Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

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Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

2011-04-01

We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Anti-Glycoprotein G Antibodies of Herpes Simplex Virus 2 Contribute to Complete Protection after Vaccination in Mice and Induce Antibody-Dependent Cellular Cytotoxicity and Complement-Mediated Cytolysis

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Staffan Görander

2014-11-01

Full Text Available We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2 of herpes simplex virus 2 (HSV-2 in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1 and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC and complement-mediated cytolysis (ACMC activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

High throughput screening for antibody induced complement-dependent cytotoxicity in early antibody discovery using homogeneous macroconfocal fluorescence imaging

NARCIS (Netherlands)

Gerritsen, Arnout F.; Bosch, Martijn; de Weers, Michel; van de Winkel, Jan G. J.; Parren, Paul W. H. I.

2010-01-01

Complement-dependent cytotoxicity (CDC) represents an important Fc-mediated effector function of antibodies and is a quality often sought in candidates for therapeutic antibody development in cancer. Antibodies inducing potent CDC are relatively rare as the ability to induce CDC is strongly

Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

DEFF Research Database (Denmark)

Lavrsen, Kirstine; Madsen, Caroline B; Rasch, Morten G

2013-01-01

to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis...

Studies on ADCC (antibody-dependent cell-mediated cytotoxicity) using sheep red blood cells as target cells, 2

International Nuclear Information System (INIS)

Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

1979-01-01

A non-specific cytotoxic mediator from effector cells (human peripheral blood leukocytes) was investigated in the ADCC (antibody-dependent cell-mediated cytotoxicity) system using antibody-coated sheep red blood cells (SRBC) as target cells. 51 Cr-labelled homologous (sheep) or heterologous (human) red blood cells were used as adjacent cells. Either crude lymphocyte fraction, phagocyte depleted fraction or granulocyte rich fraction separated from human peripheral leukocytes showed moderate cytotoxic effect on homologous adjacent cells, however no cytotoxic activity on heterologous adjacent cells was demonstrated in any leukocyte fraction. This suggests that the cytotoxic effects on homologous adjacent cells were resulted from the translocation of antibody molecules to adjacent cells from antibody-coated target cells. We concluded that the cytotoxic mechanism in this ADCC system was not mediated by non-specific soluble factors released from either human peripheral lymphocytes, monocytes or granulocytes. (author)

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

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Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

2015-01-01

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2 Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

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Masaki Kurogochi

Full Text Available Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain, and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC and complement dependent cytotoxicity (CDC. To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases, one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2, high-mannose type (Man4-9GlcNAc2, and complex type (Man3GlcNAc3-4 N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL, the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1 were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q, and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2 was performed using SKBR-3 and BT-474 as target

Simultaneous development of antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activity in irradiated mice reconstituted with bone marrow cells

International Nuclear Information System (INIS)

Sihvola, M.; Hurme, M.

1987-01-01

Spleen cells from irradiated, bone marrow-reconstituted mice were tested for their ability to mediate antibody-dependent cellular cytotoxicity against P815 target (ADCC-P815), ADCC against sheep red blood cells (ADCC-SRBC), and natural killer (NK) activity judged as YAC-1 lysis at different times after bone marrow reconstitution. Donor-derived ADCC-P815 effectors were found to appear in the spleens 10-12 days after bone marrow reconstitution simultaneously with the appearance of donor-derived NK cells. NK cells recently derived from bone marrow are known to express the Thy-1 antigen; the phenotype of the ''early'' ADCC-P815 effectors was found to be the same as that of NK cells, i.e., Thy-1+, asialo-GM1+. These data suggest that ADCC-P815 effector cells belong to the NK cell population. ADCC-SRBC, in contrast to ADCC-P815 and NK activity, was already high on Day 7 after bone marrow reconstitution. However, it was mediated partly by recipient-derived effectors. ADCC-SRBC effectors were characterized to be different from ADCC-P815 effectors

An Fc engineering approach that modulates antibody-dependent cytokine release without altering cell-killing functions.

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Kinder, Michelle; Greenplate, Allison R; Strohl, William R; Jordan, Robert E; Brezski, Randall J

2015-01-01

Cytotoxic therapeutic monoclonal antibodies (mAbs) often mediate target cell-killing by eliciting immune effector functions via Fc region interactions with cellular and humoral components of the immune system. Key functions include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, there has been increased appreciation that along with cell-killing functions, the induction of antibody-dependent cytokine release (ADCR) can also influence disease microenvironments and therapeutic outcomes. Historically, most Fc engineering approaches have been aimed toward modulating ADCC, ADCP, or CDC. In the present study, we describe an Fc engineering approach that, while not resulting in impaired ADCC or ADCP, profoundly affects ADCR. As such, when peripheral blood mononuclear cells are used as effector cells against mAb-opsonized tumor cells, the described mAb variants elicit a similar profile and quantity of cytokines as IgG1. In contrast, although the variants elicit similar levels of tumor cell-killing as IgG1 with macrophage effector cells, the variants do not elicit macrophage-mediated ADCR against mAb-opsonized tumor cells. This study demonstrates that Fc engineering approaches can be employed to uncouple macrophage-mediated phagocytic and subsequent cell-killing functions from cytokine release.

The in vivo mechanism of action of CD20 monoclonal antibodies depends on local tumor burden

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Boross, Peter; Jansen, J.H. Marco; de Haij, Simone; Beurskens, Frank J.; van der Poel, Cees E.; Bevaart, Lisette; Nederend, Maaike; Golay, Josée; van de Winkel, Jan G.J.; Parren, Paul W.H.I.; Leusen, Jeanette H.W.

2011-01-01

Background CD20 monoclonal antibodies are widely used in clinical practice. Antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and direct cell death have been suggested to be important effector functions for CD20 antibodies. However, their specific contributions to the in vivo mechanism of action of CD20 immunotherapy have not been well defined. Design and Methods Here we studied the in vivo mechanism of action of type I (rituximab and ofatumumab) and type II (HuMab-11B8) CD20 antibodies in a peritoneal, syngeneic, mouse model with EL4-CD20 cells using low and high tumor burden. Results Interestingly, we observed striking differences in the in vivo mechanism of action of CD20 antibodies dependent on tumor load. In conditions of low tumor burden, complement was sufficient for tumor killing both for type I and type II CD20 antibodies. In contrast, in conditions of high tumor burden, activating FcγR (specifically FcγRIII), active complement and complement receptor 3 were all essential for tumor killing. Our data suggest that complement-enhanced antibody-dependent cellular cytotoxicity may critically affect tumor killing by CD20 antibodies in vivo. The type II CD20 antibody 11B8, which is a poor inducer of complement activation, was ineffective against high tumor burden. Conclusions Tumor burden affects the in vivo mechanism of action of CD20 antibodies. Low tumor load can be eliminated by complement alone, whereas elimination of high tumor load requires multiple effector mechanisms. PMID:21880632

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

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Cavazzoni Andrea

2012-12-01

Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

Lymphocyte-dependent antibody-mediated cytotoxicity in Hashimoto thyroiditis

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Calder, Elizabeth A.; Penhale, W. J.; McLeman, Dena; Barnes, E. W.; Irvine, W. J.

1973-01-01

In the presence of normal human lymphocytes, decomplemented sera from twentynine out of thirty-nine patients with Hashimoto thyroiditis caused significant lysis of thyroglobulin-coated chicken red blood cells, as estimated by the release of 51Cr; the mean% specific 51Cr release being 14·1 ± 1·9 (SEM). Serum from twenty-one control subjects studied concurrently caused no significant lysis of thyroglobulin-coated chicken red blood cells; the mean% specific 51Cr release being −1·6±0·7 (SEM). The degree of cytotoxicity correlated with the titre of thyroglobulin antibodies in the serum, determined by tanned red cell haemagglutination. The active component in the Hashimoto serum was localized in the 19S fraction, was unaffected by pre-absorption with anti-human IgM serum, but was neutralized by pre-absorption with anti-human IgG serum. These findings suggest that the cytotoxic activity of serum from patients with Hashimoto thyroiditis is due to the presence of thyroglobulin antibody of the IgG class in the form of complexes, either alone or with antigen. It is postulated that non-specific lymphocytes may play an important role in the pathogenesis of Hashimoto thyroiditis, being activated by the presence in the gland of thyroglobulin antibody, either alone or in the form of complexes attached to thyroid cells. PMID:4740445

Avelumab: combining immune checkpoint inhibition and antibody-dependent cytotoxicity.

Science.gov (United States)

Hamilton, Gerhard; Rath, Barbara

2017-04-01

Immune checkpoint inhibition holds great promise for selected tumors. The human monoclonal antibody (mAB) avelumab is directed to programmed death ligand-1 (PD-L1) and is supposed to inhibit the immunosuppressive PD-L1/PD-1 interaction and, furthermore, effect antibody-dependent cytotoxicity (ADCC) lysis of tumor cells. Areas covered: This article presents an overview of the current means to activate the antitumor immune defense by targeting PD-1 or PD-L1 with mABs and their possible role in ADCC-mediated tumor cell elimination. Expert opinion: Avelumab contains a Fc region which can bind cognate receptors on immune effector cells and induce ADCC-mediated tumor cell lysis, in contrast to other mABs directed to PD-1/PD-L1 which lack the ability to trigger ADCC due to belonging to the IgG4 subclass or possessing a mutated Fc region. Preclinical and clinical data indicate that avelumab can be safely administered to cancer patients with a toxicity profile comparable to other mABs and without lysis of PD-L1-positive activated immune cells. This antibody yielded durable responses in a phase II trial in advanced Merkel cell carcinoma patients. Tumor cell lysis by avelumab prevents cells from resorting to alternative checkpoints as shown by targeting PD-1 and the upregulation of TIM-3.

Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

Science.gov (United States)

Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

2015-01-01

Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination

NARCIS (Netherlands)

Mahan, Alison E.; Jennewein, Madeleine F.; Suscovich, Todd; Dionne, Kendall; Tedesco, Jacquelynne; Chung, Amy W.; Streeck, Hendrik; Pau, Maria; Schuitemaker, Hanneke; Francis, Don; Fast, Patricia; Laufer, Dagna; Walker, Bruce D.; Baden, Lindsey; Barouch, Dan H.; Alter, Galit

2016-01-01

Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain

Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti-PD-L1 Antibody Avelumab (MSB0010718C) on Human Tumor Cells.

Science.gov (United States)

Boyerinas, Benjamin; Jochems, Caroline; Fantini, Massimo; Heery, Christopher R; Gulley, James L; Tsang, Kwong Yok; Schlom, Jeffrey

2015-10-01

Several anti-PD-1/PD-L1 monoclonal antibodies (mAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these mAbs is to inhibit PD-1 on immune cells interacting with PD-L1 on tumor cells. These mAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective mAb-mediated cancer therapies. A fully human anti-PD-L1 mAb would potentially be able to block PD-1/PD-L1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 mAb. The studies reported here demonstrate (i) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (ii) IFNγ can enhance tumor cell PD-L1 expression and, in some cases, enhance ADCC tumor cell lysis; (iii) purified NK cells are potent effectors for avelumab; (iv) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (v) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (vi) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 mAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity. ©2015 American Association for Cancer Research.

Antibody-dependent cellular cytotoxicity (ADCC) activity of a novel anti-PD-L1 antibody avelumab (MSB0010718C) on human tumor cells

Science.gov (United States)

Fantini, Massimo; Heery, Christopher R.; Gulley, James L.; Tsang, Kwong Yok; Schlom, Jeffrey

2015-01-01

Several anti-PD1/PD-L1 monoclonal antibodies (MAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these MAbs is to inhibit PD1 on immune cells interacting with PD-L1 on tumor cells. These MAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective MAb-mediated cancer therapies. A fully human anti-PD-L1 MAb would potentially be able to block PD-L1/PD1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 MAb. The studies reported here demonstrate (a) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (b) IFNγ can enhance tumor cell PD-L1 expression and in some cases enhance ADCC tumor cell lysis; (c) purified NK cells are potent effectors for avelumab; (d) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (e) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (f) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 MAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity. PMID:26014098

Antibody-dependent NK cell activation is associated with late kidney allograft dysfunction and the complement-independent alloreactive potential of donor-specific antibodies

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Tristan Legris

2016-08-01

Full Text Available Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs. The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK cells as innate immune effectors of antibody-dependent cellular cytotoxicity, but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1+ cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years. Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate (eGFR over a 1-year period (hazard ratio: 2.83. In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration

Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity

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Campana Dario

2010-10-01

Full Text Available Abstract Background The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i the small number of NK cells in peripheral blood, (ii the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii the need to activate the NK cells in order to induce NK cell mediated killing and (iv the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii their ability to kill allogeneic and autologous tumor targets by direct cytotoxitiy and by antibody-mediated cellular cytotoxicity and (iii defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing. Methods Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated. Results Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors. Conclusion These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical

A novel antibody-dependent cellular cytotoxicity epitope in gp120 is identified by two monoclonal antibodies isolated from a long-term survivor of human immunodeficiency virus type 1 infection.

Science.gov (United States)

Alsmadi, O; Herz, R; Murphy, E; Pinter, A; Tilley, S A

1997-01-01

Two monoclonal antibodies (MAbs), 42F and 43F, were isolated some 14 months apart from a single long-term survivor of human immunodeficiency virus type 1 (HIV-1) infection. These MAbs were found to be indistinguishable in terms of their isotypes, specificities, affinities, and biological activities. Both 42F and 43F directed substantial antibody-dependent cellular cytotoxicity (ADCC) against cells infected with four divergent lab-adapted strains of HIV-1, but no neutralizing activity against these strains was detectable. The ability of MAbs 42F and 43F, as well as that of MAbs against two other gp120 epitopes, to direct ADCC against uninfected CD4+ cells to which recombinant gp120SF2 had been adsorbed (i.e., "innocent bystanders") was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells. Flow cytometry analyses showed that 42F and 43F also bind to native primary isolate Envs from clades B and E expressed on cell surfaces. By direct binding and competition assays, it was demonstrated that the 42F/43F epitope lies in a domain of gp120 outside the previously described CD4-binding site and V3 loop ADCC epitope clusters. Immunoblot analysis revealed that the 42F/43F epitope is not dependent on disulfide bonds or N-linked glycans in gp120. Epitope mapping of 42F and 43F by binding to linear peptides demonstrated specificity of these MAbs for a sequence of 10 amino acids in the C5 domain comprising residues 491 to 500 (Los Alamos National Laboratory numbering for the HXB2 strain). Thus, 42F and 43F define a new ADCC epitope in gp120. Because of the relative conservation of this epitope and the fact that it appears to have been significantly immunogenic in the individual from which these MAbs were derived, it may prove to be a useful component of HIV vaccines. Furthermore, these MAbs may be used as tools to probe the potential importance of ADCC as an antiviral activity in HIV-1 infection. PMID

Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1.

Science.gov (United States)

Wang, Kening; Tomaras, Georgia D; Jegaskanda, Sinthujan; Moody, M Anthony; Liao, Hua-Xin; Goodman, Kyle N; Berman, Phillip W; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayapan, Sorachai; Kaewkungwal, Jaranit; Haynes, Barton F; Cohen, Jeffrey I

2017-10-01

The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice. IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were

A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

DEFF Research Database (Denmark)

de Jong, R. N.; Beurskens, F. J.; Verploegen, S.

2016-01-01

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology......) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce...... conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD...

Antitumor and cytotoxic properties of a humanized antibody specific for the GM3(Neu5Gc) ganglioside.

Science.gov (United States)

Dorvignit, Denise; García-Martínez, Liliana; Rossin, Aurélie; Sosa, Katya; Viera, Justo; Hernández, Tays; Mateo, Cristina; Hueber, Anne-Odile; Mesa, Circe; López-Requena, Alejandro

2015-12-01

Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors. Copyright © 2015 Elsevier GmbH. All rights reserved.

Monoclonal antibodies targeting CD38 in hematological malignancies and beyond

DEFF Research Database (Denmark)

van de Donk, Niels W C J; Janmaat, Maarten L.; Mutis, Tuna

2016-01-01

CD38 is a multifunctional cell surface protein that has receptor as well as enzyme functions. The protein is generally expressed at low levels on various hematological and solid tissues, while plasma cells express particularly high levels of CD38. The protein is also expressed in a subset of hema...... strong anti-tumor activity in preclinical models. The antibody engages diverse mechanisms of action, including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, programmed cell death, modulation of enzymatic activity...... combination therapies with existing as well as emerging therapies, which are currently evaluated in the clinic. Finally, CD38 antibodies may have a role in the treatment of diseases beyond hematological malignancies, including solid tumors and antibody-mediated autoimmune diseases. © 2016 John Wiley & Sons A....../S. Published by John Wiley & Sons Ltd....

Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab

OpenAIRE

Fujii, Rika; Friedman, Eitan R.; Richards, Jacob; Tsang, Kwong Y.; Heery, Christopher R.; Schlom, Jeffrey; Hodge, James W.

2016-01-01

Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 ch...

Higher cytotoxicity of divalent antibody-toxins than monovalent antibody-toxins

International Nuclear Information System (INIS)

Won, JaeSeon; Nam, PilWon; Lee, YongChan; Choe, MuHyeon

2009-01-01

Recombinant antibody-toxins are constructed via the fusion of a 'carcinoma-specific' antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G 4 S) between the Fab fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38] 2 ) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.

Glycoengineering of therapeutic antibodies enhances monocyte/macrophage-mediated phagocytosis and cytotoxicity.

Science.gov (United States)

Herter, Sylvia; Birk, Martina C; Klein, Christian; Gerdes, Christian; Umana, Pablo; Bacac, Marina

2014-03-01

Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive immunity. In view of the important role of phagocytic lineage cells in the mechanism of action of therapeutic Abs, we analyzed FcγR receptor-dependent effector functions of monocytes and macrophages triggered by glycoengineered (GE) Abs (having enhanced FcγRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different experimental conditions. We first defined the precise FcγR repertoire on classical and nonclassical intermediate monocytes--M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to CD16a under conditions that more closely resemble the physiologic setting.

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

Science.gov (United States)

Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

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

Science.gov (United States)

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.

The impact of HLA class I-specific killer cell immunoglobulin-like receptors on antibody-dependent natural killer cell-mediated cytotoxicity and organ allograft rejection

Directory of Open Access Journals (Sweden)

Raja Rajalingam

2016-12-01

Full Text Available Natural killer (NK cells of the innate immune system are cytotoxic lymphocytes that play important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self HLA class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIR is involved in the calibration of NK cell effector capacities during a 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.

Studies of cytotoxic antibodies against eye muscle antigens in patients with thyroid-associated ophthalmopathy

International Nuclear Information System (INIS)

Zhang, Z.-G.; Hiromatsu, Y.; Salvi, M.; Triller, H.; Bernard, N.; Wall, J.R.; Medeiros-Neto, G.; Iacona, A.; Lima, N.

1989-01-01

We have studied the prevalence and significance of cytotoxic antibodies against human eye muscle cells, as detected in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated antibody-dependent cytotoxicity (CMAC) in 51 Cr release assays, in patients with Graves' ophthalmopathy or Hashimoto's thyroiditis. A high prevalence of positive ADCC tests was found in all groups of patients with ophthalmopathy tested. Tests were positive in 64% of patients with Graves' ophthalmopathy from an area of severe iodine deficiency (Sao Paulo) and in 64% of such patients from an iodine replete area (Montreal). In patients with so-called ''euthyroid ophthalmopathy'', i.e. eye disease associated with thyroiditis, ADCC tests were positive in 75 and 38% of patients from the two areas, respectively, while tests were positive in 40 and 22%, respectively, of patients with Graves' hyperthyroidism without evident eye disease. In normal subjects, levels of 51 Cr release was always at background levels. In a group of patients from the high-iodine area, levels of antibodies in ADCC correlated positively with the intraocular pressure (mmHg) in primary position as a parameter of eye muscle dysfunction. In patients with ophthalmopathy, positive ADCC tests were assciated with antibodies to eye muscle membrane antigens of 55,65 and 95 kD as detected by immunoblotting, although the correlation was not close for any antigen. in contrast, CMAC tests were negative in all patients with ophthalmopathy. We also tested 9 mouse and 10 human monoclonal antibodies, reactive with orbital antigens in an enzyme-linked immunosorbent assay, for cytotoxic activity, in ADCC and CMAC, against eye muscle and thyroid cells. All monoclonal antibodies were of the IgM class and negative in ADCC assays. When tested in CMAC against eye muscle cells, one of 9 mouse and 5 of 8 human monoclonal antibodies showed significant activity while tests were positive in one of 9 and one of 10 monoclonal antibodies

Poly-L-arginine: Enhancing Cytotoxicity and Cellular Uptake of Doxorubicin and Necrotic Cell Death.

Science.gov (United States)

Movafegh, Bahareh; Jalal, Razieh; Mohammadi, Zobeideh; Aldaghi, Seyyede Araste

2018-04-11

Cell resistance to doxorubicin and its toxicity to healthy tissue reduce its efficiency. The use of cell penetrating peptides as drug delivery system along with doxorubicin is a strategy to reduce its side effects. In this study, the influence of poly-L-arginine on doxorubicin cytotoxicity, its cellular uptake and doxorubicin-induced apoptosis on human prostate cancer DU145 cells are assessed. The cytotoxicity of doxorubicin and poly-L-arginine, alone and in combination, in DU145 cells was evaluated at different exposure times using MTT assay. The influence of poly-L-arginine on doxorubicin delivery into cells was evaluated by fluorescence microscopy and ultraviolet spectroscopy. DAPI and ethidium bromide-acridine orange stainings, flow cytometry using annexin V/propidium iodide, western blot analysis with anti-p21 antibody and caspase-3 activity were used to examine the influence of poly-L-arginine on doxorubicin-induced cell death. Poly-L-arginine had no cytotoxicity at low concentrations and short exposure times. Poly-L-arginine increased the cytotoxic effect of doxorubicin in DU145 cells in a time-dependent manner. But no significant reduction was found in HFF cell viability. Poly-L-arginine seems to facilitate doxorubicin uptake and increase its intracellular concentration. 24 h combined treatment of cells with doxorubicin (0.5 μM) and poly-L-arginine (1 μg ml-1) caused a small increase in doxorubicin-induced apoptosis and significant elevated necrosis in DU145 cells as compared to each agent alone. Conlusion: Our results indicate that poly-L-arginine at lowest and highest concentrations act as proliferation-inducing and antiproliferative agents, respectively. Between these concentrations, poly-L-arginine increases the cellular uptake of doxorubicin and its cytotoxicity through induction of necrosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Different cellular response mechanisms contribute to the length-dependent cytotoxicity of multi-walled carbon nanotubes

Science.gov (United States)

Liu, Dun; Wang, Lijun; Wang, Zhigang; Cuschieri, Alfred

2012-07-01

To date, there has not been an agreement on the best methods for the characterisation of multi-walled carbon nanotube (MWCNT) toxicity. The length of MWCNTs has been identified as a factor in in vitro and in vivo studies, in addition to their purity and biocompatible coating. Another unresolved issue relates to the variable toxicity of MWCNTs on different cell types. The present study addressed the effects of MWCNTs' length on mammalian immune and epithelial cancer cells RAW264.7 and MCF-7, respectively. Our data confirm that MWCNTs induce cytotoxicity in a length- and cell type-dependent manner. Whereas, longer (3 to 14 μm) MWCNTs exert high toxicity, especially to RAW264.7 cells, shorter (1.5 μm) MWCNTs are significantly less cytotoxic. These findings confirm that the degree of biocompatibility of MWCNTs is closely related to their length and that immune cells appear to be more susceptible to damage by MWCNTs. Our study also indicates that MWCNT nanotoxicity should be analysed for various components of cellular response, and cytotoxicity data should be validated by the use of more than one assay system. Results from chromogenic-based assays should be confirmed by trypan blue exclusion.

Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals.

Science.gov (United States)

Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T; Ackerman, Margaret E; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

2011-07-05

In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. Copyright © 2011 Elsevier Inc. All rights reserved.

