Dimethyl Sulfoxide Protects Escherichia coli from Rapid Antimicrobial-Mediated Killing.

PubMed

Mi, Hongfei; Wang, Dai; Xue, Yunxin; Zhang, Zhi; Niu, Jianjun; Hong, Yuzhi; Drlica, Karl; Zhao, Xilin

2016-08-01

The contribution of reactive oxygen species (ROS) to antimicrobial lethality was examined by treating Escherichia coli with dimethyl sulfoxide (DMSO), an antioxidant solvent frequently used in antimicrobial studies. DMSO inhibited killing by ampicillin, kanamycin, and two quinolones and had little effect on MICs. DMSO-mediated protection correlated with decreased ROS accumulation and provided evidence for ROS-mediated programmed cell death. These data support the contribution of ROS to antimicrobial lethality and suggest caution when using DMSO-dissolved antimicrobials for short-time killing assays. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Immune Interventions to Eliminate the HIV Reservoir.

PubMed

Hsu, Denise C; Ananworanich, Jintanat

2017-10-26

Inducing HIV remission is a monumental challenge. A potential strategy is the "kick and kill" approach where latently infected cells are first activated to express viral proteins and then eliminated through cytopathic effects of HIV or immune-mediated killing. However, pre-existing immune responses to HIV cannot eradicate HIV infection due to the presence of escape variants, inadequate magnitude, and breadth of responses as well as immune exhaustion. The two major approaches to boost immune-mediated elimination of infected cells include enhancing cytotoxic T lymphocyte mediated killing and harnessing antibodies to eliminate HIV. Specific strategies include increasing the magnitude and breadth of T cell responses through therapeutic vaccinations, reversing the effects of T cell exhaustion using immune checkpoint inhibition, employing bispecific T cell targeting immunomodulatory proteins or dual-affinity re-targeting molecules to direct cytotoxic T lymphocytes to virus-expressing cells and broadly neutralizing antibody infusions. Methods to steer immune responses to tissue sites where latently infected cells are located need to be further explored. Ultimately, strategies to induce HIV remission must be tolerable, safe, and scalable in order to make a global impact.

Abrogation of TNF-mediated cytotoxicity by space flight involves protein kinase C

NASA Technical Reports Server (NTRS)

Woods, K. M.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Experiments conducted on STS-50 indicated that space flight significantly inhibited tumor necrosis factor (TNF)-mediated killing of LM929 cells compared to ground controls. In ground-based studies, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also inhibited TNF-mediated killing of LM929 cells. Therefore, we used PKC inhibitors to determine if the inhibitory effects of spaceflight on TNF-mediated cytotoxicity involved the activation of PKC. In experiments conducted onboard space shuttle mission STS-54, we saw that in the presence of the protein kinase C inhibitors H7 and H8, TNF-mediated cytotoxicity was restored to levels of those observed in the ground controls. Subsequent experiments done during the STS-57 mission tested the dose response of two protein kinase inhibitors, H7 and HA1004. We again saw that killing was restored in a dose-dependent manner, with inhibitor concentrations known to inhibit PKC being most effective. These data suggest that space flight ameliorates the action of TNF by affecting PKC in target cells.

Sibling Rivalry in Myxococcus xanthus Is Mediated by Kin Recognition and a Polyploid Prophage.

PubMed

Dey, Arup; Vassallo, Christopher N; Conklin, Austin C; Pathak, Darshankumar T; Troselj, Vera; Wall, Daniel

2016-01-19

Myxobacteria form complex social communities that elicit multicellular behaviors. One such behavior is kin recognition, in which cells identify siblings via their polymorphic TraA cell surface receptor, to transiently fuse outer membranes and exchange their contents. In addition, outer membrane exchange (OME) regulates behaviors, such as inhibition of wild-type Myxococcus xanthus (DK1622) from swarming. Here we monitored the fate of motile cells and surprisingly found they were killed by nonmotile siblings. The kill phenotype required OME (i.e., was TraA dependent). The genetic basis of killing was traced to ancestral strains used to construct DK1622. Specifically, the kill phenotype mapped to a large "polyploid prophage," Mx alpha. Sensitive strains contained a 200-kb deletion that removed two of three Mx alpha units. To explain these results, we suggest that Mx alpha expresses a toxin-antitoxin cassette that uses the OME machinery of M. xanthus to transfer a toxin that makes the population "addicted" to Mx alpha. Thus, siblings that lost Mx alpha units (no immunity) are killed by cells that harbor the element. To test this, an Mx alpha-harboring laboratory strain was engineered (by traA allele swap) to recognize a closely related species, Myxococcus fulvus. As a result, M. fulvus, which lacks Mx alpha, was killed. These TraA-mediated antagonisms provide an explanation for how kin recognition specificity might have evolved in myxobacteria. That is, recognition specificity is determined by polymorphisms in traA, which we hypothesize were selected for because OME with non-kin leads to lethal outcomes. The transition from single cell to multicellular life is considered a major evolutionary event. Myxobacteria have successfully made this transition. For example, in response to starvation, individual cells aggregate into multicellular fruiting bodies wherein cells differentiate into spores. To build fruits, cells need to recognize their siblings, and in part, this is mediated by the TraA cell surface receptor. Surprisingly, we report that TraA recognition can also involve sibling killing. We show that killing originates from a prophage-like element that has apparently hijacked the TraA system to deliver a toxin to kin. We hypothesize that this killing system has imposed selective pressures on kin recognition, which in turn has resulted in TraA polymorphisms and hence many different recognition groups. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Sibling Rivalry in Myxococcus xanthus Is Mediated by Kin Recognition and a Polyploid Prophage

PubMed Central

Dey, Arup; Vassallo, Christopher N.; Conklin, Austin C.; Pathak, Darshankumar T.; Troselj, Vera

2016-01-01

ABSTRACT Myxobacteria form complex social communities that elicit multicellular behaviors. One such behavior is kin recognition, in which cells identify siblings via their polymorphic TraA cell surface receptor, to transiently fuse outer membranes and exchange their contents. In addition, outer membrane exchange (OME) regulates behaviors, such as inhibition of wild-type Myxococcus xanthus (DK1622) from swarming. Here we monitored the fate of motile cells and surprisingly found they were killed by nonmotile siblings. The kill phenotype required OME (i.e., was TraA dependent). The genetic basis of killing was traced to ancestral strains used to construct DK1622. Specifically, the kill phenotype mapped to a large “polyploid prophage,” Mx alpha. Sensitive strains contained a 200-kb deletion that removed two of three Mx alpha units. To explain these results, we suggest that Mx alpha expresses a toxin-antitoxin cassette that uses the OME machinery of M. xanthus to transfer a toxin that makes the population “addicted” to Mx alpha. Thus, siblings that lost Mx alpha units (no immunity) are killed by cells that harbor the element. To test this, an Mx alpha-harboring laboratory strain was engineered (by traA allele swap) to recognize a closely related species, Myxococcus fulvus. As a result, M. fulvus, which lacks Mx alpha, was killed. These TraA-mediated antagonisms provide an explanation for how kin recognition specificity might have evolved in myxobacteria. That is, recognition specificity is determined by polymorphisms in traA, which we hypothesize were selected for because OME with non-kin leads to lethal outcomes. IMPORTANCE The transition from single cell to multicellular life is considered a major evolutionary event. Myxobacteria have successfully made this transition. For example, in response to starvation, individual cells aggregate into multicellular fruiting bodies wherein cells differentiate into spores. To build fruits, cells need to recognize their siblings, and in part, this is mediated by the TraA cell surface receptor. Surprisingly, we report that TraA recognition can also involve sibling killing. We show that killing originates from a prophage-like element that has apparently hijacked the TraA system to deliver a toxin to kin. We hypothesize that this killing system has imposed selective pressures on kin recognition, which in turn has resulted in TraA polymorphisms and hence many different recognition groups. PMID:26787762

The oncolytic peptide LTX-315 kills cancer cells through Bax/Bak-regulated mitochondrial membrane permeabilization.

PubMed

Zhou, Heng; Forveille, Sabrina; Sauvat, Allan; Sica, Valentina; Izzo, Valentina; Durand, Sylvère; Müller, Kevin; Liu, Peng; Zitvogel, Laurence; Rekdal, Øystein; Kepp, Oliver; Kroemer, Guido

2015-09-29

LTX-315 has been developed as an amphipathic cationic peptide that kills cancer cells. Here, we investigated the putative involvement of mitochondria in the cytotoxic action of LTX-315. Subcellular fractionation of LTX-315-treated cells, followed by mass spectrometric quantification, revealed that the agent was enriched in mitochondria. LTX-315 caused an immediate arrest of mitochondrial respiration without any major uncoupling effect. Accordingly, LTX-315 disrupted the mitochondrial network, dissipated the mitochondrial inner transmembrane potential, and caused the release of mitochondrial intermembrane proteins into the cytosol. LTX-315 was relatively inefficient in stimulating mitophagy. Cells lacking the two pro-apoptotic multidomain proteins from the BCL-2 family, BAX and BAK, were less susceptible to LTX-315-mediated killing. Moreover, cells engineered to lose their mitochondria (by transfection with Parkin combined with treatment with a protonophore causing mitophagy) were relatively resistant against LTX-315, underscoring the importance of this organelle for LTX-315-mediated cytotoxicity. Altogether, these results support the notion that LTX-315 kills cancer cells by virtue of its capacity to permeabilize mitochondrial membranes.

The oncolytic peptide LTX-315 kills cancer cells through Bax/Bak-regulated mitochondrial membrane permeabilization

PubMed Central

Zhou, Heng; Forveille, Sabrina; Sauvat, Allan; Sica, Valentina; Izzo, Valentina; Durand, Sylvère; Müller, Kevin; Liu, Peng; Zitvogel, Laurence; Rekdal, Øystein; Kepp, Oliver; Kroemer, Guido

2015-01-01

LTX-315 has been developed as an amphipathic cationic peptide that kills cancer cells. Here, we investigated the putative involvement of mitochondria in the cytotoxic action of LTX-315. Subcellular fractionation of LTX-315-treated cells, followed by mass spectrometric quantification, revealed that the agent was enriched in mitochondria. LTX-315 caused an immediate arrest of mitochondrial respiration without any major uncoupling effect. Accordingly, LTX-315 disrupted the mitochondrial network, dissipated the mitochondrial inner transmembrane potential, and caused the release of mitochondrial intermembrane proteins into the cytosol. LTX-315 was relatively inefficient in stimulating mitophagy. Cells lacking the two pro-apoptotic multidomain proteins from the BCL-2 family, BAX and BAK, were less susceptible to LTX-315-mediated killing. Moreover, cells engineered to lose their mitochondria (by transfection with Parkin combined with treatment with a protonophore causing mitophagy) were relatively resistant against LTX-315, underscoring the importance of this organelle for LTX-315-mediated cytotoxicity. Altogether, these results support the notion that LTX-315 kills cancer cells by virtue of its capacity to permeabilize mitochondrial membranes. PMID:26378049

Killing of Staphylococcus aureus via Magnetic Hyperthermia Mediated by Magnetotactic Bacteria

PubMed Central

Chen, Changyou; Chen, Linjie; Yi, Yong; Chen, Chuanfang

2016-01-01

Staphylococcus aureus is a common hospital and household pathogen. Given the emergence of antibiotic-resistant derivatives of this pathogen resulting from the use of antibiotics as general treatment, development of alternative therapeutic strategies is urgently needed. Here, we assess the feasibility of killing S. aureus cells in vitro and in vivo through magnetic hyperthermia mediated by magnetotactic bacteria that possess magnetic nanocrystals and demonstrate magnetically steered swimming. The S. aureus suspension was added to magnetotactic MO-1 bacteria either directly or after coating with anti-MO-1 polyclonal antibodies. The suspensions were then subjected to an alternating magnetic field (AMF) for 1 h. S. aureus viability was subsequently assessed through conventional plate counting and flow cytometry. We found that approximately 30% of the S. aureus cells mixed with uncoated MO-1 cells were killed after AMF treatment. Moreover, attachment between the magnetotactic bacteria and S. aureus increased the killing efficiency of hyperthermia to more than 50%. Using mouse models, we demonstrated that magnetic hyperthermia mediated by antibody-coated magnetotactic MO-1 bacteria significantly improved wound healing. These results collectively demonstrated the effective eradication of S. aureus both in vitro and in vivo, indicating the potential of magnetotactic bacterium-mediated magnetic hyperthermia as a treatment for S. aureus-induced skin or wound infections. PMID:26873320

Mycobacterium tuberculosis-Infected Hematopoietic Stem and Progenitor Cells Unable to Express Inducible Nitric Oxide Synthase Propagate Tuberculosis in Mice.

PubMed

Reece, Stephen T; Vogelzang, Alexis; Tornack, Julia; Bauer, Wolfgang; Zedler, Ulrike; Schommer-Leitner, Sandra; Stingl, Georg; Melchers, Fritz; Kaufmann, Stefan H E

2018-04-23

Persistence of Mycobacterium tuberculosis within human bone marrow stem cells has been identified as a potential bacterial niche during latent tuberculosis. Using a murine model of tuberculosis, we show here that bone marrow stem and progenitor cells containing M. tuberculosis propagated tuberculosis when transferred to naive mice, given that both transferred cells and recipient mice were unable to express inducible nitric oxide synthase, which mediates killing of intracellular bacteria via nitric oxide. Our findings suggest that bone marrow stem and progenitor cells containing M. tuberculosis propagate hallmarks of disease if nitric oxide-mediated killing of bacteria is defective.

OH radicals from the indirect actions of X-rays induce cell lethality and mediate the majority of the oxygen enhancement effect.

PubMed

Hirayama, Ryoichi; Ito, Atsushi; Noguchi, Miho; Matsumoto, Yoshitaka; Uzawa, Akiko; Kobashi, Gen; Okayasu, Ryuichi; Furusawa, Yoshiya

2013-11-01

We examined OH radical-mediated indirect actions from X irradiation on cell killing in wild-type Chinese hamster ovary cell lines (CHO and AA8) under oxic and hypoxic conditions, and compared the contribution of direct and indirect actions under both conditions. The contribution of indirect action on cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide, which suppresses indirect action by quenching OH radicals without affecting the direct action of X rays on cell killing. The contributions of indirect action on cell killing of CHO cells were 76% and 50% under oxic and hypoxic conditions, respectively, and those for AA8 cells were 85% and 47%, respectively. Therefore, the indirect action on cell killing was enhanced by oxygen during X irradiation in both cell lines tested. Oxygen enhancement ratios (OERs) at the 10% survival level (D10 or LD90) for CHO and AA8 cells were 2.68 ± 0.15 and 2.76 ± 0.08, respectively. OERs were evaluated separately for indirect and direct actions, which gave the values of 3.75 and 2.01 for CHO, and 4.11 and 1.32 for AA8 cells, respectively. Thus the generally accepted OER value of ∼3 is best understood as the average of the OER values for both indirect and direct actions. These results imply that both indirect and direct actions on cell killing require oxygen for the majority of lethal DNA damage, however, oxygen plays a larger role in indirect than for direct effects. Conversely, the lethal damage induced by the direct action of X rays are less affected by oxygen concentration.

Homologous species restriction of the complement-mediated killing of nucleated cells.

PubMed Central

Yamamoto, H; Blaas, P; Nicholson-Weller, A; Hänsch, G M

1990-01-01

The homologous restriction of complement (C) lysis is attributed to membrane proteins: decay-accelerating factor (DAF), C8 binding protein (C8bp) and P18/CD59. Since these proteins are also expressed on peripheral blood cells, species restriction was tested for in the complement-mediated killing of antibody-coated human leucocytes by human or rabbit complement. Killing was more efficient when rabbit complement was used. Preincubation of cells with an antibody to DAF abolished the difference. When C1-7 sites were first attached to the cells and either rabbit or human C8, C9 were added, the killing of monocytes and lymphocytes was equally efficient; only in polymorphonuclear neutrophils was a higher efficiency of rabbit C8, C9 seen. Thus, in contrast to haemolysis, restriction occurred predominantly at the C3 level and the action of the terminal complement components was not inhibited. Since C8bp isolated from peripheral blood cells showed essentially similar characteristics as the erythrocyte-derived C8bp, the failure of C8bp to inhibit the action of the terminal components on nucleated cells might reflect differences of the complement membrane interactions between erythrocytes or nucleated cells, respectively. Images Figure 5 PMID:1697561

[Retroviral-mediated transfer of a hygromycin phosphotransferase-thymidine kinase fusion gene into human bladder carcinoma cell].

PubMed

Ye, C; Chen, S; Pei, X; Li, L; Feng, K

1999-08-01

To evaluate the therapeutic efficacy of retroviral-mediated hygromycin phosphotransferase-thymidine kinase fusion gene (HyTK)/GCV on human bladder carcinoma cell. A retroviral expression vector pL (HyTK) SN was constructed. By using FuGENE 6-mediated transfection and "ping-pong effect" technique, high-titer of retroviral supernatant was obtained and HyTK gene was transferred into EJ cells. A retroviral vector encoding, enhanced green fluorescent protein, EGFP was used to rapidly detect the transduction efficiency. Antitumor effects were observed after GCV treatment. In vitro experiments demonstrated the EJ cells transferred by HyTK gene were killed in the GCV treatment. Non-transduced parental cells were not sensitive to GCV, but they were dead by the bystander killing of neighboring cells when mixed with EJ/HyTK cells at various ratios. In addition, this not only affect wild-type EJ cells but also cells from different bladder carcinoma cell lines. Retroviral-mediated HyTK/GCV systems were a promising suicide gene therapy for bladder carcinoma. EGFP may act as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in bladder carcinoma cells.

Multiple factors and processes involved in host cell killing by bacteriophage Mu: characterization and mapping.

PubMed

Waggoner, B T; Marrs, C F; Howe, M M; Pato, M L

1984-07-15

The regions of bacteriophage Mu involved in host cell killing were determined by infection of a lambda-immune host with 12 lambda pMu-transducing phages carrying different amounts of Mu DNA beginning at the left end. Infecting lambda pMu phages containing 5.0 (+/- 0.2) kb or less of the left end of Mu DNA did not kill the lambda-immune host, whereas lambda pMu containing 5.1 kb did kill, thus locating the right end of the kil gene between approximately 5.0 and 5.1 kb. For the Kil+ phages the extent of killing increased as the multiplicity of infection (m.o.i.) increased. In addition, killing was also affected by the presence of at least two other regions of Mu DNA: one, located between 5.1 and 5.8 kb, decreased the extent of killing; the other, located between 6.3 and 7.9 kb, greatly increased host cell killing. Killing was also assayed after lambda pMu infection of a lambda-immune host carrying a mini-Mu deleted for most of the B gene and the middle region of Mu DNA. Complementation of mini-Mu replication by infecting B+ lambda pMu phages resulted in killing of the lambda-immune, mini-Mu-containing host, regardless of the presence or absence of the Mu kil gene. The extent of host cell killing increased as the m.o.i. of the infecting lambda pMu increased, and was further enhanced by both the presence of the kil gene and the region located between 6.3 and 7.9 kb. These distinct processes of kil-mediated killing in the absence of replication and non-kil-mediated killing in the presence of replication were also observed after induction of replication-deficient and kil mutant prophages, respectively.

Mycobacterium tuberculosis-Infected Hematopoietic Stem and Progenitor Cells Unable to Express Inducible Nitric Oxide Synthase Propagate Tuberculosis in Mice

PubMed Central

Reece, Stephen T; Vogelzang, Alexis; Tornack, Julia; Bauer, Wolfgang; Zedler, Ulrike; Schommer-Leitner, Sandra; Stingl, Georg; Melchers, Fritz; Kaufmann, Stefan H E

2018-01-01

Abstract Persistence of Mycobacterium tuberculosis within human bone marrow stem cells has been identified as a potential bacterial niche during latent tuberculosis. Using a murine model of tuberculosis, we show here that bone marrow stem and progenitor cells containing M. tuberculosis propagated tuberculosis when transferred to naive mice, given that both transferred cells and recipient mice were unable to express inducible nitric oxide synthase, which mediates killing of intracellular bacteria via nitric oxide. Our findings suggest that bone marrow stem and progenitor cells containing M. tuberculosis propagate hallmarks of disease if nitric oxide-mediated killing of bacteria is defective. PMID:29471332

Requirement and Redundancy of the Src Family Kinases Fyn and Lyn in Perforin-Dependent Killing of Cryptococcus neoformans by NK Cells

PubMed Central

Oykhman, Paul; Timm-McCann, Martina; Xiang, Richard F.; Islam, Anowara; Li, Shu Shun; Stack, Danuta; Huston, Shaunna M.; Ma, Ling Ling

2013-01-01

Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase–extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse. PMID:23918783

Mediation of host immune responses after immunization of neonatal calves with a heat-killed Mycobacterium avium subsp. paratuberculosis vaccine

USDA-ARS?s Scientific Manuscript database

A major drawback of current whole-cell vaccines for Mycobacterium avium subsp. paratuberculosis(MAP) is the interference with diagnostic tests for bovine tuberculosis and paratuberculosis. The current study was designed to explore effects of immunization with a heat-killed whole cell vaccine (Mycop...

Heat-killed Lactobacillus spp. cells enhance survivals of Caenorhabditis elegans against Salmonella and Yersinia infections.

PubMed

Lee, J; Choe, J; Kim, J; Oh, S; Park, S; Kim, S; Kim, Y

2015-12-01

This study examined the effect of feeding heat-killed Lactobacillus cells on the survival of Caenorhabditis elegans nematodes after Salmonella Typhimurium and Yersinia enterocolitica infection. The feeding of heat-killed Lactobacillus plantarum 133 (LP133) and Lactobacillus fermentum 21 (LP21) cells to nematodes was shown to significantly increase the survival rate as well as stimulate the expression of pmk-1 gene that key factor for C. elegans immunity upon infection compared with control nematodes that were only fed Escherichia coli OP50 (OP50) cells. These results suggest that heat-killed LP133 and LF21 cells exert preventive or protective effects against the Gram-negative bacteria Salm. Typhimurium and Y. enterocolitica. To better understand the mechanisms underlying the LF21-mediated and LP133-mediated protection against bacterial infection in nematodes, transcriptional profiling was performed for each experimental group. These experiments showed that genes related to energy generation and ageing, regulators of insulin/IGF-1-like signalling, DAF genes, oxidation and reduction processes, the defence response and/or the innate immune response, and neurological processes were upregulated in nematodes that had been fed heat-killed Lactobacillus cells compared with nematodes that had been fed E. coli cells. In this study, the feeding of heat-killed Lactobacillus bacteria to Caenorhabditis elegans nematodes was shown to decrease infection by Gram-negative bacteria and increase the host lifespan. C. elegans has a small, well-organized genome and is an excellent in vivo model organism; thus, these results will potentially shed light on important Lactobacillus-host interactions. © 2015 The Society for Applied Microbiology.

Expression of complement membrane regulators membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59) in human malignant gliomas.

PubMed Central

Mäenpää, A.; Junnikkala, S.; Hakulinen, J.; Timonen, T.; Meri, S.

1996-01-01

Gliomas are malignant brain tumors, which, despite recent progress in surgical and radiological treatment, still have a poor prognosis. Since gliomas apparently resist immunological clearance mechanisms, we became interested in examining bow gliomas resist killing by the human complement system. The resistance of human cells to complement-mediated damage is, in large part, mediated by specific inhibitors of complement:membrane cofactor protein (CD46), decay-accelerating factor (CD55), and protectin (CD59). In the present study we examined the expression of complement regulators in 14 human glioma tumors and in 7 glioma cell lines (U251, U87, HS683, U373, U138, U118, and H2). Protectin was found to be strongly expressed by all glioma tumors and cell lines. Northern blotting analysis demonstrated the typical pattern of four to five protectin mRNAs in the glioma cells. Except for blood vessels, the expression of decay-accelerating factor was weak or absent in the tumors in situ, whereas in the cell lines its expression varied, ranging from negative to intermediate. Membrane cofactor protein was moderately expressed by all the cell lines but only weakly in the tumors. Cell-killing experiments demonstrated that the glioma cell lines were exceptionally resistant to C-mediated lysis. Five of the seven cell lines (U373, HS683, U118, U138, and H2) resisted complement lysis under conditions where most other cell lines were sensitive to killing. Neutralization experiments using specific monoclonal antibodies indicated that protectin was functionally the most important complement regulator in the glioma cells. The killing of the U87 and U251 cells could be significantly increased by a blocking anti-protectin monoclonal antibody, whereas for the other cell lines only moderate or no response was observed. The H2 cell line resisted killing by all antibodies and by complement. These results show that protectin is the most important complement regulator on human glioma cells. The exceptional complement resistance of some glioma cell lines suggests that they may utilize other, hitherto less well characterized, mechanisms to resist complement killing. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 PMID:8644856

Menadione reduction by pharmacological doses of ascorbate induces an oxidative stress that kills breast cancer cells.

PubMed

Beck, Raphaël; Verrax, Julien; Dejeans, Nicolas; Taper, Henryk; Calderon, Pedro Buc

2009-01-01

Oxidative stress generated by ascorbate-driven menadione redox cycling kills MCF7 cells by a concerted mechanism including glycolysis inhibition, loss of calcium homeostasis, DNA damage and changes in mitogen activated protein kinases (MAPK) activities. Cell death is mediated by necrosis rather than apoptosis or macroautophagy. Neither 3-methyladenine nor Z-VAD affects cytotoxicity by ascorbate/menadione (Asc/Men). BAPTA-AM, by restoring cellular capacity to reduce MTT, underlines the role of calcium in the necrotic process. Oxidative stress-mediated cell death is shown by the opposite effects of N-acetylcysteine and 3-aminotriazole. Moreover, oxidative stress induces DNA damage (protein poly-ADP-ribosylation and gamma-H2AX phosphorylation) and inhibits glycolysis. Asc/Men deactivates extracellular signal-regulated kinase (ERK) while activating p38, suggesting an additional mechanism to kill MCF7 cells. Since ascorbate is taken up by cancer cells and, due to their antioxidant enzyme deficiency, oxidative stress should affect cancer cells to a greater extent than normal cells. This differential sensitivity may have clinical applications.

Phosphatase inhibition augments anti-CD22-mediated signaling and cytotoxicity in non-hodgkin's lymphoma cells.

PubMed

O'Donnell, Robert T; Pearson, David; McKnight, Hayes C; Ma, Ya Peng; Tuscano, Joseph M

2009-07-01

CD22 is a cell-surface molecule found on most B-cell lymphomas (NHL). HB22.7 is an anti-CD22 antibody that blocks CD22 ligand binding, initiates signaling, and kills NHL cells. The SHP-1 tyrosine phosphatase is disproportionately associated with the cytoplasmic domain of CD22. Sodium orthovanadate (NaV) and dephostatin (DP) are phosphatase inhibitors. The interaction of SHP-1 with CD22 presents an opportunity to manipulate CD22-mediated signaling effects. NaV caused dose dependent killing of NHL cells in vitro; when HB22.7 was given with NaV, antibody-mediated cell death increased. NaV caused a substantial increase in CD22-mediated SAPK and ERK-1/2 activation when CD22 was crosslinked by HB22.7; NaV did not significantly affect IgM-mediated signals. Studies using Raji NHL cells stably transfected with a SHP-1 dominant negative (DN) confirmed that these observations were due to SHP-1 inhibition. The relatively specific association of SHP-1 with CD22 suggests that CD22-specific signaling may be altered by phosphatase inhibition in ways that could prove useful for anti-CD22-based immunotherapy.

Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults

PubMed Central

MacLennan, Calman A.; Gilchrist, James J.; Gordon, Melita A.; Cunningham, Adam F.; Cobbold, Mark; Goodall, Margaret; Kingsley, Robert A.; van Oosterhout, Joep J. G.; Msefula, Chisomo L.; Mandala, Wilson L.; Leyton, Denisse L.; Marshall, Jennifer L.; Gondwe, Esther N.; Bobat, Saeeda; López-Macías, Constantino; Doffinger, Rainer; Henderson, Ian R.; Zijlstra, Eduard E.; Dougan, Gordon; Drayson, Mark T.; MacLennan, Ian C. M.; Molyneux, Malcolm E.

2013-01-01

Nontyphoidal Salmonellae are a major cause of life-threatening bacteremia among HIV-infected individuals. Although cell-mediated immunity controls intracellular infection, antibody protects against Salmonella bacteremia. We report that high titer antibodies specific for Salmonella lipopolysaccharide (LPS) associate with absent Salmonella-killing in HIV-infected African adults. Killing was restored by genetically shortening LPS from target Salmonella, or removing LPS-specific antibodies from serum. Complement-mediated killing of Salmonella by healthy serum is shown to be induced specifically by antibodies against outer membrane proteins. This killing is lost when excess antibody against Salmonella LPS is added. Thus our study indicates impaired immunity against nontyphoidal Salmonella bacteremia in HIV infection results from excess inhibitory antibodies against Salmonella LPS, whilst serum killing of Salmonella is induced by antibodies against outer membrane proteins. PMID:20413503

Sildenafil (Viagra) sensitizes prostate cancer cells to doxorubicin-mediated apoptosis through CD95

PubMed Central

Das, Anindita; Durrant, David; Mitchell, Clint; Dent, Paul; Batra, Surinder K.; Kukreja, Rakesh C.

2016-01-01

We previously reported that Sildenafil enhances apoptosis and antitumor efficacy of doxorubicin (DOX) while attenuating its cardiotoxic effect in prostate cancer. In the present study, we investigated the mechanism by which sildenafil sensitizes DOX in killing of prostate cancer (PCa) cells, DU145. The death receptor Fas (APO-1 or CD95) induces apoptosis in many carcinoma cells, which is negatively regulated by anti-apoptotic molecules such as FLIP (Fas-associated death domain (FADD) interleukin-1-converting enzyme (FLICE)-like inhibitory protein). Co-treatment of PCa cells with sildenafil and DOX for 48 hours showed reduced expression of both long and short forms of FLIP (FLIP-L and -S) as compared to individual drug treatment. Over-expression of FLIP-s with an adenoviral vector attentuated the enhanced cell-killing effect of DOX and sildenafil. Colony formation assays also confirmed that FLIP-S over-expression inhibited the DOX and sildenafil-induced synergistic killing effect as compared to the cells infected with an empty vector. Moreover, siRNA knock-down of CD95 abolished the effect of sildenafil in enhancing DOX lethality in cells, but had no effect on cell killing after treatment with a single agent. Sildenafil co-treatment with DOX inhibited DOX-induced NF-κB activity by reducing phosphorylation of IκB and nuclear translocation of the p65 subunit, in addition to down regulation of FAP-1 (Fas associated phosphatase-1, a known inhibitor of CD95-mediated apoptosis) expression. This data provides evidence that the CD95 is a key regulator of sildenafil and DOX mediated enhanced cell death in prostate cancer. PMID:26716643

Electroporation driven delivery of both an IL-12 expressing plasmid and cisplatin synergizes to inhibit B16 melanoma tumor growth through an NK cell mediated tumor killing mechanism.

PubMed

Kim, Ha; Sin, Jeong-Im

2012-11-01

Combined therapy using chemotherapeutic drugs and immunotherapeutics offers some promise for treating patients with cancer. In this study, we evaluated whether cisplatin delivered by intratumoral (IT)-electroporation (EP) might enhance antitumor activity against established B16 melanoma and whether further addition of intramuscular (IM)-EP of IL-12 cDNA to IT-EP of cisplatin might augment antitumor therapeutic activity, with a focus on the underlining antitumor mechanism(s). When tumor (7 mm)-bearing animals were treated locally with cisplatin by IT-EP, they showed tumor growth inhibition significantly more than those without IT-EP. Moreover, IL-12 cDNA delivered by IM-EP was also able to inhibit tumor growth significantly more than control vector delivery. This tumor growth inhibition was mediated by NK cells, but not CD4+ T or CD8+ T cells, as determined by immune cell subset depletion and IFN-γ induction. Moreover, concurrent therapy using IT-EP of cisplatin plus IM-EP of IL-12 cDNA displayed antitumor therapeutic synergy. This therapeutic synergy appeared to be mediated by increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. Taken together, these data support that cisplatin delivery by IT-EP plus IL-12 gene delivery by IM-EP are more effective at inducing antitumor therapeutic responses through increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. This combined approach might have some implication for treating melanoma in patients.

Interactions between neutrophils and macrophages promote macrophage killing of rat muscle cells in vitro

NASA Technical Reports Server (NTRS)

Nguyen, Hal X.; Tidball, James G.

2003-01-01

Current evidence indicates that the physiological functions of inflammatory cells are highly sensitive to their microenvironment, which is partially determined by the inflammatory cells and their potential targets. In the present investigation, interactions between neutrophils, macrophages and muscle cells that may influence muscle cell death are examined. Findings show that in the absence of macrophages, neutrophils kill muscle cells in vitro by superoxide-dependent mechanisms, and that low concentrations of nitric oxide (NO) protect against neutrophil-mediated killing. In the absence of neutrophils, macrophages kill muscle cells through a NO-dependent mechanism, and the presence of target muscle cells causes a three-fold increase in NO production by macrophages, with no change in the concentration of inducible nitric oxide synthase. Muscle cells that are co-cultured with both neutrophils and macrophages in proportions that are observed in injured muscle show cytotoxicity through a NO-dependent, superoxide-independent mechanism. Furthermore, the concentration of myeloid cells that is necessary for muscle killing is greatly reduced in assays that use mixed myeloid cell populations, rather than uniform populations of neutrophils or macrophages. These findings collectively show that the magnitude and mechanism of muscle cell killing by myeloid cells are modified by interactions between muscle cells and neutrophils, between muscle cells and macrophages and between macrophages and neutrophils.

Potassium iodide potentiates antimicrobial photodynamic inactivation mediated by Rose Bengal: in vitro and in vivo studies

NASA Astrophysics Data System (ADS)

Wen, Xiang; Zhang, Xiaoshen; Szewczyk, Grzegorz; ElHussien, Ahmed; Huang, Ying-Ying; Sarna, Tadeusz; Hamblin, Michael R.

2018-02-01

Rose Bengal (RB) is a halogenated xanthene dye that has been used to mediate antimicrobial photodynamic inactivation. While highly active against Gram-positive bacteria, RB is largely inactive in killing Gram-negative bacteria. We have discovered that addition of the non-toxic salt potassium iodide (100mM) potentiates green light (540nm)-mediated killing by up to six extra logs with Gramnegative bacteria Escherichia coli and Pseudomonas aeruginosa,Gram-positive methicillin resistant Staphylococcus aureus, and fungal yeast Candida albicans. The mechanism is proposed to be singlet oxygen addition to iodide anion to form peroxyiodide, which decomposes into radicals, finally forms hydrogen peroxide and molecular iodine. The effects of these different bactericidal species can be teased apart by comparing killing in three different scenarios: (1) cells+RB+KI are mixed together then illuminated with green light; (2) cells+RB are centrifuged then KI added then green light; (3) RB+KI+green light then cells added after light. We showed that KI could potentiate RBPDT in a mouse model of skin abrasions infected with bioluminescent P.aeruginosa.

Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells

PubMed Central

Lee, Wen Shi; Richard, Jonathan; Lichtfuss, Marit; Smith, Amos B.; Park, Jongwoo; Courter, Joel R.; Melillo, Bruno N.; Sodroski, Joseph G.; Kaufmann, Daniel E.; Parsons, Matthew S.

2015-01-01

ABSTRACT Lifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4+ T cells from HIV-1+ subjects were used as targets for ADCC. These ex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1+ serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted. IMPORTANCE An HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed “shock and kill,” aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune responses within HIV-1+ individuals can efficiently eliminate the reactivated cells. HIV-1-specific antibodies can potentially eliminate cells reactivated from latency via Fc effector functions by recruiting innate immune cells. Our study highlights the potential role that antibody-dependent cellular cytotoxicity might play in antilatency cure approaches. PMID:26656700

Altered Dynamics of Candida albicans Phagocytosis by Macrophages and PMNs When Both Phagocyte Subsets Are Present

PubMed Central

Rudkin, Fiona M.; Bain, Judith M.; Walls, Catriona; Lewis, Leanne E.; Gow, Neil A. R.; Erwig, Lars P.

2013-01-01

ABSTRACT An important first line of defense against Candida albicans infections is the killing of fungal cells by professional phagocytes of the innate immune system, such as polymorphonuclear cells (PMNs) and macrophages. In this study, we employed live-cell video microscopy coupled with dynamic image analysis tools to provide insights into the complexity of C. albicans phagocytosis when macrophages and PMNs were incubated with C. albicans alone and when both phagocyte subsets were present. When C. albicans cells were incubated with only one phagocyte subtype, PMNs had a lower overall phagocytic capacity than macrophages, despite engulfing fungal cells at a higher rate once fungal cells were bound to the phagocyte surface. PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested. In contrast, when both phagocyte subsets were studied in coculture, the two cell types phagocytosed and cleared C. albicans at equal rates and were equally susceptible to killing by the fungus. The increase in macrophage susceptibility to C. albicans-mediated killing was a consequence of macrophages taking up a higher proportion of hyphal cells under these conditions. In the presence of both PMNs and macrophages, C. albicans yeast cells were predominantly cleared by PMNs, which migrated at a greater speed toward fungal cells and engulfed bound cells more rapidly. These observations demonstrate that the phagocytosis of fungal pathogens depends on, and is modified by, the specific phagocyte subsets present at the site of infection. PMID:24169578

Lysis of autologous human macrophages by lymphokine-activated killer cells: interaction of effector cell and target cell conjugates analyzed by scanning electron microscopy.

PubMed

Streck, R J; Helinski, E H; Ovak, G M; Pauly, J L

1990-09-01

Lymphokine (i.e., interleukin 2; IL-2)-activated killer (LAK) cells derived from normal human blood are known to destroy human tumor target cells. Accordingly, immunotherapy modalities using IL-2, either alone or in combination with LAK cells, have been evaluated for eradicating metastatic cancer. In studies conducted to characterize receptors on LAK cell membrane ultrastructures, we observed that LAK cells kill autologous human monocyte-derived macrophages (M phi). In these experiments, peripheral blood mononuclear cells of a healthy adult donor were cultured to generate LAK cells and autologous non-adherent M phi. Thereafter, conjugates were prepared by incubating for 3 h autologous populations of LAK cells and M phi. Examination of the conjugates by scanning electron microscopy (SEM) identified LAK cell-mediated killing of M phi. Moreover, SEM analysis of the LAK cell membrane architecture identified microvilli-like ultrastructures that provided a physical bridge that joined together the LAK cell and M phi. The immunological mechanism(s) underling LAK cell killing of autologous M phi is not known; nevertheless, these conjugates will provide a useful model to study membrane receptors on ultrastructures that mediate the initial stages of cytolysis that include target cell recognition and cell-to-cell adhesion. The results of our observations and the findings of other investigators who have also demonstrated LAK cell killing of autologous normal human leukocytes are discussed in the context of the association of IL-2 and IL-2-activated killer cells with side effects observed in ongoing clinical trials and with autoimmune disorders.

Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing

PubMed Central

Gameiro, Sofia R.; Jammed, Momodou L.; Wattenberg, Max M.; Tsang, Kwong Y.; Ferrone, Soldano; Hodge, James W.

2014-01-01

Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response. This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone. PMID:24480782

T cells raised against allogeneic HLA-A2/CD20 kill primary follicular lymphoma and acute lymphoblastic leukemia cells.

PubMed

Abrahamsen, Ingerid Weum; Kjellevoll, Synneva; Greve-Isdahl, Margrethe; Mensali, Nadia; Wälchli, Sébastien; Kumari, Shraddha; Loland, Beate Fossum; Egeland, Torstein; Kolstad, Arne; Olweus, Johanna

2012-04-15

T cells mediating a graft-versus-leukemia/lymphoma effects without causing graft-versus-host disease would greatly improve the safety and applicability of hematopoietic stem cell transplantation. We recently demonstrated that highly peptide- and HLA-specific T cells can readily be generated against allogeneic HLA-A*02:01 in complex with a peptide from the B cell-restricted protein CD20. Here, we show that such CD20-specific T cells can easily be induced from naïve precursors in cord blood, demonstrating that they do not represent cross-reactive memory cells. The cells displayed high avidity and mediated potent cytotoxic effects on cells from patients with the CD20(pos) B cell malignancies follicular lymphoma (FL) and acute lymphoblastic leukemia (ALL). However, the cytotoxicity was consistently lower for cells from two of the ALL patients. The ALL cells that were less efficiently killed did not display lower surface expression of CD20 or HLA-A*02:01, or mutations in the CD20 sequence. Peptide pulsing fully restored the levels of cytotoxicity, indicating that they are indeed susceptible to T cell-mediated killing. Adoptive transfer of CD20-specific T cells to an HLA-A*02:01(pos) patient requires an HLA-A*02:01(neg) , but otherwise HLA identical, donor. A search clarified that donors meeting these criteria can be readily identified even for patients with rare haplotypes. The results bear further promise for the clinical utility of CD20-specific T cells in B cell malignancies. Copyright © 2011 UICC.

Enhancement of dendritic cell-based vaccine potency by anti-apoptotic siRNAs targeting key pro-apoptotic proteins in cytotoxic CD8(+) T cell-mediated cell death.

PubMed

Kim, Jin Hee; Kang, Tae Heung; Noh, Kyung Hee; Bae, Hyun Cheol; Kim, Seok-Ho; Yoo, Young Do; Seong, Seung-Yong; Kim, Tae Woo

2009-01-29

Dendritic cells (DCs) have become an important measure for the treatment of malignancies. Current DC preparations, however, generate short-lived DCs because they are subject to cell death from various apoptotic pressures. Antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is one of the main obstacles to limit the DC-mediated immune priming since CTLs can recognize the target antigen expressing DCs as target cells and kill the DCs. CTLs secret perforin and serine protease granzymes during CTL killing. Perforin and serine protease granzymes induce the release of a number of mitochondrial pro-apoptotic factors, which are controlled by members of the BCL-2 family, such as BAK, BAX and BIM. FasL linking to Fas on DCs triggers the activation of caspase-8, which eventually leads to mitochondria-mediated apoptosis via truncation of BID. In this study, we tried to enhance the DC priming capacity by prolonging DC survival using anti-apoptotic siRNA targeting these key pro-apoptotic molecules in CTL killing. Human papillomavirus (HPV)-16 E7 antigen presenting DCs that were transfected with these anti-apoptotic siRNAs showed increased resistance to T cell-mediated death, leading to enhanced E7-specific CD8(+) T cell activation in vitro and in vivo. Among them, siRNA targeting BIM (siBIM) generated strongest E7-specific E7-specific CD8(+) T cell immunity. More importantly, vaccination with E7 presenting DCs transfected with siBIM was capable of generating a marked therapeutic effect in vaccinated mice. Our data indicate that ex vivo manipulation of DCs with siBIM may represent a plausible strategy for enhancing dendritic cell-based vaccine potency.

Neutrophils Kill Antibody-Opsonized Cancer Cells by Trogoptosis.

PubMed

Matlung, Hanke L; Babes, Liane; Zhao, Xi Wen; van Houdt, Michel; Treffers, Louise W; van Rees, Dieke J; Franke, Katka; Schornagel, Karin; Verkuijlen, Paul; Janssen, Hans; Halonen, Pasi; Lieftink, Cor; Beijersbergen, Roderick L; Leusen, Jeanette H W; Boelens, Jaap J; Kuhnle, Ingrid; van der Werff Ten Bosch, Jutte; Seeger, Karl; Rutella, Sergio; Pagliara, Daria; Matozaki, Takashi; Suzuki, Eiji; Menke-van der Houven van Oordt, Catharina Willemien; van Bruggen, Robin; Roos, Dirk; van Lier, Rene A W; Kuijpers, Taco W; Kubes, Paul; van den Berg, Timo K

2018-06-26

Destruction of cancer cells by therapeutic antibodies occurs, at least in part, through antibody-dependent cellular cytotoxicity (ADCC), and this can be mediated by various Fc-receptor-expressing immune cells, including neutrophils. However, the mechanism(s) by which neutrophils kill antibody-opsonized cancer cells has not been established. Here, we demonstrate that neutrophils can exert a mode of destruction of cancer cells, which involves antibody-mediated trogocytosis by neutrophils. Intimately associated with this is an active mechanical disruption of the cancer cell plasma membrane, leading to a lytic (i.e., necrotic) type of cancer cell death. Furthermore, this mode of destruction of antibody-opsonized cancer cells by neutrophils is potentiated by CD47-SIRPα checkpoint blockade. Collectively, these findings show that neutrophil ADCC toward cancer cells occurs by a mechanism of cytotoxicity called trogoptosis, which can be further improved by targeting CD47-SIRPα interactions. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

Shielding of a lipooligosaccharide IgM epitope allows evasion of neutrophil-mediated killing of an invasive strain of nontypeable Haemophilus influenzae.

PubMed

Langereis, Jeroen D; Weiser, Jeffrey N

2014-07-22

Nontypeable Haemophilus influenzae is a frequent cause of noninvasive mucosal inflammatory diseases but may also cause invasive diseases, such as sepsis and meningitis, especially in children and the elderly. Infection by nontypeable Haemophilus influenzae is characterized by recruitment of neutrophilic granulocytes. Despite the presence of a large number of neutrophils, infections with nontypeable Haemophilus influenzae are often not cleared effectively by the antimicrobial activity of these immune cells. Herein, we examined how nontypeable Haemophilus influenzae evades neutrophil-mediated killing. Transposon sequencing (Tn-seq) was used on an isolate resistant to neutrophil-mediated killing to identify genes required for its survival in the presence of human neutrophils and serum, which provided a source of complement and antibodies. Results show that nontypeable Haemophilus influenzae prevents complement-dependent neutrophil-mediated killing by expression of surface galactose-containing oligosaccharide structures. These outer-core structures block recognition of an inner-core lipooligosaccharide epitope containing glucose attached to heptose HepIII-β1,2-Glc by replacement with galactose attached to HepIII or through shielding HepIII-β1,2-Glc by phase-variable attachment of oligosaccharide chain extensions. When the HepIII-β1,2-Glc-containing epitope is expressed and exposed, nontypeable Haemophilus influenzae is opsonized by naturally acquired IgM generally present in human serum and subsequently phagocytosed and killed by human neutrophils. Clinical nontypeable Haemophilus influenzae isolates containing galactose attached to HepIII that are not recognized by this IgM are more often found to cause invasive infections. Importance: Neutrophils are white blood cells that specialize in killing pathogens and are recruited to sites of inflammation. However, despite the presence of large numbers of neutrophils in the middle ear cavity and lungs of patients with otitis media or chronic obstructive pulmonary disease, respectively, the bacterium nontypeable Haemophilus influenzae is often not effectively cleared from these locations by these immune cells. In order to understand how nontypeable Haemophilus influenzae is able to cause inflammatory diseases in the presence of neutrophils, we determined the mechanism that underlies resistance to neutrophil-mediated killing. We have shown that nontypeable Haemophilus influenzae prevents binding of antibodies of the IgM subtype through changes in their surface lipooligosaccharide structure, thereby preventing complement activation and clearance by human neutrophils. Copyright © 2014 Langereis and Weiser.

Combination of Near Infrared Light-Activated Photodynamic Therapy Mediated by Indocyanine Green with Etoposide to Treat Non-Small-Cell Lung Cancer

PubMed Central

Luo, Ting; Zhang, Qinrong; Lu, Qing-Bin

2017-01-01

Indocyanine green (ICG) has been reported as a potential near-infrared (NIR) photosensitizer for photodynamic therapy (PDT) of cancer. However the application of ICG-mediated PDT is both intrinsically and physiologically limited. Here we report a combination of ICG-PDT with a chemotherapy drug etoposide (VP-16), aiming to enhance the anticancer efficacy, to circumvent limitations of PDT using ICG, and to reduce side effects of VP-16. We found in controlled in vitro cell-based assays that this combination is effective in killing non-small-cell lung cancer cells (NSCLC, A549 cell line). We also found that the combination of ICG-PDT and VP-16 exhibits strong synergy in killing non-small-cell lung cancer cells partially through inducing more DNA double-strand breaks (DSBs), while it has a much weaker synergy in killing human normal cells (GM05757). Furthermore, by studying the treatment sequence dependence and the cytotoxicity of laser-irradiated mixtures of ICG and VP-16, we found that the observed synergy involves direct/indirect reactions between ICG and VP-16. We further propose that there exists an electron transfer reaction between ICG and VP-16 under irradiation. This study therefore shows the anticancer efficacy of ICG-PDT combined with VP-16. These findings suggest that ICG-mediated PDT may be applied in combination with the chemotherapy drug VP-16 to treat some cancers, especially the non-small-cell lung cancer. PMID:28587258

Potassium Channels Mediate Killing by Human Natural Killer Cells

NASA Astrophysics Data System (ADS)

Schlichter, Lyanne; Sidell, Neil; Hagiwara, Susumu

1986-01-01

Human natural killer (NK) cells in peripheral blood spontaneously recognize and kill a wide variety of target cells. It has been suggested that ion channels are involved in the killing process because there is a Ca-dependent stage and because killing by presensitized cytotoxic T lymphocytes, which in many respects resembles NK killing, is associated with changes in K and Na transport in the target cell. However, no direct evidence exists for ion channels in NK cells or in their target cells. Using the whole-cell variation of the patch-clamp technique, we found a voltage-dependent potassium (K+) current in NK cells. The K+ current was reduced in a dose-dependent manner by the K-channel blockers 4-aminopyridine and quinidine and by the traditional Ca-channel blockers verapamil and Cd2+. We tested the effects of ion-channel blockers on killing of two commonly used target cell lines: K562, which is derived from a human myeloid leukemia, and U937, which is derived from a human histiocytic leukemia. Killing of K562 target cells, determined in a standard 51Cr-release assay, was inhibited in a dose-dependent manner by verapamil, quinidine, Cd2+, and 4-aminopyridine at concentrations comparable to those that blocked the K+ current in NK cells. In K562 target cells only a voltage-dependent Na+ current was found and it was blocked by concentrations of tetrodotoxin that had no effect on killing. Killing of U937 target cells was also inhibited by the two ion-channel blockers tested, quinidine and verapamil. In this cell line only a small K+ current was found that was similar to the one in NK cells. We could not find any evidence of a Ca2+ current in target cells or in NK cells; therefore, our results cannot explain the Ca dependence of killing. Our findings show that there are K channels in NK cells and that these channels play a necessary role in the killing process. In contrast, the endogenous channel type in the target cell is probably not a factor in determining target cell sensitivity to natural killing.

Superoxide dismutase and catalase protect cultured hepatocytes from the cytotoxicity of acetaminophen.

PubMed

Kyle, M E; Miccadei, S; Nakae, D; Farber, J L

1987-12-31

Superoxide dismutase, catalase and mannitol prevent the killing of cultured hepatocytes by acetaminophen in the presence of an inhibitor of glutathione reductase, BCNU. Under these conditions, the cytotoxicity of acetaminophen depends upon its metabolism, since beta-naphthoflavone, an inhibitor of mixed function oxidation, prevents the cell killing. In hepatocytes made resistant to acetaminophen by pretreatment with the ferric iron chelator, deferoxamine, addition of ferric or ferrous iron restores the sensitivity to acetaminophen. In such a situation, both superoxide dismutase and catalase prevent the killing by acetaminophen in the presence of ferric iron. By contrast, catalase, but not superoxide dismutase, prevents the cell killing dependent upon addition of ferrous iron. These results document the participation of both superoxide anion and hydrogen peroxide in the killing of cultured hepatocytes by acetaminophen and suggest that hydroxyl radicals generated by an iron catalyzed Haber-Weiss reaction mediate the cell injury.

Cytolysin-dependent evasion of lysosomal killing.

PubMed

Håkansson, Anders; Bentley, Colette Cywes; Shakhnovic, Elizabeth A; Wessels, Michael R

2005-04-05

Local host defenses limit proliferation and systemic spread of pathogenic bacteria from sites of mucosal colonization. For pathogens such as streptococci that fail to grow intracellularly, internalization and killing by epithelial cells contribute to the control of bacterial growth and dissemination. Here, we show that group A Streptococcus (GAS), the agent of streptococcal sore throat and invasive soft tissue infections, evades internalization and intracellular killing by pharyngeal epithelial cells. Production of the cholesterol-binding cytotoxin streptolysin O (SLO) prevented internalization of GAS into lysosomes. In striking contrast, GAS rendered defective in production of SLO were internalized directly or rapidly transported into lysosomes, where they were killed by a pH-dependent mechanism. Because SLO is the prototype of cholesterol-dependent cytolysins produced by many Gram-positive bacteria, cytolysin-mediated evasion of lysosomal killing may be a general mechanism to protect such pathogens from clearance by host epithelial cells.

Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer.

PubMed

Lee, J-K; Sayers, T J; Back, T C; Wigginton, J M; Wiltrout, R H

2003-03-01

Lewis lung carcinoma (3LL) cells were constitutively resistant to Fas-mediated apoptosis, but overexpression of Fas on 3LL cells allowed Fas-mediated apoptosis after crosslinking with agonist anti-Fas antibody (Jo2) in vitro. Surprisingly, Fas-overexpressing 3LL cells showed enhanced in vivo tumor progression, whereas no promotion of in vivo tumor growth was observed for dominant negative (DN) Fas-overexpressing 3LL transfectants in which the cytoplasmic death domain was deleted. In addition, the promotion of in vivo tumor growth by Fas-overexpression was reduced in gld (FasL-mutation) mice compared to normal mice. These data indicate that intact Fas/FasL cell signaling is required for the promotion of in vivo tumor growth by Fas overexpression in 3LL cells. In contrast to the efficient Fas-mediated killing induced in vitro by crosslinking with anti-Fas antibody, Fas-overexpressing 3LL cells were resistant in vitro to Fas-mediated apoptosis by activated T cells or transient FasL transfection. These data suggest that agonist anti-Fas antibody and natural FasL can transmit qualitatively different signals, and crosslinking of Fas with natural FasL on 3LL cells does not deliver the expected death signal. Thus, our results demonstrate that in some cases overexpression of Fas can result in a survival advantage for tumor cells in vivo.

Effects of murine leukemia virus env gene proteins on macrophage-mediated cytotoxicity in vitro

NASA Technical Reports Server (NTRS)

Chapes, S. K.; Takemoto, L. J.; Spooner, B. S. (Principal Investigator)

1991-01-01

F5b Tumor cells were incubated with concentrated culture supernatants taken from cells resistant (F5m) or sensitive (F5b) to contact-dependent macrophage cytotoxicity. Macrophage cell line B6MP102 and murine peritoneal macrophages killed targets incubated with supernatants taken from sensitive cells but poorly killed cells incubated in supernatants isolated from resistant cells. Membranes from cells resistant to macrophage killing, F5m, were fused into F5b cells. The fused F5b cells were killed significantly less than F5b cells fused with F5b cell membranes or untreated F5b cells. The decreased killing of F5b cells corresponded to increased concentrations of gp70(a) molecules on F5b cells. Affinity purified gp70(a) was added to cytotoxicity assays but failed to inhibit macrophage cytotoxicity. P15E molecules were detectable on both F5b and F5m cells. In addition, a synthetic peptide found to exhibit the inhibitory properties of p15E was added to cytotoxicity assays. P15E synthetic peptide also did not inhibit macrophage cytotoxicity. Therefore, env gene proteins of murine leukemia virus do not appear responsible for inducing tumor cell resistance to activated macrophage contact-dependent cytotoxicity.

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins.

PubMed

García-Bayona, Leonor; Guo, Monica S; Laub, Michael T

2017-03-21

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common.

Host protective roles of type 2 immunity: Parasite killing and tissue repair, flip sides of the same coin

PubMed Central

Allen, Judith E.; Sutherland, Tara E.

2014-01-01

Metazoan parasites typically induce a type 2 immune response, characterized by T helper 2 (Th2) cells that produce the cytokines IL-4, IL-5 and IL-13 among others. The type 2 response is host protective, reducing the number of parasites either through direct killing in the tissues, or expulsion from the intestine. Type 2 immunity also protects the host against damage mediated by these large extracellular parasites as they migrate through the body. At the center of both the innate and adaptive type 2 immune response, is the IL-4Rα that mediates many of the key effector functions. Here we highlight the striking overlap between the molecules, cells and pathways that mediate both parasite control and tissue repair. We have proposed that adaptive Th2 immunity evolved out of our innate repair pathways to mediate both accelerated repair and parasite control in the face of continual assault from multicellular pathogens. Type 2 cytokines are involved in many aspects of mammalian physiology independent of helminth infection. Therefore understanding the evolutionary relationship between helminth killing and tissue repair should provide new insight into immune mechanisms of tissue protection in the face of physical injury. PMID:25028340

Casp8p41 generated by HIV protease kills CD4 T cells through direct Bak activation

PubMed Central

Sainski, Amy M.; Dai, Haiming; Natesampillai, Sekar; Pang, Yuan-Ping; Bren, Gary D.; Cummins, Nathan W.; Correia, Cristina; Meng, X. Wei; Tarara, James E.; Ramirez-Alvarado, Marina; Katzmann, David J.; Ochsenbauer, Christina; Kappes, John C.

2014-01-01

Previous studies have shown that human immunodeficiency virus (HIV) protease cleaves procaspase 8 to a fragment, termed Casp8p41, that lacks caspase activity but nonetheless contributes to T cell apoptosis. Herein, we show that Casp8p41 contains a domain that interacts with the BH3-binding groove of pro-apoptotic Bak to cause Bak oligomerization, Bak-mediated membrane permeabilization, and cell death. Levels of active Bak are higher in HIV-infected T cells that express Casp8p41. Conversely, targeted mutations in the Bak-interacting domain diminish Bak binding and Casp8p41-mediated cell death. Similar mutations in procaspase 8 impair the ability of HIV to kill infected T cells. These observations support a novel paradigm in which HIV converts a normal cellular constituent into a direct activator that functions like a BH3-only protein. PMID:25246614

Epirubicin-Adsorbed Nanodiamonds Kill Chemoresistant Hepatic Cancer Stem Cells

PubMed Central

2015-01-01

Chemoresistance is a primary cause of treatment failure in cancer and a common property of tumor-initiating cancer stem cells. Overcoming mechanisms of chemoresistance, particularly in cancer stem cells, can markedly enhance cancer therapy and prevent recurrence and metastasis. This study demonstrates that the delivery of Epirubicin by nanodiamonds is a highly effective nanomedicine-based approach to overcoming chemoresistance in hepatic cancer stem cells. The potent physical adsorption of Epirubicin to nanodiamonds creates a rapidly synthesized and stable nanodiamond–drug complex that promotes endocytic uptake and enhanced tumor cell retention. These attributes mediate the effective killing of both cancer stem cells and noncancer stem cells in vitro and in vivo. Enhanced treatment of both tumor cell populations results in an improved impairment of secondary tumor formation in vivo compared with treatment by unmodified chemotherapeutics. On the basis of these results, nanodiamond-mediated drug delivery may serve as a powerful method for overcoming chemoresistance in cancer stem cells and markedly improving overall treatment against hepatic cancers. PMID:25437772

Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jason Z.; Ke, Yuebin; Misra, Hara P.

Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone,more » a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with low NQO1 expression, mitochondria play a critical role in beta-Lp redox activation. • In cancer cells with high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1.« less

Fosfomycin enhances phagocyte-mediated killing of Staphylococcus aureus by extracellular traps and reactive oxygen species.

PubMed

Shen, Fengge; Tang, Xudong; Cheng, Wei; Wang, Yang; Wang, Chao; Shi, Xiaochen; An, Yanan; Zhang, Qiaoli; Liu, Mingyuan; Liu, Bo; Yu, Lu

2016-01-18

The successful treatment of bacterial infections is the achievement of a synergy between the host's immune defences and antibiotics. Here, we examined whether fosfomycin (FOM) could improve the bactericidal effect of phagocytes, and investigated the potential mechanisms. FOM enhanced the phagocytosis and extra- or intracellular killing of S. aureus by phagocytes. And FOM enhanced the extracellular killing of S. aureus in macrophage (MФ) and in neutrophils mediated by extracellular traps (ETs). ET production was related to NADPH oxidase-dependent reactive oxygen species (ROS). Additionally, FOM increased the intracellular killing of S. aureus in phagocytes, which was mediated by ROS through the oxidative burst process. Our results also showed that FOM alone induced S. aureus producing hydroxyl radicals in order to kill the bacterial cells in vitro. In a mouse peritonitis model, FOM treatment increased the bactericidal extra- and intracellular activity in vivo, and FOM strengthened ROS and ET production from peritoneal lavage fluid ex vivo. An IVIS imaging system assay further verified the observed in vivo bactericidal effect of the FOM treatment. This work may provide a deeper understanding of the role of the host's immune defences and antibiotic interactions in microbial infections.

Fosfomycin enhances phagocyte-mediated killing of Staphylococcus aureus by extracellular traps and reactive oxygen species

PubMed Central

Shen, Fengge; Tang, Xudong; Cheng, Wei; Wang, Yang; Wang, Chao; Shi, Xiaochen; An, Yanan; Zhang, Qiaoli; Liu, Mingyuan; Liu, Bo; Yu, Lu

2016-01-01

The successful treatment of bacterial infections is the achievement of a synergy between the host’s immune defences and antibiotics. Here, we examined whether fosfomycin (FOM) could improve the bactericidal effect of phagocytes, and investigated the potential mechanisms. FOM enhanced the phagocytosis and extra- or intracellular killing of S. aureus by phagocytes. And FOM enhanced the extracellular killing of S. aureus in macrophage (MФ) and in neutrophils mediated by extracellular traps (ETs). ET production was related to NADPH oxidase-dependent reactive oxygen species (ROS). Additionally, FOM increased the intracellular killing of S. aureus in phagocytes, which was mediated by ROS through the oxidative burst process. Our results also showed that FOM alone induced S. aureus producing hydroxyl radicals in order to kill the bacterial cells in vitro. In a mouse peritonitis model, FOM treatment increased the bactericidal extra- and intracellular activity in vivo, and FOM strengthened ROS and ET production from peritoneal lavage fluid ex vivo. An IVIS imaging system assay further verified the observed in vivo bactericidal effect of the FOM treatment. This work may provide a deeper understanding of the role of the host’s immune defences and antibiotic interactions in microbial infections. PMID:26778774

Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis.

PubMed

Bonne-Année, Sandra; Kerepesi, Laura A; Hess, Jessica A; Wesolowski, Jordan; Paumet, Fabienne; Lok, James B; Nolan, Thomas J; Abraham, David

2014-06-01

Neutrophils are multifaceted cells that are often the immune system's first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis

PubMed Central

Bonne-Année, Sandra; Kerepesi, Laura A.; Hess, Jessica A.; Wesolowski, Jordan; Paumet, Fabienne; Lok, James B.; Nolan, Thomas J.; Abraham, David

2014-01-01

Neutrophils are multifaceted cells that are often the immune system’s first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system. PMID:24642003

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins

PubMed Central

García-Bayona, Leonor; Guo, Monica S; Laub, Michael T

2017-01-01

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common. DOI: http://dx.doi.org/10.7554/eLife.24869.001 PMID:28323618

EGFR-targeted granzyme B expressed in NK cells enhances natural cytotoxicity and mediates specific killing of tumor cells.

PubMed

Oberoi, Pranav; Jabulowsky, Robert A; Bähr-Mahmud, Hayat; Wels, Winfried S

2013-01-01

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.

Medium-mediated effects increase cell killing in a human keratinocyte cell line exposed to solar-simulated radiation.

PubMed

Maguire, Alanna; Morrissey, Brian; Walsh, James E; Lyng, Fiona M

2011-01-01

The objective of this study was to investigate whether cell culture medium is a biologically relevant exposure medium that can be employed in non-ionising photobiological investigations. The effect of solar-simulated irradiation on cell culture medium and its ability to elicit cell death was studied. The role of reactive oxygen species (ROS), cell secreted factors, and the contribution of individual components of the medium were investigated. Cell death was found to be primarily mediated through the formation of ROS via riboflavin photosensitisation and degradation in the cell culture medium. Phenol red was found to significantly reduce the cell killing ability of riboflavin. Exposures in riboflavin-free medium resulted in significantly increased cell survival compared to identical exposures in riboflavin containing medium. This study has shown that solar radiation toxicity is augmented by cell culture medium due to the presence of riboflavin. Results suggest that exposures performed in phenol red-free medium may serve to increase phototoxic effects if riboflavin is present. Riboflavin-free media is recommended for solar radiation investigations to eliminate concerns regarding riboflavin photosensitisation and nutrient deprivation.

Metal Ions, Not Metal-Catalyzed Oxidative Stress, Cause Clay Leachate Antibacterial Activity

PubMed Central

Otto, Caitlin C.; Koehl, Jennifer L.; Solanky, Dipesh; Haydel, Shelley E.

2014-01-01

Aqueous leachates prepared from natural antibacterial clays, arbitrarily designated CB-L, release metal ions into suspension, have a low pH (3.4–5), generate reactive oxygen species (ROS) and H2O2, and have a high oxidation-reduction potential. To isolate the role of pH in the antibacterial activity of CB clay mixtures, we exposed three different strains of Escherichia coli O157:H7 to 10% clay suspensions. The clay suspension completely killed acid-sensitive and acid-tolerant E. coli O157:H7 strains, whereas incubation in a low-pH buffer resulted in a minimal decrease in viability, demonstrating that low pH alone does not mediate antibacterial activity. The prevailing hypothesis is that metal ions participate in redox cycling and produce ROS, leading to oxidative damage to macromolecules and resulting in cellular death. However, E. coli cells showed no increase in DNA or protein oxidative lesions and a slight increase in lipid peroxidation following exposure to the antibacterial leachate. Further, supplementation with numerous ROS scavengers eliminated lipid peroxidation, but did not rescue the cells from CB-L-mediated killing. In contrast, supplementing CB-L with EDTA, a broad-spectrum metal chelator, reduced killing. Finally, CB-L was equally lethal to cells in an anoxic environment as compared to the aerobic environment. Thus, ROS were not required for lethal activity and did not contribute to toxicity of CB-L. We conclude that clay-mediated killing was not due to oxidative damage, but rather, was due to toxicity associated directly with released metal ions. PMID:25502790

HIV-I Nef inhibitors: a novel class of HIV-specific immune adjuvants in support of a cure.

PubMed

Dekaban, Gregory A; Dikeakos, Jimmy D

2017-09-12

The success of many current vaccines relies on a formulation that incorporates an immune activating adjuvant. This will hold true for the design of a successful therapeutic HIV vaccine targeted at controlling reactivated virus following cessation of combined antiretroviral therapy (cART). The HIV accessory protein Nef functions by interfering with HIV antigen presentation through the major histocompatibility complex I (MHC-I) pathway thereby suppressing CD8 + cytotoxic T cell (CTL)-mediated killing of HIV infected cells. Thus, this important impediment to HIV vaccine success must be circumvented. This review covers our current knowledge of Nef inhibitors that may serve as immune adjuvants that will specifically restore and enhance CTL-mediated killing of reactivated HIV infected cells as part of an overall vaccine strategy to affect a cure for HIV infection.

Towards Effective Photothermal/Photodynamic Treatment Using Plasmonic Gold Nanoparticles

PubMed Central

Bucharskaya, Alla; Maslyakova, Galina; Terentyuk, Georgy; Yakunin, Alexander; Avetisyan, Yuri; Bibikova, Olga; Tuchina, Elena; Khlebtsov, Boris; Khlebtsov, Nikolai; Tuchin, Valery

2016-01-01

Gold nanoparticles (AuNPs) of different size and shape are widely used as photosensitizers for cancer diagnostics and plasmonic photothermal (PPT)/photodynamic (PDT) therapy, as nanocarriers for drug delivery and laser-mediated pathogen killing, even the underlying mechanisms of treatment effects remain poorly understood. There is a need in analyzing and improving the ways to increase accumulation of AuNP in tumors and other crucial steps in interaction of AuNPs with laser light and tissues. In this review, we summarize our recent theoretical, experimental, and pre-clinical results on light activated interaction of AuNPs with tissues and cells. Specifically, we discuss a combined PPT/PDT treatment of tumors and killing of pathogen bacteria with gold-based nanocomposites and atomic clusters, cell optoporation, and theoretical simulations of nanoparticle-mediated laser heating of tissues and cells. PMID:27517913

The role of Fas ligand and transforming growth factor beta in tumor progression: molecular mechanisms of immune privilege via Fas-mediated apoptosis and potential targets for cancer therapy.

PubMed

Kim, Ryungsa; Emi, Manabu; Tanabe, Kazuaki; Uchida, Yoko; Toge, Tetsuya

2004-06-01

Despite the fact that expression of Fas ligand (FasL) in cytotoxic T lymphocytes (CTLs) and in natural killer (NK) cells plays an important role in Fas-mediated tumor killing, During tumor progression FasL-expressing tumor cells are involved in counterattacking to kill tumor-infiltrating lymphocytes (TILs). Soluble FasL levels also increase with tumor progression in solid tumors, and this increase inhibits Fas-mediated tumor killing by CTLs and NK cells. The increased expression of FasL in tumor cells is associated with decreased expression of Fas; and the promoter region of the FASL gene is regulated by transcription factors, such as neuronal factor kappaB (NF-kappaB) and AP-1, in the tumor microenvironment. Although the ratio of FasL expression to Fas expression in tumor cells is not strongly related to the induction of apoptosis in TILs, increased expression of FasL is associated with decreased Fas levels in tumor cells that can escape immune surveillance and facilitate tumor progression and metastasis. Transforming growth factor beta (TGF-beta) is a potent growth inhibitor and has tumor-suppressing activity in the early phases of carcinogenesis. During subsequent tumor progression, the increased secretion of TGF-beta by both tumor cells and, in a paracrine fashion, stromal cells, is involved in the enhancement of tumor invasion and metastasis accompanied by immunosuppression. Herein, the authors review the clinical significance of FasL and TGF-beta expression patterns as features of immune privilege accompanying tumor progression in the tumor microenvironment. Potential strategies for identifying which molecules can serve as targets for effective antitumor therapy also are discussed. Copyright 2004 American Cancer Society.

Inhibitors of bacterial multidrug efflux pumps potentiate antimicrobial photoinactivation.

PubMed

Tegos, George P; Masago, Kayo; Aziz, Fatima; Higginbotham, Andrew; Stermitz, Frank R; Hamblin, Michael R

2008-09-01

Antimicrobial photodynamic inactivation (APDI) combines a nontoxic photoactivatable dye or photosensitizer (PS) with harmless visible light to generate singlet oxygen and reactive oxygen species that kill microbial cells. Cationic phenothiazinium dyes, such as toluidine blue O (TBO), are the only PS used clinically for APDI, and we recently reported that this class of PS are substrates of multidrug efflux pumps in both gram-positive and gram-negative bacteria. We now report that APDI can be significantly potentiated by combining the PS with an efflux pump inhibitor (EPI). Killing of Staphylococcus aureus mediated by TBO and red light is greatly increased by coincubation with known inhibitors of the major facilitator pump (NorA): the diphenyl urea INF271, reserpine, 5'-methoxyhydnocarpin, and the polyacylated neohesperidoside, ADH7. The potentiation effect is greatest in the case of S. aureus mutants that overexpress NorA and least in NorA null cells. Addition of the EPI before TBO has a bigger effect than addition of the EPI after TBO. Cellular uptake of TBO is increased by EPI. EPI increased photodynamic inactivation killing mediated by other phenothiazinium dyes, such as methylene blue and dimethylmethylene blue, but not that mediated by nonphenothiazinium PS, such as Rose Bengal and benzoporphyrin derivative. Killing of Pseudomonas aeruginosa mediated by TBO and light was also potentiated by the resistance nodulation division pump (MexAB-OprM) inhibitor phenylalanine-arginine beta-naphthylamide but to a lesser extent than for S. aureus. These data suggest that EPI could be used in combination with phenothiazinium salts and light to enhance their antimicrobial effect against localized infections.

T lymphocyte mediated lysis of mitomycin C treated Tenon’s capsule fibroblasts

PubMed Central

Crowston, J G; Chang, L H; Daniels, J T; Khaw, P T; Akbar, A N

2004-01-01

Aims: To evaluate the effect of T cell co-culture on mitomycin C treated and untreated Tenon’s capsule fibroblasts. Methods: IL-2 dependent allogeneic T cells were incubated over a monolayer of mitomycin C treated or control fibroblasts. Fibroblast numbers were evaluated by direct counts using phase contrast microscopy. To determine whether T cell mediated lysis was a consequence of MHC mismatch, co-culture experiments were repeated with autologous T cells. The effect of Fas receptor blockade was established by co-incubation with a Fas blocking (M3) antibody. Results: T cell co-culture resulted in a dramatic reduction in fibroblast survival compared to mitomycin C treatment alone (p = 0.032). T cell killing required fibroblast/lymphocyte cell to cell contact and was observed in both allogeneic and autologous co-culture experiments. Fas blocking antibodies did not significantly inhibit T cell killing (p = 0.39). Conclusion: T cells augment mitomycin C treated fibroblast death in vitro. Similar mechanisms may contribute to the cytotoxic effect of mitomycin C in vivo and account for the largely hypocellular drainage blebs that are observed clinically. PMID:14977777

T lymphocyte mediated lysis of mitomycin C treated Tenon's capsule fibroblasts.

PubMed

Crowston, J G; Chang, L H; Daniels, J T; Khaw, P T; Akbar, A N

2004-03-01

To evaluate the effect of T cell co-culture on mitomycin C treated and untreated Tenon's capsule fibroblasts. IL-2 dependent allogeneic T cells were incubated over a monolayer of mitomycin C treated or control fibroblasts. Fibroblast numbers were evaluated by direct counts using phase contrast microscopy. To determine whether T cell mediated lysis was a consequence of MHC mismatch, co-culture experiments were repeated with autologous T cells. The effect of Fas receptor blockade was established by co-incubation with a Fas blocking (M3) antibody. T cell co-culture resulted in a dramatic reduction in fibroblast survival compared to mitomycin C treatment alone (p = 0.032). T cell killing required fibroblast/lymphocyte cell to cell contact and was observed in both allogeneic and autologous co-culture experiments. Fas blocking antibodies did not significantly inhibit T cell killing (p = 0.39). T cells augment mitomycin C treated fibroblast death in vitro. Similar mechanisms may contribute to the cytotoxic effect of mitomycin C in vivo and account for the largely hypocellular drainage blebs that are observed clinically.

[Killing effects of PWZL plasmid-mediated double suicide gene on human lens epithelium cells].

