Influenza Vaccination Generates Cytokine-Induced Memory-like NK Cells:Impact of Human Cytomegalovirus Infection

Human NK cells are activated by cytokines, immune complexes, and signals transduced via activating ligands on other host cells. After vaccination, or during secondary infection, adaptive immune responses can enhance both cytokine-driven and Ab-dependent NK cell responses. However, induction of NK cells for enhanced function after in vitro exposure to innate inflammatory cytokines has also been reported and may synergize with adaptive signals to potentiate NK cell activity during infection or vaccination. To test this hypothesis, we examined the effect of seasonal influenza vaccination on NK cell function and phenotype in 52 previously unvaccinated individuals. Enhanced, IL-2-dependent, NK cell IFN-γ responses to Influenza A/California/7/2009 virus were detected up to 4 wk postvaccination and higher in human CMV (HCMV)-seronegative (HCMV(-)) individuals than in HCMV-seropositive (HCMV(+)) individuals. By comparison, robust NK cell degranulation responses were observed both before and after vaccination, due to high titers of naturally occurring anti-influenza Abs in human plasma, and did not differ between HCMV(+) and HCMV(-) subjects. In addition to these IL-2-dependent and Ab-dependent responses, NK cell responses to innate cytokines were also enhanced after influenza vaccination; this was associated with proliferation of CD57(-) NK cells and was most evident in HCMV(+) subjects. Similar enhancement of cytokine responsiveness was observed when NK cells were cocultured in vitro with Influenza A/California/7/2009 virus, and this was at least partially dependent upon IFN-αβR2. In summary, our data indicate that attenuated or live viral vaccines promote cytokine-induced memory-like NK cells and that this process is influenced by HCMV infection

Zoledronic acid-encapsulating self-assembling nanoparticles and doxorubicin: A combinatorial approach to overcome simultaneously chemoresistance and immunoresistance in breast tumors

The resistance to chemotherapy and the tumor escape from host immunosurveillance are the main causes of the failure of anthracycline-based regimens in breast cancer, where an effective chemo-immunosensitizing strategy is lacking. The clinically used aminobisphosphonate zoledronic acid (ZA) reverses chemoresistance and immunoresistance in vitro. Previously we developed a nanoparticle-based zoledronic acid-containing formulation (NZ) that allowed a higher intratumor delivery of the drug compared with free ZA in vivo. We tested its efficacy in combination with doxorubicin in breast tumors refractory to chemotherapy and immune system recognition as a new combinatorial approach to produce chemo- and immunosensitization. NZ reduced the IC50 of doxorubicin in human and murine chemoresistant breast cancer cells and restored the doxorubicin efficacy against chemo-immunoresistant tumors implanted in immunocompetent mice. By reducing the metabolic flux through the mevalonate pathway, NZ lowered the activity of Ras/ERK1/2/HIF-1α axis and the expression of P-glycoprotein, decreased the glycolysis and the mitochondrial respiratory chain, induced a cytochrome c/caspase 9/caspase 3-dependent apoptosis, thus restoring the direct cytotoxic effects of doxorubicin on tumor cell. Moreover, NZ restored the doxorubicin-induced immunogenic cell death and reversed the tumorinduced immunosuppression due to the production of kynurenine, by inhibiting the STAT3/indoleamine 2,3 dioxygenase axis. These events increased the number of dendritic cells and decreased the number of immunosuppressive T-regulatory cells infiltrating the tumors. Our work proposes the use of nanoparticle encapsulating zoledronic acid as an effective tool overcoming at the same time chemoresistance and immunoresistance in breast tumors, thanks to the effects exerted on tumor cell and tumor-infiltrating immune cells

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Impaired NK Cell Responses to Pertussis and H1N1 Influenza Vaccine Antigens in Human Cytomegalovirus-Infected Individuals.