Decreased Fc-Receptor expression on innate immune cells is associated with impaired antibody mediated cellular phagocytic activity in chronically HIV-1 infected individuals

Science.gov (United States)

Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T.; Ackerman, Margaret E.; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

2011-01-01

In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular-phagocytosis (ADCP), antibody dependent cellular-cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. PMID:21565376

Cells of the J774 macrophage cell line are primed for antibody-dependent cell-mediated cytotoxicity following exposure to γ-irradiation

International Nuclear Information System (INIS)

Duerst, R.; Werberig, K.

1991-01-01

Activation of macrophages (M phi) for host defense against tumor cells follows a sequence of priming events followed by an initiating stimulus that results in production and release of cytotoxic molecules that mediate target cell killing. The authors have developed a model to study specific macrophage cytotoxicity in vitro utilizing a cultured murine M phi cell line, J774. Specific cytotoxicity of cultured human gastrointestinal tumor cells is achieved in the presence of murine IgG2a monoclonal antibody (mAb) 17-1-A. The ability of these cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) is greatly enhanced following gamma-irradiation. ADCC can be demonstrated at mAb 17-1-A concentrations greater than or equal to 1 microgram/ml and effector/target cell ratios greater than or equal to 2. Exposure to doses greater than or equal to 10 Gy of gamma-irradiation increases ADCC threefold. Varying the duration from J774 M phi exposure to γ-irradiation until addition of antibody-coated target cells showed that the primed state for ADCC is stable for at least 8 days but approximately 24 hr is required for complete development of the primed state. mAb-dependent target cell death begins 8 hr after addition of mAb and labeled target cells to primed effector cells and is complete by 24 hr. Incubation of unirradiated J774 M phi effector cells with recombinant murine interferon-γ (rmIFN-γ) also results in enhanced ADCC, but the extent of target cell killing achieved is less than that following priming by γ-irradiation. Concomitant priming of γ-irradiated J774 M phi with rmIFN-γ increases the extent of ADCC. Further study of irradiated J774 cells may elucidate the molecular pathways utilized by M phi for achieving and maintaining the primed state for ADCC

Daratumumab: a first-in-class CD38 monoclonal antibody for the treatment of multiple myeloma

Directory of Open Access Journals (Sweden)

Larysa Sanchez

2016-06-01

Full Text Available Abstract Daratumumab is a human monoclonal antibody that targets CD38, a cell surface protein that is overexpressed on multiple myeloma (MM cells. Preclinical studies have shown that daratumumab induces MM cell death through several mechanisms, including complement-dependent cytotoxicity (CDC, antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cellular phagocytosis (ADCP, and apoptosis. Given the encouraging efficacy and acceptable safety profile of daratumumab demonstrated in clinical trials, daratumumab has emerged as a novel treatment option for myeloma and became the first monoclonal antibody approved by the FDA for the treatment of MM.

Studies of cytotoxic antibodies against eye muscle antigens in patients with thyroid-associated ophthalmopathy

Energy Technology Data Exchange (ETDEWEB)

Zhang, Z.-G.; Hiromatsu, Y.; Salvi, M.; Triller, H.; Bernard, N.; Wall, J.R. (Thyroid Research Unit, The Montreal General Hospital Research Institute, Montreal, Quebec (Canada)); Medeiros-Neto, G.; Iacona, A.; Lima, N. (Thyroid Clinic, Hospital das Clinicas, Sao Paulo (Brazil))

1989-01-01

We have studied the prevalence and significance of cytotoxic antibodies against human eye muscle cells, as detected in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated antibody-dependent cytotoxicity (CMAC) in {sup 51}Cr release assays, in patients with Graves' ophthalmopathy or Hashimoto's thyroiditis. A high prevalence of positive ADCC tests was found in all groups of patients with ophthalmopathy tested. Tests were positive in 64% of patients with Graves' ophthalmopathy from an area of severe iodine deficiency (Sao Paulo) and in 64% of such patients from an iodine replete area (Montreal). In patients with so-called ''euthyroid ophthalmopathy'', i.e. eye disease associated with thyroiditis, ADCC tests were positive in 75 and 38% of patients from the two areas, respectively, while tests were positive in 40 and 22%, respectively, of patients with Graves' hyperthyroidism without evident eye disease. In normal subjects, levels of {sup 51}Cr release was always at background levels. In a group of patients from the high-iodine area, levels of antibodies in ADCC correlated positively with the intraocular pressure (mmHg) in primary position as a parameter of eye muscle dysfunction. In patients with ophthalmopathy, positive ADCC tests were assciated with antibodies to eye muscle membrane antigens of 55,65 and 95 kD as detected by immunoblotting, although the correlation was not close for any antigen. in contrast, CMAC tests were negative in all patients with ophthalmopathy. We also tested 9 mouse and 10 human monoclonal antibodies, reactive with orbital antigens in an enzyme-linked immunosorbent assay, for cytotoxic activity, in ADCC and CMAC, against eye muscle and thyroid cells. All monoclonal antibodies were of the IgM class and negative in ADCC assays. (Abstract Truncated)

Fundamental studies on ADCC (antibody-dependent cell-mediated cytotoxicity) of human peripheral blood leukocytes using sheep red blood cells as target cells, and the effect of erythrophagocytosis

International Nuclear Information System (INIS)

Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

1979-01-01

We investigated antibody-dependent cell-mediated cytotoxicity (ADCC) of human peripheral blood leukocytes by using 51 Cr-labelled sheep red blood cells (SRBC) as target cells and anti-SRBC rabbit antibody. Lysis of SRBC was mediated by either human peripheral lymphoid cells or phagocytes (Monocytes and granulocytes). SRBC were useful as target cells in ADCC assay against human lymphoid cells, since decreased cytotoxic activity of phagocyte-contaminated crude lymphocyte fraction was recovered by elimination of contaminating phagocytes. The monocytes inhibited ADCC of lymphoid cells through phagocytosis of SRBC. This assay system may be useful for estimating not only Fc receptor-mediated cytotoxicity but also Fc receptor-mediated phagocytic activity of human peripheral blood leukocytes. (author)

How immunoglobulin G antibodies kill target cells: revisiting an old paradigm.

Science.gov (United States)

Biburger, Markus; Lux, Anja; Nimmerjahn, Falk

2014-01-01

The capacity of immunoglobulin G (IgG) antibodies to eliminate virtually any target cell has resulted in the widespread introduction of cytotoxic antibodies into the clinic in settings of cancer therapy, autoimmunity, and transplantation, for example. More recently, it has become apparent that also the protection from viral infection via IgG antibodies may require cytotoxic effector functions, suggesting that antibody-dependent cellular cytotoxicity (ADCC) directed against malignant or virally infected cells is one of the most essential effector mechanisms triggered by IgG antibodies to protect the host. A detailed understanding of the underlying molecular and cellular pathways is critical, therefore, to make full use of this antibody effector function. Several studies over the last years have provided novel insights into the effector pathways and innate immune effector cells responsible for ADCC reactions. One of the most notable outcomes of many of these reports is that cells of the mononuclear phagocytic system rather than natural killer cells are critical for removal of IgG opsonized target cells in vivo. © 2014 Elsevier Inc. All rights reserved.

Strong antitumor activities of IgG3 antibodies to a human melanoma-associated ganglioside

International Nuclear Information System (INIS)

Hellstroem, I.; Brankovan, V.; Hellstroem, K.E.

1985-01-01

Three mouse monoclonal IgG3 antibodies, 2B2, IF4, and MG-21, recognize a G/sub D3/ ganglioside antigen that is expressed at the cell surface of most human melanomas. All three antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro when tested with human lymphocytes or effector cells in a 2-hr or 4-hr 51 Cr-release test, and one antibody, MG-21, also gives strong complement-dependent cytotoxicity with human serum. Antibody 2B2, which gives ADDC also in the presence of mouse lymphocytes, inhibited the outgrowth of a human melanoma in nude mice, but antibody IF4, which showed no ADCC with mouse lymphocyte effectors, did not

Combination of two anti-CD5 monoclonal antibodies synergistically induces complement-dependent cytotoxicity of chronic lymphocytic leukaemia cells.

Science.gov (United States)

Klitgaard, Josephine L; Koefoed, Klaus; Geisler, Christian; Gadeberg, Ole V; Frank, David A; Petersen, Jørgen; Jurlander, Jesper; Pedersen, Mikkel W

2013-10-01

The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti-CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non-overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti-CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab-induced CD5 internalization and degradation. Importantly, an anti-CD5 mAb combination enhanced CDC of CLL cells when combined with the anti-CD20 mAbs rituximab and ofatumumab as well as with the anti-CD52 mAb alemtuzumab. These results suggest that an anti-CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5-expressing haematological or lymphoid malignancies. © 2013 John Wiley & Sons Ltd.

Altered tumor growth in vivo after immunization of mice with antitumor antibodies

International Nuclear Information System (INIS)

Gorczynski, R.M.; Kennedy, M.; Polidoulis, I.; Price, G.B.

1984-01-01

A comparison has been made between the growth patterns of two spontaneously appearing mammary adenocarcinomas in murine bone marrow radiation chimeras and in mice preimmunized with monoclonal antibodies (MAb) detecting embryo-associated antigenic determinants. A correlation was seen between the ability of the embryo-immunized chimeras to produce cytotoxic antibody to the tumors, as assessed by an antibody-dependent cellular cytotoxic assay, and the permissiveness of the mice for growth of a tumor transplant. In addition, mice deliberately preimmunized with cytotoxic MAb (antibody-dependent cellular cytotoxic assay) allowed more rapid growth specifically of that tumor earlier found to be most sensitive to the MAb used for immunization. By comparing the changing antigenic phenotype of tumor cells serially passaged through different immunized, nonimmunized mice, evidence was found suggesting that immunization could cause either antigen modulation of transferred tumor cells or a (transient) selective advantage to antigenically discrete subpopulations within the heterogeneous tumor population. Finally, a study has been made of the growth pattern of tumor cells transplanted into mice immunized with rabbit antibodies directed against the murine MAb. In this case, tumor growth was slowed preferentially for the tumor reactive with the specific MAb, and again, predictable changes in the antigenic spectrum of tumor cells harvested from these animals were observed. Our overall findings are interpreted in terms of the involvement of networks of antibodies reacting with embryo-associated antigens in the regulation of growth of the murine mammary adenocarcinomas studied

ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells

NARCIS (Netherlands)

Kol, Arjan; Terwisscha van Scheltinga, Anton; Pool, Martin; Gerdes, Christian; de Vries, Elisabeth; de Jong, Steven

2017-01-01

Imgatuzumab is a novel glycoengineered anti-epidermal growth factor receptor (EGFR) monoclonal antibody optimized to induce both antibody-dependent cellular cytotoxicity (ADCC) and EGFR signal transduction inhibition. We investigated antiEGFR monoclonal antibodies imgatuzumab and cetuximab-induced

Replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies.

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Peng, Bo; Wang, Liqun Rejean; Gómez-Román, Victor Raúl; Davis-Warren, Alberta; Montefiori, David C; Kalyanaraman, V S; Venzon, David; Zhao, Jun; Kan, Elaine; Rowell, Thomas J; Murthy, Krishna K; Srivastava, Indresh; Barnett, Susan W; Robert-Guroff, Marjorie

2005-08-01

A major challenge in combating the human immunodeficiency virus (HIV) epidemic is the development of vaccines capable of inducing potent, persistent cellular immunity and broadly reactive neutralizing antibody responses to HIV type 1 (HIV-1). We report here the results of a preclinical trial using the chimpanzee model to investigate a combination vaccine strategy involving sequential priming immunizations with different serotypes of adenovirus (Ad)/HIV-1(MN)env/rev recombinants and boosting with an HIV envelope subunit protein, oligomeric HIV(SF162) gp140deltaV2. The immunogenicities of replicating and nonreplicating Ad/HIV-1(MN)env/rev recombinants were compared. Replicating Ad/HIV recombinants were better at eliciting HIV-specific cellular immune responses and better at priming humoral immunity against HIV than nonreplicating Ad-HIV recombinants carrying the same gene insert. Enhanced cellular immunity was manifested by a greater frequency of HIV envelope-specific gamma interferon-secreting peripheral blood lymphocytes and better priming of T-cell proliferative responses. Enhanced humoral immunity was seen in higher anti-envelope binding and neutralizing antibody titers and better induction of antibody-dependent cellular cytotoxicity. More animals primed with replicating Ad recombinants mounted neutralizing antibodies against heterologous R5 viruses after one or two booster immunizations with the mismatched oligomeric HIV-1(SF162) gp140deltaV2 protein. These results support continued development of the replicating Ad-HIV recombinant vaccine approach and suggest that the use of replicating vectors for other vaccines may prove fruitful.

Autophagy plays a critical role in ChLym-1-induced cytotoxicity of non-hodgkin's lymphoma cells.

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Jiajun Fan

Full Text Available Autophagy is a critical mechanism in both cancer therapy resistance and tumor suppression. Monoclonal antibodies have been documented to kill tumor cells via apoptosis, antibody-dependent cellular cytotoxicity (ADCC and complement-dependent cytotoxicity (CDC. In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin's Lymphoma (NHL cells. Interestingly, inhibition of autophagy by pharmacological inhibitors (3-methyladenine and NH4Cl or genetic approaches (siRNA targeting Atg5 suppresses chLym-1-induced growth inhibition, apoptosis, ADCC and CDC in Raji cells, while induction of autophagy could accelerate cytotoxic effects of chLym-1 on Raji cells. Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1. Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2. These results elucidate the critical role of autophagy in cytotoxicity of chLym-1 antibody and suggest a potential therapeutic strategy of NHL therapy by monoclonal antibody chLym-1 in combination with autophagy inducer.

Synergistic cytotoxic effects of antibodies directed against different cell surface determinants

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Elliott, E V; Pindar, A; Stevenson, F K; Stevenson, G T [Southampton General Hospital (UK). Tenovus Research Lab.

1978-03-01

Three antibody populations were raised in rabbits against surface antigens on guinea-pig L/sub 2/C leukaemic lymphocytes: against idiotypic determinants on the lambda chain of the surface immunoglobulin, against C region determinants on the lambda chain, and against the surface antigens recognised by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L/sub 2/C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules.

Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

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Järnum, Sofia; Runström, Anna; Bockermann, Robert; Winstedt, Lena; Crispin, Max; Kjellman, Christian

2017-09-01

Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR . ©2017 American Association for Cancer Research.

Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques.

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Bialuk, Izabela; Whitney, Stephen; Andresen, Vibeke; Florese, Ruth H; Nacsa, Janos; Cecchinato, Valentina; Valeri, Valerio W; Heraud, Jean-Michel; Gordon, Shari; Parks, Robyn Washington; Montefiori, David C; Venzon, David; Demberg, Thorsten; Guroff, Marjorie Robert-; Landucci, Gary; Forthal, Donald N; Franchini, Genoveffa

2011-12-09

The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection. Published by Elsevier Ltd.

Dual antibody therapy to harness the innate anti-tumor immune response to enhance antibody targeting of tumors.

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Chester, Cariad; Marabelle, Aurelien; Houot, Roch; Kohrt, Holbrook E

2015-04-01

Cancer immunotherapy is a rapidly evolving field that offers a novel paradigm for cancer treatment: therapies focus on enhancing the immune system's innate and adaptive anti-tumor response. Early immunotherapeutics have achieved impressive clinical outcomes and monoclonal antibodies are now integral to therapeutic strategies in a variety of cancers. However, only recently have antibodies targeting innate immune cells entered clinical development. Innate immune effector cells play important roles in generating and maintaining antitumor immunity. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important innate immune mechanisms for tumor eradication. These cytolytic processes are initiated by the detection of a tumor-targeting antibody and can be augmented by activating co-stimulatory pathways or blocking inhibitory signals on innate immune cells. The combination of FDA-approved monoclonal antibodies with innate effector-targeting antibodies has demonstrated potent preclinical therapeutic synergy and early-phase combinatorial clinical trials are ongoing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function.

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Chung, Amy W; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit

2014-11-13

To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC. Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb. Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.

Cytotoxicity and cellular uptake of tri-block copolymer nanoparticles with different size and surface characteristics

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Bhattacharjee Sourav

2012-04-01

Full Text Available Abstract Background Polymer nanoparticles (PNP are becoming increasingly important in nanomedicine and food-based applications. Size and surface characteristics are often considered to be important factors in the cellular interactions of these PNP, although systematic investigations on the role of surface properties on cellular interactions and toxicity of PNP are scarce. Results Fluorescent, monodisperse tri-block copolymer nanoparticles with different sizes (45 and 90 nm and surface charges (positive and negative were synthesized, characterized and studied for uptake and cytotoxicity in NR8383 and Caco-2 cells. All types of PNP were taken up by the cells. The positive smaller PNP45 (45 nm showed a higher cytotoxicity compared to the positive bigger PNP90 (90 nm particles including reduction in mitochondrial membrane potential (ΔΨm, induction of reactive oxygen species (ROS production, ATP depletion and TNF-α release. The negative PNP did not show any cytotoxic effect. Reduction in mitochondrial membrane potential (ΔΨm, uncoupling of the electron transfer chain in mitochondria and the resulting ATP depletion, induction of ROS and oxidative stress may all play a role in the possible mode of action for the cytotoxicity of these PNP. The role of receptor-mediated endocytosis in the intracellular uptake of different PNP was studied by confocal laser scanning microscopy (CLSM. Involvement of size and charge in the cellular uptake of PNP by clathrin (for positive PNP, caveolin (for negative PNP and mannose receptors (for hydroxylated PNP were found with smaller PNP45 showing stronger interactions with the receptors than bigger PNP90. Conclusions The size and surface characteristics of polymer nanoparticles (PNP; 45 and 90 nm with different surface charges play a crucial role in cellular uptake. Specific interactions with cell membrane-bound receptors (clathrin, caveolin and mannose leading to cellular internalization were observed to depend on

Preparation and characterization of chimeric CD19 monoclonal antibody

International Nuclear Information System (INIS)

Zola, H.; Macardle, P.J.; Bradford, T.; Weedon, H.; Yasui, H.; Kurosawa, Y.

1991-01-01

CD19 antibodies have been suggested as candidates for immunological attack on leukemic and lymphoma cells of the B lineage because the antigen is restricted to the B lineage. With the potential use of FMC63 in immunotherapy in mind a mouse-human chimera was produced in which the genes coding for the VDJ region of the heavy chain and the VJ region of the light chain derive from the FMC63 mouse hybridoma, while the C region genes code for human IgG1. The genes have been transfected back into a mouse myeloma line, which secretes low levels of immunoglobulin. (Ig). This Ig was purified and biotinylated in order to determine the specificity of the antibody. The chimeric antibody has a reaction profile concordant with the original FMC63 antibody, but has the properties of a human IgG1, including the ability to fix human complement. However, the antibody is not cytotoxic in vitro in the presence of complement or cells capable of mediating antibody-dependent cellular cytotoxicity. Possible reasons for this and ways of using the antibody are discussed. 47 refs., 7 figs

A cell impedance measurement device for the cytotoxicity assay dependent on the velocity of supplied toxic fluid

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Kang, Yoon-Tae; Kim, Min-Ji; Cho, Young-Ho

2018-04-01

We present a cell impedance measurement chip capable of characterizing the toxic response of cells depending on the velocity of the supplied toxic fluid. Previous impedance-based devices using a single open-top chamber have been limited to maintaining a constant supply velocity, and devices with a single closed-top chamber present difficulties in simultaneous cytotoxicity assay for varying levels of supply velocities. The present device, capable of generating constant and multiple levels of toxic fluid velocity simultaneously within a single stepwise microchannel, performs a cytotoxicity assay dependent on toxic fluid velocity, in order to find the effective velocity of toxic fluid to cells for maximizing the cytotoxic effect. We analyze the cellular toxic response of 5% ethanol media supplied to cancer cells within a toxic fluid velocity range of 0-8.3 mm s-1. We observe the velocity-dependent cell detachment rate, impedance, and death rate. We find that the cell detachment rate decreased suddenly to 2.4% at a velocity of 4.4 mm s-1, and that the change rates of cell resistance and cell capacitance showed steep decreases to 8% and 41%, respectively, at a velocity of 5.7 mm s-1. The cell death rate and impedance fell steeply to 32% at a velocity of 5.7 mm s-1. We conclude that: (1) the present device is useful in deciding on the toxic fluid velocity effective to cytotoxicity assay, since the cellular toxic response is dependent on the velocity of toxic fluid, and; (2) the cell impedance analysis facilitates a finer cellular response analysis, showing better correlation with the cell death rate, compared to conventional visual observation. The present device, capable of performing the combinational analysis of toxic fluid velocity and cell impedance, has potential for application to the fine cellular toxicity assay of drugs with proper toxic fluid velocity.

Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies.

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Tipton, Thomas R W; Roghanian, Ali; Oldham, Robert J; Carter, Matthew J; Cox, Kerry L; Mockridge, C Ian; French, Ruth R; Dahal, Lekh N; Duriez, Patrick J; Hargreaves, Philip G; Cragg, Mark S; Beers, Stephen A

2015-03-19

Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo. © 2015 by The American Society of Hematology.

Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines.

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Collins, Denis M; Gately, Kathy; Hughes, Clare; Edwards, Connla; Davies, Anthony; Madden, Stephen F; O'Byrne, Kenneth J; O'Donovan, Norma; Crown, John

2017-09-01

Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. In the presence of effector cells with high

Machine learning methods enable predictive modeling of antibody feature:function relationships in RV144 vaccinees.

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Choi, Ickwon; Chung, Amy W; Suscovich, Todd J; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Kaewkungwal, Jaranit; O'Connell, Robert J; Francis, Donald; Robb, Merlin L; Michael, Nelson L; Kim, Jerome H; Alter, Galit; Ackerman, Margaret E; Bailey-Kellogg, Chris

2015-04-01

The adaptive immune response to vaccination or infection can lead to the production of specific antibodies to neutralize the pathogen or recruit innate immune effector cells for help. The non-neutralizing role of antibodies in stimulating effector cell responses may have been a key mechanism of the protection observed in the RV144 HIV vaccine trial. In an extensive investigation of a rich set of data collected from RV144 vaccine recipients, we here employ machine learning methods to identify and model associations between antibody features (IgG subclass and antigen specificity) and effector function activities (antibody dependent cellular phagocytosis, cellular cytotoxicity, and cytokine release). We demonstrate via cross-validation that classification and regression approaches can effectively use the antibody features to robustly predict qualitative and quantitative functional outcomes. This integration of antibody feature and function data within a machine learning framework provides a new, objective approach to discovering and assessing multivariate immune correlates.

Machine learning methods enable predictive modeling of antibody feature:function relationships in RV144 vaccinees.

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Ickwon Choi

2015-04-01

Full Text Available The adaptive immune response to vaccination or infection can lead to the production of specific antibodies to neutralize the pathogen or recruit innate immune effector cells for help. The non-neutralizing role of antibodies in stimulating effector cell responses may have been a key mechanism of the protection observed in the RV144 HIV vaccine trial. In an extensive investigation of a rich set of data collected from RV144 vaccine recipients, we here employ machine learning methods to identify and model associations between antibody features (IgG subclass and antigen specificity and effector function activities (antibody dependent cellular phagocytosis, cellular cytotoxicity, and cytokine release. We demonstrate via cross-validation that classification and regression approaches can effectively use the antibody features to robustly predict qualitative and quantitative functional outcomes. This integration of antibody feature and function data within a machine learning framework provides a new, objective approach to discovering and assessing multivariate immune correlates.

Allogeneic major histocompatibility complex-mismatched equine bone marrow-derived mesenchymal stem cells are targeted for death by cytotoxic anti-major histocompatibility complex antibodies.

