PLGA-PEI nanoparticle covered with poly(I:C) for personalised cancer immunotherapy.

Melanoma is the main cause of death among skin cancers and its incidence worldwide has been experiencing an appalling increase. However, traditional treatments lack effectiveness in advanced or metastatic patients. Immunotherapy, meanwhile, has been shown to be an effective treatment option, but the rate of cancers responding remains far from ideal. Here we have developed a personalized neoantigen peptide-based cancer vaccine by encapsulating patient derived melanoma neoantigens in polyethylenimine (PEI)-functionalised poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and coating them with polyinosinic:polycytidylic acid (poly(I:C)). We found that PLGA NPs can be effectively modified to be coated with the immunoadjuvant poly(I:C), as well as to encapsulate neoantigens. In addition, we found that both dendritic cells (DCs) and lymphocytes were effectively stimulated. Moreover, the developed NP was found to have a better immune activation profile than NP without poly(I:C) or without antigen. Our results demonstrate that the developed vaccine has a high capacity to activate the immune system, efficiently maturing DCs to present the antigen of choice and promoting the activity of lymphocytes to exert their cytotoxic function. Therefore, the immune response generated is optimal and specific for the elimination of melanoma tumour cells.

Self-assembled peptide/polymer hybrid nanoplatform for cancer immunostimulating therapies.

Integrating peptide epitopes in self-assembling materials is a successful strategy to obtain nanovaccines with high antigen density and improved efficacy. In this study, self-assembling peptides containing MAGE-A3/PADRE epitopes were designed to generate functional therapeutic nanovaccines. To achieve higher stability, peptide/polymer hybrid nanoparticles were formulated by controlled self-assembly of the engineered peptides. The nanoparticles showed good biocompatibility to both human red blood- and dendritic cells. Incubation of the nanoparticles with immature dendritic cells triggered immune effects that ultimately activated CD8 + cells. The antigen-specific and IgG antibody responses of healthy C57BL/6 mice vaccinated with the nanoparticles were analyzed. The in vivo results indicate a specific response to the nanovaccines, mainly mediated through a cellular pathway. This research indicates that the immunogenicity of peptide epitope vaccines can be effectively enhanced by developing self-assembled peptide-polymer hybrid nanostructures.

Semisynthetic Pneumococcal Glycoconjugate Nanovaccine.

Pneumococcal conjugate vaccines offer an excellent safety profile and high protection against the serotypes comprised in the vaccine. However, inclusion of protein antigens fromStreptococcus pneumoniaecombined with potent adjuvants and a suitable delivery system are expected to both extend protection to serotype strains not represented in the formulation and stimulate a broader immune response, thus more effective in young children, elderly, and immunocompromised populations. Along this line, nanoparticle (NP) delivery systems can enhance the immunogenicity of antigens by protecting them from degradation and increasing their uptake by antigen-presenting cells, as well as offering co-delivery with adjuvants. We report herein the encapsulation of a semisynthetic glycoconjugate (GC) composed of a synthetic tetrasaccharide mimicking theS. pneumoniae serotype 14 capsular polysaccharide (CP14) linked to the Pneumococcal surface protein A (PsaA) using chitosan NPs (CNPs). These GC-loaded chitosan nanoparticles (GC-CNPs) were not toxic to human monocyte-derived dendritic cells (MoDCs), showed enhanced uptake, and displayed better immunostimulatory properties in comparison to the naked GC. A comparative study was carried out in mice to evaluate the immune response elicited by the glycoconjugate-administered subcutaneously (SC), where the GC-CNPs displayed 100-fold higher IgG response as compared with the group treated with nonencapsulated GC. Overall, the study demonstrates the potential of this chitosan-based nanovaccine for efficient delivery of glycoconjugate antigens.

A ready-to-use dry powder formulation based on protamine nanocarriers for pulmonary drug delivery.

