Comparative performance of a panel of commercially available antimicrobial nanocoatings in Europe.

BACKGROUND: Bacterial resistance against the classic antibiotics is posing an increasing challenge for the prevention and treatment of infections in health care environments. The introduction of antimicrobial nanocoatings with active ingredients provides alternative measures for active killing of microorganisms, through a preventive hygiene approach. PURPOSE: The purpose of this study was to investigate the antimicrobial activity of a panel of antimicrobial coatings available on the European market. METHODS: A comparative, biased selection of commercially available antimicrobial coatings was tested for antimicrobial efficiency. Suppliers were contacted to deliver their coatings on glass and/or stainless steel substrates. In total, 23 coatings from eleven suppliers were received, which were investigated for their effect on the growth of Escherichia coli, using the International Organization for Standardization (ISO) 22196 protocol. RESULTS: The majority of nanomaterial-containing coatings (n=13) contained nanosilver (n=12), while only one had photocatalytic TiO2 as the active particle. The differences in antimicrobial activity among all of the coatings, expressed as log reduction values, varied between 1.3 and 6.6, while the variation within the nanomaterial-based group was between 2.0 and 6.2. Although nanosilver coatings were on average very effective in reducing the number of viable bacteria after challenge, the strongest log reduction (6.6) was seen with a coating that has immobilized, covalently bound quaternary ammonium salt in its matrix. Besides these two compounds, coatings containing TiO2, poly(dimethylsiloxane), triclosan, or zinc pyrithione evoked 100% killing of E. coli. CONCLUSION: Our findings indicate that nanosilver dominates the nanoparticle-based coatings and performs adequately. However, considering the unknowns in relation to ecotoxicological emission and effects, it needs further consideration before widespread application into different environments.

Inhibition of Rho-ROCK signaling induces apoptotic and non-apoptotic PS exposure in cardiomyocytes via inhibition of flippase.

Subsequent to myocardial infarction, cardiomyocytes within the infarcted areas and border zones expose phosphatidylserine (PS) in the outer plasma membrane leaflet (flip-flop). We showed earlier that in addition to apoptosis, this flip-flop can be reversible in cardiomyocytes. We now investigated a possible role for Rho and downstream effector Rho-associated kinase (ROCK) in the process of (reversible) PS exposure and apoptosis in cardiomyocytes. In rat cardiomyoblasts (H9c2 cells) and isolated adult ventricular rat cardiomyocytes Clostridium difficile Toxin B (TcdB), a Rho GTPase family inhibitor, C3 transferase (C3), a Rho(A,B,C) inhibitor and the ROCK inhibitors Y27632 and H1152 were used to inhibit Rho-ROCK signaling. PS exposure was assessed via flow cytometry and fluorescent digital imaging microscopy using annexin V. Akt expression and phosphorylation were analyzed via Western blot, and Akt activity was inhibited by wortmannin. The cellular concentration activated caspase 3 was determined as a measure of apoptosis, and flippase activity was assessed via flow cytometry using NBD-labeled PS. TcdB, C3, Y27632 and H1152 all significantly increased PS exposure. TcdB, Y27632 and H1152 all significantly inhibited phosphorylation of the anti-apoptotic protein Akt and Akt inhibition by wortmannin lead to increased PS exposure. However, only TcdB and C3, but not ROCK- or Akt inhibition led to caspase 3 activation and thus apoptosis. Notably, pancaspase inhibitor zVAD only partially inhibited TcdB-induced PS exposure indicating the existence of apoptotic and non-apoptotic PS exposure. The induced PS exposure coincided with decreased flippase activity as measured with NBD-labeled PS flip-flop. In this study, we show a regulatory role for a novel signaling route, Rho-ROCK-flippase signaling, in maintaining asymmetrical membrane phospholipid distribution in cardiomyocytes.

TLR ligands in the local treatment of established intracerebral murine gliomas.