PubMed

Yan, Xiao-ran; Wu, Hong; Yu, Hai-tao; Wang, Xiu; Zhang, Yu

2008-04-01

To investigate the killing efficiency of PWZL plasmid-mediated herpes simplex virus-thymidine kinase (TK) and E. coli cytosine deaminase (CD) on human lens epithelium cells followed by the treatment of prodrugs. PWZL plasmid was used as a vehicle, to transduce double suicide genes into the human lens epithelium in vitro, then the cells were treated with fluorocytosine (5-FC) and/or ganciclovir (GCV) at different concentrations. The cell growth of the lens epithelium cells was observed by light microscope. MTT analysis was used to estimate the cell survival rate and the bystander effect was analyzed simultaneously. The significance of difference between each group was treated by statistical tests. The CD and TK gene could be joined into PWZL plasmid successfully, and did not have any special effect on normal cells. There was no significant difference in cell viability between CD-TK transfected cells and control cells. Cell viability in cells treated with prodrugs was decreased in a time-dependent manner. At the end of the experiment, cell viability was lowest in GCV 10 mg/L +5-FC 60 mg/L group, GCV 10 mg/L + 5-FC 100 mg/L group and GCV 100 mg/L + 5-FC 100 mg/L group. There were no significant differences between these three groups (X2 = 1.25 , P > 0.01). Analysis of bystander effect indicated that the cell viability in GCV 100 mg/L + 5-FC 100 mg/L group and GCV 10 mg/L +5-FC 60 mg/L group was significantly lower than that in the controls (t = 10.26, 13.16; P < 0.01). PWZL plasmid can transfect the CD and TK genes into lens epithelium cells successfully and efficiently. CD and TK genes can be expressed steadily. Transfection of double suicide gene reduces the dosage of prodrugs required for killing cells. The combination of 5-FC with GCV shows the greatest killing effect and also has the bystander effect.

Vitamin C, a Multi-Tasking Molecule, Finds a Molecular Target in Killing Cancer Cells.

PubMed

Li, Robert

2016-03-01

Early work in the 1970s by Linus Pauling, a twice-honored Nobel laureate, led to his proposal of using high-dose vitamin C to treat cancer patients. Over the past several decades, a number of studies in animal models as well as several small-scale clinical studies have provided substantial support of Linus Pauling's early proposal. Production of reactive oxygen species (ROS) via oxidation of vitamin C appears to be a major underlying event, leading to the selective killing of cancer cells. However, it remains unclear how vitamin C selectively kills cancer cells while sparing normal cells and what the molecular targets of high-dose vitamin C are. In a recent article published in Science (2015 December 11; 350(6266):1391-6. doi: 10.1126/science.aaa5004), Yun et al. reported that vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) through an ROS-dependent mechanism. This work by Yun et al. along with other findings advances our current understanding of the molecular basis of high-dose vitamin C-mediated cancer cell killing, which will likely give an impetus to the continued research efforts aiming to further decipher the novel biochemistry of vitamin C and its unique role in cancer therapy.

Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma.

PubMed

Sun, Jingping; Law, Gabriela P; McKallip, Robert J

2012-03-01

In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44(lo)) or elevated (CD44(hi)) expression of CD44 are generated and that the CD44(hi) cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.

Both Leukotoxin and Poly-N-Acetylglucosamine Surface Polysaccharide Protect Aggregatibacter actinomycetemcomitans Cells from Macrophage Killing

PubMed Central

Venketaraman, Vishwanath; Lin, Albert K.; Le, Amy; Kachlany, Scott C.; Connell, Nancy D.; Kaplan, Jeffrey B.

2008-01-01

Two virulence factors produced by the periodontopathogen Aggregatibacter actinomycetemcomitans are leukotoxin, a secreted lipoprotein that kills human polymorphonuclear leukocytes and macrophages, and poly-N-acetylglucosamine (PGA), a surface polysaccharide that mediates intercellular adhesion, biofilm formation and detergent resistance. In this study we examined the roles of leukotoxin and PGA in protecting A. actinomycetemcomitans cells from killing by the human macrophage cell line THP-1. Monolayers of THP-1 cells were infected with single-cell suspensions of a wild-type A. actinomycetemcomitans strain, or of isogenic leukotoxin or PGA mutant strains. After 48 h, viable bacteria were enumerated by dilution plating, macrophage morphology was evaluated microscopically, and macrophage viability was measured by a Trypan blue dye exclusion assay. The number of A. actinomycetemcomitans CFUs increased approximately 2-fold in wells infected with the wild-type strain, but decreased by approximately 70–90% in wells infected with the leukotoxin and PGA mutant strains. Infection with the wild-type or leukotoxin mutant strain caused a significant decrease in THP-1 cell viability, whereas infection with the PGA mutant strain did not result in any detectable changes in THP-1 viability. Pre-treatment of wild-type A. actinomycetemcomitans cells with the PGA-hydrolyzing enzyme dispersin B rendered them sensitive to killing by THP-1 cells. We concluded that both leukotoxin and PGA are necessary for evasion of macrophage killing by A. actinomycetemcomitans. PMID:18573331

Galectin-3 Inhibits Galectin-8/Parkin-Mediated Ubiquitination of Group A Streptococcus.

PubMed

Cheng, Yi-Lin; Wu, Yan-Wei; Kuo, Chih-Feng; Lu, Shiou-Ling; Liu, Fu-Tong; Anderson, Robert; Lin, Chiou-Feng; Liu, Yi-Ling; Wang, Wan-Yu; Chen, Ying-Da; Zheng, Po-Xing; Wu, Jiunn-Jong; Lin, Yee-Shin

2017-07-25

Group A streptococcus (GAS) is an important human pathogen that causes a wide variety of cutaneous and systemic infections. Although originally thought to be an extracellular bacterium, numerous studies have demonstrated that GAS can trigger internalization into nonimmune cells to escape from immune surveillance or antibiotic-mediated killing. Epithelial cells possess a defense mechanism involving autophagy-mediated targeting and killing of GAS within lysosome-fused autophagosomes. In endothelial cells, in contrast, we previously showed that autophagy is not sufficient for GAS killing. In the present study, we showed higher galectin-3 (Gal-3) expression and lower Gal-8 expression in endothelial cells than in epithelial cells. The recruitment of Gal-3 to GAS is higher and the recruitment of Gal-8 to GAS is lower in endothelial cells than in epithelial cells. We further showed that Gal-3 promotes GAS replication and diminishes the recruitment of Gal-8 and ubiquitin, the latter of which is a critical protein for autophagy sequestration. After knockdown of Gal-3 in endothelial cells, the colocalization of Gal-8, parkin, and ubiquitin-decorated GAS is significantly increased, as is the interaction of Gal-8 and parkin, an E3 ligase. Furthermore, inhibition of Gal-8 in epithelial cells attenuates recruitment of parkin; both Gal-8 and parkin contribute to ubiquitin recruitment and GAS elimination. Animal studies confirmed that Gal-3-knockout mice develop less-severe skin damage and that GAS replication can be detected only in the air pouch and not in organs and endothelial cells. These results demonstrate that Gal-3 inhibits ubiquitin recruitment by blocking Gal-8 and parkin recruitment, resulting in GAS replication in endothelial cells. IMPORTANCE In epithelial cells, GAS can be efficiently killed within the lysosome-fused autophaosome compartment. However, we previously showed that, in spite of LC-3 recruitment, the autophagic machinery is not sufficient for GAS killing in endothelial cells. In this report, we provide the first evidence that Gal-3, highly expressed in endothelial cells, blocks the tagging of ubiquitin to GAS by inhibiting recruitment of Gal-8 and parkin, leading to an enhancement of GAS replication. We also provide the first demonstration that Gal-8 can interact with parkin, the critical E3 ligase, for resistance to intracellular bacteria by facilitating the decoration of bacteria with ubiquitin chains. Our findings reveal that differential levels of Gal-3 and Gal-8 expression and recruitment to GAS between epithelial cells and endothelial cells may contribute to the different outcomes of GAS elimination or survival and growth of GAS in these two types of cells. Copyright © 2017 Cheng et al.

BIM mediates synergistic killing of B-cell acute lymphoblastic leukemia cells by BCL-2 and MEK inhibitors.

PubMed

Korfi, K; Smith, M; Swan, J; Somervaille, T C P; Dhomen, N; Marais, R

2016-04-07

B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological disease that kills ~50% of adult patients. With the exception of some BCR-ABL1(+) patients who benefit from tyrosine kinase inhibitors, there are no effective targeted therapies for adult B-ALL patients and chemotherapy remains first-line therapy despite adverse side effects and poor efficacy. We show that, although the MEK/ERK pathway is activated in B-ALL cells driven by different oncogenes, MEK inhibition does not suppress B-ALL cell growth. However, MEK inhibition synergized with BCL-2/BCL-XL family inhibitors to suppress proliferation and induce apoptosis in B-ALL cells. We show that this synergism is mediated by the pro-apoptotic factor BIM, which is dephosphorylated as a result of MEK inhibition, allowing it to bind to and neutralize MCL-1, thereby enhancing BCL-2/BCL-XL inhibitor-induced cell death. This cooperative effect is observed in B-ALL cells driven by a range of genetic abnormalities and therefore has significant therapeutic potential.

Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis.

PubMed

San, Kaungmyat; Long, Janet; Michels, Corinne A; Gadura, Nidhi

2015-10-01

This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis

PubMed Central

San, Kaungmyat; Long, Janet; Michels, Corinne A; Gadura, Nidhi

2015-01-01

This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain. PMID:26185055

Potassium Iodide Potentiates Antimicrobial Photodynamic Inactivation Mediated by Rose Bengal in In Vitro and In Vivo Studies.

PubMed

Wen, Xiang; Zhang, Xiaoshen; Szewczyk, Grzegorz; El-Hussein, Ahmed; Huang, Ying-Ying; Sarna, Tadeusz; Hamblin, Michael R

2017-07-01

Rose bengal (RB) is a halogenated xanthene dye that has been used to mediate antimicrobial photodynamic inactivation for several years. While RB is highly active against Gram-positive bacteria, it is largely inactive in killing Gram-negative bacteria. We have discovered that addition of the nontoxic salt potassium iodide (100 mM) potentiates green light (540-nm)-mediated killing by up to 6 extra logs with the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa , the Gram-positive bacterium methicillin-resistant Staphylococcus aureus , and the fungal yeast Candida albicans The mechanism is proposed to be singlet oxygen addition to iodide anion to form peroxyiodide, which decomposes into radicals and, finally, forms hydrogen peroxide and molecular iodine. The effects of these different bactericidal species can be teased apart by comparing the levels of killing achieved in three different scenarios: (i) cells, RB, and KI are mixed together and then illuminated with green light; (ii) cells and RB are centrifuged, and then KI is added and the mixture is illuminated with green light; and (iii) RB and KI are illuminated with green light, and then cells are added after illumination with the light. We also showed that KI could potentiate RB photodynamic therapy in a mouse model of skin abrasions infected with bioluminescent P. aeruginosa . Copyright © 2017 American Society for Microbiology.

Potassium Iodide Potentiates Antimicrobial Photodynamic Inactivation Mediated by Rose Bengal in In Vitro and In Vivo Studies

PubMed Central

Wen, Xiang; Zhang, Xiaoshen; Szewczyk, Grzegorz; El-Hussein, Ahmed; Huang, Ying-Ying; Sarna, Tadeusz

2017-01-01

ABSTRACT Rose bengal (RB) is a halogenated xanthene dye that has been used to mediate antimicrobial photodynamic inactivation for several years. While RB is highly active against Gram-positive bacteria, it is largely inactive in killing Gram-negative bacteria. We have discovered that addition of the nontoxic salt potassium iodide (100 mM) potentiates green light (540-nm)-mediated killing by up to 6 extra logs with the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium methicillin-resistant Staphylococcus aureus, and the fungal yeast Candida albicans. The mechanism is proposed to be singlet oxygen addition to iodide anion to form peroxyiodide, which decomposes into radicals and, finally, forms hydrogen peroxide and molecular iodine. The effects of these different bactericidal species can be teased apart by comparing the levels of killing achieved in three different scenarios: (i) cells, RB, and KI are mixed together and then illuminated with green light; (ii) cells and RB are centrifuged, and then KI is added and the mixture is illuminated with green light; and (iii) RB and KI are illuminated with green light, and then cells are added after illumination with the light. We also showed that KI could potentiate RB photodynamic therapy in a mouse model of skin abrasions infected with bioluminescent P. aeruginosa. PMID:28438946

Neutrophils kill pulmonary endothelial cells by a hydrogen-peroxide-dependent pathway. An in vitro model of neutrophil-mediated lung injury.

PubMed

Martin, W J

1984-08-01

Neutrophil-mediated injury to lung parenchymal cells has been proposed as an important step in the pathogenesis of many acute and chronic lung disorders. As an in vitro model of neutrophil-mediated injury, this study used activated human neutrophils as effector cells in an 18-h cytotoxicity assay with 51Cr-labeled bovine pulmonary artery endothelial cells serving as target cells. Neutrophils effectively injured pulmonary endothelial cells, expressed as cytotoxic index (CI), of 63.8 +/- 5.4, and this injury could be significantly reduced by several agents, including 1% dimethyl sulfoxide (CI, 51.3 +/- 3.7), 50 micrograms/ml ascorbic acid (CI, 40.8 +/- 4.7), and especially 1,100 U/ml catalase (CI, 14.3 +/- 4.1). As cell-free models of neutrophil-mediated endothelial cell injury, H2O2 (30 microM), O2- (generated by 0.5 mU xanthine oxidase), and the myeloperoxidase-dependent (0.32 U) hypohalite ion were each capable of injuring the target cells with CI of 6.21 +/- 2.8, 53.6 +/- 5.3, and 21.2 +/- 1.5, respectively. Catalase was effective in reducing the injurious effect of each of these oxidant-generating systems (p less than 0.01, all comparisons), confirming the important role for H2O2 in the mediation of this injury. The data indicate that neutrophils are capable of killing pulmonary endothelial cells by a pathway largely dependent on the generation of H2O2, and suggest the possibility that removal of H2O2 from the alveolar structures in subjects with these disorder might be an effective future therapeutic approach.

A Lipopeptide Facilitate Induction of Mycobacterium leprae Killing in Host Cells

PubMed Central

Maeda, Yumi; Tamura, Toshiki; Fukutomi, Yasuo; Mukai, Tetsu; Kai, Masanori; Makino, Masahiko

2011-01-01

Little is known of the direct microbicidal activity of T cells in leprosy, so a lipopeptide consisting of the N-terminal 13 amino acids lipopeptide (LipoK) of a 33-kD lipoprotein of Mycobacterium leprae, was synthesized. LipoK activated M. leprae infected human dendritic cells (DCs) to induce the production of IL-12. These activated DCs stimulated autologous CD4+ or CD8+ T cells towards type 1 immune response by inducing interferon-gamma secretion. T cell proliferation was also evident from the CFSE labeling of target CD4+ or CD8+ T cells. The direct microbicidal activity of T cells in the control of M. leprae multiplication is not well understood. The present study showed significant production of granulysin, granzyme B and perforin from these activated CD4+ and CD8+ T cells when stimulated with LipoK activated, M. leprae infected DCs. Assessment of the viability of M. leprae in DCs indicated LipoK mediated T cell-dependent killing of M. leprae. Remarkably, granulysin as well as granzyme B could directly kill M. leprae in vitro. Our results provide evidence that LipoK could facilitate M. leprae killing through the production of effector molecules granulysin and granzyme B in T cells. PMID:22132248

Effects of methyltestosterone on immunity against Salmonella Pullorum in dwarf chicks.

PubMed

Li, H; Zhang, Y; Zuo, S F; Lian, Z X; Li, N

2009-12-01

This study was conducted to determine effects of methyltestosterone on innate immunity and adaptive immunity against Salmonella Pullorum in dwarf chicks. In vivo experiment, comparisons of pathological sections, viable counts of bacteria, specific antibody levels, and subsets of T lymphocytes were set forth between chicks with or without 10(-7) M methyltestosterone treatment (2 d of age through 21 d of age) and challenged with 5 x 10(8) virulent Salmonella Pullorum (7 d of age), and in vitro experiment, phagocytic and killing abilities, reactive oxygen intermediate production, and reactive nitrogen intermediate production of monocytes-macrophages treated with high (10(-8) M/10(6) cell) or physiological (10(-14) M/10(6) cell) concentration of methyltestosterone were examined after Salmonella Pullorum infection. The results showed that (1) in vivo, administration of methyltestosterone enhanced susceptibility to Salmonella Pullorum infection and depressed cellular immunity against Salmonella Pullorum, whereas it had no effect on humoral immunity in dwarf chicks; (2) in vitro, at high concentration, methyltestosterone reduced (P < 0.05) monocytes-macrophages mediated reactive oxygen intermediate-dependent killing of Salmonella Pullorum, whereas low concentration of methyltestosterone enhanced (P < 0.05) reactive oxygen intermediate-dependent killing of Salmonella Pullorum in male dwarf chicks but not in females; and (3) although challenged with Salmonella Pullorum, phagocytic ability and monocytes-macrophages mediated reactive nitrogen intermediate-dependent killing were not affected by methyltestosterone in vitro. The results indicated that methyltestosterone affected the immune response to Salmonella Pullorum in dwarf chicks by changing monocytes-macrophages mediated reactive oxygen intermediate-dependent killing and cellular immunity, and the effects were dose-dependent; furthermore, the former 2 pathways played important roles in preventing Salmonella Pullorum infection in dwarf chicks, although the mechanism needs further study.

Solitomab, an epithelial cell adhesion molecule/CD3 bispecific antibody (BiTE), is highly active against primary chemotherapy-resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo.

PubMed

English, Diana P; Bellone, Stefania; Schwab, Carlton L; Roque, Dana M; Lopez, Salvatore; Bortolomai, Ileana; Cocco, Emiliano; Bonazzoli, Elena; Chatterjee, Sudeshna; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Rutherford, Thomas J; Santin, Alessandro D

2015-02-01

Solitomab is a novel, bispecific, single-chain antibody that targets epithelial cell adhesion molecule (EpCAM) on tumor cells and also contains a cluster of differentiation 3 (CD3) (T-cell coreceptor) binding region. The authors evaluated the in vitro activity of solitomab against primary chemotherapy-resistant epithelial ovarian carcinoma cell lines as well as malignant cells in ascites. EpCAM expression was evaluated by flow cytometry in 5 primary ovarian cancer cell lines and in 42 fresh ovarian tumor cell cultures in ascites from patients with mainly advanced or recurrent, chemotherapy-resistant disease. The potential activity of solitomab against EpCAM-positive tumor cells was evaluated by flow cytometry, proliferation, and 4-hour chromium-release, cell-mediated cytotoxicity assays. EpCAM expression was detected by flow cytometry in approximately 80% of the fresh ovarian tumors and primary ovarian tumor cell lines tested. EpCAM-positive, chemotherapy-resistant cell lines were identified as resistant to natural killer cell-mediated or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean±standard error of the mean, 3.6%±0.7% of cells killed after incubation of EpCAM-positive cell lines with control bispecific antibody). In contrast, after incubation with solitomab, EpCAM-positive, chemotherapy-resistant cells became highly sensitive to T-cell cytotoxicity (mean±standard error of the mean, 28.2%±2.05% of cells killed; P<.0001) after exposure to peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with EpCAM-expressing malignant cells in ascites with solitomab resulted in a significant increase in T-cell activation markers and a reduction in the number of viable ovarian tumor cells in ascites (P<.001). Solitomab may represent a novel, potentially effective agent for the treatment of chemotherapy-resistant ovarian cancers that overexpress EpCAM. © 2014 American Cancer Society.

A major chromatin regulator determines resistance of tumor cells to T cell-mediated killing.

PubMed

Pan, Deng; Kobayashi, Aya; Jiang, Peng; Ferrari de Andrade, Lucas; Tay, Rong En; Luoma, Adrienne M; Tsoucas, Daphne; Qiu, Xintao; Lim, Klothilda; Rao, Prakash; Long, Henry W; Yuan, Guo-Cheng; Doench, John; Brown, Myles; Liu, X Shirley; Wucherpfennig, Kai W

2018-02-16

Many human cancers are resistant to immunotherapy, for reasons that are poorly understood. We used a genome-scale CRISPR-Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T cells, the central effectors of antitumor immunity. Inactivation of >100 genes-including Pbrm1 , Arid2 , and Brd7 , which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex-sensitized mouse B16F10 melanoma cells to killing by T cells. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T cells. Treatment-resistant tumors became responsive to immunotherapy when Pbrm1 was inactivated. In many human cancers, expression of PBRM1 and ARID2 inversely correlated with expression of T cell cytotoxicity genes, and Pbrm1 -deficient murine melanomas were more strongly infiltrated by cytotoxic T cells. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Selective replication of oncolytic virus M1 results in a bystander killing effect that is potentiated by Smac mimetics.

PubMed

Cai, Jing; Lin, Yuan; Zhang, Haipeng; Liang, Jiankai; Tan, Yaqian; Cavenee, Webster K; Yan, Guangmei

2017-06-27

Oncolytic virotherapy is a treatment modality that uses native or genetically modified viruses that selectively replicate in and kill tumor cells. Viruses represent a type of pathogen-associated molecular pattern and thereby induce the up-regulation of dozens of cytokines via activating the host innate immune system. Second mitochondria-derived activator of caspases (Smac) mimetic compounds (SMCs), which antagonize the function of inhibitor of apoptosis proteins (IAPs) and induce apoptosis, sensitize tumor cells to multiple cytokines. Therefore, we sought to determine whether SMCs sensitize tumor cells to cytokines induced by the oncolytic M1 virus, thus enhancing a bystander killing effect. Here, we report that SMCs potentiate the oncolytic effect of M1 in vitro, in vivo, and ex vivo. This strengthened oncolytic efficacy resulted from the enhanced bystander killing effect caused by the M1 virus via cytokine induction. Through a microarray analysis and subsequent validation using recombinant cytokines, we identified IL-8, IL-1A, and TRAIL as the key cytokines in the bystander killing effect. Furthermore, SMCs increased the replication of M1, and the accumulation of virus protein induced irreversible endoplasmic reticulum stress- and c-Jun N-terminal kinase-mediated apoptosis. Nevertheless, the combined treatment with M1 and SMCs had little effect on normal and human primary cells. Because SMCs selectively and significantly enhance the bystander killing effect and the replication of oncolytic virus M1 specifically in cancer cells, this combined treatment may represent a promising therapeutic strategy.

Escape of HIV-1-infected dendritic cells from TRAIL-mediated NK cell cytotoxicity during NK-DC cross-talk--a pivotal role of HMGB1.

PubMed

Melki, Marie-Thérèse; Saïdi, Héla; Dufour, Alexandre; Olivo-Marin, Jean-Christophe; Gougeon, Marie-Lise

2010-04-15

Early stages of Human Immunodeficiency Virus-1 (HIV-1) infection are associated with local recruitment and activation of important effectors of innate immunity, i.e. natural killer (NK) cells and dendritic cells (DCs). Immature DCs (iDCs) capture HIV-1 through specific receptors and can disseminate the infection to lymphoid tissues following their migration, which is associated to a maturation process. This process is dependent on NK cells, whose role is to keep in check the quality and the quantity of DCs undergoing maturation. If DC maturation is inappropriate, NK cells will kill them ("editing process") at sites of tissue inflammation, thus optimizing the adaptive immunity. In the context of a viral infection, NK-dependent killing of infected-DCs is a crucial event required for early elimination of infected target cells. Here, we report that NK-mediated editing of iDCs is impaired if DCs are infected with HIV-1. We first addressed the question of the mechanisms involved in iDC editing, and we show that cognate NK-iDC interaction triggers apoptosis via the TNF-related apoptosis-inducing ligand (TRAIL)-Death Receptor 4 (DR4) pathway and not via the perforin pathway. Nevertheless, once infected with HIV-1, DC(HIV) become resistant to NK-induced TRAIL-mediated apoptosis. This resistance occurs despite normal amounts of TRAIL released by NK cells and comparable DR4 expression on DC(HIV). The escape of DC(HIV) from NK killing is due to the upregulation of two anti-apoptotic molecules, the cellular-Flice like inhibitory protein (c-FLIP) and the cellular inhibitor of apoptosis 2 (c-IAP2), induced by NK-DC(HIV) cognate interaction. High-mobility group box 1 (HMGB1), an alarmin and a key mediator of NK-DC cross-talk, was found to play a pivotal role in NK-dependent upregulation of c-FLIP and c-IAP2 in DC(HIV). Finally, we demonstrate that restoration of DC(HIV) susceptibility to NK-induced TRAIL killing can be obtained either by silencing c-FLIP and c-IAP2 by specific siRNA, or by inhibiting HMGB1 with blocking antibodies or glycyrrhizin, arguing for a key role of HMGB1 in TRAIL resistance and DC(HIV) survival. These findings provide evidence for a new strategy developed by HIV to escape immune attack, they challenge the question of the involvement of HMGB1 in the establishment of viral reservoirs in DCs, and they identify potential therapeutic targets to eliminate infected DCs.

Selective cytotoxicity of transformed cells but not normal cells by a sialoglycopeptide growth regulator in the presence of tumor necrosis factor

NASA Technical Reports Server (NTRS)

Woods, K. M.; Fattaey, H.; Johnson, T. C.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

The tumor necrosis factor-alpha (TNF)-resistant, SV40-transformed, murine fibroblast cell lines, F5b and F5m, became sensitive to TNF-mediated cytolysis after treatment with a biologically active 18 kDa peptide fragment (SGP) derived from a 66-kDa parental cell surface sialoglycoprotein. Neither TNF nor the SGP alone exhibited cytotoxicity to the two SV40-transformed cell lines. However, Balb/c 3T3 cells, incubated with SGP alone or with SGP and TNF, were not killed. Therefore, SGP can selectively sensitize cells for TNF alpha-mediated cytotoxicity. This selective sensitization may be due to the previously documented ability of the SGP to selectively mediate cell cycle arrest.

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

PubMed Central

2010-01-01

Trastuzumab (Herceptin®), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab. PMID:18941743

Intracellular Serine Protease Inhibitor SERPINB4 Inhibits Granzyme M-Induced Cell Death

PubMed Central

de Koning, Pieter J. A.; Kummer, J. Alain; de Poot, Stefanie A. H.; Quadir, Razi; Broekhuizen, Roel; McGettrick, Anne F.; Higgins, Wayne J.; Devreese, Bart; Worrall, D. Margaret; Bovenschen, Niels

2011-01-01

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1′ triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×104 M−1s−1. SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death. PMID:21857942

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design.

PubMed

de Goeij, Bart E C G; Peipp, Matthias; de Haij, Simone; van den Brink, Edward N; Kellner, Christian; Riedl, Thilo; de Jong, Rob; Vink, Tom; Strumane, Kristin; Bleeker, Wim K; Parren, Paul W H I

2014-01-01

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla(TM)). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA') fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA', was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA'-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.

CD4 mimetics sensitize HIV-1-infected cells to ADCC.

PubMed

Richard, Jonathan; Veillette, Maxime; Brassard, Nathalie; Iyer, Shilpa S; Roger, Michel; Martin, Loïc; Pazgier, Marzena; Schön, Arne; Freire, Ernesto; Routy, Jean-Pierre; Smith, Amos B; Park, Jongwoo; Jones, David M; Courter, Joel R; Melillo, Bruno N; Kaufmann, Daniel E; Hahn, Beatrice H; Permar, Sallie R; Haynes, Barton F; Madani, Navid; Sodroski, Joseph G; Finzi, Andrés

2015-05-19

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.

CD4 mimetics sensitize HIV-1-infected cells to ADCC

PubMed Central

Richard, Jonathan; Veillette, Maxime; Brassard, Nathalie; Iyer, Shilpa S.; Roger, Michel; Martin, Loïc; Pazgier, Marzena; Schön, Arne; Freire, Ernesto; Routy, Jean-Pierre; Smith, Amos B.; Park, Jongwoo; Jones, David M.; Courter, Joel R.; Melillo, Bruno N.; Kaufmann, Daniel E.; Hahn, Beatrice H.; Permar, Sallie R.; Haynes, Barton F.; Madani, Navid; Sodroski, Joseph G.; Finzi, Andrés

2015-01-01

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection. PMID:25941367

MHC class I target recognition, immunophenotypes and proteomic profiles of natural killer cells within the spleens of day-14 chick embryos

USDA-ARS?s Scientific Manuscript database

Chicken natural killer (NK) cells are not well defined, so little is known about the molecular interactions controlling their activity. At day 14 of embryonic development, chick spleens are a rich source of T-cellfree CD8aa+, CD3_ cells with natural killing activity. Cell-mediated cytotoxicity assay...

Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human macrophages

PubMed Central

Rekha, Rokeya Sultana; Rao Muvva, SSV Jagadeeswara; Wan, Min; Raqib, Rubhana; Bergman, Peter; Brighenti, Susanna; Gudmundsson, Gudmundur H; Agerberth, Birgitta

2015-01-01

LL-37 is a human antimicrobial peptide (AMP) of the cathelicidin family with multiple activities including a mediator of vitamin D-induced autophagy in human macrophages, resulting in intracellular killing of Mycobacterium tuberculosis (Mtb). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent Mtb-killing can be further enhanced by 4-phenylbutyrate (PBA), also an inducer of LL-37 expression. Here, we explore a potential mechanism(s) behind PBA and LL-37-induced autophagy and intracellular killing of Mtb. Mtb infection of macrophages downregulated the expression of both the CAMP transcript and LL-37 peptide as well as certain autophagy-related genes (BECN1 and ATG5) at both the mRNA and protein levels. In addition, activation of LC3-II in primary macrophages and THP-1 cells was not detected. PBA and the active form of vitamin D3 (1,25[OH]2D3), separately or particularly in combination, were able to overcome Mtb-induced suppression of LL-37 expression. Notably, reactivation of autophagy occurred by stimulation of macrophages with PBA and promoted colocalization of LL-37 and LC3-II in autophagosomes. Importantly, PBA treatment failed to induce autophagy in Mtb-infected THP-1 cells, when the expression of LL-37 was silenced. However, PBA-induced autophagy was restored when the LL-37 knockdown cells were supplemented with synthetic LL-37. Interestingly, we have found that LL-37-induced autophagy was mediated via P2RX7 receptor followed by enhanced cytosolic free Ca2+, and activation of AMPK and PtdIns3K pathways. Altogether, these results suggest a novel activity for PBA as an inducer of autophagy, which is LL-37-dependent and promotes intracellular killing of Mtb in human macrophages. PMID:26218841

An identical miRNA of the human JC and BK polyoma viruses targets the stress-induced ligand ULBP3 to escape immune elimination.

PubMed

Bauman, Yoav; Nachmani, Daphna; Vitenshtein, Alon; Tsukerman, Pinchas; Drayman, Nir; Stern-Ginossar, Noam; Lankry, Dikla; Gruda, Raizy; Mandelboim, Ofer

2011-02-17

The human polyoma viruses JCV and BKV establish asymptomatic persistent infection in 65%-90% of humans but can cause severe illness under immunosuppressive conditions. The mechanisms by which these viruses evade immune recognition are unknown. Here we show that a viral miRNA identical in sequence between JCV and BKV targets the stress-induced ligand ULBP3, which is a protein recognized by the killer receptor NKG2D. Consequently, viral miRNA-mediated ULBP3 downregulation results in reduced NKG2D-mediated killing of virus-infected cells by natural killer (NK) cells. Importantly, when the activity of the viral miRNA was inhibited during infection, NK cells killed the infected cells more efficiently. Because NKG2D is also expressed by various T cell subsets, we propose that JCV and BKV use an identical miRNA that targets ULBP3 to escape detection by both the innate and adaptive immune systems, explaining how these viruses remain latent without being eliminated by the immune system. Copyright © 2011 Elsevier Inc. All rights reserved.

Radio-sensitization of Prostate Cancer Cells by Monensin Treatment and its associated Gene Expression Profiling Changes

NASA Technical Reports Server (NTRS)

Zhang Ye; Rohde, Larry H.; Wu, Honglu

2008-01-01

Radio-resistant or recurrent prostate cancer represents a serious health risk for approximately 20%-30% of patients treated with primary radiation therapy for clinically localized prostate cancer. Here, we investigated the effect of monensin on sensitizing radiation mediated cell killing of two radio-resistant prostate cell lines Lncap (P53+ and AR+) and PC3 (P53- and AR-). Treatment with monensin alone (5 micromoles-20 micromoles) showed a significant direct cell killing of Lncap (10-30%), but not PC3 cells. Monensin was also shown to successfully sensitize Lncap cells to X-ray radiation (2Gy-10Gy) mediated cell death, up to 50% of killing with the combined treatment. To better understand the mechanisms of radio-resistance of these two cell lines and their different response to monensin, the apoptosis related gene expression profiles in both cell lines were analyzed using cDNA PCR array. Without any treatment, PC3 showed a much higher expression level of antiapoptosis genes than Lncap in the BCL2 family, the caspase/card family and the TNF ligand/receptor family. At 2 hr after 20 micormolar monensin treatment alone, only the TRAF and CIDE family showed a greater induction in Lncap cells than in PC3. Exposures to 10 Gy X-rays alone of Lncap cells significantly induced gene expression levels in the death and death receptor domain family, the TNF ligand and receptor family, and apoptotic group of BCL2 family; whereas exposures of PC3 induced only the expression of genes in the anti-apoptosis group of CASP and CARD family. Furthermore, we selectively suppressed the expression of several anti-apoptosis genes (BCL-xl, Bcl2A1, BIRC2, BIRC3 and CASP2) in PC3 cells by using the siRNA treatment. Exposure to 10Gy X-rays alone showed an enhanced cell killing (about 15%) in BCL-x1 silenced cells, but not in cells with siRNA treatment targeting other anti-apoptosis genes. We also exposed PC3 cells to protons in the Bragg peak region to compare the effectiveness of cell killing of X-rays. Interestingly, in comparison to X-rays, protons significantly reduced the gene expression in the anti-apoptosis family, suggesting that proton treatment may be more effective for PC3 cells. As a conclusion, monensin was found to sensitize Lncap cells, but not PC3, and over-expression of Bcl-xl cells may be responsible for the radio- or chemo-resistance characteristics of PC3 cells.

Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction.

PubMed

Mathew, Stephen O; Chaudhary, Pankaj; Powers, Sheila B; Vishwanatha, Jamboor K; Mathew, Porunelloor A

2016-10-18

Prostate cancer is the most common type of cancer diagnosed and the second leading cause of cancer-related death in American men. Natural Killer (NK) cells are the first line of defense against cancer and infections. NK cell function is regulated by a delicate balance between signals received through activating and inhibitory receptors. Previously, we identified Lectin-like transcript-1 (LLT1/OCIL/CLEC2D) as a counter-receptor for the NK cell inhibitory receptor NKRP1A (CD161). Interaction of LLT1 expressed on target cells with NKRP1A inhibits NK cell activation. In this study, we have found that LLT1 was overexpressed on prostate cancer cell lines (DU145, LNCaP, 22Rv1 and PC3) and in primary prostate cancer tissues both at the mRNA and protein level. We further showed that LLT1 is retained intracellularly in normal prostate cells with minimal cell surface expression. Blocking LLT1 interaction with NKRP1A by anti-LLT1 mAb on prostate cancer cells increased the NK-mediated cytotoxicity of prostate cancer cells. The results indicate that prostate cancer cells may evade immune attack by NK cells by expressing LLT1 to inhibit NK cell-mediated cytolytic activity through LLT1-NKRP1A interaction. Blocking LLT1-NKRP1A interaction will make prostate cancer cells susceptible to killing by NK cells and therefore may be a new therapeutic option for treatment of prostate cancer.

Mechanisms of Invariant Natural Killer T Cell-Mediated Immunoregulation in Cancer

DTIC Science & Technology

2012-05-01

by mesenchymal stem cells . Intriguingly, the increased metastatic ability was dependent on the production of CCL5 by mesenchymal stem cells , which...Tubo, R., &Weinberg, R.A.(2007) Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. Vol. 449:pp557-563. Breast...can induce preferential secretion of immunosuppressive cytokines ; 2) iNKT cells inhibit effector T cell priming by killing dendritic cells that

Complement activation on B lymphocytes opsonized with rituximab or ofatumumab produces substantial changes in membrane structure preceding cell lysis.

PubMed

Beum, Paul V; Lindorfer, Margaret A; Beurskens, Frank; Stukenberg, P Todd; Lokhorst, Henk M; Pawluczkowycz, Andrew W; Parren, Paul W H I; van de Winkel, Jan G J; Taylor, Ronald P

2008-07-01

Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death.

Monoclonal TCR-redirected tumor cell killing.

PubMed

Liddy, Nathaniel; Bossi, Giovanna; Adams, Katherine J; Lissina, Anna; Mahon, Tara M; Hassan, Namir J; Gavarret, Jessie; Bianchi, Frayne C; Pumphrey, Nicholas J; Ladell, Kristin; Gostick, Emma; Sewell, Andrew K; Lissin, Nikolai M; Harwood, Naomi E; Molloy, Peter E; Li, Yi; Cameron, Brian J; Sami, Malkit; Baston, Emma E; Todorov, Penio T; Paston, Samantha J; Dennis, Rebecca E; Harper, Jane V; Dunn, Steve M; Ashfield, Rebecca; Johnson, Andy; McGrath, Yvonne; Plesa, Gabriela; June, Carl H; Kalos, Michael; Price, David A; Vuidepot, Annelise; Williams, Daniel D; Sutton, Deborah H; Jakobsen, Bent K

2012-06-01

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

Parthanatos, a messenger of death

PubMed Central

David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

2015-01-01

Poly-ADP-ribose polymerase-1 (PARP-1)'s multiple roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include, but are not limited to DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its active role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 over activation underlies cell death in experimental models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into designing PARP-1 inhibitors and understanding mechanisms downstream of PARP-1 over activation. PARP-1 overactivation may kill by depleting cellular energy through nicotinamide adenine dinucleotide (NAD+) consumption, and by releasing the cell death effector apoptosis-inducing factor (AIF). Unexpectedly, recent evidence shows that poly-ADP ribose (PAR) polymer itself, and not the consumption of NAD+ is the source of cytotoxicity. Thus, PAR polymer acts as a cell death effector downstream of PARP-1-mediated cell death signaling. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will summarize the proposed mechanisms by which PARP-1 overactivation kills. We will present evidence for parthanatos, and the questions raised by these recent findings. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 over activation. PMID:19273119

Altered dynamics of Candida albicans phagocytosis by macrophages and PMNs when both phagocyte subsets are present.

PubMed

Rudkin, Fiona M; Bain, Judith M; Walls, Catriona; Lewis, Leanne E; Gow, Neil A R; Erwig, Lars P

2013-10-29

An important first line of defense against Candida albicans infections is the killing of fungal cells by professional phagocytes of the innate immune system, such as polymorphonuclear cells (PMNs) and macrophages. In this study, we employed live-cell video microscopy coupled with dynamic image analysis tools to provide insights into the complexity of C. albicans phagocytosis when macrophages and PMNs were incubated with C. albicans alone and when both phagocyte subsets were present. When C. albicans cells were incubated with only one phagocyte subtype, PMNs had a lower overall phagocytic capacity than macrophages, despite engulfing fungal cells at a higher rate once fungal cells were bound to the phagocyte surface. PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested. In contrast, when both phagocyte subsets were studied in coculture, the two cell types phagocytosed and cleared C. albicans at equal rates and were equally susceptible to killing by the fungus. The increase in macrophage susceptibility to C. albicans-mediated killing was a consequence of macrophages taking up a higher proportion of hyphal cells under these conditions. In the presence of both PMNs and macrophages, C. albicans yeast cells were predominantly cleared by PMNs, which migrated at a greater speed toward fungal cells and engulfed bound cells more rapidly. These observations demonstrate that the phagocytosis of fungal pathogens depends on, and is modified by, the specific phagocyte subsets present at the site of infection. Extensive work investigating fungal cell phagocytosis by macrophages and PMNs of the innate immune system has been carried out. These studies have been informative but have examined this phenomenon only when one phagocyte subset is present. The current study employed live-cell video microscopy to break down C. albicans phagocytosis into its component parts and examine the effect of a single phagocyte subset, versus a mixed phagocyte population, on these individual stages. Through this approach, we identified that the rate of fungal cell engulfment and rate of phagocyte killing altered significantly when both macrophages and PMNs were incubated in coculture with C. albicans compared to the rate of either phagocyte subset incubated alone with the fungus. This research highlights the significance of studying pathogen-host cell interactions with a combination of phagocytes in order to gain a greater understanding of the interactions that occur between cells of the host immune system in response to fungal invasion.

Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells

PubMed Central

Sloan, Derek D.; Lam, Chia-Ying Kao; Irrinki, Alivelu; Liu, Liqin; Tsai, Angela; Pace, Craig S.; Kaur, Jasmine; Murry, Jeffrey P.; Balakrishnan, Mini; Moore, Paul A.; Johnson, Syd; Nordstrom, Jeffrey L.; Cihlar, Tomas; Koenig, Scott

2015-01-01

HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs. PMID:26539983

Three distinct cell phenotypes of induced-TNF cytotoxicity and their relationship to apoptosis

NASA Technical Reports Server (NTRS)

Woods, K. M.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1993-01-01

We have identified three distinct cell phenotypes with respect to the conditions under which cells became susceptible to TNF-mediated lysis. These conditions include: 1) treatment with the protein synthesis inhibitor, cycloheximide; 2) contact with activated macrophages, and 3) infection with vaccinia virus. Whereas vaccinia virus-infected 3T3 cells became sensitive to soluble TNF, F5b cells required contact with activated macrophages. We showed that the "macrophage-resistant" F5m cells did not become sensitive to TNF or to killing by activated macrophages after infection with vaccinia virus. Therefore, vaccinia infection does not sensitize all cells to TNF. We also determined the pathways of lysis for cells after sensitization. Whereas 3T3, LM929, and F5b cells were killed by the process of necrosis, F5m cells lysis was characterized by the release of low mol wt DNA fragments (apoptosis).

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

PubMed Central

Jiménez de Bagüés, M P; Elzer, P H; Jones, S M; Blasco, J M; Enright, F M; Schurig, G G; Winter, A J

1994-01-01

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis. Images PMID:7927779

Enhanced reactive oxygen species through direct copper sulfide nanoparticle-doxorubicin complexation

NASA Astrophysics Data System (ADS)

Li, Yajuan; Cupo, Michela; Guo, Liangran; Scott, Julie; Chen, Yi-Tzai; Yan, Bingfang; Lu, Wei

2017-12-01

CuS-based nanostructures loading the chemotherapeutic agent doxorubicin (DOX) exerted excellent cancer photothermal chemotherapy under multi-external stimuli. The DOX loading was generally designed through electrostatic interaction or chemical linkers. However, the interaction between DOX molecules and CuS nanoparticles has not been investigated. In this work, we use PEGylated hollow copper sulfide nanoparticles (HCuSNPs) to directly load DOX through the DOX/Cu2+ chelation process. Distinctively, the synthesized PEG-HCuSNPs-DOX release the DOX/Cu2+ complexes into surrounding environment, which generate significant reactive oxygen species (ROS) in a controlled manner by near-infrared laser. The CuS nanoparticle-mediated photothermal ablation facilitates the ROS-induced cancer cell killing effect. Our current work reveals a DOX/Cu2+-mediated ROS-enhanced cell-killing effect in addition to conventional photothermal chemotherapy through the direct CuS nanoparticle-DOX complexation.

Macrophage migration inhibitory factor deficiency is associated with impaired killing of gram-negative bacteria by macrophages and increased susceptibility to Klebsiella pneumoniae sepsis.

PubMed

Roger, Thierry; Delaloye, Julie; Chanson, Anne-Laure; Giddey, Marlyse; Le Roy, Didier; Calandra, Thierry

2013-01-15

The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIF's regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.

Macrophage-mediated trogocytosis leads to death of antibody-opsonized tumor cells

PubMed Central

Velmurugan, Ramraj; Challa, Dilip K.; Ram, Sripad; Ober, Raimund J.; Ward, E. Sally

2016-01-01

Understanding the complex behavior of effector cells such as monocytes or macrophages in regulating cancerous growth is of central importance for cancer immunotherapy. Earlier studies using CD20-specific antibodies have demonstrated that the Fcγ receptor (FcγR)-mediated transfer of the targeted receptors from tumor cells to these effector cells through trogocytosis can enable escape from antibody therapy, leading to the viewpoint that this process is pro-tumorigenic. In the current study we demonstrate that persistent trogocytic attack results in the killing of HER2-overexpressing breast cancer cells. Further, antibody engineering to increase FcγR interactions enhances this tumoricidal activity. These studies extend the complex repertoire of activities of macrophages to trogocytic-mediated cell death of HER2-overexpressing target cells and have implications for the development of effective antibody-based therapies. PMID:27226489

Matrix regulation of skeletal cell apoptosis II: role of Arg-Gly-Asp-containing peptides.

PubMed

Perlot, Robert L; Shapiro, Irving M; Mansfield, Kyle; Adams, Christopher S

2002-01-01

This investigation was based on the assumption that arg-gly-asp (RGD)-containing peptides are released from the extracellular matrix of bone and cartilage during the remodeling cycle. We asked the question: Can RGD peptides influence skeletal cell viability? Primary human osteoblasts, mouse MC-3T3-E1 cells, and chick chondrocytes were incubated with purified RGD-containing peptides and cell viability was determined. The RGD peptide did not kill osteoblasts, chondrocytes, or MC-3T3-E1 cells. In contrast, RGDS and GRGDSP peptides killed all three cell types. Osteoblast death was quite rapid, occurring within 6 h of treatment. transferase uridyl mediated nick end labeling (TUNEL) and transmission electron microscopy (TEM) analysis indicated that death was mediated by apoptosis. To learn if mitochondria transduced the death signal, cells were treated with RGDS and organelle function was evaluated using a voltage-sensitive fluorescent probe. It was observed that there was no net loss of fluorescence and, hence, it was concluded that mitochondria were not the primary effectors of the apoptotic response. Experiments were performed with enzyme inhibitors to determine the import of the caspase pathway on RGDS-mediated osteoblast apoptosis. Results of these studies, as well as a study conducted using a fluorescent substrate, pointed to caspase 3 mediating the effector stage of the apoptotic process. Finally, using a purified labeled-RGDS peptide, we showed that the molecule was not restricted by the plasma membrane because it was accumulated in the cytosolic compartment. Results of the investigation support the view that resorption of the extracellular matrix generates peptide products that can induce apoptosis of vicinal cells.

Natural Killer (NK)/melanoma cell interaction induces NK-mediated release of chemotactic High Mobility Group Box-1 (HMGB1) capable of amplifying NK cell recruitment

PubMed Central

Parodi, Monica; Pedrazzi, Marco; Cantoni, Claudia; Averna, Monica; Patrone, Mauro; Cavaletto, Maria; Spertino, Stefano; Pende, Daniela; Balsamo, Mirna; Pietra, Gabriella; Sivori, Simona; Carlomagno, Simona; Mingari, Maria Cristina; Moretta, Lorenzo; Sparatore, Bianca; Vitale, Massimo

2015-01-01

In this study we characterize a new mechanism by which Natural Killer (NK) cells may amplify their recruitment to tumors. We show that NK cells, upon interaction with melanoma cells, can release a chemotactic form of High Mobility Group Box-1 (HMGB1) protein capable of attracting additional activated NK cells. We first demonstrate that the engagement of different activating NK cell receptors, including those mainly involved in tumor cell recognition can induce the active release of HMGB1. Then we show that during NK-mediated tumor cell killing two HMGB1 forms are released, each displaying a specific electrophoretic mobility possibly corresponding to a different redox status. By the comparison of normal and perforin-defective NK cells (which are unable to kill target cells) we demonstrate that, in NK/melanoma cell co-cultures, NK cells specifically release an HMGB1 form that acts as chemoattractant, while dying tumor cells passively release a non-chemotactic HMGB1. Finally, we show that Receptor for Advanced Glycation End products is expressed by NK cells and mediates HMGB1-induced NK cell chemotaxis. Proteomic analysis of NK cells exposed to recombinant HMGB1 revealed that this molecule, besides inducing immediate chemotaxis, also promotes changes in the expression of proteins involved in the regulation of the cytoskeletal network. Importantly, these modifications could be associated with an increased motility of NK cells. Thus, our findings allow the definition of a previously unidentified mechanism used by NK cells to amplify their response to tumors, and provide additional clues for the emerging role of HMGB1 in immunomodulation and tumor immunity. PMID:26587323

Tumor Cells Surviving Exposure to Proton or Photon Radiation Share a Common Immunogenic Modulation Signature, Rendering Them More Sensitive to T Cell–Mediated Killing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gameiro, Sofia R.; Malamas, Anthony S.; Bernstein, Michael B.

Purpose: To provide the foundation for combining immunotherapy to induce tumor antigen–specific T cells with proton radiation therapy to exploit the activity of those T cells. Methods and Materials: Using cell lines of tumors frequently treated with proton radiation, such as prostate, breast, lung, and chordoma, we examined the effect of proton radiation on the viability and induction of immunogenic modulation in tumor cells by flow cytometric and immunofluorescent analysis of surface phenotype and the functional immune consequences. Results: These studies show for the first time that (1) proton and photon radiation induced comparable up-regulation of surface molecules involved in immune recognition (histocompatibilitymore » leukocyte antigen, intercellular adhesion molecule 1, and the tumor-associated antigens carcinoembryonic antigen and mucin 1); (2) proton radiation mediated calreticulin cell-surface expression, increasing sensitivity to cytotoxic T-lymphocyte killing of tumor cells; and (3) cancer stem cells, which are resistant to the direct cytolytic activity of proton radiation, nonetheless up-regulated calreticulin after radiation in a manner similar to non-cancer stem cells. Conclusions: These findings offer a rationale for the use of proton radiation in combination with immunotherapy, including for patients who have failed radiation therapy alone or have limited treatment options.« less

Mechanisms involved in synergistic anticancer immunity of anti-4-1BB and anti-CD4 therapy.

PubMed

Choi, Beom K; Kim, Young H; Kang, Woo J; Lee, Sun K; Kim, Kwang H; Shin, Su M; Yokoyama, Wayne M; Kim, Tae Y; Kwon, Byoung S

2007-09-15

Anti-4-1BB-mediated anticancer effects were potentiated by depletion of CD4+ cells in B16F10 melanoma-bearing C57BL/6 mice. Anti-4-1BB induced the expansion and differentiation of polyclonal tumor-specific CD8+ T cells into IFN-gamma-producing CD11c+CD8+ T cells. The CD4+ cell depletion was responsible for facilitating immune cell infiltration into tumor tissues and removing some regulatory barriers such as T regulatory and indoleamine-2,3-dioxygenase (IDO)+ dendritic cells. Both monoclonal antibodies (mAb) contributed to the efficient induction of MHC class I molecules on the tumor cells in vivo. The effectors that mediated the anti-4-1BB effect were NKG2D+KLRG1+CD11c+CD8+ T cells that accumulated preferentially in the tumor tissues. Blocking NKG2D reduced the therapeutic effect by 20% to 26%, which may indicate that NKG2D contributes partially to tumor killing by the differentiated CD8+ T cells. Our results indicate that the combination of the two mAbs, agonistic anti-4-1BB and depleting anti-CD4, results in enhanced production of efficient tumor-killing CTLs, facilitation of their infiltration, and production of a susceptible tumor microenvironment.

Platelets Mediate Host Defense against Staphylococcus aureus through Direct Bactericidal Activity and by Enhancing Macrophage Activities.

PubMed

Ali, Ramadan A; Wuescher, Leah M; Dona, Keith R; Worth, Randall G

2017-01-01

Platelets are the chief effector cells in hemostasis. However, recent evidence suggests they have multiple roles in host defense against infection. Reports by us and others showed that platelets functionally contribute to protection against Staphylococcus aureus infection. In the current study, the capacity of mouse platelets to participate in host defense against S. aureus infection was determined by assessing two possibilities. First, we determined the ability of platelets to kill S. aureus directly; and, second, we tested the possibility that platelets enhance macrophage phagocytosis and intracellular killing of S. aureus In this study we report evidence in support of both mechanisms. Platelets effectively killed two different strains of S. aureus. A clinical isolate of methicillin-resistant S. aureus was killed by platelets (>40% killing in 2 h) in a thrombin-dependent manner whereas a methicillin-sensitive strain was killed to equal extent but did not require thrombin. Interestingly, thrombin-stimulated platelets also significantly enhanced peritoneal macrophage phagocytosis of both methicillin-resistant S. aureus and methicillin-sensitive S. aureus by >70%, and restricted intracellular growth by >40%. Enhancement of macrophage anti-S. aureus activities is independent of contact with platelets but is mediated through releasable products, namely IL-1β. These data confirm our hypothesis that platelets participate in host defense against S. aureus both through direct killing of S. aureus and enhancing the antimicrobial function of macrophages in protection against S. aureus infection. Copyright © 2016 by The American Association of Immunologists, Inc.

Gemcitabine sensitizes lung cancer cells to Fas/FasL system-mediated killing

PubMed Central

Siena, Liboria; Pace, Elisabetta; Ferraro, Maria; Di Sano, Caterina; Melis, Mario; Profita, Mirella; Spatafora, Mario; Gjomarkaj, Mark

2014-01-01

Gemcitabine is a chemotherapy agent commonly used in the treatment of non-small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine-induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real-time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL-expressing cells was evaluated by flow cytometry, and caspase-8 and caspase-3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine-activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti-Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane-bound FasL (mFasL) and of mFasL-positive apoptotic H292 cells, as well as caspase-8 and caspase-3 cleavage. Moreover, gemcitabine increased CH11-induced caspase-8 and caspase-3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine-treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up-regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas-dependent apoptosis mediated by caspase-8 and caspase-3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine-induced lung cancer cell killing. PMID:24128051

Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion*

PubMed Central

Catalán, Elena; Jaime-Sánchez, Paula; Aguiló, Nacho; Simon, Markus M.; Froelich, Christopher J.; Pardo, Julián

2015-01-01

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB+Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB+Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB+Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB+Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB+Tc-induced death pathways. PMID:25605735

Ionophore-A23187-induced cellular cytotoxicity: a cell fragment mediated process.

PubMed Central

Nash, G S; Niedt, G W; MacDermott, R P

1980-01-01

Calcium ionophore A23187 was found to induce human white blood cells to kill human red blood cells. Optimal conditions for ionophore-induced cellular cytotoxicity (IICC) included an 18 h time period, an incubation temperature of 25 degrees, a 25:1 or 50:1 killer:target cell ratio,and a final ionophore concentration of 2 . 5 microgram/ml. WBC or granulocytes which were either frozen and thawed three times or sonicated were capable of mediating IICC. As intact cells, granulocytes (67 . 2% cytotoxicity), monocytes (34 . 8%), B cells (22 . 0%) and Null cells (19 . 3%) were effector cells but T cells (7 . 4%) were not. After fragmenting these cells, all cell types including T cells were able to mediate IICC. When cell lines (K562, Chang, and NCTC) were used as effectors, none would mediate IICC when intact. After freezing and thawing, Chang and NCTC would not mediate IICC, whereas K562 cells did. These studies may be indicative of a calcium-dependent, membrane-localized mechanism in cellular cytotoxic processes, and may provide a useful indicator system for isolation of the enzyme systems involved in cellular cytotoxicity. PMID:6773881

Selective killing of hepatocellular carcinoma HepG2 cells by three-dimensional nanographene nanoparticles based on triptycene

NASA Astrophysics Data System (ADS)

Xiong, Xiaoqin; Gan, Lu; Liu, Ying; Zhang, Chun; Yong, Tuying; Wang, Ziyi; Xu, Huibi; Yang, Xiangliang

2015-03-01

Carbon-based materials have been widely used in the biomedical fields including drug delivery and cancer therapies. In this paper, a recently synthesized three-dimensional nanographene (NG) based on triptycene self-assembles into nanoparticles which selectively kill human hepatocellular carcinoma HepG2 cells as compared to human normal liver HL7702 cells. Obvious differences in cellular accumulation, the endocytic pathway and intracellular trafficking of NG nanoparticles are observed in HepG2 cells and HL7702 cells. Further studies reveal that NG nanoparticles significantly increase the levels of reactive oxygen species (ROS) in HepG2 cells, but not in HL7702 cells. NG nanoparticle-induced ROS result in apoptosis induction and the decrease in mitochondrial membrane potential in HepG2 cells. Moreover, IKK/nuclear factor-κB (NF-κB) signaling is found to be activated by NG nanoparticle-induced ROS and serves to antagonize NG nanoparticle-induced apoptosis in HepG2 cells. Our studies show that the distinct behaviors of cellular uptake and ROS-mediated cytotoxicity are responsible for the selective killing of HepG2 cells. This study provides a foundation for understanding the mechanism of selective induction of apoptosis in cancer cells by NG nanoparticles and designing more effective chemotherapeutical agents.Carbon-based materials have been widely used in the biomedical fields including drug delivery and cancer therapies. In this paper, a recently synthesized three-dimensional nanographene (NG) based on triptycene self-assembles into nanoparticles which selectively kill human hepatocellular carcinoma HepG2 cells as compared to human normal liver HL7702 cells. Obvious differences in cellular accumulation, the endocytic pathway and intracellular trafficking of NG nanoparticles are observed in HepG2 cells and HL7702 cells. Further studies reveal that NG nanoparticles significantly increase the levels of reactive oxygen species (ROS) in HepG2 cells, but not in HL7702 cells. NG nanoparticle-induced ROS result in apoptosis induction and the decrease in mitochondrial membrane potential in HepG2 cells. Moreover, IKK/nuclear factor-κB (NF-κB) signaling is found to be activated by NG nanoparticle-induced ROS and serves to antagonize NG nanoparticle-induced apoptosis in HepG2 cells. Our studies show that the distinct behaviors of cellular uptake and ROS-mediated cytotoxicity are responsible for the selective killing of HepG2 cells. This study provides a foundation for understanding the mechanism of selective induction of apoptosis in cancer cells by NG nanoparticles and designing more effective chemotherapeutical agents. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07248k

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.

PubMed

Wang, Dachun; Quan, Yuan; Yan, Qing; Morales, John E; Wetsel, Rick A

2015-10-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. This study reports the generation of a novel β2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M-/- hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M-/- hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M-/- hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells. ©AlphaMed Press.

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells

PubMed Central

Quan, Yuan; Yan, Qing; Morales, John E.

2015-01-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. Significance This study reports the generation of a novel β2-microglobulin (B2M)−/− human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M−/− hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M−/− hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M−/− hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells. PMID:26285657

A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.

PubMed

Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi

2014-10-31

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Candida albicans Adheres to Chitin by Recognizing N-acetylglucosamine (GlcNAc).

PubMed

Ishijima, Sanae A; Yamada, Tsuyoshi; Maruyama, Naho; Abe, Shigeru

2017-01-01

The binding of Candida albicans cells to chitin was examined in a cell-binding assay. Microscopic observations indicated that both living and heat-killed Candida cells bound to chitin-coated substrates. C. albicans preferentially bound to chitin-coated plastic plates over chitosan-coated and uncoated plates. We prepared 125 I-labeled Candida cells for quantitative analysis of their binding to chitin. Heat-killed 125 I-labeled Candida cells bound to chitin-coated plates in a time-dependent manner until 1.5 hours after start of incubation at 4℃. The binding of 125 I-labeled Candida cells to chitin-coated plates was inhibited by adding unlabeled living or unlabeled heat-killed Candida cells. The binding of Candida to chitin was also reduced by addition of 25 mg/ml chitin or chitosan up to 10%. N-acetylglucosamine (GlcNAc), which is a constituent of chitin, inhibited binding of Candida to chitin in a dose-dependent manner between 12.5 and 200 mM. Glucosamine, which is a constituent of chitosan, showed no such inhibitory effect. These findings suggest that the binding of Candida to chitin may be mediated by recognition of GlcNAc.

Low susceptibility of NC/Nga mice to the lipopolysaccharide-mediated lethality with D-galactosamine sensitization and the involvement of fewer natural killer T cells.

PubMed

Koide, Naoki; Morikawa, Akiko; Odkhuu, Erdenezaya; Haque, Abedul; Badamtseren, Battuvshin; Naiki, Yoshikazu; Komatsu, Takayuki; Yoshida, Tomoaki; Yokochi, Takashi

2012-02-01

The LPS-mediated lethality of NC/Nga mice, having fewer NKT cells, was examined by using d-galactosamine (d-GalN)-sensitization. The NC/Nga mice were not killed by a simultaneous administration of d-GalN and LPS whereas all C57BL/6 (B6) control mice were killed. The injection of d-GalN and LPS failed to elevate the levels of serum alanine aminotransferase and caspase 3 in the liver tissues of NC/Nga mice. Further, the nitric oxide (NO) level of the d-GalN- and LPS-injected NC/Nga mice was much lower than those of the B6 mice. The expression of an inducible NO synthase (iNOS) was significantly reduced in the livers of NC/Nga mice. However, there was no significant difference in LPS-induced TNF-α production between B6 mice and NC/Nga mice. The NC/Nga mice had an impaired expression of IFN-γ protein and mRNA in response to d-GalN and LPS. The pretreatment with α-galactosylceramide (α-GalCer), which activates Vα14(+) NKT cells and induces the production of IFN-γ, rendered NC/Nga mice more susceptible to the LPS-mediated lethality. The livers of NC/Nga mice had fewer NKT cells compared to B6 mice. Taken together, it is suggested that the resistance of NC/Nga mice to the LPS-mediated lethality with d-GalN sensitization depended on the impaired IFN-γ production caused by fewer NKT cells and reduced NO production that followed.

How Do CD4+ T Cells Detect and Eliminate Tumor Cells That Either Lack or Express MHC Class II Molecules?

PubMed Central

Haabeth, Ole Audun Werner; Tveita, Anders Aune; Fauskanger, Marte; Schjesvold, Fredrik; Lorvik, Kristina Berg; Hofgaard, Peter O.; Omholt, Hilde; Munthe, Ludvig A.; Dembic, Zlatko; Corthay, Alexandre; Bogen, Bjarne

2014-01-01

CD4+ T cells contribute to tumor eradication, even in the absence of CD8+ T cells. Cytotoxic CD4+ T cells can directly kill MHC class II positive tumor cells. More surprisingly, CD4+ T cells can indirectly eliminate tumor cells that lack MHC class II expression. Here, we review the mechanisms of direct and indirect CD4+ T cell-mediated elimination of tumor cells. An emphasis is put on T cell receptor (TCR) transgenic models, where anti-tumor responses of naïve CD4+ T cells of defined specificity can be tracked. Some generalizations can tentatively be made. For both MHCIIPOS and MHCIINEG tumors, presentation of tumor-specific antigen by host antigen-presenting cells (APCs) appears to be required for CD4+ T cell priming. This has been extensively studied in a myeloma model (MOPC315), where host APCs in tumor-draining lymph nodes are primed with secreted tumor antigen. Upon antigen recognition, naïve CD4+ T cells differentiate into Th1 cells and migrate to the tumor. At the tumor site, the mechanisms for elimination of MHCIIPOS and MHCIINEG tumor cells differ. In a TCR-transgenic B16 melanoma model, MHCIIPOS melanoma cells are directly killed by cytotoxic CD4+ T cells in a perforin/granzyme B-dependent manner. By contrast, MHCIINEG myeloma cells are killed by IFN-γ stimulated M1-like macrophages. In summary, while the priming phase of CD4+ T cells appears similar for MHCIIPOS and MHCIINEG tumors, the killing mechanisms are different. Unresolved issues and directions for future research are addressed. PMID:24782871

Nitric oxide inhibits exocytosis of cytolytic granules from lymphokine-activated killer cells

PubMed Central

Ferlito, Marcella; Irani, Kaikobad; Faraday, Nauder; Lowenstein, Charles J.

2006-01-01

NO inhibits cytotoxic T lymphocyte killing of target cells, although the precise mechanism is unknown. We hypothesized that NO decreases exocytosis of cytotoxic granules from activated lymphocytes. We now show that NO inhibits lymphokine-activated killer cell killing of K562 target cells. Exogenous and endogenous NO decreases the release of granzyme B, granzyme A, and perforin: all contents of cytotoxic granules. NO inhibits the signal transduction cascade initiated by cross-linking of the T cell receptor that leads to granule exocytosis. In particular, we found that NO decreases the expression of Ras, a critical signaling component within the exocytic pathway. Ectopic expression of Ras prevents NO inhibition of exocytosis. Our data suggest that Ras mediates NO inhibition of lymphocyte cytotoxicity and emphasize that alterations in the cellular redox state may regulate the exocytic signaling pathway. PMID:16857739

Combined regimen of photodynamic therapy mediated by Gallium phthalocyanine chloride and Metformin enhances anti-melanoma efficacy

PubMed Central

Filip, Gabriela Adriana; Olteanu, Diana; Cenariu, Mihai; Tabaran, Flaviu; Ion, Rodica Mariana; Gligor, Lucian; Baldea, Ioana

2017-01-01

Background Melanoma therapy is challenging, especially in advanced cases, due to multiple developed tumor defense mechanisms. Photodynamic therapy (PDT) might represent an adjuvant treatment, because of its bimodal action: tumor destruction and immune system awakening. In this study, a combination of PDT mediated by a metal substituted phthalocyanine—Gallium phthalocyanine chloride (GaPc) and Metformin was used against melanoma. The study aimed to: (1) find the anti-melanoma efficacy of GaPc-PDT, (2) assess possible beneficial effects of Metformin addition to PDT, (3) uncover some of the mechanisms underlining cell killing and anti-angiogenic effects. Methods Two human lightly pigmented melanoma cell lines: WM35 and M1/15 subjected to previous Metformin exposure were treated by GaPc-PDT. Cell viability, death mechanism, cytoskeleton alterations, oxidative damage, were assessed by means of colorimetry, flowcytometry, confocal microscopy, spectrophotometry, ELISA, Western Blotting. Results GaPc proved an efficient photosensitizer. Metformin addition enhanced cell killing by mechanisms dependent on the cell line, namely apoptosis in the metastatic M1/15 and necrosis in the radial growth phase, WM35. Cell death mechanism relied on the inhibition of nuclear transcription factor (NF)-κB activation and tumor necrosis factor (TNF)—related apoptosis-inducing ligand (TRAIL) sensitization, leading to TRAIL and TNF-α induced apoptosis. Metformin diminished the anti-angiogenic effect of PDT. Conclusions Metformin addition to GaPc-PDT increased tumor cell killing through enhanced oxidative damage and induction of proapoptotic mechanisms, but altered PDT anti-angiogenic effects. General significance Combination of Metformin and PDT might represent a solution to enhance the efficacy, leading to a potential adjuvant role of PDT in melanoma therapy. PMID:28278159

Combined regimen of photodynamic therapy mediated by Gallium phthalocyanine chloride and Metformin enhances anti-melanoma efficacy.

PubMed

Tudor, Diana; Nenu, Iuliana; Filip, Gabriela Adriana; Olteanu, Diana; Cenariu, Mihai; Tabaran, Flaviu; Ion, Rodica Mariana; Gligor, Lucian; Baldea, Ioana

2017-01-01

Melanoma therapy is challenging, especially in advanced cases, due to multiple developed tumor defense mechanisms. Photodynamic therapy (PDT) might represent an adjuvant treatment, because of its bimodal action: tumor destruction and immune system awakening. In this study, a combination of PDT mediated by a metal substituted phthalocyanine-Gallium phthalocyanine chloride (GaPc) and Metformin was used against melanoma. The study aimed to: (1) find the anti-melanoma efficacy of GaPc-PDT, (2) assess possible beneficial effects of Metformin addition to PDT, (3) uncover some of the mechanisms underlining cell killing and anti-angiogenic effects. Two human lightly pigmented melanoma cell lines: WM35 and M1/15 subjected to previous Metformin exposure were treated by GaPc-PDT. Cell viability, death mechanism, cytoskeleton alterations, oxidative damage, were assessed by means of colorimetry, flowcytometry, confocal microscopy, spectrophotometry, ELISA, Western Blotting. GaPc proved an efficient photosensitizer. Metformin addition enhanced cell killing by mechanisms dependent on the cell line, namely apoptosis in the metastatic M1/15 and necrosis in the radial growth phase, WM35. Cell death mechanism relied on the inhibition of nuclear transcription factor (NF)-κB activation and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) sensitization, leading to TRAIL and TNF-α induced apoptosis. Metformin diminished the anti-angiogenic effect of PDT. Metformin addition to GaPc-PDT increased tumor cell killing through enhanced oxidative damage and induction of proapoptotic mechanisms, but altered PDT anti-angiogenic effects. Combination of Metformin and PDT might represent a solution to enhance the efficacy, leading to a potential adjuvant role of PDT in melanoma therapy.

1,25D3 potentiates cisplatin antitumor activity by p73 induction in a squamous cell carcinoma model

PubMed Central

Ma, Yingyu; Yu, Wei-Dong; Hershberger, Pamela A.; Flynn, Geraldine; Kong, Rui-Xian; Trump, Donald L.; Johnson, Candace S.

2008-01-01

1,25D3 exhibits anti-tumor activity in a variety of cancers including squamous cell carcinoma (SCC). Intrinsic resistance of SCC cells to cisplatin was observed and led to the investigation into whether 1,25D3 sensitizes SCC cells to cisplatin. Pretreatment with 1,25D3 followed by cisplatin enhanced growth inhibition in SCC cells compared with 1,25D3 alone, as assessed by cytotoxicity and in vitro clonogenic assays. In addition, 1,25D3 sensitized SCC cells to cisplatin-mediated apoptosis. Treatment of tumor-bearing C3H mice with 1,25D3 prior to cisplatin reduced clonogenic survival using in vivo excision clonogenic assay. These results were not observed in a 1,25D3-resistant SCC variant, indicating the critical role of 1,25D3 in sensitizing SCC cells to cisplatin. Further, a marked decrease in fractional tumor volume was observed when SCC tumor-bearing mice were treated with 1,25D3 prior to cisplatin as compared to either agent administered alone. Cisplatin has been shown to modulate p73 protein level in certain cancer cells. Our data showed that p73 level was not affected by cisplatin, but increased by 1,25D3 in SCC cells. Knocking down p73 by siRNA protected SCC cells against 1,25D3 and cisplatin-mediated clonogenic cell kill and apoptosis. Increasing p73 protein level by knocking down UFD2a, which mediates p73 degradation, promoted 1,25D3 and cisplatin-mediated clonogenic cell kill. These results suggest that 1,25D3 potentiates cisplatin anti-tumor activity in vitro and in vivo in a SCC model system, possibly through p73 induction and apoptosis. The combination treatment may provide a more effective therapeutic regimen in cancer treatment. PMID:18790784

Reovirus-Mediated Cytotoxicity and Enhancement of Innate Immune Responses Against Acute Myeloid Leukemia

PubMed Central

Hall, Kathryn; Scott, Karen J.; Rose, Ailsa; Desborough, Michael; Harrington, Kevin; Pandha, Hardev; Parrish, Christopher; Vile, Richard; Coffey, Matt; Bowen, David; Errington-Mais, Fiona

2012-01-01

Abstract Reovirus is a naturally occurring oncolytic virus that has shown preclinical efficacy in the treatment of a wide range of tumor types and has now reached phase III testing in clinical trials. The anti-cancer activity of reovirus has been attributed to both its direct oncolytic activity and the enhancement of anti-tumor immune responses. In this study, we have investigated the direct effect of reovirus on acute myeloid leukemia (AML) cells and its potential to enhance innate immune responses against AML, including the testing of primary samples from patients. Reovirus was found to replicate in and kill AML cell lines, and to reduce cell viability in primary AML samples. The pro-inflammatory cytokine interferon alpha (IFNα) and the chemokine (C-C motif) ligand 5 (known as RANTES [regulated upon activation, normal T-cell expressed, and secreted]) were also secreted from AML cells in response to virus treatment. In addition, reovirus-mediated activation of natural killer (NK) cells, within the context of peripheral blood mononuclear cells, stimulated their anti-leukemia response, with increased NK degranulation and IFNγ production and enhanced killing of AML targets. These data suggest that reovirus has the potential as both a direct cytotoxic and an immunotherapeutic agent for the treatment of AML. PMID:23515241

Neisseria meningitidis: pathogenesis and immunity.

PubMed

Pizza, Mariagrazia; Rappuoli, Rino

2015-02-01

The recent advances in cellular microbiology, genomics, and immunology has opened new horizons in the understanding of meningococcal pathogenesis and in the definition of new prophylactic intervention. It is now clear that Neissera meningitidis has evolved a number of surface structures to mediate interaction with host cells and a number of mechanisms to subvert the immune system and escape complement-mediated killing. In this review we report the more recent findings on meningococcal adhesion and on the bacteria-complement interaction highlighting the redundancy of these mechanisms. An effective vaccine against meningococcus B, based on multiple antigens with different function, has been recently licensed. The antibodies induced by the 4CMenB vaccine could mediate bacterial killing by activating directly the classical complement pathway or, indirectly, by preventing binding of fH on the bacterial surface and interfering with colonization. Copyright © 2014 The Author. Published by Elsevier Ltd.. All rights reserved.

Susceptibility to cytotoxic T cell lysis of cancer stem cells derived from cervical and head and neck tumor cell lines.

PubMed

Liao, Tian; Kaufmann, Andreas M; Qian, Xu; Sangvatanakul, Voramon; Chen, Chao; Kube, Tina; Zhang, Guoyou; Albers, Andreas E

2013-01-01

To explore cancer stem cell susceptibility to a host's cytotoxic T lymphocyte (CTL)-mediated immune response. We compared the susceptibility of putative CSC generated from cancer cell lines to immunologic recognition and killing by alloantigen-specific CD8(+) CTL. CSC-enriched spheroid culture-derived cells (SDC) exhibited higher expression of ALDH, ICAM1 and of stem/progenitor cell markers on all 3 tumor cell lines investigated and lower MHC class I on the cervical cancer cell line as compared to their monolayer-derived cells (MDC). The expression of ICAM1 and MHCI was upregulated by IFN-γ treatment. CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8(+) CTL lysis as compared to matched MDC. IFN-γ pretreatment resulted in over-proportionally enhanced lysis of SDC. Finally, the subset of ALDH(high) expressing SDC presented more sensitivity toward CD8(+) CTL killing than the ALDH(low) SDC. Tumor therapy resistance has been attributed to cancer stem cells (CSC). We show in vitro susceptibility of CSC to CTL-mediated lysis. Immunotherapy targeting of ALDH(+) CSC may therefore be a promising approach. Our results and method may be helpful for the development and optimization of adjuvants, as here exemplified for INF-γ, for CSC-targeted vaccines, independent of the availability of CSC-specific antigens.