The use of oral antibiotic therapy for the treatment of respiratory diseases as tuberculosis has promoted the appearance of side effects as well as resistance to these treatments. The low solubility, high metabolism, and degradation of drugs as rifabutin, have led to the use of combined and prolonged therapies, which difficult patient compliance. In this work, we develop inhalable formulations from biomaterials such as protamine to improve the therapeutic effect. Rifabutin-loaded protamine nanocapsules (NCs) were prepared by solvent displacement method and were physico-chemically characterized and evaluated for their dissolution, permeability, stability, cytotoxicity, hemocompatibility, internalization, and aerodynamic characteristics after a spray-drying procedure. Protamine NCs presented a size of around 200 nm, positive surface charge, and drug association up to 54%. They were stable as suspension under storage, as well as in biological media and as a dry powder after lyophilization in the presence of mannitol. Nanocapsules showed a good safety profile and cellular uptake with no tolerogenic effect on macrophages and showed good compatibility with red blood cells. Moreover, the aerodynamic evaluation showed a fine particle fraction deposition up to 30% and a mass median aerodynamic diameter of about 5 µm, suitable for the pulmonary delivery of therapeutics.

Dry Powder Comprised of Isoniazid-Loaded Nanoparticles of Hyaluronic Acid in Conjugation with Mannose-Anchored Chitosan for Macrophage-Targeted Pulmonary Administration in Tuberculosis.

Marketed dosage forms fail to deliver anti-tubercular drugs directly to the lungs in pulmonary Tuberculosis (TB). Therefore, nanomediated isoniazid (INH)-loaded dry powder for inhalation (Nano-DPI) was developed for macrophage-targeted delivery in TB. Mannosylated chitosan (MC) and hyaluronic acid (HA) with an affinity for the surface mannose and CD44 receptors of macrophages were used in conjugation to prepare hybrid nanosuspension by ionic gelation method using cross-linker, sodium tri-polyphosphate (TPP) followed by freeze-drying to obtain a dry powder composed of nanoparticles (INH-MC/HA NPs). Nanoformulations were evaluated for aerodynamic characteristics, cytotoxicity, hemocompatibility, macrophage phenotype analysis, and immune regulation. Cellular uptake imaging was also conducted to evaluate the uptake of NPs. The nanopowders did not pose any significant toxicity to the cells, along with good compatibility with red blood cells (RBCs). The pro-inflammatory costimulatory markers were upregulated, demonstrating the activation of T-cell response. Moreover, the NPs did not show any tolerogenic effect on the macrophages. Furthermore, confocal imaging exhibited the translocation of NPs in the cells. Altogether, the findings present that nano-DPI was found to be a promising vehicle for targeting macrophages.

Study of Plasma Anti-CD26 Autoantibody Levels in a Cohort of Treatment-Naïve Early Arthritis Patients.

In rheumatoid arthritis (RA), the identification of biomarkers to adjust treatment intensity and to correctly diagnose the disease in early stages still constitutes a challenge and, as such, novel biomarkers are needed. We proposed that autoantibodies (aAbs) against CD26 (DPP4) might have both etiological importance and clinical value. Here, we perform a prospective study of the potential diagnostic power of Anti-CD26 aAbs through their quantification in plasmas from 106 treatment-naïve early and undifferentiated AR. Clinical antibodies, Anti-CD26 aAbs, and other disease-related biomarkers were measured in plasmas obtained in the first visit from patients, which were later classified as RA and non-RA according to the American College of Rheumatology criteria. Two different isotype signatures were found among ten groups of patients, one for Anti-CD26 IgA and other for Anti-CD26 IgG and IgM isotypes, both converging in patients with arthritis (RA and Unresolved Undifferentiated Arthritis: UUA), who present elevated levels of all three isotypes. The four UUA patients, unresolved after two years, were ACPA and rheumatic factor (RF) negatives. In the whole cohort, 51.3% of ACPA/RF seronegatives were Anti-CD26 positives, and a similar frequency was observed in the seropositive RA patients. Only weak associations of the three isotypes with ESR, CRP and disease activity parameters were observed. Anti-CD26 aAbs are present in treatment-naïve early arthritis patients, including ACPA and RF seronegative individuals, suggestive of a potential pathogenic and/or biomarker role of Anti-CD26 aAbs in the development of rheumatic diseases.

Distinctive CD26 Expression on CD4 T-Cell Subsets.