Local TLR stimulation is an attractive approach to induce antitumor immunity. In this study, we compared various TLR ligands for their ability to affect murine GL261 cells in vitro and to eradicate established intracerebral murine gliomas in vivo. Our data show that GL261 cells express TLR2, TLR3, and TLR4 and respond to the corresponding TLR ligands with increasing MHC class I expression and inducing IL-6 secretion in vitro, while TLR5, TLR7, and TLR9 are essentially absent. Remarkably, CpG-oligonucleotides (CpG-ODN, TLR9) appeared to inhibit GL261 cell proliferation in a cell-type specific, but CpG-motif and TLR9-independent manner. A single intratumoral injection of CpG-ODN most effectively inhibited glioma growth in vivo and cured 80% of glioma-bearing C57BL/6 mice. Intratumoral injection of Pam3Cys-SK4 (TLR1/2) or R848 (TLR7) also produced a significant survival benefit, whereas poly(I:C) (TLR3) or purified LPS (TLR4) stimulation alone was not effective. Additional studies using TLR9(+/+) wild-type and TLR9(-/-) knockout mice revealed that the efficacy of local CpG-ODN treatment in vivo required TLR9 expression on nontumor cells. Additional experiments demonstrated increased frequencies of tumor-infiltrating IFN-gamma producing CD4(+) and CD8(+) effector T cells and a marked increase in the ratio of CD4(+) effector T cells to CD4(+)FoxP3(+) regulatory T cells upon CpG-ODN treatment. Surviving CpG-ODN treated mice were also protected from a subsequent tumor challenge without further addition of CpG-ODN. In summary, this study underlines the potency of local TLR treatment in antiglioma therapy and demonstrates that local CpG-ODN treatment most effectively restores antitumor immunity in a therapeutic murine glioma model.

Invariant natural killer T cells and immunotherapy of cancer.

Invariant CD1d restricted natural killer T (iNKT) cells are regulatory cells that express a canonical TCR-Valpha-chain (Valpha24.Jalpha18 in humans and Valpha14.Jalpha18 in mice) which recognizes glycolipid antigens presented by the monomorphic CD1d molecule. They can secrete a wide variety of both pro-inflammatory and anti-inflammatory cytokines very swiftly upon their activation. Evidence for the significance of iNKT cells in human cancer has been ambiguous. Still, the (pre-)clinical findings reviewed here, provide evidence for a distinct contribution of iNKT cells to natural anti-tumor immune responses in humans. Furthermore, clinical phase I studies that are discussed here have revealed that the infusion of cancer patients with ligand-loaded dendritic cells or cultured iNKT cells is well tolerated. We thus underscore the potential of iNKT cell based immunotherapy in conjunction with established modalities such as surgery and radiotherapy, as adjuvant therapy against carcinomas.

In vitro expanded human invariant natural killer T-cells promote functional activity of natural killer cells.

Invariant natural killer T (iNKT) cells play a pivotal role in cancer immunity through trans-activation of effector cells via swift cytokine secretion. In mice, iNKT cell activation by alpha-galactosylceramide (alpha-GC) induces potent NK cell-mediated anti-tumour effects. Here we investigated whether human iNKT cells could enhance NK cell functional activity in vitro. iNKT cell activation by alpha-GC treatment of peripheral blood mononuclear cells (PBMC) was not sufficient to enhance NK cell effector functions. However, addition of in vitro expanded iNKT cells to PBMC enhanced NK cell-mediated cytotoxicity in an alpha-GC-dependent manner. NK cell activation by iNKT cells was primarily mediated by soluble factors, and could be enhanced by the NK cell activating cytokine IL-21. These results suggest that adoptive transfer of ex vivo expanded iNKT cells will enhance NK cell function and is expected to enhance the efficacy of cancer immunotherapy, particularly in combination with IL-21 and alpha-GC.

IFN-gamma-producing human invariant NKT cells promote tumor-associated antigen-specific cytotoxic T cell responses.

CD1d-restricted invariant NKT (iNKT) cells can enhance immunity to cancer or prevent autoimmunity, depending on the cytokine profile secreted. Antitumor effects of the iNKT cell ligand alpha-galactosylceramide (alphaGC) and iNKT cell adoptive transfer have been demonstrated in various tumor models. Together with reduced numbers of iNKT cells in cancer patients, which have been linked to poor clinical outcome, these data suggest that cancer patients may benefit from therapy aiming at iNKT cell proliferation and activation. Herein we present results of investigations on the effects of human iNKT cells on Ag-specific CTL responses. iNKT cells were expanded using alphaGC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate type 1 cytokine production. Enhanced activation and increased IFN-gamma production was observed in iNKT cells, irrespective of CD4 expression, upon stimulation with IL-12-overexpressing dendritic cells. IL-12-stimulated iNKT cells strongly enhanced the MART-1 (melanoma Ag recognized by T cell 1)-specific CD8(+) CTL response, which was dependent on iNKT cell-derived IFN-gamma. Furthermore, autologous IL-12-overexpressing dendritic cells, loaded with Ag as well as alphaGC, was superior in stimulating both iNKT cells and Ag-specific CTL. This study shows that IL-12-overexpressing allogeneic dendritic cells expand IFN-gamma-producing iNKT cells, which may be more effective against tumors in vivo. Furthermore, the efficacy of autologous Ag-loaded DC vaccines may well be enhanced by IL-12 overexpression and loading with alphaGC.