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Berglund, A K; Schnabel, L V

2017-07-01

Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Controversy exists, however, over whether major histocompatibility complex (MHC)-mismatched MSCs are recognised by the recipient immune system and targeted for death by a cytotoxic antibody response. To determine if cytotoxic anti-MHC antibodies generated in vivo following MHC-mismatched MSC injections are capable of initiating complement-dependent cytotoxicity of MSCs. Experimental controlled study. Antisera previously collected at Days 0, 7, 14 and 21 post-injection from 4 horses injected with donor MHC-mismatched equine leucocyte antigen (ELA)-A2 haplotype MSCs and one control horse injected with donor MHC-matched ELA-A2 MSCs were utilised in this study. Antisera were incubated with ELA-A2 MSCs before adding complement in microcytotoxicity assays and cell death was analysed via eosin dye exclusion. ELA-A2 peripheral blood leucocytes (PBLs) were used in the assays as a positive control. Antisera from all 4 horses injected with MHC-mismatched MSCs contained antibodies that caused the death of ELA-A2 haplotype MSCs in the microcytotoxicity assays. In 2 of the 4 horses, antibodies were present as early as Day 7 post-injection. MSC death was consistently equivalent to that of ELA-A2 haplotype PBL death at all time points and antisera dilutions. Antisera from the control horse that was injected with MHC-matched MSCs did not contain cytotoxic ELA-A2 antibodies at any of the time points examined. This study examined MSC death in vitro only and utilized antisera from a small number of horses. The cytotoxic antibody response induced in recipient horses following injection with donor MHC-mismatched MSCs is capable of killing donor MSCs in vitro. These results suggest that the use of allogeneic MHC-mismatched MSCs must be cautioned against, not only for potential adverse events, but also for reduced therapeutic efficacy due to targeted MSC death. © 2016 The

Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab.

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Fujii, Rika; Friedman, Eitan R; Richards, Jacob; Tsang, Kwong Y; Heery, Christopher R; Schlom, Jeffrey; Hodge, James W

2016-06-07

Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 chordoma cell lines, first for expression of PD-L1, and in vitro for ADCC killing using NK cells and avelumab. PD-L1 expression was markedly upregulated by IFN-γ in all 4 chordoma cell lines, which significantly increased sensitivity to ADCC. Brachyury is a transcription factor that is uniformly expressed in chordoma. Clinical trials are ongoing in which chordoma patients are treated with brachyury-specific vaccines. Co-incubating chordoma cells with brachyury-specific CD8+ T cells resulted in significant upregulation of PD-L1 on the tumor cells, mediated by the CD8+ T cells' IFN-γ production, and increased sensitivity of chordoma cells to avelumab-mediated ADCC. Residential cancer stem cell subpopulations of chordoma cells were also killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell populations. These findings suggest that as a monotherapy for chordoma, avelumab may enable endogenous NK cells, while in combination with T-cell immunotherapy, such as a vaccine, avelumab may enhance NK-cell killing of chordoma cells via ADCC.

IgM-mediated opsonization and cytotoxicity in the shark.

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McKinney, E C; Flajnik, M F

1997-02-01

Two types of cytotoxic reactions have been observed using cells from the nurse shark: spontaneous cytotoxicity mediated by cells of the macrophage lineage and antibody-dependent killing carried out by a different effector cell population. Previous data showed that removal of phagocytic cells using iron particles abolished macrophage-mediated killing, but not antibody-dependent reactions. The current study used single cell assays and showed that the effector of antibody-driven reactions was the neutrophil. Surprisingly, the mechanism of killing was shown to be phagocytosis mediated by both 7S and 19S immunoglobulin M (IgM). Reactions proceeded with as little as 0.01 microg of purified 19S or 7S IgM and were complete within 4-6 h. In contrast, purified immunoglobulin did not adsorb to macrophages and had no effect on target cell binding or cytotoxicity. Pretreatment of cells with cytochalasin D abolished the phagocytic reaction, but not spontaneous cytotoxicity. These data show that antibody-mediated killing results from opsonization and phagocytosis; the mechanism of macrophage killing is currently unknown. In addition, these data show that the shark neutrophil, not the macrophage lineage, carries a receptor for Fc mu.

Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

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Sioud, Mouldy; Westby, Phuong; Vasovic, Vlada; Fløisand, Yngvar; Peng, Qian

2018-04-16

mAbs have emerged as a promising strategy for the treatment of cancer. However, in several malignancies, no effective antitumor mAbs are yet available. Identifying therapeutic mAbs that recognize common tumor antigens could render the treatment widely applicable. Here, a human single-chain variable fragment (scFv) antibody library was sequentially affinity selected against a panel of human cancer cell lines and an antibody fragment (named MS5) that bound to solid and blood cancer cells was identified. The MS5 scFv was fused to the human IgG1 Fc domain to generate an antibody (MS5-Fc fusion) that induced antibody-dependent cellular cytotoxicity and phagocytosis of cancer cells by macrophages. In addition, the MS5-Fc antibody bound to primary leukemia cells and induced antibody-dependent cellular cytotoxicity. In the majority of analyzed cancer cells, the MS5-Fc antibody induced cell surface redistribution of the receptor complexes, but not internalization, thus maximizing the accessibility of the IgG1 Fc domain to immune effector cells. In vitro stability studies showed that the MS5-Fc antibody was stable after 6 d of incubation in human serum, retaining ∼60% of its initial intact form. After intravenous injections, the antibody localized into tumor tissues and inhibited the growth of 3 different human tumor xenografts (breast, lymphoma, and leukemia). These antitumor effects were associated with tumor infiltration by macrophages and NK cells. In the Ramos B-cell lymphoma xenograft model, the MS5-Fc antibody exhibited a comparable antitumor effect as rituximab, a chimeric anti-CD20 IgG1 mAb. These results indicate that human antibodies with pan-cancer abilities can be generated from phage display libraries, and that the engineered MS5-Fc antibody could be an attractive agent for further clinical investigation.-Sioud, M., Westby, P., Vasovic, V., Fløisand, Y., Peng, Q. Development of a new high-affinity human antibody with antitumor activity against solid and

Interplay between Natural Killer Cells and Anti-HER2 Antibodies: Perspectives for Breast Cancer Immunotherapy

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Aura Muntasell

2017-11-01

Full Text Available Overexpression of the human epidermal growth factor receptor 2 (HER2 defines a subgroup of breast tumors with aggressive behavior. The addition of HER2-targeted antibodies (i.e., trastuzumab, pertuzumab to chemotherapy significantly improves relapse-free and overall survival in patients with early-stage and advanced disease. Nonetheless, considerable proportions of patients develop resistance to treatment, highlighting the need for additional and co-adjuvant therapeutic strategies. HER2-specific antibodies can trigger natural killer (NK cell-mediated antibody-dependent cellular cytotoxicity and indirectly enhance the development of tumor-specific T cell immunity; both mechanisms contributing to their antitumor efficacy in preclinical models. Antibody-dependent NK cell activation results in the release of cytotoxic granules as well as the secretion of pro-inflammatory cytokines (i.e., IFNγ and TNFα and chemokines. Hence, NK cell tumor suppressive functions include direct cytolytic killing of tumor cells as well as the regulation of subsequent antitumor adaptive immunity. Albeit tumors with gene expression signatures associated to the presence of cytotoxic lymphocyte infiltrates benefit from trastuzumab-based treatment, NK cell-related biomarkers of response/resistance to HER2-specific therapeutic antibodies in breast cancer patients remain elusive. Several variables, including (i the configuration of the patient NK cell repertoire; (ii tumor molecular features (i.e., estrogen receptor expression; (iii concomitant therapeutic regimens (i.e., chemotherapeutic agents, tyrosine kinase inhibitors; and (iv evasion mechanisms developed by progressive breast tumors, have been shown to quantitatively and qualitatively influence antibody-triggered NK cell responses. In this review, we discuss possible interventions for restoring/enhancing the therapeutic activity of HER2 therapeutic antibodies by harnessing NK cell antitumor potential through

Cellular cytotoxic response induced by highly purified multi-wall carbon nanotube in human lung cells.

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Tsukahara, Tamotsu; Haniu, Hisao

2011-06-01

Carbon nanotubes, a promising nanomaterial with unique characteristics, have applications in a variety of fields. The cytotoxic effects of carbon nanotubes are partially due to the induction of oxidative stress; however, the detailed mechanisms of nanotube cytotoxicity and their interaction with cells remain unclear. In this study, the authors focus on the acute toxicity of vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) by high-temperature thermal treatment. The authors exposed human bronchial epithelial cells (BEAS-2B) to HTT2800 and measured the cellular uptake, mitochondrial function, cellular LDH release, apoptotic signaling, reactive oxygen species (ROS) generation and pro-inflammatory cytokine release. The HTT2800-exposed cells showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. However, the exposed cells showed no obvious intracellular ROS generation. These cellular and molecular findings suggest that HTT2800 could cause a potentially adverse inflammatory response in BEAS-2B cells.

Trastuzumab mediates antibody-dependent cell-mediated cytotoxicity and phagocytosis to the same extent in both adjuvant and metastatic HER2/neu breast cancer patients.

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Petricevic, Branka; Laengle, Johannes; Singer, Josef; Sachet, Monika; Fazekas, Judit; Steger, Guenther; Bartsch, Rupert; Jensen-Jarolim, Erika; Bergmann, Michael

2013-12-12

Monoclonal antibodies (mAb), such as trastuzumab are a valuable addition to breast cancer therapy. Data obtained from neoadjuvant settings revealed that antibody-dependent cell-mediated cytotoxicity (ADCC) is a major mechanism of action for the mAb trastuzumab. Conflicting results still call into question whether disease progression, prolonged treatment or concomitant chemotherapy influences ADCC and related immunological phenomena. We analyzed the activity of ADCC and antibody-dependent cell-mediated phagocytosis (ADCP) of peripheral blood mononuclear cells (PBMCs) from human epidermal growth factor receptor 2 (HER2/neu) positive breast cancer patients receiving trastuzumab therapy either in an adjuvant (n = 13) or metastatic (n = 15) setting as well as from trastuzumab treatment-naive (t-naive) HER2/neu negative patients (n = 15). PBMCs from healthy volunteers (n = 24) were used as controls. ADCC and ADCP activity was correlated with the expression of antibody binding Fc-gamma receptor (FcγR)I (CD64), FcγRII (CD32) and FcγRIII (CD16) on CD14+ (monocytes) and CD56+ (NK) cells, as well as the expression of CD107a+ (LAMP-1) on CD56+ cells and the total amount of CD4+CD25+FOXP3+ (Treg) cells. In metastatic patients, markers were correlated with progression-free survival (PFS). ADCC activity was significantly down regulated in metastatic, adjuvant and t-naive patient cohorts as compared to healthy controls. Reduced ADCC activity was inversely correlated with the expression of CD107a on CD56+ cells in adjuvant patients. ADCC and ADCP activity of the patient cohorts were similar, regardless of treatment duration or additional chemotherapy. PFS in metastatic patients inversely correlated with the number of peripheral Treg cells. The reduction of ADCC in patients as compared to healthy controls calls for adjuvant strategies, such as immune-enhancing agents, to improve the activity of trastuzumab. However, efficacy of trastuzumab-specific ADCC and ADCP appears not to

Antibody- and TRIM21-dependent intracellular restriction of Salmonella enterica.

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Rakebrandt, Nikolas; Lentes, Sabine; Neumann, Heinz; James, Leo C; Neumann-Staubitz, Petra

2014-11-01

TRIM21 ('tripartite motif-containing protein 21', Ro52) is a ubiquitously expressed cytosolic Fc receptor, which has a potent role in protective immunity against nonenveloped viruses. TRIM21 mediates intracellular neutralisation of antibody-coated viruses, a process called ADIN (antibody-dependent intracellular neutralisation). Our results reveal a similar mechanism to fight bacterial infections. TRIM21 is recruited to the intracellular pathogen Salmonella enterica in epithelial cells early in infection. TRIM21 does not bind directly to S. enterica, but to antibodies opsonising it. Most importantly, bacterial restriction is dependent on TRIM21 as well as on the opsonisation state of the bacteria. Finally, Salmonella and TRIM21 colocalise with the autophagosomal marker LC3, and intracellular defence is enhanced in starved cells suggesting an involvement of the autophagocytic pathway. Our data extend the protective role of TRIM21 from viruses to bacteria and thereby strengthening the general role of ADIN in cellular immunity. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Differential effects of IL-2 and IL-21 on expansion of the CD4+ CD25+ Foxp3+ T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia.

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Gowda, Aruna; Ramanunni, Asha; Cheney, Carolyn; Rozewski, Darlene; Kindsvogel, Wayne; Lehman, Amy; Jarjoura, David; Caligiuri, Michael; Byrd, John C; Muthusamy, Natarajan

2010-01-01

CD4(+) CD25(+) regulatory T cells are expanded in solid and hematological malignancies including chronic lymphocytic leukemia (CLL). Several cytokines and co-stimulatory molecules are required for generation, survival and maintenance of their suppressive effect. We and others have shown direct cytotoxic effect of the novel common gamma chain cytokine interleukin (IL)-21 on primary B cells from CLL patients. Since members of this family of cytokines are known to exhibit their effects on diverse immune cells, we have examined the effects of IL-21 on CLL patient derived regulatory T cell (Treg) induction, expansion and the inhibitory effect on natural killer cells in vitro. We demonstrate here the expression of IL-21 receptor in CD4(+)CD25(High) regulatory cells from CLL patients. In contrast to IL-2, the IL-21 cytokine failed to mediate expansion of regulatory T cells or induced expression of Foxp3 in CD4(+)CD25(Intermediate) or CD4(+)CD25(Dim/-) T cells in whole blood derived from CLL patients. Interestingly, in contrast to their differential effects on expansion of the CD4(+)CD25(+)Foxp3(+)T cells, IL-2 and IL-21 exhibited a redundant role in Treg mediated suppression of NK cell mediated antibody dependent cytotoxicity function. Given the infusion related toxicities and pro-survival effect of IL-2 in CLL, these studies provide a rationale to explore IL-21 as an alternate gamma chain cytokine in CLL therapy.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

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Richardson, Simone I; Chung, Amy W; Natarajan, Harini; Mabvakure, Batsirai; Mkhize, Nonhlanhla N; Garrett, Nigel; Abdool Karim, Salim; Moore, Penny L; Ackerman, Margaret E; Alter, Galit; Morris, Lynn

2018-04-01

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

Synergistic anti-tumor therapy by a comb-like multifunctional antibody nanoarray with exceptionally potent activity

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Li, Huafei; Sun, Yun; Chen, Di; Zhao, He; Zhao, Mengxin; Zhu, Xiandi; Ke, Changhong; Zhang, Ge; Jiang, Cheng; Zhang, Li; Zhang, Fulei; Wei, Huafeng; Li, Wei

2015-10-01

Simultaneously blocking multiple mediators offers new hope for the treatment of complex diseases. However, the curative potential of current combination therapy by chronological administration of separate monoclonal antibodies (mAbs) or multi-specific mAbs is still moderate due to inconvenient manipulation, low cooperative effectors, poor pharmacokinetics and insufficient tumor accumulation. Here, we describe a facile strategy that arms distinct mAbs with cooperative effectors onto a long chain to form a multicomponent comb-like nano mAb. Unlike dissociative parental mAbs, the multifunctional mAb nanoarray (PL-RB) constructed from type I/II anti-CD20 mAbs shows good pharmacokinetics. This PL-RB simultaneously targets distinct epitopes on a single antigen (Ag) and neighboring Ags on different lymphocytes. This unique intra- and intercellular Ag cross-linking endows the multifunctional mAb nanoarray with potent apoptosis activity. The exceptional apoptosis, complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) that are synchronously evoked by the nano PL-RB are further synergistically promoted via enhanced permeability and retention (EPR), which resulted in high intratumor accumulation and excellent anti-lymphoma efficiency.

Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies.

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Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

2015-01-01

Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.

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Fan, W-Y; Wang, D-P; Wen, Q; Fan, T-J

2017-08-01

Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.

Cellular distribution of inorganic mercury and its relation to cytotoxicity in bovine kidney cell cultures

International Nuclear Information System (INIS)

Bracken, W.M.; Sharma, R.P.; Bourcier, D.R.

1984-01-01

A bovine kidney cell culture system was used to assess what relationship mercuric chloride (HgCl 2 ) uptake and subcellular distribution had to cytotoxicity. Twenty-four-hour incubations with 0.05-50 μM HgCl 2 elicited a concentration-related cytotoxicity. Cellular accumulation of 203 Hg was also concentration-related, with 1.0 nmol/10 6 cells at the IC50. Measurement of Hg uptake over the 24-h exposure period revealed a multiphasic process. Peak accumulation was attained by 1 h and was followed by extrusion and plateauing of intracellular Hg levels. Least-squares regression analysis of the cytotoxicity and cellular uptake data indicated a potential relationship between the Hg uptake and cytotoxicity. However, the subcellular distribution of Hg was not concentration-related. Mitochondria and soluble protein fractions accounted for greater than 65% of the cell-associated Hg at all concentrations. The remaining Hg was distributed between the microsomal (6-10%) and nuclear and cell debris (11-22%) fractions at all concentrations tested. Less than 20% of the total cell-associated Hg was bound with metallothionein-like protein. 31 references, 4 figures, 3 tables

Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

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Choi, Seon Young; Jang, Soo Hwa [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of); Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su [Soongsil University, Department of Chemistry (Korea, Republic of); Lee, Kangtaek [Yonsei University, Department of Chemical and Biomolecular Engineering (Korea, Republic of); Yang, Sung Ik [Kyung Hee University, College of Environment and Applied Chemistry (Korea, Republic of); Joo, Sang-Woo, E-mail: sjoo@ssu.ac.kr [Soongsil University, Department of Chemistry (Korea, Republic of); Ryu, Pan Dong; Lee, So Yeong, E-mail: leeso@snu.ac.kr [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of)

2012-12-15

Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

International Nuclear Information System (INIS)

Choi, Seon Young; Jang, Soo Hwa; Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su; Lee, Kangtaek; Yang, Sung Ik; Joo, Sang-Woo; Ryu, Pan Dong; Lee, So Yeong

2012-01-01

Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

Selective cytotoxicity of an oxygen-radical-generating enzyme conjugated to a monoclonal antibody.

Science.gov (United States)

Battelli, M G; Abbondanza, A; Tazzari, P L; Dinota, A; Rizzi, S; Grassi, G; Gobbi, M; Stirpe, F

1988-07-01

The monoclonal antibody 8A, which recognizes a human plasma cell-associated antigen, was covalently linked to xanthine oxidase in a conjugate maintaining both immunological and enzymatic properties. A significant degree of target cell lysis was obtained at an enzyme concentration that was ineffective on non-target cells and on myeloid staminal cells (CFU-GM). The cytotoxic activity was abolished by an excess of antibody, by allopurinol and by superoxide dismutase and catalase. A possible use of the conjugate for bone marrow purging in multiple myeloma patients is suggested.

ZnO nanofluids for the improved cytotoxicity and cellular uptake of doxorubicin

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Safoura Soleymani

2018-01-01

Full Text Available Objective(s: Combination anticancer therapy holds promise for improving the therapeutic efficacy of chemotherapy drugs such as doxorubicin (DOX as well as decreasing their dose-limiting side effects. Overcoming the side effects of doxorubicin (DOX is a major challenge to the effective treatment of cancer. Zinc oxide nanoparticles (ZnO NPs are emerging as potent tools for a wide variety of biomedical applications. The aim of this study was to develop a combinatorial approach for enhancing the anticancer efficacy and cellular uptake of DOX. Materials and Methods: ZnO NPs were synthesized by the solvothermal method and were characterized by X-ray diffraction (XRD, dynamic light scattering (DLS and transmission electron microscopy (TEM. ZnO NPs were dispersed in 10% bovine serum albumin (BSA and the cytotoxic effect of the resulting ZnO nanofluids was evaluated alone and in combination with DOX on DU145 cells. The influence of ZnO nanofluids on the cellular uptake of DOX and DOX-induced catalase mRNA expression were investigated by fluorescence microscopy and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR, respectively. Results: The MTT results revealed that ZnO nanofluids decreased the cell viability of DU145 cells in a timeand dose-dependent manner. Simultaneous combination treatment of DOX and ZnO nanofluid showed a significant increase in anticancer activity and the cellular uptake of DOX compared to DOX alone. Also, a time-dependent reduction of catalase mRNA expression was observed in the cells treated with ZnO nanofluids and DOX, alone and in combination with each other. Conclusion: These results indicate the role of ZnO nanofluid as a growth-inhibitory agent and a drug delivery system for DOX in DU145 cells. Thus, ZnO nanofluid could be a candidate for combination chemotherapy.

Immunological low-dose radiation modulates the pediatric medulloblastoma antigens and enhances antibody-dependent cellular cytotoxicity.

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Das, Arabinda; McDonald, Daniel; Lowe, Stephen; Bredlau, Amy-Lee; Vanek, Kenneth; Patel, Sunil J; Cheshier, Samuel; Eskandari, Ramin

2017-03-01

Immunotherapy can be an effective treatment for pediatric medulloblastoma (MB) patients. However, major subpopulations do not respond to immunotherapy, due to the lack of antigenic mutations or the immune-evasive properties of MB cells. Clinical observations suggest that radiation therapy (RT) may expand the therapeutic reach of immunotherapy. The aim of the present investigation is to study the effect of low-dose X-ray radiation (LDXR, 1 Gy) on the functional immunological responses of MB cells (DAOY, D283, and D341). Induction of MB cell death was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Production of reactive oxygen species (ROS) was measured by fluorescent probes. Changes in the expression of  human leukocyte antigen (HLA) molecules and caspase-3 activities during treatment were analyzed using Western blotting and caspase-3 assay. Western blot analysis demonstrated that LDXR upregulated the expression of HLA class I and HLA II molecules by more than 20% compared with control and high-dose (12 Gy) groups in vitro. Several of these HLA subtypes, such as MAGE C1, CD137, and ICAM-1, have demonstrated upregulation. In addition, LDXR increases ROS production in association with phosphorylation of NF-κB and cell surface expression of mAb target molecules (HER2 and VEGF). These data suggest that a combined LDXR and mAb therapy can create a synergistic effect in vitro. These results suggest that LDXR modulates HLA molecules, leading to alterations in T-cell/tumor-cell interaction and enhancement of T-cell-mediated MB cell death. Also, low-dose radiotherapy combined with monoclonal antibody therapy may one day augment the standard treatment for MB, but more investigation is needed to prove its utility as a new therapeutic combination for MB patients.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

Directory of Open Access Journals (Sweden)

Simone I Richardson

2018-04-01

Full Text Available While the induction of broadly neutralizing antibodies (bNAbs is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP, complement deposition (ADCD, cellular cytotoxicity (ADCC and cellular trogocytosis (ADCT were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.

Science.gov (United States)

Liu, Zhi; Leng, Esther C; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir; Liu, Hua; Fanslow, William; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio; Foltz, Ian N; Yan, Wei

2015-03-20

Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

Science.gov (United States)

Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

2013-01-01

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

HIV antibodies for treatment of HIV infection.

Science.gov (United States)

Margolis, David M; Koup, Richard A; Ferrari, Guido

2017-01-01

The bar is high to improve on current combination antiretroviral therapy (ART), now highly effective, safe, and simple. However, antibodies that bind the HIV envelope are able to uniquely target the virus as it seeks to enter new target cells, or as it is expressed from previously infected cells. Furthermore, the use of antibodies against HIV as a therapeutic may offer advantages. Antibodies can have long half-lives, and are being considered as partners for long-acting antiretrovirals for use in therapy or prevention of HIV infection. Early studies in animal models and in clinical trials suggest that such antibodies can have antiviral activity but, as with small-molecule antiretrovirals, the issues of viral escape and resistance will have to be addressed. Most promising, however, are the unique properties of anti-HIV antibodies: the potential ability to opsonize viral particles, to direct antibody-dependent cellular cytotoxicity (ADCC) against actively infected cells, and ultimately the ability to direct the clearance of HIV-infected cells by effector cells of the immune system. These distinctive activities suggest that HIV antibodies and their derivatives may play an important role in the next frontier of HIV therapeutics, the effort to develop treatments that could lead to an HIV cure. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Irradiation and various cytotoxic drugs enhance tyrosine phosphorylation and β1-integrin clustering in human A549 lung cancer cells in a substratum-dependent manner in vitro

International Nuclear Information System (INIS)

Cordes, N.; Beinke, C.; Beuningen, D. van; Plasswilm, L.