Immune system CD4 T-cells with high cell-surface CD26 expression show anti-tumoral properties. When engineered with a chimeric antigen receptor (CAR), they incite strong responses against solid cancers. This subset was originally associated to human CD4 T helper cells bearing the CD45R0 effector/memory phenotype and later to Th17 cells. CD26 is also found in soluble form (sCD26) in several biological fluids, and its serum levels correlate with specific T cell subsets. However, the relationship between glycoprotein sCD26 and its dipeptidyl peptidase 4 (DPP4) enzymatic activity, and cell-surface CD26 expression is not well understood. We have studied ex vivo cell-surface CD26 and in vitro surface and intracellular CD26 expression and secretome's sCD26 in cultured CD4 T cells under different polarization conditions. We show that most human CD26negative CD4 T cells in circulating lymphocytes are central memory (TCM) cells while CD26high expression is present in effector Th1, Th2, Th17, and TEM (effector memory) cells. However, there are significant percentages of Th1, Th2, Th17, and Th22 CD26 negative cells. This information may help to refine the research on CAR-Ts. The cell surface CD45R0 and CD26 levels in the different T helper subsets after in vitro polarization resemble those found ex vivo. In the secretomes of these cultures there was a significant amount of sCD26. However, in all polarizations, including Th1, the levels of sCD26 were lower (although not significantly) compared to the Th0 condition (activation without polarization). These differences could have an impact on the various physiological functions proposed for sCD26/DPP4.

A chitosan-based nanosystem as pneumococcal vaccine delivery platform.

Chitosan-based nanosystems have been described as interesting tools for antigen delivery and for enhancing the immunogenicity of nasally administered vaccines. As a possible vaccine delivery method, the chemical conjugation of chitosan nanocapsules with the Streptococcus pneumoniae cell membrane protein PsaA (pneumococcal surface adhesin A) is suggested here. The antigen PsaA, common to all pneumococcus serotypes, is expected to improve its uptake by immune cells and to activate specific T cells, generating an adaptive immune response against pneumococcus. With this aim, chitosan nanocapsules with thiol-maleimide conjugation between the polymer (chitosan) and the antigen (PsaA) were designed to enable the surface presentation of PsaA for immune cell recognition. Spherical-shaped particles, with a size of 266 ± 32 nm, positive charge of +30 ± 1 mV, and good stability profiles in simulated nasal fluids (up to 24 h) were achieved. PsaA association rates were three times higher compared with nanocapsules without covalent polymer-protein conjugation. Cytotoxicity studies in cell culture media showed non-toxic effect under 150 µg/mL concentration of nanocapsules, and subsequent studies on the maturation of immature dendritic cells in the presence of antigen-conjugated nanocapsules displayed peripheral blood mononuclear cell activation and lymphocyte differentiation after their presentation by dendritic cells. Secretion of TNFα following exposure to nanocapsules and the ability of nanocapsules to activate CD4 and CD8 T lymphocytes had also been studied. Antigen loaded nanocarrier uptake and presentation by professional presenting cells.

The mechanism of sitagliptin inhibition of colorectal cancer cell lines' metastatic functionalities.

The cell membrane glycoprotein CD26 with peptidase activity (DPP4) and/or its soluble CD26/DPP4 counterpart expression and/or activity are altered in several cancers. Its role in metastasis development was recently highlighted by the discovery of CD26+ cancer stem cell subsets and the fact that clinical DPP4 inhibitors showed antimetastatic effects in animal models. Also, diabetic patients treated with the DPP4 inhibitor sitagliptin showed greater overall survival after colorectal or lung cancer surgery than patients under other diabetic therapies. However, the mechanism of action of these inhibitors in this context is unclear. We studied the role of CD26 and its DPP4 enzymatic activity in malignant cell features such as cell-to-cell homotypic aggregation, cancer cell motility, and invasion in a panel of human colorectal cancer (CRC) cell lines, avoiding models that include the physiological role of DPP4 in chemotaxis. Present results indicate that CD26 participates in the induction of cell invasion, motility, and aggregation of CD26-positive CRC cell lines. Moreover, only invasion and motility assays, which are collagen matrix-dependent, showed a decrease upon treatment with the DPP4 inhibitor sitagliptin. Sitagliptin showed opposite effects to those of transforming growth factor-ß1 on epithelial-to-mesenchymal transition and cell cycle, but this result does not explain its CD26/DPP4-dependent effect. These results contribute to the elucidation of the molecular mechanisms behind sitagliptin inhibition of metastatic traits. At the same time, this role of sitagliptin may help to define areas of medicine where DPP4 inhibitors might be introduced. However, they also suggest that additional tools against CD26 as a target might be used or developed for metastasis prevention in addition to gliptins.