Human Salivary Protein Histatin 5 Has Potent Bactericidal Activity against ESKAPE Pathogens

PubMed Central

Du, Han; Puri, Sumant; McCall, Andrew; Norris, Hannah L.; Russo, Thomas; Edgerton, Mira

2017-01-01

ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species) pathogens have characteristic multiple-drug resistance and cause an increasing number of nosocomial infections worldwide. Peptide-based therapeutics to treat ESKAPE infections might be an alternative to conventional antibiotics. Histatin 5 (Hst 5) is a salivary cationic histidine-rich peptide produced only in humans and higher primates. It has high antifungal activity against Candida albicans through an energy-dependent, non-lytic process; but its bactericidal effects are less known. We found Hst 5 has bactericidal activity against S. aureus (60–70% killing) and A. baumannii (85–90% killing) in 10 and 100 mM sodium phosphate buffer (NaPB), while killing of >99% of P. aeruginosa, 60–80% E. cloacae and 20–60% of E. faecium was found in 10 mM NaPB. Hst 5 killed 60% of biofilm cells of P. aeruginosa, but had reduced activity against biofilms of S. aureus and A. baumannii. Hst 5 killed 20% of K. pneumonia biofilm cells but not planktonic cells. Binding and uptake studies using FITC-labeled Hst 5 showed E. faecium and E. cloacae killing required Hst 5 internalization and was energy dependent, while bactericidal activity was rapid against P. aeruginosa and A. baumannii suggesting membrane disruption. Hst 5-mediated killing of S. aureus was both non-lytic and energy independent. Additionally, we found that spermidine conjugated Hst 5 (Hst5-Spd) had improved killing activity against E. faecium, E. cloacae, and A. baumannii. Hst 5 or its derivative has antibacterial activity against five out of six ESKAPE pathogens and may be an alternative treatment for these infections. PMID:28261570

Human Salivary Protein Histatin 5 Has Potent Bactericidal Activity against ESKAPE Pathogens.

PubMed

Du, Han; Puri, Sumant; McCall, Andrew; Norris, Hannah L; Russo, Thomas; Edgerton, Mira

2017-01-01

ESKAPE ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumanni , Pseudomonas aeruginosa , and Enterobacter species) pathogens have characteristic multiple-drug resistance and cause an increasing number of nosocomial infections worldwide. Peptide-based therapeutics to treat ESKAPE infections might be an alternative to conventional antibiotics. Histatin 5 (Hst 5) is a salivary cationic histidine-rich peptide produced only in humans and higher primates. It has high antifungal activity against Candida albicans through an energy-dependent, non-lytic process; but its bactericidal effects are less known. We found Hst 5 has bactericidal activity against S. aureus (60-70% killing) and A. baumannii (85-90% killing) in 10 and 100 mM sodium phosphate buffer (NaPB), while killing of >99% of P. aeruginosa , 60-80% E. cloacae and 20-60% of E. faecium was found in 10 mM NaPB. Hst 5 killed 60% of biofilm cells of P. aeruginosa , but had reduced activity against biofilms of S. aureus and A. baumannii . Hst 5 killed 20% of K. pneumonia biofilm cells but not planktonic cells. Binding and uptake studies using FITC-labeled Hst 5 showed E. faecium and E. cloacae killing required Hst 5 internalization and was energy dependent, while bactericidal activity was rapid against P. aeruginosa and A. baumannii suggesting membrane disruption. Hst 5-mediated killing of S. aureus was both non-lytic and energy independent. Additionally, we found that spermidine conjugated Hst 5 (Hst5-Spd) had improved killing activity against E. faecium, E. cloacae , and A. baumannii . Hst 5 or its derivative has antibacterial activity against five out of six ESKAPE pathogens and may be an alternative treatment for these infections.

Inhibiting Mitophagy as a Novel Mechanism to Kill Prostate Cancer Cells

DTIC Science & Technology

2014-10-01

cells. Key mediators of the mitophagic process, specifically Parkin , dynamin- related protein-1 (Drp1), fission-1 (Fis1), and cyclophilin-D (CypD...production and was protective against chemotherapeutic-induced cell death. In contrast, Fis1 and Parkin knockdown was sufficient to sensitize LNCaP...targeting of Fis1 and Parkin may have therapeutic value as they both sensitized prostate cancer cells to the necrotic effects of doxorubicin. Finally

4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

PubMed

Tang, Jen-Yang; Huang, Hurng-Wern; Wang, Hui-Ru; Chan, Ya-Ching; Haung, Jo-Wen; Shu, Chih-Wen; Wu, Yang-Chang; Chang, Hsueh-Wei

2018-03-01

Reactive oxygen species (ROS) induction had been previously reported in 4β-hydroxywithanolide (4βHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4βHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4βHWE-treated oral cancer cells than in oral normal cells. All the 4βHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells. © 2017 Wiley Periodicals, Inc.

Immotile Active Matter: Activity from Death and Reproduction.

PubMed

Kalziqi, Arben; Yanni, David; Thomas, Jacob; Ng, Siu Lung; Vivek, Skanda; Hammer, Brian K; Yunker, Peter J

2018-01-05

Unlike equilibrium atomic solids, biofilms-soft solids composed of bacterial cells-do not experience significant thermal fluctuations at the constituent level. However, living cells stochastically reproduce and die, provoking a mechanical response. We investigate the mechanical consequences of cellular death and reproduction by measuring surface-height fluctuations of biofilms containing two mutually antagonistic strains of Vibrio cholerae that kill one another on contact via the type VI secretion system. While studies of active matter typically focus on activity via constituent mobility, here, activity is mediated by reproduction and death events in otherwise immobilized cells. Biofilm surface topography is measured in the nearly homeostatic limit via white light interferometry. Although biofilms are far from equilibrium systems, measured surface-height fluctuation spectra resemble the spectra of thermal permeable membranes but with an activity-mediated effective temperature, as predicted by Risler, Peilloux, and Prost [Phys. Rev. Lett. 115, 258104 (2015)PRLTAO0031-900710.1103/PhysRevLett.115.258104]. By comparing the activity of killer strains of V. cholerae with that of genetically modified strains that cannot kill each other and validating with individual-based simulations, we demonstrate that extracted effective temperatures increase with the amount of death and reproduction and that death and reproduction can fluidize biofilms. Together, these observations demonstrate the unique physical consequences of activity mediated by death and reproduction events.

Mechanisms of lectin and antibody-dependent polymorphonuclear leukocyte-mediated cytolysis.

PubMed

Tsunawaki, S; Ikenami, M; Mizuno, D; Yamazaki, M

1983-04-01

The mechanisms of tumor lysis by polymorphonuclear leukocytes (PMNs) were investigated. In antibody-dependent PMN-mediated cytolysis (ADPC), sensitized tumor cells were specifically lysed via Fc receptors on PMNs. On the other hand, lectin-dependent PMN-mediated cytolysis (LDPC) caused nonspecific lysis of several murine tumors after recognition of carbohydrate moieties on the cell membrane of both PMNs and tumor cells. Both ADPC and LDPC depended on glycolysis, and cytotoxicity was mediated by reactive oxygen species; LDPC was dependent on superoxide and ADPC on the myeloperoxidase system. The participation of reactive oxygen species in PMN cytotoxicity was also demonstrated by pharmacological triggering with phorbol myristate acetate. These results indicate that reactive oxygen species have an important role In tumor killing by PMNs and that ADPC and LDPC have partly different cytolytic processes as well as different recognition steps.

Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease.

PubMed

Jochmans, Dirk; Anders, Maria; Keuleers, Inge; Smeulders, Liesbeth; Kräusslich, Hans-Georg; Kraus, Günter; Müller, Barbara

2010-10-15

Current antiretroviral therapy against human immunodeficiency virus (HIV-1) reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR) related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs) can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM). Specific cytotoxicity was reverted by addition of PR inhibitors. Two of the most active compounds, VRX-480773 and GW-678248, were also tested in primary human cells and mediated cytotoxicity on HIV-1 infected peripheral blood mononuclear cells. These data present proof of concept for targeted drug induced elimination of HIV producing cells. While NNRTIs themselves may not be sufficiently potent for therapeutic application, the results provide a basis for the development of drugs exploiting this mechanism of action.

Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease

PubMed Central

2010-01-01

Background Current antiretroviral therapy against human immunodeficiency virus (HIV-1) reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR) related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs) can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Results Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM). Specific cytotoxicity was reverted by addition of PR inhibitors. Two of the most active compounds, VRX-480773 and GW-678248, were also tested in primary human cells and mediated cytotoxicity on HIV-1 infected peripheral blood mononuclear cells. Conclusion These data present proof of concept for targeted drug induced elimination of HIV producing cells. While NNRTIs themselves may not be sufficiently potent for therapeutic application, the results provide a basis for the development of drugs exploiting this mechanism of action. PMID:20950436

Engineered T cells for pancreatic cancer treatment

PubMed Central

Katari, Usha L; Keirnan, Jacqueline M; Worth, Anna C; Hodges, Sally E; Leen, Ann M; Fisher, William E; Vera, Juan F

2011-01-01

Objective Conventional chemotherapy and radiotherapy produce marginal survival benefits in pancreatic cancer, underscoring the need for novel therapies. The aim of this study is to develop an adoptive T cell transfer approach to target tumours expressing prostate stem cell antigen (PSCA), a tumour-associated antigen that is frequently expressed by pancreatic cancer cells. Methods Expression of PSCA on cell lines and primary tumour samples was confirmed by immunohistochemistry. Healthy donor- and patient-derived T cells were isolated, activated in vitro using CD3/CD28, and transduced with a retroviral vector encoding a chimeric antigen receptor (CAR) targeting PSCA. The ability of these cells to kill tumour cells was analysed by chromium-51 (Cr51) release. Results Prostate stem cell antigen was expressed on >70% of the primary tumour samples screened. Activated, CAR-modified T cells could be readily generated in clinically relevant numbers and were specifically able to kill PSCA-expressing pancreatic cancer cell lines with no non-specific killing of PSCA-negative target cells, thus indicating the potential efficacy and safety of this approach. Conclusions Prostate stem cell antigen is frequently expressed on pancreatic cancer cells and can be targeted for immune-mediated destruction using CAR-modified, adoptively transferred T cells. The safety and efficacy of this approach indicate that it deserves further study and may represent a promising novel treatment for patients with pancreatic cancer. PMID:21843265

Tailoring CD19xCD3-DART exposure enhances T-cells to eradication of B-cell neoplasms.

PubMed

Circosta, Paola; Elia, Angela Rita; Landra, Indira; Machiorlatti, Rodolfo; Todaro, Maria; Aliberti, Sabrina; Brusa, Davide; Deaglio, Silvia; Chiaretti, Sabina; Bruna, Riccardo; Gottardi, Daniela; Massaia, Massimo; Giacomo, Filomena Di; Guarini, Anna Rita; Foà, Robin; Kyriakides, Peter W; Bareja, Rohan; Elemento, Olivier; Chichili, Gurunadh R; Monteleone, Emanuele; Moore, Paul A; Johnson, Syd; Bonvini, Ezio; Cignetti, Alessandro; Inghirami, Giorgio

2018-01-01

Many patients with B-cell malignancies can be successfully treated, although tumor eradication is rarely achieved. T-cell-directed killing of tumor cells using engineered T-cells or bispecific antibodies is a promising approach for the treatment of hematologic malignancies. We investigated the efficacy of CD19xCD3 DART bispecific antibody in a broad panel of human primary B-cell malignancies. The CD19xCD3 DART identified 2 distinct subsets of patients, in which the neoplastic lymphocytes were eliminated with rapid or slow kinetics. Delayed responses were always overcome by a prolonged or repeated DART exposure. Both CD4 and CD8 effector cytotoxic cells were generated, and DART-mediated killing of CD4 + cells into cytotoxic effectors required the presence of CD8 + cells. Serial exposures to DART led to the exponential expansion of CD4 + and CD8 + cells and to the sequential ablation of neoplastic cells in absence of a PD-L1-mediated exhaustion. Lastly, patient-derived neoplastic B-cells (B-Acute Lymphoblast Leukemia and Diffuse Large B Cell Lymphoma) could be proficiently eradicated in a xenograft mouse model by DART-armed cytokine induced killer (CIK) cells. Collectively, patient tailored DART exposures can result in the effective elimination of CD19 positive leukemia and B-cell lymphoma and the association of bispecific antibodies with unmatched CIK cells represents an effective modality for the treatment of CD19 positive leukemia/lymphoma.

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone, a Triterpene Isolated from the Mushroom Astraeus hygrometricus, Involves the Induction of Protective Cell-Mediated Immunity and TLR9

PubMed Central

Mallick, Suvadip; Dutta, Aritri; Chaudhuri, Ankur; Mukherjee, Debasri; Dey, Somaditya; Halder, Subhadra; Ghosh, Joydip; Mukherjee, Debarati; Sultana, Sirin Salma; Biswas, Gunjan; Lai, Tapan Kumar; Patra, Pradyumna; Sarkar, Indranil; Chakraborty, Sibani; Saha, Bhaskar; Acharya, Krishnendu

2016-01-01

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 μg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo. Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo. PMID:26883702

Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway

PubMed Central

Ardiani, Andressa; Gameiro, Sofia R.; Kwilas, Anna R.; Donahue, Renee N.; Hodge, James W.

2014-01-01

Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and hormone-deprivation therapies is the major cause of death in patients with castration-resistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Here we show for the first time that both enzalutamide and abiraterone render prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT. PMID:25344864

Potassium Iodide Potentiates Broad-Spectrum Antimicrobial Photodynamic Inactivation Using Photofrin.

PubMed

Huang, Liyi; Szewczyk, Grzegorz; Sarna, Tadeusz; Hamblin, Michael R

2017-04-14

It is known that noncationic porphyrins such as Photofrin (PF) are effective in mediating antimicrobial photodynamic inactivation (aPDI) of Gram-positive bacteria or fungi. However, the aPDI activity of PF against Gram-negative bacteria is accepted to be extremely low. Here we report that the nontoxic inorganic salt potassium iodide (KI) at a concentration of 100 mM when added to microbial cells (10 8 /mL) + PF (10 μM hematoporphyrin equivalent) + 415 nm light (10 J/cm 2 ) can eradicate (>6 log killing) five different Gram-negative species (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Acinetobacter baumannii), whereas no killing was obtained without KI. The mechanism of action appears to be the generation of microbicidal molecular iodine (I 2 /I 3 - ) as shown by comparable bacterial killing when cells were added to the mixture after completion of illumination and light-dependent generation of iodine as detected by the formation of the starch complex. Gram-positive methicillin-resistant Staphylococcus aureus is much more sensitive to aPDI (200-500 nM PF), and in this case potentiation by KI may be mediated mainly by short-lived iodine reactive species. The fungal yeast Candida albicans displayed intermediate sensitivity to PF-aPDI, and killing was also potentiated by KI. The reaction mechanism occurs via singlet oxygen ( 1 O 2 ). KI quenched 1 O 2 luminescence (1270 nm) at a rate constant of 9.2 × 10 5 M -1 s -1 . Oxygen consumption was increased when PF was illuminated in the presence of KI. Hydrogen peroxide but not superoxide was generated from illuminated PF in the presence of KI. Sodium azide completely inhibited the killing of E. coli with PF/blue light + KI.

Tumor necrosis factor alpha mediates resistance to Trypanosoma cruzi infection in mice by inducing nitric oxide production in infected gamma interferon-activated macrophages.

PubMed Central

Silva, J S; Vespa, G N; Cardoso, M A; Aliberti, J C; Cunha, F Q

1995-01-01

Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for continuation of the parasite life cycle and for production of Chagas' disease. T. cruzi is able to replicate in nucleated cells and can be killed by activated macrophages. Gamma interferon (IFN-gamma) is one of the major stimuli for the activation of macrophages and has been shown to be a key activation factor for the killing of intracellular parasites through a mechanism dependent upon nitric oxide (NO) biosynthesis. We show that although the addition of exogenous tumor necrosis factor alpha (TNF-alpha) does not potentiate the trypanocidal activity of IFN-gamma in vitro, treatment of resistant C57BI/6 mice with an anti-TNF-alpha monoclonal antibody increased parasitemia and mortality. In addition, the anti-TNF-alpha-treated animals had decreased NO production, both in vivo and in vitro, suggesting an important role for TNF-alpha in controlling infection. In order to better understand the role of TNF-alpha in the macrophage-mediating killing of parasites, cultures of T. cruzi-infected macrophages were treated with an anti-TNF-alpha monoclonal antibody. IFN-gamma-activated macrophages failed to kill intracellular parasites following treatment with 100 micrograms of anti-TNF-alpha. In these cultures, the number of parasites released at various time points after infection was significantly increased while NO production was significantly reduced. We conclude that IFN-gamma-activated macrophages produce TNF-alpha after infection by T. cruzi and suggest that this cytokine plays a role in amplifying NO production and parasite killing. PMID:7591147

Cell-mediated immunity in herpes simplex virus-infected mice: functional analysis of lymph node cells during periods of acute and latent infection, with reference to cytotoxic and memory cells.

PubMed

Nash, A A; Quartey-Papafio, R; Wildy, P

1980-08-01

The functional characteristics of lymphoid cells were investigated during acute and latent infection of mice with herpes simplex virus (HSV). Cytotoxic T cells were found in the draining lymph node (DLN) 4 days p.i. and had reached maximum activity between 6 and 9 days. After the 12th day and during the period of latent infection (> 20 days) no cytotoxic cell activity was observed. Cytotoxic activity could only be detected when the lymphoid cells had been cultured for a period of 3 days. In general, the cell killing was specific for syngeneic infected target cells, although some killing of uninfected targets was observed. In contrast to the cytotoxic response, DLN cells responding to HSV in a proliferation assay were detected towards the end of the acute phase and at lease up to 9 months thereafter. The significance of these observations for the pathogenesis of HSV is discussed.

Trichinella spiralis: killing of newborn larvae by lung cells.

PubMed

Falduto, Guido H; Vila, Cecilia C; Saracino, María P; Calcagno, Marcela A; Venturiello, Stella M

2015-02-01

The migratory stage of Trichinella spiralis, the newborn larva (NBL), travels along the pulmonary microvascular system on its way to the skeletal muscle cells. The present work studies the capability of lung cells to kill NBL. For this purpose, in vitro cytotoxicity assays were performed using NBL, lung cell suspensions from Wistar rats, rat anti-NBL surface sera, and fresh serum as complement source. The cytotoxic activity of lung cells from rats infected on day 6 p.i. was compared with that from noninfected rats. Two and 20 h-old NBL (NBL2 and NBL20) were used as they had shown to exhibit different surface antigens altering their biological activity. Sera antibodies were analyzed by indirect immunofluorescence assay, and cell populations used in each assay were characterized by histological staining. The role of IgE in the cytotoxic attack against NBL was analyzed using heated serum. The FcεRI expression on cell suspensions was examined by flow cytometry. Results showed that lung cells were capable of killing NBL by antibody-dependent cell-mediated cytotoxicity (ADCC). Lung cells from infected animals yielded the highest mortality percentages of NBL, with NBL20 being the most susceptible to such attack. IgE yielded a critical role in the cytotoxic attack. Regarding the analysis of cell suspensions, cells from infected rats showed an increase in the percentage of eosinophils, neutrophils, and the number of cells expressing the FcεRI receptor. We conclude that lung cells are capable of killing NBL in the presence of specific antibodies, supporting the idea that the lung is one of the sites where the NBL death occurs due to ADCC.

IFN-γ Stimulated Human Umbilical-Tissue-Derived Cells Potently Suppress NK Activation and Resist NK-Mediated Cytotoxicity In Vitro

PubMed Central

Noone, Cariosa; Kihm, Anthony; O'Dea, Shirley; Mahon, Bernard P.

2013-01-01

Umbilical cord tissue represents a unique source of cells with potential for cell therapy applications for multiple diseases. Human umbilical tissue-derived cells (hUTC) are a developmentally early stage, homogenous population of cells that are HLA-ABC dim, HLA-DR negative, and lack expression of co-stimulatory molecules in the unactivated state. The lack of HLA-DR and co-stimulatory molecule expression on unactivated hUTC may account for their reduced immunogenicity, facilitating their use in allogeneic settings. However, such approaches could be confounded by host innate cells such as natural killer (NK) cells. Here, we evaluate in vitro NK cell interactions with hUTC and compare them with human mesenchymal stem cells (MSC). Our investigations show that hUTC suppress NK activation, through prostaglandin-E2 secretion in a contact-independent manner. Prestimulation of hUTC or human MSC with interferon gamma (IFN-γ) induced expression of the tryptophan degrading enzyme indoleamine 2, 3 dioxygenase, facilitating enhanced suppression. However, resting NK cells of different killer immunoglobulin-like receptor haplotypes did not kill hUTC or MSC; only activated NK cells had the ability to kill nonstimulated hUTC and, to a lesser extent, MSC. The cell killing process involved signaling through the NKG2D receptor and the perforin/granzyme pathway; this was supported by CD54 (ICAM-1) expression by hUTC. IFN-γ-stimulated hUTC or hMSC were less susceptible to NK killing; in this case, protection was associated with elevated HLA-ABC expression. These data delineate the different mechanisms in a two-way interaction between NK cells and two distinct cell therapies, hUTC or hMSC, and how these interactions may influence their clinical applications. PMID:23795941

Cisplatin or LA-12 enhance killing effects of TRAIL in prostate cancer cells through Bid-dependent stimulation of mitochondrial apoptotic pathway but not caspase-10

PubMed Central

Herůdková, Jarmila; Krkoška, Martin; Tománková, Silvie; Kahounová, Zuzana; Anděra, Ladislav; Bouchal, Jan; Kharaishvili, Gvantsa; Král, Milan; Sova, Petr; Kozubík, Alois

2017-01-01

Searching for new strategies for effective elimination of human prostate cancer cells, we investigated the cooperative cytotoxic action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and two platinum-based complexes, cisplatin or LA-12, and related molecular mechanisms. We demonstrated a notable ability of cisplatin or LA-12 to enhance the sensitivity of several human prostate cancer cell lines to TRAIL-induced cell death via an engagement of mitochondrial apoptotic pathway. This was accompanied by augmented Bid cleavage, Bak activation, loss of mitochondrial membrane potential, activation of caspase-8, -10, -9, and -3, and XIAP cleavage. RNAi-mediated silencing of Bid or Bak in Bax-deficient DU 145 cells suppressed the drug combination-induced cytotoxicity, further underscoring the involvement of mitochondrial signaling. The caspase-10 was dispensable for enhancement of cisplatin/LA-12 and TRAIL combination-induced cell death and stimulation of Bid cleavage. Importantly, we newly demonstrated LA-12-mediated enhancement of TRAIL-induced cell death in cancer cells derived from human patient prostate tumor specimens. Our results provide convincing evidence that employing TRAIL combined with cisplatin/LA-12 could contribute to more effective killing of prostate cancer cells compared to the individual action of the drugs, and offer new mechanistic insights into their cooperative anticancer action. PMID:29182622

Pathogen response-like recruitment and activation of neutrophils by sterile immunogenic dying cells drives neutrophil-mediated residual cell killing

PubMed Central

Garg, Abhishek D; Vandenberk, Lien; Fang, Shentong; Fasche, Tekele; Van Eygen, Sofie; Maes, Jan; Van Woensel, Matthias; Koks, Carolien; Vanthillo, Niels; Graf, Norbert; de Witte, Peter; Van Gool, Stefaan; Salven, Petri; Agostinis, Patrizia

2017-01-01

Innate immune sensing of dying cells is modulated by several signals. Inflammatory chemokines-guided early recruitment, and pathogen-associated molecular patterns-triggered activation, of major anti-pathogenic innate immune cells like neutrophils distinguishes pathogen-infected stressed/dying cells from sterile dying cells. However, whether certain sterile dying cells stimulate innate immunity by partially mimicking pathogen response-like recruitment/activation of neutrophils remains poorly understood. We reveal that sterile immunogenic dying cancer cells trigger (a cell autonomous) pathogen response-like chemokine (PARC) signature, hallmarked by co-release of CXCL1, CCL2 and CXCL10 (similar to cells infected with bacteria or viruses). This PARC signature recruits preferentially neutrophils as first innate immune responders in vivo (in a cross-species, evolutionarily conserved manner; in mice and zebrafish). Furthermore, key danger signals emanating from these dying cells, that is, surface calreticulin, ATP and nucleic acids stimulate phagocytosis, purinergic receptors and toll-like receptors (TLR) i.e. TLR7/8/9-MyD88 signaling on neutrophil level, respectively. Engagement of purinergic receptors and TLR7/8/9-MyD88 signaling evokes neutrophil activation, which culminates into H2O2 and NO-driven respiratory burst-mediated killing of viable residual cancer cells. Thus sterile immunogenic dying cells perform 'altered-self mimicry' in certain contexts to exploit neutrophils for phagocytic targeting of dead/dying cancer cells and cytotoxic targeting of residual cancer cells. PMID:28234357

Anti-leukemia activity of a bacterial toxin with natural specificity for LFA-1 on white blood cells

PubMed Central

Kachlany, Scott C.; Schwartz, Amy B.; Balashova, Nataliya V.; Hioe, Catarina E.; Tuen, Michael; Le, Amy; Kaur, Manpreet; Mei, Yongyi; Rao, Jia

2009-01-01

The oral bacterium, Aggregatibacter actinomycetemcomitans, produces a leukotoxin (LtxA) that is specific for white blood cells (WBCs) from humans and Old World primates by interacting with lymphocyte function antigen-1 (LFA-1) on susceptible cells. To determine if LtxA could be used as a therapeutic agent for the treatment of WBC diseases, we tested the in vitro and in vivo anti-leukemia activity of the toxin. LtxA kills human malignant WBC lines and primary leukemia cells from acute myeloid leukemia patients, but healthy peripheral blood mononuclear cells (PBMCs) are relatively resistant to LtxA-mediated cytotoxicity. Levels of LFA-1 on cell lines correlated with killing by LtxA and the toxin preferentially killed cells expressing the activated form of LFA-1. In a SCID mouse model for human leukemia, LtxA had potent therapeutic value resulting in long-term survival in LtxA-treated mice. Intravenous infusion of LtxA into a rhesus macaque resulted in a drop in WBC counts at early times post-infusion; however, red blood cells, platelets, hemoglobin and blood chemistry values remained unaffected. Thus, LtxA may be an effective and safe novel therapeutic agent for the treatment of hematologic malignancies. PMID:19747730

T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells

PubMed Central

Witkover, Aviva; Tanaka, Yuetsu; Fields, Paul; Bangham, Charles R. M.

2016-01-01

There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease. PMID:27893842

Sodium ascorbate kills Candida albicans in vitro via iron-catalyzed Fenton reaction: importance of oxygenation and metabolism

PubMed Central

Avci, Pinar; Freire, Fernanda; Banvolgyi, Andras; Mylonakis, Eleftherios; Wikonkal, Norbert M; Hamblin, Michael R

2016-01-01

Aim: Ascorbate can inhibit growth and even decrease viability of various microbial species including Candida albicans. However the optimum conditions and the mechanism of action are unclear. Materials/methodology: Candida albicans shaken for 90 min in a buffered solution of ascorbate (90 mM) gave a 5-log reduction of cell viability, while there was no killing without shaking, in growth media with different carbon sources or at 4°C. Killing was inhibited by the iron chelator 2,2′-bipyridyl. Hydroxyphenyl fluorescein probe showed the intracellular generation of hydroxyl radicals. Results/conclusion: Ascorbate-mediated killing of C. albicans depends on oxygenation and metabolism, involves iron-catalyzed generation of hydroxyl radicals via Fenton reaction and depletion of intracellular NADH. Ascorbate could serve as a component of a topical antifungal therapy. PMID:27855492

Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies.

PubMed

Friedman, Jay; Morisada, Megan; Sun, Lillian; Moore, Ellen C; Padget, Michelle; Hodge, James W; Schlom, Jeffrey; Gameiro, Sofia R; Allen, Clint T

2018-06-21

Natural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined. Tumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed. Cultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab. Tumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.

A synthetic lethal screen identifies ATR-inhibition as a novel therapeutic approach for POLD1-deficient cancers

PubMed Central

Hocke, Sandra; Guo, Yang; Job, Albert; Orth, Michael; Ziesch, Andreas; Lauber, Kirsten; De Toni, Enrico N; Gress, Thomas M.; Herbst, Andreas; Göke, Burkhard; Gallmeier, Eike

2016-01-01

The phosphoinositide 3-kinase-related kinase ATR represents a central checkpoint regulator and mediator of DNA-repair. Its inhibition selectively eliminates certain subsets of cancer cells in various tumor types, but the underlying genetic determinants remain enigmatic. Here, we applied a synthetic lethal screen directed against 288 DNA-repair genes using the well-defined ATR knock-in model of DLD1 colorectal cancer cells to identify potential DNA-repair defects mediating these effects. We identified a set of DNA-repair proteins, whose knockdown selectively killed ATR-deficient cancer cells. From this set, we further investigated the profound synthetic lethal interaction between ATR and POLD1. ATR-dependent POLD1 knockdown-induced cell killing was reproducible pharmacologically in POLD1-depleted DLD1 cells and a panel of other colorectal cancer cell lines by using chemical inhibitors of ATR or its major effector kinase CHK1. Mechanistically, POLD1 depletion in ATR-deficient cells caused caspase-dependent apoptosis without preceding cell cycle arrest and increased DNA-damage along with impaired DNA-repair. Our data could have clinical implications regarding tumor genotype-based cancer therapy, as inactivating POLD1 mutations have recently been identified in small subsets of colorectal and endometrial cancers. POLD1 deficiency might thus represent a predictive marker for treatment response towards ATR- or CHK1-inhibitors that are currently tested in clinical trials. PMID:26755646

Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12.

PubMed

Homma, Sadamu; Komita, Hideo; Sagawa, Yukiko; Ohno, Tsuneya; Toda, Gotaro

2005-08-01

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)-12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL-12 alone did not show a tumour-suppressive effect. Antitumour activity induced by DC/BNL + IL-12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL-treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)-gamma. Furthermore, CD4+ T cells killed syngeneic-irrelevant CT26 cells and even allogeneic Hepa1-6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti-Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II-positive macrophages, but not CD8(+) T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL-12. Flow cytometric analysis of tumour-infiltrating cells in mice treated with DC/BNL + IL-12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL-bearing mice treated with DC/BNL + IL-12, tumour macrophages activated by INF-gamma produced by IL-12-stimulated T cells might present BNL tumour antigens and activate DC/BNL-primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II-dependent manner, leading to perforin-mediated bystander killing of neighbouring MHC class II-negative tumour cells.

Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12

PubMed Central

Homma, Sadamu; Komita, Hideo; Sagawa, Yukiko; Ohno, Tsuneya; Toda, Gotaro

2005-01-01

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)-12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL-12 alone did not show a tumour-suppressive effect. Antitumour activity induced by DC/BNL + IL-12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL-treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)-γ. Furthermore, CD4+ T cells killed syngeneic-irrelevant CT26 cells and even allogeneic Hepa1-6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti-Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II-positive macrophages, but not CD8+ T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL-12. Flow cytometric analysis of tumour-infiltrating cells in mice treated with DC/BNL + IL-12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL-bearing mice treated with DC/BNL + IL-12, tumour macrophages activated by INF-γ produced by IL-12-stimulated T cells might present BNL tumour antigens and activate DC/BNL-primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II-dependent manner, leading to perforin-mediated bystander killing of neighbouring MHC class II-negative tumour cells. PMID:16011514

PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing.

PubMed

Akhmetzyanova, Ilseyar; Drabczyk, Malgorzata; Neff, C Preston; Gibbert, Kathrin; Dietze, Kirsten K; Werner, Tanja; Liu, Jia; Chen, Lieping; Lang, Karl S; Palmer, Brent E; Dittmer, Ulf; Zelinskyy, Gennadiy

2015-10-01

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.

Intracellular Copper Does Not Catalyze the Formation of Oxidative DNA Damage in Escherichia coli▿

PubMed Central

Macomber, Lee; Rensing, Christopher; Imlay, James A.

2007-01-01

Because copper catalyzes the conversion of H2O2 to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H2O2 than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H2O2 damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H2O2 resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H2O2-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while E. coli responds to H2O2 stress by inducing iron sequestration proteins, H2O2-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism. PMID:17189367

The PCC assay can be used to predict radiosensitivity in biopsy cultures irradiated with different types of radiation.

PubMed

Suzuki, Masao; Tsuruoka, Chizuru; Nakano, Takashi; Ohno, Tatsuya; Furusawa, Yoshiya; Okayasu, Ryuichi

2006-12-01

The aim of this study was to identify potential biomarkers for radiosensitivity using the relationship between cell killing and the yield of excess chromatin fragments detected with the premature chromosome condensation (PCC) technique. This method was applied to primary cultured cells obtained from biopsies from patients. Six primary culture biopsies were obtained from 6 patients with carcinoma of the cervix before starting radiotherapy. The cultures were irradiated with two different LET carbon-ion beams (LET = 13 keV/microm, 77.1+/-2.8 keV/microm) and 200 kV X-rays. The carbon-ion beams were produced by Heavy Ion Medical Accelerator in Chiba (HIMAC). PCC was performed using the polyethylene glycol-mediated cell fusion technique. The yield of excess chromatin fragments were measured by counting the number of unrejoined chromatin fragments detected with the PCC technique after a 24-h post-irradiation incubation period. Obtained results indicated that cultures which were more sensitive to killing were also more susceptible to the induction of excess chromatin fragments. Furthermore there was a good correlation between cell killing and excess chromatin fragments among the 6 cell cultures examined. There is also evidence that the induction of excess chromatin fragments increased with increasing LET as well as cell-killing effect in the same cell culture. The data reported here support the idea that the yield of excess chromatin fragments detected with the PCC technique might be useful for predicting the radiosensitivity of cells contained in tumor tissue, and to predict responses to different radiation types.

Enhancing effects of gamma interferon on phagocytic cell association with and killing of Trypanosoma cruzi

NASA Technical Reports Server (NTRS)

Wirth, J. J.; Kierszenbaum, F.; Sonnenfeld, G.; Zlotnik, A.

1985-01-01

Results are reported from a study of the influence gamma interferon (GIFN) and interleukin 2 (IL2) have on the capability of P388D1 cells and mouse resident peritoneal macrophages (MPM) to attach to the blood-resident parasites Trypanosoma cruzi and kill them. Cultures of trypomastigote forms of the Tulahuen strain of T. cruzi grown in bovine serum were introduced into peritoneal cells of mice, along with P388D1 cells incubated with GIFN, IL2 and both. Control cells were also maintained. Statistical analysis were then performed on data on counts of the number of dead T. Cruzi cells. The GIFN enhanced the interaction of MPM and P388D1 cells with the surface of T. Cruzi, provided the interaction was given over 12 hr to take place. A depression of the cytotoxicity of P388D1 cells was attributed to mediation by H2O2, an effect partially offset by incubation with the lymphokine GIFN.

Antineoplastic And Antiviral Properties Of Merocyanine 540

NASA Astrophysics Data System (ADS)

Sieber, Fritz

1989-03-01

Simultaneous exposure to the lipophilic photosensitizer, merocyanine 540, and light in the presence of serum (or certain serum components) and oxygen kills leukemia cells, lymphoma cells, neuroblastoma cells, cell-free enveloped viruses, cell-associated enveloped viruses, and virus-infected cells. The same treatment spares pluripotent hematopoietic stem cells, mature erythrocytes, factor VIII, von Willebrand factor, and probably other blood components. Merocyanine 540-mediated photosensitization is now being evaluated clinically as a means to eliminate residual tumor cells from autologous remission bone marrow grafts and preclinically as a means to inactivate pathogenic viruses in blood products.

The natural dietary genistein boosts bacteriophage-mediated cancer cell killing by improving phage-targeted tumor cell transduction.

PubMed

Tsafa, Effrosyni; Al-Bahrani, Mariam; Bentayebi, Kaoutar; Przystal, Justyna; Suwan, Keittisak; Hajitou, Amin

2016-08-09

Gene therapy has long been regarded as a promising treatment for cancer. However, cancer gene therapy is still facing the challenge of targeting gene delivery vectors specifically to tumors when administered via clinically acceptable non-invasive systemic routes (i.e. intravenous). The bacteria virus, bacteriophage (phage), represents a new generation of promising vectors in systemic gene delivery since their targeting can be achieved through phage capsid display ligands, which enable them to home to specific tumor receptors without the need to ablate any native eukaryotic tropism. We have previously reported a tumor specific bacteriophage vector named adeno-associated virus/phage, or AAVP, in which gene expression is under a recombinant human rAAV2 virus genome targeted to tumors via a ligand-directed phage capsid. However, cancer gene therapy with this tumor-targeted vector achieved variable outcomes ranging from tumor regression to no effect in both experimental and natural preclinical models. Herein, we hypothesized that combining the natural dietary genistein, with proven anticancer activity, would improve bacteriophage anticancer safe therapy. We show that combination treatment with genistein and AAVP increased targeted cancer cell killing by AAVP carrying the gene for Herpes simplex virus thymidine kinase (HSVtk) in 2D tissue cultures and 3D tumor spheroids. We found this increased tumor cell killing was associated with enhanced AAVP-mediated gene expression. Next, we established that genistein protects AAVP against proteasome degradation and enhances vector genome accumulation in the nucleus. Combination of genistein and phage-guided virotherapy is a safe and promising strategy that should be considered in anticancer therapy with AAVP.

Mansouramycin C kills cancer cells through reactive oxygen species production mediated by opening of mitochondrial permeability transition pore

PubMed Central

Kuang, Shan; Liu, Ge; Cao, Ruobing; Zhang, Linlin; Yu, Qiang; Sun, Chaomin

2017-01-01

Cancer is one of the deadliest diseases in the world and the search for novel anticancer agents is urgently required. Marine-derived isoquinolinequinones have exhibited promising anticancer activities. However, the exact mechanisms of cytotoxic activities of these isoquinolinequinones are poorly characterized. In this study, we investigated the anticancer effects and molecular mechanisms of mansouramycin C (Mm C), a cytotoxic isoquinolinequinone isolated from a marine streptomycete. We demonstrated that Mm C preferentially killed cancer cells and the cytotoxic effects were mediated by reactive oxygen species (ROS) generation. Mass spectrometry based proteomic analysis of Mm C-treated A549 cells revealed that many ROS-related proteins were differentially expressed. Proteomic-profiling after Mm C treatment identified oxidative phosphorylation as the most significant changes in pathways. Analysis also revealed extensive defects in mitochondrial structure and function. Furthermore, we disclosed that Mm C-induced ROS generation was caused by opening of mitochondrial permeability transition pore. Notably, Mm C synergized with sorafenib to induce cell death in A549 cells. Hence, we propose that the marine-derived natural compound Mm C is a potent inducer of the mitochondrial permeability transition and a promising anticancer drug candidate. Moreover, molecular mechanisms of Mm C shed new light on the understanding of the cytotoxic mechanisms of marine-derived isoquinolinequiones. PMID:29262621

TNF-induced target cell killing by CTL activated through cross-presentation.

PubMed

Wohlleber, Dirk; Kashkar, Hamid; Gärtner, Katja; Frings, Marianne K; Odenthal, Margarete; Hegenbarth, Silke; Börner, Carolin; Arnold, Bernd; Hämmerling, Günter; Nieswandt, Bernd; van Rooijen, Nico; Limmer, Andreas; Cederbrant, Karin; Heikenwalder, Mathias; Pasparakis, Manolis; Protzer, Ulrike; Dienes, Hans-Peter; Kurts, Christian; Krönke, Martin; Knolle, Percy A

2012-09-27

Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Hyperglycaemia does not affect antigen-specific activation and cytolytic killing by CD8+ T cells in vivo.

PubMed

Recino, Asha; Barkan, Kerry; Wong, F Susan; Ladds, Graham; Cooke, Anne; Wallberg, Maja

2017-08-31

Metabolism is of central importance for T cell survival and differentiation. It is well known that T cells cannot function in the absence of glucose, but it is less clear how they respond to excessive levels of glucose. In the present study, we investigated how increasing levels of glucose affect T-cell-mediated immune responses. We examined the effects of increased levels of glucose on CD8 + T-cell behaviour in vitro by assessing activation and cytokine production, as well as oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and intracellular signalling. In addition, we assessed in vivo proliferation, cytokine production and cytolytic activity of cells in chemically induced diabetic C57BL/6 mice. Elevated levels of glucose in in vitro cultures had modest effects on proliferation and cytokine production, while in vivo hyperglycaemia had no effect on CD8 + T-cell proliferation, interferon γ (IFNγ) production or cytolytic killing. © 2017 The Author(s).

Internalization of a C17α-alkynylestradiol-porphyrin conjugate into estrogen receptor positive MCF-7 breast cancer cells.

PubMed

Sadler, Sara; Persons, Kelly S; Jones, Graham B; Ray, Rahul

2011-08-01

We hypothesized that expression of nuclear estrogen receptor (ER) in hormone-sensitive breast cancer cells could be harnessed synergistically with the tumor-accumulating effect of porphyrins to selectively deliver estrogen-porphyrin conjugates into breast tumor cells, and preferentially kill tumor cells upon exposure to visible light. In this study we synthesized a conjugate of C(17α)-alkynylestradiol and pyropheophorbide and demonstrated that this conjugate is internalized by ER-positive MCF-7 cells while pyropheophorbide did not, suggesting an ER-mediated uptake and internalization of the conjugate by incipient nuclear ER in MCF-7 cells. This study is a direct demonstration of our hypothesis about ER-mediated internalization of estrogen-porphyrin conjugates. Copyright © 2011. Published by Elsevier Ltd.

Low doses of killed parasite in CpG elicit vigorous CD4+ T cell responses against blood-stage malaria in mice

PubMed Central

Pinzon-Charry, Alberto; McPhun, Virginia; Kienzle, Vivian; Hirunpetcharat, Chakrit; Engwerda, Christian; McCarthy, James; Good, Michael F.

2010-01-01

Development of a vaccine that targets blood-stage malaria parasites is imperative if we are to sustainably reduce the morbidity and mortality caused by this infection. Such a vaccine should elicit long-lasting immune responses against conserved determinants in the parasite population. Most blood-stage vaccines, however, induce protective antibodies against surface antigens, which tend to be polymorphic. Cell-mediated responses, on the other hand, offer the theoretical advantage of targeting internal antigens that are more likely to be conserved. Nonetheless, few of the current blood-stage vaccine candidates are able to harness vigorous T cell immunity. Here, we present what we believe to be a novel blood-stage whole-organism vaccine that, by combining low doses of killed parasite with CpG-oligodeoxynucleotide (CpG-ODN) adjuvant, was able to elicit strong and cross-reactive T cell responses in mice. Our data demonstrate that immunization of mice with 1,000 killed parasites in CpG-ODN engendered durable and cross-strain protection by inducing a vigorous response that was dependent on CD4+ T cells, IFN-γ, and nitric oxide. If applicable to humans, this approach should facilitate the generation of robust, cross-reactive T cell responses against malaria as well as antigen availability for vaccine manufacture. PMID:20628205

5-Benzylglycinyl-Amiloride Kills Proliferating and Nonproliferating Malignant Glioma Cells through Caspase-Independent Necroptosis Mediated by Apoptosis-Inducing Factor

PubMed Central

Pasupuleti, Nagarekha; Leon, Leonardo; Carraway, Kermit L.

2013-01-01

5′–Βenzylglycinyl-amiloride (UCD38B) and glycinyl-amiloride (UCD74A) are cell-permeant and cell-impermeant derivatives of amiloride, respectively, and used here to identify the cellular mechanisms of action underlying their antiglioma effects. UCD38B comparably kills proliferating and nonproliferating gliomas cells when cell cycle progression is arrested either by cyclin D1 siRNA or by acidification. Cell impermeant UCD74A inhibits plasmalemmal urokinase plasminogen activator (uPA) and the type 1 sodium-proton exchanger with potencies analogous to UCD38B, but is cytostatic. In contrast, UCD38B targets intracellular uPA causing mistrafficking of uPA into perinuclear mitochondria, reducing the mitochondrial membrane potential, and followed by the release of apoptotic inducible factor (AIF). AIF nuclear translocation is followed by a caspase-independent necroptotic cell death. Reduction in AIF expression by siRNA reduces the antiglioma cytotoxic effects of UCD38B, while not activating the caspase pathway. Ultrastructural changes shortly following treatment with UCD38B demonstrate dilation of endoplasmic reticulum (ER) and mitochondrial swelling followed by nuclear condensation within hours consistent with a necroptotic cell death differing from apoptosis and from autophagy. These drug mechanism of action studies demonstrate that UCD38B induces a cell cycle-independent, caspase-independent necroptotic glioma cell death that is mediated by AIF and independent of poly (ADP-ribose) polymerase and H2AX activation. PMID:23241369

Platinum(IV) complex LA-12 exerts higher ability than cisplatin to enhance TRAIL-induced cancer cell apoptosis via stimulation of mitochondrial pathway.

PubMed

Jelínková, Iva; Šafaříková, Barbora; Vondálová Blanářová, Olga; Skender, Belma; Hofmanová, Jiřina; Sova, Petr; Moyer, Mary Pat; Kozubík, Alois; Kolář, Zdeněk; Ehrmann, Jiří; Hyršlová Vaculová, Alena

2014-12-01

In search for novel strategies in colon cancer treatment, we investigated the unique ability of platinum(IV) complex LA-12 to efficiently enhance the killing effects of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), and compared it with the sensitizing action of cisplatin. We provide the first evidence that LA-12 primes human colon cancer cells for TRAIL-induced cytotoxicity by p53-independent activation of the mitochondrial apoptotic pathway. The cooperative action of LA-12 and TRAIL was associated with stimulation of Bax/Bak activation, drop of mitochondrial membrane potential, caspase-9 activation, and a shift of the balance among Bcl-2 family proteins in favor of the pro-apoptotic members. In contrast to cisplatin, LA-12 was a potent inducer of ERK-mediated Noxa and BimL protein upregulation, and more effectively enhanced TRAIL-induced apoptosis in the absence of Bax. The cooperative action of LA-12 and TRAIL was augmented following the siRNA-mediated silencing of Mcl-1 in both Bax proficient/deficient cells. We newly demonstrated that LA-12 induced ERK-mediated c-Myc upregulation, and proved that c-Myc silencing inhibited the mitochondrial activation and apoptosis in colon cancer cells treated with LA-12 and TRAIL. The LA-12-mediated sensitization to TRAIL-induced apoptosis was demonstrated in several colon cancer cell lines, further underscoring the general relevance of our findings. The selective action of LA-12 was documented by preferential priming of cancer but not normal colon cancer cells to TRAIL killing effects. Our work highlights the promising potential of LA-12 over cisplatin to enhance the colon cancer cell sensitivity to TRAIL-induced apoptosis, and provides new mechanistic insights into their cooperative action. Copyright © 2014 Elsevier Inc. All rights reserved.

Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response.

PubMed

Delledonne, M; Zeier, J; Marocco, A; Lamb, C

2001-11-06

Nitric oxide (NO) and reactive oxygen intermediates (ROIs) play key roles in the activation of disease resistance mechanisms both in animals and plants. In animals NO cooperates with ROIs to kill tumor cells and for macrophage killing of bacteria. Such cytotoxic events occur because unregulated NO levels drive a diffusion-limited reaction with O(2)(-) to generate peroxynitrite (ONOO(-)), a mediator of cellular injury in many biological systems. Here we show that in soybean cells unregulated NO production at the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate hypersensitive cell death. The HR is triggered only by balanced production of NO and ROIs. Moreover, hypersensitive cell death is activated after interaction of NO not with O(2)- but with H(2)O(2) generated from O(2)(-) by superoxide dismutase. Increasing the level of O(2)(-) reduces NO-mediated toxicity, and ONOO(-) is not a mediator of hypersensitive cell death. During the HR, superoxide dismutase accelerates O(2)(-) dismutation to H(2)O(2) to minimize the loss of NO by reaction with O(2)(-) and to trigger hypersensitive cell death through NO/H(2)O(2) cooperation. However, O(2)(-) rather than H(2)O(2) is the primary ROI signal for pathogen induction of glutathione S-transferase, and the rates of production and dismutation of O(2)(-) generated during the oxidative burst play a crucial role in the modulation and integration of NO/H(2)O(2) signaling in the HR. Thus although plants and animals use a similar repertoire of signals in disease resistance, ROIs and NO are deployed in strikingly different ways to trigger host cell death.

Redox-Mediated and Ionizing-Radiation-Induced Inflammatory Mediators in Prostate Cancer Development and Treatment

PubMed Central

Miao, Lu; Holley, Aaron K.; Zhao, Yanming; St. Clair, William H.

2014-01-01

Abstract Significance: Radiation therapy is widely used for treatment of prostate cancer. Radiation can directly damage biologically important molecules; however, most effects of radiation-mediated cell killing are derived from the generated free radicals that alter cellular redox status. Multiple proinflammatory mediators can also influence redox status in irradiated cells and the surrounding microenvironment, thereby affecting prostate cancer progression and radiotherapy efficiency. Recent Advances: Ionizing radiation (IR)–generated oxidative stress can regulate and be regulated by the production of proinflammatory mediators. Depending on the type and stage of the prostate cancer cells, these proinflammatory mediators may lead to different biological consequences ranging from cell death to development of radioresistance. Critical Issues: Tumors are heterogeneous and dynamic communication occurs between stromal and prostate cancer cells, and complicated redox-regulated mechanisms exist in the tumor microenvironment. Thus, antioxidant and anti-inflammatory strategies should be carefully evaluated for each patient at different stages of the disease to maximize therapeutic benefits while minimizing unintended side effects. Future Directions: Compared with normal cells, tumor cells are usually under higher oxidative stress and secrete more proinflammatory mediators. Thus, redox status is often less adaptive in tumor cells than in their normal counterparts. This difference can be exploited in a search for new cancer therapeutics and treatment regimes that selectively activate cell death pathways in tumor cells with minimal unintended consequences in terms of chemo- and radio-resistance in tumor cells and toxicity in normal tissues. Antioxid. Redox Signal. 20, 1481–1500. PMID:24093432

Prophylactic immunization against experimental leishmaniasis. III. Protection against fatal Leishmania tropica infection induced by irradiated promastigotes involves Lyt-1/sup +/2/sup -/ T cells that do not mediate cutaneous DTH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liew, F.Y.; Howard, J.G.; Hale, C.

1984-01-01

Protective immunity against fatal L. tropica infection in genetically vulnerable BALB/c mice can be induced by prophylactic immunization with irradiated promastigotes even when heat-killed. Such immunity is adoptively transferable transiently into intact or durably into sub-lethally irradiated (200 or 550 rad) syngeneic recipients by splenic T but not B cells. The effector T cells are of the Lyt-1/sup +/2/sup -/ phenotype, devoid of demonstrable cytotoxic activity. The immune splenic T cell population expresses specific helper activity for antibody synthesis. A causal role for helper T cells in this capacity, however, seems unlikely, because it was shown that antibody does notmore » determine the protective immunity against L. tropica. The immunized donors show no detectable cutaneous DTH or its early memory recall in response to live or killed promastigotes or a soluble L. tropica antigen preparation. Spleen, lymph node, and peritoneal exudate cells from protectively immunized donors similarly fail to transfer DTH locally or systemically. These cells also lack demonstrable suppressive activity against the expression or induction of DTH to L. tropica. Thus, protection against L. tropica induced by prophylactic i.v. immunization with irradiated promastigotes appears to be conferred by Lyt-1/sup +/2/sup -/ T cells that are distinguishable from T cells mediating either both DTH and T help, or cytotoxicity.« less

Structural basis of human β-cell killing by CD8+ T cells in Type 1 diabetes

PubMed Central

Bulek, Anna M.; Cole, David K.; Skowera, Ania; Dolton, Garry; Gras, Stephanie; Madura, Florian; Fuller, Anna; Miles, John J.; Gostick, Emma; Price, David A.; Drijfhout, Jan W.; Knight, Robin R.; Huang, Guo C.; Lissin, Nikolai; Molloy, Peter E.; Wooldridge, Linda; Jakobsen, Bent K.; Rossjohn, Jamie; Peakman, Mark; Rizkallah, Pierre J.; Sewell, Andrew K.

2011-01-01

The structural characteristics of autoreactive-T cell receptor (TCR) engagement of major histocompatability (MHC) class II-restricted self-antigens is established, but how autoimmune-TCRs interact with self-MHC class I has been unclear. We examined how CD8+ T cells kill human islet β-cells, in Type-1 diabetes, via autoreactive-TCR (1E6) recognition of an HLA-A*0201-restricted glucose-sensitive preproinsulin peptide. Rigid ‘lock-and-key’ binding underpinned the 1E6-HLA-A*0201-peptide interaction, whereby 1E6 docked similarly to most MHCI-restricted TCRs. However, this interaction was extraordinarily weak, due to limited contacts with MHCI. TCR binding was highly peptide-centric, dominated by two CDR3-loop-encoded residues, acting as an ‘aromatic-cap’, over the peptide MHCI (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8+ T cell-mediated autoreactivity. PMID:22245737

Multi-agent chemotherapy overcomes glucocorticoid resistance conferred by a BIM deletion polymorphism in pediatric acute lymphoblastic leukemia.

PubMed

Soh, Sheila Xinxuan; Lim, Joshua Yew Suang; Huang, John W J; Jiang, Nan; Yeoh, Allen Eng Juh; Ong, S Tiong

2014-01-01

A broad range of anti-cancer agents, including glucocorticoids (GCs) and tyrosine kinase inhibitors (TKIs), kill cells by upregulating the pro-apoptotic BCL2 family member, BIM. A common germline deletion in the BIM gene was recently shown to favor the production of non-apoptotic BIM isoforms, and to predict inferior responses in TKI-treated chronic myeloid leukemia (CML) and EGFR-driven lung cancer patients. Given that both in vitro and in vivo GC resistance are predictive of adverse outcomes in acute lymphoblastic leukemia (ALL), we hypothesized that this polymorphism would mediate GC resistance, and serve as a biomarker of poor response in ALL. Accordingly, we used zinc finger nucleases to generate ALL cell lines with the BIM deletion, and confirmed the ability of the deletion to mediate GC resistance in vitro. In contrast to CML and lung cancer, the BIM deletion did not predict for poorer clinical outcome in a retrospective analysis of 411 pediatric ALL patients who were uniformly treated with GCs and chemotherapy. Underlying the lack of prognostic significance, we found that the chemotherapy agents used in our cohort (vincristine, L-asparaginase, and methotrexate) were each able to induce ALL cell death in a BIM-independent fashion, and resensitize BIM deletion-containing cells to GCs. Together, our work demonstrates how effective therapy can overcome intrinsic resistance in ALL patients, and suggests the potential of using combinations of drugs that work via divergent mechanisms of cell killing to surmount BIM deletion-mediated drug resistance in other cancers.

Enhancement of neutrophil autophagy by an IVIG preparation against multidrug-resistant bacteria as well as drug-sensitive strains.

PubMed

Itoh, Hiroshi; Matsuo, Hidemasa; Kitamura, Naoko; Yamamoto, Sho; Higuchi, Takeshi; Takematsu, Hiromu; Kamikubo, Yasuhiko; Kondo, Tadakazu; Yamashita, Kouhei; Sasada, Masataka; Takaori-Kondo, Akifumi; Adachi, Souichi

2015-07-01

Autophagy occurs in human neutrophils after the phagocytosis of multidrug-resistant bacteria and drug-sensitive strains, including Escherichia coli and Pseudomonas aeruginosa. The present study detected autophagy by immunoblot analysis of LC3B conversion, by confocal scanning microscopic examination of LC3B aggregate formation and by transmission electron microscopic examination of bacteria-containing autophagosomes. Patients with severe bacterial infections are often treated with IVIG alongside antimicrobial agents. Here, we showed that IVIG induced neutrophil-mediated phagocytosis of multidrug-resistant strains. Compared with untreated neutrophils, neutrophils exposed to IVIG showed increased levels of bacterial cell killing, phagocytosis, O(2)(-) release, MPO release, and NET formation. IVIG also increased autophagy in these cells. Inhibiting the late phase of autophagy (fusion of lysosomes with autophagosomes) with bafilomycin A1-reduced, neutrophil-mediated bactericidal activity. These findings indicate that autophagy plays a critical role in the bactericidal activity mediated by human neutrophils. Furthermore, the autophagosomes within the neutrophils contained bacteria only and their organelles only, or both bacteria and their organelles, a previously undocumented observation. Taken together, these results suggest that the contents of neutrophil autophagosomes may be derived from specific autophagic systems, which provide the neutrophil with an advantage. Thus, IVIG promotes the neutrophil-mediated killing of multidrug-resistant bacteria as well as drug-sensitive strains. © Society for Leukocyte Biology.

Enhancement of neutrophil autophagy by an IVIG preparation against multidrug-resistant bacteria as well as drug-sensitive strains

PubMed Central

Itoh, Hiroshi; Matsuo, Hidemasa; Kitamura, Naoko; Yamamoto, Sho; Higuchi, Takeshi; Takematsu, Hiromu; Kamikubo, Yasuhiko; Kondo, Tadakazu; Yamashita, Kouhei; Sasada, Masataka; Takaori-Kondo, Akifumi; Adachi, Souichi

2015-01-01

Autophagy occurs in human neutrophils after the phagocytosis of multidrug-resistant bacteria and drug-sensitive strains, including Escherichia coli and Pseudomonas aeruginosa. The present study detected autophagy by immunoblot analysis of LC3B conversion, by confocal scanning microscopic examination of LC3B aggregate formation and by transmission electron microscopic examination of bacteria-containing autophagosomes. Patients with severe bacterial infections are often treated with IVIG alongside antimicrobial agents. Here, we showed that IVIG induced neutrophil-mediated phagocytosis of multidrug-resistant strains. Compared with untreated neutrophils, neutrophils exposed to IVIG showed increased levels of bacterial cell killing, phagocytosis, O2− release, MPO release, and NET formation. IVIG also increased autophagy in these cells. Inhibiting the late phase of autophagy (fusion of lysosomes with autophagosomes) with bafilomycin A1-reduced, neutrophil-mediated bactericidal activity. These findings indicate that autophagy plays a critical role in the bactericidal activity mediated by human neutrophils. Furthermore, the autophagosomes within the neutrophils contained bacteria only and their organelles only, or both bacteria and their organelles, a previously undocumented observation. Taken together, these results suggest that the contents of neutrophil autophagosomes may be derived from specific autophagic systems, which provide the neutrophil with an advantage. Thus, IVIG promotes the neutrophil-mediated killing of multidrug-resistant bacteria as well as drug-sensitive strains. PMID:25908735

Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis.

PubMed

Ebert, Gregor; Allison, Cody; Preston, Simon; Cooney, James; Toe, Jesse G; Stutz, Michael D; Ojaimi, Samar; Baschuk, Nikola; Nachbur, Ueli; Torresi, Joseph; Silke, John; Begley, C Glenn; Pellegrini, Marc

2015-05-05

We have shown that cellular inhibitor of apoptosis proteins (cIAPs) impair clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. A key question, with profound therapeutic implications, is whether this finding can be translated to the development of drugs that promote elimination of infected cells. Drug inhibitors of cIAPs were developed as cancer therapeutics to promote TNF-mediated tumor killing. These drugs are also known as Smac mimetics, because they mimic the action of the endogenous protein Smac/Diablo that antagonizes cIAP function. Here, we show using an immunocompetent mouse model of chronic HBV infection that birinapant and other Smac mimetics are able to rapidly reduce serum HBV DNA and serum HBV surface antigen, and they promote the elimination of hepatocytes containing HBV core antigen. The efficacy of Smac mimetics in treating HBV infection is dependent on their chemistry, host CD4(+) T cells, and TNF. Birinapant enhances the ability of entecavir, an antiviral nucleoside analog, to reduce viral DNA production in HBV-infected animals. These results indicate that birinapant and other Smac mimetics may have efficacy in treating HBV infection and perhaps, other intracellular infections.

Killing mediated spatial structure in V. Cholerae biofilms

NASA Astrophysics Data System (ADS)

Yanni, David

Most bacteria live in biofilms, which are implicated in 60 - 80 % of microbial infections in the body. The spatial structure of a biofilm confers advantages to its member-cells, such as antibiotic resistance, and is strongly affected by competition between strains and taxa. However, A complete picture of how competition affects the self-organized structure of these complex, far-from-equilibrium systems, is yet to emerge. To that end, we investigate phase separation dynamics driven by T6SS-facilitated bacterial warfare in a system composed of two strains of mutually antagonistic V. cholerae. T6SS is a contact mediated killing mechanism present in 25 % of all gram negative bacteria, and has been shown by recent work to play a major role in the spatial assortment of biofilms. T6SS events induce lysis, causing variations in local mechanical pressure, and acting as thermalizing events. We study cells immobilized in biofilms at the air-solid interface, so our experimental system represents a different type active matter, wherein activity is due to cell death and reproduction, not mobility. Here, we show how that activity imposes a constraint of minimal curvature on strain-strain interfaces; an effective Laplace pressure is characterized which governs interfacial dynamics.

Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

PubMed Central

Balagué, Cristina; Noya, Francisco; Alemany, Ramon; Chow, Louise T.; Curiel, David T.

2001-01-01

Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells. PMID:11462032

The mechanism of T-cell mediated cytotoxicity. VI. T-cell projections and their role in target cell killing.

PubMed Central

Sanderson, C J; Glauert, A M

1979-01-01

Electron micrographs of material fixed during the first 10 min of a T-cell cytotoxic system showed T-cell projections and T-cell burrowing into target cells. These observations were made possible by using a system with a very high rate of killing. The projections vary in shape and size, and can push deeply into the target cell, distorting organelles in their path, including the nucleus. The projections contain fine fibrillar material, to the exclusion of organelles. They push the target cell membrane in front of them to form pockets approximating to the shape of the projection. Areas of close contact occur between the projections and the target cell membrane, particularly at the leading edges. The likelihood that these projections develop as a result of contact with specific antigen, and are involved in the cytotoxic mechanism is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 PMID:311336

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage.

PubMed

Barker, Catherine R; McNamara, Anne V; Rackstraw, Stephen A; Nelson, David E; White, Mike R; Watson, Alastair J M; Jenkins, John R

2006-01-01

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90-topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death.

Mesenchymal stem cells express serine protease inhibitor to evade the host immune response

PubMed Central

El Haddad, Najib; Heathcote, Dean; Moore, Robert; Yang, Sunmi; Azzi, Jamil; Mfarrej, Bechara; Atkinson, Mark; Sayegh, Mohamed H.; Lee, Jeng-Shin; Ashton-Rickardt, Philip G.

2011-01-01

Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow–derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6−/−) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6−/− MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell–mediated death in vitro. In addition, SPI6−/− MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases. PMID:21076046

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage

PubMed Central

Barker, Catherine R.; McNamara, Anne V.; Rackstraw, Stephen A.; Nelson, David E.; White, Mike R.; Watson, Alastair J. M.; Jenkins, John R.

2006-01-01

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90–topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death. PMID:16504968

Combating rituximab resistance by inducing ceramide/lysosome-involved cell death through initiation of CD20-TNFR1 co-localization

PubMed Central

Zhang, Fan; Yang, Junlan; Li, Huafei; Liu, Moyan; Zhang, Jie; Zhao, Lichao; Wang, Lingxiong; LingHu, RuiXia; Feng, Fan; Gao, Xudong; Dong, Biqin; Liu, Xiaohan; Zi, Jian; Zhang, Weijing; Hu, Yi; Pan, Jingkun; Tian, Lei; Hu, Yazuo; Han, Zhitao; Zhang, Honghong; Wang, Xiaoning; Zhao, Lei

2016-01-01

ABSTRACT Despite the success of CD20 antibody rituximab in immunotherapy, acquired resistance is one of the prime obstacles for the successful treatment of B-cell malignancies. There is an urgent need to intensify efforts against resistance in cancer treatment. Growing evidence indicated that lysosomes may form an “Achilles heel” for cancer cells by sensitizing them to death pathways. Here, we uncover an important role of CD20 in initiation of ceramide/lysosomal membrane permeabilization (LMP)-mediated cell death, showing that colocalization of CD20-TNFR1 after type II CD20 antibody ligation can stimulate de novo ceramide synthesis by ceramide synthase and consequently induce remarkable lysosomal permeabilization (LMP) and lysosome-mediated cell death. Further studies show that the potent lysosome-mediated cell death induced by CD20 antibodies exhibits a profound killing effect against both rituximab-sensitive and -resistant (RR) lymphoma. Furthermore, engineering of rituximab by introducing a point mutation endows it with the ability to induce potent ceramide/LMP-mediated cell death in both RR lymphoma and primary B-cell malignancies from patients with rituximab-refractory, suggesting the potential clinical application to combat rituximab resistance. PMID:27467962

HIV envelope-mediated, CCR5/α4β7-dependent killing of CD4-negative γδ T cells which are lost during progression to AIDS.

PubMed

Li, Haishan; Pauza, C David

2011-11-24

HIV infects and replicates in CD4+ T cells but effects on host immunity and disease also involve depletion, hyper-activation, and modification of CD4-negative cell populations. In particular, the depletion of CD4-negative γδ T cells is common to all HIV+ individuals. We found that soluble or cell-associated envelope glycoproteins from CCR5-tropic strains of HIV could bind, activates the p38-caspase pathway, and induce the death of γδ cells. Envelope binding requires integrin α4β7 and chemokine receptor CCR5 which are at high levels and form a complex on the γδ T cell membrane. This receptor complex facilitated V3 loop binding to CCR5 in the absence of CD4-induced conformational changes. Cell death was increased by antigen stimulation after exposure to envelope glycoprotein. Direct signaling by envelope glycoprotein killed CD4-negative γδ T cells and reproduced a defect observed in all patients with HIV disease.

Farnesyltransferase inhibitor tipifarnib inhibits Rheb prenylation and stabilizes Bax in acute myelogenous leukemia cells

PubMed Central

Ding, Husheng; McDonald, Jennifer S.; Yun, Seongseok; Schneider, Paula A.; Peterson, Kevin L.; Flatten, Karen S.; Loegering, David A.; Oberg, Ann L.; Riska, Shaun M.; Huang, Shengbing; Sinicrope, Frank A.; Adjei, Alex A.; Karp, Judith E.; Meng, X. Wei; Kaufmann, Scott H.

2014-01-01

Although farnesyltransferase inhibitors have shown promising activity in relapsed lymphoma and sporadic activity in acute myelogenous leukemia, their mechanism of cytotoxicity is incompletely understood, making development of predictive biomarkers difficult. In the present study, we examined the action of tipifarnib in human acute myelogenous leukemia cell lines and clinical samples. In contrast to the Ras/MEK/ERK pathway-mediated Bim upregulation that is responsible for tipifarnib-induced killing of malignant lymphoid cells, inhibition of Rheb-induced mTOR signaling followed by dose-dependent upregulation of Bax and Puma occurred in acute myelogenous leukemia cell lines undergoing tipifarnib-induced apoptosis. Similar Bax and Puma upregulation occurred in serial bone marrow samples harvested from a subset of acute myelogenous leukemia patients during tipifarnib treatment. Expression of FTI-resistant Rheb M184L, like knockdown of Bax or Puma, diminished tipifarnib-induced killing. Further analysis demonstrated that increased Bax and Puma levels reflect protein stabilization rather than increased gene expression. In U937 cells selected for tipifarnib resistance, neither inhibition of signaling downstream of Rheb nor Bax and Puma stabilization occurred. Collectively, these results not only identify a pathway downstream from Rheb that contributes to tipifarnib cytotoxicity in human acute myelogenous leukemia cells, but also demonstrate that FTI-induced killing of lymphoid versus myeloid cells reflects distinct biochemical mechanisms downstream of different farnesylated substrates. (ClinicalTrials.gov identifier NCT00602771) PMID:23996484

Intravital imaging of CTLs killing islet cells in diabetic mice

PubMed Central

Coppieters, Ken; Amirian, Natalie; von Herrath, Matthias

2011-01-01

Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing β cells in the pancreatic islets, which are essentially mini-organs embedded in exocrine tissue. CTLs are considered to have a predominant role in the autoimmune destruction underlying T1D. Visualization of CTL-mediated killing of β cells would provide new insight into the pathogenesis of T1D, but has been technically challenging to achieve. Here, we report our use of intravital 2-photon imaging in mice to visualize the dynamic behavior of a virally expanded, diabetogenic CTL population in the pancreas at cellular resolution. Following vascular arrest and extravasation, CTLs adopted a random motility pattern throughout the compact exocrine tissue and displayed unimpeded yet nonlinear migration between anatomically nearby islets. Upon antigen encounter within islets, a confined motility pattern was acquired that allowed the CTLs to scan the target cell surface. A minority of infiltrating CTLs subsequently arrested at the β cell junction, while duration of stable CTL–target cell contact was on the order of hours. Slow-rate killing occurred in the sustained local presence of substantial numbers of effector cells. Collectively, these data portray the kinetics of CTL homing to and between antigenic target sites as a stochastic process at the sub-organ level and argue against a dominant influence of chemotactic gradients. PMID:22133877

The natural dietary genistein boosts bacteriophage-mediated cancer cell killing by improving phage-targeted tumor cell transduction

PubMed Central

Tsafa, Effrosyni; Al-Bahrani, Mariam; Bentayebi, Kaoutar; Przystal, Justyna; Suwan, Keittisak; Hajitou, Amin

2016-01-01

Gene therapy has long been regarded as a promising treatment for cancer. However, cancer gene therapy is still facing the challenge of targeting gene delivery vectors specifically to tumors when administered via clinically acceptable non-invasive systemic routes (i.e. intravenous). The bacteria virus, bacteriophage (phage), represents a new generation of promising vectors in systemic gene delivery since their targeting can be achieved through phage capsid display ligands, which enable them to home to specific tumor receptors without the need to ablate any native eukaryotic tropism. We have previously reported a tumor specific bacteriophage vector named adeno-associated virus/phage, or AAVP, in which gene expression is under a recombinant human rAAV2 virus genome targeted to tumors via a ligand-directed phage capsid. However, cancer gene therapy with this tumor-targeted vector achieved variable outcomes ranging from tumor regression to no effect in both experimental and natural preclinical models. Herein, we hypothesized that combining the natural dietary genistein, with proven anticancer activity, would improve bacteriophage anticancer safe therapy. We show that combination treatment with genistein and AAVP increased targeted cancer cell killing by AAVP carrying the gene for Herpes simplex virus thymidine kinase (HSVtk) in 2D tissue cultures and 3D tumor spheroids. We found this increased tumor cell killing was associated with enhanced AAVP-mediated gene expression. Next, we established that genistein protects AAVP against proteasome degradation and enhances vector genome accumulation in the nucleus. Combination of genistein and phage-guided virotherapy is a safe and promising strategy that should be considered in anticancer therapy with AAVP. PMID:27437775

Serum killing of Ureaplasma parvum shows serovar-determined susceptibility for normal individuals and common variable immuno-deficiency patients.

PubMed

Beeton, Michael L; Daha, Mohamed R; El-Shanawany, Tariq; Jolles, Stephen R; Kotecha, Sailesh; Spiller, O Brad

2012-02-01

Many Gram-negative bacteria, unlike Gram-positive, are directly lysed by complement. Ureaplasma can cause septic arthritis and meningitis in immunocompromised individuals and induce premature birth. Ureaplasma has no cell wall, cannot be Gram-stain classified and its serum susceptibility is unknown. Survival of Ureaplasma serovars (SV) 1, 3, 6 and 14 (collectively Ureaplasma parvum) were measured following incubation with normal or immunoglobulin-deficient patient serum (relative to heat-inactivated controls). Blocking monoclonal anti-C1q antibody and depletion of calcium, immunoglobulins, or lectins were used to determine the complement pathway responsible for killing. Eighty-three percent of normal sera killed SV1, 67% killed SV6 and 25% killed SV14; greater killing correlating to strong immunoblot identification of anti-Ureaplasma antibodies; killing was abrogated following ProteinA removal of IgG1. All normal sera killed SV3 in a C1q-dependent fashion, irrespective of immunoblot identification of anti-Ureaplasma antibodies; SV3 killing was unaffected by total IgG removal by ProteinG, where complement activity was retained. Only one of four common variable immunodeficient (CVID) patient sera failed to kill SV3, despite profound IgM and IgG deficiency for all; however, killing of SV3 and SV1 was restored with therapeutic intravenous immunoglobulin therapy. Only the classical complement pathway mediated Ureaplasma-cidal activity, sometimes in the absence of observable immunoblot reactive bands. Copyright © 2011 Elsevier GmbH. All rights reserved.

T lymphocyte-mediated protection against Pseudomonas aeruginosa infection in granulocytopenic mice.

PubMed Central

Powderly, W G; Pier, G B; Markham, R B

1986-01-01

BALB/c mice immunized with Pseudomonas aeruginosa immunotype 1 polysaccharide develop protective T cell immunity to bacterial challenge. In vitro, T cells from immunized mice kill P. aeruginosa by production of a bactericidal lymphokine. The present study demonstrates that adoptive transfer of T cells from immunized BALB/c mice to granulocytopenic mice resulted in 97% survival on challenge with P. aeruginosa, compared with 17% survival with adoptive transfer of T cells from nonimmune BALB/c mice. This protection is specifically elicited by reexposure to the original immunizing antigen; adoptive recipients cannot withstand challenge with immunotype 3 P. aeruginosa. However, the adoptive recipients do survive simultaneous infection with both P. aeruginosa immunotypes 1 and 3. Adoptive transfer of T cells from the congenic CB.20 mice, which are unable to kill P. aeruginosa in vitro, provides only 20% protection to granulocytopenic mice. These studies indicate that transfer of specific immune T lymphocytes can significantly enhance the resistance to P. aeruginosa infection in granulocytopenic mice. PMID:2426306

Gene therapy of uterine leiomyoma: adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir treatment inhibits growth of human and rat leiomyoma cells in vitro and in a nude mouse model.

PubMed

Salama, S A; Kamel, M; Christman, G; Wang, H Q; Fouad, H M; Al-Hendy, A

2007-01-01

Uterine leiomyomas (LM) affect a high percentage of reproductive-age women. They develop as discrete, well-defined tumors that are easily accessible with imaging techniques--making this disease ideal for localized gene therapy approaches. In this study, we determined the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene transfer in combination with ganciclovir (Ad-TK/GCV) as a potential therapy for LM. Rat ELT-3 LM cells and human LM cells were transfected with different multiplicity of infections (10-100 plaque forming units [PFU]/cell) of Ad-TK and treated with GCV (5, 10, or 20 microg/ml) for 5 days. To test the bystander effect, Ad-TK-transfected ELT-3 cells (100 PFU/cell) or LM cells (10 PFU/cell) were cocultured with corresponding nontransfected cells at increasing percentages and treated with GCV followed by cell counting. In ELT-3 cells transfected with Ad-TK/GCV (10, 20, 50, or 100 PFU/cell), the cell count was reduced by 24, 42, 77, and 87%, respectively, compared with the control cells (transfected with Ad-Lac Z/GCV). Similarly, in LM cells transfected with Ad-TK/GCV (10, 50, or 100 PFU/cell), the cell count was reduced by 31, 62, and 82%, respectively, compared with the control. A strong bystander effect was noted in both ELT-3 and LM cells with significant killing (p = 0.001) at a ratio of infected:uninfected cells of only 1:99 and maximal killing at 1:4. This study demonstrates the potential efficacy of the Ad-TK/GCV gene therapy approach as a viable nonsurgical alternative treatment for uterine LM.