2004-01-01

Background and purpose: interactions of cells with a substratum, especially extracellular matrix proteins, initiate clustering of integrin receptors in the cell membrane. This process represents the initial step for the activation of signaling pathways regulating survival, proliferation, differentiation, adhesion, and migration, and could, furthermore, be important for cellular resistance-mediating mechanisms against radiation or cytotoxic drugs. The lack of data elucidating the impact of irradiation or cytotoxic drugs on this important phenomenon led to this study on human A549 lung cancer cells in vitro. Material and methods: the human lung carcinoma cell line A549 grown on polystyrene or fibronectin (FN) was irradiated with 0-8 Gy or treated with cisplatin (0.1-50 μM), paclitaxel (0.1-50 nM), or mitomycin (0.1-50 μM). Colony formation assays, immunofluorescence staining in combination with activation of integrin clustering using anti-β 1 -integrin antibodies (K20), and Western blotting for tyrosine phosphorylation under treatment of cells with the IC 50 for irradiation (2 Gy; IC 50 = 2.2 Gy), cisplatin (2 μM), paclitaxel (5 nM), or mitomycin (7 μM) were performed. Results: attachment of cells to FN resulted in a significantly reduced radio- and chemosensitivity compared to polystyrene. The clustering of β 1 -integrins examined by immunofluorescence staining was only stimulated by irradiation, cisplatin, paclitaxel, or mitomycin in case of cell attachment to FN. By contrast, tyrosine phosphorylation, as one of the major events following β 1 -integrin clustering, showed a 3.7-fold, FN-related enhancement, and treatment of cells with the IC 50 of radiation, cisplatin, paclitaxel, or mitomycin showed a substratum-dependent induction. Conclusion: for the first time, a strong influence of irradiation and a variety of cytotoxic drugs on the clustering of β 1 -integrins could be shown. This event is a prerequisite for tyrosine phosphorylation and, thus, the

Cellular defense against UVB-induced phototoxicity by cytosolic NADP+-dependent isocitrate dehydrogenase

International Nuclear Information System (INIS)

Jo, Seung-Hee; Lee, So-Hyun; Suk Chun, Hang; Min Lee, Su; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

2002-01-01

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury

Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase.

Science.gov (United States)

Jo, Seung-Hee; Lee, So-Hyun; Chun, Hang Suk; Lee, Su Min; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

2002-03-29

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury. (c)2002 Elsevier Science (USA).

Preclinical Characterization of a Novel Monoclonal Antibody NEO-201 for the Treatment of Human Carcinomas

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Massimo Fantini

2018-01-01

Full Text Available NEO-201 is a novel humanized IgG1 monoclonal antibody that was derived from an immunogenic preparation of tumor-associated antigens from pooled allogeneic colon tumor tissue extracts. It was found to react against a variety of cultured human carcinoma cell lines and was highly reactive against the majority of tumor tissues from many different carcinomas, including colon, pancreatic, stomach, lung, and breast cancers. NEO-201 also exhibited tumor specificity, as the majority of normal tissues were not recognized by this antibody. Functional assays revealed that treatment with NEO-201 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC and complement-dependent cytotoxicity (CDC against tumor cells. Furthermore, the growth of human pancreatic xenograft tumors in vivo was largely attenuated by treatment with NEO-201 both alone and in combination with human peripheral blood mononuclear cells as an effector cell source for ADCC. In vivo biodistribution studies in human tumor xenograft-bearing mice revealed that NEO-201 preferentially accumulates in the tumor but not organ tissue. Finally, a single-dose toxicity study in non-human primates demonstrated safety and tolerability of NEO-201, as a transient decrease in circulating neutrophils was the only related adverse effect observed. These findings indicate that NEO-201 warrants clinical testing as both a novel diagnostic and therapeutic agent for the treatment of a broad variety of carcinomas.

A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

Science.gov (United States)

Fujii, Rika; Schlom, Jeffrey; Hodge, James W

2018-05-01

OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

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Kim, Sun Yee; Park, Jeen-Woo

2003-03-01

Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

Evaluation of the Cytotoxicity and Genotoxicity of Flavonolignans in Different Cellular Models

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Michal Bijak

2017-12-01

Full Text Available Flavonolignans are the main components of silymarin, which represents 1.5–3% of the dry fruit weight of Milk thistle (Silybum marianum L. Gaernt.. In ancient Greece and Romania, physicians and herbalists used the Silybum marianum to treat a range of liver diseases. Besides their hepatoprotective action, silymarin flavonolignans have many other healthy properties, such as anti-platelet and anti-inflammatory actions. The aim of this study was to evaluate the toxic effect of flavonolignans on blood platelets, peripheral blood mononuclear cells (PBMCs and human lung cancer cell line—A549—using different molecular techniques. We established that three major flavonolignans: silybin, silychristin and silydianin, in concentrations of up to 100 µM, have neither a cytotoxic nor genotoxic effect on blood platelets, PMBCs and A549. We also saw that silybin and silychristin have a protective effect on cellular mitochondria, observed as a reduction of spontaneous mitochondrial DNA (mtDNA damage in A549, measured as mtDNA copies, and mtDNA lesions in ND1 and ND5 genes. Additionally, we observed that flavonolignans increase the blood platelets’ mitochondrial membrane potential and reduce the generation of reactive oxygen species in blood platelets. Our current findings show for the first time that the three major flavonolignans, silybin, silychristin and silydianin, do not have any cytotoxicity and genotoxicity in various cellular models, and that they actually protect cellular mitochondria. This proves that the antiplatelet and anti-inflammatory effect of these compounds is part of our molecular health mechanisms.

Macrophage and NK-mediated killing of precursor-B acute lymphoblastic leukemia cells targeted with a-fucosylated anti-CD19 humanized antibodies.

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Matlawska-Wasowska, K; Ward, E; Stevens, S; Wang, Y; Herbst, R; Winter, S S; Wilson, B S

2013-06-01

This work reports the tumoricidal effects of a novel investigational humanized anti-CD19 monoclonal antibody (Medi-551). An a-fucosylated antibody with increased affinity for human FcγRIIIA, Medi-551 is shown to mediate both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly (>5 h) and thus remain accessible to effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human natural killer (NK) cells and macrophages against precursor-B (pre-B) acute lymphoblastic leukemia (ALL) cell lines and pediatric patient blasts. Fluorescent imaging studies document immunological synapses formed between anti-CD19-bound target leukemia cells and effector cells and capture the kinetics of both NK-mediated killing and macrophage phagocytosis. Genetic polymorphisms in FcγRIIIA-158F/V modulate in vitro activities of effector cells, with FcγRIIIA-158V homozygotes or heterozygotes showing the strongest activity. Medi-551 treatment of severe combined immunodeficiency (SCID) mice engrafted with human pre-B cells led to prolonged animal survival and markedly reduced disease burden in blood, liver and bone marrow. These data show that anti-CD19 antibodies effectively recruit immune cells to pre-B ALL cells and support a move forward to early phase trials in this disease.

Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors

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Floyd Hassenrück

2018-01-01

Full Text Available 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.

The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles

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Fröhlich E

2012-11-01

Full Text Available Eleonore FröhlichCenter for Medical Research, Medical University of Graz, Graz, AustriaAbstract: Many types of nanoparticles (NPs are tested for use in medical products, particularly in imaging and gene and drug delivery. For these applications, cellular uptake is usually a prerequisite and is governed in addition to size by surface characteristics such as hydrophobicity and charge. Although positive charge appears to improve the efficacy of imaging, gene transfer, and drug delivery, a higher cytotoxicity of such constructs has been reported. This review summarizes findings on the role of surface charge on cytotoxicity in general, action on specific cellular targets, modes of toxic action, cellular uptake, and intracellular localization of NPs. Effects of serum and intercell type differences are addressed. Cationic NPs cause more pronounced disruption of plasma-membrane integrity, stronger mitochondrial and lysosomal damage, and a higher number of autophagosomes than anionic NPs. In general, nonphagocytic cells ingest cationic NPs to a higher extent, but charge density and hydrophobicity are equally important; phagocytic cells preferentially take up anionic NPs. Cells do not use different uptake routes for cationic and anionic NPs, but high uptake rates are usually linked to greater biological effects. The different uptake preferences of phagocytic and nonphagocytic cells for cationic and anionic NPs may influence the efficacy and selectivity of NPs for drug delivery and imaging.Keywords: endocytosis, plasma membrane, lysosomes, polystyrene particles, quantum dots, dendrimers

Structure-cytotoxicity relationships for dietary flavonoids

DEFF Research Database (Denmark)

Breinholt, V.; Dragsted, L.O.

1998-01-01

The cytotoxicity of a large series of dietary flavonoids was tested in a non-tumorigenic mouse and two human cancer cell lines, using the neutral red dye exclusion assay. All compounds tested exhibited a concentration-dependent cytotoxic action in the employed cell lines. The relative cytotoxicity...... of the flavonoids, however, Tvas found to vary greatly among the different cell Lines. With a few exceptions, the investigated flavonoids were more cytotoxic to the human cancer cell lines, than the mouse cell line. The differences in cytotoxicity were accounted for in part by differences in cellular uptake...... and metabolic capacity among the different cell types. In 3T3 cells fairly consistent structure-cytotoxicity relationships were found. The most cytotoxic structures tested in 3T3 cells were flavonoids with adjacent 3',4' hydroxy groups on the B-ring, such as luteolin, quercetin, myricetin, fisetin, eriodictyol...

Antibody drug conjugates and bystander killing: is antigen-dependent internalisation required?

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Staudacher, Alexander H; Brown, Michael P

2017-12-05

Antibody drug conjugates (ADCs) employ the exquisite specificity of tumour-specific monoclonal antibodies (mAb) for the targeted delivery of highly potent cytotoxic drugs to the tumour site. The chemistry of the linker, which connects the drug to the mAb, determines how and when the drug is released from the mAb. This, as well as the chemistry of the drug, can dictate whether the drug can diffuse into surrounding cells, resulting in 'bystander killing'. Initially, any bystander killing mechanism of action of an ADC was understood to involve an essential sequence of steps beginning with surface antigen targeting, internalisation, intracellular linker cleavage, drug release, and diffusion of drug away from the targeted cell. However, recent studies indicate that, depending on the linker and drug combination, this mechanism may not be essential and ADCs can be cleaved extracellularly or via other mechanisms. In this minireview, we will examine the role of bystander killing by ADCs and explore the emerging evidence of how this can occur independently of internalisation.

Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control.

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Margaret E Ackerman

2016-01-01

Full Text Available Elite controllers (ECs represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune-recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.

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.

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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

Cytotoxicity and cellular uptake of pyrimidine nucleosides for imaging herpes simplex type-1 thymidine kinase (HSV-1 TK) expression in mammalian cells

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Morin, Kevin W.; Duan Weili; Xu Lihua; Zhou Aihua; Moharram, Sameh; Knaus, Edward E.; McEwan, Alexander J.B.; Wiebe, Leonard I. E-mail: leonard.wiebe@ualberta.ca

2004-07-01

In vivo transfer of the herpes simplex virus type-1 thymidine kinase (HSV-1 TK) gene, with subsequent administration of antiviral drugs such as ganciclovir, has emerged as a promising gene therapy protocol for treating proliferative disorders. The in vitro cytotoxicities (IC{sub 50}) for two series of 5-iodo- and (E)-5-(2-iodovinyl)-substituted 2'-deoxy- and 2'-deoxy-2'-fluoro-pyrimidine nucleosides ranged from millimolar to low nanomolar concentrations in mammalian tumor cell lines (KBALB; R-970-5; 143B; EMT-6) and their counterparts engineered to express HSV-1 TK (KBALB-STK; 143B-LTK). Their HSV-1 TK selectivity indices ranged from one (nonselective) to one million (highly selective) based on cytotoxicity, with FIRU being the least toxic to all cell lines, and FIAU being most toxic. HSV-1 TK selectivity, based on uptake, ranged from 10 to 140, with IVDU being most selective for HSV-1 TK expressing cells, followed by IVFRU, FIRU, FIAU, IVFAU and finally IUDR. Phosphorylation of [{sup 125}I]FIAU led to incorporation of the radiolabel into nucleic acids, whereas IVFRU and FIRU radioactivity was trapped primarily in the nucleotide pool. These data indicate that cytotoxicity does not depend on initial metabolic trapping (e.g., phosphorylation), but on elaboration of the mononucleotides to more cytotoxic anabolites. Lipophilicities and nucleoside transport rates of the six nucleosides tested were within narrow ranges. This supports the premise that cellular biochemistry, and not cellular bioavailability, is responsible for the observed broad range of cytotoxicity and trapping. In vivo biodistribution studies with 5-[{sup 125}I]iodo-2'-fluoro-2'-deoxyribouridine (FIRU), 5-[{sup 125}I]iodo-2'-fluoro-2'-deoxyarabinouridine (FIAU) and (E)-5-(2-[{sup 125}I]iodovinyl)-2'-fluoro-2'-deoxyuridine (IVFRU) demonstrate selective accumulation of all three radiotracers in HSV-1 TK-expressing KBABK-STK tumors, compared to their very low

Health and Cellular Impacts of Air Pollutants: From Cytoprotection to Cytotoxicity

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Karine Andreau

2012-01-01

Full Text Available Air pollution as one of the ravages of our modern societies is primarily linked to urban centers, industrial activities, or road traffic. These atmospheric pollutants have been incriminated in deleterious health effects by numerous epidemiological and in vitro studies. Environmental air pollutants are a heterogeneous mixture of particles suspended into a liquid and gaseous phase which trigger the disruption of redox homeostasis—known under the term of cellular oxidative stress—in relation with the establishment of inflammation and cell death via necrosis, apoptosis, or autophagy. Activation or repression of the apoptotic process as an adaptative response to xenobiotics might lead to either acute or chronic toxicity. The purpose of this paper is to highlight the central role of oxidative stress induced by air pollutants and to focus on the subsequent cellular impacts ranging from cytoprotection to cytotoxicity by decreasing or stimulating apoptosis, respectively.

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

International Nuclear Information System (INIS)

Ristic, Biljana; Bosnjak, Mihajlo; Arsikin, Katarina; Mircic, Aleksandar; Suzin-Zivkovic, Violeta; Bogdanovic, Andrija; Perovic, Vladimir; Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

2014-01-01

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade. �

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

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Ristic, Biljana [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Bosnjak, Mihajlo [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Arsikin, Katarina [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Mircic, Aleksandar; Suzin-Zivkovic, Violeta [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Bogdanovic, Andrija [Clinic for Hematology, Clinical Centre of Serbia, School of Medicine, University of Belgrade, Belgrade (Serbia); Perovic, Vladimir [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Trajkovic, Vladimir, E-mail: vtrajkovic@med.bg.ac.rs [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Harhaji-Trajkovic, Ljubica, E-mail: buajk@yahoo.com [Institute for Biological Research, University of Belgrade, Belgrade, Despot Stefan Blvd. 142, 11000 Belgrade (Serbia)

2014-08-01

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade. �

Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

DEFF Research Database (Denmark)

Møller-Larsen, A; Brudek, T; Petersen, T

2013-01-01

on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used...

Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies

NARCIS (Netherlands)

Burgess, Janette K.; Lopez, Jose A.; Gaudry, Leonie E.; Chong, Beng H.

2000-01-01

The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because

High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines.

Science.gov (United States)

Martinez, Emily M; Klebanoff, Samuel D; Secrest, Stephanie; Romain, Gabrielle; Haile, Samuel T; Emtage, Peter C R; Gilbert, Amy E

2018-04-01

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects.

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Johannes S Gach

Full Text Available Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5 boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART. Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC. We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir.

Cellular uptake and cytotoxic potential of respirable bentonite particles with different quartz contents and chemical modifications in human lung fibroblasts

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Geh, Stefan; Rettenmeier, Albert W.; Dopp, Elke [University Hospital, Institute of Hygiene and Occupational Medicine, Essen (Germany); Yuecel, Raif [University Hospital, Institute of Cell Biology (Cancer Research), Essen (Germany); Duffin, Rodger [Institute of Environmental Health Research (IUF), Duesseldorf (Germany); University of Edinburgh, ELEGI COLT Lab, Scotland (United Kingdom); Albrecht, Catrin; Borm, Paul J.A. [Institute of Environmental Health Research (IUF), Duesseldorf (Germany); Armbruster, Lorenz [Verein fuer Technische Sicherheit und Umweltschutz e.V., Gotha (Germany); Raulf-Heimsoth, Monika; Bruening, Thomas [Research Institute for Occupational Medicine of the Institutions for Statutory Accident Insurance and Prevention (BGFA), Bochum (Germany); Hoffmann, Eik [University of Rostock, Institute of Biology, Department of Cell Biology and Biosystems Technology, Rostock (Germany)

2006-02-01

Considering the biological reactivity of pure quartz in lung cells, there is a strong interest to clarify the cellular effects of respirable siliceous dusts, like bentonites. In the present study, we investigated the cellular uptake and the cytotoxic potential of bentonite particles (Oe< 10 {mu}m) with an {alpha}-quartz content of up to 6% and different chemical modifications (activation: alkaline, acidic, organic) in human lung fibroblasts (IMR90). Additionally, the ability of the particles to induce apoptosis in IMR90-cells and the hemolytic activity was tested. All bentonite samples were tested for endotoxins with the in vitro-Pyrogen test and were found to be negative. Cellular uptake of particles by IMR90-cells was studied by transmission electron microscopy (TEM). Cytotoxicity was analyzed in IMR90-cells by determination of viable cells using flow cytometry and by measuring of the cell respiratory activity. Induced apoptotic cells were detected by AnnexinV/Propidiumiodide-staining and gel electrophoresis. Our results demonstrate that activated bentonite particles are better taken up by IMR90-cells than untreated (native) bentonite particles. Also, activated bentonite particles with a quartz content of 5-6% were more cytotoxic than untreated bentonites or bentonites with a quartz content lower than 4%. The bentonite samples induced necrotic as well as apoptotic cell death. In general, bentonites showed a high membrane-damaging potential shown as hemolytic activity in human erythrocytes. We conclude that cellular effects of bentonite particles in human lung cells are enhanced after chemical treatment of the particles. The cytotoxic potential of the different bentonites is primarily characterized by a strong lysis of the cell membrane. (orig.)

CD44v10, osteopontin and lymphoma growth retardation by a CD44v10-specific antibody.

Science.gov (United States)

Megaptche, Amelie Pajip; Erb, Ulrike; Büchler, Markus Wolfgang; Zöller, Margot

2014-09-01

Blockade of CD44 is considered a therapeutic option for the elimination of leukemia-initiating cells. However, the application of anti-panCD44 can be burdened by severe side effects. We determined whether these side effects could be avoided by replacing anti-panCD44 with CD44 variant isoform (CD44v)-specific antibodies in CD44v-positive hematological malignancies using the EL4 thymoma and CD44v10-transfected EL4 (EL4-v10) as models. Subcutaneous growth of EL4 and EL4-v10 was equally well inhibited by the anti-panCD44 and anti-CD44v10 antibodies, respectively. Ex vivo analysis indicated that natural killer cytotoxicity and antibody-dependent cellular cytotoxicity were the main effector mechanisms. Under local inflammation, the efficacy of anti-CD44v10 prolonged the survival time twofold compared with untreated, EL4-v10 tumor-bearing mice, and this was due to inflammation-induced expression of osteopontin (OPN). A high level of OPN in EL4-v10 tumors supported leukocyte recruitment and tumor-infiltrating T-cell activation. Taken together, in hematological malignancies expressing CD44v, anti-panCD44 can be replaced by CD44v-specific antibodies without a loss in efficacy. Furthermore, CD44v10-specific antibodies appear particularly advantageous in cutaneous leukemia therapy, as CD44v10 binding of OPN drives leukocyte recruitment and activation.

Cisplatin Induces a Mitochondrial-ROS Response That Contributes to Cytotoxicity Depending on Mitochondrial Redox Status and Bioenergetic Functions

Science.gov (United States)

Marullo, Rossella; Werner, Erica; Degtyareva, Natalya; Moore, Bryn; Altavilla, Giuseppe; Ramalingam, Suresh S.; Doetsch, Paul W.

2013-01-01

Cisplatin is one of the most effective and widely used anticancer agents for the treatment of several types of tumors. The cytotoxic effect of cisplatin is thought to be mediated primarily by the generation of nuclear DNA adducts, which, if not repaired, cause cell death as a consequence of DNA replication and transcription blockage. However, the ability of cisplatin to induce nuclear DNA (nDNA) damage per se is not sufficient to explain its high degree of effectiveness nor the toxic effects exerted on normal, post-mitotic tissues. Oxidative damage has been observed in vivo following exposure to cisplatin in several tissues, suggesting a role for oxidative stress in the pathogenesis of cisplatin-induced dose-limiting toxicities. However, the mechanism of cisplatin-induced generation of ROS and their contribution to cisplatin cytotoxicity in normal and cancer cells is still poorly understood. By employing a panel of normal and cancer cell lines and the budding yeast Saccharomyces cerevisiae as model system, we show that exposure to cisplatin induces a mitochondrial-dependent ROS response that significantly enhances the cytotoxic effect caused by nDNA damage. ROS generation is independent of the amount of cisplatin-induced nDNA damage and occurs in mitochondria as a consequence of protein synthesis impairment. The contribution of cisplatin-induced mitochondrial dysfunction in determining its cytotoxic effect varies among cells and depends on mitochondrial redox status, mitochondrial DNA integrity and bioenergetic function. Thus, by manipulating these cellular parameters, we were able to enhance cisplatin cytotoxicity in cancer cells. This study provides a new mechanistic insight into cisplatin-induced cell killing and may lead to the design of novel therapeutic strategies to improve anticancer drug efficacy. PMID:24260552

Anti-influenza Hyperimmune Immunoglobulin Enhances Fc-functional Antibody Immunity during Human Influenza Infection.

Science.gov (United States)

Vanderven, Hillary A; Wragg, Kathleen; Ana-Sosa-Batiz, Fernanda; Kristensen, Anne B; Jegaskanda, Sinthujan; Wheatley, Adam K; Wentworth, Deborah; Wines, Bruce D; Hogarth, P Mark; Rockman, Steve; Kent, Stephen J

2018-05-31

New treatments for severe influenza are needed. Passive transfer of influenza-specific hyperimmune pooled immunoglobulin (Flu-IVIG) boosts neutralising antibody responses to past strains in influenza-infected subjects. The effect of Flu-IVIG on antibodies with Fc-mediated functions, which may target diverse influenza strains, is unclear. We studied the capacity of Flu-IVIG, relative to standard IVIG, to bind to Fc receptors and mediate antibody-dependent cellular cytotoxicity in vitro. The effect of Flu-IVIG infusion, compared to placebo infusion, was examined in serial plasma samples from 24 subjects with confirmed influenza infection in the INSIGHT FLU005 pilot study. Flu-IVIG contains higher concentrations of Fc-functional antibodies than IVIG against a diverse range of influenza hemagglutinins. Following infusion of Flu-IVIG into influenza-infected subjects, a transient increase in Fc-functional antibodies was present for 1-3 days against infecting and non-infecting strains of influenza. Flu-IVIG contains antibodies with Fc-mediated functions against influenza virus and passive transfer of Flu-IVIG increases anti-influenza Fc-functional antibodies in the plasma of influenza-infected subjects. Enhancement of Fc-functional antibodies to a diverse range of influenza strains suggests that Flu-IVIG infusion could prove useful in the context of novel influenza virus infections, when there may be minimal or no neutralising antibodies in the Flu-IVIG preparation.