Chronically stimulated mouse invariant NKT cell lines have a preserved capacity to enhance protection against experimental tumor metastases.

In pre-clinical models, CD1d restricted invariant Natural Killer T (iNKT) cells play a pivotal role in natural anti-tumor immune responses, mainly by trans-activating cells of both the innate and adaptive arms via swift and potent cytokine secretion. We have previously reported that patients with a severely reduced circulating iNKT cell pool have a poor clinical response to radio therapy of head and neck squamous cell carcinoma. Therefore, these patients might benefit from an immunotherapeutic approach aimed at the increase of circulating levels of iNKT cells. Furthermore, we have generated both human and mouse iNKT cell lines, and demonstrated that they had retained the capacity to release both Th1 and Th2 type cytokines even after long-term in vitro expansion using alpha-galactosylceramide (alphaGalCer) pulsed dendritic cells (DC). Here, we establish, in a pre-clinical tumor model that the large scale long lived polyclonal iNKT cell lines we generated have a preserved capacity to evoke an in vivo cytokine storm upon adoptive transfer, independently of supplemental alphaGalCer administration. This results in an augmented NK cell mediated protection against B16.F10 experimental lung metastases in vivo. These findings underscore the potential of autologous adoptive transfer of ex vivo expanded iNKT cells as a strategy to enhance immunotherapeutic modalities for the treatment of cancer patients.

CD4(+)CD25hi regulatory T-cell frequency correlates with persistence of human papillomavirus type 16 and T helper cell responses in patients with cervical intraepithelial neoplasia.

CD4(+)CD25(hi)CTLA4(+)FoxP3(+) regulatory T cells (Treg) have been shown to maintain immune tolerance against self antigens and increased circulating frequencies have been reported in various types of cancers. Circulating invariant natural killer T-cells (iNKT) are reduced in cancer patients and low iNKT frequency is related to poor prognosis. It is not yet clear whether high Treg numbers and low iNKT cell numbers pose an increased risk for the progression of premalignant lesions or whether Treg and iNKT cell numbers are influenced by dysplasia. We therefore studied prospectively the relation between iNKT cell and Treg frequencies and the natural course of human papillomavirus type 16 (HPV16) induced pre-malignant cervical dysplasia in 82 patients who participated in a nonintervention cohort study of women with abnormal cytology. Treg frequencies were significantly increased in women who had persistent HPV16 infection. Within the HPV16 persistence group there was no difference in Treg frequencies among patients who developed a CIN3 lesion and patients who did not progress to CIN3. Furthermore, Treg frequencies were increased in patients who had detectable HPV16 E7 specific IL-2 producing T-helper cells, which suggests a causal role of HPV infection in Treg development in parallel with HPV16 specific T helper cells. No evidence was found for a role for iNKT cells in persistence of HPV16 and progression of HPV16 induced CIN lesions. However, HPV-persistence-associated Tregs may explain the inefficacy of concomitant persistence associated immunity and may contribute to subsequent progression to neoplasia.

Generation and sustained expansion of mouse spleen invariant NKT cell lines with preserved cytokine releasing capacity.