Cannabinoids increase lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1.

PubMed

Haustein, Maria; Ramer, Robert; Linnebacher, Michael; Manda, Katrin; Hinz, Burkhard

2014-11-15

Cannabinoids have been shown to promote the expression of the intercellular adhesion molecule 1 (ICAM-1) on lung cancer cells as part of their anti-invasive and antimetastatic action. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study addressed the impact of cannabinoid-induced ICAM-1 on cancer cell adhesion to lymphokine-activated killer (LAK) cells and LAK cell-mediated cytotoxicity. Cannabidiol (CBD), a non-psychoactive cannabinoid, enhanced the susceptibility of cancer cells to adhere to and subsequently be lysed by LAK cells, with both effects being reversed by a neutralizing ICAM-1 antibody. Increased cancer cell lysis by CBD was likewise abrogated when CBD-induced ICAM-1 expression was blocked by specific siRNA or by antagonists to cannabinoid receptors (CB1, CB2) and to transient receptor potential vanilloid 1. In addition, enhanced killing of CBD-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen-1 (LFA-1) suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. ICAM-1-dependent pro-killing effects were further confirmed for the phytocannabinoid Δ(9)-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), a hydrolysis-stable endocannabinoid analogue. Finally, each cannabinoid elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less pronounced (CBD, THC) or absent (MA) ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate cannabinoid-induced upregulation of ICAM-1 on lung cancer cells to be responsible for increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of cannabinoids. Copyright © 2014 Elsevier Inc. All rights reserved.

A High-Throughput Assay for Screening of Natural Products that Enhanced Tumoricidal Activity of NK Cells.

PubMed

Gong, Chenyuan; Ni, Zhongya; Yao, Chao; Zhu, Xiaowen; Ni, Lulu; Wang, Lixin; Zhu, Shiguo

2015-01-01

Recently, immunotherapy has shown a lot of promise in cancer treatment and different immune cell types are involved in this endeavor. Among different immune cell populations, NK cells are also an important component in unleashing the therapeutic activity of immune cells. Therefore, in order to enhance the tumoricidal activity of NK cells, identification of new small-molecule natural products is important. Despite the availability of different screening methods for identification of natural products, a simple, economic and high-throughput method is lacking. Hence, in this study, we have developed a high-throughput assay for screening and indentifying natural products that can enhance NK cell-mediated killing of cancer cells. We expanded human NK cell population from human peripheral blood mononuclear cells (PBMCs) by culturing these PBMCs with membrane-bound IL-21 and CD137L engineered K562 cells. Next, expanded NK cells were co-cultured with non-small cell lung cancer (NSCLC) cells with or without natural products and after 24 h of co-culturing, harvested supernatants were analyzed for IFN-γ secretions by ELISA method. We screened 502 natural products and identified that 28 candidates has the potential to induce IFN-γ secretion by NK cells to varying degrees. Among the 28 natural product candidates, we further confirmed and analyzed the potential of one molecule, andrographolide. It actually increased IFN-γ secretion by NK cells and enhanced NK cell-mediated killing of NSCLC cells. Our results demonstrated that this IFN-γ based high-throughput assay for screening of natural products for NK cell tumoricidal activity is a simple, economic and reliable method.

Immotile Active Matter: Activity from Death and Reproduction

NASA Astrophysics Data System (ADS)

Kalziqi, Arben; Yanni, David; Thomas, Jacob; Ng, Siu Lung; Vivek, Skanda; Hammer, Brian K.; Yunker, Peter J.

2018-01-01

Unlike equilibrium atomic solids, biofilms—soft solids composed of bacterial cells—do not experience significant thermal fluctuations at the constituent level. However, living cells stochastically reproduce and die, provoking a mechanical response. We investigate the mechanical consequences of cellular death and reproduction by measuring surface-height fluctuations of biofilms containing two mutually antagonistic strains of Vibrio cholerae that kill one another on contact via the type VI secretion system. While studies of active matter typically focus on activity via constituent mobility, here, activity is mediated by reproduction and death events in otherwise immobilized cells. Biofilm surface topography is measured in the nearly homeostatic limit via white light interferometry. Although biofilms are far from equilibrium systems, measured surface-height fluctuation spectra resemble the spectra of thermal permeable membranes but with an activity-mediated effective temperature, as predicted by Risler, Peilloux, and Prost [Phys. Rev. Lett. 115, 258104 (2015), 10.1103/PhysRevLett.115.258104]. By comparing the activity of killer strains of V. cholerae with that of genetically modified strains that cannot kill each other and validating with individual-based simulations, we demonstrate that extracted effective temperatures increase with the amount of death and reproduction and that death and reproduction can fluidize biofilms. Together, these observations demonstrate the unique physical consequences of activity mediated by death and reproduction events.

A heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage-mediated, vascular-targeted suicide gene transfer.

PubMed

Trepel, Martin; Stoneham, Charlotte A; Eleftherohorinou, Hariklia; Mazarakis, Nicholas D; Pasqualini, Renata; Arap, Wadih; Hajitou, Amin

2009-08-01

Suicide gene transfer is the most commonly used cytotoxic approach in cancer gene therapy; however, a successful suicide gene therapy depends on the generation of efficient targeted systemic gene delivery vectors. We recently reported that selective systemic delivery of suicide genes such as herpes simplex virus thymidine kinase (HSVtk) to tumor endothelial cells through a novel targeted adeno-associated virus/phage vector leads to suppression of tumor growth. This marked effect has been postulated to result primarily from the death of cancer cells by hypoxia following the targeted disruption of tumor blood vessels. Here, we investigated whether an additional mechanism of action is involved. We show that there is a heterotypic "bystander" effect between endothelial cells expressing the HSVtk suicide gene and tumor cells. Treatment of cocultures of HSVtk-transduced endothelial cells and non-HSVtk-transduced tumor cells with ganciclovir results in the death of both endothelial and tumor cells. Blocking of this effect by 18alpha-glycyrrhetinic acid indicates that gap junctions between endothelial and tumor cells are largely responsible for this phenomenon. Moreover, the observed bystander killing is mediated by connexins 43 and 26, which are expressed in endothelial and tumor cell types. Finally, this heterotypic bystander effect is accompanied by a suppression of tumor growth in vivo that is independent of primary gene transfer into host-derived tumor vascular endothelium. These findings add an alternative nonmutually exclusive and potentially synergistic cytotoxic mechanism to cancer gene therapy based on targeted adeno-associated virus/phage and further support the promising role of nonmalignant tumor stromal cells as therapeutic targets.

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

PubMed

Sarkar, Saheli; Sabhachandani, Pooja; Ravi, Dashnamoorthy; Potdar, Sayalee; Purvey, Sneha; Beheshti, Afshin; Evens, Andrew M; Konry, Tania

2017-01-01

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

The Herpes Simplex Virus Type 1 Latency-Associated Transcript Can Protect Neuron-Derived C1300 and Neuro2A Cells from Granzyme B-Induced Apoptosis and CD8 T-Cell Killing▿

PubMed Central

Jiang, Xianzhi; Alami Chentoufi, Aziz; Hsiang, Chinhui; Carpenter, Dale; Osorio, Nelson; BenMohamed, Lbachir; Fraser, Nigel W.; Jones, Clinton; Wechsler, Steven L.

2011-01-01

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only HSV-1 gene transcript abundantly expressed throughout latency. LAT null mutants have a significantly reduced reactivation phenotype. LAT's antiapoptosis activity is the major LAT factor involved in supporting the wild-type reactivation phenotype. During HSV-1 latency, some ganglionic neurons are surrounded by CD8 T cells, and it has been proposed that these CD8 T cells help maintain HSV-1 latency by suppressing viral reactivations. Surprisingly, despite injection of cytotoxic lytic granules by these CD8 T cells into latently infected neurons, neither apoptosis nor neuronal cell death appears to occur. We hypothesized that protection of latently infected neurons against cytotoxic CD8 T-cell killing is due to LAT's antiapoptosis activity. Since CD8 T-cell cytotoxic lytic granule-mediated apoptosis is critically dependent on granzyme B (GrB), we examined LAT's ability to block GrB-induced apoptosis. We report here that (i) LAT can interfere with GrB-induced apoptosis in cell cultures, (ii) LAT can block GrB-induced cleavage (activation) of caspase-3 both in cell culture and in a cell-free in vitro cell extract assay, and (iii) LAT can protect C1300 and Neuro2A cells from cytotoxic CD8 T-cell killing in vitro. These findings support the hypothesis that LAT's antiapoptosis activity can protect latently infected neurons from being killed by CD8 T-cell lytic granules in vivo. PMID:21177822

Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1–AKT interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Xian-Ying; Chen, Wei; Fan, Jun-Ting

2013-02-15

In the present paper, we examined the effects of a natural cyclopeptide RA-V on human breast cancer cells and the underlying mechanisms. RA-V significantly inhibited the growth of human breast cancer MCF-7, MDA-MB-231 cells and murine breast cancer 4T1 cells. In addition, RA-V triggered mitochondrial apoptotic pathway which was indicated by the loss of mitochondrial membrane potential, the release of cytochrome c, and the activation of caspase cascade. Further study showed that RA-V dramatically inhibited phosphorylation of AKT and 3-phosphoinositide dependent protein kinase 1 (PDK1) in MCF-7 cells. Moreover, RA-V disrupted the interaction between PDK1 and AKT in MCF-7 cells.more » Furthermore, RA-V-induced apoptosis could be enhanced by phosphatidylinositol 3-kinase inhibitor or attenuated by over-expression of AKT in all the three kinds of breast cancer cells. Taken together, this study shows that RA-V, which can induce mitochondria-mediated apoptosis, exerts strong anti-tumor activity against human breast cancer. The underlying anti-cancer mechanism of RA-V is related to the blockage of the interaction between PDK1 and AKT. - Highlights: ► Plant cyclopeptide RA-V kills human breast cancer cells. ► RA-V triggered mitochondrial apoptotic pathway in human breast cancer cells. ► RA-V inhibited phosphorylation of AKT and PDK1 in breast cancer MCF-7 cells. ► Its mechanism is related to the blockage of the interaction between PDK1 and AKT.« less

The interaction of gamma delta T cells with activated macrophages is a property of the V gamma 1 subset.

PubMed

Dalton, Jane E; Pearson, Jayne; Scott, Phillip; Carding, Simon R

2003-12-15

Immunoregulation is an emerging paradigm of gammadelta T cell function. The mechanisms by which gammadelta T cells mediate this function, however, are not clear. Studies have identified a direct role for gammadelta T cells in resolving the host immune response to infection, by eliminating populations of activated macrophages. The aim of this study was to identify macrophage-reactive gammadelta T cells and establish the requirements/outcomes of macrophage-gammadelta T cell interactions during the immune response to the intracellular bacterium, Listeria monocytogenes (Lm). Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRdelta(-/-) mice were incubated with splenocytes from naive and Lm-infected alphabeta/gammadelta T cell-deficient and wild-type mice, the ability to bind macrophages was shown to be restricted to gammadelta T cells and the GV5S1 (Vgamma1) subset of gammadelta T cells. Macrophage adherence resulted in a 4- to 10-fold enrichment of Vgamma1(+) T cells. Enrichment of Vgamma1 T cells was dependent upon the activation status of macrophages, but independent of the activation status of gammadelta T cells. Vgamma1 T cells were cytotoxic for activated macrophages with both the binding to and killing of macrophages being TCR dependent because anti-TCRgammadelta Abs inhibited both Vgamma1 binding and killing activities. These studies establish the identity of macrophage cytotoxic gammadelta T cells, the conditions under which this interaction occurs, and the outcome of this interaction. These findings are concordant with the involvement of Vgamma1 T cells in macrophage homeostasis during the resolution of pathogen-mediated immune responses.

Role of Acinetobactin-Mediated Iron Acquisition Functions in the Interaction of Acinetobacter baumannii Strain ATCC 19606T with Human Lung Epithelial Cells, Galleria mellonella Caterpillars, and Mice

PubMed Central

Gaddy, Jennifer A.; Arivett, Brock A.; McConnell, Michael J.; López-Rojas, Rafael; Pachón, Jerónimo

2012-01-01

Acinetobacter baumannii, which causes serious infections in immunocompromised patients, expresses high-affinity iron acquisition functions needed for growth under iron-limiting laboratory conditions. In this study, we determined that the initial interaction of the ATCC 19606T type strain with A549 human alveolar epithelial cells is independent of the production of BasD and BauA, proteins needed for acinetobactin biosynthesis and transport, respectively. In contrast, these proteins are required for this strain to persist within epithelial cells and cause their apoptotic death. Infection assays using Galleria mellonella larvae showed that impairment of acinetobactin biosynthesis and transport functions significantly reduces the ability of ATCC 19606T cells to persist and kill this host, a defect that was corrected by adding inorganic iron to the inocula. The results obtained with these ex vivo and in vivo approaches were validated using a mouse sepsis model, which showed that expression of the acinetobactin-mediated iron acquisition system is critical for ATCC 19606T to establish an infection and kill this vertebrate host. These observations demonstrate that the virulence of the ATCC 19606T strain depends on the expression of a fully active acinetobactin-mediated system. Interestingly, the three models also showed that impairment of BasD production results in an intermediate virulence phenotype compared to those of the parental strain and the BauA mutant. This observation suggests that acinetobactin intermediates or precursors play a virulence role, although their contribution to iron acquisition is less relevant than that of mature acinetobactin. PMID:22232188

A defucosylated bispecific multivalent molecule exhibits broad HIV-1 neutralizing activity and enhanced ADCC against reactivated HIV-1 latently infected cells.

PubMed

Kong, Desheng; Wang, Yan; Ji, Ping; Li, Wei; Ying, Tianlei; Huang, Jinghe; Wang, Chen; Wu, Yanling; Wang, Yanping; Chen, Weizao; Hao, Yanling; Hong, Kunxue; Shao, Yiming; Dimitrov, Dimiter S; Jiang, Shibo; Ma, Liying

2018-05-11

Current treatments cannot completely eradicate HIV-1 owing to the presence of latently infected cells which harbor transcriptionally silent HIV-1. However, defucosylated antibodies can readily kill latently infected cells after their activation to express envelope glycoprotein (Env) through antibody-dependent cellular cytotoxicity (ADCC). We herein aimed to test a defucosylated bispecific multivalent molecule consisting of domain-antibody and single-domain CD4, LSEVh-LS-F, for its HIV-1 neutralizing activity and ADCC against the reactivated latently infected cells, compared with the non-defucosylated molecule LSEVh-LS. LSEVh-LS-F's neutralizing activity against a panel of newly characterized Chinese HIV-1 clinical isolates was assessed by using TZM-bl- and PBMC-based assays. LSEVh-LS-F-mediated ADCC in the presence of NK cells against cell lines that stably express Env proteins, HIV-1-infected cells and LRA-reactivated HIV-1 latent cells, was measured using a lactate dehydrogenase (LDH) cytotoxicity assay or flow cytometry. LSEVh-LS-F and LSEVh-LS were equally effective in neutralized infection of all HIV-1 isolates tested with IC50 and IC90 values 3∼4-fold lower than those of VRC01. LSEVh-LS-F was more effective in NK-mediated killing of HIV-1 Env-expressing cell lines, HIV-1-infected cells, latency reactivation agents-reactivated ACH2 cells, and reactivated latently infected resting CD4 T cell line as well as resting CD4 T lymphocytes isolated from patients receiving highly active anti-retroviral therapy (HAART). LSEVh-LS-F exhibits broad HIV-1 neutralizing activity and enhanced ADCC against HIV-1-infected cells, reactivated latently infected cell lines and primary CD4 T cells, thus being a promising candidate therapeutic for eradicating the HIV-1 reservoir.

Neutrophil Extracellular Trap (NET)-Mediated Killing of Pseudomonas aeruginosa: Evidence of Acquired Resistance within the CF Airway, Independent of CFTR

PubMed Central

Young, Robert L.; Malcolm, Kenneth C.; Kret, Jennifer E.; Caceres, Silvia M.; Poch, Katie R.; Nichols, David P.; Taylor-Cousar, Jennifer L.; Saavedra, Milene T.; Randell, Scott H.; Vasil, Michael L.; Burns, Jane L.; Moskowitz, Samuel M.; Nick, Jerry A.

2011-01-01

The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa. PMID:21909403

Involvement of NK cells against tumors and parasites.

PubMed

Papazahariadou, M; Athanasiadis, G I; Papadopoulos, E; Symeonidou, I; Hatzistilianou, M; Castellani, M L; Bhattacharya, K; Shanmugham, L N; Conti, P; Frydas, S

2007-01-01

Host resistance against pathogens depends on a complex interplay of innate and adaptive immune mechanisms. Acting as an early line of defence, the immune system includes activation of neutrophils, tissue macrophages, monocytes, dendritic cells, eosinophils and natural killer (NK) cells. NK cells are lymphoid cells that can be activated without previous stimulation and are therefore like macrophages in the first line of defence against tumor cells and a diverse range of pathogens. NK cells mediate significant activity and produce high levels of proinflammatory cytokines in response to infection. Their cytotoxicity production is induced principally by monocyte-, macrophage- and dendritic cell-derived cytokines, but their activation is also believed to be cytokine-mediated. Recognition of infection by NK cells is accomplished by numerous activating and inhibitory receptors on the NK cells' surface that selectively trigger the cytolytic activity in a major histocompability complex-independent manner. NK cells have trypanocidal activity of fibroblast cells and mediate direct destruction of extracellular epimastigote and trypomastigote forms of T. cruzi and T. lewisi in vitro; moreover, they kill plasmodia-infected erythrocytes directly through cell-cell interaction. This review provides a more detailed analysis of how NK cells recognize and respond to parasites and how they mediate cytotoxicity against tumor cells. Also the unique role of NK cells in innate immunity to infection and the relationship between parasites and carcinogenesis are discussed.

Mapping of binding epitopes of a human decay-accelerating factor monoclonal antibody capable of enhancing rituximab-mediated complement-dependent cytotoxicity.

PubMed

Guo, Bo; Ma, Zheng-wei; Li, Hua; Xu, Gui-lian; Zheng, Ping; Zhu, Bo; Wu, Yu-Zhang; Zou, Qiang

2008-08-01

Complement-dependent cytotoxicity (CDC) is thought to be one of the most important mechanisms of action of therapeutic monoclonal antibodies (mAbs). The decay-accelerating factor (DAF) overexpressed in certain tumors limits the CDC effect of the therapeutic anticancer antibodies. The use of DAF blocking antibodies targeted specifically at cancer cells in combination with immunotherapeutic mAbs of cancer may improve the therapeutic effect in cancer patients. In this study, the lysis of Raji cells mediated by CDC was determined after blocking DAF function by anti-DAF polyclonal antibody and 3 mAbs (DG3, DG9, DA11) prepared in our laboratory, respectively, in the presence of the anti-CD20 chimeric mAb rituximab. The binding domains of the three anti-DAF mAbs were identified using yeast surface display technique, and the mimic epitopes of mAb DG3 were screened from a random phage-display nonapeptide library. The results showed that blocking DAF function by anti-DAF polyclonal antibody enhanced complement-mediated killing of Raji cells. Among the 3 mAbs against DAF, only DG3 was found to be able to remarkably enhance the CDC effect of the therapeutic mAb rituximab. DG3 bound to the third short consensus repeat (SCR) of DAF. Binding of DG3 to immobilized DAF was inhibited by mimic epitope peptides screened from the peptide library. Our results suggest that a higher level of DAF expressed by certain tumor cells is significant to abolish the CDC effect of therapeutic anticancer antibodies, and mAbs binding to SCR3 can enhance the complement-mediated killing of Raji cells. It is of significance to identify the DAF epitopes required in inhibiting CDC not only for better understanding of the relationship between the structure and function of DAF, but also for designing and developing anti-DAF mAbs capable of enhancing CDC.

Bispecific antibody targets multiple Pseudomonas aeruginosa evasion mechanisms in the lung vasculature.

PubMed

Thanabalasuriar, Ajitha; Surewaard, Bas Gj; Willson, Michelle E; Neupane, Arpan S; Stover, Charles K; Warrener, Paul; Wilson, George; Keller, Ashley E; Sellman, Bret R; DiGiandomenico, Antonio; Kubes, Paul

2017-06-01

Pseudomonas aeruginosa is a major cause of severe infections that lead to bacteremia and high patient mortality. P. aeruginosa has evolved numerous evasion and subversion mechanisms that work in concert to overcome immune recognition and effector functions in hospitalized and immunosuppressed individuals. Here, we have used multilaser spinning-disk intravital microscopy to monitor the blood-borne stage in a murine bacteremic model of P. aeruginosa infection. P. aeruginosa adhered avidly to lung vasculature, where patrolling neutrophils and other immune cells were virtually blind to the pathogen's presence. This cloaking phenomenon was attributed to expression of Psl exopolysaccharide. Although an anti-Psl mAb activated complement and enhanced neutrophil recognition of P. aeruginosa, neutrophil-mediated clearance of the pathogen was suboptimal owing to a second subversion mechanism, namely the type 3 secretion (T3S) injectisome. Indeed, T3S prevented phagosome acidification and resisted killing inside these compartments. Antibody-mediated inhibition of the T3S protein PcrV did not enhance bacterial phagocytosis but did enhance killing of the few bacteria ingested by neutrophils. A bispecific mAb targeting both Psl and PcrV enhanced neutrophil uptake of P. aeruginosa and also greatly increased inhibition of T3S function, allowing for phagosome acidification and bacterial killing. These data highlight the need to block multiple evasion and subversion mechanisms in tandem to kill P. aeruginosa.

Cheating, facilitation and cooperation regulate the effectiveness of phage-encoded exotoxins as antipredator molecules.

PubMed

Aijaz, Iqbal; Koudelka, Gerald B

2018-04-19

Temperate phage encoded Shiga toxin (Stx) kills the bacterivorous predator, Tetrahymena thermophila, providing Stx + Escherichia coli with a survival advantage over Stx - cells. Although bacterial death accompanies Stx release, since bacteria grow clonally the fitness benefits of predator killing accrue to the kin of the sacrificed organism, meaning Stx-mediated protist killing is a form of self-destructive cooperation. We show here that the fitness benefits of Stx production are not restricted to the kin of the phage-encoding bacteria. Instead, nearby "free loading" bacteria, irrespective of their genotype, also reap the benefit of Stx-mediated predator killing. This finding indicates that the phage-borne Stx exotoxin behaves as a public good. Stx is encoded by a mobile phage. We find that Stx-encoding phage can use susceptible bacteria in the population as surrogates to enhance toxin and phage production. Moreover, our findings also demonstrate that engulfment and concentration of Stx-encoding and susceptible Stx - bacteria in the Tetrahymena phagosome enhances the transfer of Stx-encoding temperate phage from the host to the susceptible bacteria. This transfer increases the population of cooperating bacteria within the community. Since these bacteria now encode Stx, the predation-stimulated increase in phage transfer increases the population of toxin encoding bacteria in the environment. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Annatto Constituent Cis-Bixin Has Selective Antimyeloma Effects Mediated by Oxidative Stress and Associated with Inhibition of Thioredoxin and Thioredoxin Reductase

PubMed Central

Tibodeau, Jennifer D.; Isham, Crescent R.

2010-01-01

Abstract In pursuit of the anticancer effects of seeds of the rain forest plant Bixa orellana (annatto), we found that its constituent cis-bixin induced cytotoxicity in a wide variety of tumor cell lines (IC50 values from 10 to 50 μM, 24-h exposures) and, importantly, also selectively killed freshly collected patient multiple myeloma cells and highly drug-resistant multiple myeloma cell lines. Mechanistic studies indicated that cis-bixin–induced cytotoxicity was greatly attenuated by co-treatment with glutathione or N-acetylcysteine (NAC); whereas fluorescence-activated cell sorting (FACS) assays using the cell-permeable dyes 5-(and-6) chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), or dihydroethidium demonstrated that cis-bixin rapidly induced cellular reactive oxygen species (ROS) in dose- and time-dependent fashions, collectively implicating ROS as contributory to cis-bixin–induced cytotoxicity. In pursuit of potential contributors to ROS imposition by cis-bixin, we found that cis-bixin inhibited both thioredoxin (Trx) and thioredoxin reductase (TrxR1) activities at concentrations comparable to those required for cytotoxicity, implicating the inhibition of these redox enzymes as potentially contributing to its ability to impose cellular ROS and to kill cancer cells. Collectively, our studies indicate that the annatto constituent cis-bixin has intriguing selective antimyeloma activity that appears to be mediated through effects on redox signaling. Antioxid. Redox Signal. 13, 987–997. PMID:20170403

Comparative study of in vitro and in vivo drug effects on cell-mediated cytotoxicity.

PubMed Central

Borel, J F

1976-01-01

Cell-mediated cytolysis (CMC) was assayed in a system using spleen cells from mice (C57BL/6) sensitized with allogeneic tumour cells (DBA/2 mastocytoma P-815). Anti-inflammatory drugs, immunosuppressives, inhibitors of cell division and other agents were investigated for their capacity to inhibit CMC in three different ways. First, inhibition of CMC after in vitro addition of drug was observed with corticosteroids, some immunosuppressives and inhibitors of cell division. Secondly, suppression of CMC after a single drug administration to sensitized mice shortly before being killed was found with corticosteroids, several immunosuppressives and irradiation. Thirdly, prevention of development of CMC by repeated drug treatment (immunosuppressive schedule) was achieved with most immunosuppressives and cytostatic drugs. Non-steroidal anti-inflammatory drugs were inactive in these tests. Correlation of effects between the three procedures was very poor and it is suggested that various mechanisms may be involved in the different assays. PMID:824198

Salinomycin kills cancer stem cells by sequestering iron in lysosomes

NASA Astrophysics Data System (ADS)

Mai, Trang Thi; Hamaï, Ahmed; Hienzsch, Antje; Cañeque, Tatiana; Müller, Sebastian; Wicinski, Julien; Cabaud, Olivier; Leroy, Christine; David, Amandine; Acevedo, Verónica; Ryo, Akihide; Ginestier, Christophe; Birnbaum, Daniel; Charafe-Jauffret, Emmanuelle; Codogno, Patrice; Mehrpour, Maryam; Rodriguez, Raphaël

2017-10-01

Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.

Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Zhendong, E-mail: zdyu@hotmail.com; Wang, Hao; Zhang, Libin

CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrugmore » system.« less

Platelet factor 4 activity against P. falciparum and its translation to nonpeptidic mimics as antimalarials.

PubMed

Love, Melissa S; Millholland, Melanie G; Mishra, Satish; Kulkarni, Swapnil; Freeman, Katie B; Pan, Wenxi; Kavash, Robert W; Costanzo, Michael J; Jo, Hyunil; Daly, Thomas M; Williams, Dewight R; Kowalska, M Anna; Bergman, Lawrence W; Poncz, Mortimer; DeGrado, William F; Sinnis, Photini; Scott, Richard W; Greenbaum, Doron C

2012-12-13

Plasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV). PF4 rapidly accumulates only within infected erythrocytes and is required for parasite killing in infected erythrocyte-platelet cocultures. To exploit this antimalarial mechanism, we tested a library of small, nonpeptidic mimics of HDPs (smHDPs) and identified compounds that kill P. falciparum by rapidly lysing the parasite DV while sparing the erythrocyte plasma membrane. Lead smHDPs also reduced parasitemia in a murine malaria model. Thus, identifying host molecules that control parasite growth can further the development of related molecules with therapeutic potential. Copyright © 2012 Elsevier Inc. All rights reserved.

Neutrophils Are Central to Antibody-Mediated Protection against Genital Chlamydia.

PubMed

Naglak, Elizabeth K; Morrison, Sandra G; Morrison, Richard P

2017-10-01

Determining the effector populations involved in humoral protection against genital chlamydia infection is crucial to development of an effective chlamydial vaccine. Antibody has been implicated in protection studies in multiple animal models, and we previously showed that the passive transfer of immune serum alone does not confer immunity in the mouse. Using the Chlamydia muridarum model of genital infection, we demonstrate a protective role for both Chlamydia -specific immunoglobulin G (IgG) and polymorphonuclear neutrophils and show the importance of an antibody/effector cell interaction in mediating humoral immunity. While neutrophils were found to contribute significantly to antibody-mediated protection in vivo , natural killer (NK) cells were dispensable for protective immunity. Furthermore, gamma interferon (IFN-γ)-stimulated primary peritoneal neutrophils (PPNs) killed chlamydiae in vitro in an antibody-dependent manner. The results from this study support the view that an IFN-γ-activated effector cell population cooperates with antibody to protect against genital chlamydia and establish neutrophils as a key effector cell in this response. Copyright © 2017 Naglak et al.

First demonstration of gold nanorods-mediated photodynamic therapeutic destruction of tumors via near infra-red light activation.

PubMed

Vankayala, Raviraj; Huang, Yu-Kuan; Kalluru, Poliraju; Chiang, Chi-Shiun; Hwang, Kuo Chu

2014-04-24

Previously, a large volume of papers reports that gold nanorods (Au NRs) are able to effectively kill cancer cells upon high laser doses (usually 808 nm, 1-48 W/cm²) irradiation, leading to hyperthermia-induced destruction of cancer cells, i.e, photothermal therapy (PTT) effects. Combination of Au NRs-mediated PTT and organic photosensitizers-mediated photodynamic therapy (PDT) were also reported to achieve synergistic PTT and PDT effects on killing cancer cells. Herein, we demonstrate for the first time that Au NRs alone can sensitize formation of singlet oxygen (¹O₂) and exert dramatic PDT effects on complete destrcution of tumors in mice under very low LED/laser doses of single photon NIR (915 nm, <130 mW/cm²) light excitation. By changing the NIR light excitation wavelengths, Au NRs-mediated phototherapeutic effects can be switched from PDT to PTT or combination of both. Both PDT and PTT effects were confirmed by measurements of reactive oxygen species (ROS) and heat shock protein (HSP 70), singlet oxygen sensor green (SOSG) sensing, and sodium azide quenching in cellular experiments. In vivo mice experiments further show that the PDT effect via irradiation of Au NRs by 915 nm can destruct the B16F0 melanoma tumor in mice far more effectively than doxorubicin (a clinically used anti-cancer drug) as well as the PTT effect (via irradiation of Au NRs by 780 nm light). In addition, we show that Au NRs can emit single photon-induced fluorescence to illustrate their in vivo locations/distribution. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pulmonary Regnase-1 orchestrates the interplay of epithelium and adaptive immune systems to protect against pneumonia.

PubMed

Nakatsuka, Yoshinari; Vandenbon, Alexis; Mino, Takashi; Yoshinaga, Masanori; Uehata, Takuya; Cui, Xiaotong; Sato, Ayuko; Tsujimura, Tohru; Suzuki, Yutaka; Sato, Atsuyasu; Handa, Tomohiro; Chin, Kazuo; Sawa, Teiji; Hirai, Toyohiro; Takeuchi, Osamu

2018-04-25

Inhaled pathogens including Pseudomonas aeruginosa initially encounter airway epithelial cells (AECs), which are poised to evoke cell-intrinsic innate defense, affecting second tier of hematopoietic cell-mediated immune reaction. However, it is largely unknown how pulmonary immune responses mediated by a variety of immune cells are coordinated. Here we show that Regnase-1, an endoribonuclease expressed in AECs and immune cells, plays an essential role in coordinating innate responses and adaptive immunity against P. aeruginosa infection. Intratracheal treatment of mice with heat-killed P. aeruginosa resulted in prolonged disappearance of Regnase-1 consistent with sustained expression of Regnase-1 target inflammatory genes, whereas the transcription factor NF-κB was only transiently activated. AEC-specific deletion of Regnase-1 not only augmented innate defenses against P. aeruginosa but also enhanced secretion of Pseudomonas-specific IgA and Th17 accumulation in the lung, culminating in conferring significant resistance against P. aeruginosa re-infection in vivo. Although Regnase-1 directly controls distinct sets of genes in each of AECs and T cells, degradation of Regnase-1 in both cell types is beneficial for maximizing acquired immune responses. Collectively, these results demonstrate that Regnase-1 orchestrates AEC-mediated and immune cell-mediated host defense against pulmonary bacterial infection.

Interactions between macrophage/Kupffer cells and hepatocytes in surgical sepsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

West, M.A.

Experiments were performed to investigate the role of Kupffer cell/macrophage interactions with hepatocytes in modulating liver function during infections using direct in vitro cocultivation of rat macrophages or Kupffer cells with rat hepatocytes. Protein synthesis was assayed as a sensitive indicator of integrated hepatocellular function by measuring {sup 3}H-leucine incorporation into hepatocyte protein. Septic stimuli such as lipoploysaccharide and killed bacteria were added to cocultures of hepatocytes and macrophages or Kupffer cells and the responses compared to hepatocytes alone. Information about the types of proteins synthesized by hepatocytes under various culture conditions was determined using polyacrylamide gel electrophoresis and autoradiography.more » These experiments showed that septic stimuli alter the amount and type of protein synthesized by hepatocytes and had no direct effect on hepatocytes in the absence of macrophages or Kupffer cells. The mediator(s) appears to be a heat labile, soluble monokine(s) which is distinct from interleukin-1 or tumor necrosis factor. The important role of Kupffer cells/macrophages in mediating alterations in hepatocellular function in sepsis may ultimately improve patient care.« less

Nanoparticle Delivery of Artesunate Enhances the Anti-tumor Efficiency by Activating Mitochondria-Mediated Cell Apoptosis

NASA Astrophysics Data System (ADS)

Liu, Rui; Yu, Xiwei; Su, Chang; Shi, Yijie; Zhao, Liang

2017-06-01

Artemisinin and its derivatives were considered to exert a broad spectrum of anti-cancer activities, and they induced significant anti-cancer effects in tumor cells. Artemisinin and its derivatives could be absorbed quickly, and they were widely distributed, selectively killing tumor cells. Since low concentrations of artesunate primarily depended on oncosis to induce cell death in tumor cells, its anti-tumor effects were undesirable and limited. To obtain better anti-tumor effects, in this study, we took advantage of a new nanotechnology to design novel artesunate-loaded bovine serum albumin nanoparticles to achieve the mitochondrial accumulation of artesunate and induce mitochondrial-mediated apoptosis. The results showed that when compared with free artesunate's reliance on oncotic death, artesunate-loaded bovine serum albumin nanoparticles showed higher cytotoxicity and their significant apoptotic effects were induced through the distribution of artesunate in the mitochondria. This finding indicated that artesunate-loaded bovine serum albumin nanoparticles damaged the mitochondrial integrity and activated mitochondrial-mediated cell apoptosis by upregulating apoptosis-related proteins and facilitating the rapid release of cytochrome C.

The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer.

PubMed

Borgeaud, Sandrine; Metzger, Lisa C; Scrignari, Tiziana; Blokesch, Melanie

2015-01-02

Natural competence for transformation is a common mode of horizontal gene transfer and contributes to bacterial evolution. Transformation occurs through the uptake of external DNA and its integration into the genome. Here we show that the type VI secretion system (T6SS), which serves as a predatory killing device, is part of the competence regulon in the naturally transformable pathogen Vibrio cholerae. The T6SS-encoding gene cluster is under the positive control of the competence regulators TfoX and QstR and is induced by growth on chitinous surfaces. Live-cell imaging revealed that deliberate killing of nonimmune cells via competence-mediated induction of T6SS releases DNA and makes it accessible for horizontal gene transfer in V. cholerae. Copyright © 2015, American Association for the Advancement of Science.

Selfish restriction modification genes: resistance of a resident R/M plasmid to displacement by an incompatible plasmid mediated by host killing.

PubMed

Naito, Y; Naito, T; Kobayashi, I

1998-01-01

Previous work from this laboratory demonstrated that plasmids carrying a type II restriction-modification gene complex are not easily lost from their bacterial host because plasmid-free segregant cells are killed through chromosome cleavage. Here, we have followed the course of events that takes place when an Escherichia coli rec BC sbcA strain carrying a plasmid coding for the PaeR7I restriction-modification (R/M) gene complex is transformed by a plasmid with an identical origin of replication. The number of transformants that appeared was far fewer than with the restriction-minus (r-) control. Most of the transformants were very small. After prolonged incubation, the number and the size of the colonies increased, but this increase never attained the level of the r- control. Most of the transformed colonies retained the drug-resistance of the resident, r+ m+ plasmid. These results indicate that post-segregational host killing occurs when a plasmid bearing an R/M gene complex is displaced by an incompatible plasmid. Such cell killing eliminates the competitor plasmid along with the host and, thus, would allow persistence of the R/M plasmid in the neighboring, clonal host cells in nature. This phenomenon is reminiscent of mammalian apoptosis and other forms of altruistic cell death strategy against infection. This type of resistance to displacement was also studied in a wild type Escherichia coli strain that was normal for homologous recombination (rec+). A number of differences between the recBC sbcA strain and the rec+ strain were observed and these will be discussed.

Altering calcium influx for selective destruction of breast tumor.

PubMed

Yu, Han-Gang; McLaughlin, Sarah; Newman, Mackenzie; Brundage, Kathleen; Ammer, Amanda; Martin, Karen; Coad, James

2017-03-04

Human triple-negative breast cancer has limited therapeutic choices. Breast tumor cells have depolarized plasma membrane potential. Using this unique electrical property, we aim to develop an effective selective killing of triple-negative breast cancer. We used an engineered L-type voltage-gated calcium channel (Cec), activated by membrane depolarization without inactivation, to induce excessive calcium influx in breast tumor cells. Patch clamp and flow cytometry were used in testing the killing selectivity and efficiency of human breast tumor cells in vitro. Bioluminescence and ultrasound imaging were used in studies of human triple-negative breast cancer cell MDA-MB-231 xenograft in mice. Histological staining, immunoblotting and immunohistochemistry were used to investigate mechanism that mediates Cec-induced cell death. Activating Cec channels expressed in human breast cancer MCF7 cells produced enormous calcium influx at depolarized membrane. Activating the wild-type Cav1.2 channels expressed in MCF7 cells also produced a large calcium influx at depolarized membrane, but this calcium influx was diminished at the sustained membrane depolarization due to channel inactivation. MCF7 cells expressing Cec died when the membrane potential was held at -10 mV for 1 hr, while non-Cec-expressing MCF7 cells were alive. MCF7 cell death was 8-fold higher in Cec-expressing cells than in non-Cec-expressing cells. Direct injection of lentivirus containing Cec into MDA-MB-231 xenograft in mice inhibited tumor growth. Activated caspase-3 protein was detected only in MDA-MB-231 cells expressing Cec, along with a significantly increased expression of activated caspase-3 in xenograft tumor treated with Cec. We demonstrated a novel strategy to induce constant calcium influx that selectively kills human triple-negative breast tumor cells.

Sublytic complement protects prostate cancer cells from tumour necrosis factor-α-induced cell death.

PubMed

Liu, L; Li, W; Li, Z; Kirschfink, M

2012-08-01

Inflammation is a critical component of tumour progression. Although complement and tumour necrosis factor (TNF)-α potentially exert significant anti-tumour effects, both mediators may also promote tumour progression. It has been demonstrated that sublytic complement confers resistance on tumour cells not only against lytic complement, but also other danger molecules such as perforin. In low concentrations, TNF promotes survival of malignant cells rather than exerting cytotoxic activity. In this study, we tested if sublytic complement is able to interfere with TNF-mediated tumour cell killing. Our results demonstrate that either subcytotoxic concentrations of TNF or sublytic complement rescue prostate carcinoma cells (DU145) from TNF-α-mediated cell death. Upon pretreatment with low-dose TNF-α, but not upon pre-exposure to sublytic complement, TNF resistance was associated with the down-regulation of TNF receptor 1 (TNF-R1) expression. Complement-induced protection against TNF-mediated apoptosis accompanied the induction of anti-apoptotic proteins [B cell leukaemia/lymphoma (Bcl)-2 and Bcl-xL] at an early stage followed by inhibition of the TNF-induced decrease in the amount of Bcl-2 and Bcl-xL. Cell protection also accompanied the inhibition of caspase-8 activation, poly (ADP-ribose) polymerase (PARP)-1 cleavage and the activation of nuclear factor (NF)-κB. Our data extend our current view on the induction of tumour cell resistance against cytotoxic mediators supporting the role of the tumour microenvironment in mediating protection against the anti-cancer immune response. © 2012 The Authors. Clinical and Experimental Immunology © 2012 British Society for Immunology.

Histone deacetylase inhibitor SAHA mediates mast cell death and epigenetic silencing of constitutively active D816V KIT in systemic mastocytosis.

PubMed

Lyberg, Katarina; Ali, Hani Abdulkadir; Grootens, Jennine; Kjellander, Matilda; Tirfing, Malin; Arock, Michel; Hägglund, Hans; Nilsson, Gunnar; Ungerstedt, Johanna

2017-02-07

Systemic mastocytosis (SM) is a clonal bone marrow disorder, where therapeutical options are limited. Over 90% of the patients carry the D816V point mutation in the KIT receptor that renders this receptor constitutively active. We assessed the sensitivity of primary mast cells (MC) and mast cell lines HMC1.2 (D816V mutated), ROSA (KIT WT) and ROSA (KIT D816V) cells to histone deacetylase inhibitor (HDACi) treatment. We found that of four HDACi, suberoyl anilide hydroxamic acid (SAHA) was the most effective in killing mutated MC. SAHA downregulated KIT, followed by major MC apoptosis. Primary SM patient MC cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas primary MC from healthy subjects were less affected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive SM, with almost 100% cell death among MC from the mast cell leukemia patient. Additionally, ROSA (KIT D816V) was more affected by HDACi than ROSA (KIT WT) cells. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/H3 decreased significantly in the KIT region. This epigenetic silencing was seen only in the KIT region and not in control genes upstream and downstream of KIT, indicating that the downregulation of KIT is exerted by specific epigenetic silencing. In conclusion, KIT D816V mutation sensitized MC to HDACi mediated killing, and SAHA may be of value as specific treatment for SM, although the specific mechanism of action requires further investigation.

Histone deacetylase inhibitor SAHA mediates mast cell death and epigenetic silencing of constitutively active D816V KIT in systemic mastocytosis

PubMed Central

Lyberg, Katarina; Ali, Hani Abdulkadir; Grootens, Jennine; Kjellander, Matilda; Tirfing, Malin; Arock, Michel; Hägglund, Hans

2017-01-01

Systemic mastocytosis (SM) is a clonal bone marrow disorder, where therapeutical options are limited. Over 90% of the patients carry the D816V point mutation in the KIT receptor that renders this receptor constitutively active. We assessed the sensitivity of primary mast cells (MC) and mast cell lines HMC1.2 (D816V mutated), ROSA (KIT WT) and ROSA (KIT D816V) cells to histone deacetylase inhibitor (HDACi) treatment. We found that of four HDACi, suberoyl anilide hydroxamic acid (SAHA) was the most effective in killing mutated MC. SAHA downregulated KIT, followed by major MC apoptosis. Primary SM patient MC cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas primary MC from healthy subjects were less affected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive SM, with almost 100% cell death among MC from the mast cell leukemia patient. Additionally, ROSA (KIT D816V) was more affected by HDACi than ROSA (KIT WT) cells. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/H3 decreased significantly in the KIT region. This epigenetic silencing was seen only in the KIT region and not in control genes upstream and downstream of KIT, indicating that the downregulation of KIT is exerted by specific epigenetic silencing. In conclusion, KIT D816V mutation sensitized MC to HDACi mediated killing, and SAHA may be of value as specific treatment for SM, although the specific mechanism of action requires further investigation. PMID:28038453

The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

PubMed

Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

2013-04-25

The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.

NK Cells with KIR2DS2 Immunogenotype Have a Functional Activation Advantage To Efficiently Kill Glioblastoma and Prolong Animal Survival

PubMed Central

Gras Navarro, Andrea; Kmiecik, Justyna; Leiss, Lina; Zelkowski, Mateusz; Engelsen, Agnete; Bruserud, Øystein; Zimmer, Jacques; Enger, Per Øyvind

2014-01-01

Glioblastomas (GBMs) are lethal brain cancers that are resistant to current therapies. We investigated the cytotoxicity of human allogeneic NK cells against patient-derived GBM in vitro and in vivo, as well as mechanisms mediating their efficacy. We demonstrate that KIR2DS2 immunogenotype NK cells were more potent killers, notwithstanding the absence of inhibitory killer Ig–like receptor (KIR)-HLA ligand mismatch. FACS-sorted and enriched KIR2DS2+ NK cell subpopulations retained significantly high levels of CD69 and CD16 when in contact with GBM cells at a 1:1 ratio and highly expressed CD107a and secreted more soluble CD137 and granzyme A. In contrast, KIR2DS2− immunogenotype donor NK cells were less cytotoxic against GBM and K562, and, similar to FACS-sorted or gated KIR2DS2− NK cells, significantly diminished CD16, CD107a, granzyme A, and CD69 when in contact with GBM cells. Furthermore, NK cell–mediated GBM killing in vitro depended upon the expression of ligands for the activating receptor NKG2D and was partially abrogated by Ab blockade. Treatment of GBM xenografts in NOD/SCID mice with NK cells from a KIR2DS2+ donor lacking inhibitory KIR-HLA ligand mismatch significantly prolonged the median survival to 163 d compared with vehicle controls (log-rank test, p = 0.0001), in contrast to 117.5 d (log-rank test, p = 0.0005) for NK cells with several inhibitory KIR-HLA ligand mismatches but lacking KIR2DS2 genotype. Significantly more CD56+CD16+ NK cells from a KIR2DS2+ donor survived in nontumor-bearing brains 3 wk after infusion compared with KIR2DS2− NK cells, independent of their proliferative capacity. In conclusion, KIR2DS2 identifies potent alloreactive NK cells against GBM that are mediated by commensurate, but dominant, activating signals. PMID:25381437

Increased ICAM-1 Expression in Transformed Human Oral Epithelial Cells: Molecular Mechanism and Functional Role in Peripheral Blood Mononuclear Cell Adhesion and Lymphokine-Activated-Killer Cell Cytotoxicity

PubMed Central

Huang, George T.-J.; Zhang, Xinli; Park, No-Hee

2012-01-01

The intercellular adhesion molecule-1 (ICAM-1, CD54) serves as a counter-receptor for the β2-integrins, LFA-1 and Mac-1, which are expressed on leukocytes. Although expression of ICAM-1 on tumor cells has a role in tumor progression and development, information on ICAM-1 expression and its role in oral cancer has not been established. Normal human oral keratinocytes (NHOK), human papilloma virus (HPV)-immortalized human oral keratinocyte lines (HOK-16B, HOK-18A, and HOK-18C), and six human oral neoplastic cell lines (HOK-16B-BaP-T1, SCC-4, SCC-9, HEp-2, Tu-177 and 1483) were used to study ICAM-1 expression and its functional role in vitro. Our results demonstrated that NHOK express negligible levels of ICAM-1, whereas immortalized human oral keratinocytes and cancer cells express significantly higher levels of ICAM-1, except for HOK-16B-BaP-T1 and HEp-2. Altered mRNA half-lives did not fully account for the increased accumulation of ICAM-1 mRNA. Adhesion of peripheral blood mononuclear cells (PBMC) to epithelial cells correlated with cell surface ICAM-1 expression levels. This adhesion was inhibited by antibodies specific for either ICAM-1 or LFA-1/Mac-1, suggesting a role for these molecules in adhesion. In contrast, lymphokine-activated-killer (LAK) cell cytotoxic killing of epithelial cells did not correlate with ICAM-1 levels or with adhesion. Nonetheless, within each cell line, blocking of ICAM-1 or LFA-1/Mac-1 reduced LAK cells killing, suggesting that ICAM-1 is involved in mediating this killing. PMID:10938387

The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation

PubMed Central

Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P.; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S.

2013-01-01

The adaptor molecule signaling lymphocytic activation molecule–associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase–mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)–induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell–mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA–mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS. PMID:23430111

In vitro cytotoxicity of galvanically coupled magnesium-titanium particles on human osteosarcoma SAOS2 cells: A potential cancer therapy.

PubMed

Kim, Jua; Gilbert, Jeremy L

2018-04-10

Osteosarcoma is a malignant bone cancer that occurs mostly in children and young adults. This study investigated the cytotoxicity of Mg and Mg-Ti microparticles to human osteosarcoma cells. Osteosarcoma cells were killed in a dosage-dependent manner when cells, with a cell seeding density of 30,000 cells/cm 2 , were cultured with 0 to 2500 µg/mL of Mg or Mg-Ti in cell culture media for 24-72 h. Mg-Ti killed cells more effectively, where 1250 µg/mL of Mg-Ti killed cells completely by 24 h, while 2500 µg/mL of Mg killed nearly all cells, but not all. Killing due to particle corrosion occurred mostly during the first 24 h, and so the percent cell viability between 24 and 72 h showed not much variability. However, the measurement of live and dead cell numbers, over the timeframe of 24-72 h, showed more insight, such as cell recovery. If particle concentrations were low, the number of live cells increased after 24 h, indicating cell proliferation. If particle concentrations were high, the number of live cells either remained steady or decreased, indicating cell quiescence or continued killing, respectively. Increase in the number of dead cells also indicated killing, while plateau meant discontinued killing. In addition, repeated killing of recovered cells exhibited the same dose-dependent killing profile as the initial experiment, implying little development of cell resistance to treatment. These results, together, show that osteosarcoma cells are susceptible to killing by way of exposure to corroding particles, showing highly effective killing using the galvanic couple of Mg-Ti. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc.

Killing of Pseudomonas aeruginosa by Chicken Cathelicidin-2 Is Immunogenically Silent, Preventing Lung Inflammation In Vivo

PubMed Central

Coorens, Maarten; Banaschewski, Brandon J. H.; Baer, Brandon J.; Yamashita, Cory; van Dijk, Albert; Veldhuizen, Ruud A. W.; Veldhuizen, Edwin J. A.

2017-01-01

ABSTRACT The development of antibiotic resistance by Pseudomonas aeruginosa is a major concern in the treatment of bacterial pneumonia. In the search for novel anti-infective therapies, the chicken-derived peptide cathelicidin-2 (CATH-2) has emerged as a potential candidate, with strong broad-spectrum antimicrobial activity and the ability to limit inflammation by inhibiting Toll-like receptor 2 (TLR2) and TLR4 activation. However, as it is unknown how CATH-2 affects inflammation in vivo, we investigated how CATH-2-mediated killing of P. aeruginosa affects lung inflammation in a murine model. First, murine macrophages were used to determine whether CATH-2-mediated killing of P. aeruginosa reduced proinflammatory cytokine production in vitro. Next, a murine lung model was used to analyze how CATH-2-mediated killing of P. aeruginosa affects neutrophil and macrophage recruitment as well as cytokine/chemokine production in the lung. Our results show that CATH-2 kills P. aeruginosa in an immunogenically silent manner both in vitro and in vivo. Treatment with CATH-2-killed P. aeruginosa showed reduced neutrophil recruitment to the lung as well as inhibition of cytokine and chemokine production, compared to treatment with heat- or gentamicin-killed bacteria. Together, these results show the potential for CATH-2 as a dual-activity antibiotic in bacterial pneumonia, which can both kill P. aeruginosa and prevent excessive inflammation. PMID:28947647

Natural killing and antibody-dependent cellular cytotoxicity are independent immune functions in the Minnesota miniature swine.

PubMed

Koren, H S; Amos, D B; Kim, Y B

1978-10-01

Peripheral blood lymphocytes from Minnesota miniature pigs were tested for natural killing (NK) and antibody-dependent cellular cytotoxicity (ADCC) in a 2- to 4-hr 51Cr release assay against human myeloid and lymphoid tumor target cells. Adult specific pathogen-free and germfree animals exhibited normal levels of activity in both assays. In addition, the NK and ADCC activities of peripheral blood lymphocytes from colostrum-deprived newborn piglets were examined. These animals were obtained by hysterectomy and previously shown to be immunologically "virgin." We found that these newborn piglets exhibited normal ADCC but lacked NK activity. The differences in the ontogeny of the two activities suggest that they are distinct. Preliminary effector cell characterization studies suggest that: (i) NK and ADCC in the pig are physically not separable; (ii) the majority of the cytotoxic activity on a cell-per-cell basis is mediated by the non-T lymphocyte fraction; and (iii) the rosetted T cells, which account for about 60% of the total pig peripheral blood lymphocytes, have low but demonstrable cytotoxic activity as well.

NAADP Activates Two-Pore Channels on T Cell Cytolytic Granules to Stimulate Exocytosis and Killing

PubMed Central

Davis, Lianne C.; Morgan, Anthony J.; Chen, Ji-Li; Snead, Charlotte M.; Bloor-Young, Duncan; Shenderov, Eugene; Stanton-Humphreys, Megan N.; Conway, Stuart J.; Churchill, Grant C.; Parrington, John; Cerundolo, Vincenzo; Galione, Antony

2012-01-01

Summary A cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Ca2+-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. Although inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum activates the store-operated Ca2+-influx pathway that is necessary for exocytosis, it is not a sufficient stimulus [1–4]. Here we identify the Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and its recently identified molecular target, two-pore channels (TPCs) [5–7], as being important for T cell receptor signaling in CTLs. We demonstrate that cytolytic granules are not only reservoirs of cytolytic proteins but are also the acidic Ca2+ stores mobilized by NAADP via TPC channels on the granules themselves, so that TPCs migrate to the immunological synapse upon CTL activation. Moreover, NAADP activates TPCs to drive exocytosis in a way that is not mimicked by global Ca2+ signals induced by IP3 or ionomycin, suggesting that critical, local Ca2+ nanodomains around TPCs stimulate granule exocytosis. Hence, by virtue of the NAADP/TPC pathway, cytolytic granules generate Ca2+ signals that lead to their own exocytosis and to cell killing. This study highlights a selective role for NAADP in stimulating exocytosis crucial for immune cell function and may impact on stimulus-secretion coupling in wider cellular contexts. PMID:23177477

Anti-CD30-targeted gold nanoparticles for photothermal therapy of L-428 Hodgkin’s cell

PubMed Central

Qu, Xiaochao; Yao, Cuiping; Wang, Jing; Li, Zheng; Zhang, Zhenxi

2012-01-01

Purpose Due to the efficient bioconjugation and highly photothermal effect, gold nanoparticles can stain receptor-overexpressing cancer cells through specific targeting of ligands to receptors, strongly absorb specific light and efficiently convert it into heat based on the property of surface plasmon resonance, and then induce the localized protein denaturation and cell death. Methods Two gold nanoparticle–antibody conjugates, gold-BerH2 antibody (anti-CD30 receptor) and gold-ACT1 antibody (anti-CD25-receptor), were synthesized. Gold-BerH2 conjugates can specifically bind to the surface of L-428 Hodgkin’s cells, and gold-ACT1 conjugates were used for the control. The gold nanoparticle-induced L-428 cell-killing experiments were implemented with different experimental parameters. Results At a relatively low concentration of gold and short incubation time, the influence of cytotoxicity of gold on cell viability can be overlooked. Under laser irradiation at suitable power, the high killing efficiency of gold-targeted L-428 cells was achieved, but little damage was done to nontargeted cancer cells. Conclusion Gold nanoparticle-mediated photothermal therapy provides a relatively safe therapeutic technique for cancer treatment. PMID:23269868

Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells

PubMed Central

Choi, Paul J.; Mitchison, Timothy J.

2013-01-01

Cytotoxic lymphocytes eliminate virus-infected and cancerous cells by immune recognition and killing through the perforin-granzyme pathway. Traditional killing assays measure average target cell lysis at fixed times and high effector:target ratios. Such assays obscure kinetic details that might reveal novel physiology. We engineered target cells to report on granzyme activity, used very low effector:target ratios to observe potential serial killing, and performed low magnification time-lapse imaging to reveal time-dependent statistics of natural killer (NK) killing at the single-cell level. Most kills occurred during serial killing, and a single NK cell killed up to 10 targets over a 6-h assay. The first kill was slower than subsequent kills, especially on poor targets, or when NK signaling pathways were partially inhibited. Spatial analysis showed that sequential kills were usually adjacent. We propose that NK cells integrate signals from the previous and current target, possibly by simultaneous contact. The resulting burst kinetics and spatial coordination may control the activity of NK cells in tissues. PMID:23576740

The complex pathophysiology of acquired aplastic anaemia

PubMed Central

Zeng, Y; Katsanis, E

2015-01-01

Immune-mediated destruction of haematopoietic stem/progenitor cells (HSPCs) plays a central role in the pathophysiology of acquired aplastic anaemia (aAA). Dysregulated CD8+ cytotoxic T cells, CD4+ T cells including T helper type 1 (Th1), Th2, regulatory T cells and Th17 cells, natural killer (NK) cells and NK T cells, along with the abnormal production of cytokines including interferon (IFN)-γ, tumour necrosis factor (TNF)-α and transforming growth factor (TGF)-β, induce apoptosis of HSPCs, constituting a consistent and defining feature of severe aAA. Alterations in the polymorphisms of TGF-β, IFN-γ and TNF-α genes, as well as certain human leucocyte antigen (HLA) alleles, may account for the propensity to immune-mediated killing of HSPCs and/or ineffective haematopoiesis. Although the inciting autoantigens remain elusive, autoantibodies are often detected in the serum. In addition, recent studies provide genetic and molecular evidence that intrinsic and/or secondary deficits in HSPCs and bone marrow mesenchymal stem cells may underlie the development of bone marrow failure. PMID:25683099

Chaperone-mediated autophagy degrades mutant p53

PubMed Central

Vakifahmetoglu-Norberg, Helin; Kim, Minsu; Xia, Hong-guang; Iwanicki, Marcin P.; Ofengeim, Dimitry; Coloff, Jonathan L.; Pan, Lifeng; Ince, Tan A.; Kroemer, Guido; Brugge, Joan S.; Yuan, Junying

2013-01-01

Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells. PMID:23913924

Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells

PubMed Central

Sung, Julia A.M.; Pickeral, Joy; Liu, Liqin; Stanfield-Oakley, Sherry A.; Lam, Chia-Ying Kao; Garrido, Carolina; Pollara, Justin; LaBranche, Celia; Bonsignori, Mattia; Moody, M. Anthony; Yang, Yinhua; Parks, Robert; Archin, Nancie; Allard, Brigitte; Kirchherr, Jennifer; Kuruc, JoAnn D.; Gay, Cynthia L.; Cohen, Myron S.; Ochsenbauer, Christina; Soderberg, Kelly; Liao, Hua-Xin; Montefiori, David; Moore, Paul; Johnson, Syd; Koenig, Scott; Haynes, Barton F.; Nordstrom, Jeffrey L.; Margolis, David M.; Ferrari, Guido

2015-01-01

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell–mediated clearance of HIV-1–infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity–mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4+ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8+ T cell clearance of CD4+ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected–patient resting CD4+ T cells. Moreover, DARTs mediated CD8+ T cell clearance of HIV from resting CD4+ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals. PMID:26413868

In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages

PubMed Central

Cockburn, Ian A.; Amino, Rogerio; Kelemen, Reka K.; Kuo, Scot C.; Tse, Sze-Wah; Radtke, Andrea; Mac-Daniel, Laura; Ganusov, Vitaly V.; Zavala, Fidel; Ménard, Robert

2013-01-01

CD8+ T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8+ T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8+ T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8+ T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8+ T cells and signaling by G protein-coupled receptors, although CD8+ T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8+ T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8+ T cells. PMID:23674673

CADM1/TSLC1 Identifies HTLV-1-Infected Cells and Determines Their Susceptibility to CTL-Mediated Lysis

PubMed Central

Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.

2016-01-01

Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax–at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax–HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax−CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+CADM1– cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax–CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus. PMID:27105228

Recognition and killing of brain tumor stem-like initiating cells by CD8+ cytolytic T cells.

PubMed

Brown, Christine E; Starr, Renate; Martinez, Catalina; Aguilar, Brenda; D'Apuzzo, Massimo; Todorov, Ivan; Shih, Chu-Chih; Badie, Behnam; Hudecek, Michael; Riddell, Stanley R; Jensen, Michael C

2009-12-01

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population.

Release of nitric oxide during the T cell-independent pathway of macrophage activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beckerman, K.P.; Rogers, H.W.; Corbett, J.A.

1993-02-01

Immunodeficient mice are remarkably resistant to Listeria monocytogenes (LM) infection. The authors examined the role that nitric oxide (NO) plays in the CB-17/lcr SCID (SCID) response to LM. SCID spleen cells produced large quantities of NO (as measured by nitrite formation) when incubated in the presence of heat-killed LM. NO produced large quantities of nitrite in response to LM, but only in the presence of IFN-[gamma]. The production of NO induced by LM was not affected by neutralizing antibodies to TNF or IL-1. The production of NO was inhibited by addition of either of two inhibitors of NO synthase, N[supmore » G]-monomethyl arginine, or aminoguanidine. In a different situation, NK cells that were stimulated by TNF and Listeria products to release IFN-[gamma] did not produce NO. Macrophages cultured with IFN-[gamma] killed live LM. This increased killing of LM was significantly inhibited by amino-guanidine. In vivo, administration of aminoguanidine resulted in a marked increase in the mortality and spleen bacterial loads of LM-infected SCID or immunocompetent control mice. It is concluded that NO is a critical effector molecule of T cell-independent natural resistence of LM as studied in the SCID mouse, and that the NO-mediated response is essential for both SCID and immunocompetent host to survive after LM infection. 37 refs., 7 figs.« less

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

PubMed Central

Calow, Jenny; Bockau, Ulrike; Struwe, Weston B.; Nowaczyk, Marc M.; Loser, Karin; Crispin, Max

2016-01-01

ABSTRACT Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity. PMID:27594301

Effect of space flight on cell-mediated immunity

NASA Technical Reports Server (NTRS)

Mandel, A. D.; Balish, E.

1977-01-01

The cell-mediated immune response to Listeria monocytogenes was studied in rats subjected to 20 days of flight aboard the Soviet biosatellite Kosmos 7820. Groups of rats were immunized with 1,000,000 formalin-killed Listeria suspended in Freunds Complete Adjuvant, 5 days prior to flight. Immunized rats subjected to the same environmental factors as the flight rats, except flight itself, and immunized and nonimmunized rats held in a normal animal colony served as controls. Following recovery, lymphocyte cultures were harvested from spleens of all rats, cultured in vitro in the presence of L. monocytogenes antigens, Phytohemagglutinin, Conconavlin A, or purified protein derivative (PPD), and measured for their uptake of H-3-thymidine. Although individual rats varied considerably, all flight and immunized control rats gave a blastogenic response to the Listeria antigens and PPD. With several mitogens, the lymphocytes of flight rats showed a significantly increased blastogenic response over the controls. The results of this study do not support a hypothesis of a detrimental effect of space flight on cell-mediated immunity. The data suggest a possible suppressive effect of stress and gravity on an in vitro correlate of cell-mediated immunity.

Adoptive transfer of natural killer cells promotes the anti-tumor efficacy of T cells.

PubMed

Goding, Stephen R; Yu, Shaohong; Bailey, Lisa M; Lotze, Michael T; Basse, Per H

2017-04-01

The density of NK cells in tumors correlates positively with prognosis in many types of cancers. The average number of infiltrating NK cells is, however, quite modest (approximately 30 NK cells/sq.mm), even in tumors deemed to have a "high" density of infiltrating NK cells. It is unclear how such low numbers of tumor-infiltrating NK cells can influence outcome. Here, we used ovalbumin-expressing tumor cell lines and TCR transgenic, OVA-specific cytotoxic T lymphocytes (OT-I-CTLs) to determine whether the simultaneous attack by anti-tumor CTLs and IL-2-activated NK (A-NK) cells synergistically increases the overall tumor cell kill and whether upregulation of tumor MHC class-I by NK cell-derived interferon-gamma (IFNγ) improves tumor-recognition and kill by anti-tumor CTLs. At equal E:T ratios, A-NK cells killed OVA-expressing tumor cells better than OT-I-CTLs. The cytotoxicity against OVA-expressing tumor cells increased by combining OT-I-CTLs and A-NK cells, but the increase was additive rather than synergistic. A-NK cells adenovirally-transduced to produce IL-12 (A-NK IL-12 ) produced high amounts of IFNγ. The addition of a low number of A-NK IL-12 cells to OT-I-CTLs resulted in a synergistic, albeit modest, increase in overall cytotoxicity. Pre-treatment of tumor cells with NK cell-conditioned medium increased tumor MHC expression and sensitivity to CTL-mediated killing. Pre-treatment of CTLs with NK cell-conditioned medium had no effect on CTL cytotoxicity. In vivo, MHC class-I expression by OVA-expressing B16 melanoma lung metastases increased significantly within 24-48h after adoptive transfer of A-NK IL-12 cells. OT-I-CTLs and A-NK IL-12 cells localized selectively and equally well into OVA-expressing B16 lung metastases and treatment of mice bearing 7-days-old OVA-B16 lung metastases with both A-NK IL-12 cells and OT-I-CTLs lead to a significant prolongation of survival. Thus, an important function of tumor-infiltrating NK cells may be to increase tumor cell expression of MHC class-I through secretion of IFNγ, to prepare them for recognition by tumor-specific CTLs. Copyright © 2016 Elsevier Inc. All rights reserved.

Innate NK cells and macrophages recognize and reject allogeneic nonself in vivo via different mechanisms.

PubMed

Liu, Wentao; Xiao, Xiang; Demirci, Gulcin; Madsen, Joren; Li, Xian C

2012-03-15

Both innate and adaptive immune cells are involved in the allograft response. But how the innate immune cells respond to allotransplants remains poorly defined. In the current study, we examined the roles of NK cells and macrophages in recognizing and rejecting allogeneic cells in vivo. We found that in naive mice NK cells are the primary effector cells in the killing of allogeneic cells via "missing self" recognition. However, in alloantigen-presensitized mice, NK cells are dispensable. Instead, macrophages become alloreactive and readily recognize and reject allogeneic nonself. This effect requires help from activated CD4(+) T cells and involves CD40/CD40L engagement, because blocking CD40/CD40L interactions prevents macrophage-mediated rejection of allogeneic cells. Conversely, actively stimulating CD40 triggers macrophage-mediated rejection in the absence of CD4(+) T cells. Importantly, alloantigen-primed and CD4(+) T cell-helped macrophages (licensed macrophages) exhibit potent regulatory function in vivo in an acute graft-versus-host disease model. Together, our data uncover an important role for macrophages in the alloimmune response and may have important clinical implications.

Apoptin towards safe and efficient anticancer therapies.

PubMed

Backendorf, Claude; Noteborn, Mathieu H M

2014-01-01

The chicken anemia virus derived protein apoptin harbors cancer-selective cell killing characteristics, essentially based on phosphorylation-mediated nuclear transfer in cancer cells and efficient cytoplasmic degradation in normal cells. Here, we describe a growing set of preclinical experiments underlying the promises of the anti-cancer potential of apoptin. Various non-replicative oncolytic viral vector systems have revealed the safety and efficacy of apoptin. In addition, apoptin enhanced the oncolytic potential of adenovirus, parvovirus and Newcastle disease virus vectors. Intratumoral injection of attenuated Salmonella typhimurium bacterial strains and plasmid-based systems expressing apoptin resulted in significant tumor regression. In-vitro and in-vivo experiments showed that recombinant membrane-transferring PTD4- or TAT-apoptin proteins have potential as a future anticancer therapeutics. In xenografted hepatoma and melanoma mouse models PTD4-apoptin protein entered both cancer and normal cells, but only killed cancer cells. Combinatorial treatment of PTD4-apoptin with various (chemo)therapeutic compounds revealed an additive or even synergistic effect, reducing the side effects of the single (chemo)therapeutic treatment. Degradable polymeric nanocapsules harboring MBP-apoptin fusion-protein induced tumor-selective cell killing in-vitro and in-vivo and revealed the potential of polymer-apoptin protein vehicles as an anticancer agent.Besides its direct use as an anticancer therapeutic, apoptin research has also generated novel possibilities for drug design. The nuclear location domains of apoptin are attractive tools for targeting therapeutic compounds into the nucleus of cancer cells. Identification of cancer-related processes targeted by apoptin can potentially generate novel drug targets. Recent breakthroughs important for clinical applications are reported inferring apoptin-based clinical trials as a feasible reality.

Truncated Autoinducing Peptide Conjugates Selectively Recognize and Kill Staphylococcus aureus.

PubMed

Tsuchikama, Kyoji; Shimamoto, Yasuhiro; Anami, Yasuaki

2017-06-09

The accessory gene regulator (agr) of Staphylococcus aureus coordinates various pathogenic events and is recognized as a promising therapeutic target for virulence control. S. aureus utilizes autoinducing peptides (AIPs), cyclic-peptide signaling molecules, to mediate the agr system. Despite the high potency of synthetic AIP analogues in agr inhibition, the potential of AIP molecules as a delivery vehicle for antibacterial agents remains unexplored. Herein, we report that truncated AIP scaffolds can be fused with fluorophore and cytotoxic photosensitizer molecules without compromising their high agr inhibitory activity, binding affinity to the receptor AgrC, or cell specificity. Strikingly, a photosensitizer-AIP conjugate exhibited 16-fold greater efficacy in a S. aureus cell-killing assay than a nontargeting analogue. These findings highlight the potential of truncated AIP conjugates as useful chemical tools for in-depth biological studies and as effective anti-S. aureus agents.

Minimization of bacterial size allows for complement evasion and is overcome by the agglutinating effect of antibody

PubMed Central

Dalia, Ankur B.; Weiser, Jeffrey N.

2011-01-01

SUMMARY The complement system, which functions by lysing pathogens directly or by promoting their uptake by phagocytes, is critical for controlling many microbial infections. Here we show that in Streptococcus pneumoniae, increasing bacterial chain length sensitizes this pathogen to complement deposition and subsequent uptake by human neutrophils. Consistent with this, we show that minimizing chain length provides wild-type bacteria with a competitive advantage in vivo in a model of systemic infection. Investigating how the host overcomes this virulence strategy, we find that antibody promotes complement-dependent opsonophagocytic killing of Streptococcus pneumoniae and lysis of Haemophilus influenzae independent of Fc-mediated effector functions. Consistent with the agglutinating effect of antibody, F(ab′)2 but not Fab could promote this effect. Therefore, increasing pathogen size, whether by natural changes in cellular morphology or via antibody-mediated agglutination, promotes complement-dependent killing. These observations have broad implications for how cell size and morphology can affect virulence among pathogenic microbes. PMID:22100164

Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy.

PubMed

Portillo, Jose-Andres C; Muniz-Feliciano, Luis; Lopez Corcino, Yalitza; Lee, So Jung; Van Grol, Jennifer; Parsons, Sarah J; Schiemman, William P; Subauste, Carlos S

2017-10-01

Targeting of Toxoplasma gondii by autophagy is an effective mechanism by which host cells kill the protozoan. Thus, the parasite must avoid autophagic targeting to survive. Here we show that the mammalian cytoplasmic molecule Focal Adhesion Kinase (FAK) becomes activated during invasion of host cells. Activated FAK appears to accompany the formation of the moving junction (as assessed by expression the parasite protein RON4). FAK activation was inhibited by approaches that impaired β1 and β3 integrin signaling. FAK caused activation of Src that in turn mediated Epidermal Growth Factor Receptor (EGFR) phosphorylation at the unique Y845 residue. Expression of Src-resistant Y845F EGFR mutant markedly inhibited ROP16-independent activation of STAT3 in host cells. Activation of FAK, Y845 EGFR or STAT3 prevented activation of PKR and eIF2α, key stimulators of autophagy. Genetic or pharmacologic inhibition of FAK, Src, EGFR phosphorylation at Y845, or STAT3 caused accumulation of the autophagy protein LC3 and LAMP-1 around the parasite and parasite killing dependent on autophagy proteins (ULK1 and Beclin 1) and lysosomal enzymes. Parasite killing was inhibited by expression of dominant negative PKR. Thus, T. gondii activates a FAK→Src→Y845-EGFR→STAT3 signaling axis within mammalian cells, thereby enabling the parasite to survive by avoiding autophagic targeting through a mechanism likely dependent on preventing activation of PKR and eIF2α.

Insufficient natural killer cell responses against retroviruses: how to improve NK cell killing of retrovirus-infected cells.

PubMed

Littwitz-Salomon, Elisabeth; Dittmer, Ulf; Sutter, Kathrin

2016-11-08

Natural killer (NK) cells belong to the innate immune system and protect against cancers and a variety of viruses including retroviruses by killing transformed or infected cells. They express activating and inhibitory receptors on their cell surface and often become activated after recognizing virus-infected cells. They have diverse antiviral effector functions like the release of cytotoxic granules, cytokine production and antibody dependent cellular cytotoxicity. The importance of NK cell activity in retroviral infections became evident due to the discovery of several viral strategies to escape recognition and elimination by NK cells. Mutational sequence polymorphisms as well as modulation of surface receptors and their ligands are mechanisms of the human immunodeficiency virus-1 to evade NK cell-mediated immune pressure. In Friend retrovirus infected mice the virus can manipulate molecular or cellular immune factors that in turn suppress the NK cell response. In this model NK cells lack cytokines for optimal activation and can be functionally suppressed by regulatory T cells. However, these inhibitory pathways can be overcome therapeutically to achieve full activation of NK cell responses and ultimately control dissemination of retroviral infection. One effective approach is to modulate the crosstalk between NK cells and dendritic cells, which produce NK cell-stimulating cytokines like type I interferons (IFN), IL-12, IL-15, and IL-18 upon retrovirus sensing or infection. Therapeutic administration of IFNα directly increases NK cell killing of retrovirus-infected cells. In addition, IL-2/anti-IL-2 complexes that direct IL-2 to NK cells have been shown to significantly improve control of retroviral infection by NK cells in vivo. In this review, we describe novel approaches to improve NK cell effector functions in retroviral infections. Immunotherapies that target NK cells of patients suffering from viral infections might be a promising treatment option for the future.

Antagonists of growth hormone-releasing hormone receptor induce apoptosis specifically in retinoblastoma cells.