NK cells contribute to postvaccination immune responses after activation by IL-2 from Ag-specific memory T cells or by cross-linking of the low-affinity IgG receptor, CD16, by Ag-Ab immune complexes. Sensitivity of NK cells to these signals from the adaptive immune system is heterogeneous and influenced by their stage of differentiation. CD56(dim)CD57(+) NK cells are less responsive to IL-2 and produce less IFN-γ in response to T cell-mediated activation than do CD56(bright) or CD56(dim)CD57(-) NK cells. Conversely, NK cell cytotoxicity, as measured by degranulation, is maintained across the CD56(dim) subsets. Human CMV (HCMV), a highly prevalent herpes virus causing lifelong, usually latent, infections, drives the expansion of the CD56(dim)CD57(+)NKG2C(+) NK cell population, skewing the NK cell repertoire in favor of cytotoxic responses at the expense of cytokine-driven responses. We hypothesized, therefore, that HCMV seropositivity would be associated with altered NK cell responses to vaccine Ags. In a cross-sectional study of 152 U.K. adults, with HCMV seroprevalence rate of 36%, we find that HCMV seropositivity is associated with lower NK cell IFN-γ production and degranulation after in vitro restimulation with pertussis or H1N1 influenza vaccine Ags. Higher expression of CD57/NKG2C and lower expression of IL-18Rα on NK cells from HCMV seropositive subjects do not fully explain these impaired responses, which are likely the result of multiple receptor-ligand interactions. This study demonstrates for the first time, to our knowledge, that HCMV serostatus influences NK cell contributions to adaptive immunity and raises important questions regarding the impact of HCMV infection on vaccine efficacy

Insights in the chemical components of liposomes responsible for P-glycoprotein inhibition.

In this work we investigated how the surface charge and the presence of polyethylene glycol (PEG) on liposome carriers affect the delivery of the encapsulated doxorubicin in P-glycoprotein (Pgp)-overexpressing cells. We found that neutral net charge was critical to favour the liposome uptake and decrease the Vmax of doxorubicin efflux. PEG-coating was necessary to increase the Km of doxorubicin for Pgp. In particular the PEGylated phospholipid present in neutral liposomes, i.e. PEGylated distearoyl-phosphatidylethanolamine (DSPE-PEG), was a Pgp allosteric inhibitor, increased doxorubicin Km and inhibited Pgp ATPase activity. Site-directed mutagenesis experiments suggested that the domain centred around glycine 185 of Pgp was necessary for these inhibitory properties of DSPE-PEG and PEGylated neutral liposomes. We conclude that both surface charge and PEGylation must be considered to optimize the doxorubicin delivery within chemoresistant cells. DSPE-PEG-enriched particles may represent promising tools for therapeutic and diagnostic applications in tissues with high levels of Pgp

Morpho-anatomical adaptations of Potamogeton polygonus (Potamogetonaceae) to lotic and lentic environments

Abstract Aquatic macrophytes show great phenotypic plasticity and are able to occupy environments with different physicochemical conditions. The present study aimed to characterize morphology and anatomical structure of the pondweed, Potamogeton polygonus Cham. & Schltdl., and to identify adaptive modifications of the plant in lotic and lentic environments. Sampling was carried out in Palmas and General Carneiro, Paraná state, southern Brazil. Ten individuals from each locality were collected. Morpho-anatomical characteristics of the roots, stems and leaves were measured. The anatomical structure was analyzed with light microscopy and scanning electron microscopy. Significant morphological and anatomical adaptive differences were observed between plants of the two environments

Self-assembling nanoparticles encapsulating zoledronic acid revert multidrug resistance in cancer cells

The overexpression of ATP binding cassette (ABC) transporters makes tumor cells simultaneously resistant to several cytotoxic drugs. Impairing the energy metabolism of multidrug resistant (MDR) cells is a promising chemosensitizing strategy, but many metabolic modifiers are too toxic in vivo. We previously observed that the aminobisphosphonate zoledronic acid inhibits the activity of hypoxia inducible factor-1α (HIF-1α), a master regulator of cancer cell metabolism. Free zoledronic acid, however, reaches low intratumor concentration. We synthesized nanoparticle formulations of the aminobisphosphonate that allow a higher intratumor delivery of the drug. We investigated whether they are effective metabolic modifiers and chemosensitizing agents against human MDR cancer cells in vitro and in vivo. At not toxic dosage, nanoparticles carrying zoledronic acid chemosensitized MDR cells to a broad spectrum of cytotoxic drugs, independently of the type of ABC transporters expressed. The nanoparticles inhibited the isoprenoid synthesis and the Ras/ERK1/2-driven activation of HIF-1α, decreased the transcription and activity of glycolytic enzymes, the glucose flux through the glycolysis and tricarboxylic acid cycle, the electron flux through the mitochondrial respiratory chain, the synthesis of ATP. So doing, they lowered the ATP-dependent activity of ABC transporters, increasing the chemotherapy efficacy in vitro and in vivo. These effects were more pronounced in MDR cells than in chemosensitive ones and were due to the inhibition of farnesyl pyrophosphate synthase (FPPS), as demonstrated in FPPS-silenced tumors. Our work proposes nanoparticle formulations of zoledronic acid as the first not toxic metabolic modifiers, effective against MDR tumors