CD26-Related Serum Biomarkers: sCD26 Protein, DPP4 Activity, and Anti-CD26 Isotype Levels in a Colorectal Cancer-Screening Context.

Current screening trials are showing reduction in colorectal cancer incidence and mortality. However, participation rates are often low, and blood-based tests could complement existing screening strategies. CD26 protein (sCD26) and its dipeptidyl peptidase IV (DPP4) enzymatic activity in circulation have been proposed as biomarkers for colorectal cancer and other diseases. However, changes in sCD26 and DPP4 levels show complex degrees of correlation, and their physiological or pathophysiological role is unclear. The aim of this study was to analyse if anti-CD26 autoantibodies are related to sCD26 and DPP4 and to determine their relevance in a context of colorectal cancer screening for complementing the value of sCD26 and DPP4 as biomarkers. These biomarkers were measured in a large prospective cohort (n = 497, except the anti-CD26 antibodies, evaluated in 125 samples) that included a subgroup of individuals that were positive for the faecal immunological occult blood test (FIT) (n = 86) and underwent a colonoscopy (n = 47). We confirmed for the first time higher DPP4 activity in men compared to women (Student's t test, p = 0.002), though this difference between sexes was not seen for serum sCD26 protein. These biomarkers correlated (R = 0.246, p = 0.003) only in women. Correlations were found between anti-CD26 isotypes but not with DPP4 activity or sCD26 concentration, except for a negative correlation only in men between anti-CD26 IgA isotype and sCD26 (R = -0.232, p = 0.044), and an almost significant negative correlation between anti-CD26 IgG and sCD26 limited to FIT-positive men. Interestingly, patients with advanced adenomas displayed the most elevated mean levels of anti-CD26 IgA, IgM, and particularly IgG (Mann-Whitney U test, p = 0.030) in comparison with the other FIT positives without adenomas, and these levels did not correlate with sCD26 or its DPP4 activity. Our preliminary results suggest that the combination of these measures using sex as confounder could perhaps be used as biomarkers for colorectal disease. It also suggests that events affecting the gut influence the levels of anti-CD26 antibodies, which show little or no effect in antigen clearance. These findings should be confirmed in a larger cohort of individuals with colonoscopy. The physiological origin of the sex differences observed should be further addressed.

Design of polymeric nanocapsules to improve their lympho-targeting capacity.

Aim: To design lympho-targeted nanocarriers with the capacity to enhance the activity of associated drugs/antigens whose target is within the lymphatic system. Materials & methods: Inulin (INU)-based nanocapsules (NCs), negatively charged and positively charged chitosan NCs were prepared by the solvent displacement techniques. The NCs were produced in two sizes: small (70 nm) and medium (170-250 nm). Results:In vitro results indicated that small NCs interacted more efficiently with dendritic cells than the larger ones. The study of the NCs biodistribution in mice, using 3D reconstruction of the popliteal lymph node, showed that small INU NCs have the greatest access and uniform accumulation in different subsets of resident immune cells. Conclusion: Small and negatively charged INU NCs have a potential as lympho-targeted antigen/drug nanocarriers.

Oral hygiene might prevent cancer.