PubMed

Chu, Wai Kit; Law, Ka Sin; Chan, Sun On; Yam, Jason Cheuk Sing; Chen, Li Jia; Zhang, Hao; Cheung, Herman S; Block, Norman L; Schally, Andrew V; Pang, Chi Pui

2016-12-13

Retinoblastoma (RB) is the most common intraocular cancer in children worldwide. Current treatments mainly involve combinations of chemotherapies, cryotherapies, and laser-based therapies. Severe or late-stage disease may require enucleation or lead to fatality. Recently, RB has been shown to arise from cone precursor cells, which have high MDM2 levels to suppress p53-mediated apoptosis. This finding leads to the hypothesis that restoring apoptosis mechanisms in RBs could specifically kill the cancer cells without affecting other retinal cells. We have previously reported involvement of an extrapituitary signaling pathway of the growth hormone-releasing hormone (GHRH) in the retina. Here we show that the GHRH receptor (GHRH-R) is highly expressed in RB cells but not in other retinal cells. We induced specific apoptosis with two different GHRH-R antagonists, MIA-602 and MIA-690. Importantly, these GHRH-R antagonists do not trigger apoptosis in other retinal cells such as retinal pigmented epithelial cells. We delineated the gene expression profiles regulated by GHRH-R antagonists and found that cell proliferation genes and apoptotic genes are down- and up-regulated, respectively. Our results reveal the involvement of GHRH-R in survival and proliferation of RB and demonstrate that GHRH-R antagonists can specifically kill the RB cells.

Zika Virus Escapes NK Cell Detection by Upregulating Major Histocompatibility Complex Class I Molecules.

PubMed

Glasner, Ariella; Oiknine-Djian, Esther; Weisblum, Yiska; Diab, Mohammad; Panet, Amos; Wolf, Dana G; Mandelboim, Ofer

2017-11-15

NK cells are innate lymphocytes that participate in many immune processes encompassing cancer, bacterial and fungal infection, autoimmunity, and even pregnancy and that specialize in antiviral defense. NK cells express inhibitory and activating receptors and kill their targets when activating signals overpower inhibitory signals. The NK cell inhibitory receptors include a uniquely diverse array of proteins named killer cell immunoglobulin-like receptors (KIRs), the CD94 family, and the leukocyte immunoglobulin-like receptor (LIR) family. The NK cell inhibitory receptors recognize mostly major histocompatibility complex (MHC) class I (MHC-I) proteins. Zika virus has recently emerged as a major threat due to its association with birth defects and its pandemic potential. How Zika virus interacts with the immune system, and especially with NK cells, is unclear. Here we show that Zika virus infection is barely sensed by NK cells, since little or no increase in the expression of activating NK cell ligands was observed following Zika infection. In contrast, we demonstrate that Zika virus infection leads to the upregulation of MHC class I proteins and consequently to the inhibition of NK cell killing. Mechanistically, we show that MHC class I proteins are upregulated via the RIGI-IRF3 pathway and that this upregulation is mediated via beta interferon (IFN-β). Potentially, countering MHC class I upregulation during Zika virus infection could be used as a prophylactic treatment against Zika virus. IMPORTANCE NK cells are innate lymphocytes that recognize and eliminate various pathogens and are known mostly for their role in controlling viral infections. NK cells express inhibitory and activating receptors, and they kill or spare their targets based on the integration of inhibitory and activating signals. Zika virus has recently emerged as a major threat to humans due to its pandemic potential and its association with birth defects. The role of NK cells in Zika virus infection is largely unknown. Here we demonstrate that Zika virus infection is almost undetected by NK cells, as evidenced by the fact that the expression of activating ligands for NK cells is not induced following Zika infection. We identified a mechanism whereby Zika virus sensing via the RIGI-IRF3 pathway resulted in IFN-β-mediated upregulation of MHC-I molecules and inhibition of NK cell activity. Countering MHC class I upregulation and boosting NK cell activity may be employed as prophylactic measures to combat Zika virus infection. Copyright © 2017 American Society for Microbiology.

Zika Virus Escapes NK Cell Detection by Upregulating Major Histocompatibility Complex Class I Molecules

PubMed Central

Glasner, Ariella; Oiknine-Djian, Esther; Weisblum, Yiska; Diab, Mohammad; Panet, Amos; Wolf, Dana G.

2017-01-01

ABSTRACT NK cells are innate lymphocytes that participate in many immune processes encompassing cancer, bacterial and fungal infection, autoimmunity, and even pregnancy and that specialize in antiviral defense. NK cells express inhibitory and activating receptors and kill their targets when activating signals overpower inhibitory signals. The NK cell inhibitory receptors include a uniquely diverse array of proteins named killer cell immunoglobulin-like receptors (KIRs), the CD94 family, and the leukocyte immunoglobulin-like receptor (LIR) family. The NK cell inhibitory receptors recognize mostly major histocompatibility complex (MHC) class I (MHC-I) proteins. Zika virus has recently emerged as a major threat due to its association with birth defects and its pandemic potential. How Zika virus interacts with the immune system, and especially with NK cells, is unclear. Here we show that Zika virus infection is barely sensed by NK cells, since little or no increase in the expression of activating NK cell ligands was observed following Zika infection. In contrast, we demonstrate that Zika virus infection leads to the upregulation of MHC class I proteins and consequently to the inhibition of NK cell killing. Mechanistically, we show that MHC class I proteins are upregulated via the RIGI-IRF3 pathway and that this upregulation is mediated via beta interferon (IFN-β). Potentially, countering MHC class I upregulation during Zika virus infection could be used as a prophylactic treatment against Zika virus. IMPORTANCE NK cells are innate lymphocytes that recognize and eliminate various pathogens and are known mostly for their role in controlling viral infections. NK cells express inhibitory and activating receptors, and they kill or spare their targets based on the integration of inhibitory and activating signals. Zika virus has recently emerged as a major threat to humans due to its pandemic potential and its association with birth defects. The role of NK cells in Zika virus infection is largely unknown. Here we demonstrate that Zika virus infection is almost undetected by NK cells, as evidenced by the fact that the expression of activating ligands for NK cells is not induced following Zika infection. We identified a mechanism whereby Zika virus sensing via the RIGI-IRF3 pathway resulted in IFN-β-mediated upregulation of MHC-I molecules and inhibition of NK cell activity. Countering MHC class I upregulation and boosting NK cell activity may be employed as prophylactic measures to combat Zika virus infection. PMID:28878071

Impaired removal of Vβ8(+) lymphocytes aggravates colitis in mice deficient for B cell lymphoma-2-interacting mediator of cell death (Bim).

PubMed

Leucht, K; Caj, M; Fried, M; Rogler, G; Hausmann, M

2013-09-01

We investigated the role of B cell lymphoma (BCL)-2-interacting mediator of cell death (Bim) for lymphocyte homeostasis in intestinal mucosa. Lymphocytes lacking Bim are refractory to apoptosis. Chronic colitis was induced in Bim-deficient mice (Bim(-/-) ) with dextran sulphate sodium (DSS). Weight loss and colonoscopic score were increased significantly in Bim(-/-) mice compared to wild-type mice. As Bim is induced for the killing of autoreactive cells we determined the role of Bim in the regulation of lymphocyte survival at mucosal sites. Upon chronic dextran sulphate sodium (DSS)-induced colitis, Bim(-/-) animals exhibited an increased infiltrate of lymphocytes into the mucosa compared to wild-type mice. The number of autoreactive T cell receptor (TCR) Vβ8(+) lymphocytes was significantly higher in Bim(-/-) mice compared to wild-type controls. Impaired removal of autoreactive lymphocytes in Bim(-/-) mice upon chronic DSS-induced colitis may therefore contribute to aggravated mucosal inflammation. © 2013 British Society for Immunology.

Ca(2+)-mediated prostaglandin E2 induction reduces haematoporphyrin-derivative-induced cytotoxicity of T24 human bladder transitional carcinoma cells in vitro.

PubMed Central

Penning, L C; Keirse, M J; VanSteveninck, J; Dubbelman, T M

1993-01-01

The effects of haematoporphyrin-derivative-mediated photodynamic treatment on arachidonic acid metabolism and its relation to clonogenicity have been studied in human bladder-tumour cells. Photodynamic treatment resulted in a transient release of arachidonic acid-derived compounds; prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) especially were strongly increased. This release was reduced by chelation of intracellular Ca2+ with Quin-2 or by lowering the extracellular Ca2+ concentration in the medium with EGTA, presumably resulting in inhibition of phospholipase A2. A similar reduction was obtained when indomethacin, an inhibitor of the cyclo-oxygenase pathway, was added prior to light exposure. These three treatments enhanced the photosensitivity, as revealed by the clonogenicity assay. Incubation with PGE2 prior to light exposure, but not with TXB2, protected against reproductive-cell death. The results of these experiments suggest that Ca(2+)-mediated activation of cyclo-oxygenase, resulting in increased levels of PGE2, participates in a cellular-defence mechanism against photodynamic cell killing. PMID:8503851

Hypoxia Induced Impairment of NK Cell Cytotoxicity against Multiple Myeloma Can Be Overcome by IL-2 Activation of the NK Cells

PubMed Central

Sarkar, Subhashis; Germeraad, Wilfred T. V.; Rouschop, Kasper M. A.; Steeghs, Elisabeth M. P.; van Gelder, Michel; Bos, Gerard M. J.; Wieten, Lotte

2013-01-01

Background Multiple Myeloma (MM) is an incurable plasma cell malignancy residing within the bone marrow (BM). We aim to develop allogeneic Natural Killer (NK) cell immunotherapy for MM. As the BM contains hypoxic regions and the tumor environment can be immunosuppressive, we hypothesized that hypoxia inhibits NK cell anti-MM responses. Methods NK cells were isolated from healthy donors by negative selection and NK cell function and phenotype were examined at oxygen levels representative of hypoxic BM using flowcytometry. Additionally, NK cells were activated with IL-2 to enhance NK cell cytotoxicity under hypoxia. Results Hypoxia reduced NK cell killing of MM cell lines in an oxygen dependent manner. Under hypoxia, NK cells maintained their ability to degranulate in response to target cells, though, the percentage of degranulating NK cells was slightly reduced. Adaptation of NK- or MM cells to hypoxia was not required, hence, the oxygen level during the killing process was critical. Hypoxia did not alter surface expression of NK cell ligands (HLA-ABC, -E, MICA/B and ULBP1-2) and receptors (KIR, NKG2A/C, DNAM-1, NCRs and 2B4). It did, however, decrease expression of the activating NKG2D receptor and of intracellular perforin and granzyme B. Pre-activation of NK cells by IL-2 abrogated the detrimental effects of hypoxia and increased NKG2D expression. This emphasized that activated NK cells can mediate anti-MM effects, even under hypoxic conditions. Conclusions Hypoxia abolishes the killing potential of NK cells against multiple myeloma, which can be restored by IL-2 activation. Our study shows that for the design of NK cell-based immunotherapy it is necessary to study biological interactions between NK- and tumor cells also under hypoxic conditions. PMID:23724099

The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection.

PubMed

Rajalingam, Raja

2016-01-01

Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell

PubMed Central

Wang, Qianli; Zhang, Junping; Guo, Zhongwu

2007-01-01

To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine. PMID:17892942

Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell.

PubMed

Wang, Qianli; Zhang, Junping; Guo, Zhongwu

2007-12-15

To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine.

Beyond the Matrix: The Many Non-ECM Ligands for Integrins

PubMed Central

LaFoya, Bryce; Munroe, Jordan A.; Miyamoto, Alison; Detweiler, Michael A.; Crow, Jacob J.; Gazdik, Tana

2018-01-01

The traditional view of integrins portrays these highly conserved cell surface receptors as mediators of cellular attachment to the extracellular matrix (ECM), and to a lesser degree, as coordinators of leukocyte adhesion to the endothelium. These canonical activities are indispensable; however, there is also a wide variety of integrin functions mediated by non-ECM ligands that transcend the traditional roles of integrins. Some of these unorthodox roles involve cell-cell interactions and are engaged to support immune functions such as leukocyte transmigration, recognition of opsonization factors, and stimulation of neutrophil extracellular traps. Other cell-cell interactions mediated by integrins include hematopoietic stem cell and tumor cell homing to target tissues. Integrins also serve as cell-surface receptors for various growth factors, hormones, and small molecules. Interestingly, integrins have also been exploited by a wide variety of organisms including viruses and bacteria to support infectious activities such as cellular adhesion and/or cellular internalization. Additionally, the disruption of integrin function through the use of soluble integrin ligands is a common strategy adopted by several parasites in order to inhibit blood clotting during hematophagy, or by venomous snakes to kill prey. In this review, we strive to go beyond the matrix and summarize non-ECM ligands that interact with integrins in order to highlight these non-traditional functions of integrins. PMID:29393909

High-Precision Photothermal Ablation Using Biocompatible Palladium Nanoparticles and Laser Scanning Microscopy

PubMed Central

2018-01-01

Herein, we report a straightforward method for the scalable preparation of Pd nanoparticles (Pd-NPs) with reduced inherent cytotoxicity and high photothermal conversion capacity. These Pd-NPs are rapidly taken up by cells and able to kill labeled cancer cells upon short exposure to near-infrared (NIR) light. Following cell treatment with Pd-NPs, ablated areas were patterned with high precision by laser scanning microscopy, allowing one to perform cell migration assays with unprecedented accuracy. Using coherent Raman microscopy, cells containing Pd-NPs were simultaneously ablated and imaged. This novel methodology was combined with intravital imaging to mediate microablation of cancerous tissue in tumor xenografts in mice. PMID:29320154

Preclinical in vitro and in vivo studies to examine the potential use of photodynamic therapy in the treatment of osteomyelitis

NASA Astrophysics Data System (ADS)

Bisland, Stuart K.; Chien, Claudia; Wilson, Brian C.; Burch, Shane

2005-04-01

Osteomyelitis can lead to severe morbidity and even death resulting from an acute or chronic inflammation of the bone and contiguous structures due to fungal or bacterial infection. Incidence approximates 1 in 1,000 neonates and 1 in 5,000 children in the United States annually and increases up to 0.36% and 16% in adults with diabetes or sickle cell anaemia, respectively. Current regiments of treatment include antibiotics and/or surgery. However, the increasing number of antibiotic resistant pathogens suggests that alternate strategies are required. We are investigating photodynamic therapy (PDT) as one such alternate treatment for osteomyelitis using a bioluminescent strain of biofilm-producing staphylococcus aureus (SA) grown onto kirschner wires (K-wire). SA-coated K-wires were exposed to methylene blue (MB) or 5-aminolevulinic acid (ALA)-mediated PDT either in vitro or following implant into the tibial medullary cavity of Sprague-Dawley rats. The progression of SA biofilm was monitored non-invasively using bioluminescence and expressed as a percentage of the signal for each sample immediately prior to treatment. SA infections were subject to PDT 10 days post inoculation. Treatment comprised administration of ALA (300 mg/Kg) intraperitoneally followed 4 hr later by light (635 +/- 10 nm; 38 or 75 J/cm2) delivered transcutaneously via an optical fiber placed onto the tibia. In vitro, MB and ALA displayed similar cell kill with >= 4log10 cell kill. In vivo, ALA-mediated PDT inhibited biofilm implants in bone. These results confirm that MB or ALA-mediated PDT have potential to treat SA cultures grown in vitro or in vivo using an animal model of osteomyelitis.

The TRK1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, Histatin 5.

PubMed

Baev, Didi; Rivetta, Alberto; Vylkova, Slavena; Sun, Jianing N; Zeng, Ge-Fei; Slayman, Clifford L; Edgerton, Mira

2004-12-31

The principal feature of killing of Candida albicans and other pathogenic fungi by the catonic protein Histatin 5 (Hst 5) is loss of cytoplasmic small molecules and ions, including ATP and K(+), which can be blocked by the anion channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. We constructed C. albicans strains expressing one, two, or three copies of the TRK1 gene in order to investigate possible roles of Trk1p (the organism's principal K(+) transporter) in the actions of Hst 5. All measured parameters (Hst 5 killing, Hst 5-stimulated ATP efflux, normal Trk1p-mediated K(+) ((86)Rb(+)) influx, and Trk1p-mediated chloride conductance) were similarly reduced (5-7-fold) by removal of a single copy of the TRK1 gene from this diploid organism and were fully restored by complementation of the missing allele. A TRK1 overexpression strain of C. albicans, constructed by integrating an additional TRK1 gene into wild-type cells, demonstrated cytoplasmic sequestration of Trk1 protein, along with somewhat diminished toxicity of Hst 5. These results could be produced either by depletion of intracellular free Hst 5 due to sequestered binding, or to cooperativity in Hst 5-protein interactions at the plasma membrane. Furthermore, Trk1p-mediated chloride conductance was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in all of the tested strains, strongly suggesting that the TRK1 protein provides the essential pathway for ATP loss and is the critical effector for Hst 5 toxicity in C. albicans.

Bisphosphonates inactivate human EGFRs to exert antitumor actions

PubMed Central

Yuen, Tony; Stachnik, Agnes; Iqbal, Jameel; Sgobba, Miriam; Gupta, Yogesh; Lu, Ping; Colaianni, Graziana; Ji, Yaoting; Zhu, Ling-Ling; Kim, Se-Min; Li, Jianhua; Liu, Peng; Izadmehr, Sudeh; Sangodkar, Jaya; Bailey, Jack; Latif, Yathin; Mujtaba, Shiraz; Epstein, Solomon; Davies, Terry F.; Bian, Zhuan; Zallone, Alberta; Aggarwal, Aneel K.; Haider, Shozeb; New, Maria I.; Sun, Li; Narla, Goutham; Zaidi, Mone

2014-01-01

Bisphosphonates are the most commonly prescribed medicines for osteoporosis and skeletal metastases. The drugs have also been shown to reduce cancer progression, but only in certain patient subgroups, suggesting that there is a molecular entity that mediates bisphosphonate action on tumor cells. Using connectivity mapping, we identified human epidermal growth factor receptors (human EGFR or HER) as a potential new molecular entity for bisphosphonate action. Protein thermal shift and cell-free kinase assays, together with computational modeling, demonstrated that N-containing bisphosphonates directly bind to the kinase domain of HER1/2 to cause a global reduction in downstream signaling. By doing so, the drugs kill lung, breast, and colon cancer cells that are driven by activating mutations or overexpression of HER1. Knocking down HER isoforms thus abrogates cell killing by bisphosphonates, establishing complete HER dependence and ruling out a significant role for other receptor tyrosine kinases or the enzyme farnesyl pyrophosphate synthase. Consistent with this finding, colon cancer cells expressing low levels of HER do not respond to bisphosphonates. The results suggest that bisphosphonates can potentially be repurposed for the prevention and therapy of HER family-driven cancers. PMID:25453081

Aag DNA Glycosylase Promotes Alkylation-Induced Tissue Damage Mediated by Parp1

PubMed Central

Calvo, Jennifer A.; Moroski-Erkul, Catherine A.; Lake, Annabelle; Eichinger, Lindsey W.; Shah, Dharini; Jhun, Iny; Limsirichai, Prajit; Bronson, Roderick T.; Christiani, David C.; Meira, Lisiane B.; Samson, Leona D.

2013-01-01

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag −/− mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage. PMID:23593019

Nuclear thioredoxin-1 is required to suppress cisplatin-mediated apoptosis of MCF-7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Xiao-Ping; Liu, Shou; Tang, Wen-Xin

2007-09-21

Different cell line with increased thioredoxin-1 (Trx-1) showed a decreased or increased sensitivity to cell killing by cisplatin. Recently, several studies found that the subcellular localization of Trx-1 is closely associated with its functions. In this study, we explored the association of the nuclear Trx-1 with the cisplatin-mediated apoptosis of breast cancer cells MCF-7. Firstly, we found that higher total Trx-1 accompanied by no change of nuclear Trx-1 can not influence apoptosis induced by cisplatin in MCF-7 cells transferred with Trx-1 cDNA. Secondly, higher nuclear Trx-1 accompanied by no change of total Trx-1 can protect cells from apoptosis induced bymore » cisplatin. Thirdly, high nuclear Trx-1 involves in the cisplatin-resistance in cisplatin-resistive cells. Meanwhile, we found that the mRNA level of p53 is closely correlated with the level of nuclear Trx-1. In summary, we concluded that the nuclear Trx-1 is required to resist apoptosis of MCF-7 cells induced by cisplatin, probably through up-regulating the anti-apoptotic gene, p53.« less

The complex pathophysiology of acquired aplastic anaemia.

PubMed

Zeng, Y; Katsanis, E

2015-06-01

Immune-mediated destruction of haematopoietic stem/progenitor cells (HSPCs) plays a central role in the pathophysiology of acquired aplastic anaemia (aAA). Dysregulated CD8(+) cytotoxic T cells, CD4(+) T cells including T helper type 1 (Th1), Th2, regulatory T cells and Th17 cells, natural killer (NK) cells and NK T cells, along with the abnormal production of cytokines including interferon (IFN)-γ, tumour necrosis factor (TNF)-α and transforming growth factor (TGF)-β, induce apoptosis of HSPCs, constituting a consistent and defining feature of severe aAA. Alterations in the polymorphisms of TGF-β, IFN-γ and TNF-α genes, as well as certain human leucocyte antigen (HLA) alleles, may account for the propensity to immune-mediated killing of HSPCs and/or ineffective haematopoiesis. Although the inciting autoantigens remain elusive, autoantibodies are often detected in the serum. In addition, recent studies provide genetic and molecular evidence that intrinsic and/or secondary deficits in HSPCs and bone marrow mesenchymal stem cells may underlie the development of bone marrow failure. © 2015 British Society for Immunology.

Accelerated killing of cancer cells using a multifunctional single-walled carbon nanotube-based system for targeted drug delivery in combination with photothermal therapy.

PubMed

Jeyamohan, Prashanti; Hasumura, Takashi; Nagaoka, Yutaka; Yoshida, Yasuhiko; Maekawa, Toru; Kumar, D Sakthi

2013-01-01

The photothermal effect of single-walled carbon nanotubes (SWCNTs) in combination with the anticancer drug doxorubicin (DOX) for targeting and accelerated destruction of breast cancer cells is demonstrated in this paper. A targeted drug-delivery system was developed for selective killing of breast cancer cells with polyethylene glycol biofunctionalized and DOX-loaded SWCNTs conjugated with folic acid. In our work, in vitro drug-release studies showed that the drug (DOX) binds at physiological pH (pH 7.4) and is released only at a lower pH, ie, lysosomal pH (pH 4.0), which is the characteristic pH of the tumor environment. A sustained release of DOX from the SWCNTs was observed for a period of 3 days. SWCNTs have strong optical absorbance in the near-infrared (NIR) region. In this special spectral window, biological systems are highly transparent. Our study reports that under laser irradiation at 800 nm, SWCNTs exhibited strong light-heat transfer characteristics. These optical properties of SWCNTs open the way for selective photothermal ablation in cancer therapy. It was also observed that internalization and uptake of folate-conjugated NTs into cancer cells was achieved by a receptor-mediated endocytosis mechanism. Results of the in vitro experiments show that laser was effective in destroying the cancer cells, while sparing the normal cells. When the above laser effect was combined with DOX-conjugated SWCNTs, we found enhanced and accelerated killing of breast cancer cells. Thus, this nanodrug-delivery system, consisting of laser, drug, and SWCNTs, looks to be a promising selective modality with high treatment efficacy and low side effects for cancer therapy.

Dynein Separately Partners with NDE1 and Dynactin To Orchestrate T Cell Focused Secretion.

PubMed

Nath, Shubhankar; Christian, Laura; Tan, Sarah Youngsun; Ki, Sanghee; Ehrlich, Lauren I R; Poenie, Martin

2016-09-15

Helper and cytotoxic T cells accomplish focused secretion through the movement of vesicles toward the microtubule organizing center (MTOC) and translocation of the MTOC to the target contact site. In this study, using Jurkat cells and OT-I TCR transgenic primary murine CTLs, we show that the dynein-binding proteins nuclear distribution E homolog 1 (NDE1) and dynactin (as represented by p150(Glued)) form mutually exclusive complexes with dynein, exhibit nonoverlapping distributions in target-stimulated cells, and mediate different transport events. When Jurkat cells expressing a dominant negative form of NDE1 (NDE1-enhanced GFP fusion) were activated by Staphylococcus enterotoxin E-coated Raji cells, NDE1 and dynein failed to accumulate at the immunological synapse (IS) and MTOC translocation was inhibited. Knockdown of NDE1 in Jurkat cells or primary mouse CTLs also inhibited MTOC translocation and CTL-mediated killing. In contrast to NDE1, knockdown of p150(Glued), which depleted the alternative dynein/dynactin complex, resulted in impaired accumulation of CTLA4 and granzyme B-containing intracellular vesicles at the IS, whereas MTOC translocation was not affected. Depletion of p150(Glued) in CTLs also inhibited CTL-mediated lysis. We conclude that the NDE1/Lissencephaly 1 and dynactin complexes separately mediate two key components of T cell-focused secretion, namely translocation of the MTOC and lytic granules to the IS, respectively. Copyright © 2016 by The American Association of Immunologists, Inc.

Expression of Fas ligand by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer.

PubMed

Bennett, M W; O'connell, J; O'sullivan, G C; Roche, D; Brady, C; Kelly, J; Collins, J K; Shanahan, F

1999-02-01

Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a "Fas counterattack" against antitumour immune effector cells may contribute to tumour immune escape. To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. Thirty paraffin wax embedded human gastric adenocarcinomas. FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.

Temporins A and B Stimulate Migration of HaCaT Keratinocytes and Kill Intracellular Staphylococcus aureus

PubMed Central

Di Grazia, Antonio; Luca, Vincenzo; Segev-Zarko, Li-av T.; Shai, Yechiel

2014-01-01

The growing number of microbial pathogens resistant to available antibiotics is a serious threat to human life. Among them is the bacterium Staphylococcus aureus, which colonizes keratinocytes, the most abundant cell type in the epidermis. Its intracellular accumulation complicates treatments against resulting infections, mainly due to the limited diffusion of conventional drugs into the cells. Temporins A (Ta) and B (Tb) are short frog skin antimicrobial peptides (AMPs). Despite extensive studies regarding their antimicrobial activity, very little is known about their activity on infected cells or involvement in various immunomodulatory functions. Here we show that Tb kills both ATCC-derived and multidrug-resistant clinical isolates of S. aureus within infected HaCaT keratinocytes (80% and 40% bacterial mortality, respectively) at a nontoxic concentration, i.e., 16 μM, whereas a weaker effect is displayed by Ta. Furthermore, the peptides prevent killing of keratinocytes by the invading bacteria. Further studies revealed that both temporins promote wound healing in a monolayer of HaCaT cells, with front speed migrations of 19 μm/h and 12 μm/h for Ta and Tb, respectively. Migration is inhibited by mitomycin C and involves the epidermal growth factor receptor (EGFR) signaling pathway. Finally, confocal fluorescence microscopy indicated that the peptides diffuse into the cells. By combining antibacterial and wound-healing activities, Ta and Tb may act as multifunctional mediators of innate immunity in humans. Particularly, their nonendogenous origin may reduce microbial resistance to them as well as the risk of autoimmune diseases in mammals. PMID:24514087

Stem-like tumor-initiating cells isolated from IL13Rα2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells.

PubMed

Brown, Christine E; Starr, Renate; Aguilar, Brenda; Shami, Andrew F; Martinez, Catalina; D'Apuzzo, Massimo; Barish, Michael E; Forman, Stephen J; Jensen, Michael C

2012-04-15

To evaluate IL13Rα2 as an immunotherapeutic target for eliminating glioma stem-like cancer initiating cells (GSC) of high-grade gliomas, with particular focus on the potential of genetically engineered IL13Rα2-specific primary human CD8(+) CTLs (IL13-zetakine(+) CTL) to target this therapeutically resistant glioma subpopulation. A panel of low-passage GSC tumor sphere (TS) and serum-differentiated glioma lines were expanded from patient glioblastoma specimens. These glioblastoma lines were evaluated for expression of IL13Rα2 and for susceptibility to IL13-zetakine(+) CTL-mediated killing in vitro and in vivo. We observed that although glioma IL13Rα2 expression varies between patients, for IL13Rα2(pos) cases this antigen was detected on both GSCs and more differentiated tumor cell populations. IL13-zetakine(+) CTL were capable of efficient recognition and killing of both IL13Rα2(pos) GSCs and IL13Rα2(pos) differentiated cells in vitro, as well as eliminating glioma-initiating activity in an orthotopic mouse tumor model. Furthermore, intracranial administration of IL13-zetakine(+) CTL displayed robust antitumor activity against established IL13Rα2(pos) GSC TS-initiated orthotopic tumors in mice. Within IL13Rα2 expressing high-grade gliomas, this receptor is expressed by GSCs and differentiated tumor populations, rendering both targetable by IL13-zetakine(+) CTLs. Thus, our results support the potential usefullness of IL13Rα2-directed immunotherapeutic approaches for eradicating therapeutically resistant GSC populations. ©2012 AACR.

Parthanatos, a messenger of death.

PubMed

David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

2009-01-01

Poly-ADP-ribose polymerase-1 (PARP-1)'s roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 overactivation underlies cell death in models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into understanding mechanisms downstream of PARP-1 overactivation. Recent evidence shows that poly-ADP ribose (PAR) polymer itself can act as a cell death effector downstream of PARP-1. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will present evidence and questions raised by these recent findings, and summarize the proposed mechanisms by which PARP-1 overactivation kills. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 overactivation.

Colony-Stimulating Factor 1 Receptor Antagonists Sensitize Human Immunodeficiency Virus Type 1-Infected Macrophages to TRAIL-Mediated Killing

PubMed Central

Cunyat, Francesc; Rainho, Jennifer N.; West, Brian; Swainson, Louise; McCune, Joseph M.

2016-01-01

ABSTRACT Strategies aimed at eliminating persistent viral reservoirs from HIV-1-infected individuals have focused on CD4+ T-cell reservoirs. However, very little attention has been given to approaches that could promote elimination of tissue macrophage reservoirs. HIV-1 infection of macrophages induces phosphorylation of colony-stimulating factor 1 receptor (CSF-1R), which confers resistance to apoptotic pathways driven by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), thereby promoting viral persistence. In this study, we assessed whether CSF-1R antagonists (PLX647, PLX3397, and PLX5622) restored apoptotic sensitivity of HIV-1-infected macrophages in vitro. PLX647, PLX3397, and PLX5622 at clinically relevant concentrations blocked the activation of CSF-1R and reduced the viability of infected macrophages, as well as the extent of viral replication. Our data show that strategies targeting monocyte colony-stimulating factor (MCSF) signaling could be used to promote elimination of HIV-1-infected myeloid cells and to contribute to the elimination of persistent viral reservoirs. IMPORTANCE As the HIV/AIDS research field explores approaches to eliminate HIV-1 in individuals on suppressive antiviral therapy, those approaches will need to eliminate both CD4+ T-cell and myeloid cell reservoirs. Most of the attention has focused on CD4+ T-cell reservoirs, and scant attention has been paid to myeloid cell reservoirs. The distinct nature of the infection in myeloid cells versus CD4+ T cells will likely dictate different approaches in order to achieve their elimination. For CD4+ T cells, most strategies focus on promoting virus reactivation to promote immune-mediated clearance and/or elimination by viral cytopathicity. Macrophages resist viral cytopathic effects and CD8+ T-cell killing. Therefore, we have explored clearance strategies that render macrophages sensitive to viral cytopathicity. This research helps inform the design of strategies to promote clearance of the macrophage reservoir in infected individuals on suppressive antiviral therapy. PMID:27122585

CD4+ T cell-mediated rejection of MHC class II-positive tumor cells is dependent on antigen secretion and indirect presentation on host APCs.

PubMed

Haabeth, Ole Audun Werner; Fauskanger, Marte; Manzke, Melanie; Lundin, Katrin U; Corthay, Alexandre; Bogen, Bjarne; Tveita, Anders Aune

2018-05-11

Tumor-specific CD4+ T cells have been shown to mediate efficient anti-tumor immune responses against cancer. Such responses can occur through direct binding to MHC class II (MHC II)-expressing tumor cells or indirectly via activation of professional antigen-presenting cells (APC) that take up and present the tumor antigen. We have previously shown that CD4+ T cells reactive against an epitope within the Ig light chain variable region of a murine B cell lymphoma can reject established tumors. Given the presence of MHC II molecules at the surface of lymphoma cells, we investigated whether MHC II-restricted antigen presentation on tumor cells alone was required for rejection. Variants of the A20 B lymphoma cell line that either secreted or intracellularly retained different versions of the tumor-specific antigen revealed that antigen secretion by the MHC II-expressing tumor cells was essential both for the priming and effector phase of CD4+ T cell-driven anti-tumor immune responses. Consistent with this, genetic ablation of MHC II in tumor cells, both in the case of B lymphoma and B16 melanoma, did not preclude rejection of tumors by tumor antigen-specific CD4+ T cells in vivo. These findings demonstrate that MHC class II expression on tumor cells themselves is not required for CD4+ T cell-mediated rejection, and that indirect display on host APC is sufficient for effective tumor elimination. These results support the importance of tumor-infiltrating APC as mediators of tumor cell killing by CD4+ T cells. Copyright ©2018, American Association for Cancer Research.

The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection

PubMed Central

Rajalingam, Raja

2016-01-01

Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR–HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants. PMID:28066408

A Multimodal System with Synergistic Effects of Magneto-Mechanical, Photothermal, Photodynamic and Chemo Therapies of Cancer in Graphene-Quantum Dot-Coated Hollow Magnetic Nanospheres

PubMed Central

Wo, Fangjie; Xu, Rujiao; Shao, Yuxiang; Zhang, Zheyu; Chu, Maoquan; Shi, Donglu; Liu, Shupeng

2016-01-01

In this study, a multimodal therapeutic system was shown to be much more lethal in cancer cell killing compared to a single means of nano therapy, be it photothermal or photodynamic. Hollow magnetic nanospheres (HMNSs) were designed and synthesized for the synergistic effects of both magneto-mechanical and photothermal cancer therapy. By these combined stimuli, the cancer cells were structurally and physically destroyed with the morphological characteristics distinctively different from those by other therapeutics. HMNSs were also coated with the silica shells and conjugated with carboxylated graphene quantum dots (GQDs) as a core-shell composite: HMNS/SiO2/GQDs. The composite was further loaded with an anticancer drug doxorubicin (DOX) and stabilized with liposomes. The multimodal system was able to kill cancer cells with four different therapeutic mechanisms in a synergetic and multilateral fashion, namely, the magnetic field-mediated mechanical stimulation, photothermal damage, photodynamic toxicity, and chemotherapy. The unique nanocomposites with combined mechanical, chemo, and physical effects will provide an alternative strategy for highly improved cancer therapy efficiency. PMID:26941842

A Multimodal System with Synergistic Effects of Magneto-Mechanical, Photothermal, Photodynamic and Chemo Therapies of Cancer in Graphene-Quantum Dot-Coated Hollow Magnetic Nanospheres.

PubMed

Wo, Fangjie; Xu, Rujiao; Shao, Yuxiang; Zhang, Zheyu; Chu, Maoquan; Shi, Donglu; Liu, Shupeng

2016-01-01

In this study, a multimodal therapeutic system was shown to be much more lethal in cancer cell killing compared to a single means of nano therapy, be it photothermal or photodynamic. Hollow magnetic nanospheres (HMNSs) were designed and synthesized for the synergistic effects of both magneto-mechanical and photothermal cancer therapy. By these combined stimuli, the cancer cells were structurally and physically destroyed with the morphological characteristics distinctively different from those by other therapeutics. HMNSs were also coated with the silica shells and conjugated with carboxylated graphene quantum dots (GQDs) as a core-shell composite: HMNS/SiO2/GQDs. The composite was further loaded with an anticancer drug doxorubicin (DOX) and stabilized with liposomes. The multimodal system was able to kill cancer cells with four different therapeutic mechanisms in a synergetic and multilateral fashion, namely, the magnetic field-mediated mechanical stimulation, photothermal damage, photodynamic toxicity, and chemotherapy. The unique nanocomposites with combined mechanical, chemo, and physical effects will provide an alternative strategy for highly improved cancer therapy efficiency.

The Yin/Yan of CCL2: a minor role in neutrophil anti-tumor activity in vitro but a major role on the outgrowth of metastatic breast cancer lesions in the lung in vivo.

PubMed

Lavender, Nicole; Yang, Jinming; Chen, Sheau-Chiann; Sai, Jiqing; Johnson, C Andrew; Owens, Philip; Ayers, Gregory D; Richmond, Ann

2017-01-31

The role of the chemokine CCL2 in breast cancer is controversial. While CCL2 recruits and activates pro-tumor macrophages, it is also reported to enhance neutrophil-mediated anti-tumor activity. Moreover, loss of CCL2 in early development enhances breast cancer progression. To clarify these conflicting findings, we examined the ability of CCL2 to alter naïve and tumor entrained neutrophil production of ROS, release of granzyme-B, and killing of tumor cells in multiple mouse models of breast cancer. CCL2 was delivered intranasally in mice to elevate CCL2 levels in the lung and effects on seeding and growth of breast tumor cells were evaluated. The TCGA data base was queried for relationship between CCL2 expression and relapse free survival of breast cancer patients and compared to subsets of breast cancer patients. Even though each of the tumor cell lines studied produced approximately equal amounts of CCL2, exogenous delivery of CCL2 to co-cultures of breast tumor cells and neutrophils enhanced the ability of tumor-entrained neutrophils (TEN) to kill the less aggressive 67NR variant of 4T1 breast cancer cells. However, exogenous CCL2 did not enhance naïve or TEN neutrophil killing of more aggressive 4T1 or PyMT breast tumor cells. Moreover, this anti-tumor activity was not observed in vivo. Intranasal delivery of CCL2 to BALB/c mice markedly enhanced seeding and outgrowth of 67NR cells in the lung and increased the recruitment of CD4+ T cells and CD8+ central memory T cells into lungs of tumor bearing mice. There was no significant increase in the recruitment of CD19+ B cells, or F4/80+, Ly6G+ and CD11c + myeloid cells. CCL2 had an equal effect on CD206+ and MHCII+ populations of macrophages, thus balancing the pro- and anti-tumor macrophage cell population. Analysis of the relationship between CCL2 levels and relapse free survival in humans revealed that overall survival is not significantly different between high CCL2 expressing and low CCL2 expressing breast cancer patients grouped together. However, examination of the relationship between high CCL2 expressing basal-like, HER2+ and luminal B breast cancer patients revealed that higher CCL2 expressing tumors in these subgroups have a significantly higher probability of surviving longer than those expressing low CCL2. While our in vitro data support a potential anti-tumor role for CCL2 in TEN neutrophil- mediated tumor killing in poorly aggressive tumors, intranasal delivery of CCL2 increased CD4+ T cell recruitment to the pre-metastatic niche of the lung and this correlated with enhanced seeding and growth of tumor cells. These data indicate that effects of CCL2/CCR2 antagonists on the intratumoral leukocyte content should be monitored in ongoing clinical trials using these agents.

Radiation quality dependence of signal transmission and bystander induced cell killing

NASA Astrophysics Data System (ADS)

Esposito, Giuseppe; Bertolotti, Alessia; Facoetti, Angelica; Grande, Sveva; Mariotti, Luca; Ottolenghi, Andrea; Ranza, Elena; Simone, Giustina; Sorrentino, Eugenio; Antonella Tabocchini, Maria

Low dose radiobiological studies have shown effects, observable in cells that are in the vicinity of irradiated cells, which are due to the release by irradiated cells of several cellular mediators among which Reactive Oxygen and Nitrogen Species (ROS, NRS), and cytokines are likely to play a key role. Despite the large number in the literature of studies on bystander effects induced by ionizing radiation the results are still conflicting, and further studies are therefore needed on the possible underlying mechanisms. The dependence on radiation quality deserve particular attention because bystander mechanisms are probably more important with high-LET irradi-ations, where many cells are not hit (bystander). Moreover, due to the different patterns of energy deposition, the cellular response to low LET and high LET radiation can be different. Understanding whether these cells can contribute to the adverse effects of low radiation doses in a radiation quality-dependent fashion might have important implications in risk estimates for both cancer induction and non-cancer diseases. In this context, we addressed to the study of the bystander induced cell killing after incubation with "conditioned medium" from primary human fibroblasts irradiated with 0.1 and 0.5 Gy of α-particles or γ-rays. Medium transfer was performed after 1h incubation from irradiation. The results have confirmed a reduction in clonogenic survival after incubation with medium from α-irradiated cells, independently of the dose; similar results were obtained after γ-irradiation, although in this case a slight dose depen-dence could be envisaged. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were measured in the conditioned medium collected up to 20 hours after irradiation with α-particles and γ-rays in the dose-range of 0.1-1.0 Gy, in parallel with evaluation of their receptor expression in irradi-ated and bystander cells. Concerning IL-6, we observed the strongest modulation of its release at 20 hours post exposure, whereas IL-8 release was significantly increased at shorter times, i.e. 5-7 hours after irradiation. The expression of their receptors was modulated in both irradiated and bystander cells, although it is not apparently correlated with the relative interleukin re-lease. In order to investigate possible correlation between NRS and cytokines as early and late mediators in the signalling chain leading to bystander induced cell killing, experiments were performed using c-PTIO, a well known scavengers of RNS. In these experiments conditioned medium taken after 1h or 5h from α-particle irradiated cells was used. The results obtained after 5h in the absence of c-PTIO didn't show any further decrease in clonogenic survival of bystander cells. The presence of the scavenger seems to reduce, the bystander induced cell killing indicating that RNS are involved in the transduction of the bystander signal. Experi-ments, performed with both c-PTIO and DMSO, a scavenger of hydroxyl radicals, showed that RNS and ROS play a role in some cytokine pathways (interleukins release and their receptor expression) activated by irradiation. ACKNOWLEDGEMENTS This work was partially supported by the European Commission (EC Contract FP6-36465, "NOTE")

Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate.

PubMed

Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K; Liu, Peter; Pantua, Homer; Abbas, Alexander R; Nickerson, Nicholas N; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min; Whitfield, Chris; Kapadia, Sharookh B

2017-05-23

Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro , the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for "group 2" capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. IMPORTANCE Uropathogenic E. coli (UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistance in vitro and for complement-mediated bacterial clearance in vivo This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of "group 2" capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for murein lipoprotein in complex interactions between different outer membrane biogenesis pathways and further highlight the importance of lipoprotein assembly and transport in bacterial pathogenesis. Copyright © 2017 Diao et al.

RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by CD8+ T cells

PubMed Central

Duewell, P; Steger, A; Lohr, H; Bourhis, H; Hoelz, H; Kirchleitner, S V; Stieg, M R; Grassmann, S; Kobold, S; Siveke, J T; Endres, S; Schnurr, M

2014-01-01

Pancreatic cancer is characterized by a microenvironment suppressing immune responses. RIG-I-like helicases (RLH) are immunoreceptors for viral RNA that induce an antiviral response program via the production of type I interferons (IFN) and apoptosis in susceptible cells. We recently identified RLH as therapeutic targets of pancreatic cancer for counteracting immunosuppressive mechanisms and apoptosis induction. Here, we investigated immunogenic consequences of RLH-induced tumor cell death. Treatment of murine pancreatic cancer cell lines with RLH ligands induced production of type I IFN and proinflammatory cytokines. In addition, tumor cells died via intrinsic apoptosis and displayed features of immunogenic cell death, such as release of HMGB1 and translocation of calreticulin to the outer cell membrane. RLH-activated tumor cells led to activation of dendritic cells (DCs), which was mediated by tumor-derived type I IFN, whereas TLR, RAGE or inflammasome signaling was dispensable. Importantly, CD8α+ DCs effectively engulfed apoptotic tumor material and cross-presented tumor-associated antigen to naive CD8+ T cells. In comparison, tumor cell death mediated by oxaliplatin, staurosporine or mechanical disruption failed to induce DC activation and antigen presentation. Tumor cells treated with sublethal doses of RLH ligands upregulated Fas and MHC-I expression and were effectively sensitized towards Fas-mediated apoptosis and cytotoxic T lymphocyte (CTL)-mediated lysis. Vaccination of mice with RLH-activated tumor cells induced protective antitumor immunity in vivo. In addition, MDA5-based immunotherapy led to effective tumor control of established pancreatic tumors. In summary, RLH ligands induce a highly immunogenic form of tumor cell death linking innate and adaptive immunity. PMID:25012502

Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis

PubMed Central

Wang, Wei; Wang, Wei-Hua; Azadzoi, Kazem M.; Su, Ning; Dai, Peng; Sun, Jianbin; Wang, Qin; Liang, Ping; Zhang, Wentao; Lei, Xiaoying; Yan, Zhen; Yang, Jing-Hua

2016-01-01

Viruses induce double-stranded RNA (dsRNA) in the host cells. The mammalian system has developed dsRNA-dependent recognition receptors such as RLRs that recognize the long stretches of dsRNA as PAMPs to activate interferon-mediated antiviral pathways and apoptosis in severe infection. Here we report an efficient antiviral immune response through dsRNA-dependent RLR receptor-mediated necroptosis against infections from different classes of viruses. We demonstrated that virus-infected A549 cells were efficiently killed in the presence of a chimeric RLR receptor, dsCARE. It measurably suppressed the interferon antiviral pathway but promoted IL-1β production. Canonical cell death analysis by morphologic assessment, phosphatidylserine exposure, caspase cleavage and chemical inhibition excluded the involvement of apoptosis and consistently suggested RLR receptor-mediated necroptosis as the underlying mechanism of infected cell death. The necroptotic pathway was augmented by the formation of RIP1-RIP3 necrosome, recruitment of MLKL protein and the activation of cathepsin D. Contributing roles of RIP1 and RIP3 were confirmed by gene knockdown. Furthermore, the necroptosis inhibitor necrostatin-1 but not the pan-caspase inhibitor zVAD impeded dsCARE-dependent infected cell death. Our data provides compelling evidence that the chimeric RLR receptor shifts the common interferon antiviral responses of infected cells to necroptosis and leads to rapid death of the virus-infected cells. This mechanism could be targeted as an efficient antiviral strategy. PMID:26935990

Kill and spread the word: stimulation of antitumor immune responses in the context of radiotherapy.

PubMed

Gaipl, Udo S; Multhoff, Gabriele; Scheithauer, Heike; Lauber, Kirsten; Hehlgans, Stefanie; Frey, Benjamin; Rödel, Franz

2014-01-01

Besides the direct, targeted effects of ionizing irradiation (x-ray) on cancer cells, namely DNA damage and cell death induction, indirect, nontargeted ones exist, which are mediated in large part by the immune system. Immunogenic forms of tumor cell death induced by x-ray, including immune modulating danger signals like the heat shock protein 70, adenosine triphosphate, and high-mobility group box 1 protein are presented. Further, antitumor effects exerted by cells of the innate (natural killer cells) as well as adaptive immune system (T cells activated by dendritic cells) are outlined. Tumor cell death inhibiting molecules such as survivin are introduced as suitable target for molecularly tailored therapies in combination with x-ray. Finally, reasonable combinations of immune therapies with radiotherapy are discussed.

IL-15 converts human intestinal intraepithelial lymphocytes to CD94+ producers of IFN-γ and IL-10, the latter promoting Fas ligand-mediated cytotoxicity

PubMed Central

Ebert, Ellen C

2005-01-01

Intestinal intraepithelial lymphocytes (IELs), T-cell receptor αβ+ CD8+ T cells located between epithelial cells, are thought to contribute to Fas ligand (FL)-mediated epithelial cell death in coeliac disease, a condition characterized by excess interleukin-15 (IL-15). This study evaluates the effects of prolonged IL-15 stimulation on IELs. Human IELs were obtained from jejunal mucosa from gastric bypass operations for morbid obesity and cultured for 3 or 10 days with IL-15. As the culture progressed, an increasing number of IELs became CD94+ and produced massive quantities of interferon-γ (IFN-γ) and IL-10. There was a steady rate of transcription with no feedback regulation. Few chronically activated IELs produced IL-2, IL-4, or tumour necrosis factor-α (TNF-α). To determine whether the accumulation of IL-10 affected IEL functions, endogenous IL-10 was neutralized by antibody during culture with IL-15. This manipulation reduced expression of CD94, NKG2D, and FL as well as FL-mediated killing of Jurkat cells by IELs. It did not affect perforin or TNF-α expression or the associated cytotoxic activities. This study shows that IL-15 induces the development of CD94+ IELs containing IFN-γ and IL-10, and that endogenous IL-10 promotes FL-mediated cytotoxicity. PMID:15819704

A novel factor H-Fc chimeric immunotherapeutic molecule against Neisseria gonorrhoeae

PubMed Central

Shaughnessy, Jutamas; Gulati, Sunita; Agarwal, Sarika; Unemo, Magnus; Ohnishi, Makoto; Su, Xia-Hong; Monks, Brian G.; Visintin, Alberto; Madico, Guillermo; Lewis, Lisa A.; Golenbock, Douglas T.; Reed, George W.; Rice, Peter A.; Ram, Sanjay

2015-01-01

Neisseria gonorrhoeae (Ng), the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to almost every conventional antibiotic. There is an urgent need to develop novel therapies against gonorrhea. Many pathogens, including Ng, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains 18-20 (FH18-20). We explored the utility of fusing FH18-20 with IgG Fc (FH18-20/Fc) to create a novel anti-infective immunotherapeutic. FH18-20 also binds to select host glycosaminoglycans to limit unwanted complement activation on host cells. To identify mutation(s) in FH18-20 that eliminated complement activation on host cells, yet maintained binding to Ng, we created four mutations in domains 19 or 20 described in atypical hemolytic uremic syndrome that prevented binding of mutated fH to human erythrocytes. One of the mutant proteins (D to G at position 1119 in domain 19; FHD1119G/Fc) facilitated complement-dependent killing of gonococci similar to unmodified FH18-20/Fc, but unlike FH18-20/Fc, did not lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15 sialylated clinical Ng isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10/15 (67%) strains and enhanced C3 deposition (≥10-fold above baseline levels) on each of the five isolates not directly killed by complement. FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistant strain by human polymorphonuclear neutrophils. FHD1119G/Fc administered intravaginally significantly reduced the duration and burden of gonococcal infection in the mouse vaginal colonization model. FHD1119G/Fc represents a novel immunotherapeutic against multidrug-resistant Ng. PMID:26773149

Mechanism of killing of streptococcus mutans by light-activated drugs

NASA Astrophysics Data System (ADS)

Burns, Tracy; Wilson, Michael; Pearson, G. J.

1996-01-01

Recent studies have shown that cariogenic bacteria can be killed when exposed to low power laser light in the presence of a photosensitizing agent. The purpose of this study was to determine the mechanism by which the cariogenic bacterium Streptococcus mutans can be killed by toluidine blue O and helium neon laser light. To determine whether membrane damage occurred, suspensions of sensitized S. mutans were exposed to a 7.3 mW HeNe laser for 30 mins and samples removed every 5 mins. Survivors were enumerated by viable counting on tryptone soya agar plates and cell free filtrates were assayed for phosphate and (beta) -galactosidase. Lipid peroxidation was assessed by assaying for malondialdehyde, a by- product of lipid peroxidation. The role of oxygen and reactive oxygen species was studied by exposing sensitized bacteria to laser light (1) under different atmospheric conditions, (2) in the presence of deuterium oxide, and (3) in the presence of inhibitors of reactive oxygen species. Following exposure of sensitizede S. mutans to 13.2 J of HeNe laser light, 2.6 nmoles of phosphate and 228 nmoles of (beta) -galactosidase were detected in the cell free filtrates. Ten micrometers oles of malondialdehyde were also detected. When the sensitized bacteria were exposed to laser light under anaerobic conditions there was no significant decrease in the viable count compared to a 60% kill in the presence of oxygen. In the presence of D2O there was a 15-fold increase in the numbers of bacteria killed. O.1 M methionine and 0.5 M sodium azide each afforded 98% protection from lethal photosensitization. These results imply that lethal photosensitization results from membrane damage due to lipid peroxidation and that reactive oxygen species are mediators of this process.

Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

PubMed

Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Osuna-Pérez, Jesús; Torres-Torresano, Mónica; Zorita, Virgina; Martínez-Riaño, Ana; Boccasavia, Viola; Borroto, Aldo; Martínez Del Hoyo, Gloria; González-Granado, José María; Alarcón, Balbino; Sánchez-Madrid, Francisco; Veiga, Esteban

2017-11-17

Bacterial phagocytosis and antigen cross-presentation to activate CD8 + T cells are principal functions of professional antigen presenting cells. However, conventional CD4 + T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 + T cells, which proliferate and become cytotoxic in response. CD4 + T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 + T cells with low PD-1 expression. Moreover, transphagocytic CD4 + T cells induce protective anti-tumour immune responses by priming CD8 + T cells, highlighting the potential of CD4 + T cells as a tool for cancer immunotherapy.

Targeting CD123 in acute myeloid leukemia using a T-cell–directed dual-affinity retargeting platform

PubMed Central

Al-Hussaini, Muneera; Rettig, Michael P.; Ritchey, Julie K.; Karpova, Darja; Uy, Geoffrey L.; Eissenberg, Linda G.; Gao, Feng; Eades, William C.; Bonvini, Ezio; Chichili, Gurunadh R.; Moore, Paul A.; Johnson, Syd; Collins, Lynne

2016-01-01

T-cell–directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123+ AML. PMID:26531164

Critical role of type 1 cytokines in controlling initial infection with Burkholderia mallei.

PubMed

Rowland, Caroline A; Lertmemongkolchai, Ganjana; Bancroft, Alison; Haque, Ashraful; Lever, M Stephen; Griffin, Kate F; Jackson, Matthew C; Nelson, Michelle; O'Garra, Anne; Grencis, Richard; Bancroft, Gregory J; Lukaszewski, Roman A

2006-09-01

Burkholderia mallei is a gram-negative bacterium which causes the potentially fatal disease glanders in humans; however, there is little information concerning cell-mediated immunity to this pathogen. The role of gamma interferon (IFN-gamma) during B. mallei infection was investigated using a disease model in which infected BALB/c mice normally die between 40 and 60 days postinfection. IFN-gamma knockout mice infected with B. mallei died within 2 to 3 days after infection, and there was uncontrolled bacterial replication in several organs, demonstrating the essential role of IFN-gamma in the innate immune response to this pathogen. Increased levels of IFN-gamma, interleukin-6 (IL-6), and monocyte chemoattractant protein 1 were detected in the sera of immunocompetent mice in response to infection, and splenic mRNA expression of IFN-gamma, IL-6, IL-12p35, and IL-27 was elevated 24 h postinfection. The effects of IL-18, IL-27, and IL-12 on stimulation of the rapid IFN-gamma production were investigated in vitro by analyzing IFN-gamma production in the presence of heat-killed B. mallei. IL-12 was essential for IFN-gamma production in vitro; IL-18 was also involved in induction of IFN-gamma, but IL-27 was not required for IFN-gamma production in response to heat-killed B. mallei. The main cellular sources of IFN-gamma were identified in vitro as NK cells, CD8+ T cells, and TCRgammadelta T cells. Our data show that B. mallei is susceptible to cell-mediated immune responses which promote expression of type 1 cytokines. This suggests that development of effective vaccines against glanders should target the production of IFN-gamma.

Live Candida albicans suppresses production of reactive oxygen species in phagocytes.

PubMed

Wellington, Melanie; Dolan, Kristy; Krysan, Damian J

2009-01-01

Production of reactive oxygen species (ROS) is an important aspect of phagocyte-mediated host responses. Since phagocytes play a crucial role in the host response to Candida albicans, we examined the ability of Candida to modulate phagocyte ROS production. ROS production was measured in the murine macrophage cell line J774 and in primary phagocytes using luminol-enhanced chemiluminescence. J774 cells, murine polymorphonuclear leukocytes (PMN), human monocytes, and human PMN treated with live C. albicans produced significantly less ROS than phagocytes treated with heat-killed C. albicans. Live C. albicans also suppressed ROS production in murine bone marrow-derived macrophages from C57BL/6 mice, but not from BALB/c mice. Live C. albicans also suppressed ROS in response to external stimuli. C. albicans and Candida glabrata suppressed ROS production by phagocytes, whereas Saccharomyces cerevisiae stimulated ROS production. The cell wall is the initial point of contact between Candida and phagocytes, but isolated cell walls from both heat-killed and live C. albicans stimulated ROS production. Heat-killed C. albicans has increased surface exposure of 1,3-beta-glucan, a cell wall component that can stimulate phagocytes. To determine whether surface 1,3-beta-glucan exposure accounted for the difference in ROS production, live C. albicans cells were treated with a sublethal dose of caspofungin to increase surface 1,3-beta-glucan exposure. Caspofungin-treated C. albicans was fully able to suppress ROS production, indicating that suppression of ROS overrides stimulatory signals from 1,3-beta-glucan. These studies indicate that live C. albicans actively suppresses ROS production in phagocytes in vitro, which may represent an important immune evasion mechanism.

Live Candida albicans Suppresses Production of Reactive Oxygen Species in Phagocytes▿ †

PubMed Central

Wellington, Melanie; Dolan, Kristy; Krysan, Damian J.

2009-01-01

Production of reactive oxygen species (ROS) is an important aspect of phagocyte-mediated host responses. Since phagocytes play a crucial role in the host response to Candida albicans, we examined the ability of Candida to modulate phagocyte ROS production. ROS production was measured in the murine macrophage cell line J774 and in primary phagocytes using luminol-enhanced chemiluminescence. J774 cells, murine polymorphonuclear leukocytes (PMN), human monocytes, and human PMN treated with live C. albicans produced significantly less ROS than phagocytes treated with heat-killed C. albicans. Live C. albicans also suppressed ROS production in murine bone marrow-derived macrophages from C57BL/6 mice, but not from BALB/c mice. Live C. albicans also suppressed ROS in response to external stimuli. C. albicans and Candida glabrata suppressed ROS production by phagocytes, whereas Saccharomyces cerevisiae stimulated ROS production. The cell wall is the initial point of contact between Candida and phagocytes, but isolated cell walls from both heat-killed and live C. albicans stimulated ROS production. Heat-killed C. albicans has increased surface exposure of 1,3-β-glucan, a cell wall component that can stimulate phagocytes. To determine whether surface 1,3-β-glucan exposure accounted for the difference in ROS production, live C. albicans cells were treated with a sublethal dose of caspofungin to increase surface 1,3-β-glucan exposure. Caspofungin-treated C. albicans was fully able to suppress ROS production, indicating that suppression of ROS overrides stimulatory signals from 1,3-β-glucan. These studies indicate that live C. albicans actively suppresses ROS production in phagocytes in vitro, which may represent an important immune evasion mechanism. PMID:18981256

Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir.

PubMed

Mujib, Shariq; Liu, Jun; Rahman, A K M Nur-Ur; Schwartz, Jordan A; Bonner, Phil; Yue, Feng Yun; Ostrowski, Mario A

2017-08-15

Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, and PGT145) are best at recognizing HIV-1-infected CD4 T cells. However, only PG9 and PG16 and their combinations with other bnAbs sufficiently induced the elimination of HIV-1-infected CD4 T cells by ADCML, ADCC, or both. Notably, CD4 binding site antibodies VRC01, 3BNC117, and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared toward the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we conducted a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics toward the development of a cure for HIV/AIDS. IMPORTANCE Several antibodies capable of neutralizing the majority of circulating HIV-1 strains have been identified to date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and eradication of HIV-1 in infected humans remains uncertain. In this study, we tested the ability of bnAbs to directly recognize and eliminate primary human CD4 T cells infected with diverse HIV-1 strains representative of the global epidemic by antibody-dependent pathways. We also tested several combinations of bnAbs in our assays in order to maximize the clearance of infected cells. We show that the ability of bnAbs to identify and kill infected cells is highly variable and that only a few of them are able to exert this function. Our data will help guide the formulation of bnAbs to test in future human trials aimed at the development of a cure. Copyright © 2017 American Society for Microbiology.

Piper and Vismia species from Colombian Amazonia differentially affect cell proliferation of hepatocarcinoma cells.

PubMed

Lizcano, Leandro J; Siles, Maite; Trepiana, Jenifer; Hernández, M Luisa; Navarro, Rosaura; Ruiz-Larrea, M Begoña; Ruiz-Sanz, José Ignacio

2014-12-30

There is an increasing interest to identify plant-derived natural products with antitumor activities. In this work, we have studied the effects of aqueous leaf extracts from Amazonian Vismia and Piper species on human hepatocarcinoma cell toxicity. Results showed that, depending on the cell type, the plants displayed differential effects; thus, Vismia baccifera induced the selective killing of HepG2, while increasing cell growth of PLC-PRF and SK-HEP-1. In contrast, these two last cell lines were sensitive to the toxicity by Piper krukoffii and Piper putumayoense, while the Piperaceae did not affect HepG2 growth. All the extracts induced cytotoxicity to rat hepatoma McA-RH7777, but were innocuous (V. baccifera at concentrations < 75 µg/mL) or even protected cells from basal death (P. putumayoense) in primary cultures of rat hepatocytes. In every case, cytotoxicity was accompanied by an intracellular accumulation of reactive oxygen species (ROS). These results provide evidence for the anticancer activities of the studied plants on specific cell lines and suggest that cell killing could be mediated by ROS, thus involving mechanisms independent of the plants free radical scavenging activities. Results also support the use of these extracts of the Vismia and Piper genera with opposite effects as a model system to study the mechanisms of the antitumoral activity against different types of hepatocarcinoma.

Augmented IFN-γ and TNF-α Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL-10

PubMed Central

Bui, Vickie T.; Tseng, Han-Ching; Kozlowska, Anna; Maung, Phyu Ou; Kaur, Kawaljit; Topchyan, Paytsar; Jewett, Anahid

2015-01-01

Our previous reports demonstrated that the magnitude of natural killer (NK) cell-mediated cytotoxicity correlate directly with the stage and level of differentiation of tumor cells. In addition, we have shown previously that activated NK cells inhibit growth of cancer cells through induction of differentiation, resulting in the resistance of tumor cells to NK cell-mediated cytotoxicity through secreted cytokines, as well as direct NK-tumor cell contact. In this report, we show that in comparison to IL-2 + anti-CD16mAb-treated NK cells, activation of NK cells by probiotic bacteria (sAJ2) in combination with IL-2 and anti-CD16mAb substantially decreases tumor growth and induces maturation, differentiation, and resistance of oral squamous cancer stem cells, MIA PaCa-2 stem-like/poorly differentiated pancreatic tumors, and healthy stem cells of apical papillae through increased secretion of IFN-γ and TNF-α, as well as direct NK-tumor cell contact. Tumor resistance to NK cell-mediated killing induced by IL-2 + anti-CD16mAb + sAJ2-treated NK cells is induced by combination of IFN-γ and TNF-α since antibodies to both, and not each cytokine alone, were able to restore tumor sensitivity to NK cells. Increased surface expression of CD54, B7H1, and MHC-I on NK-differentiated tumors was mediated by IFN-γ since the addition of anti-IFN-γ abolished their increase and restored the ability of NK cells to trigger cytokine and chemokine release; whereas differentiated tumors inhibited cytokine release by the NK cells. Monocytes synergize with NK cells in the presence of probiotic bacteria to induce regulated differentiation of stem cells through secretion of IL-10 resulting in resistance to NK cell-mediated cytotoxicity and inhibition of cytokine release. Therefore, probiotic bacteria condition activated NK cells to provide augmented differentiation of cancer stem cells resulting in inhibition of tumor growth, and decreased inflammatory cytokine release. PMID:26697005

Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms

PubMed Central

Jackson, Desmond N.; Yang, Lin; Wu, ShiBiao; Kennelly, Edward J.

2015-01-01

Xanthochymol and garcinol, isoprenylated benzophenones purified from Garcinia xanthochymus fruits, showed multiple activities against Candida albicans biofilms. Both compounds effectively prevented emergence of fungal germ tubes and were also cytostatic, with MICs of 1 to 3 μM. The compounds therefore inhibited development of hyphae and subsequent biofilm maturation. Xanthochymol treatment of developing and mature biofilms induced cell death. In early biofilm development, killing had the characteristics of apoptosis, including externalization of phosphatidyl serine and DNA fragmentation, as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) fluorescence. These activities resulted in failure of biofilm maturation and hyphal death in mature biofilms. In mature biofilms, xanthochymol and garcinol caused the death of biofilm hyphae, with 50% effective concentrations (EC50s) of 30 to 50 μM. Additionally, xanthochymol-mediated killing was complementary with fluconazole against mature biofilms, reducing the fluconazole EC50 from >1,024 μg/ml to 13 μg/ml. Therefore, xanthochymol has potential as an adjuvant for antifungal treatments as well as in studies of fungal apoptosis. PMID:26195512

In vivo inhibition of tryptophan catabolism reorganizes the tuberculoma and augments immune-mediated control of Mycobacterium tuberculosis

PubMed Central

Gautam, Uma S.; Foreman, Taylor W.; Bucsan, Allison N.; Veatch, Ashley V.; Alvarez, Xavier; Adekambi, Toidi; Golden, Nadia A.; Gentry, Kaylee M.; Doyle-Meyers, Lara A.; Didier, Peter J.; Blanchard, James L.; Kousoulas, K. Gus; Lackner, Andrew A.; Kalman, Daniel; Rengarajan, Jyothi; Khader, Shabaana A.; Kaushal, Deepak

2018-01-01

Mycobacterium tuberculosis continues to cause devastating levels of mortality due to tuberculosis (TB). The failure to control TB stems from an incomplete understanding of the highly specialized strategies that M. tuberculosis utilizes to modulate host immunity and thereby persist in host lungs. Here, we show that M. tuberculosis induced the expression of indoleamine 2,3-dioxygenase (IDO), an enzyme involved in tryptophan catabolism, in macrophages and in the lungs of animals (mice and macaque) with active disease. In a macaque model of inhalation TB, suppression of IDO activity reduced bacterial burden, pathology, and clinical signs of TB disease, leading to increased host survival. This increased protection was accompanied by increased lung T cell proliferation, induction of inducible bronchus-associated lymphoid tissue and correlates of bacterial killing, reduced checkpoint signaling, and the relocation of effector T cells to the center of the granulomata. The enhanced killing of M. tuberculosis in macrophages in vivo by CD4+ T cells was also replicated in vitro, in cocultures of macaque macrophages and CD4+ T cells. Collectively, these results suggest that there exists a potential for using IDO inhibition as an effective and clinically relevant host-directed therapy for TB. PMID:29255022

Arachidonic acid, but not its metabolites, is essential for FcγR-stimulated intracellular killing of Staphylococcus aureus by human monocytes

PubMed Central

Zheng, L; Zomerdijk, T P L; Van Den Barselaar, M T; Geertsma, M F; Van Furth, R; Nibbering, P H

1999-01-01

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcγ receptor (FcγR)-mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcγR-stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo-oxygenase and lipoxygenase, markedly suppressed the FcγR-mediated killing process. The production of O−2 by monocytes upon FcγR cross-linking was inhibited by 4-bromophenacyl bromide in a dose-dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen-dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcγR cross-linking. Furthermore, FcγR cross-linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcγR-mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcγR-stimulated intracellular killing of S. aureus by monocytes. PMID:10233682

Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fuchs, Dominik; Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg; Daniel, Volker

2010-04-16

Leukemia stem cells are known to exhibit multidrug resistance by expression of ATP-binding cassette (ABC) transporters which constitute transmembrane proteins capable of exporting a wide variety of chemotherapeutic drugs from the cytosol. We show here that human promyeloblastic leukemia KG-1a cells exposed to the histone deacetylase inhibitor phenylbutyrate resemble many characteristics of leukemia stem cells, including expression of functional ABC transporters such as P-glycoprotein, BCRP and MRP8. Consequently, KG-1a cells display resistance to the induction of apoptosis by various chemotherapeutic drugs. Resistance to apoptosis induction by chemotherapeutic drugs can be reversed by cyclosporine A, which effectively inhibits the activity ofmore » P-glycoprotein and BCRP, thus demonstrating ABC transporter-mediated drug resistance in KG-1a cells. However, KG-1a are highly sensitive to apoptosis induction by salinomycin, a polyether ionophore antibiotic that has recently been shown to kill human breast cancer stem cell-like cells and to induce apoptosis in human cancer cells displaying multiple mechanisms of drug and apoptosis resistance. Whereas KG-1a cells can be adapted to proliferate in the presence of apoptosis-inducing concentrations of bortezomib and doxorubicin, salinomycin does not permit long-term adaptation of the cells to apoptosis-inducing concentrations. Thus, salinomycin should be regarded as a novel and effective agent for the elimination of leukemia stem cells and other tumor cells exhibiting ABC transporter-mediated multidrug resistance.« less

Genome-wide protective response used by group A Streptococcus to evade destruction by human polymorphonuclear leukocytes.

PubMed

Voyich, Jovanka M; Sturdevant, Daniel E; Braughton, Kevin R; Kobayashi, Scott D; Lei, Benfang; Virtaneva, Kimmo; Dorward, David W; Musser, James M; DeLeo, Frank R

2003-02-18

Group A Streptococcus (GAS) evades polymorphonuclear leukocyte (PMN) phagocytosis and killing to cause human disease, including pharyngitis and necrotizing fasciitis (flesh-eating syndrome). We show that GAS genes differentially regulated during phagocytic interaction with human PMNs comprise a global pathogen-protective response to innate immunity. GAS prophage genes and genes involved in virulence, oxidative stress, cell wall biosynthesis, and gene regulation were up-regulated during PMN phagocytosis. Genes encoding novel secreted proteins were up-regulated, and the proteins were produced during human GAS infections. We discovered an essential role for the Ihk-Irr two-component regulatory system in evading PMN-mediated killing and promoting host-cell lysis, processes that would facilitate GAS pathogenesis. Importantly, the irr gene was highly expressed during human GAS pharyngitis. We conclude that a complex pathogen genetic program circumvents human innate immunity to promote disease. The gene regulatory program revealed by our studies identifies previously undescribed potential vaccine antigens and targets for therapeutic interventions designed to control GAS infections.

HDAC inhibition induces HIV-1 protein and enables immune-based clearance following latency reversal

PubMed Central

Wu, Guoxin; Swanson, Michael; Talla, Aarthi; Graham, Donald; Strizki, Julie; Gorman, Daniel; Barnard, Richard J.O.; Blair, Wade; Søgaard, Ole S.; Tolstrup, Martin; Østergaard, Lars; Rasmussen, Thomas A.; Sekaly, Rafick-Pierre; Archin, Nancie M.; Hazuda, Daria J.; Howell, Bonnie J.

2017-01-01

Promising therapeutic approaches for eradicating HIV include transcriptional activation of provirus from latently infected cells using latency-reversing agents (LRAs) and immune-mediated clearance to purge reservoirs. Accurate detection of cells capable of producing viral antigens and virions, and the measurement of clearance of infected cells, is essential to assessing therapeutic efficacy. Here, we apply enhanced methodology extending the sensitivity limits for the rapid detection of subfemtomolar HIV gag p24 capsid protein in CD4+ T cells from ART-suppressed HIV+ individuals, and we show viral protein induction following treatment with LRAs. Importantly, we demonstrate that clinical administration of histone deacetylase inhibitors (HDACis; vorinostat and panobinostat) induced HIV gag p24, and ex vivo stimulation produced sufficient viral antigen to elicit immune-mediated cell killing using anti-gp120/CD3 bispecific antibody. These findings extend beyond classical nucleic acid endpoints, which are confounded by the predominance of mutated, defective proviruses and, of paramount importance, enable assessment of cells making HIV protein that can now be targeted by immunological approaches. PMID:28814661

Cytotoxic effect of galvanically coupled magnesium-titanium particles.

PubMed

Kim, Jua; Gilbert, Jeremy L

2016-01-01

Recent work has shown that reduction reactions at metallic biomaterial surfaces can induce significant killing of cells in proximity to the surface. To exploit this phenomenon for therapeutic purposes, for example, for cancer tumor killing or antibacterial effects (amongst other applications), magnesium metal particles, galvanically coupled to titanium by sputtering, have been evaluated for their cell-killing capability (i.e. cytotoxicity). Magnesium (Mg) particles large enough to prevent particle phagocytosis were investigated, so that only electrochemical reactions, and not particle toxicity per se, caused cytotoxic effects. Titanium (Ti) coated magnesium particles, as well as magnesium-only particles were introduced into MC3T3-E1 mouse pre-osteoblast cell cultures over a range of particle concentrations, and cells were observed to die in a dosage-dependent manner. Ti-coated magnesium particles killed more cells at lower particle concentration than magnesium alone (P<0.05), although the pH measured for magnesium and magnesium-titanium had no significant difference at similar particle concentrations. Complete cell killing occurred at 750μg/ml and 1500μg/ml for Mg-Ti and Mg, respectively. Thus, this work demonstrates that galvanically coupled Mg-Ti particles have a significant cell killing capability greater than Mg alone. In addition, when the pH associated with complete killing with particles was created using NaOH only (no particles), then the percentage of cells killed was significantly less (P<0.05). Together, these findings show that pH is not the sole factor associated with cell killing and that the electrochemical reactions, including the reduction reactions, play an important role. Reduction reactions on galvanically coupled Mg-Ti and Mg particles may generate reactive oxygen intermediates that are able to kill cells in close proximity to the particles and this approach may lead to potential therapies for infection and cancer. This paper demonstrates that during active corrosion of both Mg and Mg-Ti particles cells cultured with the particles are killed in a dose-dependent particle concentration fashion. Additionally, galvanically-coupled magnesium-titanium microparticles kill cells more effectively than magnesium particles alone. The killing effect was shown to not be due to pH shifts since no differences were seen for different particle types and pH adjusted medium without particles did not exhibit the same level of killing. The significance of this work is the recognition of this killing effect with Mg particles and the potential therapeutic applications in infection control and cancer treatment that this process may provide. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

CAR-T cells are serial killers

PubMed Central

Davenport, Alexander J; Jenkins, Misty R; Ritchie, David S; Prince, H Miles; Trapani, Joseph A; Kershaw, Michael H; Darcy, Phillip K; Neeson, Paul J

2015-01-01

Chimeric antigen receptor (CAR) T cells have enjoyed unprecedented clinical success against haematological malignancies in recent years. However, several aspects of CAR T cell biology remain unknown. We recently compared CAR and T cell receptor (TCR)-based killing in the same effector cell and showed that CAR T cells can not only efficiently kill single tumor targets, they can also kill multiple tumor targets in a sequential manner. Single and serial killing events were not sustained long term due to CAR down-regulation after 20 hours. PMID:26587330

CAR-T cells are serial killers.

PubMed

Davenport, Alexander J; Jenkins, Misty R; Ritchie, David S; Prince, H Miles; Trapani, Joseph A; Kershaw, Michael H; Darcy, Phillip K; Neeson, Paul J

2015-12-01

Chimeric antigen receptor (CAR) T cells have enjoyed unprecedented clinical success against haematological malignancies in recent years. However, several aspects of CAR T cell biology remain unknown. We recently compared CAR and T cell receptor (TCR)-based killing in the same effector cell and showed that CAR T cells can not only efficiently kill single tumor targets, they can also kill multiple tumor targets in a sequential manner. Single and serial killing events were not sustained long term due to CAR down-regulation after 20 hours.