Localization, kinetics and metabolism of labelled monoclonal antibodies on a cellular level

International Nuclear Information System (INIS)

Steinstraesser, A.; Kuhlmann, L.; Zimmer, M.; Schwarz, A.

1988-01-01

In order to gain insight into the mechanisms, the localization, kinetics and metabolism of preparations labelled with 131 J and 111 In were examined on a cellular level. Micro-autoradiography for histological assessment of the storage tissue in the organs was complemented by cytological examination methods for assessing the extent of internalisation of the antibodies, and the metabolism of the antibodies in the cytosol fraction could be followed up by chromatography. One of the major results is that even with the complete antibody, accumulation in the liver cells proceeds very rapidly and protein degradation is practically completed within twenty-four hours. In the tumor, however, internalisation plays a minor part (about 80 p.c. of the antibodies remain bound to the membrane). Rapid accumulation of the antibodies by the tubulus epithelium of the kidney causes the intensive image of the renal scintiscan. (orig./MG) [de

Daratumumab-mediated lysis of primary multiple myeloma cells is enhanced in combination with the human anti-KIR antibody IPH2102 and lenalidomide

DEFF Research Database (Denmark)

Nijhof, I. S.; Lammerts van Bueren, J. J.; van Kessel, B.

2015-01-01

Despite recent treatment improvements, multiple myeloma remains an incurable disease. Since antibody-dependent cell-mediated cytotoxicity is an important effector mechanism of daratumumab, we explored the possibility of improving daratumumab-mediated cell-mediated cytotoxicity by blocking natural...... killer cell inhibitory receptors with the human monoclonal anti-KIR antibody IPH2102, next to activation of natural killer cells with the immune modulatory drug lenalidomide. In 4-hour antibody-dependent cell-mediated cytotoxicity assays, IPH2102 did not induce lysis of multiple myeloma cell lines...... effective treatment strategies can be designed for multiple myeloma by combining daratumumab with agents that independently modulate natural killer cell function....

Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

Science.gov (United States)

Romain, Gabrielle; Senyukov, Vladimir; Rey-Villamizar, Nicolas; Merouane, Amine; Kelton, William; Liadi, Ivan; Mahendra, Ankit; Charab, Wissam; Georgiou, George; Roysam, Badrinath; Lee, Dean A.

2014-01-01

The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia. PMID:25232058

An alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of Tregs to induce immune responses in humans with advanced cancer.

Science.gov (United States)

Morse, Michael A; Hobeika, Amy C; Osada, Takuya; Berglund, Peter; Hubby, Bolyn; Negri, Sarah; Niedzwiecki, Donna; Devi, Gayathri R; Burnett, Bruce K; Clay, Timothy M; Smith, Jonathan; Lyerly, H Kim

2010-09-01

Therapeutic anticancer vaccines are designed to boost patients' immune responses to tumors. One approach is to use a viral vector to deliver antigen to in situ DCs, which then activate tumor-specific T cell and antibody responses. However, vector-specific neutralizing antibodies and suppressive cell populations such as Tregs remain great challenges to the efficacy of this approach. We report here that an alphavirus vector, packaged in virus-like replicon particles (VRP) and capable of efficiently infecting DCs, could be repeatedly administered to patients with metastatic cancer expressing the tumor antigen carcinoembryonic antigen (CEA) and that it overcame high titers of neutralizing antibodies and elevated Treg levels to induce clinically relevant CEA-specific T cell and antibody responses. The CEA-specific antibodies mediated antibody-dependent cellular cytotoxicity against tumor cells from human colorectal cancer metastases. In addition, patients with CEA-specific T cell responses exhibited longer overall survival. These data suggest that VRP-based vectors can overcome the presence of neutralizing antibodies to break tolerance to self antigen and may be clinically useful for immunotherapy in the setting of tumor-induced immunosuppression.

Cytotoxicity of Phenol Red in Toxicity Assays for Carbon Nanoparticles

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Chunhai Fan

2012-09-01

Full Text Available To explore the novel properties of carbon nanoparticles (CNPs in nanotoxicity assays, the adsorption of phenol red (a pH indicator for culture medium by multi-walled carbon nanotubes (MWNTs and three kinds of carbon blacks (CBs with nanosize, and its effects on cytotoxicity were studied. Results indicated that the phenol red adsorbed and delivered into cells by CBs was responsible for the toxicity to Hela cells in the medium without serum. The cellular uptake of phenol red was verified using 125I-labeling techniques. The size-dependent cytotoxicity of CBs was found to closely correlate to adsorption of phenol red, cellular uptake of phenol red-CB complexes and the amount of phenol red delivered into the cells by CBs. Although the CBs were either nontoxic or slightly toxic, as vehicles of phenol red, they played an essential role in the cytotoxicity induced by phenol red. However, MWNTs showed an intrinsic cytotoxicity independent of phenol red. The implications associated with these findings are discussed.

A rapid fluorometric method for semiautomated determination of cytotoxicity and cellular proliferation of human tumor cell lines in microculture.

Science.gov (United States)

Larsson, R; Nygren, P

1989-01-01

A fluorometric method for the determination of cellular growth and cytotoxicity of human tumor cell lines in 96-well microculture plates is described. The assay is based on the combined use of the DNA-binding dye Hoechst 33342 and the fluorogenic substrate fluorescein diacetate (FDA). Hoechst 33342 undergoes a dramatic enhancement of fluorescence when specifically intercalated with cellular DNA, whereas the FDA fluorescence is dependent on cellular hydrolysis of the non-fluorescent substrate into its fluorescent product. Fluorescence from both dyes was linearly related to the density of freshly seeded cells (6 x 10(3)-1 x 10(5)/well) and correlated well with physical cell count of cells under normal culture conditions as well as in response to the vinca alkaloid vincristine. However, the amount of FDA fluorescence produces and retained by the cultures was clearly dependent on the fraction of intact and viable cells, whereas the fluorescence reported by Hoechst 33342 was not. The assay was found to be simple, reliable and many samples could be analysed in a short period of time with minimal waste of cells and biological reagents. Apart from giving an estimate of cell density, the protocol described also provides a separate index of viability which in certain situations may be of importance for distinguishing between cytocidal and cytostatic drug actions. The method may be well suited for several applications, including the large scale screening for antitumor activity of compounds with potential cytocidal or cytostatic actions.

Non-Neutralizing Antibodies Directed against HIV and Their Functions

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Luzia M. Mayr

2017-11-01

Full Text Available B cells produce a plethora of anti-HIV antibodies (Abs but only few of them exhibit neutralizing activity. This was long considered a profound limitation for the enforcement of humoral immune responses against HIV-1 infection, especially since these neutralizing Abs (nAbs are extremely difficult to induce. However, increasing evidence shows that additional non-neutralizing Abs play a significant role in decreasing the viral load, leading to partial and sometimes even total protection. Mechanisms suspected to participate in protection are numerous. They involve the Fc domain of Abs as well as their Fab part, and consequently the induced Ab isotype will be determinant for their functions, as well as the quantity and quality of the Fc-receptors (FcRs expressed on immune cells. Fc-mediated inhibitory functions, such as Ab-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, aggregation, and even immune activation have been proposed. However, as for nAbs, the non-neutralizing activities are limited to a subset of anti-HIV Abs. An improved in-depth characterization of the Abs displaying these functional responses is required for the development of new vaccination strategies, which aim to selectively trigger the B cells able to induce the right functional Ab combinations both at the right place and at the right time. This review summarizes our current knowledge on non-neutralizing functional inhibitory Abs and discusses the potential benefit of inducing them via vaccination. We also provide new insight into the roles of the FcγR-mediated Ab therapeutics in clinical trials for HIV diseases.

The cellular basis of skin injury after cytotoxic insult

International Nuclear Information System (INIS)

Potten, C.S.

1986-01-01

It is concluded that although the major target in terms of radiation damage is undoubtedly the epidermis, the skin is a complex tissue made up of many inter-dependent components each of which may constitute an important secondary target. Damage to each component has been considered at the cellular level. The precise inter-relationships and interdependencies remain somewhat obscure. Even within one site, the epidermis, a comprehensive cellular explanation of the various post-irradiation changes is difficult. Substantial bibliography. (UK)

Cytotoxicity and cellular uptake of doxorubicin and its formamidine derivatives in HL60 sensitive and HL60/MX2 resistant cells.

Science.gov (United States)

Kik, Krzysztof; Wasowska-Lukawska, Malgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

2009-04-01

In this work a comparison was made of the cytotoxicity and cellular uptake of doxorubicin (DOX) and two of its derivatives containing a formamidino group (-N=CH-N<) at the 3' position with morpholine (DOXM) or hexamethyleneimine (DOXH) ring. All tests were performed in doxorubicin-sensitive HL60 and -resistant HL60/MX2 cells which are known for the presence of altered topoisomerase II. Cytotoxic activity of DOX toward HL60/MX2 cells was about 195 times lower when compared with the sensitive HL60 cell line. DOXM and DOXH were approximately 20 times more active in resistant cells than DOX. It was found that the uptake of DOX was lower in resistant cells by about 16%, while that of DOXM and DOXH was lower by about 36% and 19%, respectively. Thus the changes in the cellular uptake of anthracyclines are not associated with the fact that cytotoxicity of DOXM and DOXH exceed the cytotoxicity of DOX. Experiments in cell-free system containing human topoisomerase II showed that topoisomerase II is not inhibited by DOXM and DOXH. Formamidinoanthracyclines may be more useful than parent drugs in therapy against tumor cells with altered topoisomerase II activity.

Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation.

Science.gov (United States)

van Spriel, A B; Leusen, J H; van Egmond, M; Dijkman, H B; Assmann, K J; Mayadas, T N; van de Winkel, J G

2001-04-15

Receptors for human immunoglobulin (Ig)G and IgA initiate potent cytolysis of antibody (Ab)-coated targets by polymorphonuclear leukocytes (PMNs). Mac-1 (complement receptor type 3, CD11b/CD18) has previously been implicated in receptor cooperation with Fc receptors (FcRs). The role of Mac-1 in FcR-mediated lysis of tumor cells was characterized by studying normal human PMNs, Mac-1-deficient mouse PMNs, and mouse PMNs transgenic for human FcR. All PMNs efficiently phagocytosed Ab-coated particles. However, antibody-dependent cellular cytotoxicity (ADCC) was abrogated in Mac-1(-/-) PMNs and in human PMNs blocked with anti-Mac-1 monoclonal Ab (mAb). Mac-1(-/-) PMNs were unable to spread on Ab-opsonized target cells and other Ab-coated surfaces. Confocal laser scanning and electron microscopy revealed a striking difference in immunologic synapse formation between Mac-1(-/-) and wild-type PMNs. Also, respiratory burst activity could be measured outside membrane-enclosed compartments by using Mac-1(-/-) PMNs bound to Ab-coated tumor cells, in contrast to wild-type PMNs. In summary, these data document an absolute requirement of Mac-1 for FcR-mediated PMN cytotoxicity toward tumor targets. Mac-1(-/-) PMNs exhibit defective spreading on Ab-coated targets, impaired formation of immunologic synapses, and absent tumor cytolysis.

Effect of radiotherapy on lymphocyte cytotoxicity in vitro

Energy Technology Data Exchange (ETDEWEB)

Wasserman, J; Melen, B [Central Microbiological Laboratory, Stockholm County Council (Sweden); Blomgren, H; Glas, U; Perlmann, P

1975-11-01

The cytotoxic functions of highly purified blood lymphocytes from patients with breast cancer were studied before and after radiotherapy. Addition of PHA or of rabbit antibodies to target cells (chicken erythrocytes) were chosen as two means of inducing lymphocyte cytotoxicity in vitro. The proportion of T and non-T lymphocytes was determined by means of E and EAC rosette tests. The antibody-induced cytotoxicity of lymphocytes decreased following radiotherapy while that mediated by PHA remained unchanged. There was some reduction in the percentage of EAC rosette-forming cells. These results, as well as earlier observations, suggest that the decrease in the peripheral blood of the proportion of lymphocytes with receptors for activated complement is responsible for changes in the antibody-mediated lymphocyte cytotoxicity.

CD47 limits antibody dependent phagocytosis against non-malignant B cells.

Science.gov (United States)

Gallagher, Sandra; Turman, Sean; Lekstrom, Kristen; Wilson, Susan; Herbst, Ronald; Wang, Yue

2017-05-01

Recent studies have demonstrated the importance of CD47 in protecting malignant B cells from antibody dependent cellular phagocytosis (ADCP). Combined treatment of anti-CD47 and -CD20 antibodies synergistically augment elimination of tumor B cells in xenograft mouse models. This has led to the development of novel reagents that can potentially enhance killing of malignant B cells in patients. B cell depleting therapy is also a promising treatment for autoimmune patients. In the current study, we aimed to investigate whether or not CD47 protects non-malignant B cells from ADCP. We show that CD47 is expressed on all B cells in mice, with the highest level on plasma cells in bone marrow and spleen. Although its expression is dispensable for B cell development in mice, CD47 on B cells limits antibody mediated phagocytosis. B cell depletion following in vivo anti-CD19 treatment is more efficient in CD47-/- mice than in wild type mice. In vitro, both naïve and activated B cells from CD47-/- mice are more sensitive to ADCP than wild type B cells. Lastly, we show in an ADCP assay that blocking CD47 can enhance anti-CD19 antibody mediated phagocytosis of wild type B cells. These results suggest that in addition to its already demonstrated benefit in cancer, targeting CD47 may be used as an adjunct in combination with B cell depletion antibodies for treatment of autoimmune diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Human Cytomegalovirus Infection Increases Both Antibody- and Non–Antibody-Dependent Cellular Reactivity by Natural Killer Cells

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Clive M. Michelo, PhD

2017-12-01

Conclusions. With regard to organ transplantation, these data suggest that CMV infection enhances NK cell alloreactivity, which may pose an additional adverse effect on graft survival, especially in the presence of donor specific antibodies.

Nanosilica induced dose-dependent cytotoxicity and cell type-dependent multinucleation in HepG2 and L-02 cells

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Yu, Yongbo [Capital Medical University, Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children’s Hospital (China); Duan, Junchao; Li, Yang; Yu, Yang; Hu, Hejing; Wu, Jing; Zhang, Yannan; Li, Yanbo; CaixiaGuo; Zhou, Xianqing; Sun, Zhiwei, E-mail: zwsun@ccmu.edu.cn [Capital Medical University, School of Public Health (China)

2016-11-15

The prevalent exposure to nanosilica gained concerns about health effects of these particles on human beings. Although nanosilica-induced multinucleation has been confirmed previously, the underlying mechanism was still not clear; this study was to investigate the origination of multinucleated cells caused by nanosilica (62 nm) in both HepG2 and L-02 cells. Cell viability and cellular uptake was determined by MTT assay and transmission electron microscope (TEM), respectively. Giemsa staining was applied to detect multinucleation. To clarify the origination of multinucleated cells, fluorescent probes, PKH26 and PKH67, time-lapse observation were further conducted by confocal microscopy. Results indicated that nanosilica particles were internalized into cells and induced cytotoxicity in a dose-dependent manner. Quantification analysis showed that nanosilica significantly increased the rates of binucleated and multinucleated cells, which suggested mitotic catastrophe induction. Moreover, dynamic visualization verified that multinucleation resulted from cell fusion in HepG2 cells not in L-02 cells after nanosilica exposure, suggesting cell type-dependent multinucleation formation. Both multinucleation and cell fusion were involved in genetic instability, which emphasized the significance to explore the multinucleation induced by nanosilica via environmental, occupational and consumer product exposure.

Immunoglobulin G1 Allotype Influences Antibody Subclass Distribution in Response to HIV gp140 Vaccination

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Sven Kratochvil

2017-12-01

Full Text Available Antibody subclasses exhibit extensive polymorphisms (allotypes that could potentially impact the quality of HIV-vaccine induced B cell responses. Allotypes of immunoglobulin (Ig G1, the most abundant serum antibody, have been shown to display altered functional properties in regard to serum half-life, Fc-receptor binding and FcRn-mediated mucosal transcytosis. To investigate the potential link between allotypic IgG1-variants and vaccine-generated humoral responses in a cohort of 14 HIV vaccine recipients, we developed a novel protocol for rapid IgG1-allotyping. We combined PCR and ELISA assays in a dual approach to determine the IgG1 allotype identity (G1m3 and/or G1m1 of trial participants, using human plasma and RNA isolated from PBMC. The IgG1-allotype distribution of our participants mirrored previously reported results for caucasoid populations. We observed elevated levels of HIV gp140-specific IgG1 and decreased IgG2 levels associated with the G1m1-allele, in contrast to G1m3 carriers. These data suggest that vaccinees homozygous for G1m1 are predisposed to develop elevated Ag-specific IgG1:IgG2 ratios compared to G1m3-carriers. This elevated IgG1:IgG2 ratio was further associated with higher FcγR-dimer engagement, a surrogate for potential antibody-dependent cellular cytotoxicity (ADCC and antibody-dependent cellular phagocytosis (ADCP function. Although preliminary, these results suggest that IgG1 allotype may have a significant impact on IgG subclass distribution in response to vaccination and associated Fc-mediated effector functions. These results have important implications for ongoing HIV vaccine efficacy studies predicated on engagement of FcγR-mediated cellular functions including ADCC and ADCP, and warrant further investigation. Our novel allotyping protocol provides new tools to determine the potential impact of IgG1 allotypes on vaccine efficacy.

Modulation in vitro and in vivo of cytotoxicity but not cellular levels of doxorubicin by the calmodulin inhibitor trifluoperazine is dependent on the level of resistance.

Science.gov (United States)

Ganapathi, R.; Schmidt, H.; Grabowski, D.; Melia, M.; Ratliff, N.

1988-01-01

The role of the calmodulin inhibitor trifluoperazine (TFP) in modulating the cellular levels and cytotoxicity in vitro and antitumour effects in vivo of doxorubicin (DOX), was evaluated in progressively DOX-resistant (5- to 40-fold) sublines of B16-BL6 mouse melanoma. In parental-sensitive B16-BL6 cells treated for 3 h, the IC50 of DOX was 0.1 microgram ml-1, and a less than 2-fold enhancement in DOX cell kill in the presence of a noncytotoxic concentration of 5 microM TFP was observed. However, in the DOX-resistant sublines, the IC50 was 0.7 to 5.0 micrograms ml-1 DOX in the absence of 5 microM TFP and 0.3 to 0.7 microgram ml-1 DOX in the presence of 5 microM TFP. The 2- to 7.5-fold decrease in the IC50 of DOX in the presence of 5 microM TFP, was dependent on the level of DOX-resistance in the various sublines. Compared to parental-sensitive cells, a 2-fold decrease in DOX-accumulation was evident only in the 40-fold DOX-resistant subline. Further, maximal enhancement (50%) of cellular DOX accumulation in the presence of 5 microM TFP was observed only in the 40-fold resistant cells treated with 5.0 micrograms ml-1 DOX. Retention of DOX in the 40-fold resistant subline was only 20% lower than similarly treated sensitive cells, and the inclusion of TFP increased DOX retention less than 10-15%. Antitumour studies in mice with experimental pulmonary metastases revealed that although DOX and DOX plus TFP had similar antitumour activity with the parental sensitive B16-BL6 cells, the combination of DOX plus TFP was significantly more effective than DOX alone with the DOX-resistant sublines. No overt toxicity was observed in normal mice treated with doses of TFP, DOX or DOX plus TFP used for in vivo chemotherapy studies. Results from this study suggest that gross cellular DOX levels do not appear to correlate with the magnitude of resistance, and the effects of TFP in modulating DOX resistance is possibly due to mechanisms other than mere alterations in cellular drug

Cytotoxicity of cadmium-containing quantum dots based on a study using a microfluidic chip

International Nuclear Information System (INIS)

Zheng Xiannuo; Weng Lixing; Tian Jing; Wang Lianhui; Wu Lei; Jin Qinghui; Zhao Jianlong

2012-01-01

There is a lack of reliable nanotoxicity assays available for monitoring and quantifying multiple cellular events in cultured cells. In this study, we used a microfluidic chip to systematically investigate the cytotoxicity of three kinds of well-characterized cadmium-containing quantum dots (QDs) with the same core but different shell structures, including CdTe core QDs, CdTe/CdS core–shell QDs, and CdTe/CdS/ZnS core–shell–shell QDs, in HEK293 cells. Using the microfluidic chip combined with fluorescence microscopy, multiple QD-induced cellular events including cell morphology, viability, proliferation, and QD uptake were simultaneously analysed. The three kinds of QDs showed significantly different cytotoxicities. The CdTe QDs, which are highly toxic to HEK293 cells, resulted in remarkable cellular and nuclear morphological changes, a dose-dependent decrease in cell viability, and strong inhibition of cell proliferation; the CdTe/CdS QDs were moderately toxic but did not significantly affect the proliferation of HEK293 cells; while the CdTe/CdS/ZnS QDs had no detectable influence on cytotoxicity with respect to cell morphology, viability, and proliferation. Our data indicated that QD cytotoxicity was closely related to their surface structures and specific physicochemical properties. This study also demonstrated that the microfluidic chip could serve as a powerful tool to systematically evaluate the cytotoxicity of nanoparticles in multiple cellular events. (paper)

Cytotoxicity of cadmium-containing quantum dots based on a study using a microfluidic chip

Science.gov (United States)

Zheng, Xiannuo; Tian, Jing; Weng, Lixing; Wu, Lei; Jin, Qinghui; Zhao, Jianlong; Wang, Lianhui

2012-02-01

There is a lack of reliable nanotoxicity assays available for monitoring and quantifying multiple cellular events in cultured cells. In this study, we used a microfluidic chip to systematically investigate the cytotoxicity of three kinds of well-characterized cadmium-containing quantum dots (QDs) with the same core but different shell structures, including CdTe core QDs, CdTe/CdS core-shell QDs, and CdTe/CdS/ZnS core-shell-shell QDs, in HEK293 cells. Using the microfluidic chip combined with fluorescence microscopy, multiple QD-induced cellular events including cell morphology, viability, proliferation, and QD uptake were simultaneously analysed. The three kinds of QDs showed significantly different cytotoxicities. The CdTe QDs, which are highly toxic to HEK293 cells, resulted in remarkable cellular and nuclear morphological changes, a dose-dependent decrease in cell viability, and strong inhibition of cell proliferation; the CdTe/CdS QDs were moderately toxic but did not significantly affect the proliferation of HEK293 cells; while the CdTe/CdS/ZnS QDs had no detectable influence on cytotoxicity with respect to cell morphology, viability, and proliferation. Our data indicated that QD cytotoxicity was closely related to their surface structures and specific physicochemical properties. This study also demonstrated that the microfluidic chip could serve as a powerful tool to systematically evaluate the cytotoxicity of nanoparticles in multiple cellular events.

Lack of protection following passive transfer of polyclonal highly functional low-dose non-neutralizing antibodies.

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Anne-Sophie Dugast

Full Text Available Recent immune correlates analysis from the RV144 vaccine trial has renewed interest in the role of non-neutralizing antibodies in mediating protection from infection. While neutralizing antibodies have proven difficult to induce through vaccination, extra-neutralizing antibodies, such as those that mediate antibody-dependent cellular cytotoxicity (ADCC, are associated with long-term control of infection. However, while several non-neutralizing monoclonal antibodies have been tested for their protective efficacy in vivo, no studies to date have tested the protective activity of naturally produced polyclonal antibodies from individuals harboring potent ADCC activity. Because ADCC-inducing antibodies are highly enriched in elite controllers (EC, we passively transferred highly functional non-neutralizing polyclonal antibodies, purified from an EC, to assess the potential impact of polyclonal non-neutralizing antibodies on a stringent SHIV-SF162P3 challenge in rhesus monkeys. Passive transfer of a low-dose of ADCC inducing antibodies did not protect from infection following SHIV-SF162P3 challenge. Passively administered antibody titers and gp120-specific, but not gp41-specific, ADCC and antibody induced phagocytosis (ADCP were detected in the majority of the monkeys, but did not correlate with post infection viral control. Thus these data raise the possibility that gp120-specific ADCC activity alone may not be sufficient to control viremia post infection but that other specificities or Fc-effector profiles, alone or in combination, may have an impact on viral control and should be tested in future passive transfer experiments.