Invariant Natural Killer T (iNKT) cells are CD1d restricted innate lymphoid cells with an invariant T cell receptor (TCR) alpha chain gene rearrangement (Valpha24-Jalpha18 in human and Valpha14-Jalpha18 in mouse). iNKT cells play a pivotal role in anti-tumor immune responses via cytokine mediated transactivation of various cells which mediate innate and adaptive immune responses. Here we describe, to our knowledge for the first time, the generation of long-term mouse spleen derived iNKT cell lines. We found that dendritic cells (DC) derived from the D1 line, but not Mf4/4 macrophages, loaded with the artificial iNKT cell ligand alpha-Galactosylceramide (alphaGalCer) could be employed to expand iNKT cells in vitro. Furthermore, exogenously added IL-7, but not IL-2 or IL-15 had a pronounced additive effect on iNKT cell expansion. Using this method up to 10(8) iNKT cells could be obtained from one spleen within 12 to 14 weeks, and cell lines could be continued for up to 24 months. Importantly, the iNKT cell lines had retained the capacity to swiftly secrete substantial amounts of both T helper (Th) 1 and Th2 cytokines upon activation. In conclusion we have generated iNKT cell lines with high yields that can be maintained for up to 24 months, by repeated stimulation using alpha-GalCer loaded D1.DC and IL-7. These in vitro expanded iNKT cells preserved the capacity to swiftly produce both Th1 and Th2 type cytokines and are currently being utilized in pre-clinical adoptive transfer models to identify and optimize the characteristics of therapeutically effective iNKT cells in an anti-tumor setting.

Low levels of circulating invariant natural killer T cells predict poor clinical outcome in patients with head and neck squamous cell carcinoma.

PURPOSE: Evading antitumor immune responses is an important aspect of the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Invariant CD1d-restricted natural killer T (iNKT) cells play an allegedly pivotal role in such responses via transactivation of immune effector cells. It has been reported that iNKT cells are reduced in peripheral blood of cancer patients compared with healthy controls. Here, we investigated whether the extent of this deficiency affected disease outcome in HNSCC patients. PATIENTS AND METHODS: In a prospective study, circulating iNKT cell numbers were evaluated in 47 patients before radiotherapy. Patients were stratified in three groups based on iNKT cell levels, and clinical data were obtained during a median follow-up period of 31 months. RESULTS: A small, compared with an intermediate or large, circulating iNKT cell fraction was significantly associated with decreased 3-year overall survival rate (39% v 75% and 92%, respectively), disease-specific survival rate (43% v 87% and 92%, respectively), and locoregional control rate (31% v 74% and 92%, respectively) in HNSCC patients. Cox regression revealed that the iNKT cell level, as well as clinical T stage, was an independent prognostic parameter even after correction for the confounding effect of age. CONCLUSION: A severe circulating iNKT cell deficiency was related to poor clinical outcome in HNSCC patients, suggesting their critical contribution to antitumor immune responses. Furthermore, screening for iNKT cell levels may be useful for determining which patients can benefit from immunotherapeutic adjuvant therapies aimed at reconstitution of the circulating iNKT cell pool.

Cutting edge: Rapid recovery of NKT cells upon institution of highly active antiretroviral therapy for HIV-1 infection.

CD1d-restricted NKT cells play important regulatory roles in various immune responses and are rapidly and selectively depleted upon infection with HIV-1. The cause of this selective depletion is incompletely understood, although it is in part due to the high susceptibility of CD4+ NKT cells to direct infection and subsequent cell death by HIV-1. Here, we demonstrate that highly active antiretroviral therapy (HAART) results in the rapid recovery of predominantly CD4(-) NKT cells with kinetics that are strikingly similar to those of mainstream T cells. As it is well known that the early recovery of mainstream T cells in response to HAART is due to their redistribution from tissues to the circulation, our data suggest that the selective depletion of circulating NKT cells is likely due to a combination of cell death and tissue sequestration and indicates that HAART can improve immune functions by reconstituting both conventional T cells and immunoregulatory NKT cells.

Peripheral blood IFN-gamma-secreting Valpha24+Vbeta11+ NKT cell numbers are decreased in cancer patients independent of tumor type or tumor load.