[Experimental research in vitro of TK/GCV system for osteosarcoma MG-63 cell damage].

PubMed

Zhang, Hua-Dong; Lu, Zhi; Feng, Yi; Liu, Xiao-Li; Hou, Hui-Ming

2014-03-01

To study the killing effects of the liposome-mediated thymidine kinase (TK)/ganciclovir (GCV) system on MG-63 osteosarcoma (OS) cells and its bystander effects. Liposome-mediated TK gene transfected into MG-63 OS cells, the efficiency of transfection was analyzed by flow cytometry and observed under inverted fluorescence microscope. Non-transfected osteosarcoma MG-63 cells were divided into three groups,in the experimental group 1 transfected TK/GCV cells cultured in solutiona liquid mixture by supernatant by 1/10,1/7,1/5,1/2 ratio to original broth; in the experimental group 2 transfected cells cultured in solutiona liquid mixture of supernatant filtered through 0.22 microm filter by 1/10,1/7, 1/5, 1/2 ratio to original broth, in control group the transfection cells cultured in original culture solution. Cell growth inhibition rate and osteosarcoma cell sensitivity to TK/GCV system were measured by MTT assay in each group. The TK gene was transfected into MG-63 OS cells successfully by liposome-mediated, flow cytometry instrument detection TK gene transfection cell transfection efficiency can reach 75.5%. Six days later the MTT assay showed that in the experimental group 1 inhibition rate of all concentration ratio of the mixed culture fluid were statistically significant as compared with the control group (P < 0.05), and in the experimental group 2 that of the 1/10 and 1/7 of concentration ratio of mixed culture medium was not statistically significant as compared with the control group (P > 0.05). TK gene transfected MG-63 cells increased with the the GCV concentration,the cell apoptosis rate increased. The experiment demonstrated that the MG-63 OS cells are sensitive to the liposome-mediated TK/GCV system and bystander effects are significant.

Current update of adoptive immunotherapy using cytokine-induced killer cells to eliminate malignant gliomas.

PubMed

Ryu, Je Il; Han, Myung Hoon; Cheong, Jin Hwan; Kim, Jae Min; Kim, Choong Hyun

2017-03-01

The therapeutic outcome for those with malignant glioma is poor, even though diverse therapeutic modalities have been developed. Immunotherapy has emerged as a therapeutic approach for malignant gliomas, making it possible to selectively treat tumors while sparing normal tissue. Here, we review clinical trials of adoptive immunotherapy approaches for malignant gliomas. We also describe a clinical trial that examined the efficacy and safety of autologous cytokine-induced killer (CIK) cells along with concomitant chemoradiotherapy for newly diagnosed glioblastoma. These CIK cells identify and kill autologous tumor cells. This review focuses on the use of adoptive immunotherapy for malignant gliomas and reviews the current literature on the concept of antitumor activity mediated by CIK cells.

Immunogenicity of Nontypeable Haemophilus influenzae Outer Membrane Vesicles and Protective Ability in the Chinchilla Model of Otitis Media.

PubMed

Winter, Linda E; Barenkamp, Stephen J

2017-10-01

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are enriched in several outer membrane components, including major and minor outer membrane proteins and lipooligosaccharide. We assessed the functional activity of nontypeable Haemophilus influenzae (NTHi) OMV-specific antisera and the protective ability of NTHi OMVs as vaccine antigens in the chinchilla otitis media model. OMVs were purified from three HMW1/HMW2-expressing NTHi strains, two of which were also engineered to overexpress Hia proteins. OMV-specific antisera raised in guinea pigs were assessed for their ability to mediate killing of representative NTHi in an opsonophagocytic assay. The three OMV-specific antisera mediated killing of 18 of 65, 24 of 65, and 30 of 65 unrelated HMW1/HMW2-expressing NTHi strains. Overall, they mediated killing of 39 of 65 HMW1/HMW2-expressing strains. The two Hia-expressing OMV-specific antisera mediated killing of 17 of 25 and 14 of 25 unrelated Hia-expressing NTHi strains. Overall, they mediated killing of 20 of 25 Hia-expressing strains. OMVs from prototype NTHi strain 12 were used to immunize chinchillas and the course of middle ear infection was monitored following intrabullar challenge with the homologous strain. All control animals developed culture-positive otitis media, as did two of three HMW1/HMW2-immunized animals. All OMV-immunized animals, with or without supplemental HMW1/HMW2 immunization, were completely protected against otitis media. NTHi OMVs are the first immunogens examined in this model that provided complete protection with sterile immunity after NTHi strain 12 challenge. These data suggest that NTHi OMVs hold significant potential as components of protective NTHi vaccines, possibly in combination with HMW1/HMW2 proteins. Copyright © 2017 American Society for Microbiology.

Liposomes loaded with bioactive lipids enhance antibacterial innate immunity irrespective of drug resistance.

PubMed

Poerio, Noemi; Bugli, Francesca; Taus, Francesco; Santucci, Marilina B; Rodolfo, Carlo; Cecconi, Francesco; Torelli, Riccardo; Varone, Francesco; Inchingolo, Riccardo; Majo, Fabio; Lucidi, Vincenzina; Mariotti, Sabrina; Nisini, Roberto; Sanguinetti, Maurizio; Fraziano, Maurizio

2017-03-27

Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA- or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections.

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity

PubMed Central

Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Liu, Lin; Figini, Mariangela; Powell, Daniel J.

2015-01-01

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate “on-target” toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and “on-target off-tumor” toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs. PMID:26101914

Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion

PubMed Central

Ralston, Katherine S.; Solga, Michael D.; Mackey-Lawrence, Nicole M.; Somlata; Bhattacharya, Alok; Petri, William A.

2014-01-01

Summary paragraph Entamoeba histolytica is the causative agent of amoebiasis, a potentially fatal diarrheal disease in the developing world. The parasite was named “histolytica” for its ability to destroy host tissues, which is most likely driven by direct killing of human cells. The mechanism of human cell killing has been unclear, though the accepted model was that the parasites use secreted toxic effectors to kill cells prior to ingestion1. Here we report the surprising discovery that amoebae kill by biting off and ingesting distinct pieces of living human cells, resulting in intracellular calcium elevation and eventual cell death. After cell killing, amoebae detach and cease ingestion. Ingestion of bites is required for cell killing, and also contributes to invasion of intestinal tissue. The internalization of bites of living human cells is reminiscent of trogocytosis (Greek trogo–, nibble) observed between immune cells2–6, but amoebic trogocytosis differs since it results in death. The ingestion of live cell material and the rejection of corpses illuminate a stark contrast to the established model of dead cell clearance in multicellular organisms7. These findings change the paradigm for tissue destruction in amoebiasis and suggest an ancient origin of trogocytosis as a form of intercellular exchange. PMID:24717428

CD4+ T-Cell-Independent Secondary Immune Responses to Pneumocystis Pneumonia

PubMed Central

de la Rua, Nicholas M.; Samuelson, Derrick R.; Charles, Tysheena P.; Welsh, David A.; Shellito, Judd E.

2016-01-01

Pneumocystis pneumonia is a major cause of morbidity and mortality among immunocompromised patients, especially in the context of HIV/AIDS. In the murine model of Pneumocystis pneumonia, CD4+ T-cells are required for clearance of a primary infection of Pneumocystis, but not the memory recall response. We hypothesized that the memory recall response in the absence of CD4+ T-cells is mediated by a robust memory humoral response, CD8+ T-cells, and IgG-mediated phagocytosis by alveolar macrophages. To investigate the role of CD8+ T-cells and alveolar macrophages in the immune memory response to Pneumocystis, mice previously challenged with Pneumocystis were depleted of CD8+ T-cells or alveolar macrophages prior to re-infection. Mice depleted of CD4+ T-cells prior to secondary challenge cleared Pneumocystis infection within 48 h identical to immunocompetent mice during a secondary memory recall response. However, loss of CD8+ T-cells or macrophages prior to the memory recall response significantly impaired Pneumocystis clearance. Specifically, mice depleted of CD8+ T-cells or alveolar macrophages had significantly higher fungal burden in the lungs. Furthermore, loss of alveolar macrophages significantly skewed the lung CD8+ T-cell response toward a terminally differentiated effector memory population and increased the percentage of IFN-γ+ CD8+ T-cells. Finally, Pneumocystis-infected animals produced significantly more bone marrow plasma cells and Pneumocystis-specific IgG significantly increased macrophage-mediated killing of Pneumocystis in vitro. These data suggest that secondary immune memory responses to Pneumocystis are mediated, in part, by CD8+ T-cells, alveolar macrophages, and the production of Pneumocystis-specific IgG. PMID:27242785

Biomcompatible gold nanorods conjugated with photosensitizers assisted for photostability and photodestructive ability

NASA Astrophysics Data System (ADS)

Kuo, Wen-Shuo; Chen, Shean-Jen

2012-02-01

Light-exposure-mediated higher temperatures that markedly accelerate the degradation of indocyanine green (ICG) in aqueous solutions by thermal decomposition have been a serious medical problem. In this work, we present the example of using gold nanorods (Au NRs) simultaneously serving as photodynamic and photothermal agents to destroy malignant cells. Au NRs were successfully conjugated with hydrophilic photosensitizer, indocyanine green (ICG), to achieve photodynamic therapy (PDT) and photothermal therapy (PTT). We also demonstrated that Au NRs conjugated with ICG displayed high chemical stability and acted as a promising diagnostic probe. Due to its stability even via higher temperatures mediated by laser irradiation, the combination of PDT and PTT proved to be efficiently killing cancer cells as compared to PDT or PTT treatment alone and enhanced the effectiveness of photodestruction and was demonstrated to enhance its photostability.

Intracellular delivery of peptide cargos using iron oxide based nanoparticles: studies on antitumor efficacy of a BCL-2 converting peptide, NuBCP-9

NASA Astrophysics Data System (ADS)

Kumar, Manoj; Singh, Gurpal; Sharma, Sapna; Gupta, Dikshi; Bansal, Vivek; Arora, Vikas; Bhat, Madhusudan; Srivastava, Sandeep K.; Sapra, Sameer; Kharbanda, Surender; Dinda, Amit K.; Singh, Harpal

2014-11-01

Delivering peptides into cells targeting the undruggable oncoproteins is an emerging area in cancer therapeutics. Here we report a novel nanoparticle-based delivery system that can transport therapeutic cargos to the intracellular sites without the need for a cell transduction or penetration domain (CPP). In the present study, we have used iron oxide nanoparticles to deliver an oncopeptide, NuBCP-9, targeting the BCL-2 BH3 domain. Citric acid/2-bromo 2-methylpropanoic acid (CA/BMPA)-capped SPIONs were used to immobilize and deliver the NuBCP-9 peptide to the cancer cells without any noticeable off-target effects. Our results have demonstrated that NuBCP-9-SPIONs efficiently penetrate into cancer cells and bind to its intracellular target protein BCL-2. Moreover, significant inhibition of proliferation and substantial induction of cell death were observed when cancer cells were treated with NuBCP-9-SPIONs at different time intervals. Importantly, the IC50 values for killing of breast cancer cells with NuBCP-9-SPIONs were much lower compared to cells treated with the NuBCP-9 peptide linked with a CPP (Arg-8; NuBCP-9-R8). Molecular and biochemical analyses further supported that NuBCP-9-SPIONs killed breast cancer cells by apoptosis-mediated mechanisms. Furthermore, our data demonstrated that administration of NuBCP-9-SPIONs to mice bearing Ehrlich ascites tumors (EAT) was associated with loss of tumorigenicity and extensive apoptosis in tumor tissues. Taken together, these findings show that a non-CPP-tagged peptide can be successfully delivered to undruggable intracellular oncotargets using SPIONs.Delivering peptides into cells targeting the undruggable oncoproteins is an emerging area in cancer therapeutics. Here we report a novel nanoparticle-based delivery system that can transport therapeutic cargos to the intracellular sites without the need for a cell transduction or penetration domain (CPP). In the present study, we have used iron oxide nanoparticles to deliver an oncopeptide, NuBCP-9, targeting the BCL-2 BH3 domain. Citric acid/2-bromo 2-methylpropanoic acid (CA/BMPA)-capped SPIONs were used to immobilize and deliver the NuBCP-9 peptide to the cancer cells without any noticeable off-target effects. Our results have demonstrated that NuBCP-9-SPIONs efficiently penetrate into cancer cells and bind to its intracellular target protein BCL-2. Moreover, significant inhibition of proliferation and substantial induction of cell death were observed when cancer cells were treated with NuBCP-9-SPIONs at different time intervals. Importantly, the IC50 values for killing of breast cancer cells with NuBCP-9-SPIONs were much lower compared to cells treated with the NuBCP-9 peptide linked with a CPP (Arg-8; NuBCP-9-R8). Molecular and biochemical analyses further supported that NuBCP-9-SPIONs killed breast cancer cells by apoptosis-mediated mechanisms. Furthermore, our data demonstrated that administration of NuBCP-9-SPIONs to mice bearing Ehrlich ascites tumors (EAT) was associated with loss of tumorigenicity and extensive apoptosis in tumor tissues. Taken together, these findings show that a non-CPP-tagged peptide can be successfully delivered to undruggable intracellular oncotargets using SPIONs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr04504a

Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress

PubMed Central

Thompson, Scott M.; Callstrom, Matthew R.; Jondal, Danielle E.; Butters, Kim A.; Knudsen, Bruce E.; Anderson, Jill L.; Lien, Karen R.; Sutor, Shari L.; Lee, Ju-Seog; Thorgeirsson, Snorri S.; Grande, Joseph P.; Roberts, Lewis R.; Woodrum, David A.

2016-01-01

Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2–3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin. PMID:27611696

Killing of targets by effector CD8 T cells in the mouse spleen follows the law of mass action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganusov, Vitaly V

2009-01-01

In contrast with antibody-based vaccines, it has been difficult to measure the efficacy of T cell-based vaccines and to correlate the efficacy of CD8 T cell responses with protection again viral infections. In part, this difficulty is due to poor understanding of the in vivo efficacy of CD8 T cells produced by vaccination. Using a: recently developed experimental method of in vivo cytotoxicity we have investigated quantitative aspects of killing of peptide-pulsed targets by effector and memory CD8 T cells, specific to three epitopes of lymphocytic choriomeningitis virus (LCMV), in the mouse spleen. By analyzing data on killing of targetsmore » with varying number of epitope-specific effector and memory CD8 T cells, we find that killing of targets by effectors follows the law of mass-action, that is the death rate of peptide-pulsed targets is proportional to the frequency of CTLs in the spleen. In contrast, killing of targets by memory CD8 T cells does not follow the mass action law because the death rate of targets saturates at high frequencies of memory CD8 T cells. For both effector and memory cells, we also find little support for the killing term that includes the decrease of the death rate of targets with target cell density. Interestingly, our analysis suggests that at low CD8 T cell frequencies, memory CD8 T cells on the per capita basis are more efficient at killing peptide-pulsed targets than effectors, but at high frequencies, effectors are more efficient killers than memory T cells. Comparison of the estimated killing efficacy of effector T cells with the value that is predicted from theoretical physics and based on motility of T cells in lymphoid tissues, suggests that limiting step in the killing of peptide-pulsed targets is delivering the lethal hit and not finding the target. Our results thus form a basis for quantitative understanding of the process of killing of virus-infected cells by T cell responses in tissues and can be used to correlate the phenotype of vaccine-induced memory CD8 T cells with their killing efficacy in vivo.« less

Targeting the Epidermal Growth Factor Receptor Can Counteract the Inhibition of Natural Killer Cell Function Exerted by Colorectal Tumor-Associated Fibroblasts

PubMed Central

Costa, Delfina; Venè, Roberta; Benelli, Roberto; Romairone, Emanuele; Scabini, Stefano; Catellani, Silvia; Rebesco, Barbara; Mastracci, Luca; Grillo, Federica; Minghelli, Simona; Loiacono, Fabrizio; Zocchi, Maria Raffaella; Poggi, Alessandro

2018-01-01

Mesenchymal stromal cells (MSC) present in the tumor microenvironment [usually named tumor-associated fibroblasts (TAF)] can exert immunosuppressive effects on T and natural killer (NK) lymphocytes, favoring tumor immune escape. We have analyzed this mechanism in colorectal carcinoma (CRC) and found that co-culture of NK cells with TAF can prevent the IL-2-mediated NKG2D upregulation. This leads to the impairment of NKG2D-mediated recognition of CRC cells, sparing the NK cell activation through DNAM1 or FcγRIIIA (CD16). In situ, TAF express detectable levels of epidermal growth factor receptor (EGFR); thus, the therapeutic anti-EGFR humanized antibody cetuximab can trigger the antibody-dependent cellular cytotoxicity of TAF, through the engagement of FcγRIIIA on NK cells. Importantly, in the tumor, we found a lymphoid infiltrate containing NKp46+CD3− NK cells, enriched in CD16+ cells. This population, sorted and cultured with IL-2, could be triggered via CD16 and via NKG2D. Of note, ex vivo NKp46+CD3− cells were able to kill autologous TAF; in vivo, this might represent a control mechanism to reduce TAF-mediated regulatory effect on NK cell function. Altogether, these findings suggest that MSC from the neoplastic mucosa (TAF) of CRC patients can downregulate the immune cell recognition of CRC tumor cells. This immunosuppression can be relieved by the anti-EGFR antibody used in CRC immunotherapy. PMID:29910806

High vancomycin MICs within the susceptible range in Staphylococcus aureus bacteraemia isolates are associated with increased cell wall thickness and reduced intracellular killing by human phagocytes.

PubMed

Falcón, Rocío; Martínez, Alba; Albert, Eliseo; Madrid, Silvia; Oltra, Rosa; Giménez, Estela; Soriano, Mario; Vinuesa, Víctor; Gozalbo, Daniel; Gil, María Luisa; Navarro, David

2016-05-01

Vancomycin minimum inhibitory concentrations (MICs) at the upper end of the susceptible range for Staphylococcus aureus have been associated with poor clinical outcomes of bloodstream infections. We tested the hypothesis that high vancomycin MICs in S. aureus bacteraemia isolates are associated with increased cell wall thickness and suboptimal bacterial internalisation or lysis by human phagocytes. In total, 95 isolates were evaluated. Original vancomycin MICs were determined by Etest. The susceptibility of S. aureus isolates to killing by phagocytes was assessed in a human whole blood assay. Internalisation of bacterial cells by phagocytes was investigated by flow cytometry. Cell wall thickness was evaluated by transmission electron microscopy. Genotypic analysis of S. aureus isolates was performed using a DNA microarray system. Vancomycin MICs were significantly higher (P=0.006) in isolates that were killed suboptimally (killing index <60%) compared with those killed efficiently (killing index >70%) and tended to correlate inversely (P=0.08) with the killing indices. Isolates in both killing groups were internalised by human neutrophils and monocytes with comparable efficiency. The cell wall was significantly thicker (P=0.03) in isolates in the low killing group. No genotypic differences were found between the isolates in both killing groups. In summary, high vancomycin MICs in S. aureus bacteraemia isolates were associated with increased cell wall thickness and reduced intracellular killing by phagocytes. Copyright © 2016 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function*

PubMed Central

Ren, Li; Campbell, Amanda; Fang, Huiqing; Gautam, Shalini; Elavazhagan, Saranya; Fatehchand, Kavin; Mehta, Payal; Stiff, Andrew; Reader, Brenda F.; Mo, Xiaokui; Byrd, John C.; Carson, William E.; Butchar, Jonathan P.; Tridandapani, Susheela

2016-01-01

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function. PMID:26627823

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function.

PubMed

Ren, Li; Campbell, Amanda; Fang, Huiqing; Gautam, Shalini; Elavazhagan, Saranya; Fatehchand, Kavin; Mehta, Payal; Stiff, Andrew; Reader, Brenda F; Mo, Xiaokui; Byrd, John C; Carson, William E; Butchar, Jonathan P; Tridandapani, Susheela

2016-02-05

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Disulfide bond disrupting agents activate the unfolded protein response in EGFR- and HER2-positive breast tumor cells

PubMed Central

Law, Mary E.; Davis, Bradley J.; Bartley, Ashton N.; Higgins, Paul J.; Kilberg, Michael S.; Santostefano, Katherine E.; Terada, Naohiro; Heldermon, Coy D.; Castellano, Ronald K.; Law, Brian K.

2017-01-01

Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus, new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors, and “Triple-Negative” Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor, Progesterone Receptor, and HER2. A significant fraction of TNBCs overexpress the HER2 family member Epidermal Growth Factor Receptor (EGFR). Thus agents that selectively kill EGFR+ and HER2+ tumors would provide new options for breast cancer therapy. We previously identified a class of compounds we termed Disulfide bond Disrupting Agents (DDAs) that selectively kill EGFR+ and HER2+ breast cancer cells in vitro and blocked the growth of HER2+ breast tumors in an animal model. DDA-dependent cytotoxicity was found to correlate with downregulation of HER1-3 and Akt dephosphorylation. Here we demonstrate that DDAs activate the Unfolded Protein Response (UPR) and that this plays a role in their ability to kill EGFR+ and HER2+ cancer cells. The use of breast cancer cell lines ectopically expressing EGFR or HER2 and pharmacological probes of UPR revealed all three DDA responses: HER1-3 downregulation, Akt dephosphorylation, and UPR activation, contribute to DDA-mediated cytotoxicity. Significantly, EGFR overexpression potentiates each of these responses. Combination studies with DDAs suggest that they may be complementary with EGFR/HER2-specific receptor tyrosine kinase inhibitors and mTORC1 inhibitors to overcome drug resistance. PMID:28423644

Enhanced anti-tumor activity by the combination of a conditionally replicating adenovirus mediated interleukin-24 and dacarbazine against melanoma cells via induction of apoptosis.

PubMed

Jiang, Guan; Liu, Yan-Qun; Wei, Zhi-Ping; Pei, Dong-Sheng; Mao, Li-Jun; Zheng, Jun-Nian

2010-08-28

Malignant melanoma is one of the most lethal and aggressive human malignancies. It is notoriously resistant to all of the current therapeutic modalities, including chemotherapy. Suppressed apoptosis and extraordinary invasiveness are the distinctive features that contribute to the malignancy of melanoma. Dacarbazine (DTIC) has been considered as the gold standard for melanoma treatment with a response rate of 15-20%. Unfortunately, the resistance to this chemotherapeutic agent occurs frequently. ZD55-IL-24 is a selective conditionally replicating adenovirus that can mediate the expression of interleukin-24 (IL-24) gene, which has a strong anti-tumor effect. In this study, we hypothesized that a combination of ZD55-IL-24-mediated gene virotherapy and chemotherapy using DTIC would produce an increased cytotoxicity against human melanoma cells in comparison with these agents alone. Our results showed that the combination of ZD55-IL-24 and DTIC significantly enhanced the anti-tumor activity by more effectively inducing apoptosis in melanoma cells than either agent used alone without any overlapping toxicity against normal cells. This additive or synergistic effect of ZD55-IL-24 in combination with DTIC in killing human malignant melanoma cells implies a promising novel approach for melanoma therapy. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements.

PubMed

Sanders, Anne M; Stehle, John R; Blanks, Michael J; Riedlinger, Gregory; Kim-Shapiro, Jung W; Monjazeb, Arta M; Adams, Jonathan M; Willingham, Mark C; Cui, Zheng

2010-03-31

Spontaneous Regression/Complete Resistant (SR/CR) mice are a colony of cancer-resistant mice that can detect and rapidly destroy malignant cells with innate cellular immunity, predominately mediated by granulocytes. Our previous studies suggest that several effector mechanisms, such as perforin, granzymes, or complements, may be involved in the killing of cancer cells. However, none of these effector mechanisms is known as critical for granulocytes. Additionally, it is unclear which effector mechanisms are required for the cancer killing activity of specific leukocyte populations and the survival of SR/CR mice against the challenges of lethal cancer cells. We hypothesized that if any of these effector mechanisms was required for the resistance to cancer cells, its functional knockout in SR/CR mice should render them sensitive to cancer challenges. This was tested by cross breeding SR/CR mice into the individual genetic knockout backgrounds of perforin (Prf-/-), superoxide (Cybb-/), or inducible nitric oxide (Nos2-/). SR/CR mice were bred into individual Prf-/-, Cybb-/-, or Nos2-/- genetic backgrounds and then challenged with sarcoma 180 (S180). Their overall survival was compared to controls. The cancer killing efficiency of purified populations of macrophages and neutrophils from these immunodeficient mice was also examined. When these genetically engineered mice were challenged with cancer cells, the knockout backgrounds of Prf-/-, Cybb-/-, or Nos2-/- did not completely abolish the SR/CR cancer resistant phenotype. However, the Nos2-/- background did appear to weaken the resistance. Incidentally, it was also observed that the male mice in these immunocompromised backgrounds tended to be less cancer-resistant than SR/CR controls. Despite the previously known roles of perforin, superoxide or nitric oxide in the effector mechanisms of innate immune responses, these effector mechanisms were not required for cancer-resistance in SR/CR mice. The resistance was functional when any one of these effector mechanisms was completely absent, except some noticeably reduced penetrance, but not abolishment, of the phenotype in the male background in comparison to female background. These results also indicate that some other effector mechanism(s) of granulocytes may be involved in the killing of cancer cells in SR/CR mice.

An artemisinin-mediated ROS evolving and dual protease light-up nanocapsule for real-time imaging of lysosomal tumor cell death.

PubMed

Huang, Liwei; Luo, Yingping; Sun, Xian; Ju, Huangxian; Tian, Jiangwei; Yu, Bo-Yang

2017-06-15

Lysosomes are critical organelles for cellular homeostasis and can be used as potential targets to kill tumor cells from inside. Many photo-therapeutic methods have been developed to overproduce reactive oxygen species (ROS) to trigger lysosomal membrane permeabilization (LMP)-associated cell death pathway. However, these technologies rely on extra irradiation to activate the photosensitizers, which limits the applications in treating deep seated tumors and widespread metastatic lesions. This work reports a multifunctional nanocapsule to achieve targeted lysosomal tumor cell death without irradiation and real-time monitoring of drug effect through encapsulating artemisinin and dual protease light-up nanoprobe in a folate-functionalized liposome. The nanocapsule can be specifically uptaken by tumor cells via folate receptor-mediated endocytosis to enter lysosomes, in which artemisinin reacts with ferrous to generate ROS for LMP-associated cell death. By virtue of confocal fluorescence imaging, the artemisinin location in lysosome, ROS-triggered LMP and ultimate cell apoptosis can be visualized with the cathepsin B and caspase-3 activatable nanoprobe. Notably, the artemisinin-mediated ROS evolving for tumor therapy and real-time therapeutic monitoring were successfully implemented by living imaging in tumor-bearing mice, which broaden the nanocapsule for in vivo theranostics and may offer new opportunities for precise medicine. Copyright © 2016 Elsevier B.V. All rights reserved.

Mitochondrial protection impairs BET bromodomain inhibitor-mediated cell death and provides rationale for combination therapeutic strategies.

PubMed

Lasorsa, E; Smonksey, M; Kirk, J S; Rosario, S; Hernandez-Ilizaliturri, F J; Ellis, L

2015-12-10

Inhibitors of the bromodomain and extraterminal domain family (BETI) have recently entered phase I clinical trials. In patients with advanced leukemia's, potent antileukemia activity was displayed with minimum dose-limiting toxicity. In preclinical models of hematological malignancies, including aggressive B-cell lymphomas, BETI induced cell-cycle arrest and apoptosis. However, the underlying cell death mechanisms are still not well understood. Dissecting the mechanisms required by BETI to mediate cell death would provide strong direction on how to best utilize BETI to treat patients with aggressive hematological malignancies. Herein, we provide understanding of the molecular mechanisms underlying BETI-mediated cell death using I-BET762. Induction of cell death occurred in primary murine and human B-cell lymphomas through apoptosis. Genetic dissection using Eμ-myc B-cell lymphoma compound mutants demonstrated that I-BET762-induced apoptosis does not require the p53 pathway. Furthermore, deletion of Apaf1, and thus the absence of a functional apoptosome, is associated with a delayed drug response but do not provide long-term resistance. Prolonged treatment of this model in fact fails to suppress the therapeutic efficacy of the drug and is associated with biochemical features of autophagy. However, lack of mitochondrial permeability completely inhibited I-BET762-mediated tumor cell death, indicating mitochondrial damage as key events for its activity. Combination of I-BET762 with BH3-only mimetics ABT-263 or obatoclax, restored sensitivity to I-BET762 lymphoma killing; however, success was determined by expression of Bcl-2 family antiapoptotic proteins. Our study provides critical insight for clinical decisions regarding the appropriate strategy for using BETI as a single agent or in combination to treat patients with aggressive B-cell lymphomas.

Stat6 Promotes Intestinal Tumorigenesis in a Mouse Model of Adenomatous Polyposis by Expansion of MDSCs and Inhibition of Cytotoxic CD8 Response.

PubMed

Jayakumar, Asha; Bothwell, Alfred L M

2017-08-01

Intestinal tumorigenesis in the ApcMin/+ model is initiated by aberrant activation of Wnt pathway. Increased IL-4 expression in human colorectal cancer tissue and growth of colon cancer cell lines implied that IL-4-induced Stat6-mediated tumorigenic signaling likely contributes to intestinal tumor progression in ApcMin/+ mice. Stat6 also appears to promote expansion of myeloid-derived suppressor cells (MDSCs) cells. MDSCs promote polyp formation in the ApcMin/+ model. Hence, Stat6 could have a broad role in coordinating both polyp cell proliferation and MDSC expansion. We found that IL-4-induced Stat6-mediated proliferation of intestinal epithelial cells is augmented by platelet-derived growth factor-BB, a tumor-promoting growth factor. To determine whether polyp progression in ApcMin/+ mice is dependent on Stat6 signaling, we disrupted Stat6 in this model. Total polyps in the small intestine were fewer in ApcMin/+ mice lacking Stat6. Furthermore, proliferation of polyp epithelial cells was reduced, indicating that Stat6 in part controlled polyp formation. Stat6 also promoted expansion of MDSCs in the spleen and lamina propria of ApcMin/+ mice, implying regulation of antitumor T-cell response. More CD8 cells and reduced PD-1 expression on CD4 cells correlated with reduced polyps. In addition, a strong CD8-mediated cytotoxic response led to killing of tumor cells in Stat6-deficient ApcMin/+ mice. Therefore, these findings show that Stat6 has an oncogenic role in intestinal tumorigenesis by promoting polyp cell proliferation and immunosuppressive mediators, and preventing an active cytotoxic process. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Using natural products to promote caspase-8-dependent cancer cell death.

PubMed

Tewary, Poonam; Gunatilaka, A A Leslie; Sayers, Thomas J

2017-02-01

The selective killing of cancer cells without toxicity to normal nontransformed cells is an idealized goal of cancer therapy. Thus, there has been much interest in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a protein that appears to selectively kill cancer cells. TRAIL has been reported to trigger apoptosis and under some circumstances, an alternate death signaling pathway termed necroptosis. The relative importance of necroptosis for cell death induction in vivo is under intensive investigation. Nonetheless, many cancer cells (particularly those freshly isolated from cancer patients) are highly resistant to TRAIL-mediated cell death. Therefore, there is an underlying interest in identifying agents that can be combined with TRAIL to improve its efficacy. There are numerous reports in which combination of TRAIL with standard antineoplastic drugs has resulted in enhanced cancer cell death in vitro. However, many of these chemotherapeutic drugs are nonspecific and associated with adverse effects, which raise serious concerns for cancer therapy in patients. By contrast, natural products have been shown to be safer and efficacious alternatives. Recently, a number of studies have suggested that certain natural products when combined with TRAIL can enhance cancer cell death. In this review, we highlight molecular pathways that might be targeted by various natural products to promote cell death, and focus on our recent work with withanolides as TRAIL sensitizers. Finally, we will suggest synergistic approaches for combining active withanolides with various forms of immunotherapy to promote cancer cell death and an effective antitumor immune response.

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

PubMed

Winkler, Mark T; Bushey, Ryan T; Gottlin, Elizabeth B; Campa, Michael J; Guadalupe, Eross S; Volkheimer, Alicia D; Weinberg, J Brice; Patz, Edward F

2017-01-01

Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

Methylene blue photodynamic therapy induces selective and massive cell death in human breast cancer cells.

PubMed

Dos Santos, Ancély F; Terra, Letícia F; Wailemann, Rosangela A M; Oliveira, Talita C; Gomes, Vinícius de Morais; Mineiro, Marcela Franco; Meotti, Flávia Carla; Bruni-Cardoso, Alexandre; Baptista, Maurício S; Labriola, Leticia

2017-03-15

Breast cancer is the main cause of mortality among women. The disease presents high recurrence mainly due to incomplete efficacy of primary treatment in killing all cancer cells. Photodynamic therapy (PDT), an approach that causes tissue destruction by visible light in the presence of a photosensitizer (Ps) and oxygen, appears as a promising alternative therapy that could be used adjunct to chemotherapy and surgery for curing cancer. However, the efficacy of PDT to treat breast tumours as well as the molecular mechanisms that lead to cell death remain unclear. In this study, we assessed the cell-killing potential of PDT using methylene blue (MB-PDT) in three breast epithelial cell lines that represent non-malignant conditions and different molecular subtypes of breast tumours. Cells were incubated in the absence or presence of MB and irradiated or not at 640 nm with 4.5 J/cm 2 . We used a combination of imaging and biochemistry approaches to assess the involvement of classical autophagic and apoptotic pathways in mediating the cell-deletion induced by MB-PDT. The role of these pathways was investigated using specific inhibitors, activators and gene silencing. We observed that MB-PDT differentially induces massive cell death of tumour cells. Non-malignant cells were significantly more resistant to the therapy compared to malignant cells. Morphological and biochemical analysis of dying cells pointed to alternative mechanisms rather than classical apoptosis. MB-PDT-induced autophagy modulated cell viability depending on the cell model used. However, impairment of one of these pathways did not prevent the fatal destination of MB-PDT treated cells. Additionally, when using a physiological 3D culture model that recapitulates relevant features of normal and tumorous breast tissue morphology, we found that MB-PDT differential action in killing tumour cells was even higher than what was detected in 2D cultures. Finally, our observations underscore the potential of MB-PDT as a highly efficient strategy which could use as a powerful adjunct therapy to surgery of breast tumours, and possibly other types of tumours, to safely increase the eradication rate of microscopic residual disease and thus minimizing the chance of both local and metastatic recurrence.

Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model.

PubMed

Swift, Brenna E; Williams, Brent A; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

2012-07-01

Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted.

In Search of the E. coli Compounds that Change the Antibiotic Production Pattern of Streptomyces coelicolor During Inter-species Interaction.

PubMed

Mavituna, Ferda; Luti, Khalid Jaber Kadhum; Gu, Lixing

2016-08-01

The aim of this work was to investigate the interaction between E.coli and Streptomyces coelicolor A3 (2) for the increased production of undecylprodigiosin and identify the E. coli actives mediating this inter-species interaction. The antibiotics of interest were the red-pigmented undecylprodigiosin and blue-pigmented actinorhodin. Pure cultures of S. coelicolor in a defined medium produced higher concentrations of actinorhodin compared to those of undecylprodigiosin. The latter however, is more important due to its immunosuppressive and antitumor properties. As a strategy to increase undecylprodigiosin production, we added separately, live cells and heat-killed cells of E. coli C600, and the cell-free supernatant of E. coli culture to S. coelicolor cultures in shake flasks. The interaction with live cells of E. coli altered the antibiotic production pattern and undecylprodigiosin production was enhanced by 3.5-fold compared to the pure cultures of S. coelicolor and actinorhodin decreased by 15-fold. The heat-killed cells of E. coli however, had no effect on antibiotic production. In all cases, growth and glucose consumption of S. coelicolor remained almost the same as those observed in the pure culture indicating that the changes in antibiotic production were not due to nutritional stress. Results with cell-free supernatant of E. coli culture indicated that the interaction between S. coelicolor and E. coli was mediated via diffusible molecule(s). Using a set of extraction procedures and agar-well diffusion bioassays, we isolated and preliminarily identified a class of compounds. For the preliminary verification, we added the compound which was the common chemical structural moiety in this class of compounds to the pure S. coelicolor cultures. We observed similar effects on antibiotic production as with the live E. coli cells and their supernatant indicating that this class of compounds secreted by E. coli indeed could act as actives during interspecies interaction and increase the production of undecylprodigiosin. Copyright © 2016 Elsevier Inc. All rights reserved.

Extracellular Fibrils of Pathogenic Yeast Cryptococcus gattii Are Important for Ecological Niche, Murine Virulence and Human Neutrophil Interactions

PubMed Central

Springer, Deborah J.; Ren, Ping; Raina, Ramesh; Dong, Yimin; Behr, Melissa J.; McEwen, Bruce F.; Bowser, Samuel S.; Samsonoff, William A.; Chaturvedi, Sudha; Chaturvedi, Vishnu

2010-01-01

Cryptococcus gattii, an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical and temperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used Arabidopsis thaliana plants and plant-derived substrates to model C. gattii in its natural habitat. Yeast cells readily colonized scratch-wounded plant leaves and formed distinctive extracellular fibrils (40–100 nm diameter ×500–3000 nm length). Extracellular fibrils were observed on live plants and plant-derived substrates by scanning electron microscopy (SEM) and by high voltage- EM (HVEM). Only encapsulated yeast cells formed extracellular fibrils as a capsule-deficient C. gattii mutant completely lacked fibrils. Cells deficient in environmental sensing only formed disorganized extracellular fibrils as apparent from experiments with a C. gattii STE12α mutant. C. gattii cells with extracellular fibrils were more virulent in murine model of pulmonary and systemic cryptococcosis than cells lacking fibrils. C. gattii cells with extracellular fibrils were also significantly more resistant to killing by human polymorphonuclear neutrophils (PMN) in vitro even though these PMN produced elaborate neutrophil extracellular traps (NETs). These observations suggest that extracellular fibril formation could be a structural adaptation of C. gattii for cell-to-cell, cell-to-substrate and/or cell-to- phagocyte communications. Such ecological adaptation of C. gattii could play roles in enhanced virulence in mammalian hosts at least initially via inhibition of host PMN– mediated killing. PMID:20539754

Individual motile CD4+ T cells can participate in efficient multi-killing through conjugation to multiple tumor cells

PubMed Central

Liadi, Ivan; Singh, Harjeet; Romain, Gabrielle; Rey-Villamizar, Nicolas; Merouane, Amine; Adolacion, Jay R T.; Kebriaei, Partow; Huls, Helen; Qiu, Peng; Roysam, Badrinath; Cooper, Laurence J.N.; Varadarajan, Navin

2015-01-01

T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multi-killing via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that while CAR4 cells can participate in killing and multi-killing, they do so at slower rates, likely due to the lower Granzyme B content. Significantly, in both sets of T cells, a minor sub-population of individual T cells identified by their high motility, demonstrated efficient killing of single tumor cells. By comparing both the multi-killer and single killer CAR+ T cells it appears that the propensity and kinetics of T-cell apoptosis was modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multi-killing should be evaluated in the context of their ability to resist activation induced cell death (AICD). We anticipate that TIMING may be utilized to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. PMID:25711538

Fascin-1 knock-down of human glioma cells reduces their microvilli/filopodia while improving their susceptibility to lymphocyte-mediated cytotoxicity

PubMed Central

Hoa, Neil T; Ge, Lisheng; Erickson, Kate L; Kruse, Carol A; Cornforth, Andrew N; Kuznetsov, Yurii; McPherson, Alex; Martini, Filippo; Jadus, Martin R

2015-01-01

Cancer cells derived from Glioblastoma multiforme possess membranous protrusions allowing these cells to infiltrate surrounding tissue, while resisting lymphocyte cytotoxicity. Microvilli and filopodia are supported by actin filaments cross-linked by fascin. Fascin-1 was genetically silenced within human U251 glioma cells; these knock-down glioma cells lost their microvilli/filopodia. The doubling time of these fascin-1 knock-down cells was doubled that of shRNA control U251 cells. Fascin-1 knock-down cells lost their transmigratory ability responding to interleukin-6 or insulin-like growth factor-1. Fascin-1 silenced U251 cells were more easily killed by cytolytic lymphocytes. Fascin-1 knock-down provides unique opportunities to augment glioma immunotherapy by simultaneously targeting several key glioma functions: like cell transmigration, cell division and resisting immune responses. PMID:25901196

CXCR1-mediated Neutrophil Degranulation and Fungal Killing Promotes Candida Clearance and Host Survival

PubMed Central

Swamydas, Muthulekha; Gao, Ji-Liang; Break, Timothy J.; Johnson, Melissa D.; Jaeger, Martin; Rodriguez, Carlos A.; Lim, Jean K.; Green, Nathaniel M.; Collar, Amanda L.; Fischer, Brett G.; Lee, Chyi-Chia Richard; Perfect, John R.; Alexander, Barbara D.; Kullberg, Bart-Jan; Netea, Mihai G.; Murphy, Philip M.; Lionakis, Michail S.

2016-01-01

Systemic Candida albicans infection causes high morbidity and mortality and is now the leading cause of nosocomial bloodstream infection in the US. Neutropenia is a major risk factor for poor outcome in infected patients; however, the molecular factors that mediate neutrophil trafficking and effector function during infection are poorly defined. Here, using a mouse model of systemic candidiasis, we found that the neutrophil-selective CXC chemokine receptor Cxcr1 and its ligand, Cxcl5, are highly induced in the Candida-infected kidney, the target organ in the model. To investigate the role of Cxcr1 in antifungal host defense in vivo, we generated Cxcr1−/− mice and analyzed their immune response to Candida. Mice lacking Cxcr1 exhibited decreased survival with enhanced Candida growth in the kidney and renal failure. Surprisingly, increased susceptibility of Cxcr1−/− mice to systemic candidiasis was not due to impaired neutrophil trafficking from the blood into the infected kidney but was the result of defective killing of the fungus by neutrophils that exhibited a cell-intrinsic decrease in degranulation. In humans, the mutant CXCR1 allele CXCR1-T276 results in impaired neutrophil degranulation and fungal killing and was associated with increased risk of disseminated candidiasis in infected patients. Together, our data demonstrate a biological function for mouse Cxcr1 in vivo and indicate that CXCR1-dependent neutrophil effector function is a critical innate protective mechanism of fungal clearance and host survival in systemic candidiasis. PMID:26791948

CCR-2 neutralization augments murine fresh BMC activation by Staphylococcus aureus via two distinct mechanisms: at the level of ROS production and cytokine response.

PubMed

Nandi, Ajeya; Bishayi, Biswadev

2017-05-01

CCR-2 signaling regulates recruitment of monocytes from the bone marrow into the bloodstream and then to sites of infection. We sought to determine whether CCL-2/CCR-2 signaling is involved in the killing of Staphylococcus aureus by murine bone marrow cells (BMCs). The intermittent link of reactive oxygen species (ROS)-NF-κB/p38-MAPK-mediated CCL-2 production in CCR-2 signaling prompted us to determine whether neutralization of CCR-2 augments the response of murine fresh BMCs (FBMCs) after S. aureus infection. It was observed that anti-CCR-2 Ab-treated FBMCs released fewer ROS on encountering S. aureus infection than CCR-2 non-neutralized FBMCs, also correlating with reduced killing of S. aureus in CCR-2 neutralized FBMCs. Staphylococcal catalase and SOD were also found to play a role in protecting S. aureus from the ROS-mediated killing of FBMC. S. aureus infection of CCR-2 intact FBMCs pre-treated with either NF-κB or p-38-MAPK blocker induced less CCL-2, suggesting that NF-κB or p-38-MAPK is required for CCL-2 production by FBMCs. Moreover, blocking of CCR-2 along with NF-κB or p-38-MAPK resulted in elevated CCL-2 production and reduced CCR-2 expression. Inhibition of CCR-2 impairs the response of murine BMCs to S. aureus infection by attenuation ROS production and modulating the cytokine response.

Host and Pathogen Copper-Transporting P-Type ATPases Function Antagonistically during Salmonella Infection.

PubMed

Ladomersky, Erik; Khan, Aslam; Shanbhag, Vinit; Cavet, Jennifer S; Chan, Jefferson; Weisman, Gary A; Petris, Michael J

2017-09-01

Copper is an essential yet potentially toxic trace element that is required by all aerobic organisms. A key regulator of copper homeostasis in mammalian cells is the copper-transporting P-type ATPase ATP7A, which mediates copper transport from the cytoplasm into the secretory pathway, as well as copper export across the plasma membrane. Previous studies have shown that ATP7A-dependent copper transport is required for killing phagocytosed Escherichia coli in a cultured macrophage cell line. In this investigation, we expanded on these studies by generating Atp7a LysMcre mice, in which the Atp7a gene was specifically deleted in cells of the myeloid lineage, including macrophages. Primary macrophages isolated from Atp7a LysMcre mice exhibit decreased copper transport into phagosomal compartments and a reduced ability to kill Salmonella enterica serovar Typhimurium compared to that of macrophages isolated from wild-type mice. The Atp7a LysMcre mice were also more susceptible to systemic infection by S Typhimurium than wild-type mice. Deletion of the S Typhimurium copper exporters, CopA and GolT, was found to decrease infection in wild-type mice but not in the Atp7a LysMcre mice. These studies suggest that ATP7A-dependent copper transport into the phagosome mediates host defense against S Typhimurium, which is counteracted by copper export from the bacteria via CopA and GolT. These findings reveal unique and opposing functions for copper transporters of the host and pathogen during infection. Copyright © 2017 American Society for Microbiology.

A Sequential Model of Host Cell Killing and Phagocytosis by Entamoeba histolytica

PubMed Central

Sateriale, Adam; Huston, Christopher D.

2011-01-01

The protozoan parasite Entamoeba histolytica is responsible for invasive intestinal and extraintestinal amebiasis. The virulence of Entamoeba histolytica is strongly correlated with the parasite's capacity to effectively kill and phagocytose host cells. The process by which host cells are killed and phagocytosed follows a sequential model of adherence, cell killing, initiation of phagocytosis, and engulfment. This paper presents recent advances in the cytolytic and phagocytic processes of Entamoeba histolytica in context of the sequential model. PMID:21331284

Relationship between antibody susceptibility and lipopolysaccharide O-antigen characteristics of invasive and gastrointestinal nontyphoidal Salmonellae isolates from Kenya.

PubMed

Onsare, Robert S; Micoli, Francesca; Lanzilao, Luisa; Alfini, Renzo; Okoro, Chinyere K; Muigai, Anne W; Revathi, Gunturu; Saul, Allan; Kariuki, Samuel; MacLennan, Calman A; Rondini, Simona

2015-03-01

Nontyphoidal Salmonellae (NTS) cause a large burden of invasive and gastrointestinal disease among young children in sub-Saharan Africa. No vaccine is currently available. Previous reports indicate the importance of the O-antigen of Salmonella lipopolysaccharide for virulence and resistance to antibody-mediated killing. We hypothesised that isolates with more O-antigen have increased resistance to antibody-mediated killing and are more likely to be invasive than gastrointestinal. We studied 192 NTS isolates (114 Typhimurium, 78 Enteritidis) from blood and stools, mostly from paediatric admissions in Kenya 2000-2011. Isolates were tested for susceptibility to antibody-mediated killing, using whole adult serum. O-antigen structural characteristics, including O-acetylation and glucosylation, were investigated. Overall, isolates were susceptible to antibody-mediated killing, but S. Enteritidis were less susceptible and expressed more O-antigen than Typhimurium (p<0.0001 for both comparisons). For S. Typhimurium, but not Enteritidis, O-antigen expression correlated with reduced sensitivity to killing (r = 0.29, 95% CI = 0.10-0.45, p = 0.002). Both serovars expressed O-antigen populations ranging 21-33 kDa average molecular weight. O-antigen from most Typhimurium were O-acetylated on rhamnose and abequose residues, while Enteritidis O-antigen had low or no O-acetylation. Both Typhimurium and Enteritidis O-antigen were approximately 20%-50% glucosylated. Amount of S. Typhimurium O-antigen and O-antigen glucosylation level were inversely related. There was no clear association between clinical presentation and antibody susceptibility, O-antigen level or other O-antigen features. Kenyan S. Typhimurium and Enteritidis clinical isolates are susceptible to antibody-mediated killing, with degree of susceptibility varying with level of O-antigen for S. Typhimurium. This supports the development of an antibody-inducing vaccine against NTS for Africa. No clear differences were found in the phenotype of isolates from blood and stool, suggesting that the same isolates can cause invasive disease and gastroenteritis. Genome studies are required to understand whether invasive and gastrointestinal isolates differ at the genotypic level.

Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis

PubMed Central

Chen, Xin; He, Wan-ting; Hu, Lichen; Li, Jingxian; Fang, Yuan; Wang, Xin; Xu, Xiaozheng; Wang, Zhuo; Huang, Kai; Han, Jiahuai

2016-01-01

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis. PMID:27573174

Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis.

PubMed

Chen, Xin; He, Wan-Ting; Hu, Lichen; Li, Jingxian; Fang, Yuan; Wang, Xin; Xu, Xiaozheng; Wang, Zhuo; Huang, Kai; Han, Jiahuai

2016-09-01

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis.

Influence of anaesthetics on tumour-cell kill and repopulation in B16 melanoma treated with melphalan.

PubMed Central

Peacock, J. H.; Stephens, T. C.

1978-01-01

The influence of anaesthetics on the in vivo response of B16 melanoma to melphalan was studied using an in vitro cell-survival assay. Three anaesthetics were used, Saffan (Althesin) Sagatal (Nembutal) and Hypnorm. When Saffan was administered to tumour-bearing animals before melphalan there was a significant increase in tumour-cell kill. This effect was not observed with Sagatal or Hypnorm. Maximum increase in tumour-cell kill was achieved when Saffan was administered about 1 h before melphalan, and was dependent on Saffan dose. Clonogenic tumour-cell repopulation after melphalan was rapid (TD - 1 day) and the rate was similar from 2 levels of cell kill. When Saffan was combined with melphalan the repopulation rate was the same as with melphalan alone, and the increased cell kill was reflected in increased growth delay. The in vitro response of B16 melanoma cells to melphalan was unaltered by pretreatment with, or simultaneous exposure to Saffan. The results suggest that the mechanism of the enhanced cell kill in vivo is probably due to an indirect systemic effect, rather than a direct effect on the tumour cells. PMID:743490

In vitro susceptibility of Mycobacterium leprae to oxygen-mediated damage.

PubMed

Dhople, A M

1996-01-01

In order to evaluate factors responsible for the failure of Mycobacterium leprae to multiply in cell-free cultures in vitro studies were undertaken to determine the possible poisoning of the organism by hydroxide and superoxide radicals produced in the growth medium. The superoxide dismutase activity was very low, 10% of the levels found in armadillo cells, while measured activity of catalase and glutathione peroxidase was negligible. Susceptibility of M. leprae to hydrogen peroxide was enhanced by potassium iodide but not by lactoperoxidase. The addition of high amounts of catalase completely prevented hydrogen peroxide-mediated killing of M. leprae. Superoxide generated by the action of xanthine oxidase on xanthine was lethal to M. leprae, but superoxide dismutase added to the reaction mixture gave significant protection. Thus superoxide radicals may be a major cause for the sudden termination of growth of M. leprae in primary cultures and also for failure of subcultures.

Induction of cell death by pyropheophorbide-α methyl ester-mediated photodynamic therapy in lung cancer A549 cells.

PubMed

Tu, Ping-Hua; Huang, Wen-Jun; Wu, Zhan-Ling; Peng, Qing-Zhen; Xie, Zhi-Bin; Bao, Ji; Zhong, Ming-Hua

2017-03-01

Pyropheophorbide-α methyl ester (MPPa) was a promising photosensitizer with stable chemical structure, strong absorption, higher tissue selectivity and longer activation wavelengths. The present study investigated the effect of MPPa-mediated photodynamic treatment on lung cancer A549 cells as well as the underlying mechanisms. Cell Counting Kit-8 was employed for cell viability assessment. Reactive oxygen species levels were determined by fluorescence microscopy and flow cytometry. Cell morphology was evaluated by Hoechst staining and transmission electron microscopy. Mitochondrial membrane potential, cellular apoptosis and cell cycle distribution were evaluated flow-cytometrically. The protein levels of apoptotic effectors were examined by Western blot. We found that the photocytotoxicity of MPPa showed both drug- and light- dose dependent characteristics in A549 cells. Additionally, MPPa-PDT caused cell apoptosis by reducing mitochondrial membrane potential, increasing reactive oxygen species (ROS) production, inducing caspase-9/caspase-3 signaling activation as well as cell cycle arrest at G 0 /G 1 phase. These results suggested that MPPa-PDT mainly kills cells by apoptotic mechanisms, with overt curative effects, indicating that MPPa should be considered a potent photosensitizer for lung carcinoma treatment. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Selective killing of cancer cells by iron oxide nanoparticles mediated through reactive oxygen species via p53 pathway

NASA Astrophysics Data System (ADS)

Ahamed, Maqusood; Alhadlaq, Hisham A.; Khan, M. A. Majeed; Akhtar, Mohd. Javed

2013-01-01

Iron oxide (Fe3O4) nanoparticles (NPs) are increasingly recognized for their utility in biomedical applications. However, little is known about the anticancer activity of Fe3O4 NPs. This study was designed to investigate whether Fe3O4 NPs induced toxicity in a cell-specific manner and determine the possible mechanisms of toxicity caused by Fe3O4 NPs in cancer cells. Fe3O4 NPs used in this study were synthesized by green method using α- d-glucose as a reducing agent. Prepared Fe3O4 NPs were spherical in shape with a smooth surface, were fairly distributed, and had an average diameter of 23 nm. Cytotoxicity of Fe3O4 NPs was examined against two types of cancer cells (human hepatocellular carcinoma HepG2 and human lung adenocarcinoma A549) and two normal cells (human lung fibroblast IMR-90 and rat hepatocytes). Fe3O4 NPs exerted distinct effects on cell viability via killing of cancer cells while posing no toxicity on normal cells. Fe3O4 NPs were found to induce depletion of glutathione and induction of reactive oxygen species (ROS) in both types of cancer cells (HepG2 and A549). Further, co-exposure of ascorbic acid significantly attenuated the Fe3O4 NPs-induced oxidative stress. The mRNA levels of tumor suppressor gene p53 and apoptotic genes (caspase-3 and caspase-9) were up-regulated in both types of cancer cells due to Fe3O4 NPs exposure. Protein level of p53, along with the higher activity of caspase-3 and caspase-9 enzymes, was also up-regulated by Fe3O4 NPs. Taken together, our data demonstrated that Fe3O4 NPs selectively induced apoptosis in cancer cells (HepG2 and A549) through up-regulation of p53 that might be mediated by ROS through which most of the anticancer drugs trigger apoptosis. The present study warrants further investigation on anticancer activity of Fe3O4 NPs in relevant animal models.

Cancer hyperthermia using magnetic nanoparticles.

PubMed

Kobayashi, Takeshi

2011-11-01

Magnetic-nanoparticle-mediated intracellular hyperthermia has the potential to achieve localized tumor heating without any side effects. The technique consists of targeting magnetic nanoparticles to tumor tissue followed by application of an external alternating magnetic field that induces heat through Néel relaxation loss of the magnetic nanoparticles. The temperature in tumor tissue is increased to above 43°C, which causes necrosis of cancer cells, but does not damage surrounding normal tissue. Among magnetic nanoparticles available, magnetite has been extensively studied. Recent years have seen remarkable advances in magnetite-nanoparticle-mediated hyperthermia; both functional magnetite nanoparticles and alternating-magnetic-field generators have been developed. In addition to the expected tumor cell death, hyperthermia treatment has also induced unexpected biological responses, such as tumor-specific immune responses as a result of heat-shock protein expression. These results suggest that hyperthermia is able to kill not only local tumors exposed to heat treatment, but also tumors at distant sites, including metastatic cancer cells. Currently, several research centers have begun clinical trials with promising results, suggesting that the time may have come for clinical applications. This review describes recent advances in magnetite nanoparticle-mediated hyperthermia. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Eradication of Pseudomonas aeruginosa cells by cathodic electrochemical currents delivered with graphite electrodes.

PubMed

Niepa, Tagbo H R; Wang, Hao; Gilbert, Jeremy L; Ren, Dacheng

2017-03-01

Antibiotic resistance is a major challenge to the treatment of bacterial infections associated with medical devices and biomaterials. One important intrinsic mechanism of such resistance is the formation of persister cells that are phenotypic variants of microorganisms and highly tolerant to antibiotics. Recently, we reported a new approach to eradicating persister cells of Pseudomonas aeruginosa using low-level direct electrochemical current (DC) and synergy with the antibiotic tobramycin. To further understand the underlying mechanism and develop this technology toward possible medical applications, we investigated the electricidal activities of non-metallic biomaterial on persister and biofilm cells of P. aeruginosa using graphite-based TGON™ 805 electrodes. We employed both single and dual chamber systems to compare electrochemical factors of TGON and stainless steel 304 electrodes. The results revealed that TGON-based treatments were highly effective against P. aeruginosa persister cells. In the single chamber system, complete eradication of planktonic persister cells (corresponding to a 7-log killing) was achieved with 70μA/cm 2 DC using TGON electrodes within 40min of treatment, while the cell viability in biofilms was reduced by 2 logs within 1h. The killing effects were dose and time dependent with higher current densities requiring less time. Moreover, reduction reactions were found more effective than oxidation reactions, confirming that metal cations are not indispensable, although they may facilitate cell killing. The findings of this study can help develop electrochemical technologies to eradicate persister and biofilm cells for more effective treatment of medical device and biomaterial associated infections. Infections associated with medical devices and biomaterials present a major challenge due to high-level tolerance of microbes to conventional antibiotics. It is well established that such tolerance is due to the formation of dormant persister cells and multicellular structures known as biofilms. Recent studies have demonstrated electrochemical treatment as a promising alternative to eradicate bacterial infections, since the killing mechanism is independent of the growth phase of bacterial cells, but relies on various electrochemical species interplaying during the treatment. The current study investigated major bactericidal properties of the electrochemical currents mediated via TGON, a carbon-based electrode material. Up to total eradication of Pseudomonas aeruginosa persister cells was achieved. The new knowledge of electrochemical properties and the bioactivity of TGON may help develop new methods/devices to eradicate bacterial infections by delivering safe levels of electrochemical currents. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Activation of the cholinergic anti-inflammatory pathway by GTS-21 attenuates cisplatin-induced acute kidney injury in mice

PubMed Central

Chatterjee, Prodyot K.; Yeboah, Michael M.; Solanki, Malvika H.; Kumar, Gopal; Xue, Xiangying; Pavlov, Valentin A.; Al-Abed, Yousef

2017-01-01

Acute kidney injury (AKI) is the most common side effect of cisplatin, a widely used chemotherapy drug. Although AKI occurs in up to one third of cancer patients receiving cisplatin, effective renal protective strategies are lacking. Cisplatin targets renal proximal tubular epithelial cells leading to inflammation, reactive oxygen species, tubular cell injury, and eventually cell death. The cholinergic anti-inflammatory pathway is a vagus nerve-mediated reflex that suppresses inflammation via α7 nicotinic acetylcholine receptors (α7nAChRs). Our previous studies demonstrated the renoprotective and anti-inflammatory effects of cholinergic agonists, including GTS-21. Therefore, we examined the effect of GTS-21 on cisplatin-induced AKI. Male C57BL/6 mice received either saline or GTS-21 (4mg/kg, i.p.) twice daily for 4 days before cisplatin and treatment continued through euthanasia; 3 days post-cisplatin mice were euthanized and analyzed for markers of renal injury. GTS-21 significantly reduced cisplatin-induced renal dysfunction and injury (p<0.05). GTS-21 significantly attenuated renal Ptgs2/COX-2 mRNA and IL-6, IL-1β, and CXCL1 protein expression, as well as neutrophil infiltration after cisplatin. GTS-21 blunted cisplatin-induced renal ERK1/2 activation, as well as renal ATP depletion and apoptosis (p<0.05). GTS-21 suppressed the expression of CTR1, a cisplatin influx transporter and enhanced the expression of cisplatin efflux transporters MRP2, MRP4, and MRP6 (p<0.05). Using breast, colon, and lung cancer cell lines we showed that GTS-21 did not inhibit cisplatin’s tumor cell killing activity. GTS-21 protects against cisplatin-AKI by attenuating renal inflammation, ATP depletion and apoptosis, as well as by decreasing renal cisplatin influx and increasing efflux, without impairing cisplatin-mediated tumor cell killing. Our results support further exploring the cholinergic anti-inflammatory pathway for preventing cisplatin-induced AKI. PMID:29190774

Synergy between tobramycin and trivalent chromium ion in electrochemical control of Pseudomonas aeruginosa.

PubMed

Niepa, Tagbo H R; Wang, Hao; Dabrowiak, James C; Gilbert, Jeremy L; Ren, Dacheng

2016-05-01

We recently demonstrated that the effectiveness of tobramycin (Tob), an aminoglycoside, against antibiotic-tolerant persister cells of Pseudomonas aeruginosa can be enhanced by electrochemical factors generated from direct currents (DC). Supplementation of Ni(II), Cr(III) and Fe(II) during carbon-mediated DC treatment revealed that these metal cations promote killing of persister cells in the presence of tobramycin, which led to our hypothesis that specific interactions between Tob and some metal ions contribute to the synergistic killing of persister cells. In this study, the interactions between selected metal cations and Tob were investigated using (1)H-(13)C HSQC NMR. Increase in the concentration of Cr(III) (in the form of [CrCl2(H2O)4](+)) in solutions containing Tob was found to shift the HSQC NMR peaks of Tob to new positions, suggesting the formation of a Cr(III)-Tob complex. Crystal field effects and electrochemical properties of the complex were further studied using UV-visible spectroscopy and cyclic voltammetry, which led to the finding that the Cr(III)-Tob complex has increased affinity with negatively charged nucleic acids. These findings are helpful for understanding the mechanism of electrochemical control of bacterial cells and for developing more effective antimicrobial therapies based on aminoglycosides and electrochemical species released from various metallic biomaterials. Medical device associated infections present a major challenge to healthcare and the quality of life of affected individuals. This problem is further exacerbated by the emergence of multidrug resistant pathogens. Thus, alternative methods for microbial control are urgently needed. Recently, we reported synergy between tobramycin and low-level electrochemical currents generated using stainless steel electrodes in killing bacterial persister cells, a dormant population with high-level intrinsic tolerance to antibiotics. In this article, we describe how electrically-induced interaction between aminoglycosides and certain metal cations enhance the potency of tobramycin in bacterial killing. The findings will help design new methods for controlling infections through electrochemical disruption of cellular function and associated drug resistance. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Distinct single-cell morphological dynamics under beta-lactam antibiotics

PubMed Central

Yao, Zhizhong; Kahne, Daniel; Kishony, Roy

2012-01-01

Summary The bacterial cell wall is conserved in prokaryotes, stabilizing cells against osmotic stress. Beta-lactams inhibit cell wall synthesis and induce lysis through a bulge-mediated mechanism; however, little is known about the formation dynamics and stability of these bulges. To capture processes of different timescales, we developed an imaging platform combining automated image analysis with live cell microscopy at high time resolution. Beta-lactam killing of Escherichia coli cells proceeded through four stages: elongation, bulge formation, bulge stagnation and lysis. Both the cell wall and outer membrane (OM) affect the observed dynamics; damaging the cell wall with different beta-lactams and compromising OM integrity cause different modes and rates of lysis. Our results show that the bulge formation dynamics is determined by how the cell wall is perturbed. The OM plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows recovery upon drug removal. PMID:23103254

Bactericidal effects of various concentrations of enrofloxacin, florfenicol, tilmicosin phosphate, and tulathromycin on clinical isolates of Mannheimia haemolytica.

PubMed

Blondeau, Joseph M; Shebelski, Shantelle D; Hesje, Christine K

2015-10-01

To determine bactericidal effects of enrofloxacin, florfenicol, tilmicosin, and tulathromycin on clinical isolates of Mannheimia haemolytica at various bacterial densities and drug concentrations. 4 unique isolates of M haemolytica recovered from clinically infected cattle. Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined for each drug and isolate. Mannheimia haemolytica suspensions (10(6) to 10(9) CFUs/mL) were exposed to the determined MIC and MPC and preestablished maximum serum and tissue concentrations of each drug. Log10 reduction in viable cells (percentage of cells killed) was measured at various points. Bacterial killing at the MIC was slow and incomplete. After 2 hours of isolate exposure to the MPC and maximum serum and tissue concentrations of the tested drugs, 91% to almost 100% cell killing was achieved with enrofloxacin, compared with 8% growth to 93% cell killing with florfenicol, 199% growth to 63% cell killing with tilmicosin, and 128% growth to 43% cell killing with tulathromycin over the range of inoculum tested. For all drugs, killing of viable organisms was evident at all bacterial densities tested; however, killing was more substantial at the MPC and maximum serum and tissue drug concentrations than at the MIC and increased with duration of drug exposure. Rank order of drugs by killing potency was enrofloxacin, florfenicol, tilmicosin, and tulathromycin. Findings suggested that antimicrobial doses that equaled or exceeded the MPC provided rapid killing of M haemolytica by the tested drugs, decreasing opportunities for antimicrobial-resistant subpopulations of bacteria to develop during drug exposure.

Intracellular cavitation as a mechanism of short-pulse laser injury to the retinal pigment epithelium

NASA Astrophysics Data System (ADS)

Kelly, Michael William

This research was primarily motivated to determine the retinal injury mechanism from ultra-short pulse (<1ns) lasers. The American National Standards Institute, ANSI, standards for safe retinal exposures, and mechanisms for injury, are established for pulse durations longer than 1 ns. Little data exists for shorter pulse durations. High temperatures and pressures, generated within pigmented melanosomes, leads to mechanically mediated injury for such exposures. We used nanosecond time resolved imaging to evaluate transient photo-mechanical effects on isolated melanosomes, pigmented cell cultures, and the retinal pigment epithelium, RPE, ex-vivo. Exposures between 20 ns and 100 fs were performed. We developed a unique ex-vivo model to examine transient events directly on the RPE. Evaluation of cell viability was accomplished in real time, minutes after the exposure. The threshold for cavitation (bubble formation) around single melanosomes corresponded with the threshold for intracellular cavitation and cell killing, in the nanosecond and picosecond domain. Shock waves, formed around melanosomes following sub-nanosecond exposures, did not affect the mechanism for cell killing at threshold. Although the wavelength was increased for shorter exposures (3 ps, 300 fs, and 100 fs) the threshold for intracellular cavitation decreased. All results were compared with data collected by others, using live animal models.

Subinhibitory Doses of Aminoglycoside Antibiotics Induce Changes in the Phenotype of Mycobacterium abscessus

PubMed Central

Tsai, Sheng-Hui; Lai, Hsin-Chih

2015-01-01

Subinhibitory doses of antibiotics have been shown to cause changes in bacterial morphology, adherence ability, and resistance to antibiotics. In this study, the effects of subinhibitory doses of aminoglycoside antibiotics on Mycobacterium abscessus were investigated. The treatment of M. abscessus cells with subinhibitory doses of amikacin was found to change their colony from a smooth to a rough morphotype and increase their ability to adhere to a polyvinylchloride plate, aggregate in culture, and resist phagocytosis and killing by macrophages. M. abscessus cells treated with a subinhibitory dose of amikacin also became more potent in Toll-like receptor 2 (TLR-2) stimulation, leading to increased tumor necrosis factor alpha (TNF-α) production by macrophages. The MAB_3508c gene was shown to play a role in mediating these phenotypic changes, as its expression in M. abscessus cells was increased when they were treated with a subinhibitory dose of amikacin. In addition, overexpression of MAB_3508c in M. abscessus cells caused changes similar to those induced by subinhibitory doses of amikacin, including a switch from smooth to rough colony morphology, increased ability to aggregate in liquid culture, decreased motility, and increased resistance to killing by macrophages. These findings suggest the importance of using sufficient doses of antibiotics for the treatment of M. abscessus infections. PMID:26195529

Differential Fc-receptor engagement drives an anti-tumor vaccinal effect

PubMed Central

DiLillo, David J.; Ravetch, Jeffrey V.

2015-01-01

Summary Passively-administered anti-tumor mAbs rapidly kill tumor targets via FcγR-mediated cytotoxicity (ADCC), a short-term process. However, anti-tumor mAb treatment can also induce a vaccinal effect, in which mAb-mediated tumor death induces a long-term anti-tumor cellular immune response. To determine how such responses are generated, we utilized a murine model of an anti-tumor vaccinal effect against a model neoantigen. We demonstrate that FcγR expression by CD11c+ antigen-presenting cells is required to generate anti-tumor T cell responses upon ADCC-mediated tumor clearance. Using FcγR-humanized mice, we demonstrate that anti-tumor huIgG1 must engage hFcγRIIIA on macrophages to mediate ADCC, but also engage hFcγRIIA, the sole hFcγR expressed by human DCs, to generate a potent vaccinal effect. Thus, while next-generation anti-tumor antibodies with enhanced binding to only hFcγRIIIA are now in clinical use, ideal anti-tumor antibodies must be optimized for both cytotoxic effects as well as hFcγRIIA engagement on DCs to stimulate long-term anti-tumor cellular immunity. PMID:25976835

Reactive oxygen species mediated ginsenoside Rg3- and Rh2-induced apoptosis in hepatoma cells through mitochondrial signaling pathways.

PubMed

Park, Hye-Min; Kim, Shang-Jin; Kim, Jin-Shang; Kang, Hyung-Sub

2012-08-01

Panax ginseng (P. ginseng) has anti-cancer effects in several cancer models. Ginsenosides are the main bioactive components in P. ginseng. Korean red ginseng (KRG) extract can potently kill various cancer cells and ginsenosides Rg3 (GRg3) and Rh2 (GRh2) are the primary ginsenosides in KRG. This study was carried out to examine whether KRG and its primary ginsenosides (GRg3 and GRh2) affect apoptosis of human hepatocellular carcinoma cells (Hep3B). KRG, GRg3 and GRh2 have obvious cytotoxic and apoptotic effects in Hep3B cells as evidenced by a decrease in cell viability and mitochondria membrane potential, but an increase in LDH release. In the mitochondria-mediated apoptosis pathway, KRG, GRg3 and GRh2 have the ability to stimulate the release of mitochondrial cytochrome c, activation of caspase-3 and Bax protein, inhibition of Bcl-2 protein and production of intracellular reactive oxygen species in Hep3B cells. These results suggest that KRG, GRg3 and GRh2 may induce apoptosis by direct activation of the mitochondrial pathway. Copyright © 2012 Elsevier Ltd. All rights reserved.

Caspase inhibitors protect neurons by enabling selective necroptosis of inflamed microglia.

PubMed

Fricker, Michael; Vilalta, Anna; Tolkovsky, Aviva M; Brown, Guy C

2013-03-29

Microglia are resident brain macrophages, which can cause neuronal loss when activated in infectious, ischemic, traumatic, and neurodegenerative diseases. Caspase-8 has both prodeath and prosurvival roles, mediating apoptosis and/or preventing RIPK1-mediated necroptosis depending on cell type and stimulus. We found that inflammatory stimuli (LPS, lipoteichoic acid, or TNF-α) caused an increase in caspase-8 IETDase activity in primary rat microglia without inducing apoptosis. Inhibition of caspase-8 with either Z-VAD-fmk or IETD-fmk resulted in necrosis of activated microglia. Inhibition of caspases with Z-VAD-fmk did not kill non-activated microglia, or astrocytes and neurons in any condition. Necrostatin-1, a specific inhibitor of RIPK1, prevented microglial caspase inhibition-induced death, indicating death was by necroptosis. In mixed cerebellar cultures of primary neurons, astrocytes, and microglia, LPS induced neuronal loss that was prevented by inhibition of caspase-8 (resulting in microglial necroptosis), and neuronal death was restored by rescue of microglia with necrostatin-1. We conclude that the activation of caspase-8 in inflamed microglia prevents their death by necroptosis, and thus, caspase-8 inhibitors may protect neurons in the inflamed brain by selectively killing activated microglia.

Live and heat-killed Lactobacillus spp. interfere with Streptococcus mutans and Streptococcus oralis during biofilm development on titanium surface.

PubMed

Ciandrini, E; Campana, R; Baffone, W

2017-06-01

This research investigates the ability of live and heat-killed (HK) Lactic Acid Bacteria (LAB) to interfere with Streptococcus mutans ATCC 25175 and Streptococcus oralis ATCC 9811 during biofilm formation. Eight Lactobacillus spp. and two oral colonizers, pathogenic Streptococcus mutans and resident Streptococcus oralis, were characterized for their aggregation abilities, cell surface properties and biofilm formation ability on titanium surface. Then, the interference activity of selected live and HK Lactobacillus spp. during S. mutans and S. oralis biofilm development were performed. The cell-free culture supernatants (CFCS) anti-biofilm activity was also determined. LAB possess good abilities of auto-aggregation (from 14.19 to 28.97%) and of co-aggregation with S. oralis. The cell-surfaces characteristics were most pronounced in S. mutans and S. oralis, while the highest affinities to xylene and chloroform were observed in Lactobacillus rhamnosus ATCC 53103 (56.37%) and Lactobacillus paracasei B21060 (43.83%). S. mutans and S. oralis developed a biofilm on titanium surface, while LAB showed a limited or no ability to create biofilm. Live and HK L. rhamnosus ATCC 53103 and L. paracasei B21060 inhibited streptococci biofilm formation by competition and displacement mechanisms with no substantial differences. The CFCSs of both LAB strains, particularly the undiluted one of L. paracasei B21060, decreased S. mutans and S. oralis biofilm formation. This study evidenced the association of LAB aggregation abilities and cell-surface properties with the LAB-mediated inhibition of S. mutans and S. oralis biofilm formation. Lactobacilli showed different mechanisms of action and peculiar strain-specific characteristics, maintained also in the heat-killed LAB. Copyright © 2017 Elsevier Ltd. All rights reserved.

220D-F2 from Rubus ulmifolius kills Streptococcus pneumoniae planktonic cells and pneumococcal biofilms.