Absence of cytotoxic antibody to human immunodeficiency virus-infected cells in humans and its induction in animals after infection or immunization with purified envelope glycoprotein gp120

International Nuclear Information System (INIS)

Nara, P.L.; Robey, W.G.; Gonda, M.A.; Carter, S.G.; Fischinger, P.J.

1987-01-01

The presence of antibody-dependent complement-mediated cytotoxicity (ACC) was assessed in humans and chimpanzees, which are capable of infection with human immunodeficiency virus isolate HTLV-IIIb, and examined in the goat after immunization with the major viral glycoprotein (gp120) of HTLV-IIIb. In infected humans no antibody mediating ACC was observed regardless of the status of disease. Even healthy individuals with high-titer, broadly reactive, neutralizing antibodies has no ACC. In contrast, chimpanzees infected with HTLV-IIIb, from whom virus could be isolated, not only had neutralizing antibody but also antibodies broadly reactive in ACC, even against distantly related human immunodeficiency virus isolates, as well as against their own reisolated virus. In the goat, the gp120 of HTLV-IIIb induced a highly type-specific response as measured by both ACC and flow cytofluorometry of live infected H9 cells. Normal human cells were not subject to ACC by animal anti-HTLV-III gp120-specific sera. Induction of ACC and neutralizing antibody were closely correlated in the animal experimental models but not in humans. The presence of ACC in gp120-inoculated goats and HTLV-III-infected chimpanzees represent a qualitative difference that may be important in the quest for the elicitation of a protective immunity in humans

Cellular pharmacodynamics of the cytotoxic guanidino-containing drug CHS 828. Comparison with methylglyoxal-bis(guanylhydrazone).

Science.gov (United States)

Ekelund, S; Sjöholm, A; Nygren, P; Binderup, L; Larsson, R

2001-04-20

N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828) is a new guanidino-containing compound with antitumoral activity both in vitro and in vivo. Its activity profile differs from those of standard cytotoxic drugs but the mechanism of action is not yet fully understood. CHS 828 is presently in early phase I and II clinical trials. In the present study, the pharmacodynamic effects at the cellular level of CHS 828 was compared to another compound containing two guanidino groups, methylglyoxal-bis(guanylhydrazone) (MGBG). MGBG is known to inhibit the synthesis of polyamines, which are important in, e.g., proliferation and macromolecular synthesis. The concentration-response relationship of CHS 828 closely resembled that of MGBG and the drugs were similar with respect to inhibition of DNA and protein synthesis. On the other hand, CHS 828 induced a significant increase in cellular metabolism while MGBG did not. The cytotoxic effect of MGBG was reversed by the addition of exogenous polyamines, while that of CHS 828 was unaffected. Unlike MGBG, there was also no effect of CHS 828 on the levels of decarboxylating enzymes in the polyamine biosynthesis. In conclusion, CHS 828 does not appear to share any major mechanisms of action with the polyamine synthesis inhibitor MGBG. Further studies will be required to define the exact mechanism of action of CHS 828.

Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation

International Nuclear Information System (INIS)

Milgram, S.; Carriere, M.; Thiebault, C.; Malaval, L.; Gouget, B.

2008-01-01

Bone is one of the main retention organs for uranium (U) and lead (Pb). The clinical effects of U or Pb poisoning are well known: acute and chronic intoxications impair bone formation. However, only few studies dealt with the cellular and molecular mechanisms of their toxicity. The purpose of this study was to investigate acute cytotoxicity of U and Pb and their phenotypic effects on rat and human osteoblasts, the cells responsible for bone formation. The most likely species of the toxicants in contact with cells after blood contamination were selected for cell exposure. Results showed that the cytotoxic effect of U and Pb is highly dependent on their speciation. Thus, Pb was cytotoxic when left free in the exposure medium or when complexed with carbonate, cystein or citrate, but not when complexed with albumin or phosphate, under an insoluble form. U was cytotoxic whatever its speciation, but differences in sensitivity were observed as a function of speciation. Population growth recovery could be obtained after exposure to low doses of U or Pb, except for some U-carbonate complexes which had irreversible effects whatever the dose. The activation of two markers of bone formation and mineralization, osteocalcin and bone sialoprotein (BSP), was observed after exposure to non-toxic doses or non-toxic species of U or Pb while their inhibition was observed after toxic exposure to both metals. This work provides new elements to better understand the complex mechanisms of U and Pb toxicity to osteoblasts. Our results also illustrate the importance of a strictly controlled speciation of the metals in toxicological studies

Peripheral neuropathies associated with antibodies directed to intracellular neural antigens.

Science.gov (United States)

Antoine, J-C

2014-10-01

Antibodies directed to intracellular neural antigens have been mainly described in paraneoplastic peripheral neuropathies and mostly includes anti-Hu and anti-CV2/CRMP5 antibodies. These antibodies occur with different patterns of neuropathy. With anti-Hu antibody, the most frequent manifestation is sensory neuronopathy with frequent autonomic involvement. With anti-CV2/CRMP5 the neuropathy is more frequently sensory and motor with an axonal or mixed demyelinating and axonal electrophysiological pattern. The clinical pattern of these neuropathies is in keeping with the cellular distribution of HuD and CRMP5 in the peripheral nervous system. Although present in high titer, these antibodies are probably not directly responsible for the neuropathy. Pathological and experimental studies indicate that cytotoxic T-cells are probably the main effectors of the immune response. These disorders contrast with those in which antibodies recognize a cell surface antigen and are probably responsible for the disease. The neuronal cell death and axonal degeneration which result from T-cell mediated immunity explains why treating these disorders remains challenging. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Determining the size and concentration dependence of gold nanoparticles in vitro cytotoxicity (IC50) test using WST-1 assay

International Nuclear Information System (INIS)

Rosli, Nur Shafawati binti; Rahman, Azhar Abdul; Aziz, Azlan Abdul; Shamsuddin, Shaharum

2015-01-01

Gold nanoparticles (AuNPs) received a great deal of attention for biomedical applications, especially in diagnostic imaging and therapeutics. Even though AuNPs have potential benefits in biomedical applications, the impact of AuNPs on human and environmental health still remains unclear. The use of AuNPs which is a high-atomic-number materials, provide advantages in terms of radiation dose enhancement. However, before this can become a clinical reality, cytotoxicity of the AuNPs has to be carefully evaluated. Cytotoxicity test is a rapid, standardized test that is very sensitive to determine whether the nanoparticles produced are harmful or benign on cellular components. In this work the size and concentration dependence of AuNPs cytotoxicity in breast cancer cell lines (MCF-7) are tested by using WST-1 assay. The sizes of AuNPs tested were 13 nm, 50 nm, and 70 nm. The cells were seeded in the 96-well plate and were treated with different concentrations of AuNPs by serial dilution for each size of AuNPs. The high concentration of AuNPs exhibit lower cell viability compared to low concentration of AuNPs. We quantified the toxicity of AuNPs in MCF-7 cell lines by determining the IC 50 values in WST-1 assays. The IC 50 values (inhibitory concentrations that effected 50% growth inhibition) of 50 nm AuNPs is lower than 13 nm and 70 nm AuNPs. Mean that, 50nm AuNPs are more toxic to the MCF-7 cells compared to smaller and larger sizes AuNPs. The presented results clearly indicate that the cytotoxicity of AuNPs depend not only on the concentration, but also the size of the nanoparticles

Antitumor effect of novel anti-podoplanin antibody NZ-12 against malignant pleural mesothelioma in an orthotopic xenograft model.

Science.gov (United States)

Abe, Shinji; Kaneko, Mika Kato; Tsuchihashi, Yuki; Izumi, Toshihiro; Ogasawara, Satoshi; Okada, Naoto; Sato, Chiemi; Tobiume, Makoto; Otsuka, Kenji; Miyamoto, Licht; Tsuchiya, Koichiro; Kawazoe, Kazuyoshi; Kato, Yukinari; Nishioka, Yasuhiko

2016-09-01

Podoplanin (aggrus) is highly expressed in several types of cancers, including malignant pleural mesothelioma (MPM). Previously, we developed a rat anti-human podoplanin mAb, NZ-1, and a rat-human chimeric anti-human podoplanin antibody, NZ-8, derived from NZ-1, which induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against podoplanin-positive MPM cell lines. In this study, we showed the antitumor effect of NZ-1, NZ-8, and NZ-12, a novel rat-human chimeric anti-human podoplanin antibody derived from NZ-1, in an MPM orthotopic xenograft SCID mouse model. Treatment with NZ-1 and rat NK (CD161a(+) ) cells inhibited the growth of tumors and the production of pleural effusion in NCI-H290/PDPN or NCI-H226 orthotopic xenograft mouse models. NZ-8 and human natural killer (NK) (CD56(+) ) cells also inhibited tumor growth and pleural effusion in MPM orthotopic xenograft mice. Furthermore, NZ-12 induced potent ADCC mediated by human MNC, compared with either NZ-1 or NZ-8. Antitumor effects were observed following treatment with NZ-12 and human NK (CD56(+) ) cells in MPM orthotopic xenograft mice. In addition, combined immunotherapy using the ADCC activity of NZ-12 mediated by human NK (CD56(+) ) cells with pemetrexed, led to enhanced antitumor effects in MPM orthotopic xenograft mice. These results strongly suggest that combination therapy with podoplanin-targeting immunotherapy using both NZ-12 and pemetrexed might provide an efficacious therapeutic strategy for the treatment of MPM. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Correlation of particle properties with cytotoxicity and cellular uptake of hydroxyapatite nanoparticles in human gastric cancer cells

Energy Technology Data Exchange (ETDEWEB)

Cui, Xinhui [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Liang, Tong [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Liu, Changsheng [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Yuan, Yuan, E-mail: yyuan@ecust.edu.cn [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Qian, Jiangchao, E-mail: jiangchaoqian@ecust.edu.cn [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)

2016-10-01

Three types of hydroxyapatite nanoparticles (HAPNs) were synthesized employing a sonochemistry-assisted microwave method by changing microwave power (from 200 to 300 W) or using calcination treatment: L200 (200 W, lyophilization), L300 (300 W, lyophilization) and C200 (200 W, lyophilization & calcination). Their physiochemical properties were characterized and correlated with cytotoxicity to human gastric cancer cells (MGC80-3). The major differences among these HAPN preparations were their size and specific surface area, with the L200 showing a smaller size and higher specific surface area. Although all HAPNs inhibited cell proliferation and induced apoptosis of cancer cells, L200 exhibited the greatest toxicity. All types of HAPNs were internalized through energy-dependent pathways, but the L200 nanoparticles were more efficiently uptaken by MGC80-3 cells. Inhibitor studies with dynasore and methyl-β-cyclodextrin suggested that caveolae-mediated endocytosis and, to a much lesser extent, clathrin-mediated endocytosis, were involved in cellular uptake of the various preparations, whereas the inhibition of endocytosis was more obvious for L200. Using fluorescein isothiocyanate-labeled HAPNs and laser-scanning confocal microscopy, we found that all forms of nanoparticles were present in the cytoplasm, and some L200 HAPNs were even found within nuclei. Treatment with all HAPN preparations led to the increase in the intracellular calcium level with the highest level detected for L200. - Highlights: • Three types of HAPNs (L200, L300 and C200) were synthesized employing a sonochemistry-assisted microwave method. • L200 exhibited the greatest cytotoxicity to human gastric cancer (MGC80-3) cells. • L200 showed a smaller size and higher specific surface area. • The L200 nanoparticles were more efficiently uptaken by MGC80-3 cells through energy-dependent pathways. • L200 caused the most significant increase in the intracellular calcium level.

Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro*

Science.gov (United States)

Liu, Ling; Jacobsen, Frederick W.; Everds, Nancy; Zhuang, Yao; Yu, Yan Bin; Li, Nianyu; Clark, Darcey; Nguyen, Mai Phuong; Fort, Madeline; Narayanan, Padma; Kim, Kei; Stevenson, Riki; Narhi, Linda; Gunasekaran, Kannan; Bussiere, Jeanine L.

2017-01-01

The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem. 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20+ human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro. These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions. PMID:27994063

A perillyl alcohol-conjugated analog of 3-bromopyruvate without cellular uptake dependency on monocarboxylate transporter 1 and with activity in 3-BP-resistant tumor cells.

Science.gov (United States)

Chen, Thomas C; Yu, Jiali; Nouri Nigjeh, Eslam; Wang, Weijun; Myint, Phyo Thazin; Zandi, Ebrahim; Hofman, Florence M; Schönthal, Axel H

2017-08-01

The anticancer agent 3-bromopyruvate (3-BP) is viewed as a glycolytic inhibitor that preferentially kills glycolytic cancer cells through energy depletion. However, its cytotoxic activity is dependent on cellular drug import through transmembrane monocarboxylate transporter 1 (MCT-1), which restricts its anticancer potential to MCT-1-positive tumor cells. We created and characterized an MCT-1-independent analog of 3-BP, called NEO218. NEO218 was synthesized by covalently conjugating 3-BP to perillyl alcohol (POH), a natural monoterpene. The responses of various tumor cell lines to treatment with either compound were characterized in the presence or absence of supplemental pyruvate or antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH). Drug effects on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme activity were investigated by mass spectrometric analysis. The development of 3-BP resistance was investigated in MCT-1-positive HCT116 colon carcinoma cells in vitro. Our results show that NEO218: (i) pyruvylated GAPDH on all 4 of its cysteine residues and shut down enzymatic activity; (ii) severely lowered cellular ATP content below life-sustaining levels, and (iii) triggered rapid necrosis. Intriguingly, supplemental antioxidants effectively prevented cytotoxic activity of NEO218 as well as 3-BP, but supplemental pyruvate powerfully protected cells only from 3-BP, not from NEO218. Unlike 3-BP, NEO218 exerted its potent cytotoxic activity irrespective of cellular MCT-1 status. Treatment of HCT116 cells with 3-BP resulted in prompt development of resistance, based on the emergence of MCT-1-negative cells. This was not the case with NEO218, and highly 3-BP-resistant cells remained exquisitely sensitive to NEO218. Thus, our study identifies a mechanism by which tumor cells develop rapid resistance to 3-BP, and presents NEO218 as a superior agent not subject to this cellular defense. Furthermore, our results offer alternative interpretations of previously

In vitro assessment of cytotoxic activities of Lachesis muta muta snake venom.

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Stephanie Stransky

2018-04-01

Full Text Available Envenomation by the bushmaster snake Lachesis muta muta is considered severe, characterized by local effects including necrosis, the main cause of permanent disability. However, cellular mechanisms related to cell death and tissue destruction, triggered by snake venoms, are poorly explored. The purpose of this study was to investigate the cytotoxic effect caused by L. m. muta venom in normal human keratinocytes and to identify the cellular processes involved in in cellulo envenomation. In order to investigate venom effect on different cell types, Alamar Blue assay was performed to quantify levels of cellular metabolism as a readout of cell viability. Apoptosis, necrosis and changes in mitochondrial membrane potential were evaluated by flow cytometry, while induction of autophagy was assessed by expression of GFP-LC3 and analyzed using fluorescence microscopy. The cytotoxic potential of the venom is shown by reduced cell viability in a concentration-dependent manner. It was also observed the sequential appearance of cells undergoing autophagy (by 6 hours, apoptosis and necrosis (12 and 24 hours. Morphologically, incubation with L. m. muta venom led to a significant cellular retraction and formation of cellular aggregates. These results indicate that L. m. muta venom is cytotoxic to normal human keratinocytes and other cell lines, and this toxicity involves the integration of distinct modes of cell death. Autophagy as a cell death mechanism, in addition to apoptosis and necrosis, can help to unravel cellular pathways and mechanisms triggered by the venom. Understanding the mechanisms that underlie cellular damage and tissue destruction will be useful in the development of alternative therapies against snakebites.

Determining the size and concentration dependence of gold nanoparticles in vitro cytotoxicity (IC{sub 50}) test using WST-1 assay

Energy Technology Data Exchange (ETDEWEB)

Rosli, Nur Shafawati binti; Rahman, Azhar Abdul [School of Physics, Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); Aziz, Azlan Abdul [School of Physics, Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); Nano-Biotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); Shamsuddin, Shaharum [Nano-Biotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

2015-04-24

Gold nanoparticles (AuNPs) received a great deal of attention for biomedical applications, especially in diagnostic imaging and therapeutics. Even though AuNPs have potential benefits in biomedical applications, the impact of AuNPs on human and environmental health still remains unclear. The use of AuNPs which is a high-atomic-number materials, provide advantages in terms of radiation dose enhancement. However, before this can become a clinical reality, cytotoxicity of the AuNPs has to be carefully evaluated. Cytotoxicity test is a rapid, standardized test that is very sensitive to determine whether the nanoparticles produced are harmful or benign on cellular components. In this work the size and concentration dependence of AuNPs cytotoxicity in breast cancer cell lines (MCF-7) are tested by using WST-1 assay. The sizes of AuNPs tested were 13 nm, 50 nm, and 70 nm. The cells were seeded in the 96-well plate and were treated with different concentrations of AuNPs by serial dilution for each size of AuNPs. The high concentration of AuNPs exhibit lower cell viability compared to low concentration of AuNPs. We quantified the toxicity of AuNPs in MCF-7 cell lines by determining the IC{sub 50} values in WST-1 assays. The IC{sub 50} values (inhibitory concentrations that effected 50% growth inhibition) of 50 nm AuNPs is lower than 13 nm and 70 nm AuNPs. Mean that, 50nm AuNPs are more toxic to the MCF-7 cells compared to smaller and larger sizes AuNPs. The presented results clearly indicate that the cytotoxicity of AuNPs depend not only on the concentration, but also the size of the nanoparticles.

Protective immunization with B16 melanoma induces antibody response and not cytotoxic T cell response

International Nuclear Information System (INIS)

Sarzotti, M.; Sriyuktasuth, P.; Klimpel, G.R.; Cerny, J.

1986-01-01

C57BL/6 mice immunized with three intraperitoneal injections of syngeneic, irradiated B16 melanoma cells, became resistant to B16 tumor challenge. Immunized mice had high levels of serum antibody against a membrane antigen of B16 cells. The B16 antigen recognized by the anti-B16 sera formed a major band of 90 KD in gel electrophoresis. The anti-B16 antibody was partially protective when mixed with B16 cells and injected into normal recipient mice. Surprisingly, B16 resistance mice were incapable of generating cytotoxic T cells (CTL) specific for the B16 tumor. Both spleen and lymph node cell populations from immunized mice did not generate B16-specific CTL. Allogeneic mice (DBA/2 or C3H) were also unable to generate B16-specific CTL: however, alloreactive CTL produced in these strains of mice by immunization with C57BL/6 lymphocytes, did kill B16 target cells. Interestingly, spleen cells from syngeneic mice immunized with B16 tumor produced 6-fold more interleukin-2 (IL-2) than normal spleen cells, in vitro. These data suggest that immunization with B16 tumor activates a helper subset of T cells (for antibody and IL-2 production) but not the effector CTL response

Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo.

Science.gov (United States)

Chang, De-Kuan; Moniz, Raymond J; Xu, Zhongyao; Sun, Jiusong; Signoretti, Sabina; Zhu, Quan; Marasco, Wayne A

2015-06-11

Carbonic anhydrase (CA) IX is a surface-expressed protein that is upregulated by the hypoxia inducible factor (HIF) and represents a prototypic tumor-associated antigen that is overexpressed on renal cell carcinoma (RCC). Therapeutic approaches targeting CAIX have focused on the development of CAIX inhibitors and specific immunotherapies including monoclonal antibodies (mAbs). However, current in vivo mouse models used to characterize the anti-tumor properties of fully human anti-CAIX mAbs have significant limitations since the role of human effector cells in tumor cell killing in vivo is not directly evaluated. The role of human anti-CAIX mAbs on CAIX(+) RCC tumor cell killing by immunocytes or complement was tested in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) as well as on CAIX(+) RCC cellular motility, wound healing, migration and proliferation. The in vivo therapeutic activity mediated by anti-CAIX mAbs was determined by using a novel orthotopic RCC xenograft humanized animal model and analyzed by histology and FACS staining. Our studies demonstrate the capacity of human anti-CAIX mAbs that inhibit CA enzymatic activity to result in immune-mediated killing of RCC, including nature killer (NK) cell-mediated ADCC, CDC, and macrophage-mediated ADCP. The killing activity correlated positively with the level of CAIX expression on RCC tumor cell lines. In addition, Fc engineering of anti-CAIX mAbs was shown to enhance the ADCC activity against RCC. We also demonstrate that these anti-CAIX mAbs inhibit migration of RCC cells in vitro. Finally, through the implementation of a novel orthotopic RCC model utilizing allogeneic human peripheral blood mononuclear cells in NOD/SCID/IL2Rγ(-/-) mice, we show that anti-CAIX mAbs are capable of mediating human immune response in vivo including tumor infiltration of NK cells and activation of T cells, resulting in

Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the Antibody-Dependent Enhancement of Ebola Virus Infection.

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Wakako Furuyama

2016-12-01

Full Text Available Antibody-dependent enhancement (ADE of Ebola virus (EBOV infection has been demonstrated in vitro, raising concerns about the detrimental potential of some anti-EBOV antibodies. ADE has been described for many viruses and mostly depends on the cross-linking of virus-antibody complexes to cell surface Fc receptors, leading to enhanced infection. However, little is known about the molecular mechanisms underlying this phenomenon. Here we show that Fcγ-receptor IIa (FcγRIIa-mediated intracellular signaling through Src family protein tyrosine kinases (PTKs is required for ADE of EBOV infection. We found that deletion of the FcγRIIa cytoplasmic tail abolished EBOV ADE due to decreased virus uptake into cellular endosomes. Furthermore, EBOV ADE, but not non-ADE infection, was significantly reduced by inhibition of the Src family protein PTK pathway, which was also found to be important to promote phagocytosis/macropinocytosis for viral uptake into endosomes. We further confirmed a significant increase of the Src phosphorylation mediated by ADE. These data suggest that antibody-EBOV complexes bound to the cell surface FcγRIIa activate the Src signaling pathway that leads to enhanced viral entry into cells, providing a novel perspective for the general understanding of ADE of virus infection.

Fc engineering of anti-Nectin-2 antibody improved thrombocytopenic adverse event in monkey.

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Tsutomu Oshima

Full Text Available Nectin-2 is a transmembrane glycoprotein which is involved in the process of Ca2+-independent cell-cell adhesion. In our previous study, we have demonstrated that Nectin-2 is over-expressed in breast and ovarian cancer tissues by using gene expression analysis and immunohistochemistry. Furthermore, we discovered multiple anti-Nectin-2 fully human monoclonal antibodies which inhibited tumor growth in in vivo subcutaneous xenograft models with antibody-dependent cellular cytotoxicity (ADCC as the principal mechanism of action. In this report, we assessed the toxicity of Y-443, a fully human IgG1/kappa anti-Nectin-2 monoclonal antibody exhibiting strong in vitro ADCC and in vivo anti-tumor activity in cynomolgus monkeys (Macaca fascicularis (Cynos. Unexpectedly, upon administration, Y-443 induced strong thrombocytopenia through Nectin-2 expressed on Cyno platelets, presumably followed by phagocytosis in the mononuclear phagocytic system. To mitigate the adverse safety profile, we mutated the Fc region of Y-443 to reduce the Fc binding activity to Fcγ receptor I, which is the primary receptor for phagocytosis on macrophages. Moreover, we further engineered the Fc through defucosylation to maintain ADCC activity. The resultant Fc engineered antibody, termed Y-634, demonstrated diminished thrombocytopenia in Cyno toxicological studies and maintained anti-tumor activity in a mouse xenograft model. These findings suggest that Y-634 may have a therapeutic potential for the treatment of Nectin-2 positive cancers, and moreover, Fc engineering is a potential mitigation strategy to ameliorate safety liabilities in antibody induced thrombocytopenia while maintaining antibody potency.