Many evidences support that species from the Human Oral Microbiome Database such as Fusobacterium nucleatum or Bacteroides, linked previously to periodontitis and appendicitis, play a role in colorectal cancer (CRC), including metastasis. These typically oral species are invasive anaerobes that form biofilms in their virulent state. Aspirin (a NSAID) has been recently included into routine CRC prevention rationale. NSAIDs can prevent the growth of neoplastic lesions by inhibiting COX enzymes and another set of recently identified COX-independent targets, which include the WNT, AMPK and MTOR signaling pathways, the crosstalk between nucleoli and NF-κB transcriptional activity in apoptosis, and the biochemistry of platelets. These are signaling pathways related to tumor-promoting inflammation. In this process, pathogens or simple deregulation of the microbiota play an important role in CRC. Aspirin and other NSAIDs are efficient inhibitors of biofilm formation and able to control periodontitis development preventing inflammation related to the microbiota of the gingival tissue, so its seems plausible to include this pathway in the mechanisms that aspirin uses to prevent CRC. We propose arguments suggesting that current oral hygiene methods and other future developments against periodontitis might prevent CRC and probably other cancers, alone or in combination with other options; and that the multidisciplinary studies needed to prove this hypothesis might be relevant for cancer prevention.

IC-Tagging methodology applied to the expression of viral glycoproteins and the difficult-to-express membrane-bound IGRP autoantigen.

We have previously developed a methodology to produce protein microspheres (MS) that can be loaded with proteins of interest in living cells through their C or N-terminal tagging with the so-called IC-Tag. The IC-Tagging method has many applications ranging from the production of immobilized enzymes for industrial use to the production of subunit vaccines due to its intrinsic adjuvancy. Here we show the adaptation of the IC-Tagging to work inside the endoplasmic reticulum and bacteria, allowing us to produce properly modified viral glycoproteins. Additionally, we were able to express the Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), whose expression remained elusive to date possibly due to its toxicity when over-expressed. IGRP is an antigen of enormous pharmaceutical interest as it is specifically targeted during the autoimmune response taking place in both the Non-Obese Diabetic (NOD) mice and type 1 diabetes (T1D) patients leading to the destruction of insulin-producing beta cells.

Functional assessment of the BMPR2 gene in lymphoblastoid cell lines from Graves' disease patients.

In this study, we analysed the possible influence of the c.419-43delT BMPR2 variant in patients with Graves' disease (GD), in a molecular basis, focusing our efforts on possible alterations in the mRNA processing and synthesis. The molecular assessment of this variant in patients with GD would shed light on the association between the BMPR2 gene and the disease. The variant was detected in 18%, 55% and 10% of patients with pulmonary arterial hypertension, GD and in general population, respectively. Patients with GD fold change showed increased BMPR2 expression when matched against the controls, with a mean of 4.21 ± 1.73 (P = 0.001); BMPR2 was overexpressed in the analysed cell cycle stages. Fold change analysis of variant carriers and non-carriers showed slight overexpression and differences between phases, but none of them were statistically significant. BMPR2 expression was confirmed in the lymphoblastoid cell lines (LCLs) with a molecular weight of 115 kD, and no differences between variant carriers and non-carriers were detected. To conclude, the BMPR2 variant c.419-19delT appears in high frequency in patients with GD, and independently of its presence, BMPR2 is overexpressed in the LCLs from the GD patients tested. This increase could be paired with the described decreased expression of transforming growth factor-ß1 in thyroid tissue from patients with GD.

Anti-CD26 autoantibodies are involved in rheumatoid arthritis and show potential clinical interest.

OBJECTIVES: Rheumatoid arthritis (RA) patients show low serum levels of the Ag dipeptidyl peptidase IV (DPP-IV/CD26), both soluble CD26 (sCD26) concentration and its DPP-IV activity. The aim of this study was to test if anti-DPP-IV/CD26 Abs (Anti-CD26) cleared sCD26. DESIGN & METHODS: Serum Anti-CD26 and Total titers (as comparison) of isotypes IgA, IgM and IgG as well as sCD26 concentration and DPP-IV activity were measured in a cohort of RA patients undergoing different biological and non-biological therapies (n=105) and controls (n=50). RESULTS: Anti-CD26 levels were increased approximately two-fold for each isotype in RA, were not related to the sCD26 clearance, showed several correlations with disease activity parameters, were significantly higher in smokers and they were not ACPA. Anti-CD26 Igs showed high diagnostic power (82% sensitivity and 96% specificity) and their levels differed amongst the different groups of patients stratified by the type of therapy. CONCLUSIONS: As DPP-IV/CD26 is associated to factors triggering RA in the lung and periodontal tissue, these results suggest that Anti-CD26 isotypes may participate in pathogenesis and may be useful as biomarkers for earlier diagnosis and/or precision medicine.