PubMed

Talekar, Sharmila J; Chochua, Sopio; Nelson, Katie; Klugman, Keith P; Quave, Cassandra L; Vidal, Jorge E

2014-01-01

Streptococcus pneumoniae (pneumococcus) forms organized biofilms to persist in the human nasopharynx. This persistence allows the pneumococcus to produce severe diseases such as pneumonia, otitis media, bacteremia and meningitis that kill nearly a million children every year. While bacteremia and meningitis are mediated by planktonic pneumococci, biofilm structures are present during pneumonia and otitis media. The global emergence of S. pneumoniae strains resistant to most commonly prescribed antibiotics warrants further discovery of alternative therapeutics. The present study assessed the antimicrobial potential of a plant extract, 220D-F2, rich in ellagic acid, and ellagic acid derivatives, against S. pneumoniae planktonic cells and biofilm structures. Our studies first demonstrate that, when inoculated together with planktonic cultures, 220D-F2 inhibited the formation of pneumococcal biofilms in a dose-dependent manner. As measured by bacterial counts and a LIVE/DEAD bacterial viability assay, 100 and 200 µg/ml of 220D-F2 had significant bactericidal activity against pneumococcal planktonic cultures as early as 3 h post-inoculation. Quantitative MIC's, whether quantified by qPCR or dilution and plating, showed that 80 µg/ml of 220D-F2 completely eradicated overnight cultures of planktonic pneumococci, including antibiotic resistant strains. When preformed pneumococcal biofilms were challenged with 220D-F2, it significantly reduced the population of biofilms 3 h post-inoculation. Minimum biofilm inhibitory concentration (MBIC)50 was obtained incubating biofilms with 100 µg/ml of 220D-F2 for 3 h and 6 h of incubation. 220D-F2 also significantly reduced the population of pneumococcal biofilms formed on human pharyngeal cells. Our results demonstrate potential therapeutic applications of 220D-F2 to both kill planktonic pneumococcal cells and disrupt pneumococcal biofilms.

40 CFR 180.1325 - Heat-killed Burkholderia spp. strain A396 cells and spent fermentation media exemption from the...

Code of Federal Regulations, 2014 CFR

2014-07-01

... A396 cells and spent fermentation media exemption from the requirement of a tolerance. 180.1325 Section...-killed Burkholderia spp. strain A396 cells and spent fermentation media exemption from the requirement of...-killed Burkholderia spp. strain A396 cells and spent fermentation media in or on all food commodities...

Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta.

PubMed

Nicolay, Nils H; Carter, Rebecca; Hatch, Stephanie B; Schultz, Niklas; Prevo, Remko; McKenna, W Gillies; Helleday, Thomas; Sharma, Ricky A

2012-11-01

DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt's lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control.

BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

PubMed Central

Mahajan, Indra M.; Chen, Miao-Der; Muro, Israel; Robertson, John D.; Wright, Casey W.; Bratton, Shawn B.

2014-01-01

Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax−/−Bak−/− cells and better than either Bid−/− or dominant-negative caspase-9-expressing cells. Only Bim−/− and Bax−/−Bak−/− cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid−/− cells, it readily did so in Bim−/− cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1−/− cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-XL with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members. PMID:24427286

Stepwise cytoskeletal polarization as a series of checkpoints in innate but not adaptive cytolytic killing

NASA Astrophysics Data System (ADS)

Wülfing, Christoph; Purtic, Bozidar; Klem, Jennifer; Schatzle, John D.

2003-06-01

Cytolytic killing is a major effector mechanism in the elimination of virally infected and tumor cells. The innate cytolytic effectors, natural killer (NK) cells, and the adaptive effectors, cytotoxic T cells (CTL), despite differential immune recognition, both use the same lytic mechanism, cytolytic granule release. Using live cell video fluorescence microscopy in various primary cell models of NK cell and CTL killing, we show here that on tight target cell contact, a majority of the NK cells established cytoskeletal polarity required for effective lytic function slowly or incompletely. In contrast, CTLs established cytoskeletal polarity rapidly. In addition, NK cell killing was uniquely sensitive to minor interference with cytoskeletal dynamics. We propose that the stepwise NK cell cytoskeletal polarization constitutes a series of checkpoints in NK cell killing. In addition, the use of more deliberate progression to effector function to compensate for inferior immune recognition specificity provides a mechanistic explanation for how the same effector function can be used in the different functional contexts of the innate and adaptive immune response.

The Listeriolysin O PEST-like Sequence Co-opts AP-2-Mediated Endocytosis to Prevent Plasma Membrane Damage during Listeria Infection.

PubMed

Chen, Chen; Nguyen, Brittney N; Mitchell, Gabriel; Margolis, Shally R; Ma, Darren; Portnoy, Daniel A

2018-06-13

Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of Listeria monocytogenes from a phagosome, enabling growth of the bacteria in the host cell cytosol. LLO contains a PEST-like sequence that prevents it from killing infected cells, but the mechanism involved is unknown. We found that the LLO PEST-like sequence was necessary to mediate removal of LLO from the interior face of the plasma membrane, where it coalesces into discrete puncta. LLO interacts with Ap2a2, an adaptor protein involved in endocytosis, via its PEST-like sequence, and Ap2a2-dependent endocytosis is required to prevent LLO-induced cytotoxicity. An unrelated PEST-like sequence from a human G protein-coupled receptor (GPCR), which also interacts with Ap2a2, could functionally complement the PEST-like sequence in L. monocytogenes LLO. These data revealed that LLO co-opts the host endocytosis machinery to protect the integrity of the host plasma membrane during L. monocytogenes infection. Copyright © 2018 Elsevier Inc. All rights reserved.

Cryptococcus Neoformans Modulates Extracellular Killing by Neutrophils

PubMed Central

Qureshi, Asfia; Grey, Angus; Rose, Kristie L.; Schey, Kevin L.; Del Poeta, Maurizio

2011-01-01

We recently established a key role for host sphingomyelin synthase (SMS) in regulating the killing activity of neutrophils against Cryptococcus neoformans. In this paper, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and natural killer (NK) cells (Tgε26 mice). To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in vitro and in vivo. We show that unlike Candida albicans, neither the presence nor the capsule size of C. neoformans cells have any effect on neutrophil viability. Interestingly, melanized C. neoformans cells totally abrogated the killing activity of neutrophils. We monitored how exposure of neutrophils to C. neoformans cells would interfere with any further killing activity of the conditioned medium and found that pre-incubation with live but not “heat-killed” fungal cells significantly inhibits further killing activity of the medium. We then studied whether activation of SMS at the site of C. neoformans infection is dependent on T and NK cells. Using matrix-assisted laser desorption–ionization tissue imaging in infected lung we found that similar to previous observations in the isogenic wild-type CBA/J mice, SM 16:0 levels are significantly elevated at the site of infection in mice lacking T and NK cells, but only at early time points. This study highlights that C. neoformans may negatively regulate the killing activity of neutrophils and that SMS activation in neutrophils appears to be partially independent of T and/or NK cells. PMID:21960987

Membrane oxidation in cell delivery and cell killing applications

PubMed Central

Wang, Ting-Yi; Libardo, M. Daben J.; Angeles-Boza, Alfredo M.; Pellois, Jean-Philippe

2018-01-01

Cell delivery or cell killing processes often involve the crossing or disruption of cellular membranes. We review how, by modifying the composition and properties of membranes, membrane oxidation can be exploited to enhance the delivery of macromolecular cargos into live human cells. We also describe how membrane oxidation can be utilized to achieve efficient killing of bacteria by antimicrobial peptides. Finally, we present recent evidence highlighting how membrane oxidation is intimately engaged in natural biological processes such as antigen delivery in dendritic cells and in the killing of bacteria by human macrophages. Overall, the insights that have been recently gained in this area should facilitate the development of more effective delivery technologies and antimicrobial therapeutic approaches. PMID:28355059

Mechanisms and functions of lysosome positioning

PubMed Central

Pu, Jing; Guardia, Carlos M.; Keren-Kaplan, Tal

2016-01-01

ABSTRACT Lysosomes have been classically considered terminal degradative organelles, but in recent years they have been found to participate in many other cellular processes, including killing of intracellular pathogens, antigen presentation, plasma membrane repair, cell adhesion and migration, tumor invasion and metastasis, apoptotic cell death, metabolic signaling and gene regulation. In addition, lysosome dysfunction has been shown to underlie not only rare lysosome storage disorders but also more common diseases, such as cancer and neurodegeneration. The involvement of lysosomes in most of these processes is now known to depend on the ability of lysosomes to move throughout the cytoplasm. Here, we review recent findings on the mechanisms that mediate the motility and positioning of lysosomes, and the importance of lysosome dynamics for cell physiology and pathology. PMID:27799357

Impaired natural killer cell self-education and "missing-self" responses in Ly49-deficient mice.

PubMed

Bélanger, Simon; Tu, Megan M; Rahim, Mir Munir Ahmed; Mahmoud, Ahmad B; Patel, Rajen; Tai, Lee-Hwa; Troke, Angela D; Wilhelm, Brian T; Landry, Josette-Renée; Zhu, Qinzhang; Tung, Kenneth S; Raulet, David H; Makrigiannis, Andrew P

2012-07-19

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.

Human Periodontal Stem Cells Release Specialized Proresolving Mediators and Carry Immunomodulatory and Prohealing Properties Regulated by Lipoxins

PubMed Central

Cianci, Eleonora; Recchiuti, Antonio; Trubiani, Oriana; Diomede, Francesca; Marchisio, Marco; Miscia, Sebastiano; Colas, Romain A.; Dalli, Jesmond; Serhan, Charles N.

2016-01-01

Unresolved inflammation and tissue destruction are underlying mechanisms of periodontitis, which is linked to dysregulated polymorphonuclear neutrophil (PMN) functions. Lipoxin A4 (LXA4) is a specialized proresolving lipid mediator (SPM) that dampens excessive inflammation, promotes resolution, and protects from leukocyte-mediated tissue damage. Human periodontal ligament stem cells (hPDLSCs) represent key players during tissue regeneration and may contribute to resolution of inflammation; thus, they may represent a promising tool in regenerative dentistry. In the present study, we investigated the actions of hPDLSCs on PMN apoptosis and antimicrobial functions, and determined the impact of LXA4 on hPDLSCs. hPDLSCs significantly reduced apoptosis and stimulated microbicidal activity of human PMNs, via both cell-cell interactions and paracrine mechanisms. Lipid mediator metabololipidomics analysis demonstrated that hPDLSCs biosynthesize SPMs, including resolvin D1, D2, D5, and D6; protectin D1; maresins; and LXB4; as well as prostaglandins D2, E2, and F2α. LXA4 significantly enhanced proliferation, migration, and wound healing capacity of hPDLSCs through the activation of its cognate receptor ALX/FPR2, expressed on hPDLSCs. Together, these results demonstrate that hPDLSCs modulate PMN functions, and provide the first evidence that stem cells generate SPM and that the LXA4-ALX/FPR2 axis regulates regenerative functions of hPDLSCs by a novel receptor-mediated mechanism. Significance These findings uncovered unappreciated features of stem cells from the periodontal ligament, supporting the notion that these cells may act as master regulators of pathophysiological events through the release of mediators that promote the resolution of inflammation and bacterial killing. The study also demonstrated that it is possible to modulate important functions of periodontal stem cells using lipoxin A4, a potent endogenous stop signal of inflammation. Thus, this study revealed an unappreciated anti-inflammatory proregenerative circuit that may be exploited to combat periodontal pathologies using resident stem cells. Moreover, the data may represent a more general template to explain the immunomodulatory functions of stem cells. PMID:26607175

Synthesis and Evaluation of Folate-Conjugated Phenanthraquinones for Tumor-Targeted Oxidative Chemotherapy

PubMed Central

Kumar, Ajay; Chelvam, Venkatesh; Sakkarapalayam, Mahalingam; Li, Guo; Sanchez-Cruz, Pedro; Piñero, Natasha S.; Low, Philip S.; Alegria, Antonio E.

2016-01-01

Almost all cells are easily killed by exposure to potent oxidants. Indeed, major pathogen defense mechanisms in both animal and plant kingdoms involve production of an oxidative burst, where host defense cells show an invading pathogen with reactive oxygen species (ROS). Although cancer cells can be similarly killed by ROS, development of oxidant-producing chemotherapies has been limited by their inherent nonspecificity and potential toxicity to healthy cells. In this paper, we describe the targeting of an ROS-generating molecule selectively to tumor cells using folate as the tumor-targeting ligand. For this purpose, we exploit the ability of 9,10-phenanthraquinone (PHQ) to enhance the continuous generation of H2O2 in the presence of ascorbic acid to establish a constitutive source of ROS within the tumor mass. We report here that incubation of folate receptor-expressing KB cells in culture with folate-PHQ plus ascorbate results in the death of the cancer cells with an IC50 of ~10 nM (folate-PHQ). We also demonstrate that a cleavable spacer linking folate to PHQ is significantly inferior to a noncleavable spacer, in contrast to most other folate-targeted therapeutic agents. Unfortunately, no evidence for folate-PHQ mediated tumor regression in murine tumor models is obtained, suggesting that unanticipated impediments to generation of cytotoxic quantities of ROS in vivo are encountered. Possible mechanisms and potential solutions to these unanticipated results are offered. PMID:27066312

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden

PubMed Central

Martin, Erik W.; Buzza, Marguerite S.; Driesbaugh, Kathryn H.; Liu, Shihui; Fortenberry, Yolanda M.; Leppla, Stephen H.; Antalis, Toni M.

2015-01-01

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent. PMID:26392335

The effect of cell density, proximity, and time on the cytotoxicity of magnesium and galvanically coupled magnesium-titanium particles in vitro.

PubMed

Kim, Jua; Gilbert, Jeremy L

2018-05-01

Magnesium (Mg) and galvanically coupled magnesium-titanium (Mg-Ti) particles in vitro have been reported previously to kill cells in a dosage-dependent manner. Mg-Ti particles kill cells more effectively than Mg alone, due to the galvanic effect of Mg and Ti. This study further investigated the in vitro cytotoxicity of Mg and Mg-Ti in terms of particle concentration, cell density, time, and proximity. Cell density has an effect on cell viability only at low particle concentrations (below 250 µg/mL), where cell viability dropped only for lower cell densities (5000-10,000 cells/cm 2 ) and not for higher cell densities (20,000-30,000 cells/cm 2 ), showing that the particles cannot kill if there are more cells present. Cytotoxicity of Mg and Mg-Ti particles is quick and temporary, where the particles kill cells only during particle corrosion (first 24 h). Depending on the percentage of surviving cells, particle concentrations, and ongoing corrosion activity, the remaining live cells either proliferated and recovered, or just remained viable and quiescent. The particle killing is also proximity-dependent, where cell viability was significantly higher for cells far away from the particles (greater than ∼1 mm) compared to those close to the particles (less than ∼1 mm). Although the increase of pH does affect cell viability negatively, it is not the sole killing factor since cell viability is significantly dependent on particle type and proximity but not pH. Mg and Mg-Ti particles used in this study are large enough to prevent direct cell phagocytosis so that the cell killing effect may be attributed to solely electrochemical reactions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1428-1439, 2018. © 2018 Wiley Periodicals, Inc.

A Novel Polyphenol Conjugate Sensitizes Cisplatin-Resistant Head and Neck Cancer Cells to Cisplatin via Nrf2 Inhibition.

PubMed

Kim, Eun Hye; Jang, Hyejin; Roh, Jong-Lyel

2016-11-01

Many cancer cells show acquired resistance to chemotherapeutic agents, such as cisplatin. This is a major cause of cancer treatment failure, and novel agents to overcome resistance are thus urgently required. A novel synthetic polyphenol conjugate, (E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (DPP-23), selectively kills tumor cells via the reactive oxygen species (ROS)-mediated unfolded protein response. We investigated the ability of DPP-23 to overcome cisplatin resistance in head and neck cancer (HNC) cells and further clarified its molecular mechanisms of action. Cisplatin-resistant HNC cell lines and their parental and other HNC cell lines were used. The effects of cisplatin and DPP-23 were assessed alone and in combination in HNC and normal cells using cell viability, cell cycle, and cell death assays, by measuring glutathione (GSH), ROS, and protein levels, and via preclinical mouse studies. DPP-23 induced selective cell death in HNC cells, including cisplatin-resistant HNC cells, but spared normal cells, via cellular GSH depletion and ROS accumulation. The effect was blocked by the antioxidant N-acetyl-L-cysteine. DPP-23 activated p53 and its related cell death pathways via a robust accumulation of cellular ROS that involved inhibition of nuclear factor erythroid 2-related factor 2 antioxidant defense mechanisms. Thus, DPP-23 significantly overcame cisplatin resistance in HNC cells in vitro and in vivo As a promising anticancer strategy, ROS generation and subsequent selective cancer cell killing by DPP-23 might help to overcome cisplatin resistance in HNC. Mol Cancer Ther; 15(11); 2620-9. ©2016 AACR. ©2016 American Association for Cancer Research.

Study of cell killing effect on S180 by ultrasound activating protoporphyrin IX.

PubMed

Wang, Xiao Bing; Liu, Quan Hong; Wang, Pan; Tang, Wei; Hao, Qiao

2008-04-01

The present study was initiated to investigate the potential biological mechanism of cell killing effect on isolate sarcoma 180 (S180) cells induced by ultrasound activating protoporphyrin IX (PPIX). S180 cells were exposed to ultrasound for 30s duration, at a frequency of 2.2 MHz and an acoustic power of 3 W/cm(2) in the presence of 120 microM PPIX. The viability of cells was evaluated using trypan blue staining. The generation of oxygen free radicals in cell suspensions was detected immediately after treatment using a reactive oxygen detection kit. A copper reagent colorimetry method was used to measure the level of FFAs released into cell suspensions by the process of cell damage induced by ultrasound and PPIX treatment. Oxidative stress was assessed by measuring the activities of key antioxidant enzymes (i.e., SOD, CAT, GSH-PX) in S180 tumor cells. Treatment with ultrasound and PPIX together increased the cell damage rate to 50.91%, while treatment with ultrasound alone gave a cell damage rate to 24.24%, and PPIX alone kept this rate unchanged. Colorimetry and enzymatic chemical methods showed that the level of FFAs in cell suspension increased significantly after the treatment, while the activity of all the above enzymes decreased in tumor cells at different levels, and were associated with the generation of oxygen free radicals in cell suspension after treatment. The results indicate that oxygen free radicals may play an important role in improving the membrane lipid peroxidation, degrading membrane phospholipids to release FFAs, and decreasing the activities of the key antioxidant enzymes in cells. This biological mechanism might be involved in mediating the effects on S180 cells and resulting in the cell damage seen with SDT.

An Acidic Microenvironment Increases NK Cell Killing of Cryptococcus neoformans and Cryptococcus gattii by Enhancing Perforin Degranulation

PubMed Central

Islam, Anowara; Li, Shu Shun; Oykhman, Paul; Timm-McCann, Martina; Huston, Shaunna M.; Stack, Danuta; Xiang, Richard F.; Kelly, Margaret M.; Mody, Christopher H.

2013-01-01

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment. PMID:23853583

ATM-NFκB axis-driven TIGAR regulates sensitivity of glioma cells to radiomimetics in the presence of TNFα.

PubMed

Sinha, S; Ghildiyal, R; Mehta, V S; Sen, E

2013-05-02

Gliomas are resistant to radiation therapy, as well as to TNFα induced killing. Radiation-induced TNFα triggers Nuclear factor κB (NFκB)-mediated radioresistance. As inhibition of NFκB activation sensitizes glioma cells to TNFα-induced apoptosis, we investigated whether TNFα modulates the responsiveness of glioma cells to ionizing radiation-mimetic Neocarzinostatin (NCS). TNFα enhanced the ability of NCS to induce glioma cell apoptosis. NCS-mediated death involved caspase-9 activation, reduction of mitochondrial copy number and lactate production. Death was concurrent with NFκB, Akt and Erk activation. Abrogation of Akt and NFκB activation further potentiated the death inducing ability of NCS in TNFα cotreated cells. NCS-induced p53 expression was accompanied by increase in TP53-induced glycolysis and apoptosis regulator (TIGAR) levels and ATM phosphorylation. siRNA-mediated knockdown of TIGAR abrogated NCS-induced apoptosis. While DN-IκB abrogated NCS-induced TIGAR both in the presence and absence of TNFα, TIGAR had no effect on NFκB activation. Transfection with TIGAR mutant (i) decreased apoptosis and γH2AX foci formation (ii) decreased p53 (iii) elevated ROS and (iv) increased Akt/Erk activation in cells cotreated with NCS and TNFα. Heightened TIGAR expression was observed in GBM tumors. While NCS induced ATM phosphorylation in a NFκB independent manner, ATM inhibition abrogated TIGAR and NFκB activation. Metabolic gene profiling indicated that TNFα affects NCS-mediated regulation of several genes associated with glycolysis. The existence of ATM-NFκB axis that regulate metabolic modeler TIGAR to overcome prosurvival response in NCS and TNFα cotreated cells, suggests mechanisms through which inflammation could affect resistance and adaptation to radiomimetics despite concurrent induction of death.

ATM-NFκB axis-driven TIGAR regulates sensitivity of glioma cells to radiomimetics in the presence of TNFα

PubMed Central

Sinha, S; Ghildiyal, R; Mehta, V S; Sen, E

2013-01-01

Gliomas are resistant to radiation therapy, as well as to TNFα induced killing. Radiation-induced TNFα triggers Nuclear factor κB (NFκB)-mediated radioresistance. As inhibition of NFκB activation sensitizes glioma cells to TNFα-induced apoptosis, we investigated whether TNFα modulates the responsiveness of glioma cells to ionizing radiation-mimetic Neocarzinostatin (NCS). TNFα enhanced the ability of NCS to induce glioma cell apoptosis. NCS-mediated death involved caspase-9 activation, reduction of mitochondrial copy number and lactate production. Death was concurrent with NFκB, Akt and Erk activation. Abrogation of Akt and NFκB activation further potentiated the death inducing ability of NCS in TNFα cotreated cells. NCS-induced p53 expression was accompanied by increase in TP53-induced glycolysis and apoptosis regulator (TIGAR) levels and ATM phosphorylation. siRNA-mediated knockdown of TIGAR abrogated NCS-induced apoptosis. While DN-IκB abrogated NCS-induced TIGAR both in the presence and absence of TNFα, TIGAR had no effect on NFκB activation. Transfection with TIGAR mutant (i) decreased apoptosis and γH2AX foci formation (ii) decreased p53 (iii) elevated ROS and (iv) increased Akt/Erk activation in cells cotreated with NCS and TNFα. Heightened TIGAR expression was observed in GBM tumors. While NCS induced ATM phosphorylation in a NFκB independent manner, ATM inhibition abrogated TIGAR and NFκB activation. Metabolic gene profiling indicated that TNFα affects NCS-mediated regulation of several genes associated with glycolysis. The existence of ATM-NFκB axis that regulate metabolic modeler TIGAR to overcome prosurvival response in NCS and TNFα cotreated cells, suggests mechanisms through which inflammation could affect resistance and adaptation to radiomimetics despite concurrent induction of death. PMID:23640457

The biology of NK cells and their receptors affects clinical outcomes after hematopoietic cell transplantation (HCT).

PubMed

Foley, Bree; Felices, Martin; Cichocki, Frank; Cooley, Sarah; Verneris, Michael R; Miller, Jeffrey S

2014-03-01

Natural killer (NK) cells were first identified for their capacity to reject bone marrow allografts in lethally irradiated mice without prior sensitization. Subsequently, human NK cells were detected and defined by their non-major histocompatibility complex (MHC)-restricted cytotoxicity toward transformed or virally infected target cells. Karre et al. later proposed 'the missing self hypothesis' to explain the mechanism by which self-tolerant cells could kill targets that had lost self MHC class I. Subsequently, the receptors that recognize MHC class I to mediate tolerance in the host were identified on NK cells. These class I-recognizing receptors contribute to the acquisition of function by a dynamic process known as NK cell education or licensing. In the past, NK cells were assumed to be short lived, but more recently NK cells have been shown to mediate immunologic memory to secondary exposures to cytomegalovirus infection. Because of their ability to lyse tumors with aberrant MHC class I expression and to produce cytokines and chemokines upon activation, NK cells may be primed by many stimuli, including viruses and inflammation, to contribute to a graft-versus-tumor effect. In addition, interactions with other immune cells support the therapeutic potential of NK cells to eradicate tumor and to enhance outcomes after hematopoietic cell transplantation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Dioscin augments HSV-tk-mediated suicide gene therapy for melanoma by promoting connexin-based intercellular communication

PubMed Central

Li, Bin; Wu, Yingya; Liu, Xijuan; Tan, Yuhui; Du, Biaoyan

2017-01-01

Suicide gene therapy is a promising strategy against melanoma. However, the low efficiency of the gene transfer technique can limit its application. Our preliminary data showed that dioscin, a glucoside saponin, could upregulate the expression of connexins Cx26 and Cx43, major components of gap junctions, in melanoma cells. We hypothesized that dioscin may increase the bystander effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) through increasing the formation of gap junctions. Further analysis showed that dioscin indeed could increase the gap junctional intercellular communication in B16 melanoma cells, resulting in more efficient GCV-induced bystander killing in B16tk cells. By contrast, overexpression of dominant negative Cx43 impaired the cell-cell communication of B16 cells and subsequently weakened the bystander effect of HSV-tk/GCV gene therapy. In vivo, combination treatment with dioscin and GCV of tumor-bearing mice with 30% positive B16tk cells and 70% wild-type B16 cells caused a significant reduction in tumor volume and weight compared to treatment with GCV or dioscin alone. Taken together, these results demonstrated that dioscin could augment the bystander effect of the HSV-tk/GCV system through increasing connexin-mediated gap junction coupling. PMID:27903977

Novel cancer vaccines prepared by anchoring cytokines to tumor cells avoiding gene transfection

NASA Astrophysics Data System (ADS)

Nizard, Philippe; Gross, David-Alexandre; Chenal, Alexandre; Beaumelle, Bruno; Kosmatopoulos, Konstadinos; Gillet, Daniel

2002-06-01

Cytokines have a strong potential for triggering anticancer immunity if released in the tumor microenvironment. Successful vaccines have been engineered using tumor cells genetically modified to secrete the cytokines. Unfortunately, this approach remains difficult and hazardous to perform in the clinic. We describe a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This consists in anchoring recombinant cytokines to the membrane of killed tumor cells. Attachment is mediated by a fragment of diphtheria toxin (T) genetically connected to the cytokine. It is triggered by an acid pH pulse. The method was applied to IL-2, a potent anti-tumor cytokine. IL-2 anchored to the surface of tumor cells by the T anchor retained its IL-2 activity and remained exposed several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective anti-tumor immunity mediated by tumor-specific cytotoxic T lymphocytes. This procedure presents several advantages as compared to the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and eliminates the safety problems connected with viral vectors while allowing the control of the amount of cytokines delivered with the vaccine.

Glutamate-mediated protection of crayfish glial cells from PDT-induced apoptosis

NASA Astrophysics Data System (ADS)

Rudkovskii, M. V.; Romanenko, N. P.; Berezhnaya, E. V.; Kovaleva, V. D.; Uzdensky, A. B.

2011-03-01

Photodynamic treatment that causes intense oxidative stress and kills cells is currently used in neurooncology. However, along with tumor it damages surrounding healthy neurons and glial cells. In order to study the possible role of glutamate-related signaling pathways in photodynamic injury of neurons and glia, we investigated photodynamic effect of alumophthalocyanine Photosens on isolated crayfish stretch receptor that consists of a single neuron surrounded by glial cells. The laser diode (670 nm, 0.4 W/cm2) was used for dye photoexcitation. Application of glutamate increased photodynamically induced necrosis of neurons and glial cells but significantly decreased glial apoptosis. The natural neuroglial mediator N-acetylaspartylglutamate, which releases glutamate after cleavage in the extracellular space by glutamate carboxypeptidase II, also inhibited photoinduced apoptosis. Inhibition of glutamate carboxypeptidase II, oppositely, enhanced apoptosis of glial cells. These data confirm the anti-apoptotic activity of glutamate. Application of NMDA or inhibition of NMDA receptors by MK801 did not influence photodynamic death of neurons and glial cells that indicated nonparticipation of NMDA receptors in these processes. Inhibition of metabotropic glutamate receptors by AP-3 decreased PDT-induced apoptosis. One can suggest that crayfish neurons naturally secrete NAAG, which being cleaved by GCOP produces glutamate. Glutamate prevents photoinduced apoptosis of glial cells possibly through metabotropic but not ionotropic glutamate receptors.

First Phase I human clinical trial of a killed whole-HIV-1 vaccine: demonstration of its safety and enhancement of anti-HIV antibody responses.

PubMed

Choi, Eunsil; Michalski, Chad J; Choo, Seung Ho; Kim, Gyoung Nyoun; Banasikowska, Elizabeth; Lee, Sangkyun; Wu, Kunyu; An, Hwa-Yong; Mills, Anthony; Schneider, Stefan; Bredeek, U Fritz; Coulston, Daniel R; Ding, Shilei; Finzi, Andrés; Tian, Meijuan; Klein, Katja; Arts, Eric J; Mann, Jamie F S; Gao, Yong; Kang, C Yong

2016-11-28

Vaccination with inactivated (killed) whole-virus particles has been used to prevent a wide range of viral diseases. However, for an HIV vaccine this approach has been largely negated due to inherent safety concerns, despite the ability of killed whole-virus vaccines to generate a strong, predominantly antibody-mediated immune response in vivo. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence for the Env signal peptide with that of honeybee melittin signal peptide to produce a less virulent and more replication efficient virus. This genetically modified virus (gmHIV-1 NL4-3 ) was inactivated and formulated as a killed whole-HIV vaccine, and then used for a Phase I human clinical trial (Trial Registration: Clinical Trials NCT01546818). The gmHIV-1 NL4-3 was propagated in the A3.01 human T cell line followed by virus purification and inactivation with aldrithiol-2 and γ-irradiation. Thirty-three HIV-1 positive volunteers receiving cART were recruited for this observer-blinded, placebo-controlled Phase I human clinical trial to assess the safety and immunogenicity. Genetically modified and killed whole-HIV-1 vaccine, SAV001, was well tolerated with no serious adverse events. HIV-1 NL4-3 -specific PCR showed neither evidence of vaccine virus replication in the vaccine virus-infected human T lymphocytes in vitro nor in the participating volunteers receiving SAV001 vaccine. Furthermore, SAV001 with adjuvant significantly increased the pre-existing antibody response to HIV-1 proteins. Antibodies in the plasma of vaccinees were also found to recognize HIV-1 envelope protein on the surface of infected cells as well as showing an enhancement of broadly neutralizing antibodies inhibiting tier I and II of HIV-1 B, D, and A subtypes. The killed whole-HIV vaccine, SAV001, is safe and triggers anti-HIV immune responses. It remains to be determined through an appropriate trial whether this immune response prevents HIV infection.

DNA Double-Strand Break Repair as Determinant of Cellular Radiosensitivity to Killing and Target in Radiation Therapy

PubMed Central

Mladenov, Emil; Magin, Simon; Soni, Aashish; Iliakis, George

2013-01-01

Radiation therapy plays an important role in the management of a wide range of cancers. Besides innovations in the physical application of radiation dose, radiation therapy is likely to benefit from novel approaches exploiting differences in radiation response between normal and tumor cells. While ionizing radiation induces a variety of DNA lesions, including base damages and single-strand breaks, the DNA double-strand break (DSB) is widely considered as the lesion responsible not only for the aimed cell killing of tumor cells, but also for the general genomic instability that leads to the development of secondary cancers among normal cells. Homologous recombination repair (HRR), non-homologous end-joining (NHEJ), and alternative NHEJ, operating as a backup, are the major pathways utilized by cells for the processing of DSBs. Therefore, their function represents a major mechanism of radiation resistance in tumor cells. HRR is also required to overcome replication stress – a potent contributor to genomic instability that fuels cancer development. HRR and alternative NHEJ show strong cell-cycle dependency and are likely to benefit from radiation therapy mediated redistribution of tumor cells throughout the cell-cycle. Moreover, the synthetic lethality phenotype documented between HRR deficiency and PARP inhibition has opened new avenues for targeted therapies. These observations make HRR a particularly intriguing target for treatments aiming to improve the efficacy of radiation therapy. Here, we briefly describe the major pathways of DSB repair and review their possible contribution to cancer cell radioresistance. Finally, we discuss promising alternatives for targeting DSB repair to improve radiation therapy and cancer treatment. PMID:23675572

Homoharringtonine combined with aclarubicin and cytarabine synergistically induces apoptosis in t(8;21) leukemia cells and triggers caspase-3-mediated cleavage of the AML1-ETO oncoprotein.

PubMed

Cao, Jiang; Feng, Hao; Ding, Ning-Ning; Wu, Qing-Yun; Chen, Chong; Niu, Ming-Shan; Chen, Wei; Qiu, Ting-Ting; Zhu, Hong-Hu; Xu, Kai-Lin

2016-11-01

Homoharringtonine combined with aclarubicin and cytarabine (HAA) is a highly effective treatment for acute myeloid leukemia (AML), especially for t(8;21) AML. However, the underlying mechanisms by which HAA kills t(8;21) AML cells remain unclear. In this study, SKNO-1 and Kasumi-1 cells with t(8;21) were used. Compared with individual or pairwise administration of homoharringtonine, aclarubicin, or cytarabine, HAA showed the strongest inhibition of growth and induction of apoptosis in SKNO-1 and Kasumi-1 cells. HAA caused cleavage of the AML1-ETO (AE) oncoprotein to form truncated AE (ΔAE). Pretreatment with the caspase-3 inhibitor caspase-3 inhibitor Q-DEVD-OPh (QDO) not only suppressed HAA-induced apoptosis but also abrogated the cleavage of AE and generation of ΔAE. These results suggest that HAA synergistically induces apoptosis in t(8;21) leukemia cells and triggers caspase-3-mediated cleavage of the AML1-ETO oncoprotein, thus providing direct evidence for the strong activity of HAA toward t(8;21) AML. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Dual-Mode Imaging-Guided Synergistic Chemo- and Magnetohyperthermia Therapy in a Versatile Nanoplatform To Eliminate Cancer Stem Cells.

PubMed

Tang, Jinglong; Zhou, Huige; Liu, Jiaming; Liu, Jing; Li, Wanqi; Wang, Yuqing; Hu, Fan; Huo, Qing; Li, Jiayang; Liu, Ying; Chen, Chunying

2017-07-19

Cancer stem cells (CSCs) have been identified as a new target for therapy in diverse cancers. Traditional therapies usually kill the bulk of cancer cells, but are often unable to effectively eliminate CSCs, which may lead to drug resistance and cancer relapse. Herein, we propose a novel strategy: fabricating multifunctional magnetic Fe 3 O 4 @PPr@HA hybrid nanoparticles and loading it with the Notch signaling pathway inhibitor N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycinet-butylester (DAPT) to eliminate CSCs. Hyaluronic acid ligands greatly enhance the accumulation of the hybrid nanoparticles in the tumor site and in the CSCs. Both hyaluronase in the tumor microenvironment and the magnetic hyperthermia effect of the inner magnetic core can accelerate the release of DAPT. This controlled release of DAPT in the tumor site further enhances the ability of the combination of chemo- and magnetohyperthermia therapy to eliminate cancer stem cells. With the help of polypyrrole-mediated photoacoustic and Fe 3 O 4 -mediated magnetic resonance imaging, the drug release can be precisely monitored in vivo. This versatile nanoplatform enables effective elimination of the cancer stem cells and monitoring of the drugs.

Induction of mitochondrial-mediated apoptosis by Morinda citrifolia (Noni) in human cervical cancer cells.

PubMed

Gupta, Rakesh Kumar; Banerjee, Ayan; Pathak, Suajta; Sharma, Chandresh; Singh, Neeta

2013-01-01

Cervical cancer is the second most common cause of cancer in women and has a high mortality rate. Cisplatin, an antitumor agent, is generally used for its treatment. However, the administration of cisplatin is associated with side effects and intrinsic resistance. Morinda citrifolia (Noni), a natural plant product, has been shown to have anti-cancer properties. In this study, we used Noni, cisplatin, and the two in combination to study their cytotoxic and apoptosis-inducing effects in cervical cancer HeLa and SiHa cell lines. We demonstrate here, that Noni/Cisplatin by themselves and their combination were able to induce apoptosis in both these cell lines. Cisplatin showed slightly higher cell killing as compared to Noni and their combination showed additive effects. The observed apoptosis appeared to be mediated particularly through the up-regulation of p53 and pro-apoptotic Bax proteins, as well as down- regulation of the anti-apoptotic Bcl-2, Bcl-XL proteins and survivin. Augmentation in the activity of caspase-9 and -3 was also observed, suggesting the involvement of the intrinsic mitochondrial pathway of apoptosis for both Noni and Cisplatin in HeLa and SiHa cell lines.

Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

PubMed

Chahroudi, A; Silvestri, G; Feinberg, M B

2003-10-01

Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

Autocrine Complement Inhibits IL10-Dependent T-Cell Mediated Antitumor Immunity to Promote Tumor Progression

PubMed Central

Wang, Yu; Sun, Sheng-Nan; Liu, Qing; Yu, Yang-Yang; Guo, Jian; Wang, Kun; Xing, Bao-Cai; Zheng, Qing-Feng; Campa, Michael J.; Patz, Edward F.; Li, Shi-You; He, You-Wen

2016-01-01

In contrast to its inhibitory effects on many cells, IL-10 activates CD8+ tumor infiltrating lymphocytes (TILs) and enhances their antitumor activity. However, CD8+ TILs do not routinely express IL-10 as autocrine complement C3 inhibits IL-10 production through complement receptors C3aR and C5aR. CD8+ TILs from C3-deficient mice, however, express IL-10 and exhibit enhanced effector function. C3-deficient mice are resistant to tumor development in a T cell- and IL-10-dependent manner; human TILs expanded with IL-2 plus IL-10 increase the killing of primary tumors in vitro compared to IL-2 treated TILs. Complement-mediated inhibition of antitumor immunity is independent of the PD-1/PD-L1 immune checkpoint pathway. Our findings suggest that complement receptors C3aR and C5aR expressed on CD8+ TILs represent a novel class of immune checkpoints that could be targeted for tumor immunotherapy. Moreover, incorporation of IL-10 in the expansion of TILs and in gene-engineered T cells for adoptive cell therapy enhances their antitumor efficacy. PMID:27297552

Ly49H signaling through DAP10 is essential for optimal natural killer cell responses to mouse cytomegalovirus infection

PubMed Central

Orr, Mark T.; Sun, Joseph C.; Hesslein, David G.T.; Arase, Hisashi; Phillips, Joseph H.; Takai, Toshiyuki

2009-01-01

The activating natural killer (NK) cell receptor Ly49H recognizes the mouse cytomegalovirus (MCMV) m157 glycoprotein expressed on the surface of infected cells and is required for protection against MCMV. Although Ly49H has previously been shown to signal via DAP12, we now show that Ly49H must also associate with and signal via DAP10 for optimal function. In the absence of DAP12, DAP10 enables Ly49H-mediated killing of m157-bearing target cells, proliferation in response to MCMV infection, and partial protection against MCMV. DAP10-deficient Ly49H+ NK cells, expressing only Ly49H–DAP12 receptor complexes, are partially impaired in their ability to proliferate during MCMV infection, display diminished ERK1/2 activation, produce less IFN-γ upon Ly49H engagement, and demonstrate reduced control of MCMV infection. Deletion of both DAP10 and DAP12 completely abrogates Ly49H surface expression and control of MCMV infection. Thus, optimal NK cell–mediated immunity to MCMV depends on Ly49H signaling through both DAP10 and DAP12. PMID:19332875

Infection by Toxoplasma gondii Specifically Induces Host c-Myc and the Genes This Pivotal Transcription Factor Regulates

PubMed Central

Franco, Magdalena; Shastri, Anjali J.

2014-01-01

Toxoplasma gondii infection has previously been described to cause dramatic changes in the host transcriptome by manipulating key regulators, including STATs, NF-κB, and microRNAs. Here, we report that Toxoplasma tachyzoites also mediate rapid and sustained induction of another pivotal regulator of host cell transcription, c-Myc. This induction is seen in cells infected with all three canonical types of Toxoplasma but not the closely related apicomplexan parasite Neospora caninum. Coinfection of cells with both Toxoplasma and Neospora still results in an increase in the level of host c-Myc, showing that c-Myc is actively upregulated by Toxoplasma infection (rather than repressed by Neospora). We further demonstrate that this upregulation may be mediated through c-Jun N-terminal protein kinase (JNK) and is unlikely to be a nonspecific host response, as heat-killed Toxoplasma parasites do not induce this increase and neither do nonviable parasites inside the host cell. Finally, we show that the induced c-Myc is active and that transcripts dependent on its function are upregulated, as predicted. Hence, c-Myc represents an additional way in which Toxoplasma tachyzoites have evolved to specifically alter host cell functions during intracellular growth. PMID:24532536

Stem cell plasticity enables hair regeneration following Lgr5+ cell loss.

PubMed

Hoeck, Joerg D; Biehs, Brian; Kurtova, Antonina V; Kljavin, Noelyn M; de Sousa E Melo, Felipe; Alicke, Bruno; Koeppen, Hartmut; Modrusan, Zora; Piskol, Robert; de Sauvage, Frederic J

2017-06-01

Under injury conditions, dedicated stem cell populations govern tissue regeneration. However, the molecular mechanisms that induce stem cell regeneration and enable plasticity are poorly understood. Here, we investigate stem cell recovery in the context of the hair follicle to understand how two molecularly distinct stem cell populations are integrated. Utilizing diphtheria-toxin-mediated cell ablation of Lgr5 + (leucine-rich repeat-containing G-protein-coupled receptor 5) stem cells, we show that killing of Lgr5 + cells in mice abrogates hair regeneration but this is reversible. During recovery, CD34 + (CD34 antigen) stem cells activate inflammatory response programs and start dividing. Pharmacological attenuation of inflammation inhibits CD34 + cell proliferation. Subsequently, the Wnt pathway controls the recovery of Lgr5 + cells and inhibition of Wnt signalling prevents Lgr5 + cell and hair germ recovery. Thus, our study uncovers a compensatory relationship between two stem cell populations and the underlying molecular mechanisms that enable hair follicle regeneration.

Autoradiographic and histopathological studies of boric acid-mediated BNCT in hepatic VX2 tumor-bearing rabbits: Specific boron retention and damage in tumor and tumor vessels.

PubMed

Yang, C H; Lin, Y T; Hung, Y H; Liao, J W; Peir, J J; Liu, H M; Lin, Y L; Liu, Y M; Chen, Y W; Chuang, K S; Chou, F I

2015-12-01

Hepatoma is a malignant tumor that responds poorly to conventional therapies. Boron neutron capture therapy (BNCT) may provide a better way for hepatoma therapy. In this research, (10)B-enriched boric acid (BA, 99% (10)B) was used as the boron drug. A multifocal hepatic VX2 tumor-bearing rabbit model was used to study the mechanisms of BA-mediated BNCT. Autoradiography demonstrated that BA was selectively targeted to tumors and tumor vessels. Histopathological examination revealed the radiation damage to tumor-bearing liver was concentrated in the tumor regions during BNCT treatment. The selective killing of tumor cells and the destruction of the blood vessels in tumor masses may be responsible for the success of BA-mediated BNCT for liver tumors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cytotoxic Killing and Immune Evasion by Repair

NASA Astrophysics Data System (ADS)

Chan, Cliburn; George, Andrew J. T.; Stark, Jaroslav

2007-07-01

The interaction between the immune system and pathogens is a complex one, with pathogens constantly developing new ways of evading destruction by the immune system. The immune system's task is made even harder when the pathogen in question is an intra-cellular one (such as a virus or certain bacteria) and it is necessary to kill the infected host cell in order to eliminate the pathogen. This causes damage to the host, and such killing therefore needs to be carefully controlled, particularly in tissues with poor regenerative potential, or those involved in the immune response itself. Host cells therefore possess repair mechanisms which can counteract killing by immune cells. These in turn can be subverted by pathogens which up-regulate the resistance of infected cells to killing. In this paper, we explore the hypothesis that this repair process plays an important role in determining the efficacy of evasion and escape from immune control. We model a situation where cytotoxic T lymphocytes (CTL) and natural killer (NK) cells kill pathogen-infected and tumour cells by directed secretion of preformed granules containing perforin and granzymes. Resistance to such killing can be conferred by the expression of serine protease inhibitors (serpins). These are utilized by several virally infected and tumour cells, as well as playing a role in the protection of host bystander, immune and immuneprivileged cells. We build a simple stochastic model of cytotoxic killing, where serpins can neutralize granzymes stoichiometrically by forming an irreversible complex, and the survival of the cell is determined by the balance between serpin depletion and replenishment, which in its simplest form is equivalent to the well known shot noise process. We use existing analytical results for this process, and additional simulations to analyse the effects of repair on cytotoxic killing. We then extend the model to the case of a replicating target cell population, which gives a branching process coupled to shot noise. We show how the process of repair can have a major impact on the dynamics of pathogen evasion and escape of tumour cells from immune surveillance

Galectin-9 Signaling through TIM-3 Is Involved in Neutrophil-Mediated Gram-Negative Bacterial Killing: An Effect Abrogated within the Cystic Fibrosis Lung

PubMed Central

Vega-Carrascal, Isabel; Bergin, David A.; McElvaney, Oliver J.; McCarthy, Cormac; Banville, Nessa; Pohl, Kerstin; Hirashima, Mitsuomi; Kuchroo, Vijay K.; Reeves, Emer P.; McElvaney, Noel G.

2016-01-01

The T cell Ig and mucin domain–containing molecule (TIM) family of receptors have emerged as potential therapeutic targets to correct abnormal immune function in chronic inflammatory conditions. TIM-3 serves as a functional receptor in structural cells of the airways and via the ligand galectin-9 (Gal-9) can modulate the inflammatory response. The aim of this study was to investigate TIM-3 expression and function in neutrophils, focusing on its potential role in cystic fibrosis (CF) lung disease. Results revealed that TIM-3 mRNA and protein expression values of circulating neutrophils were equal between healthy controls (n = 20) and people with CF (n = 26). TIM-3 was detected on resting neutrophil membranes by FACS analysis, and expression levels significantly increased post IL-8 or TNF-α exposure (p < 0.05). Our data suggest a novel role for TIM-3/Gal-9 signaling involving modulation of cytosolic calcium levels. Via TIM-3 interaction, Gal-9 induced neutrophil degranulation and primed the cell for enhanced NADPH oxidase activity. Killing of Pseudomonas aeruginosa was significantly increased upon bacterial opsonization with Gal-9 (p < 0.05), an effect abrogated by blockade of TIM-3 receptors. This mechanism appeared to be Gram-negative bacteria specific and mediated via Gal-9/ LPS binding. Additionally, we have demonstrated that neutrophil TIM-3/Gal-9 signaling is perturbed in the CF airways due to proteolytic degradation of the receptor. In conclusion, results suggest a novel neutrophil defect potentially contributing to the defective bacterial clearance observed in the CF airways and suggest that manipulation of the TIM-3 signaling pathway may be of therapeutic value in CF, preferably in conjunction with antiprotease treatment. PMID:24477913

Decursin enhances TRAIL-induced apoptosis through oxidative stress mediated- endoplasmic reticulum stress signalling in non-small cell lung cancers.

PubMed

Kim, Jaekwang; Yun, Miyong; Kim, Eun-Ok; Jung, Deok-Beom; Won, Gunho; Kim, Bonglee; Jung, Ji Hoon; Kim, Sung-Hoon

2016-03-01

The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL-induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy. Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and calcusyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting. Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL-resistant non-small-cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer-binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down-regulated expression of survivin and Bcl-xL in TRAIL-resistant NSCLC cells. ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL-resistant NSCLC cell lines, partly via up-regulation of DR5. © 2015 The British Pharmacological Society.

Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors

PubMed Central

Knödgen, Eva; Göckeritz, Elisa; Vondey, Verena; Neumann, Lars; Herter, Sylvia; Klein, Christian; Hallek, Michael

2018-01-01

The antibody-dependent cell-mediated cytotoxicity (ADCC) of the anti-CD20 monoclonal antibodies (mAbs) rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI) was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 μM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism. PMID:29750146

The Cek1‑mediated MAP kinase pathway regulates exposure of α‑1,2 and β‑1,2‑mannosides in the cell wall of Candida albicans modulating immune recognition.

PubMed

Román, E; Correia, I; Salazin, A; Fradin, C; Jouault, T; Poulain, D; Liu, F-T; Pla, J

2016-07-03

The Cek1 MAP kinase (MAPK) mediates vegetative growth and cell wall biogenesis in the fungal pathogen Candida albicans. Alterations in the fungal cell wall caused by a defective Cek1‑mediated signaling pathway leads to increased β‑1,3‑glucan exposure influencing dectin‑1 fungal recognition by immune cells. We show here that cek1 cells also display an increased exposure of α‑1,2 and β‑1,2‑mannosides (α‑M and β‑M), a phenotype shared by strains defective in the activating MAPKK Hst7, suggesting a general defect in cell wall assembly. cek1 cells display walls with loosely bound material as revealed by transmission electron microscopy and are sensitive to tunicamycin, an inhibitor of N‑glycosylation. Transcriptomal analysis of tunicamycin treated cells revealed a differential pattern between cek1 and wild type cells which involved mainly cell wall and stress related genes. Mapping α‑M and β‑M epitopes in the mannoproteins of different cell wall fractions (CWMP) revealed an important shift in the molecular weight of the mannan derived from mutants defective in this MAPK pathway. We have also assessed the role of galectin‑3, a member of a β‑galactoside‑binding protein family shown to bind to and kill C. albicans through β‑M recognition, in the infection caused by cek1 mutants. Increased binding of cek1 to murine macrophages was shown to be partially blocked by lactose. Galectin-3(-/-) mice showed increased resistance to fungal infection, although galectin-3 did not account for the reduced virulence of cek1 mutants in a mouse model of systemic infection. All these data support a role for the Cek1‑mediated pathway in fungal cell wall maintenance, virulence and antifungal discovery.

An in vitro investigation of immunomodulatory properties of Lactobacillus plantarum and L. delbrueckii cells and their extracellular polysaccharides

PubMed Central

KISHIMOTO, Mana; NOMOTO, Ryohei; MIZUNO, Masashi; OSAWA, Ro

2017-01-01

Many probiotic lactobacilli and their extracellular polysaccharides (EPS) have beneficial immunological properties. However, it is unclear how they elicit the host immune response. We thus investigated the immunological properties of UV-killed Lactobacillus delbrueckii TU-1 and L. plantarum KM-9 cells as well as their extracellular polysaccharides (EPSs). High-performance liquid chromatography and ion exchange chromatography analyses showed that their EPSs differ in sugar composition and sugar fractionation. The immunological properties were evaluated in a semi-intestinal model using a Transwell co-culture system that employed human intestinal epithelial (Caco-2) cells on the apical side and murine macrophage (RAW264.7) cells on the basolateral side. The UV-killed cells and EPSs were added to the apical side to allow direct contact with Caco-2 cells and incubated for 6 hr. After incubation, the amounts of tumor necrosis factor-α and several cytokines released by RAW264.7 or Caco-2 cells were quantified by cytotoxic activity on L929 cells (murine fibrosarcoma cell line) and quantitative reverse-transcriptase PCR. We found that the UV-killed cells and their EPSs had immunological effects on RAW264.7 cells via Caco-2 cells. The RAW264.7 cells showed different cytokine production profiles when treated with UV-killed cells and EPSs. The UV-killed cells and EPSs promoted a Th1-type cellular response. Furthermore, we found that the UV-killed cells sent positive signals through Toll-like receptor (TLR) 2. Meanwhile, neither EPS sent a positive signal through TLR4 and TLR2. This evidence suggests that both UV-killed cells of the lactobacillus strains and their EPSs trigger a Th1-type immune response in a human host, with the former triggering the response via the TLRs expressed on its epithelium and the latter employing a mechanism yet to be determined, possibly involving a novel receptor that is designed to recognize specific patterns of repeating sugar in the EPSs. PMID:28748131

An in vitro investigation of immunomodulatory properties of Lactobacillus plantarum and L. delbrueckii cells and their extracellular polysaccharides.

PubMed

Kishimoto, Mana; Nomoto, Ryohei; Mizuno, Masashi; Osawa, Ro

2017-01-01

Many probiotic lactobacilli and their extracellular polysaccharides (EPS) have beneficial immunological properties. However, it is unclear how they elicit the host immune response. We thus investigated the immunological properties of UV-killed Lactobacillus delbrueckii TU-1 and L. plantarum KM-9 cells as well as their extracellular polysaccharides (EPSs). High-performance liquid chromatography and ion exchange chromatography analyses showed that their EPSs differ in sugar composition and sugar fractionation. The immunological properties were evaluated in a semi-intestinal model using a Transwell co-culture system that employed human intestinal epithelial (Caco-2) cells on the apical side and murine macrophage (RAW264.7) cells on the basolateral side. The UV-killed cells and EPSs were added to the apical side to allow direct contact with Caco-2 cells and incubated for 6 hr. After incubation, the amounts of tumor necrosis factor-α and several cytokines released by RAW264.7 or Caco-2 cells were quantified by cytotoxic activity on L929 cells (murine fibrosarcoma cell line) and quantitative reverse-transcriptase PCR. We found that the UV-killed cells and their EPSs had immunological effects on RAW264.7 cells via Caco-2 cells. The RAW264.7 cells showed different cytokine production profiles when treated with UV-killed cells and EPSs. The UV-killed cells and EPSs promoted a Th1-type cellular response. Furthermore, we found that the UV-killed cells sent positive signals through Toll-like receptor (TLR) 2. Meanwhile, neither EPS sent a positive signal through TLR4 and TLR2. This evidence suggests that both UV-killed cells of the lactobacillus strains and their EPSs trigger a Th1-type immune response in a human host, with the former triggering the response via the TLRs expressed on its epithelium and the latter employing a mechanism yet to be determined, possibly involving a novel receptor that is designed to recognize specific patterns of repeating sugar in the EPSs.

Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model

PubMed Central

Swift, Brenna E.; Williams, Brent A.; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A.; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

2012-01-01

Background Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. Design and Methods The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Results Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89–99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. Conclusions This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted. PMID:22271890

Improbability of Effective Vaccination Against Human Immunodeficiency Virus Because of Its Intracellular Transmission and Rectal Portal of Entry

NASA Astrophysics Data System (ADS)

Sabin, Albert B.

1992-09-01

The worldwide effort to produce a vaccine against AIDS continues to disregard the fact that even human immunodeficiency virus (HIV)-specific neutralizing antibodies and cell-mediated immunity are ineffective against virus within cells without viral antigens on the cell membrane-and that much of HIV infection is transmitted in this manner. According to a recent report, a simian immunodeficiency virus vaccine that protected monkeys against an intravenous challenge with cell-free virus was, as predicted, ineffective against an intravenous challenge with the same amount of virus in infected cells. Moreover, antibody and HIV have been found to coexist in cell-free plasma from asymptomatic and symptomatic patients. Excluding direct introduction of HIV into the bloodstream, the most common and efficient form of transmission of HIV infection is by receptive anal intercourse, and semen contains large numbers of infected cells per milliliter. Recent reports showing that colorectal cells can be persistently infected by HIV and that HIV RNA and cDNA are present in the cells of the colon of dead AIDS patients indicate that either cell-free or intracellular HIV has the capacity to multiply at the portal of entry in the colorectal area without interference from neutralizing antibodies. The available data provide no basis for testing any HIV vaccine in human beings either before or after infection. The main challenge is to find a way to kill cells with chromosomally integrated HIV cDNA without harming normal cells, perhaps by identifying repressor proteins that might be produced by the cells with integrated HIV cDNA and thus could become specific targets for cell-killing drugs.

Interleukin-12 up-regulates perforin- and Fas-mediated lymphokine-activated killer activity by intestinal intraepithelial lymphocytes

PubMed Central

EBERT, E C

2004-01-01

Human intraepithelial lymphocytes (IELs) comprise a unique compartment of memory T cell receptor (TCR)-αβ+CD8+ T lymphocytes interspersed between intestinal epithelial cells. They develop potent lymphokine-activated killer (LAK) activity with interleukin (IL)-15, a cytokine that is found in excess in certain mucosal inflammatory states. IL-12, released by activated antigen-presenting cells, is known to potentiate perforin-induced cytotoxicity. This study evaluates the mechanism by which IL-12 up-regulates LAK activity. When IELs were stimulated with IL-15, the CD94+ IEL subset expanded and carried out cytotoxic activity in redirected lysis against P815 cells as well as Fas ligand (FL)- and tumour necrosis factor (TNF)-α-mediated lysis of Jurkat and WEHI cells, respectively. IL-12 enhanced the perforin- and FL-, but not TNF-α-mediated events. In addition, the up-regulated killing of HT-29 cells by IL-12 was reduced by concanamycin (which targets perforin) and antibody neutralizing FL but not by anti-TNF-α antibody. Furthermore, IL-12 augmented IL-15-stimulated release of serine esterases as well as expression of perforin and FL by IELs, but not TNF-α. This study shows that LAK activity, carried out by the CD94+ IELs, involves perforin, FL and TNF-α. IL-12 up-regulates the first two mechanisms of action, showing for the first time its effect on FL production and lytic activity. PMID:15498035

Vaccination for treatment and prevention of cancer in animal models.

PubMed

Cavallo, Federica; Offringa, Rienk; van der Burg, Sjoerd H; Forni, Guido; Melief, Cornelis J M

2006-01-01

Two approaches to immunological intervention in tumor-host interactions in mouse models are discussed in this review. The first is described with reference to experiments in which CD8(+) T lymphocytes are used to kill established transplantable tumors. Peptides and their optimal presentation by dendritic cells and intervention in immune regulatory mechanisms are the key issues for efficient induction of T-killer cell-mediated tumor eradication. The time frame of tumor therapy and the threat imposed by tumor growth in transplantable models and cancer patients require the induction of a robust T-cell reaction. Prevention of the progression of small preneoplastic lesions, on the other hand, requires the significant and prolonged immune protection sought in the second approach. This is based on antibody production and the coordinated activation of multiple low-avidity cell-mediated mechanisms elicited by DNA vaccination in genetically modified cancer-prone mice, transgenic for a mutant Her-2/neu growth factor receptor expressed at the plasma membrane surface of preneoplastic mammary gland epithelial cells. Vaccination with appropriate DNA formulations results in prolonged immune inhibition of the progression of preneoplastic mammary lesions but is ineffective against established tumors. The use of molecularly defined adjuvants and intervention in immune regulatory mechanisms are critical in both the elicitation of an effective T-cell mediated reaction required for tumor debulking in the first set of models and the induction by vaccination of a sustained immune memory able to prevent the expansion of preneoplastic lesions in genetically cancer-prone mice.

CD40 dependent exacerbation of immune mediated hepatitis by hepatic CD11b+ Gr-1+ myeloid derived suppressor cells in tumor bearing mice

PubMed Central

Kapanadze, Tamar; Medina-Echeverz, José; Gamrekelashvili, Jaba; Weiss, Jonathan M.; Wiltrout, Robert H.; Kapoor, Veena; Hawk, Nga; Terabe, Masaki; Berzofsky, Jay A.; Manns, Michael P.; Wang, Ena; Marincola, Francesco M.; Korangy, Firouzeh; Greten, Tim F.

2015-01-01

Immunosuppressive CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) accumulate in the livers of tumor-bearing mice. We studied hepatic MDSC in two murine models of immune mediated hepatitis. Unexpectedly, treatment of tumor bearing mice with Concanavalin A or α-Galactosylceramide resulted in increased ALT and AST serum levels in comparison to tumor free mice. Adoptive transfer of hepatic MDSC into naïve mice exacerbated Concanavalin A induced liver damage. Hepatic CD11b+Gr-1+ cells revealed a polarized pro-inflammatory gene signature after Concanavalin A treatment. An interferon gamma- dependent up-regulation of CD40 on hepatic CD11b+Gr-1+ cells along with an up-regulation of CD80, CD86, and CD1d after Concanavalin A treatment was observed. Concanavalin A treatment resulted in a loss of suppressor function by tumor-induced CD11b+Gr-1+ MDSC as well as enhanced reactive oxygen species-mediated hepatotoxicity. CD40 knockdown in hepatic MDSC led to increased arginase activity upon Concanavalin A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40−/− tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased reactive oxygen species production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSC act as pro-inflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner. PMID:25616156

Lysosomes as Oxidative Targets for Cancer Therapy.

PubMed

Dielschneider, Rebecca F; Henson, Elizabeth S; Gibson, Spencer B

2017-01-01

Lysosomes are membrane-bound vesicles that contain hydrolases for the degradation and recycling of essential nutrients to maintain homeostasis within cells. Cancer cells have increased lysosomal function to proliferate, metabolize, and adapt to stressful environments. This has made cancer cells susceptible to lysosomal membrane permeabilization (LMP). There are many factors that mediate LMP such as Bcl-2 family member, p53; sphingosine; and oxidative stress which are often altered in cancer. Upon lysosomal disruption, reactive oxygen species (ROS) levels increase leading to lipid peroxidation, mitochondrial dysfunction, autophagy, and reactive iron. Cathepsins are also released causing degradation of macromolecules and cellular structures. This ultimately kills the cancer cell through different types of cell death (apoptosis, autosis, or ferroptosis). In this review, we will explore the contributions lysosomes play in inducing cell death, how this is regulated by ROS in cancer, and how lysosomotropic agents might be utilized to treat cancers.

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors.

PubMed

Kushwaha, Ambuj K; Apolis, Liana; Ito, Daisuke; Desai, Sanjay A

2018-05-03

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad-selectivity channel known as the plasmodial surface anion channel, increased Ca ++ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca ++ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N-hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca ++ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca ++ permeability, suggesting involvement of parasite-encoded proteins trafficked to the host membrane. A high-throughput chemical screen identified the first Ca ++ transport inhibitors active against Plasmodium-infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca ++ ] is consistent with parasite killing specifically via action on one or more Ca ++ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca ++ transport and may be starting points for new antimalarial drugs. © 2018 John Wiley & Sons Ltd.

A Nuclease from Streptococcus mutans Facilitates Biofilm Dispersal and Escape from Killing by Neutrophil Extracellular Traps

PubMed Central

Liu, Jia; Sun, Luping; Liu, Wei; Guo, Lihong; Liu, Zhaohui; Wei, Xi; Ling, Junqi

2017-01-01

Streptococcus mutans is the primary etiologic agent of dental caries and occasionally infective endocarditis, with the ability to form biofilms and disperse cells into distal sites to exacerbate and spread infection. In this study, we identified a nuclease (DeoC) as a S. mutans biofilm dispersal modulating factor through microarray analysis. In vitro assays revealed a dispersal defect of a deoC deletion mutant, and functional studies with purified protein were indicative of the biofilm dispersal activity of DeoC. Neutrophils are a key host response factor restraining bacterial spreading through the formation of neutrophil extracellular traps (NETs), which consist of a nuclear DNA backbone associated with antimicrobial peptides. Therefore, we hypothesized that the dispersed S. mutans might utilize DeoC to degrade NETs and escape killing by the immune system. It was found that S. mutans induced NET formation upon contact with neutrophils, while the presence of NETs in turn enhanced the deoC expression of S. mutans. Fluorescence microscopy inspection showed that deoC deletion resulted in a decreased NET degradation ability of S. mutans and enhanced susceptibility to neutrophil killing. Data obtained from this study assigned two important roles for DeoC in S. mutans: contributing to the spread of infection through mediating biofilm dispersal, and facilitating the escape of S. mutans from neutrophil killing through NET degradation. PMID:28401067

A Nuclease from Streptococcus mutans Facilitates Biofilm Dispersal and Escape from Killing by Neutrophil Extracellular Traps.

PubMed

Liu, Jia; Sun, Luping; Liu, Wei; Guo, Lihong; Liu, Zhaohui; Wei, Xi; Ling, Junqi

2017-01-01

Streptococcus mutans is the primary etiologic agent of dental caries and occasionally infective endocarditis, with the ability to form biofilms and disperse cells into distal sites to exacerbate and spread infection. In this study, we identified a nuclease (DeoC) as a S. mutans biofilm dispersal modulating factor through microarray analysis. In vitro assays revealed a dispersal defect of a deoC deletion mutant, and functional studies with purified protein were indicative of the biofilm dispersal activity of DeoC. Neutrophils are a key host response factor restraining bacterial spreading through the formation of neutrophil extracellular traps (NETs), which consist of a nuclear DNA backbone associated with antimicrobial peptides. Therefore, we hypothesized that the dispersed S. mutans might utilize DeoC to degrade NETs and escape killing by the immune system. It was found that S. mutans induced NET formation upon contact with neutrophils, while the presence of NETs in turn enhanced the deoC expression of S. mutans . Fluorescence microscopy inspection showed that deoC deletion resulted in a decreased NET degradation ability of S. mutans and enhanced susceptibility to neutrophil killing. Data obtained from this study assigned two important roles for DeoC in S. mutans : contributing to the spread of infection through mediating biofilm dispersal, and facilitating the escape of S. mutans from neutrophil killing through NET degradation.

Herpes Simplex Virus-based gene Therapy Enhances the Efficacy of Mitomycin-C in the Treatment of Human Bladder Transitional Cell Carcinoma

PubMed Central

Mullerad, Michael; Bochner, Bernard H.; Adusumilli, Prasad S.; Bhargava, Amit; Kikuchi, Eiji; Hui-Ni, Chen; Kattan, Michael W.; Chou, Ting-Chao; Fong, Yuman

2005-01-01

Purpose Oncolytic replication-competent herpes simplex virus type-1 (HSV) mutants have the ability to replicate in and kill malignant cells. We have previously demonstrated the ability of replication-competent HSV to control bladder cancer growth in an orthotopic murine model. We hypothesized that a combination of a chemotherapeutic agent used for intravesical treatment - mitomycin-C (MMC) - and oncolytic HSV would exert a synergistic effect in the treatment of human transitional cell carcinoma (TCC). Materials and Methods We used the mutant HSV NV1066, which is deleted for viral genes ICP0 and ICP4 and selectively infects cancer cells, to treat TCC lines, KU19-19 and SKUB. Cell survival was determined by lactate dehydrogenase (LDH) assay for each agent as well as for drug-viral combinations from days 1 to 5. The isobologram method and the combination index method of Chou-Talalay were used to assess for synergistic effect. Results NV1066 enhanced MMC mediated cytotoxicity at all combinations tested for both KU19-19 and SKUB. Combination of both agents demonstrated a synergistic effect and allowed dose reduction by 12 and 10.4 times (NV1066) and by 3 and 156 times (MMC) in the treatment of KU19-19 and SKUB respectively, while achieving an estimated 90% cell kill. Conclusion These data provide the cellular basis for the clinical investigation of combined mitomycin-C and oncolytic HSV therapy in the treatment of bladder cancer. PMID:16006968

Protein Deiminase 4 and CR3 Regulate Aspergillus fumigatus and β-Glucan-Induced Neutrophil Extracellular Trap Formation, but Hyphal Killing Is Dependent Only on CR3.

PubMed

Clark, Heather L; Abbondante, Serena; Minns, Martin S; Greenberg, Elyse N; Sun, Yan; Pearlman, Eric

2018-01-01

Neutrophil extracellular trap (NET) formation requires chromatin decondensation before nuclear swelling and eventual extracellular release of DNA, which occurs together with nuclear and cytoplasmic antimicrobial proteins. A key mediator of chromatin decondensation is protein deiminase 4 (PAD4), which catalyzes histone citrullination. In the current study, we examined the role of PAD4 and NETosis following activation of neutrophils by A. fumigatus hyphal extract or cell wall β-glucan (curdlan) and found that both induced NET release by human and murine neutrophils. Also, using blocking antibodies to CR3 and Dectin-1 together with CR3-deficient CD18 -/- and Dectin-1 -/- murine neutrophils, we found that the β-glucan receptor CR3, but not Dectin-1, was required for NET formation. NETosis was also dependent on NADPH oxidase production of reactive oxygen species (ROS). Using an antibody to citrullinated histone 3 (H3Cit) as an indicator of PAD4 activity, we show that β-glucan stimulated NETosis occurs in neutrophils from C57BL/6, but not PAD4 -/- mice. Similarly, a small molecule PAD4 inhibitor (GSK484) blocked NET formation by human neutrophils. Despite these observations, the ability of PAD4 -/- neutrophils to release calprotectin and kill A. fumigatus hyphae was not significantly different from C57BL/6 neutrophils, whereas CD18 -/- neutrophils exhibited an impaired ability to perform both functions. We also detected H3Cit in A. fumigatus infected C57BL/6, but not PAD4 -/- corneas; however, we found no difference between C57BL/6 and PAD4 -/- mice in either corneal disease or hyphal killing. Taken together, these findings lead us to conclude that although PAD4 together with CR3-mediated ROS production is required for NET formation in response to A. fumigatus , PAD4-dependent NETosis is not required for A. fumigatus killing either in vitro or during infection.

The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence

PubMed Central

Childers, Delma S.; Raziunaite, Ingrida; Mol Avelar, Gabriela; Mackie, Joanna; Budge, Susan; Stead, David; Gow, Neil A. R.; Lenardon, Megan D.; Ballou, Elizabeth R.; MacCallum, Donna M.; Brown, Alistair J. P.

2016-01-01

Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is “Crabtree positive”, displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for yeasts in environmental niches, the more flexible carbon assimilation strategies offered by Crabtree negativity enhance the ability of yeasts to colonize and infect the mammalian host. PMID:27073846

Cell-to-Cell Heterogeneity in Cortical Tension Specifies Curvature of Contact Surfaces in Caenorhabditis elegans Embryos

PubMed Central

Fujita, Masashi; Onami, Shuichi

2012-01-01

In the two-cell stage embryos of Caenorhabditis elegans, the contact surface of the two blastomeres forms a curve that bulges from the AB blastomere to the P1 blastomere. This curve is a consequence of the high intracellular hydrostatic pressure of AB compared with that of P1. However, the higher pressure in AB is intriguing because AB has a larger volume than P1. In soap bubbles, which are a widely used model of cell shape, a larger bubble has lower pressure than a smaller bubble. Here, we reveal that the higher pressure in AB is mediated by its higher cortical tension. The cell fusion experiments confirmed that the curvature of the contact surface is related to the pressure difference between the cells. Chemical and genetic interferences showed that the pressure difference is mediated by actomyosin. Fluorescence imaging indicated that non-muscle myosin is enriched in the AB cortex. The cell killing experiments provided evidence that AB but not P1 is responsible for the pressure difference. Computer simulation clarified that the cell-to-cell heterogeneity of cortical tensions is indispensable for explaining the pressure difference. This study demonstrates that heterogeneity in surface tension results in significant deviations of cell behavior compared to simple soap bubble models, and thus must be taken into consideration in understanding cell shape within embryos. PMID:22253922

Powerful bacterial killing by buckwheat honeys is concentration-dependent, involves complete DNA degradation and requires hydrogen peroxide.

PubMed

Brudzynski, Katrina; Abubaker, Kamal; Wang, Tony

2012-01-01

Exposure of bacterial cells to honey inhibits their growth and may cause cell death. Our previous studies showed a cause-effect relationship between hydroxyl radical generated from honey hydrogen peroxide and growth arrest. Here we explored the role of hydroxyl radicals as inducers of bacterial cells death. The bactericidal effect of ·OH on antibiotic-resistant clinical isolates of MRSA and VRE and standard bacterial strains of E. coli and B. subtiles was examined using a broth microdilution assay supplemented with 3'-(p-aminophenyl) fluorescein (APF) as the ·OH trap, followed by colony enumeration. Bactericidal activities of eight honeys (six varieties of buckwheat, blueberry and manuka honeys) were analyzed. The MBC/MIC ratio ≤4 and the killing curves indicated that honeys exhibited powerful, concentration-dependent bactericidal effect. The extent of killing depended on the ratio of honey concentration to bacterial load, indicating that honey dose was critical for its bactericidal efficacy. The killing rate and potency varied between honeys and ranged from over a 6-log(10) to 4-log(10) CFU/ml reduction of viable cells, equivalent to complete bacterial eradication. The maximal killing was associated with the extensive degradation of bacterial DNA. Honey concentration at which DNA degradation occurred correlated with cell death observed in the concentration-dependent cell-kill on agar plates. There was no quantitative relationship between the ·OH generation by honey and bactericidal effect. At the MBC, where there was no surviving cells and no DNA was visible on agarose gels, the ·OH levels were on average 2-3x lower than at Minimum Inhibitory Concentration (MICs) (p < 0.0001). Pre-treatment of honey with catalase, abolished the bactericidal effect. This raised possibilities that either the abrupt killing prevented accumulation of ·OH (dead cells did not generate ·OH) or that DNA degradation and killing is the actual footprint of ·OH action. In conclusion, honeys of buckwheat origin exhibited powerful, concentration-dependent bactericidal effect. The killing and DNA degradation showed a cause-effect relationship. Hydrogen peroxide was an active part of honey killing mechanism.

Parasite-mediated nuclear factor κB regulation in lymphoproliferation caused by Theileria parva infection

PubMed Central

Palmer, Guy H.; Machado, Joel; Fernandez, Paula; Heussler, Volker; Perinat, Therese; Dobbelaere, Dirk A. E.

1997-01-01

Infection of cattle with the protozoan Theileria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor κB (NFκB) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NFκB activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the IκB molecules which normally sequester NFκB in the cytoplasm and that continuous degradation requires viable parasites. Using T. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of IκBα. However, IκBα reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NFκB-mediated positive feedback loop which restores cytoplasmic IκBα. In contrast, T. parva mediated continuous degradation of IκBβ resulting in persistently low cytoplasmic IκBβ levels. Normal IκBβ levels were only restored following T. parva killing, indicating that viable parasites are required for IκBβ degradation. Treatment of T. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both IκB degradation and consequent enhanced expression of NFκB dependent genes. However treatment using the antioxidant N-acetylcysteine had no effect on either IκB levels or NFκB activation, indicating that the parasite subverts the normal IκB regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function. PMID:9356483

PDE5 Inhibitors Enhance Celecoxib Killing in Multiple Tumor Types

PubMed Central

BOOTH, LAURENCE; ROBERTS, JANE L.; CRUICKSHANKS, NICHOLA; TAVALLAI, SEYEDMEHRAD; WEBB, TIMOTHY; SAMUEL, PETER; CONLEY, ADAM; BINION, BRITTANY; YOUNG, HAROLD F.; POKLEPOVIC, ANDREW; SPIEGEL, SARAH; DENT, PAUL

2015-01-01

The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. PMID:25303541

Repression of GSK3 restores NK cell cytotoxicity in AML patients

PubMed Central

Parameswaran, Reshmi; Ramakrishnan, Parameswaran; Moreton, Stephen A.; Xia, Zhiqiang; Hou, Yongchun; Lee, Dean A.; Gupta, Kalpana; deLima, Marcos; Beck, Rose C.; Wald, David N.

2016-01-01

Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy. PMID:27040177

LET and ion-species dependence for cell killing and mutation induction in normal human fibroblasts.