Natural killer T (NKT) cells are CD1d-restricted lymphoid cells and are characterized by an invariant T-cell receptor, which in humans consists of a Valpha24 chain paired with a Vbeta11 chain. These cells are known for their rapid production of large amounts of cytokines (e.g., IFN-gamma and IL-4), thereby modulating other cells of the immune system such as T cells, NK cells and dendritic cells. NKT cells have been reported to play important regulatory roles in many immune responses, including antitumor immune responses. Here, we demonstrate an age-dependent decrease in circulating Valpha24(+)Vbeta11(+) NKT cell numbers in both healthy controls and cancer patients and demonstrate that in both groups females have higher NKT cell levels compared to males. In a large group of 120 cancer patients, we show that circulating Valpha24(+)Vbeta11(+) NKT cell numbers are about 50% lower than in age- and gender-matched healthy controls and that this decrease is independent of tumor type or tumor load. This decrease was not restored upon tumor removal by means of surgery or radiotherapy. Even though the percentage of NKT cells that secrete IFN-gamma, as detected by ELISPOT, is normal in cancer patients, the absolute number of circulating IFN-gamma-secreting NKT cells is reduced. Together, our results suggest that the reduced circulating Valpha24(+)Vbeta11(+) NKT cell numbers in cancer patients are not affected by tumor load, but might actually reflect a risk factor for tumor development, e.g., by hampering efficient tumor immunosurveillance.

Circulating Valpha24+Vbeta11+ NKT cell numbers and dendritic cell CD1d expression in hepatitis C virus infected patients.

CD1d-restricted natural killer T (NKT) cells are involved in the regulation of various immune responses, and have been shown to inhibit viral replication in animal hepatitis models when activated by the glycolipid alpha-galactosylceramide (alpha-GalCer, KRN7000). Previous studies have indicated that alpha-GalCer-induced activation of the immune system requires both CD1d expression by antigen-presenting cells as well as (normal) numbers of NKT cells. Discrepancies exist over circulating numbers of human invariant Valpha24+Vbeta11+ NKT cells during hepatitis C virus (HCV) infection. Here, by cross-sectional analysis and longitudinal analysis of patients undergoing effective combination antiviral therapy, we demonstrate that circulating Valpha24+Vbeta11+ NKT cell numbers are not decreased during active HCV infection. Importantly, as we also show that CD1d is expressed at comparable levels by peripheral blood monocytes and CD1c+ myeloid dendritic cells (DC) of healthy individuals and HCV-infected patients, these data indicate that all ingredients for evaluating the antiviral effects of the Valpha24+Vbeta11+ NKT cell ligand alpha-GalCer in HCV-infected patients are present.

The immunoregulatory role of CD1d-restricted natural killer T cells in disease.

Natural killer T (NKT) cells constitute a T cell subpopulation that shares several characteristics with NK cells. NKT cells are characterized by a narrow T cell antigen receptor (TCR) repertoire, recognize glycolipid antigen in the context of the monomorphic CD1d antigen-presenting molecule, and have the unique capacity to rapidly produce large amounts of both T helper (Th) 1 and Th2 cytokines. Important roles of NKT cells have now been demonstrated in the regulation of autoimmune, allergic, antimicrobial, and antitumor immune responses. Here, we review the immunoregulatory role of NKT cells in disease and discuss NKT cell based immunotherapeutic strategies.

Polarization of Valpha24+ Vbeta11+ natural killer T cells of healthy volunteers and cancer patients using alpha-galactosylceramide-loaded and environmentally instructed dendritic cells.

CD1d-restricted natural killer T (NKT) cells play important regulatory roles in various immune responses. NKT cell-derived T helper (Th) 1 cytokines are important in the induction of antitumor immune responses in mice. Because the CD1d-restricted Valpha24(+) Vbeta11(+) NKT cell population in cancer patients is decreased both in size and in its capacity to secrete IFN-gamma, therapeutic strategies based on reconstitution of type 1 polarized Valpha24(+) Vbeta11(+) NKT cells merit additional investigation. Here, we report the simultaneous strong expansion and type 1 polarization of human invariant Valpha24(+) Vbeta11(+) NKT cells using alpha-galactosylceramide-loaded type 1 dendritic cells and interleukin 15. Type 1 polarized Valpha24(+) Vbeta11(+) NKT cells produced high levels of IFN-gamma, tumor necrosis factor alpha, and granulocyte macrophage colony-stimulating factor, and induced strong cytotoxicity in Jurkat cells in an alpha-galactosylceramide-dependent manner. Importantly, the cytokine profile of Valpha24(+) Vbeta11(+) NKT cells that were initially expanded under Th2 polarizing conditions could be reversed to a Th1 cytokine profile, indicating the plasticity of the cytokine profile of the human adult Valpha24(+) Vbeta11(+) NKT cell population.