Dengue-Immune Humans Have Higher Levels of Complement-Independent Enhancing Antibody than Complement-Dependent Neutralizing Antibody.

Science.gov (United States)

Yamanaka, Atsushi; Konishi, Eiji

2017-09-25

Dengue is the most important arboviral disease worldwide. We previously reported that most inhabitants of dengue-endemic countries who are naturally immune to the disease have infection-enhancing antibodies whose in vitro activity does not decrease in the presence of complement (complement-independent enhancing antibodies, or CiEAb). Here, we compared levels of CiEAb and complement-dependent neutralizing antibodies (CdNAb) in dengue-immune humans. A typical antibody dose-response pattern obtained in our assay system to measure the balance between neutralizing and enhancing antibodies showed both neutralizing and enhancing activities depending on serum dilution factor. The addition of complement to the assay system increased the activity of neutralizing antibodies at lower dilutions, indicating the presence of CdNAb. In contrast, similar dose-response curves were obtained with and without complement at higher dilutions, indicating higher levels of CiEAb than CdNAb. For experimental support for the higher CiEAb levels, a cocktail of mouse monoclonal antibodies against dengue virus type 1 was prepared. The antibody dose-response curves obtained in this assay, with or without complement, were similar to those obtained with human serum samples when a high proportion of D1-V-3H12 (an antibody exhibiting only enhancing activity and thus a model for CiEAb) was used in the cocktail. This study revealed higher-level induction of CiEAb than CdNAb in humans naturally infected with dengue viruses.

T-cell activation. VI. Inhibitory and stimulatory effects of anti-major histocompatibility complex class I antibodies in allogeneic mixed lymphocyte culture

DEFF Research Database (Denmark)

Röpke, M; Röpke, C; Claesson, Mogens Helweg

1993-01-01

Murine T splenocytes stimulated in primary allogeneic mixed lymphocyte culture (MLC) were incubated with soluble anti-major histocompatibility complex (MHC) class I monoclonal antibodies. These antibodies induced inhibition in the cytotoxicity of the responding population and this inhibition...... was not dependent on the domain on class I molecules recognized by the antibodies. Cross-reactivity of the antibodies between the responder and stimulating cell population caused a marked reduction in the inhibitory effect compared to systems where no such cross-reactivity was present. Saturating levels...... of the antibodies caused a reduction in generation of T-cell cytotoxicity, whereas low concentrations stimulated the same response. These results demonstrate that the MHC class I molecules of T cells are of significant importance in antigen-induced signal transduction....

Recombinant IgG1 Fc hexamers block cytotoxicity and pathological changes in experimental in vitro and rat models of neuromyelitis optica.

Science.gov (United States)

Tradtrantip, Lukmanee; Felix, Christian M; Spirig, Rolf; Morelli, Adriana Baz; Verkman, A S

2018-05-01

Intravenous human immunoglobulin G (IVIG) may have therapeutic benefit in neuromyelitis optica spectrum disorders (herein called NMO), in part because of the anti-inflammatory properties of the IgG Fc region. Here, we evaluated recombinant Fc hexamers consisting of the IgM μ-tailpiece fused with the Fc region of human IgG1. In vitro, the Fc hexamers prevented cytotoxicity in aquaporin-4 (AQP4) expressing cells and in rat spinal cord slice cultures exposed to NMO anti-AQP4 autoantibody (AQP4-IgG) and complement, with >500-fold greater potency than IVIG or monomeric Fc fragments. Fc hexamers at low concentration also prevented antibody-dependent cellular cytotoxicity produced by AQP4-IgG and natural killer cells. Serum from rats administered a single intravenous dose of Fc hexamers at 50 mg/kg taken at 8 h did not produce complement-dependent cytotoxicity when added to AQP4-IgG-treated AQP4-expressing cell cultures. In an experimental rat model of NMO produced by intracerebral injection of AQP4-IgG, Fc hexamers at 50 mg/kg administered before and at 12 h after AQP4-IgG fully prevented astrocyte injury, complement activation, inflammation and demyelination. These results support the potential therapeutic utility of recombinant IgG1 Fc hexamers in AQP4-IgG seropositive NMO. Copyright © 2018 Elsevier Ltd. All rights reserved.

Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells

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Xie Y

2016-07-01

Full Text Available Yuexia Xie,1,2,* Dejun Liu,3,* Chenlei Cai,1,* Xiaojing Chen,1 Yan Zhou,1 Liangliang Wu,1 Yongwei Sun,3 Huili Dai,1,2 Xianming Kong,1,2 Peifeng Liu1,2 1Central Laboratory, 2State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, 3Department of Biliary-Pancreatic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: The application of Fe3O4 nanoparticles (NPs has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mecha­nisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm. Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application. Keywords: hepatoma cells, nanoparticles, cytotoxicity, mechanism, oxidative stress

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

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Mark T Winkler

Full Text Available 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.

Acquired agranulocytosis with granulocyte specific cytotoxic autoantibody.

Science.gov (United States)

Blaschke, J; Goeken, N E; Thompson, J S; Dick, F R; Gingrich, R D

1979-05-01

Multiple infections and severe neutropenia were found in a previously healthy 29 year old man with no history of similar syndromes in the family, drug ingestion or exposure to environmental toxins. There was no evidence at the time of presentation of diseases previously associated with agranulocytosis (e.g., neoplasia, thyrotoxicosis, chronic infection, collagen-vascular disease or leukoagglutinating antibody). His serum contained a nonagglutinating, complement-dependent, cytotoxic antibody, however, reactive with peripheral blood granulocytes from 35 per cent of normal donors. The neutropenia was not affected by steroids but resolved promptly after splenectomy. Microscopic examination of the spleen revealed ingestion of polymorphonuclear leukocytes by splenic macrophages. Family studies indicated that the target antigen was non-HLA and that the antibody was not absorbed by lymphocytes or platelets. We conclude that the agranulocytosis was autoimmune in origin and suggest that similar myeloid-specific immune responses could influence granulocyte tranfusion and bone marrow transplantation by alloimmune "rejection" that would not be avoided by matching only for HLA specificities.

The cyto- and genotoxicity of organotin compounds is dependent on the cellular uptake capability

International Nuclear Information System (INIS)

Dopp, E.; Hartmann, L.M.; Recklinghausen, U. von; Florea, A.M.; Rabieh, S.; Shokouhi, B.; Hirner, A.V.; Obe, G.; Rettenmeier, A.W.

2007-01-01

Organotin compounds have been widely used as stabilizers and anti-fouling agents with the result that they are ubiquitously distributed in the environment. Organotins accumulate in the food chain and potential effects on human health are disquieting. It is not known as yet whether cell surface adsorption or accumulation within the cell, or indeed both is a prerequisite for the toxicity of organotin compounds. In this study, the alkylated tin derivatives monomethyltin trichloride (MMT), dimethyltin dichloride (DMT), trimethyltin chloride (TMT) and tetramethyltin (TetraMT) were investigated for cyto- and genotoxic effects in CHO-9 cells in relation to the cellular uptake. To identify genotoxic effects, induction of micronuclei (MN), chromosome aberrations (CA) and sister chromatid exchanges (SCE) were analyzed and the nuclear division index (NDI) was calculated. The cellular uptake was assessed using ICP-MS analysis. The toxicity of the tin compounds was also evaluated after forced uptake by electroporation. Our results show that uptake of the organotin compounds was generally low but dose-dependent. Only weak genotoxic effects were observed after exposure of cells to DMT and TMT. MMT and TetraMT were negative in the test systems. After forced uptake by electroporation MMT, DMT and TMT induced significant DNA damage at non-cytotoxic concentrations. The results presented here indicate a considerable toxicological potential of some organotin species but demonstrate clearly that the toxicity is modulated by the cellular uptake capability

The cellular bases of antibody responses during dengue virus infection

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Juan Carlos Yam-Puc

2016-06-01

Full Text Available Dengue virus (DENV is one of the most significant human viral pathogens transmitted by mosquitoes and can cause from an asymptomatic disease to mild undifferentiated fever, classical dengue, and severe dengue. Neutralizing memory antibody (Ab responses are one of the most important mechanisms that counteract reinfections and are therefore the main aim of vaccination. However, it has also been proposed that in dengue, some of these class-switched (IgG memory Abs might worsen the disease. Although these memory Abs derive from B cells by T-cell dependent processes, we know rather little about the (acute, chronic or memory B cell responses and the complex cellular mechanisms generating these Abs during DENV infections.This review aims to provide an updated and comprehensive perspective of the B cell responses during DENV infection, starting since the very early events like the cutaneous DENV entrance and the arrival into draining lymph nodes, to the putative B cell activation, proliferation and germinal centers (GCs formation (the source of affinity-matured class-switched memory Abs, till the outcome of GC reactions such as the generation of plasmablasts, Ab-secreting plasma cells and memory B cells. We discuss topics very poorly explored such as the possibility of B cell infection by DENV or even activation-induced B cell death. The current information about the nature of the Ab responses to DENV is also illustrated.

Anti-cytotoxic T lymphocyte antigen-4 antibodies in melanoma

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Tosti G

2013-10-01

Full Text Available Giulio Tosti, Emilia Cocorocchio, Elisabetta PennacchioliDivisione Melanomi e Sarcomi, Istituto Europeo di Oncologia, Milano, ItalyAbstract: Approaches aimed at enhancement of the tumor specific response have provided proof for the rationale of immunotherapy in cancer, both in animal models and in humans. Ipilimumab, an anti-cytotoxic T lymphocyte antigen-4 (CTLA-4 antibody, is a new generation immunotherapeutic agent that has shown activity in terms of disease free and overall survival in metastatic melanoma patients. Its use was approved by the US Food and Drug Administration in March 2011 to treat patients with late stage melanoma that has spread or that cannot be removed by surgery. The mechanism of action of CTLA-4 antibodies in the activation of an antitumor immune response and selected clinical studies of ipilimumab in advanced melanoma patients are discussed. Ipilimumab treatment has been associated with immune related adverse events due to T-cell activation and proliferation. Most of these serious adverse effects are associated with the gastrointestinal tract and include severe diarrhea and colitis. The relationship between immune related adverse events and antitumor activity associated with ipilimumab was explored in clinical studies. Potential biomarkers predictive for clinical response and survival in patients treated with anti-CTLA-4 therapy are presently under investigation. Besides the conventional patterns of response and stable disease as defined by standard Response Evaluation Criteria in Solid Tumors criteria, in subsets of patients, ipilimumab has shown patterns of delayed clinical activity which were associated with an improved overall survival. For this reason a new set of response criteria for tumor immunotherapy has been proposed, which was termed immune related response criteria. These new criteria are presently used to better analyze clinical activity of immunotherapeutic regimens. Ipilimumab is currently under

In vitro experimental (211)At-anti-CD33 antibody therapy of leukaemia cells overcomes cellular resistance seen in vivo against gemtuzumab ozogamicin.

Science.gov (United States)

Petrich, Thorsten; Korkmaz, Zekiye; Krull, Doris; Frömke, Cornelia; Meyer, Geerd J; Knapp, Wolfram H

2010-05-01

Monoclonal anti-CD33 antibodies conjugated with toxic calicheamicin derivative (gemtuzumab ozogamicin, GO) are a novel therapy option for acute myeloid leukaemia (AML). Key prognostic factors for patients with AML are high CD33 expression on the leukaemic cells and the ability to overcome mechanisms of resistance to cytotoxic chemotherapies, including drug efflux or other mechanisms decreasing apoptosis. Alpha particle-emitting radionuclides overwhelm such anti-apoptotic mechanisms by producing numerous DNA double-stranded breaks (DSBs) accompanied by decreased DNA repair. We labelled anti-CD33 antibodies with the alpha-emitter (211)At and compared survival of leukaemic HL-60 and K-562 cells treated with the (211)At-labelled antibodies, GO or unlabelled antibodies as controls. We also measured caspase-3/7 activity, DNA fragmentation and necrosis in HL-60 cells after treatment with the different antibodies or with free (211)At. The mean labelling ratio of (211)At-labelled antibodies was 1:1,090 +/- 364 (range: 1:738-1:1,722) in comparison to 2-3:1 for GO. Tumour cell binding of (211)At-anti-CD33 was high in the presence of abundant CD33 expression and could be specifically blocked by unlabelled anti-CD33. (211)At-anti-CD33 decreased survival significantly more than did GO at comparable dilution (1:1,000). No significant differences in induction of apoptosis or necrosis or DNA DSB or in decreased survival were observed after (211)At-anti-CD33 (1:1,090) versus GO (1:1) treatment. Our results suggest that (211)At is a promising, highly cytotoxic radioimmunotherapy in CD33-positive leukaemia and kills tumour cells more efficiently than does calicheamicin-conjugated antibody. Labelling techniques leading to higher chemical yield and specific activities must be developed to increase (211)At-anti-CD33 therapeutic effects.

PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity

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Maria Gato-Cañas

2017-08-01

Full Text Available PDL1 blockade produces remarkable clinical responses, thought to occur by T cell reactivation through prevention of PDL1-PD1 T cell inhibitory interactions. Here, we find that PDL1 cell-intrinsic signaling protects cancer cells from interferon (IFN cytotoxicity and accelerates tumor progression. PDL1 inhibited IFN signal transduction through a conserved class of sequence motifs that mediate crosstalk with IFN signaling. Abrogation of PDL1 expression or antibody-mediated PDL1 blockade strongly sensitized cancer cells to IFN cytotoxicity through a STAT3/caspase-7-dependent pathway. Moreover, somatic mutations found in human carcinomas within these PDL1 sequence motifs disrupted motif regulation, resulting in PDL1 molecules with enhanced protective activities from type I and type II IFN cytotoxicity. Overall, our results reveal a mode of action of PDL1 in cancer cells as a first line of defense against IFN cytotoxicity.

Dependence of anaphylactic histamine release from rat mast cells on cellular energy metabolism

DEFF Research Database (Denmark)

Johansen, Torben

1981-01-01

The relation between anaphylactic histamine release and the adenosine triphosphate (ATP) content of the mast cells was studied. The cells were incubated with glycolytic (2-deoxyglucose) and respiratory inhibitors (antimycin A and oligomycin) in order to decrease the ATP content of the cells prior...... to initiation of the release process by the antigen-antibody reaction. The secretory capacity of mast cells was less related to the cellular level of ATP at the time of activation of the release process by the antigen-antibody reaction than to the rate of cellular energy supply. Furthermore, mast cells were...... pretreated with 2-deoxyglucose. The release of histamine from these cells was reduced when respiratory inhibitors were added to the cell suspension 5 to 20 sec after exposure of the cells to antigen. This may indicate that the secretory process requires energy, and it seems necessary that energy should...

Real-time impedimetric monitoring of Poly(ethylenimine)s-mediated cytotoxicity during gene transfection

DEFF Research Database (Denmark)

Caviglia, Claudia; Carminati, Marco; Heiskanen, Arto

Poly(ethylenimine)s (PEIs) are able to condense DNA and RNA into stable toroidal and globular nanostructures (polyplexes) and are among the most efficient and promising synthetic transfectants, but they induce severe cytotoxicity. The mechanisms of PEI-mediated cytotoxicity have not been fully...... delineated but PEI toxicity appears to predominantly depend on membrane perturbing effects in cellular compartments in which they accumulate. Electrochemical Impedance Spectroscopy (EIS) is used as a non-invasive biophysical approach for the investigation of the electrical properties of biological materials...

Tubulin Inhibitor-Based Antibody-Drug Conjugates for Cancer Therapy

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Hao Chen

2017-08-01

Full Text Available Antibody-drug conjugates (ADCs are a class of highly potent biopharmaceutical drugs generated by conjugating cytotoxic drugs with specific monoclonal antibodies through appropriate linkers. Specific antibodies used to guide potent warheads to tumor tissues can effectively reduce undesired side effects of the cytotoxic drugs. An in-depth understanding of antibodies, linkers, conjugation strategies, cytotoxic drugs, and their molecular targets has led to the successful development of several approved ADCs. These ADCs are powerful therapeutics for cancer treatment, enabling wider therapeutic windows, improved pharmacokinetic/pharmacodynamic properties, and enhanced efficacy. Since tubulin inhibitors are one of the most successful cytotoxic drugs in the ADC armamentarium, this review focuses on the progress in tubulin inhibitor-based ADCs, as well as lessons learned from the unsuccessful ADCs containing tubulin inhibitors. This review should be helpful to facilitate future development of new generations of tubulin inhibitor-based ADCs for cancer therapy.

Long-term outcomes of kidney transplantation across a positive complement-dependent cytotoxicity crossmatch.

Science.gov (United States)

Riella, Leonardo V; Safa, Kassem; Yagan, Jude; Lee, Belinda; Azzi, Jamil; Najafian, Nader; Abdi, Reza; Milford, Edgar; Mah, Helen; Gabardi, Steven; Malek, Sayeed; Tullius, Stefan G; Magee, Colm; Chandraker, Anil

2014-06-27

More than 30% of potential kidney transplant recipients have pre-existing anti-human leukocyte antigen antibodies. This subgroup has significantly lower transplant rates and increased mortality. Desensitization has become an important tool to overcome this immunological barrier. However, limited data is available regarding long-term outcomes, in particular for the highest risk group with a positive complement-dependent cytotoxicity crossmatch (CDC XM) before desensitization. Between 2002 and 2010, 39 patients underwent living-kidney transplantation across a positive CDC XM against their donors at our center. The desensitization protocol involved pretransplant immunosuppression, plasmapheresis, and low-dose intravenous immunoglobulin±rituximab. Measured outcomes included patient survival, graft survival, renal function, rates of rejection, infection, and malignancy. The mean and median follow-up was 5.2 years. Patient survival was 95% at 1 year, 95% at 3 years, and 86% at 5 years. Death-censored graft survival was 94% at 1 year, 88% at 3 years, and 84% at 5 years. Uncensored graft survival was 87% at 1 year, 79% at 3 years, and 72% at 5 years. Twenty-four subjects (61%) developed acute antibody-mediated rejection of the allograft and one patient lost her graft because of hyperacute rejection. Infectious complications included pneumonia (17%), BK nephropathy (10%), and CMV disease (5%). Skin cancer was the most prevalent malignancy in 10% of patients. There were no cases of lymphoproliferative disorder. Mean serum creatinine was 1.7±1 mg/dL in functioning grafts at 5 years after transplantation. Despite high rates of early rejection, desensitization in living-kidney transplantation results in acceptable 5-year patient and graft survival rates.

Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

International Nuclear Information System (INIS)

Hirano, Seishiro; Fujitani, Yuji; Furuyama, Akiko; Kanno, Sanae

2010-01-01

Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC 50 of MWCNT was 12 μg/ml, whereas that of asbestos (crocidolite) was 678 μg/ml. Over the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 μg/ml. BEAS-2B cells were exposed to 2, 5, or 10 μg/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-κB or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-κB was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-κB, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.

Carboxylesterase-dependent cytotoxicity of dibasic esters (DBE) in rat nasal explants.

Science.gov (United States)

Trela, B A; Bogdanffy, M S

1991-02-01

Dibasic esters (DBE) are a solvent mixture of dimethyl adipate (DMA), dimethyl glutarate (DMG), and dimethyl succinate (DMS) used in the paint and coating industry. Subchronic inhalation toxicity studies have demonstrated that DBE induce a mild degeneration of the olfactory, but not the respiratory, epithelium of the rat nasal cavity. Carboxylesterase-mediated hydrolysis of the individual dibasic esters is more efficient in olfactory than in respiratory mucosal homogenates. In the present study, an in vitro system of cultured rat nasal explants was utilized to determine if DBE toxicity is dependent on a metabolic activation by nonspecific carboxylesterase. Explants from both the olfactory and the respiratory regions of the female rat nasal cavity were incubated for 2 hr in Williams' medium E containing 10-100 mM DMA, DMG, or DMS. DBE caused a dose-related increase in nasal explant acid phosphatase release, a biochemical index of cytotoxicity. HPLC analysis demonstrated parallel increases in the carboxylesterase-mediated formation of monomethyl ester metabolites. Diacid metabolite production in the nasal explant system was not entirely concentration-dependent. Metabolite concentrations and acid phosphatase release were generally greater in olfactory than respiratory tissues. DBE-induced cytotoxicity and acid metabolite production were markedly attenuated in nasal tissue excised from rats which were pretreated with bis(p-nitrophenyl)phosphate, a carboxylesterase inhibitor. This study presents a viable in vitro method for assessing organic ester cytotoxicity in the rat nasal cavity. It was shown that DBE are weak nasal toxicants under the conditions of this system. It was further demonstrated that DBE toxicity is dependent on a carboxylesterase-mediated activation. A similar mechanism was proposed for the nasal toxicity induced by other organic esters following inhalation exposure.

Antibody-drug conjugates: Promising and efficient tools for targeted cancer therapy.

Science.gov (United States)

Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Majidi, Jafar

2018-09-01

Over the recent decades, the use of antibody-drug conjugates (ADCs) has led to a paradigm shift in cancer chemotherapy. Antibody-based treatment of various human tumors has presented dramatic efficacy and is now one of the most promising strategies used for targeted therapy of patients with a variety of malignancies, including hematological cancers and solid tumors. Monoclonal antibodies (mAbs) are able to selectively deliver cytotoxic drugs to tumor cells, which express specific antigens on their surface, and has been suggested as a novel category of agents for use in the development of anticancer targeted therapies. In contrast to conventional treatments that cause damage to healthy tissues, ADCs use mAbs to specifically attach to antigens on the surface of target cells and deliver their cytotoxic payloads. The therapeutic success of future ADCs depends on closely choosing the target antigen, increasing the potency of the cytotoxic cargo, improving the properties of the linker, and reducing drug resistance. If appropriate solutions are presented to address these issues, ADCs will play a more important role in the development of targeted therapeutics against cancer in the next years. We review the design of ADCs, and focus on how ADCs can be exploited to overcome multiple drug resistance (MDR). © 2018 Wiley Periodicals, Inc.

Cytokine, antibody and proliferative cellular responses elicited by Taenia solium calreticulin upon experimental infection in hamsters.

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Fela Mendlovic

Full Text Available Taenia solium causes two diseases in humans, cysticercosis and taeniosis. Tapeworm carriers are the main risk factor for neurocysticercosis. Limited information is available about the immune response elicited by the adult parasite, particularly the induction of Th2 responses, frequently associated to helminth infections. Calreticulin is a ubiquitous, multifunctional protein involved in cellular calcium homeostasis, which has been suggested to play a role in the regulation of immune responses. In this work, we assessed the effect of recombinant T. solium calreticulin (rTsCRT on the cytokine, humoral and cellular responses upon experimental infection in Syrian Golden hamsters (Mesocricetus auratus. Animals were infected with T. solium cysticerci and euthanized at different times after infection. Specific serum antibodies, proliferative responses in mesenteric lymph nodes and spleen cells, as well as cytokines messenger RNA (mRNA were analyzed. The results showed that one third of the infected animals elicited anti-rTsCRT IgG antibodies. Interestingly, mesenteric lymph node (MLN cells from either infected or non-infected animals did not proliferate upon in vitro stimulation with rTsCRT. Additionally, stimulation with a tapeworm crude extract resulted in increased expression of IL-4 and IL-5 mRNA. Upon stimulation, rTsCRT increased the expression levels of IL-10 in spleen and MLN cells from uninfected and infected hamsters. The results showed that rTsCRT favors a Th2-biased immune response characterized by the induction of IL-10 in mucosal and systemic lymphoid organs. Here we provide the first data on the cytokine, antibody and cellular responses to rTsCRT upon in vitro stimulation during taeniasis.