PubMed

Tsuruoka, Chizuru; Suzuki, Masao; Fujitaka, Kazunobu

2003-10-01

We have been studying LET and ion species dependence of RBE values in cell killing and mutation induction. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon (290 Mev/u and 135 Mev/u), neon (230 Mev/u and 400 Mev/u), silicon (490 Mev/u) and iron (500 Mev/u) ion beams, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences (NIRS). Cell killing effect was detected as reproductive cell death using a colony formation assay. Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies. The RBE-LET curves of cell killing and mutation induction were different each ion beam. So, we plotted RBE for cell killing and mutation induction as function of Z*2/beta2 instead of LET. RBE-Z*2/beta2 curves of cell killing indicated that the discrepancy of RBE-LET curves was reconciled each ion species. But RBE-Z*2/beta2 curves of mutation induction didn't corresponded between carbon- and silicon-ion beams. These results suggested that different biological endpoints may be suitable for different physical parameter, which represent the track structure of energy deposition of ion beams.

Inflammatory Ly6Chigh Monocytes Protect against Candidiasis through IL-15-Driven NK Cell/Neutrophil Activation.

PubMed

Domínguez-Andrés, Jorge; Feo-Lucas, Lidia; Minguito de la Escalera, María; González, Leticia; López-Bravo, María; Ardavín, Carlos

2017-06-20

Neutrophils play a crucial role in defense against systemic candidiasis, a disease associated with a high mortality rate in patients receiving immunosuppressive therapy, although the early immune mechanisms that boost the candidacidal activity of neutrophils remain to be defined in depth. Here, we used a murine model of systemic candidiasis to explore the role of inflammatory Ly6C high monocytes in NK cell-mediated neutrophil activation during the innate immune response against C. albicans. We found that efficient anti-Candida immunity required a collaborative response between the spleen and kidney, which relied on type I interferon-dependent IL-15 production by spleen inflammatory Ly6C high monocytes to drive efficient activation and GM-CSF release by spleen NK cells; this in turn was necessary to boost the Candida killing potential of kidney neutrophils. Our findings unveil a role for IL-15 as a critical mediator in defense against systemic candidiasis and hold promise for the design of IL-15-based antifungal immunotherapies. Copyright © 2017 Elsevier Inc. All rights reserved.

NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing

PubMed Central

Hsu, Hsiang-Ting; Viswanath, Dixita I.; Önfelt, Björn

2016-01-01

Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein- and integrin signal–dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector–target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific “bystander” killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells. PMID:27903610

Arginine-assisted immobilization of silver nanoparticles on ZnO nanorods: an enhanced and reusable antibacterial substrate without human cell cytotoxicity

NASA Astrophysics Data System (ADS)

Agnihotri, Shekhar; Bajaj, Geetika; Mukherji, Suparna; Mukherji, Soumyo

2015-04-01

Silver-based hybrid nanomaterials are gaining interest as potential alternatives for conventional antimicrobial agents. Herein, we present a simple, facile and eco-friendly approach for the deposition of silver nanoparticles (AgNPs) on ZnO nanorods, which act as a nanoreactor for in situ synthesis and as an immobilizing template in the presence of arginine. The presence of arginine enhanced the stability of ZnO deposition on the glass substrate by hindering the dissolution of zinc under alkaline conditions. Various Ag/ZnO hybrid nanorod (HNR) samples were screened to obtain a high amount of silver immobilization on the ZnO substrate. Ag/ZnO HNRs displayed potent antibacterial ability and could achieve 100% kill for both Escherichia coli and Bacillus subtilis strains under various test conditions. The hybrid material mediated its dual mode of antibacterial action through direct contact-killing and release of silver ions/nanoparticles and showed superior bactericidal performance compared to pure ZnO nanorods and colloidal AgNPs. No significant decline in antibacterial efficacy was observed even after the same substrate was repeatedly reused multiple times. Interestingly, the amount of Ag and Zn release was much below their maximal limit in drinking water, thus preventing potential health hazards. Immobilized AgNPs showed no cytotoxic effects on the human hepatocarcinoma cell line (HepG2). Moreover, treating cells with the antibacterial substrate for 24 hours did not lead to significant generation of reactive oxygen species (ROS). The good biocompatibility and bactericidal efficacy would thus make it feasible to utilize this immobilization strategy for preparing new-generation antibacterial coatings.Silver-based hybrid nanomaterials are gaining interest as potential alternatives for conventional antimicrobial agents. Herein, we present a simple, facile and eco-friendly approach for the deposition of silver nanoparticles (AgNPs) on ZnO nanorods, which act as a nanoreactor for in situ synthesis and as an immobilizing template in the presence of arginine. The presence of arginine enhanced the stability of ZnO deposition on the glass substrate by hindering the dissolution of zinc under alkaline conditions. Various Ag/ZnO hybrid nanorod (HNR) samples were screened to obtain a high amount of silver immobilization on the ZnO substrate. Ag/ZnO HNRs displayed potent antibacterial ability and could achieve 100% kill for both Escherichia coli and Bacillus subtilis strains under various test conditions. The hybrid material mediated its dual mode of antibacterial action through direct contact-killing and release of silver ions/nanoparticles and showed superior bactericidal performance compared to pure ZnO nanorods and colloidal AgNPs. No significant decline in antibacterial efficacy was observed even after the same substrate was repeatedly reused multiple times. Interestingly, the amount of Ag and Zn release was much below their maximal limit in drinking water, thus preventing potential health hazards. Immobilized AgNPs showed no cytotoxic effects on the human hepatocarcinoma cell line (HepG2). Moreover, treating cells with the antibacterial substrate for 24 hours did not lead to significant generation of reactive oxygen species (ROS). The good biocompatibility and bactericidal efficacy would thus make it feasible to utilize this immobilization strategy for preparing new-generation antibacterial coatings. Electronic supplementary information (ESI) available: Synthesis of ZnO nanorods, instrumentation details, contact killing of Ag/ZnO. See DOI: 10.1039/c4nr06913g

Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

PubMed Central

Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

2011-01-01

Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

A genome-wide loss-of-function screen identifies SLC26A2 as a novel mediator of TRAIL resistance

PubMed Central

Dimberg, Lina Y.; Towers, Christina G.; Behbakht, Kian; Hotz, Taylor J.; Kim, Jihye; Fosmire, Susan; Porter, Christopher C.; Tan, Aik-Choon; Thorburn, Andrew; Ford, Heide L.

2017-01-01

TNF-related apoptosis inducing ligand (TRAIL) is a potent death-inducing ligand that mediates apoptosis through the extrinsic pathway and serves as an important endogenous tumor suppressor mechanism. Because tumor cells are often killed by TRAIL and normal cells are not, drugs that activate the TRAIL pathway have been thought to have potential clinical value. However, to date, most TRAIL-related clinical trials have largely failed due to the tumor cells having intrinsic or acquired resistance to TRAIL-induced apoptosis. Previous studies to identify resistance mechanisms have focused on targeted analysis of the canonical apoptosis pathway and other known regulators of TRAIL receptor signaling. To identify novel mechanisms of TRAIL resistance in an unbiased way, we performed a genome wide shRNA screen for genes that regulate TRAIL sensitivity in sub-lines that had been selected for acquired TRAIL resistance. This screen identified previously unknown mediators of TRAIL resistance including Angiotensin II Receptor 2, Crk-like protein, T-Box Transcription Factor 2 and solute carrier family 26 member 2 (SLC26A2). SLC26A2 downregulates the TRAIL receptors, DR4 and DR5, and this downregulation is associated with resistance to TRAIL. Its expression is high in numerous tumor types compared to normal cells, and in breast cancer, SLC26A2 is associated with a significant decrease in relapse free survival. PMID:28108622

Parasiticidal activity of bovine lactoperoxidase against Toxoplasma gondii.

PubMed

Tanaka, Tetsuya; Murakami, Shin; Kumura, Haruto; Igarashi, Ikuo; Shimazaki, Kei-Ichi

2006-10-01

Toxoplasma gondii is an obligatory intracellular parasitic protozoan transmitted via the ingestion of raw, infected meat that causes congenital infections. In a cell-free environment, virulent Toxoplasma was strikingly resistant to H2O2. The activity of H2O2 or H2O2 generated by glucose-glucose oxidase against the resistant tachyzoite stage of pathogenic T. gondii was enhanced by adding KI and bovine lactoperoxidase (bLPO), referred to here as the bLPO system. Replacing bLPO (heme content, 90%) with recombinant bLPO (heme content, 6%) did not enhance the parasiticidal activity with KI and H2O2. These results indicated that heme contributed to the enzyme activity and resulted in the killing of tachyzoites of T. gondii. Tachyzoites treated with the bLPO system also lost the ability to penetrate the mouse fibroblast cell line (NIH/3T3), and could be killed intracellularly after exposure by bLPO to a mouse macrophage cell line (J774A.1). These findings suggested that toxicity was mediated through small amounts of H2O2 generated by phagocytic events in naive macrophages, and by the peroxidative activity of bLPO. Our observations suggest that the bLPO system could help prevent the development of Toxoplasmosis in humans after ingesting raw, infected meat.

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus and in vitro properties.

PubMed

Anderson, J; Byrne, J A; Schreiber, R; Patterson, S; Oldstone, M B

1985-02-01

We have generated lymphocytic choriomeningitis virus-specific, H-2-restricted cytotoxic thymus-derived lymphocyte (CTL) clones. By using these reagents in several in vitro assays with infected target cells, we show that CTLs by themselves prevent the release of infectious virus into culture fluids and significantly lower the titers of infectious virus previously produced. This ability of cloned CTLs is not influenced by monensin. However, monensin does abrogate the ability of CTLs from spleens of mice primed 6 to 8 days previously with virus to kill virus-infected syngeneic targets. When tested for the participation of lymphokines in this system, the CTLs proliferate when reacted with syngeneic lymphocytic choriomeningitis virus-infected macrophages but fail to make interleukin-2. These CTLs make gamma interferon when reacted with syngeneic virus-infected targets. However, the production of interferon does not directly correlate with CTL-mediated killing. The number of H-2K and D molecules expressed on the target cell surface is not altered during the course of lymphocytic choriomeningitis virus infection. Electron microscopy shows finger-like projections of the CTL clone thrust into the infected cell and lesions bearing an internal diameter of approximately 15 nm in those membranes, illustrating the lytic process.

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

PubMed Central

Arimilli, Subhashini; Damratoski, Brad E.; G.L., Prasad

2015-01-01

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-γ, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-γ, TNF-α, and MIP-1α were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest. PMID:25650834

Methods to evaluate cytotoxicity and immunosuppression of combustible tobacco product preparations.

PubMed

Arimilli, Subhashini; Damratoski, Brad E; G L, Prasad

2015-01-10

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-γ, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-γ, TNF-α, and MIP-1α were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

The neuropeptide substance P stimulates the effector functions of platelets.

PubMed Central

Damonneville, M; Monté, D; Auriault, C; Capron, A

1990-01-01

Sensory neuropeptides, such as substance P, appear as potent mediators of various immunological reactions, and inhibit or stimulate a wide range of functions of immune inflammatory cells. Platelets were recently shown to participate as effector cells in an IgE or lymphokine-dependent killing of parasites. Substance P and its carboxy-terminal fragment SP (4-11) induce the cytotoxic activity of platelets towards the larvae of Schistosoma mansoni, respectively, by 90% and 40%, whereas the modified C terminal SP, the SP-free acid, exhibits no effect on the platelets. The neuropeptide effects occur at low doses (10(-8) M), are specific as shown by inhibition studies with a substance P antagonist, the D-SP. Binding data obtained after flow cytofluorometry with FITC-SP lead to the conclusion that SP binds specifically to about 20% of the homogenous population of platelets. Moreover, IgE could modulate the SP-dependent functions of platelets since the pre-incubation with myeloma human IgE or with AP2 monoclonal antibodies--known to inhibit the IgE-dependent killing of these cells-leads to a dramatic decrease of the SP dependent cytotoxic activity of platelets towards the larvae. These findings identify a potent mechanism for nervous system regulation of host defence responses. PMID:1696868

The Viral Transcription Group Determines the HLA Class I Cellular Immune Response Against Human Respiratory Syncytial Virus*

PubMed Central

Johnstone, Carolina; Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Infantes, Susana; Lemonnier, François A.; David, Chella S.; Admon, Arie; López, Daniel

2015-01-01

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design. PMID:25635267

Nafcillin enhances innate immune-mediated killing of methicillin-resistant Staphylococcus aureus.

PubMed

Sakoulas, George; Okumura, Cheryl Y; Thienphrapa, Wdee; Olson, Joshua; Nonejuie, Poochit; Dam, Quang; Dhand, Abhay; Pogliano, Joseph; Yeaman, Michael R; Hensler, Mary E; Bayer, Arnold S; Nizet, Victor

2014-02-01

Based on in vitro synergy studies, the addition of nafcillin to daptomycin was used to treat refractory methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Daptomycin is a de facto cationic antimicrobial peptide in vivo, with antistaphylococcal mechanisms reminiscent of innate host defense peptides (HDPs). In this study, the effects of nafcillin on HDP activity against MRSA were examined in vitro and in vivo. Exposures to β-lactam antimicrobials in general, and nafcillin in particular, significantly increased killing of S. aureus by selected HDPs from keratinocytes, neutrophils, and platelets. This finding correlated with enhanced killing of MRSA by whole blood, neutrophils, and keratinocytes after growth in nafcillin. Finally, nafcillin pretreatment ex vivo reduced MRSA virulence in a murine subcutaneous infection model. Despite the lack of direct activity against MRSA, these studies show potent, consistent, and generalized nafcillin-mediated "sensitization" to increased killing of MRSA by various components of the innate host response. The use of nafcillin as adjunctive therapy in MRSA bacteremia merits further study and should be considered in cases refractory to standard therapy. Nafcillin has been used as adjunctive therapy to clear persistent MRSA bacteremia. Nafcillin enhances killing of MRSA by a cadre of innate host defense peptides. Nafcillin increases binding of human cathelicidin LL-37 to the MRSA membrane. Nafcillin enhances killing of MRSA by neutrophils. Nafcillin reduces virulence of MRSA in a murine subcutaneous infection model.

Loss of SLP-76 expression within myeloid cells confers resistance to neutrophil-mediated tissue damage while maintaining effective bacterial killing.

PubMed

Clemens, Regina A; Lenox, Laurie E; Kambayashi, Taku; Bezman, Natalie; Maltzman, Jonathan S; Nichols, Kim E; Koretzky, Gary A

2007-04-01

The Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is an adaptor molecule critical for immunoreceptor and integrin signaling in multiple hemopoietic lineages. We showed previously that SLP-76 is required for neutrophil function in vitro, including integrin-induced adhesion and production of reactive oxygen intermediates, and to a lesser extent, FcgammaR-induced calcium flux and reactive oxygen intermediate production. It has been difficult to determine whether SLP-76 regulates neutrophil responses in vivo, because Slp-76(-/-) mice exhibit marked defects in thymocyte and vascular development, as well as platelet and mast cell function. To circumvent these issues, we generated mice with targeted loss of SLP-76 expression within myeloid cells. Neutrophils obtained from these animals failed to respond to integrin activation in vitro, similar to Slp-76(-/-) cells. Despite these abnormalities, SLP-76-deficient neutrophils migrated normally in vivo in response to Staphylococcus aureus infection and efficiently cleared micro-organisms. Interestingly, SLP-76-deficient neutrophils did not induce a robust inflammatory response in the localized Shwartzman reaction. Collectively, these data suggest that disruption of integrin signaling via loss of SLP-76 expression differentially impairs neutrophil functions in vivo, with preservation of migration and killing of S. aureus but reduction in LPS-induced tissue damage and vascular injury.

Radiogenetic therapy: strategies to overcome tumor resistance.

PubMed

Marples, B; Greco, O; Joiner, M C; Scott, S D

2003-01-01

The aim of cancer gene therapy is to selectively kill malignant cells at the tumor site, by exploiting traits specific to cancer cells and/or solid tumors. Strategies that take advantage of biological features common to different tumor types are particularly promising, since they have wide clinical applicability. Much attention has focused on genetic methods that complement radiotherapy, the principal treatment modality, or that exploit hypoxia, the most ubiquitous characteristic of most solid cancers. The goal of this review is to highlight two promising gene therapy methods developed specifically to target the tumor volume that can be readily used in combination with radiotherapy. The first approach uses radiation-responsive gene promoters to control the selective expression of a suicide gene (e.g., herpes simplex virus thymidine kinase) to irradiated tissue only, leading to targeted cell killing in the presence of a prodrug (e.g., ganciclovir). The second method utilizes oxygen-dependent promoters to produce selective therapeutic gene expression and prodrug activation in hypoxic cells, which are refractive to conventional radiotherapy. Further refining of tumor targeting can be achieved by combining radiation and hypoxia responsive elements in chimeric promoters activated by either and dual stimuli. The in vitro and in vivo studies described in this review suggest that the combination of gene therapy and radiotherapy protocols has potential for use in cancer care, particularly in cases currently refractory to treatment as a result of inherent or hypoxia-mediated radioresistance.

A Drosera-bioinspired hydrogel for catching and killing cancer cells

PubMed Central

Li, Shihui; Chen, Niancao; Gaddes, Erin R.; Zhang, Xiaolong; Dong, Cheng; Wang, Yong

2015-01-01

A variety of bioinspired materials have been successfully synthesized to mimic the sophisticated structures or functions of biological systems. However, it is still challenging to develop materials with multiple functions that can be performed synergistically or sequentially. The purpose of this work was to demonstrate a novel bioinspired hydrogel that can interact with cancer cells, functionally similar to Drosera in catching and killing prey. This hydrogel had two layers with the top one functionalized with oligonucleotide aptamers and the bottom one functionalized with double-stranded DNA. The results show that the top hydrogel layer was able to catch target cells with high efficiency and specificity, and that the bottom hydrogel layer could sequester doxorubicin (Dox) for sustained drug release. Importantly, the released Dox could kill 90% of the cells after 1-h residence of the cells on the hydrogel. After the cell release, this bifunctional hydrogel could be regenerated for continuous cell catching and killing. Therefore, the data presented in this study has successfully demonstrated the potential of developing a material system with the functions of attracting, catching and killing diseased cells (e.g., circulating tumor cells) or even invading microorganisms (e.g., bacteria). PMID:26396063

Glycocalyx Engineering Reveals a Siglec-Based Mechanism for NK Cell Immunoevasion

PubMed Central

Hudak, Jason E.; Canham, Stephen M.; Bertozzi, Carolyn R.

2013-01-01

The increase of cell surface sialic acid is a characteristic shared by many tumor types. A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here, we show that increasing sialylated glycans on cancer cells inhibits human NK cell activation through the recruitment of Siglec-7. Key to these findings was the use of glycopolymers end-functionalized with phospholipids, which enable the introduction of synthetically defined glycans onto cancer cell surfaces. Remodeling the sialylation status of cancer cells affected the susceptibility to NK cell cytotoxicity via Siglec-7 engagement in a variety of tumor types. These results support a model in which hypersialylation offers a selective advantage to tumor cells under pressure from NK immunosurveillance by increasing Siglec ligands. We also exploited this finding to protect allogeneic and xenogeneic primary cells from NK-mediated killing suggesting the potential of Siglecs as therapeutic targets in cell transplant therapy. PMID:24292068

Radiation-induced equilibrium is a balance between tumor cell proliferation and T cell-mediated killing

PubMed Central

Liang, Hua; Deng, Liufu; Chmura, Steven; Burnette, Byron; Liadis, Nicole; Darga, Thomas; Beckett, Michael A.; Lingen, Mark W.; Witt, MaryEllyn; Weichselbaum, Ralph R.; Fu, Yang-Xin

2013-01-01

Local failures following radiation therapy are multifactorial and the contributions of the tumor and the host are complex. Current models of tumor equilibrium suggest that a balance exists between cell birth and cell death due to insufficient angiogenesis, immune effects, or intrinsic cellular factors. We investigated whether host immune responses contribute to radiation induced tumor equilibrium in animal models. We report an essential role for immune cells and their cytokines in suppressing tumor cell regrowth in two experimental animal model systems. Depletion of T cells or neutralization of interferon-gamma reversed radiation-induced equilibrium leading to tumor regrowth. We also demonstrate that PD-L1 blockade augments T cell responses leading to rejection of tumors in radiation induced equilibrium. We identify an active interplay between tumor cells and immune cells that occurs in radiation-induced tumor equilibrium and suggest a potential role for disruption of the PD-L1/PD-1 axis in increasing local tumor control. PMID:23630355

Dynamic visualization the whole process of cytotoxic T lymphocytes killing the B16 tumor cells in vitro

NASA Astrophysics Data System (ADS)

Qi, Shuhong; Zhang, Zhihong

2016-03-01

Cytotoxic T lymphocytes (CTLs) played a key role in the immune system to destroy the tumor cells. Although some mechanisms of CTLs killing the tumor cells are revealed already, the dynamic information of CTLs interaction with tumor cells are still not known very clearly. Here we used confocal microscopy to visualize the whole process of CTLs killing the tumor cells in vitro. The imaging data showed that CTLs destroyed the target tumor cells rapidly and efficiently. Several CTLs surrounded one or some tumor cells and the average time for CTLs destroying one tumor cell is just a few minutes in vitro. The study displayed the temporal events of CTLs interacting with tumor cells at the beginning and finally killing them and directly presented the efficient tumor cell cytotoxicity of the CTLs. The results helped us to deeply understand the mechanism of the CTLs destroying the tumor cells and to develop the cancer immunotherapy.

Inhibition by sulfonamides of the candidacidal activity of human neutrophils.

PubMed

Lehrer, R I

1971-12-01

Sulfonamides reduced substantially the ability of normal human neutrophils to kill strains of Candida albicans and Candida tropicalis, and impaired to a lesser extent their activity against Staphylococcus aureus 502A and Serratia marcescens. Sulfonamides also inhibited (a) iodination of Candida cells by normal neutrophils; (b) candidacidal activity in cell-free systems containing purified human myeloperoxidase, hydrogen peroxide, and potassium iodide; and (c) accumulation of molecular iodine in analogously constructed cell-free systems. In contrast to these effects on reactions catalyzed by myeloperoxidase, sulfonamides exerted relatively little effect on the levels of microbicidal activity manifested by human neutrophils that lacked myeloperoxidase. Sulfonamides appear to influence the function of human neutrophils predominantly by interfering with myeloperoxidase-mediated pathways. Certain basic and clinical implications of these data are discussed.

A comparison of the cytological effects of three hypoxic cell radiosensitizers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spunberg, J.J.; Geard, C.R.; Rutledge-Freeman, M.H.

1982-07-01

Misonidazole has entered Phase III clinical trials as a hypoxic cell radiosensitizer. Neurotoxocity is the major dose-limiting factor and has prompted the development of two further compounds with reduced lipophilicity and shorter half-life in vivo. Aside from the short-term problem of neurotoxicity, other potential long-term consequences should be considered. Such is the purpose of this investigation where the cytological effects of three radiosensitizers upon oxic and hypoxic Chinese hamster V-79 cells have been examined. Two newer compounds, desmethylmisonidazole and Stanford Research compound 2508, were compared with their clinically used predecessor misonidazole. Under aerated conditions, cell killing was increased with SR-2508more » in a concentration and time dependent manner, so as to exceed by more than three times the level produced by the other two drugs at 5 mM for 72 hours.Cell progression into mitosis was also markedly reduced by as much as 1/10,000 of control values. However, as the three compounds induced similar frequencies of sister chromatid exchange (SCE) and chromosome aberration, the enhanced cytotoxic effect of SR-2508 appears to be mediated via an interphase rather than a post-mitotic cell death. Cells were made hypoxic and treated with the three drugs for 4 hr, then mitoses sequentially collected for 16 hr. The three compounds produced similar levels of cell killing, slowing of cell cycle progression, SCE's and chromosome aberrations, with cycle-specific effect on S and G-I phase cells for SCE induction. These results indicate that desmethylmisonidazole and misonidazole have similar cytotoxic and clastogenic properties under oxic and hypoxic conditions. SR-2508 is relatively more toxic to aerated cells and may deserve close clinical observation for toxicity to normal tissues.« less

Interleukin-32 induces the differentiation of monocytes into macrophage-like cells.

PubMed

Netea, Mihai G; Lewis, Eli C; Azam, Tania; Joosten, Leo A B; Jaekal, Jun; Bae, Su-Young; Dinarello, Charles A; Kim, Soo-Hyun

2008-03-04

After emigration from the bone marrow to the peripheral blood, monocytes enter tissues and differentiate into macrophages, the prototype scavenger of the immune system. By ingesting and killing microorganisms and removing cellular debris, macrophages also process antigens as a first step in mounting a specific immune response. IL-32 is a cytokine inducing proinflammatory cytokines and chemokines via p38-MAPK and NF-kappaB. In the present study, we demonstrate that IL-32 induces differentiation of human blood monocytes as well as THP-1 leukemic cells into macrophage-like cells with functional phagocytic activity for live bacteria. Muramyl dipepide (MDP), the ligand for the intracellular nuclear oligomerization domain (NOD) 2 receptor, has no effect on differentiation alone but augments the monocyte-to-macrophage differentiation by IL-32. Unexpectedly, IL-32 reversed GM-CSF/IL-4-induced dendritic cell differentiation to macrophage-like cells. Whereas the induction of TNFalpha, IL-1beta, and IL-6 by IL-32 is mediated by p38-MAPK, IL-32-induced monocyte-to-macrophage differentiation is mediated through nonapoptotic, caspase-3-dependent mechanisms. Thus, IL-32 not only contributes to host responses through the induction of proinflammatory cytokines but also directly affects specific immunity by differentiating monocytes into macrophage-like cells.

Size-Dependent Effects of Gold Nanoparticles Uptake on Maturation and Antitumor Functions of Human Dendritic Cells In Vitro

PubMed Central

Tomić, Sergej; Đokić, Jelena; Vasilijić, Saša; Ogrinc, Nina; Rudolf, Rebeka; Pelicon, Primož; Vučević, Dragana; Milosavljević, Petar; Janković, Srđa; Anžel, Ivan; Rajković, Jelena; Rupnik, Marjan Slak; Friedrich, Bernd; Čolić, Miodrag

2014-01-01

Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and photo-thermal therapy, but without enough evidence on their potentially adverse immunological effects. Using a model of human dendritic cells (DCs), we showed that 10 nm- and 50 nm-sized GNPs (GNP10 and GNP50, respectively) were internalized predominantly via dynamin-dependent mechanisms, and they both impaired LPS-induced maturation and allostimulatory capacity of DCs, although the effect of GNP10 was more prominent. However, GNP10 inhibited LPS-induced production of IL-12p70 by DCs, and potentiated their Th2 polarization capacity, while GNP50 promoted Th17 polarization. Such effects of GNP10 correlated with a stronger inhibition of LPS-induced changes in Ca2+ oscillations, their higher number per DC, and more frequent extra-endosomal localization, as judged by live-cell imaging, proton, and electron microscopy, respectively. Even when released from heat-killed necrotic HEp-2 cells, GNP10 inhibited the necrotic tumor cell-induced maturation and functions of DCs, potentiated their Th2/Th17 polarization capacity, and thus, impaired the DCs' capacity to induce T cell-mediated anti-tumor cytotoxicity in vitro. Therefore, GNP10 could potentially induce more adverse DC-mediated immunological effects, compared to GNP50. PMID:24802102

Single-hit mechanism of tumour cell killing by radiation.

PubMed

Chapman, J D

2003-02-01

To review the relative importance of the single-hit mechanism of radiation killing for tumour response to 1.8-2.0 Gy day(-1) fractions and to low dose-rate brachytherapy. Tumour cell killing by ionizing radiation is well described by the linear-quadratic equation that contains two independent components distinguished by dose kinetics. Analyses of tumour cell survival curves that contain six or more dose points usually provide good estimates of the alpha- and beta-inactivation coefficients. Superior estimates of tumour cell intrinsic radiosensitivity are obtained when synchronized populations are employed. The characteristics of single-hit inactivation of tumour cells are reviewed and compared with the characteristics of beta-inactivation. Potential molecular targets associated with single-hit inactivation are discussed along with strategies for potentiating cell killing by this mechanism. The single-hit mechanism of tumour cell killing shows no dependence on dose-rate and, consequently, no evidence of sublethal damage repair. It is uniquely potentiated by high linear-energy-transfer radiation, exhibits a smaller oxygen enhancement ratio and exhibits a larger indirect effect by hydroxyl radicals than the beta-mechanism. alpha-inactivation coefficients vary slightly throughout interphase but mitotic cells exhibit extremely high alpha-coefficients in the range of those observed for lymphocytes and some repair-deficient cells. Evidence is accumulating to suggest that chromatin in compacted form could be a radiation-hypersensitive target associated with single-hit radiation killing. Analyses of tumour cell survival curves demonstrate that it is the single-hit mechanism (alpha) that determines the majority of cell killing after doses of 2Gy and that this mechanism is highly variable between tumour cell lines. The characteristics of single-hit inactivation are qualitatively and quantitatively distinct from those of beta-inactivation. Compacted chromatin in tumour cells should be further investigated as a radiation-hypersensitive target that could be modulated for therapeutic advantage.

STAT3 inhibition as a therapeutic strategy for leukemia.

PubMed

Kanna, Rubashruti; Choudhary, Gaurav; Ramachandra, Nandini; Steidl, Ulrich; Verma, Amit; Shastri, Aditi

2017-11-22

Leukemia is characterized by selective overgrowth of malignant hematopoietic stem cells (HSC's) that interfere with HSC differentiation. Cytoreductive chemotherapy can kill rapidly dividing cancerous cells but cannot eradicate the malignant HSC pool leading to relapses. Leukemic stem cells have several dysregulated pathways and the Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) pathway are prominent among them. STAT3 is an important transcription factor that regulates cell growth, proliferation, and inhibits apoptosis. High STAT3 expression in leukemia has been associated with an increased risk for relapse and decreased overall survival. Multiple strategies for interfering with STAT3 activity in leukemic cells include inhibition of STAT3 phosphorylation, interfering with STAT3 interactions, preventing nuclear transfer, inhibiting transcription and causing interference in STAT: DNA binding. A better understanding of key interactions and upstream mediators of STAT3 activity will help facilitate the development of effective cancer therapies and may result in durable remissions.

Direct imaging of macrophage activation during PDT treatment

NASA Astrophysics Data System (ADS)

Song, Sheng; Zhou, Feifan; Chen, Wei R.; Xing, Da

2012-03-01

Mounting evidence describes a more complex progress of macrophage activation during photodynamic therapy (PDT), which performing distinct immunological functions and different physiologies on surrounding cells and tissues. Macrophage-targeted PDT has been applied in the selective killing of cells involved in inflammation and tumor. We have previously shown that PDT-mediated tumor cells apoptosis can induce a higher level immune response than necrosis, and enhance the macrophage activation. However, the molecular mechanism of macrophage activation during PDT-induced apoptotic cells (AC) still unclear. Here, we use confocal microscopy to image the phagocytosis of tumor cells by macrophages. We also observed that PDT-treated AC can activate Toll-like receptors (TLRs) which are present on macrophages surface. Besides, the increase in nitric oxide (NO) formation in macrophages was detected in real time by a laser scanning microscopy. This study provided more details for understanding the molecular mechanism of the immune response induced by PDT-treated AC.

Generation of B-cell chronic lymphocytic leukemia (B-CLL)-reactive T-cell lines and clones from HLA class I-matched donors using modified B-CLL cells as stimulators: implications for adoptive immunotherapy.

PubMed

Hoogendoorn, M; Wolbers, J Olde; Smit, W M; Schaafsma, M R; Barge, R M Y; Willemze, R; Falkenburg, J H F

2004-07-01

Allogeneic stem cell transplantation following reduced-intensity conditioning is being evaluated in patients with advanced B-cell chronic lymphocytic leukemia (B-CLL). The curative potential of this procedure is mediated by donor-derived alloreactive T cells, resulting in a graft-versus-leukemia effect. However, B-CLL may escape T-cell-mediated immune reactivity since these cells lack expression of costimulatory molecules. We examined the most optimal method to transform B-CLL cells into efficient antigen-presenting cells (APC) using activating cytokines, by triggering toll-like receptors (TLRs) using microbial pathogens and by CD40 stimulation with CD40L-transfected fibroblasts. CD40 activation in the presence of IL-4 induced strongest upregulation of costimulatory and adhesion molecules on B-CLL cells and induced the production of high amounts of IL-12 by the leukemic cells. In contrast to primary B-CLL cells as stimulator cells, these malignant APCs were capable of inducing the generation of B-CLL-reactive CD8(+) CTL lines and clones from HLA class I-matched donors. These CTL lines and clones recognized and killed primary B-CLL as well as patient-derived lymphoblasts, but not donor cells. These results show the feasibility of ex vivo generation of B-CLL-reactive CD8(+) CTLs. This opens new perspectives for adoptive immunotherapy, following allogeneic stem cell transplantation in patients with advanced B-CLL.

Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors1

PubMed Central

Bruckheimer, Elizabeth M; Fazenbaker, Christine A; Gallagher, Sandra; Mulgrew, Kathy; Fuhrmann, Stacy; Coffman, Karen T; Walsh, William; Ready, Shannon; Cook, Kim; Damschroder, Melissa; Kinch, Michael; Kiener, Peter A; Woods, Rob; Gao, Changshou; Dall'Acqua, William; Wu, Herren; Coats, Steven

2009-01-01

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated ∼80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells. PMID:19484140

IgG4 subclass antibodies impair antitumor immunity in melanoma

PubMed Central

Karagiannis, Panagiotis; Gilbert, Amy E.; Josephs, Debra H.; Ali, Niwa; Dodev, Tihomir; Saul, Louise; Correa, Isabel; Roberts, Luke; Beddowes, Emma; Koers, Alexander; Hobbs, Carl; Ferreira, Silvia; Geh, Jenny L.C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Blower, Philip J.; Mitchell, Tracey; Fear, David J.; Spicer, James F.; Lacy, Katie E.; Nestle, Frank O.; Karagiannis, Sophia N.

2013-01-01

Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10–driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4+-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell–mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches. PMID:23454746

G protein-coupled estrogen receptor mediates the up-regulation of fatty acid synthase induced by 17β-estradiol in cancer cells and cancer-associated fibroblasts.

PubMed

Santolla, Maria Francesca; Lappano, Rosamaria; De Marco, Paola; Pupo, Marco; Vivacqua, Adele; Sisci, Diego; Abonante, Sergio; Iacopetta, Domenico; Cappello, Anna Rita; Dolce, Vincenza; Maggiolini, Marcello

2012-12-21

Activation of lipid metabolism is an early event in carcinogenesis and a central hallmark of many tumors. Fatty acid synthase (FASN) is a key lipogenic enzyme catalyzing the terminal steps in the de novo biogenesis of fatty acids. In cancer cells, FASN may act as a metabolic oncogene, given that it confers growth and survival advantages to these cells, whereas its inhibition effectively and selectively kills tumor cells. Hormones such as estrogens and growth factors contribute to the transcriptional regulation of FASN expression also through the activation of downstream signaling and a cross-talk among diverse transduction pathways. In this study, we demonstrate for the first time that 17β-estradiol (E2) and the selective GPER ligand G-1 regulate FASN expression and activity through the GPER-mediated signaling, which involved the EGF receptor/ERK/c-Fos/AP1 transduction pathway, as ascertained by using specific pharmacological inhibitors, performing gene-silencing experiments and ChIP assays in breast SkBr3, colorectal LoVo, hepatocarcinoma HepG2 cancer cells, and breast cancer-associated fibroblasts. In addition, the proliferative effects induced by E2 and G-1 in these cells involved FASN as the inhibitor of its activity, named cerulenin, abolished the growth response to both ligands. Our data suggest that GPER may be included among the transduction mediators involved by estrogens in regulating FASN expression and activity in cancer cells and cancer-associated fibroblasts that strongly contribute to cancer progression.

Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta

PubMed Central

Sharma, Ricky A.

2012-01-01

DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt’s lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control. PMID:22822095

Recent Advances in Allograft Vasculopathy

PubMed Central

Merola, Jonathan; Jane-Wit, Daniel D.; Pober, Jordan S.

2017-01-01

Purpose of review Despite considerable advances in controlling acute rejection, the longevity of cardiac and renal allografts remains significantly limited by chronic rejection in the form of allograft vasculopathy (AV). This review discusses recently reported mechanistic insights of AV pathogenesis as well recent clinical evaluations of new therapeutic approaches. Recent findings Although adaptive immunity is the major driver of AV, natural killer cells mediate vasculopathic changes in a transplanted mouse heart following treatment with donor-specific antibody (DSA). However, NK cells may also dampen chronic inflammatory responses by killing donor-derived tissue-resident CD4 T cells that provide help to host B cells, the source of DSA. DSA may directly contribute to vascular inflammation by inducing intracellular signaling cascades that upregulate leukocyte adhesion molecules, facilitating recruitment of neutrophils and monocytes. DSA-mediated complement activation additionally enhances endothelial alloimmunogenicity through activation of non-canonical NF-κB signaling. New clinical studies evaluating mTOR and proteasome inhibitors to target these pathways have been reported. Summary AV is a pathology resultant from several innate and adaptive alloimmune responses. Mechanistic insights from preclinical studies have identified agents that are currently being investigated in clinical trials. PMID:27898462

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor.

PubMed

Gilmore, Andrew P; Valentijn, Anthony J; Wang, Pengbo; Ranger, Ann M; Bundred, Nigel; O'Hare, Michael J; Wakeling, Alan; Korsmeyer, Stanley J; Streuli, Charles H

2002-08-02

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.

Doxorubicin-loaded magnetic nanoparticle clusters for chemo-photothermal treatment of the prostate cancer cell line PC3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Weibing; Zheng, Xinmin; Shen, Shun

2015-10-16

In addition to the conventional cancer treatment such as radiotherapy, chemotherapy and surgical management, nanomedicine-based approaches have attracted widespread attention in recent years. In this paper, a promising nanocarrier, magnetic nanoparticle clusters (MNCs) as porous materials which provided enough room on the surface, was developed for loading chemotherapeutic agent of doxorubicin (DOX). Moreover, MNCs are a good near-infrared (NIR) photothermal mediator. Thus, MNCs have great potential both in photothermal therapy (PTT) and drug delivery for chemo-photothermal therapy of cancer. We firstly explored the destruction of prostate cancer in vitro by the combination of PTT and chemotherapy using DOX@MNCs. Upon NIR irradiationmore » at 808 nm, more cancer cells were killed when PC3 cells incubated with DOX@MNCs, owing to both MNCs-mediated photothermal ablation and cytotoxicity of light-triggered DOX release. Compared with PTT or chemotherapy alone, the chemo-photothermal therapy by DOX@MNCs showed a synergistically higher therapeutic efficacy. - Highlights: • MNCs have great potential both in photothermal therapy and drug delivery. • DOX@MNCs were used for chemo-photothermal therapy of prostate cancer cells. • DOX@MNCs showed a synergistically higher therapeutic efficacy.« less

Leukocyte function-associated antigen-1-dependent lysis of Fas+ (CD95+/Apo-1+) innocent bystanders by antigen-specific CD8+ CTL.

PubMed

Kojima, H; Eshima, K; Takayama, H; Sitkovsky, M V

1997-09-15

Exquisite specificity toward Ag-bearing cells (cognate targets) is one of the most important properties of CD8+ CTL-mediated cytotoxicity. Using highly Ag-specific CD8+ CTL lines and clones, which spare noncognate, Ag-free targets, we found that in the presence of Ag-bearing targets the CTL acquire the ability to lyse noncognate target cells (bystanders). It is shown that the unexpectedly rapid and efficient lysis of bystanders by Ag-activated CTL is mediated by a Fas ligand (FasL)/Fas-based mechanism and does not depend on perforin. The CTL lysed Fas-expressing bystanders, but spared the Fas-negative or anti-Fas mAb-resistant bystander cells. Accordingly, the FasL-deficient gld/gld CTL did not kill bystanders, while perforin-deficient CTL did. Unlike anti-Fas mAb-induced cell death, the lysis of bystanders was not only FasL/Fas dependent but also required adhesion molecule LFA-1 on the surface of the activated CTL. Lysis of bystanders is viewed as acceptable "collateral" damage, but the persistent presence of activated CTL could result in immunopathologies involving functional Fas-expressing tissues.

Photodynamic therapy with fullerenes in vivo: reality or a dream?

PubMed

Sharma, Sulbha K; Chiang, Long Y; Hamblin, Michael R

2011-12-01

Photodynamic therapy (PDT) employs the combination of nontoxic photosensitizers and visible light that is absorbed by the chromophore to produce long-lived triplet states that can carry out photochemistry in the presence of oxygen to kill cells. The closed carbon-cage structure found in fullerenes can act as a photosensitizer, especially when functionalized to impart water solubility. Although there are reports of the use of fullerenes to carry out light-mediated destruction of viruses, microorganisms and cancer cells in vitro, the use of fullerenes to mediate PDT of diseases such as cancer and infections in animal models is less well developed. It has recently been shown that fullerene PDT can be used to save the life of mice with wounds infected with pathogenic Gram-negative bacteria. Fullerene PDT has also been used to treat mouse models of various cancers including disseminated metastatic cancer in the peritoneal cavity. In vivo PDT with fullerenes represents a new application in nanomedicine.

Curcumin analog cytotoxicity against breast cancer cells: exploitation of a redox-dependent mechanism.

PubMed

Sun, Aiming; Lu, Yang J; Hu, Haipeng; Shoji, Mamoru; Liotta, Dennis C; Snyder, James P

2009-12-01

A series of novel curcumin analogs, symmetrical dienones, were previously shown to possess cytotoxic, anti-angiogenic and anti-tumor activities. Analogs 1 (EF24) and 2 (EF31) share the dienone scaffold and serve as Michael acceptors. We propose that the anti-cancer effects of 1 and 2 are mediated in part by redox-mediated induction of apoptosis. In order to support this concept, 1 and 2 were treated with L-glutathione (GSH) and cysteine-containing dipeptides under mild conditions to form colorless water-soluble adducts, which were identified by LC/MS. Comparison of the cytotoxic action of 1, 2 and the corresponding conjugates, 1-(GSH)(2) and 2-(GSH)(2), illustrated that the two classes of compounds exhibit essentially identical cell killing capabilities. Compared with the yellow, somewhat light sensitive and nearly water insoluble compounds 1 and 2, the glutathione conjugates represent a promising new series of stable and soluble anti-tumor pro-drugs.

RITA inhibits multiple myeloma cell growth through induction of p53-mediated caspase-dependent apoptosis and synergistically enhances nutlin-induced cytotoxic responses.

PubMed

Saha, Manujendra N; Jiang, Hua; Mukai, Asuka; Chang, Hong

2010-11-01

Mutations or deletions of p53 are relatively rare in multiple myeloma (MM), at least in newly diagnosed patients. Thus, restoration of p53 tumor suppressor function in MM by blocking the inhibitory role of murine double minute 2 (MDM2) is a promising and applicable therapeutic strategy. RITA and nutlin are two new classes of small molecule MDM2 inhibitors that prevent the p53-MDM2 interaction. Earlier reports showed p53-dependent activity of RITA in solid tumors as well as in leukemias. We and others recently described nutlin-induced apoptosis in MM cells, but it remains unclear whether RITA exerts antimyeloma activity. Here, we found that RITA activates the p53 pathway and induces apoptosis in MM cell lines and primary MM samples, preferentially killing myeloma cells. The activation of p53 induced by RITA was mediated through modulation of multiple apoptotic regulatory proteins, including upregulation of a proapoptotic protein (NOXA), downregulation of an antiapoptotic protein, Mcl-1, and activation of caspases through extrinsic pathways. Moreover, a number of key p53-mediated apoptotic target genes were identified by gene expression profiling and further validated by quantitative real-time PCR. Importantly, the combination of RITA with nutlin displayed a strong synergism on growth inhibition with the combination index ranging from 0.56 to 0.82 in MM cells. Our data support further clinical evaluation of RITA as a potential novel therapeutic intervention in MM. ©2010 AACR.

Identification of the lipopolysaccharide modifications controlled by the Salmonella PmrA/PmrB system mediating resistance to Fe(III) and Al(III)

PubMed Central

Nishino, Kunihiko; Hsu, Fong-Fu; Turk, John; Cromie, Michael J; Wösten, Marc M S M; Groisman, Eduardo A

2006-01-01

Iron is an essential metal but can be toxic in excess. While several homeostatic mechanisms prevent oxygen-dependent killing promoted by Fe(II), little is known about how cells cope with Fe(III), which kills by oxygen-independent means. Several Gram-negative bacterial species harbour a regulatory system – termed PmrA/PmrB – that is activated by and required for resistance to Fe(III). We now report the identification of the PmrA-regulated determinants mediating resistance to Fe(III) and Al(III) in Salmonella enterica serovar Typhimurium. We establish that these determinants remodel two regions of the lipopolysaccharide, decreasing the negative charge of this major constituent of the outer membrane. Remodelling entails the covalent modification of the two phosphates in the lipid A region with phosphoethanolamine and 4-aminoarabinose, which has been previously implicated in resistance to polymyxin B, as well as dephosphorylation of the Hep(II) phosphate in the core region by the PmrG protein. A mutant lacking the PmrA-regulated Fe(III) resistance genes bound more Fe(III) than the wild-type strain and was defective for survival in soil, suggesting that these PmrA-regulated lipopolysaccharide modifications aid Salmonella's survival and spread in non-host environments. PMID:16803591

Mechanisms of Dendritic Cell Lysosomal Killing of Cryptococcus

NASA Astrophysics Data System (ADS)

Hole, Camaron R.; Bui, Hoang; Wormley, Floyd L.; Wozniak, Karen L.

2012-10-01

Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that disseminates to the CNS causing fatal meningitis in immunocompromised patients. Dendritic cells (DCs) phagocytose C. neoformans following inhalation. Following uptake, cryptococci translocate to the DC lysosomal compartment and are killed by oxidative and non-oxidative mechanisms. DC lysosomal extracts kill cryptococci in vitro; however, the means of antifungal activity remain unknown. Our studies determined non-oxidative antifungal activity by DC lysosomal extract. We examined DC lysosomal killing of cryptococcal strains, anti-fungal activity of purified lysosomal enzymes, and mechanisms of killing against C. neoformans. Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes. Purified lysosomal enzymes, specifically cathepsin B, inhibited cryptococcal growth. Interestingly, cathepsin B combined with its enzymatic inhibitors led to enhanced cryptococcal killing. Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment. Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death.

Effects of Surotomycin on Clostridium difficile Viability and Toxin Production In Vitro

PubMed Central

Bouillaut, Laurent; McBride, Shonna; Schmidt, Diane J.; Suarez, José M.; Tzipori, Saul; Mascio, Carmela; Chesnel, Laurent

2015-01-01

The increasing incidence and severity of infection by Clostridium difficile have stimulated attempts to develop new antimicrobial therapies. We report here the relative abilities of two antibiotics (metronidazole and vancomycin) in current use for treating C. difficile infection and of a third antimicrobial, surotomycin, to kill C. difficile cells at various stages of development and to inhibit the production of the toxin proteins that are the major virulence factors. The results indicate that none of the drugs affects the viability of spores at 8× MIC or 80× MIC and that all of the drugs kill exponential-phase cells when provided at 8× MIC. In contrast, none of the drugs killed stationary-phase cells or inhibited toxin production when provided at 8× MIC and neither vancomycin nor metronidazole killed stationary-phase cells when provided at 80× MIC. Surotomycin, on the other hand, did kill stationary-phase cells when provided at 80× MIC but did so without inducing lysis. PMID:25941230

Bacterial exopolysaccharide based magnetic nanoparticles: a versatile nanotool for cancer cell imaging, targeted drug delivery and synergistic effect of drug and hyperthermia mediated cancer therapy.

PubMed

Sivakumar, Balasubramanian; Aswathy, Ravindran Girija; Sreejith, Raveendran; Nagaoka, Yutaka; Iwai, Seiki; Suzuki, Masashi; Fukuda, Takahiro; Hasumura, Takashi; Yoshida, Yasuhiko; Maekawa, Toru; Sakthikumar, Dasappan Nair

2014-06-01

Microbial exopolysaccharides (EPSs) are highly heterogeneous polymers produced by fungi and bacteria that have garnered considerable attention and have remarkable potential in various fields, including biomedical research. The necessity of biocompatible materials to coat and stabilize nanoparticles is highly recommended for successful application of the same in biomedical regime. In our study we have coated magnetic nanoparticles (MNPs) with two bacterial EPS-mauran (MR) and gellan gum (GG). The biocompatibility of EPS coated MNPs was enhanced and we have made it multifunctional by attaching targeting moiety, folate and with encapsulation of a potent anticancerous drug, 5FU. We have conjugated an imaging moiety along with nanocomposite to study the effective uptake of nanoparticles. It was also observed that the dye labeled folate targeted nanoparticles could effectively enter into cancer cells and the fate of nanoparticles was tracked with Lysotracker. The biocompatibility of EPS coated MNPs and synergistic effect of magnetic hyperthermia and drug for enhanced antiproliferation of cancer cells was also evaluated. More than 80% of cancer cells was killed within a period of 60 min when magnetic hyperthermia (MHT) was applied along with drug loaded EPS coated MNPs, thus signifying the combined effect of drug loaded MNPs and MHT. Our results suggests that MR and GG coated MNPs exhibited excellent biocompatibility with low cell cytotoxicity, high therapeutic potential, and superparamagnetic behavior that can be employed as prospective candidates for bacterial EPS based targeted drug delivery, cancer cell imaging and for MHT for killing cancer cells within short period of time.

Penile cancer

MedlinePlus

Cancer - penis; Squamous cell cancer - penis; Glansectomy; Partial penectomy ... cancer may include: Chemotherapy -- uses medicines to kill cancer cells Radiation -- uses high-powered x-rays to kill ...

9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Feline Rhinotracheitis Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus. Feline Rhinotracheitis Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed...

9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Feline Rhinotracheitis Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus. Feline Rhinotracheitis Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed...

9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Feline Rhinotracheitis Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus. Feline Rhinotracheitis Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed...

9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Feline Rhinotracheitis Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus. Feline Rhinotracheitis Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed...

9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Feline Rhinotracheitis Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus. Feline Rhinotracheitis Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed...

TOFA (5-tetradecyl-oxy-2-furoic acid) reduces fatty acid synthesis, inhibits expression of AR, neuropilin-1 and Mcl-1 and kills prostate cancer cells independent of p53 status.

PubMed

Guseva, Natalya V; Rokhlin, Oskar W; Glover, Rebecca A; Cohen, Michael B

2011-07-01

A key player in prostate cancer development and progression is the androgen receptor (AR). Tumor-associated lipogenesis can protect cancer cells from carcinogenic- and therapeutic-associated treatments. Increased synthesis of fatty acids and cholesterol is regulated by androgens through induction of several genes in androgen-responsive cancer cells. Acetyl-CoA-carboxylase-α (ACCA) is a key enzyme in the regulation of fatty acids synthesis. Here we show that AR binds in vivo to intron regions of human ACCA gene. We also show that the level of ACCA protein in LNCaP depends on AR expression and that DHT treatment increases ACCA expression and fatty acid synthesis. Inhibition of ACCA by TOFA (5-tetradecyl-oxy-2-furoic acid) decreases fatty acid synthesis and induces caspase activation and cell death in most PCa cell lines. Our data suggest that TOFA can kill cells via the mitochondrial pathway since we found cytochrome c release after TOFA treatment in androgen sensitive cell lines. The results also imply that the pro-apoptotic effect of TOFA may be mediated via a decrease of neuropilin-1(NRP1) and Mcl-1expression. We have previously reported that Mcl-1 is under AR regulation and plays an important role in resistance to drug-induced apoptosis in prostate cancer cells, and NRP1 is known to regulate Mcl-1 expression. Here, we show for the first time that NRP1 expression is under AR control. Taken together, our data suggest that TOFA is a potent cell death inducing agent in prostate cancer cells.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

PubMed

Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

2014-09-01

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

HematoPorphyrin Monomethyl Ether polymer contrast agent for ultrasound/photoacoustic dual-modality imaging-guided synergistic high intensity focused ultrasound (HIFU) therapy

NASA Astrophysics Data System (ADS)

Yan, Sijing; Lu, Min; Ding, Xiaoya; Chen, Fei; He, Xuemei; Xu, Chunyan; Zhou, Hang; Wang, Qi; Hao, Lan; Zou, Jianzhong

2016-08-01

This study is to prepare a hematoporphyrin monomethyl ether (HMME)-loaded poly(lactic-co-glycolic acid) (PLGA) microcapsules (HMME/PLGA), which could not only function as efficient contrast agent for ultrasound (US)/photoacoustic (PA) imaging, but also as a synergistic agent for high intensity focused ultrasound (HIFU) ablation. Sonosensitizer HMME nanoparticles were integrated into PLGA microcapsules with the double emulsion evaporation method. After characterization, the cell-killing and cell proliferation-inhibiting effects of HMME/PLGA microcapsules on ovarian cancer SKOV3 cells were assessed. The US/PA imaging-enhancing effects and synergistic effects on HIFU were evaluated both in vitro and in vivo. HMME/PLGA microcapsules were highly dispersed with well-defined spherical morphology (357 ± 0.72 nm in diameter, PDI = 0.932). Encapsulation efficiency and drug-loading efficiency were 58.33 ± 0.95% and 4.73 ± 0.15%, respectively. The HMME/PLGA microcapsules remarkably killed the SKOV3 cells and inhibited the cell proliferation, significantly enhanced the US/PA imaging results and greatly enhanced the HIFU ablation effects on ovarian cancer in nude mice by the HMME-mediated sono-dynamic chemistry therapy (SDT). HMME/PLGA microcapsules represent a potential multifunctional contrast agent for HIFU diagnosis and treatment, which might provide a novel strategy for the highly efficient imaging-guided non-invasive HIFU synergistic therapy for cancers by SDT in clinic.

HematoPorphyrin Monomethyl Ether polymer contrast agent for ultrasound/photoacoustic dual-modality imaging-guided synergistic high intensity focused ultrasound (HIFU) therapy

PubMed Central

Yan, Sijing; LU, Min; Ding, Xiaoya; Chen, Fei; He, Xuemei; Xu, Chunyan; Zhou, Hang; Wang, Qi; Hao, Lan; Zou, Jianzhong

2016-01-01

This study is to prepare a hematoporphyrin monomethyl ether (HMME)-loaded poly(lactic-co-glycolic acid) (PLGA) microcapsules (HMME/PLGA), which could not only function as efficient contrast agent for ultrasound (US)/photoacoustic (PA) imaging, but also as a synergistic agent for high intensity focused ultrasound (HIFU) ablation. Sonosensitizer HMME nanoparticles were integrated into PLGA microcapsules with the double emulsion evaporation method. After characterization, the cell-killing and cell proliferation-inhibiting effects of HMME/PLGA microcapsules on ovarian cancer SKOV3 cells were assessed. The US/PA imaging-enhancing effects and synergistic effects on HIFU were evaluated both in vitro and in vivo. HMME/PLGA microcapsules were highly dispersed with well-defined spherical morphology (357 ± 0.72 nm in diameter, PDI = 0.932). Encapsulation efficiency and drug-loading efficiency were 58.33 ± 0.95% and 4.73 ± 0.15%, respectively. The HMME/PLGA microcapsules remarkably killed the SKOV3 cells and inhibited the cell proliferation, significantly enhanced the US/PA imaging results and greatly enhanced the HIFU ablation effects on ovarian cancer in nude mice by the HMME-mediated sono-dynamic chemistry therapy (SDT). HMME/PLGA microcapsules represent a potential multifunctional contrast agent for HIFU diagnosis and treatment, which might provide a novel strategy for the highly efficient imaging-guided non-invasive HIFU synergistic therapy for cancers by SDT in clinic. PMID:27535093

Depletion of tyrosyl DNA phosphodiesterase 2 activity enhances etoposide-mediated double-strand break formation and cell killing.

PubMed

Kont, Yasemin Saygideger; Dutta, Arijit; Mallisetty, Apurva; Mathew, Jeena; Minas, Tsion; Kraus, Christina; Dhopeshwarkar, Priyanka; Kallakury, Bhaskar; Mitra, Sankar; Üren, Aykut; Adhikari, Sanjay

2016-07-01

DNA topoisomerase 2 (Top2) poisons, including common anticancer drugs etoposide and doxorubicin kill cancer cells by stabilizing covalent Top2-tyrosyl-DNA 5'-phosphodiester adducts and DNA double-strand breaks (DSBs). Proteolytic degradation of the covalently attached Top2 leaves a 5'-tyrosylated blocked termini which is removed by tyrosyl DNA phosphodiesterase 2 (TDP2), prior to DSB repair through non-homologous end joining (NHEJ). Thus, TDP2 confers resistance of tumor cells to Top2-poisons by repairing such covalent DNA-protein adducts, and its pharmacological inhibition could enhance the efficacy of Top2-poisons. We discovered NSC111041, a selective inhibitor of TDP2, by optimizing a high throughput screening (HTS) assay for TDP2's 5'-tyrosyl phosphodiesterase activity and subsequent validation studies. We found that NSC111041 inhibits TDP2's binding to DNA without getting intercalated into DNA and enhanced etoposide's cytotoxicity synergistically in TDP2-expressing cells but not in TDP2 depleted cells. Furthermore, NSC111041 enhanced formation of etoposide-induced γ-H2AX foci presumably by affecting DSB repair. Immuno-histochemical analysis showed higher TDP2 expression in a sub-set of different type of tumor tissues. These findings underscore the feasibility of clinical use of suitable TDP2 inhibitors in adjuvant therapy with Top2-poisons for a sub-set of cancer patients with high TDP2 expression. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

PubMed

Morita, Chisato; Sumioka, Ryuichi; Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg(2+) and Ca(2+) for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.

Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

PubMed Central

Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg2+ and Ca2+ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression. PMID:25084357

HematoPorphyrin Monomethyl Ether polymer contrast agent for ultrasound/photoacoustic dual-modality imaging-guided synergistic high intensity focused ultrasound (HIFU) therapy.

PubMed

Yan, Sijing; Lu, Min; Ding, Xiaoya; Chen, Fei; He, Xuemei; Xu, Chunyan; Zhou, Hang; Wang, Qi; Hao, Lan; Zou, Jianzhong

2016-08-18

This study is to prepare a hematoporphyrin monomethyl ether (HMME)-loaded poly(lactic-co-glycolic acid) (PLGA) microcapsules (HMME/PLGA), which could not only function as efficient contrast agent for ultrasound (US)/photoacoustic (PA) imaging, but also as a synergistic agent for high intensity focused ultrasound (HIFU) ablation. Sonosensitizer HMME nanoparticles were integrated into PLGA microcapsules with the double emulsion evaporation method. After characterization, the cell-killing and cell proliferation-inhibiting effects of HMME/PLGA microcapsules on ovarian cancer SKOV3 cells were assessed. The US/PA imaging-enhancing effects and synergistic effects on HIFU were evaluated both in vitro and in vivo. HMME/PLGA microcapsules were highly dispersed with well-defined spherical morphology (357 ± 0.72 nm in diameter, PDI = 0.932). Encapsulation efficiency and drug-loading efficiency were 58.33 ± 0.95% and 4.73 ± 0.15%, respectively. The HMME/PLGA microcapsules remarkably killed the SKOV3 cells and inhibited the cell proliferation, significantly enhanced the US/PA imaging results and greatly enhanced the HIFU ablation effects on ovarian cancer in nude mice by the HMME-mediated sono-dynamic chemistry therapy (SDT). HMME/PLGA microcapsules represent a potential multifunctional contrast agent for HIFU diagnosis and treatment, which might provide a novel strategy for the highly efficient imaging-guided non-invasive HIFU synergistic therapy for cancers by SDT in clinic.

Allogeneic killing by earthworm effector cells.

PubMed

Suzuki, M M; Cooper, E L

1995-01-01

We observed spontaneous allogeneic cytotoxicity by coelomocytes (Lumbricus terrestris) using three assays: trypan blue, lactate dehydrogenase release and chromium-51 release. Cell-cell contact may not be essential to effect cytotoxicity, since killing of allogeneic cells occurred in pooled allogeneic coelomic fluid derived from worms raised in two different geographic locales. We observed no significant spontaneous cytotoxicity against autogeneic target coelomocytes haptenated with 2,4,6-trinitrobenzene sulfonic acid; however, coelomocytes effected significant spontaneous cytotoxicity against haptenated allogeneic targets. These results support the view that earthworm coelomocytes can act as effector cells that can specifically kill nonself target cells.

Contact Killing of Bacteria on Copper Is Suppressed if Bacterial-Metal Contact Is Prevented and Is Induced on Iron by Copper Ions

PubMed Central

Mathews, Salima; Hans, Michael

2013-01-01

Bacteria are rapidly killed on copper surfaces, and copper ions released from the surface have been proposed to play a major role in the killing process. However, it has remained unclear whether contact of the bacteria with the copper surface is also an important factor. Using laser interference lithography, we engineered copper surfaces which were covered with a grid of an inert polymer which prevented contact of the bacteria with the surface. Using Enterococcus hirae as a model organism, we showed that the release of ionic copper from these modified surfaces was not significantly reduced. In contrast, killing of bacteria was strongly attenuated. When E. hirae cells were exposed to a solid iron surface, the loss of cell viability was the same as on glass. However, exposing cells to iron in the presence of 4 mM CuSO4 led to complete killing in 100 min. These experiments suggest that contact killing proceeds by a mechanism whereby the metal-bacterial contact damages the cell envelope, which, in turn, makes the cells susceptible to further damage by copper ions. PMID:23396344

Mesophilic Aeromonas sp. serogroup O:11 resistance to complement-mediated killing.

PubMed Central

Merino, S; Rubires, X; Aguilar, A; Albertí, S; Hernandez-Allés, S; Benedí, V J; Tomas, J M

1996-01-01

The complement activation by and resistance to complement-mediated killing of Aeromonas sp. strains from serogroup O:11 were investigated by using different wild-type strains (with an S-layer characteristic of this serogroup) and their isogenic mutants characterized for their surface components (S-layer and lipopolysaccharide [LPS]). All of the Aeromonas sp. serogroup O:11 wild-type strains are unable to activate complement, which suggested that the S-layer completely covered the LPS molecules. We found that the classical complement pathway is involved in serum killing of susceptible Aeromonas sp. mutant strains of serogroup O11, while the alternative complement pathway seems not to be involved, and that the complement activation seems to be independent of antibody. The smooth mutant strains devoid of the S-layer (S-layer isogenic mutants) or isogenic LPS mutant strains with a complete or rather complete LPS core (also without the S-layer) are able to activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b is rapidly degraded, and therefore the lytic membrane attack complex (C5b-9) is not formed. Isogenic LPS rough mutants with an incomplete LPS core are serum sensitive because they bind more C3b than the resistant strains, the C3b is not completely degraded, and therefore the lytic complex (C5b-9) is formed. PMID:8945581

ω-Hydroxyemodin limits staphylococcus aureus quorum sensing-mediated pathogenesis and inflammation.

PubMed

Daly, Seth M; Elmore, Bradley O; Kavanaugh, Jeffrey S; Triplett, Kathleen D; Figueroa, Mario; Raja, Huzefa A; El-Elimat, Tamam; Crosby, Heidi A; Femling, Jon K; Cech, Nadja B; Horswill, Alexander R; Oberlies, Nicholas H; Hall, Pamela R

2015-04-01

Antibiotic-resistant pathogens are a global health threat. Small molecules that inhibit bacterial virulence have been suggested as alternatives or adjuncts to conventional antibiotics, as they may limit pathogenesis and increase bacterial susceptibility to host killing. Staphylococcus aureus is a major cause of invasive skin and soft tissue infections (SSTIs) in both the hospital and community settings, and it is also becoming increasingly antibiotic resistant. Quorum sensing (QS) mediated by the accessory gene regulator (agr) controls virulence factor production essential for causing SSTIs. We recently identified ω-hydroxyemodin (OHM), a polyhydroxyanthraquinone isolated from solid-phase cultures of Penicillium restrictum, as a suppressor of QS and a compound sought for the further characterization of the mechanism of action. At concentrations that are nontoxic to eukaryotic cells and subinhibitory to bacterial growth, OHM prevented agr signaling by all four S. aureus agr alleles. OHM inhibited QS by direct binding to AgrA, the response regulator encoded by the agr operon, preventing the interaction of AgrA with the agr P2 promoter. Importantly, OHM was efficacious in a mouse model of S. aureus SSTI. Decreased dermonecrosis with OHM treatment was associated with enhanced bacterial clearance and reductions in inflammatory cytokine transcription and expression at the site of infection. Furthermore, OHM treatment enhanced the immune cell killing of S. aureus in vitro in an agr-dependent manner. These data suggest that bacterial disarmament through the suppression of S. aureus QS may bolster the host innate immune response and limit inflammation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

PubMed Central

Alpert, Michael D.; Heyer, Lisa N.; Williams, David E. J.; Harvey, Jackson D.; Greenough, Thomas; Allhorn, Maria

2012-01-01

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4+ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays. PMID:22933282

Nafcillin Enhances Innate Immune-Mediated Killing of Methicillin-Resistant Staphylococcus aureus

PubMed Central

Sakoulas, George; Okumura, Cheryl Y.; Thienphrapa, Wdee; Olson, Joshua; Nonejuie, Poochit; Dam, Quang; Dhand, Abhay; Pogliano, Joseph; Yeaman, Michael R.; Hensler, Mary E.; Bayer, Arnold S.; Nizet, Victor

2014-01-01

Based on in vitro synergy studies, the addition of nafcillin to daptomycin was used to treat refractory methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Daptomycin is a de facto cationic antimicrobial peptide in vivo, with antistaphylococcal mechanisms reminiscent of innate host defense peptides (HDPs). In this study, the effects of nafcillin on HDP activity against MRSA were examined in vitro and in vivo. Exposures to β-lactam antimicrobials in general, and nafcillin in particular, significantly increased killing of S. aureus by selected HDPs from, keratinocytes, neutrophils and platelets. This finding correlated with enhanced killing of MRSA by whole blood, neutrophils and keratinocytes after growth in nafcillin. Finally, nafcillin pretreatment ex vivo reduced MRSA virulence in a murine subcutaneous infection model. Despite the lack of direct activity against MRSA, these studies show potent, consistent, and generalized nafcillin-mediated ‘sensitization’ to increased killing of MRSA by various components of the innate host response. The use of nafcillin as adjunctive therapy in MRSA bacteremia merits further study and should be considered in cases refractory to standard therapy. PMID:24297496

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

PubMed Central

Dai, Heqiao; Liu, Jianying; Malkas, Linda H.; Catalano, Jennifer; Alagharu, Srilakshmi; Hickey, Robert J.

2009-01-01

Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC50 of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases α, δ and ε is 15, 45 and 125 μM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC50 =88 μM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2–13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr (III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication correlates with the genotoxic and cytotoxic effects of this metal ion; and promotes cell killing via inhibition of the DNA polymerase activity mediating the DNA replication and repair processes utilized by human cells. PMID:19371627

Selective Killing Effects of Cold Atmospheric Pressure Plasma with NO Induced Dysfunction of Epidermal Growth Factor Receptor in Oral Squamous Cell Carcinoma.

PubMed

Lee, Jung-Hwan; Om, Ji-Yeon; Kim, Yong-Hee; Kim, Kwang-Mahn; Choi, Eun-Ha; Kim, Kyoung-Nam

2016-01-01

The aim of this study is to investigate the effects of cold atmospheric pressure plasma (CAP)-induced radicals on the epidermal growth factor receptor (EGFR), which is overexpressed by oral squamous cell carcinoma, to determine the underlying mechanism of selective killing. CAP-induced highly reactive radicals were observed in both plasma plume and cell culture media. The selective killing effect was observed in oral squamous cell carcinoma compared with normal human gingival fibroblast. Degradation and dysfunction of EGFRs were observed only in the EGFR-overexpressing oral squamous cell carcinoma and not in the normal cell. Nitric oxide scavenger pretreatment in cell culture media before CAP treatment rescued above degradation and dysfunction of the EGFR as well as the killing effect in oral squamous cell carcinoma. CAP may be a promising cancer treatment method by inducing EGFR dysfunction in EGFR-overexpressing oral squamous cell carcinoma via nitric oxide radicals.

Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villa, Nancy Y.; Bartee, Eric; Mohamed, Mohamed R.

2010-06-05

Myxoma (MYXV) and vaccinia (VACV) viruses have recently emerged as potential oncolytic agents that can infect and kill different human cancer cells. Although both are structurally similar, it is unknown whether the pathway(s) used by these poxviruses to enter and cause oncolysis in cancer cells are mechanistically similar. Here, we compared the entry of MYXV and VACV-WR into various human cancer cells and observed significant differences: 1 - low-pH treatment accelerates fusion-mediated entry of VACV but not MYXV, 2 - the tyrosine kinase inhibitor genistein inhibits entry of VACV, but not MYXV, 3 - knockdown of PAK1 revealed that itmore » is required for a late stage event downstream of MYXV entry into cancer cells, whereas PAK1 is required for VACV entry into the same target cells. These results suggest that VACV and MYXV exploit different mechanisms to enter into human cancer cells, thus providing some rationale for their divergent cancer cell tropisms.« less

CD22 Ligands on a Natural N-Glycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells.

PubMed

Peng, Wenjie; Paulson, James C

2017-09-13

CD22 is a sialic acid-binding immunoglobulin-like lectin (Siglec) that is highly expressed on B-cells and B cell lymphomas, and is a validated target for antibody and nanoparticle based therapeutics. However, cell targeted therapeutics are limited by their complexity, heterogeneity, and difficulties in production. We describe here a chemically defined natural N-linked glycan scaffold that displays high affinity CD22 glycan ligands and outcompetes the natural ligand for the receptor, resulting in single molecule binding to CD22 and endocytosis into cells. Binding affinity is increased by up to 1500-fold compared to the monovalent ligand, while maintaining the selectivity for hCD22 over other Siglecs. Conjugates of these multivalent ligands with auristatin and saporin toxins are efficiently internalized via hCD22 resulting in killing of B-cell lymphoma cells. This single molecule ligand targeting strategy represents an alternative to antibody- and nanoparticle-mediated approaches for delivery of agents to cells expressing CD22 and other Siglecs.

Suppressive effects of chlorphenesin on lymphocyte function in mice and humans.

PubMed

Stites, D P; Brecher, G; Schmidt, L; Berger, F M

1979-12-01

The immunosuppressive action of chlorphenesin was investigated in a wide variety of in vitro assays for cellular immunity in humans and mice. Chlorphenesin, at doses of 20-50 micrograms/ml, inhibited mitogenic responses of both mouse and human B and T cells. These doses did not kill cells exposed to the drug for 72 hr. Mixed lymphocyte reactions in inbred strains of mice and in unrelated humans were also inhibited at concentrations of about 50 micrograms/ml. However, the generation of cytotoxic T cells in cell-mediated lympholysis assays was not inhibited to the same degree as proliferation in mixed lymphocyte reaction and the cytotoxic potential of presensitized mouse T cells for allogeneic targets was totally unaffected. These studies suggest that chlorphenesin may have a broad spectrum of suppressive effects both on T and B cells and that the predominant inhibition of proliferative responses in these cells may reduce the expansion of clones of immunocompetent cells in vivo.

Effects of verteporfin-mediated photodynamic therapy on endothelial cells

NASA Astrophysics Data System (ADS)

Kraus, Daniel; Chen, Bin

2015-03-01

Photodynamic therapy (PDT) is a treatment modality in which cytotoxic reactive oxygen species are generated from oxygen and other biological molecules when a photosensitizer is activated by light. PDT has been approved for the treatment of cancers and age-related macular degeneration (AMD) due to its effectiveness in cell killing and manageable normal tissue complications. In this study, we characterized the effects of verteporfin-PDT on SVEC mouse endothelial cells and determined its underlying cell death mechanisms. We found that verteporfin was primarily localized in mitochondria and endoplasmic reticulum (ER) in SVEC cells. Light treatment of photosensitized SVEC cells induced a rapid onset of cell apoptosis. In addition to significant structural damages to mitochondria and ER, verteporfin-PDT caused substantial degradation of ER signaling molecules, suggesting ER stress. These results demonstrate that verteporfin-PDT triggered SVEC cell apoptosis by both mitochondrial and ER stress pathways. Results from this study may lead to novel therapeutic approaches to enhance PDT outcome.

9 CFR 113.210 - Feline Calicivirus Vaccine, Killed Virus.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Feline Calicivirus Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.210 Feline Calicivirus Vaccine, Killed Virus. Feline Calicivirus Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed which...

9 CFR 113.203 - Feline Panleukopenia Vaccine, Killed Virus.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Feline Panleukopenia Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.203 Feline Panleukopenia Vaccine, Killed Virus. Feline Panleukopenia Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed which...

9 CFR 113.216 - Bovine Rhinotracheitis Vaccine, Killed Virus.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Bovine Rhinotracheitis Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.216 Bovine Rhinotracheitis Vaccine, Killed Virus. Infectious Bovine Rhinotracheitis Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed...

9 CFR 113.203 - Feline Panleukopenia Vaccine, Killed Virus.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Feline Panleukopenia Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.203 Feline Panleukopenia Vaccine, Killed Virus. Feline Panleukopenia Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed which...

9 CFR 113.216 - Bovine Rhinotracheitis Vaccine, Killed Virus.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Bovine Rhinotracheitis Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.216 Bovine Rhinotracheitis Vaccine, Killed Virus. Infectious Bovine Rhinotracheitis Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed...

9 CFR 113.210 - Feline Calicivirus Vaccine, Killed Virus.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Feline Calicivirus Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.210 Feline Calicivirus Vaccine, Killed Virus. Feline Calicivirus Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed which...

9 CFR 113.203 - Feline Panleukopenia Vaccine, Killed Virus.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Feline Panleukopenia Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.203 Feline Panleukopenia Vaccine, Killed Virus. Feline Panleukopenia Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed which...

9 CFR 113.216 - Bovine Rhinotracheitis Vaccine, Killed Virus.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Bovine Rhinotracheitis Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.216 Bovine Rhinotracheitis Vaccine, Killed Virus. Infectious Bovine Rhinotracheitis Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed...

9 CFR 113.210 - Feline Calicivirus Vaccine, Killed Virus.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Feline Calicivirus Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.210 Feline Calicivirus Vaccine, Killed Virus. Feline Calicivirus Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed which...

9 CFR 113.210 - Feline Calicivirus Vaccine, Killed Virus.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Feline Calicivirus Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.210 Feline Calicivirus Vaccine, Killed Virus. Feline Calicivirus Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed which...

9 CFR 113.203 - Feline Panleukopenia Vaccine, Killed Virus.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Feline Panleukopenia Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.203 Feline Panleukopenia Vaccine, Killed Virus. Feline Panleukopenia Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed which...