In patients after stroke a significantly lower increment in CD62P surface expression (p < 0.01) and higher increments in both CD40L platelet surface expression (p < 0.01) and monocyte-platelet aggregate percentage (p < 0.01) were found at every studied time point, as compared with the control groups. Plasma levels of soluble CD62P (sCD62P) and soluble CD40L (sCD40L) were increased in stroke subjects in both the acute and the subacute phase of the stroke and they dropped to levels observed in controls at day 90 after the ischaemic incident. In all studied groups a positive correlation was noted between plasma levels of sCD62P and sCD40L. In conclusion, while at 3-month follow-up the levels of soluble forms normalize in stroke patients, the profile of platelet reactivity in response to activation with TRAP differs from that observed in the controls despite the secondary stroke prevention.

Solubility of Methane in Water: Some Useful Results for Hydrate Nucleation.

In this paper, the solubility of methane in water along the 400 bar isobar is determined by computer simulations using the TIP4P/Ice force field for water and a simple LJ model for methane. In particular, the solubility of methane in water when in contact with the gas phase and the solubility of methane in water when in contact with the hydrate has been determined. The solubility of methane in a gas-liquid system decreases as temperature increases. The solubility of methane in a hydrate-liquid system increases with temperature. The two curves intersect at a certain temperature that determines the triple point T3 at a certain pressure. We also determined T3 by the three-phase direct coexistence method. The results of both methods agree, and we suggest 295(2) K as the value of T3 for this system. We also analyzed the impact of curvature on the solubility of methane in water. We found that the presence of curvature increases the solubility in both the gas-liquid and hydrate-liquid systems. The change in chemical potential for the formation of hydrate is evaluated along the isobar using two different thermodynamic routes, obtaining good agreement between them. It is shown that the driving force for hydrate nucleation under experimental conditions is higher than that for the formation of pure ice when compared at the same supercooling. We also show that supersaturation (i.e., concentrations above those of the planar interface) increases the driving force for nucleation dramatically. The effect of bubbles can be equivalent to that of an additional supercooling of about 20 K. Having highly supersaturated homogeneous solutions makes possible the spontaneous formation of the hydrate at temperatures as high as 285 K (i.e., 10K below T3). The crucial role of the concentration of methane for hydrate formation is clearly revealed. Nucleation of the hydrate can be either impossible or easy and fast depending on the concentration of methane which seems to play the leading role in the understanding of the kinetics of hydrate formation.

Fertility Counseling in BRCA1/2-Mutated Women with Breast Cancer and Healthy Individuals.

Breast cancer is the most commonly diagnosed cancer worldwide and the fifth leading cause of cancer death. In 2020, there were 2.3 million new cases, and 685,000 women died from it. Breast cancer among young women under 40 years of age accounts for 5% to 10% of all cases of this cancer. The greater availability of multi-gene sequence analysis by next-generation sequencing has improved diagnosis and, consequently, the possibility of using appropriate therapeutic approaches in BRCA1/2 gene mutation carriers. Treatment of young breast cancer patients affects their reproductive potential by reducing ovarian reserve. It can lead to reversible or permanent premature menopause, decreased libido, and other symptoms of sex hormone deficiency. This requires that, in addition to oncological treatment, patients are offered genetic counseling, oncofertility, psychological assistance, and sexological counseling. Given the number of BRCA1/2 gene mutation carriers among young breast cancer patients, but also thanks to growing public awareness, among their healthy family members planning offspring, the possibility of benefiting from preimplantation testing and performing cancer-risk-reduction procedures: RRM (risk-reducing mastectomy) and RRSO (risk-reducing salpingo-oophorectomy) significantly increase the chance of a genetically burdened person living a healthy life and giving birth to a child not burdened by the parent's germline mutation. The goal of this paper is to show methods and examples of fertility counselling for BRCA1/2 gene mutation carriers, including both patients already affected by cancer and healthy individuals.

Complementary and Alternative Therapies in Oncology.

Cancer is the second leading cause of death worldwide, after cardiovascular diseases. Increasing patients' awareness and providing easier access to public information result in greater interest in alternative anticancer or unproven supportive therapies. Fear of cancer and limited trust in the treating physician are also important reasons leading patients to seek these methods. Trust and good communication are essential to achieving truthful collaboration between physicians and patients. Given the popularity of CAM, better knowledge about these alternative practices may help oncologists discuss this issue with their patients. This article objectively reviews the most common unconventional therapies used by cancer patients.

The Usefulness of Prognostic Tools in Breast Cancer Patients with Brain Metastases.

BACKGROUND: Determining the proper therapy is challenging in breast cancer (BC) patients with brain metastases (BM) due to the variability of an individual's prognosis and the variety of treatment options available. Several prognostic tools for BC patients with BM have been proposed. Our review summarizes the current knowledge on this topic. METHODS: We searched PubMed for prognostic tools concerning BC patients with BM, published from January 1997 (since the Radiation Therapy Oncology Group developed) to December 2021. Our criteria were limited to adults with newly diagnosed BM regardless of the presence or absence of any leptomeningeal metastases. RESULTS: 31 prognostic tools were selected: 13 analyzed mixed cohorts with some BC cases and 18 exclusively analyzed BC prognostic tools. The majority of prognostic tools in BC patients with BM included: the performance status, the age at BM diagnosis, the number of BM (rarely the volume), the primary tumor phenotype/genotype and the extracranial metastasis status as a result of systemic therapy. The prognostic tools differed in their specific cut-off values. CONCLUSION: Prognostic tools have variable precision in determining the survival of BC patients with BM. Advances in local and systemic treatment significantly affect survival, therefore, it is necessary to update the survival indices used depending on the type and period of treatment.

Incorporation of Toll-Like Receptor Ligands and Inflammasome Stimuli in GM3 Liposomes to Induce Dendritic Cell Maturation and T Cell Responses.

Cancer vaccination aims to activate immunity towards cancer cells and can be achieved by delivery of cancer antigens together with immune stimulatory adjuvants to antigen presenting cells (APC). APC maturation and antigen processing is a subsequent prerequisite for T cell priming and anti-tumor immunity. In order to specifically target APC, nanoparticles, such as liposomes, can be used for the delivery of antigen and adjuvant. We have previously shown that liposomal inclusion of the ganglioside GM3, an endogenous ligand for CD169, led to robust uptake by CD169-expressing APC and resulted in strong immune responses when supplemented with a soluble adjuvant. To minimize the adverse effects related to a soluble adjuvant, immune stimulatory molecules can be incorporated in liposomes to achieve targeted delivery of both antigen and adjuvant. In this study, we incorporated TLR4 (MPLA) or TLR7/8 (3M-052) ligands in combination with inflammasome stimuli, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) or muramyl dipeptide (MDP), into GM3 liposomes. Incorporation of TLR and inflammasome ligands did not interfere with the uptake of GM3 liposomes by CD169-expressing cells. GM3 liposomes containing a TLR ligand efficiently matured human and mouse dendritic cells in vitro and in vivo, while inclusion of PGPC or MDP had minor effects on maturation. Immunization with MPLA-containing GM3 liposomes containing an immunogenic synthetic long peptide stimulated CD4+ and CD8+ T cell responses, but additional incorporation of either PGPC or MDP did not translate into stronger immune responses. In conclusion, our study indicates that TLRL-containing GM3 liposomes are effective vectors to induce DC maturation and T cell priming and thus provide guidance for further selection of liposomal components to optimally stimulate anti-cancer immune responses.