Apportioning Blame: Autoreactive CD4+ and CD8+ T Cells in Type 1 Diabetes.

Type 1 diabetes (T1D) is one of the most studied archetypal organ-specific autoimmune diseases. Although many clinical, epidemiological, and pathological characteristics have been described, there are still important issues which need to be resolved as these will have a major impact on the development of future antigen-specific immunotherapies. An important question relates to T lymphocytes in the development of the disease, in particular their role in the destruction of insulin-producing beta cells. Since the discovery that certain class II histocompatibility complex molecules (HLA) are linked to the development of T1D, much research has focused on CD4+ helper T lymphocytes; however, recent studies highlight class I HLA molecules as an independent risk factor; hence, research into the role played by CD8+ cytotoxic T lymphocytes has gained momentum. In this review, we summarize recent studies clarifying the role played by both sets of autoreactive T lymphocytes in T1D, discuss the targets recognized by these cells and their phenotype in T1D patients. Finally, we will examine the possible generation of regulatory CD8+ T lymphocytes upon different immuno-intervention strategies.

Human peripheral blood mononuclear cell in vitro system to test the efficacy of food bioactive compounds: Effects of polyunsaturated fatty acids and their relation with BMI.

SCOPE: To analyse the usefulness of isolated human peripheral blood mononuclear cells (PBMC) to rapidly/easily reflect n-3 long-chain polyunsaturated fatty acid (LCPUFA) effects on lipid metabolism/inflammation gene profile, and evaluate if these effects are body mass index (BMI) dependent. METHODS AND RESULTS: PBMC from normoweight (NW) and overweight/obese (OW/OB) subjects were incubated with physiological doses of docosahexaenoic (DHA), eicosapentaenoic acid (EPA), or their combination. PBMC reflected increased beta-oxidation-like capacity (CPT1A expression) in OW/OB but only after DHA treatment. However, insensitivity to n-3 LCPUFA was evident in OW/OB for lipogenic genes: both PUFA diminished FASN and SREBP1C expression in NW, but no effect was observed for DHA in PBMC from high-BMI subjects. This insensitivity was also evident for inflammation gene profile: all treatments inhibited key inflammatory genes in NW; nevertheless, no effect was observed in OW/OB after DHA treatment, and EPA effect was impaired. SLC27A2, IL6 and TNFα PBMC expression analysis resulted especially interesting to determine obesity-related n-3 LCPUFA insensitivity. CONCLUSION: A PBMC-based human in vitro system reflects n-3 LCPUFA effects on lipid metabolism/inflammation which is impaired in OW/OB. These results confirm the utility of PBMC ex vivo systems for bioactive-compound screening to promote functional food development and to establish appropriate dietary strategies for obese population.

CD26 Expression on T Helper Populations and sCD26 Serum Levels in Patients with Rheumatoid Arthritis.

We studied dipeptidyl peptidase IV (DPP-IV, CD26) expression in different T helper cells and serum soluble DPP-IV/sCD26 levels in rheumatoid arthritis (RA) patients, correlated these with disease activity score (DAS), and examined how they were affected by different therapies, conventional or biological (anti-TNF, anti-CD20 and anti-IL6R or Ig-CTLA4). The percentage of CD4+CD45R0+CD26- cells was greatly reduced in patients (up to 50%) when compared with healthy subjects. Three other subsets of CD4 cells, including a CD26high Th1-associated population, changed variably with therapies. Data from these subsets (frequency and staining density) significantly correlated with DAS28 or DAS28 components but different in each group of patients undergoing the different therapies. Th17 and Th22 subsets were implicated in RA as independent CCR4+ and CCR4- populations each, with distinct CD26 expression, and were targeted with varying efficiency by each therapy. Serum DPP-IV activity rather than sCD26 levels was lower in RA patients compared to healthy donors. DPP-IV and sCD26 serum levels were found related to specific T cell subsets but not to disease activity. We conclude that, according to their CD26 expression, different cell subsets could serve to monitor RA course, and an uncharacterized T helper CD26- subset, not targeted by therapies, should be monitored for early diagnosis.