Nanoparticles for the delivery of zoledronic acid to prostate cancer cells: A comparative analysis through time lapse video-microscopy technique.

Time-lapse live cell imaging is a powerful tool for studying the responses of cells to drugs. Zoledronic acid (ZOL) is the most potent aminobiphosphonate able to induce cell growth inhibition at very low concentrations. The lack of clear evidence of ZOL-induced anti-cancer effects is likely due to its unfavorable pharmacokinetic profile. The use of nanotechnology-based formulations allows overcoming these limitations in ZOL pharmaco-distribution. Recently, stealth liposomes (LIPOs) and new self-assembly PEGylated nanoparticles (NPs) encapsulating ZOL were developed. Both the delivery systems showed promising anticancer activity in vitro and in vivo. In this work, we investigated the cytostatic effect of these novel formulations (LIPOs and NPs) compared with free ZOL on 2 different prostate cancer cell lines, PC 3 and DU 145 and on prostate epithelial primary cells EPN using time lapse video-microscopy (TLVM). In PC3 cells, free ZOL showed a significant anti-proliferative effect but this effect was lower than that induced by LIPOs and NPs encapsulating ZOL; moreover, LIPO-ZOL was more potent in inducing growth inhibition than NP-ZOL. On the other hand, LIPO-ZOL slightly enhanced the free ZOL activity on growth inhibition of DU 145, while the anti-proliferative effect of NP-ZOL was not statistically relevant. These novel formulations did not induce anti-proliferative effects on EPN cells. Finally, we evaluated cytotoxic effects on DU145 where, LIPO-ZOL induced the highest cytotoxicity compared with NP-ZOL and free ZOL. In conclusion, ZOL can be transformed in a powerful anticancer agent, if administered with nanotechnology-based formulations without damaging the healthy tissues

Zoledronic acid-encapsulating self-assembling nanoparticles and doxorubicin: a combinatorial approach to overcome simultaneously chemoresistance and immunoresistance in breast tumors

The resistance to chemotherapy and the tumor escape from host immunosurveillance are the main causes of the failure of anthracycline-based regimens in breast cancer, where an effective chemo-immunosensitizing strategy is lacking. The clinically used aminobisphosphonate zoledronic acid (ZA) reverses chemoresistance and immunoresistance in vitro. Previously we developed a nanoparticle-based zoledronic acid-containing formulation (NZ) that allowed a higher intratumor delivery of the drug compared with free ZA in vivo. We tested its efficacy in combination with doxorubicin in breast tumors refractory to chemotherapy and immune system recognition as a new combinatorial approach to produce chemo- and immunosensitization. NZ reduced the IC50 of doxorubicin in human and murine chemoresistant breast cancer cells and restored the doxorubicin efficacy against chemo-immunoresistant tumors implanted in immunocompetent mice. By reducing the metabolic flux through the mevalonate pathway, NZ lowered the activity of Ras/ERK1/2/HIF-1? axis and the expression of P-glycoprotein, decreased the glycolysis and the mitochondrial respiratory chain, induced a cytochrome c/caspase 9/caspase 3-dependent apoptosis, thus restoring the direct cytotoxic effects of doxorubicin on tumor cell. Moreover, NZ restored the doxorubicin-induced immunogenic cell death and reversed the tumorinduced immunosuppression due to the production of kynurenine, by inhibiting the STAT3/indoleamine 2,3 dioxygenase axis. These events increased the number of dendritic cells and decreased the number of immunosuppressive T-regulatory cells infiltrating the tumors. Our work proposes the use of nanoparticle encapsulating zoledronic acid as an effective tool overcoming at the same time chemoresistance and immunoresistance in breast tumors, thanks to the effects exerted on tumor cell and tumor-infiltrating immune cells