Daratumumab depletes CD38sup>+> immune-regulatory cells, promotes T-cell expansion, and skews T-cell repertoire in multiple myeloma

DEFF Research Database (Denmark)

Krejcik, Jakub; Casneuf, Tineke; Nijhof, Inger S

2016-01-01

target non-plasma cells that express CD38, which prompted evaluation of daratumumab's effects on CD38-positive immune subpopulations. Peripheral blood (PB) and bone marrow (BM) from patients with relapsed/refractory myeloma from two daratumumab monotherapy studies were analyzed before and during therapy......Daratumumab targets CD38-expressing myeloma cells through a variety of immune-mediated mechanisms (complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis) and direct apoptosis with cross-linking. These mechanisms may also...... and at relapse. Regulatory B cells (Bregs) and myeloid-derived suppressor cells (MDSCs), previously shown to express CD38, were evaluated for immunosuppressive activity and daratumumab sensitivity in the myeloma setting. A novel subpopulation of regulatory T cells (Tregs) expressing CD38 was identified...

In vitro experimental {sup 211}At-anti-CD33 antibody therapy of leukaemia cells overcomes cellular resistance seen in vivo against gemtuzumab ozogamicin

Energy Technology Data Exchange (ETDEWEB)

Petrich, Thorsten; Korkmaz, Zekiye; Krull, Doris; Meyer, Geerd J.; Knapp, Wolfram H. [Hanover University School of Medicine, Department of Nuclear Medicine, Hanover (Germany); Froemke, Cornelia [Hanover University School of Medicine, Department of Biometry, Hanover (Germany)

2010-05-15

Monoclonal anti-CD33 antibodies conjugated with toxic calicheamicin derivative (gemtuzumab ozogamicin, GO) are a novel therapy option for acute myeloid leukaemia (AML). Key prognostic factors for patients with AML are high CD33 expression on the leukaemic cells and the ability to overcome mechanisms of resistance to cytotoxic chemotherapies, including drug efflux or other mechanisms decreasing apoptosis. Alpha particle-emitting radionuclides overwhelm such anti-apoptotic mechanisms by producing numerous DNA double-stranded breaks (DSBs) accompanied by decreased DNA repair. We labelled anti-CD33 antibodies with the alpha-emitter {sup 211}At and compared survival of leukaemic HL-60 and K-562 cells treated with the {sup 211}At-labelled antibodies, GO or unlabelled antibodies as controls. We also measured caspase-3/7 activity, DNA fragmentation and necrosis in HL-60 cells after treatment with the different antibodies or with free {sup 211}At. The mean labelling ratio of {sup 211}At-labelled antibodies was 1:1,090 {+-} 364 (range: 1:738-1:1,722) in comparison to 2-3:1 for GO. Tumour cell binding of {sup 211}At-anti-CD33 was high in the presence of abundant CD33 expression and could be specifically blocked by unlabelled anti-CD33. {sup 211}At-anti-CD33 decreased survival significantly more than did GO at comparable dilution (1:1,000). No significant differences in induction of apoptosis or necrosis or DNA DSB or in decreased survival were observed after {sup 211}At-anti-CD33 (1:1,090) versus GO (1:1) treatment. Our results suggest that {sup 211}At is a promising, highly cytotoxic radioimmunotherapy in CD33-positive leukaemia and kills tumour cells more efficiently than does calicheamicin-conjugated antibody. Labelling techniques leading to higher chemical yield and specific activities must be developed to increase {sup 211}At-anti-CD33 therapeutic effects. (orig.)

Combination with a defucosylated anti-HM1.24 monoclonal antibody plus lenalidomide induces marked ADCC against myeloma cells and their progenitors.

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Takeshi Harada

Full Text Available The immunomodulatory drug lenalidomide (Len has drawn attention to potentiate antibody-dependent cellular cytotoxicity (ADCC-mediated immunotherapies. We developed the defucosylated version (YB-AHM of humanized monoclonal antibody against HM1.24 (CD317 overexpressed in multiple myeloma (MM cells. In this study, we evaluated ADCC by YB-AHM and Len in combination against MM cells and their progenitors. YB-AHM was able to selectively kill via ADCC MM cells in bone marrow samples from patients with MM with low effector/target ratios, which was further enhanced by treatment with Len. Interestingly, Len also up-regulated HM1.24 expression on MM cells in an effector-dependent manner. HM1.24 was found to be highly expressed in a drug-resistant clonogenic "side population" in MM cells; and this combinatory treatment successfully reduced SP fractions in RPMI 8226 and KMS-11 cells in the presence of effector cells, and suppressed a clonogenic potential of MM cells in colony-forming assays. Collectively, the present study suggests that YB-AHM and Len in combination may become an effective therapeutic strategy in MM, warranting further study to target drug-resistant MM clonogenic cells.

Effect of producer cell line on functional activity of anti-D monoclonal antibodies destined for prevention of rhesus sensitization.

Science.gov (United States)

Olovnikova, N I; Ershler, M A; Belkina, E V; Nikolaeva, T L; Miterev, G Yu

2009-04-01

The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

Gold Nanocluster-Mediated Cellular Death under Electromagnetic Radiation.

Science.gov (United States)

Cifuentes-Rius, Anna; Ivask, Angela; Das, Shreya; Penya-Auladell, Nuria; Fabregas, Laura; Fletcher, Nicholas L; Houston, Zachary H; Thurecht, Kristofer J; Voelcker, Nicolas H

2017-11-29

Gold nanoclusters (Au NCs) have become a promising nanomaterial for cancer therapy because of their biocompatibility and fluorescent properties. In this study, the effect of ultrasmall protein-stabilized 2 nm Au NCs on six types of mammalian cells (fibroblasts, B-lymphocytes, glioblastoma, neuroblastoma, and two types of prostate cancer cells) under electromagnetic radiation is investigated. Cellular association of Au NCs in vitro is concentration-dependent, and Au NCs have low intrinsic toxicity. However, when Au NC-incubated cells are exposed to a 1 GHz electromagnetic field (microwave radiation), cell viability significantly decreases, thus demonstrating that Au NCs exhibit specific microwave-dependent cytotoxicity, likely resulting from localized heating. Upon i.v. injection in mice, Au NCs are still present at 24 h post administration. Considering the specific microwave-dependent cytotoxicity and low intrinsic toxicity, our work suggests the potential of Au NCs as effective and safe nanomedicines for cancer therapy.

Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

NARCIS (Netherlands)

Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

1985-01-01

T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

Size-dependent cytotoxicity of yttrium oxide nanoparticles on primary osteoblasts in vitro

Energy Technology Data Exchange (ETDEWEB)

Zhou, Guoqiang, E-mail: zhougq1982@163.com; Li, Yunfei; Ma, Yanyan; Liu, Zhu; Cao, Lili; Wang, Da; Liu, Sudan; Xu, Wenshi; Wang, Wenying [Hebei University, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science (China)

2016-05-15

Yttrium oxide nanoparticles are an excellent host material for the rare earth metals and have high luminescence efficiency providing a potential application in photodynamic therapy and biological imaging. In this study, the effects of yttrium oxide nanoparticles with four different sizes were investigated using primary osteoblasts in vitro. The results demonstrated that the cytotoxicity generated by yttrium oxide nanoparticles depended on the particle size, and smaller particles possessed higher toxicological effects. For the purpose to elucidate the relationship between reactive oxygen species generation and cell damage, cytomembrane integrity, intracellular reactive oxygen species level, mitochondrial membrane potential, cell apoptosis rate, and activity of caspase-3 in cells were then measured. Increased reactive oxygen species level was also observed in a size-dependent way. Thus, our data demonstrated that exposure to yttrium oxide nanoparticles resulted in a size-dependent cytotoxicity in cultured primary osteoblasts, and reactive oxygen species generation should be one possible damage pathway for the toxicological effects produced by yttrium oxide particles. The results may provide useful information for more rational applications of yttrium oxide nanoparticles in the future.

The enhanced cytotoxicity of misonidazole in the thiol depleted state - An oxygen dependent mechanism

International Nuclear Information System (INIS)

Tuttle, S.W.; Varnes, M.E.; Donahue, L.; Biaglow, J.E.

1985-01-01

Incubating A549 cells in the presence of L-buthionine-S, R-sulfoximine and misonidazole under aerobic conditions results in lowered rates of cell growth and greater cytotoxicity than is seen with either drug alone. The authors previously demonstrated the accumulation of hydrogen peroxide from cells treated with misonidazole following the inhibition of GSH-peroxidase with thiol depleting agents. They hypothesize that the enhancement of misonidazole toxicity by L-BSO results from the increased exposure to hydrogen peroxide, and the possible formation of the highly reactive hydroxyl radical in the presence of trace metals via Fenton chemistry. Support for this hypothesis comes from their observations that addition of radical scavengers (such as SOD and catalase) and nutritional antioxidants (vitamin E) to the culture medium will partially inhibit the cytotoxic effects. Further work is being done to measure the products of reaction of toxic oxygen species with cellular macromolecules, i.e. lipids

Synthesis, cytotoxicity, cellular uptake and influence on eicosanoid metabolism of cobalt-alkyne modified fructoses in comparison to auranofin and the cytotoxic COX inhibitor Co-ASS.

Science.gov (United States)

Ott, Ingo; Koch, Thao; Shorafa, Hashem; Bai, Zhenlin; Poeckel, Daniel; Steinhilber, Dieter; Gust, Ronald

2005-06-21

Propargylhexacarbonyldicobalt complexes with fructopyranose ligands were prepared and investigated for cytotoxicity in the MCF-7 human breast cancer cell line. The antiproliferative effects depended on the presence of isopropylidene protecting groups in the carbohydrate ligand and correlated with the cellular concentration of the complexes. IC(50) values of > 20 microM demonstrated that the fructose derivatives were only moderately active compared to the references auranofin and the aspirin (ASS) derivative [2-acetoxy(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS). In continuation of our studies on the mode of action of cobalt-alkyne complexes we studied the influence of the compounds on the formation of 12-HHT (COX-1 product) and 12-HETE (12-LOX product) by human platelets as an indication of the interference in the eicosanoid metabolism, which is discussed as a target system of cytostatics. Co-ASS was an efficient COX-1 inhibitor without LOX inhibitory activity and auranofin inhibited both COX-1 and 12-LOX eicosanoid production. The missing activity of the fructopyranose complexes at the 12-LOX and the only moderate effects at COX-1 indicate that COX/LOX inhibition may be in part responsible for the pharmacological effects of auranofin and Co-ASS but not for those of the fructopyranose complexes.

Surface-dependent cytotoxicity on bacteria as a model for environmental stress of halloysite nanotubes

Energy Technology Data Exchange (ETDEWEB)

Choi, Hyo-Jick, E-mail: hyojickchoi@gmail.com [National Institute for Nanotechnology, Nanotechnology Accelerator and Department of Chemical and Materials Engineering, University of Alberta (Canada); Stazak, Theodore J. [School of Energy, Environmental, Biological and Medical Engineering, University of Cincinnati (United States); Montemagno, Carlo D., E-mail: montemag@ualberta.ca [National Institute for Nanotechnology, Nanotechnology Accelerator and Department of Chemical and Materials Engineering, University of Alberta (Canada)

2013-10-15

This study examined the cytotoxicity of halloysite nanotubes (HNTs) by investigating physiological responses of Escherichia coli, from cell growth to protein expression. Surfaces of HNTs were modified by amine functionalization (NH{sub 2}-HNTs) or bovine serum albumin (BSA) coating and their cytotoxicity levels were compared with that of non-modified HNTs (Bare-HNTs). Bare- and NH{sub 2}-HNTs exhibited accelerated cell death rates at {>=}0.5 mg/ml of HNTs. It was also found that concentration as low as 0.01 mg/ml of HNTs exerted significant toxic effects on the bacterial cells. Cellular viability, metabolic activity, and DNA replication all decreased with increasing concentrations of Bare- and NH{sub 2}-HNTs. In contrast, 0.01 mg/ml of BSA-coated HNTs (BSA-HNTs) coated showed no evidence of cytotoxicity. Even at concentrations {<=}0.1 mg/ml, the cytocompatibility of BSA-HNTs was significantly better than those of Bare- and NH{sub 2}-HNTs, which was confirmed by the observation of (i) the same or similar levels of cell proliferation and cell viability to the control, and (ii) higher levels of metabolic activity and plasmid DNA replication than those of Bare- and NH{sub 2}-HNTs. In addition, higher ranaspumin-2 protein yield was observed from bacterial culture supplemented with BSA-HNTs (100, 83, and 80 % of yield at 0.01, 0.05, and 0.1 mg/ml, respectively, relative to the control). This work showed that the increase of bacterial cytotoxicity of HNTs correlated well with elevating HNT concentration and that surface modification of HNTs with amine functional group and BSA coating was an effective strategy to reduce cytotoxicity up to 0.1 mg/ml of HNTs.

Evaluation of Cytotoxic Effects of Different Concentrations of Porous Hollow Au Nanoparticles (PHAuNPs) on Cells

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Rao, S.; Tata, U.; Lin, V.K.; Chiao, J.C.; Huang, Ch.; Hao, Y.; Wu, P.; Arora, N.; Ahn, J.

2014-01-01

Nanoparticles (NPs) have been introduced as a suitable alternative in many in vivo bio applications. The risks of utilizing nanoparticles continue to be an ongoing research. Furthermore, the various chemicals used in their synthesis influence the cytotoxic effects of nanoparticles. We have investigated the cytotoxicity of Porous Hollow Au Nanoparticles (PHAuNPs) on cancer cell lines PC-3, PC-3ML, and MDA-MB-231 and the normal cell line PNT1A. Cell proliferation for the different cells in the presence of different concentrations of the PHAuNPs was assessed after 24 hours and 72 hours of incubation using MTT assay. The study also included the cytotoxic evaluation of pegylated PHAuNPs. Identical cell seeding densities, particle concentrations, and incubation times were employed for these two types of Au nanoparticles. Our results indicated that (1) impact on cell proliferation was concentration dependent and was different for the different cell types without cellular necrosis and (b) cellular proliferation might be impacted more based on the cell line.

Dose-dependent cytotoxicity of clinically relevant cobalt nanoparticles and ions on macrophages in vitro

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Kwon, Young-Min; Xia Zhidao; Glyn-Jones, Sion; Beard, David; Gill, Harinderjit S; Murray, David W, E-mail: young-min.kwon@ndos.ox.ac.u [Nuffield Department of Orthopaedic Surgery, University of Oxford, Oxford OX3 7LD (United Kingdom)

2009-04-15

Despite the satisfactory short-term implant survivorship of metal-on-metal hip resurfacing arthroplasty, periprosthetic soft-tissue masses such as pseudotumours are being increasingly reported. Cytotoxic effects of cobalt or chromium have been suggested to play a role in its aetiology. The aim of this study was to investigate the effects of clinically relevant metal nanoparticles and ions on the viability of macrophages in vitro. A RAW 264.7 murine macrophage cell line was cultured in the presence of either: (1) cobalt, chromium and titanium nanoparticles sized 30-35 nm; or (2) cobalt sulphate and chromium chloride. Two methods were used to quantify cell viability: Alamar Blue assay and Live/Dead assay. The cytotoxicity was observed only with cobalt. Cobalt nanoparticles and ions demonstrated dose-dependent cytotoxic effects on macrophages in vitro: the cytotoxic concentrations of nanoparticles and ions were 1 x 10{sup 12} particles ml{sup -1} and 1000 {mu}M, respectively. The high concentration of cobalt nanoparticles required for cytotoxicity of macrophages in vitro suggests that increased production of cobalt nanoparticles in vivo, due to excessive MoM implant wear, may lead to local adverse biological effects. Therefore, cytotoxicity of high concentrations of metal nanoparticles phagocytosed by macrophages located in the periprosthetic tissues may be an important factor in pathogenesis of pseudotumours.

Clinical Cancer Therapy by NK Cells via Antibody-Dependent Cell-Mediated Cytotoxicity

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Kory L. Alderson

2011-01-01

Full Text Available Natural killer (NK cells are powerful effector cells that can be directed to eliminate tumor cells through tumor-targeted monoclonal antibodies (mAbs. Some tumor-targeted mAbs have been successfully applied in the clinic and are included in the standard of care for certain malignancies. Strategies to augment the antitumor response by NK cells have led to an increased understanding of how to improve their effector responses. Next-generation reagents, such as molecularly modified mAbs and mAb-cytokine fusion proteins (immunocytokines, ICs designed to augment NK-mediated killing, are showing promise in preclinical and some clinical settings. Continued research into the antitumor effects induced by NK cells and tumor-targeted mAbs suggests that additional intrinsic and extrinsic factors may influence the antitumor response. Therefore more research is needed that focuses on evaluating which NK cell and tumor criteria are best predictive of a clinical response and which combination immunotherapy regimens to pursue for distinct clinical settings.

Characterization of a Novel Humanized Anti-CD20 Antibody with Potent Anti-Tumor Activity against Non-Hodgkin's Lymphoma

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Haifeng Zhang

2013-09-01

Full Text Available Background: Rituximab, a mouse Fab and human Fc chimeric antibody, has been widely used to treat Non-Hodgkin's lymphoma (NHL. However, only 48% of patients respond to the treatment and complete response rate is below 10%. Also, immunogenicity was reported in 17-20% patients receiving the treatment, making it unsuitable for long term diseases such as autoimmune disorders. It has been a hot research field to “humanize” rituximab toward improved efficacy and reduced immunogenicity. Methods: In this study, an advanced antibody humanization technology was applied to the sequence of the anti-CD20 antibody 2B8, its sequence of which was based on the original murine monoclonal antibody of rituximab in Roche. The complementarity-determining regions (CDRs of the humanized antibodies were further optimized through computer-aided molecular dock. Results: Five novel humanized anti-CD20 antibodies 1-5(1635, 1534, 3637, 1634 and 1536 were generated and their immunogenicity was significantly decreased when compared to rituximab. The novel humanized anti-CD20 antibodies 1-5 retained the binding activity of their murine counterpart, as demonstrated by the fluorescence-activated cell-sorting analysis (FACS. When compared to rituximab, the humanized antibodies still have the similar properties on both complement-dependent cytotoxicity (CDC and antibody-dependent cell-mediated cytotoxicity (ADCC. Furthermore, its anti-tumor efficacy in xenograft model is comparable to that of rituximab. Conclusion: The humanized anti-CD20 antibodies 1-5 have lower immunogenicity than rituximab. And at the same time, they still retain the anti-tumor effect both in vitro and vivo.

Bispecific antibodies and their use in applied research

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Harshit Verma

Full Text Available Bispecific antibodies (BsAb can, by virtue of combining two binding specificities, improve the selectivity and efficacy of antibody-based treatment of human disease. Antibodies with two distinct binding specificities have great potential for a wide range of clinical applications as targeting agents for in vitro and in vivo immunodiagnosis, therapy and for improving immunoassays. They have shown great promise for targeting cytotoxic effector cells, delivering radionuclides, toxins or cytotoxic drugs to specific targets, particularly tumour cells. The development of BsAb research goes through three main stages: chemical cross linking of murine-derived monoclonal antibody, hybrid hybridomas and engineered BsAb. This article is providing the potential applications of bispecific antibodies. [Vet World 2012; 5(12.000: 775-780

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143 in chronic lymphocytic leukemia

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Manoj K. Kashyap

2017-05-01

Full Text Available Abstract Background The CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL. Methods Patient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model. Results PF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD. PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC and complement-dependent cytotoxicity activity (CDC. PF-06747143 had significant combinatorial effect with standard of care (SOC agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A, ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy

Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib.

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Neeson, Paul J; Hsu, Andy K; Chen, Yin R; Halse, Heloise M; Loh, Joanna; Cordy, Reece; Fielding, Kate; Davis, Joanne; Noske, Josh; Davenport, Alex J; Lindqvist-Gigg, Camilla A; Humphreys, Robin; Tai, Tsin; Prince, H Miles; Trapani, Joseph A; Smyth, Mark J; Ritchie, David S

2015-09-01

There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8 + T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.

PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

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Seo, Kang-Sik; Hwang, Byung-Doo; Kim, Jong-Seok; Park, Ji-Hoon; Song, Kyoung-Sub; Yun, Eun-Jin; Park, Jong-Il; Kweon, Gi Ryang; Yoon, Wan-Hee; Lim, Kyu

2014-01-01

Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells. Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively. We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G 1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner. These results suggest that the synergy between PMA and apicularen A is involved by

Antibody drug conjugates - Trojan horses in the war on cancer.

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Iyer, U; Kadambi, V J

2011-01-01

Antibody drug conjugates (ADCs) consist of an antibody attached to a cytotoxic drug by means of a linker. ADCs provide a way to couple the specificity of a monoclonal antibody (mAb) to the cytotoxicity of a small-molecule drug and, therefore, are promising new therapies for cancer. ADCs are prodrugs that are inactive in circulation but exert their cytotoxicity upon binding to the target cancer cell. Earlier unsuccessful attempts to generate ADCs with therapeutic value have emphasized the important role each component plays in determining the efficacy and safety of the final ADC. Scientific advances in engineering antibodies for maximum efficacy as anticancer agents, identification of highly cytotoxic molecules, and generation of linkers with increased stability in circulation have all contributed to the development of the many ADCs that are currently in clinical trials. This review discusses parameters that guide the selection of the components of an ADC to increase its therapeutic window, provides a brief look at ADCs currently in clinical trials, and discusses future challenges in this field. Copyright © 2011. Published by Elsevier Inc.

Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays.

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Guo, Xiaoqing; Li, Yan; Yan, Jian; Ingle, Taylor; Jones, Margie Yvonne; Mei, Nan; Boudreau, Mary D; Cunningham, Candice K; Abbas, Mazhar; Paredes, Angel M; Zhou, Tong; Moore, Martha M; Howard, Paul C; Chen, Tao

2016-11-01

The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having three different sizes and two different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA) and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronuclei from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materials also resulted in concentration-dependent cytotoxicity and significantly increased micronucleus frequency. With both the MLA and micronucleus assays, the smaller the AgNPs, the greater the cytotoxicity and genotoxicity. The coatings had less effect on the relative genotoxicity of AgNPs than the particle size. Loss of heterozygosity analysis of the induced Tk mutants indicated that the types of mutations induced by AgNPs were different from those of ionic silver. These results suggest that AgNPs induce cytotoxicity and genotoxicity in a size- and coating-dependent manner. Furthermore, while the MLA and in vitro micronucleus assay (in both types of cells) are useful to quantitatively measure the genotoxic potencies of AgNPs, the Ames test cannot.

Monoclonal antibodies for radioimmunodetection of tumours and for targeting

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Baldwin, R.W.; Embleton, M.J.; Pimm, M.V.

1983-01-01

A monoclonal antibody 791T/36 prepared against human osteogenic sarcoma has been used to detect primary and metastatic colorectal carcinomas by external imaging of patients following injection of 131 I-labelled antibody. In 10 of 11 patients radiolabelled 791T/36 antibody localized in tumours, the tumour/non tumour ratio of radioactivity ranging from 1.5:1 to 8.1. 791T/36 antibody was also evaluated for its potential for targeting anti-tumour agents including cytotoxic drugs (Vindesine) and immunomodulating agents (interferon). Vindesine-791T/36 conjugates were preferentially cytotoxic in vitro for target cells expressing the 791T/36 anti-body defined antigen. Also interferon conjugated to 791T/36 antibody, like free interferon activated peripheral blood natural killer cell activity. These in vitro tests together with related studies on antibody localization in vivo indicate the potential of monoclonal antibody targeting of anti-tumour agents

Monoclonal antibodies for treating cancer

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Dillman, R.O.

1989-01-01

The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references

Antibody-Conjugated Nanoparticles for Biomedical Applications

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Manuel Arruebo

2009-01-01

Full Text Available Nanoscience and Nanotechnology have found their way into the fields of Biotechnology and Medicine. Nanoparticles by themselves offer specific physicochemical properties that they do not exhibit in bulk form, where materials show constant physical properties regardless of size. Antibodies are nanosize biological products that are part of the specific immune system. In addition to their own properties as pathogens or toxin neutralizers, as well as in the recruitment of immune elements (complement, improving phagocytosis, cytotoxicity antibody dependent by natural killer cells, etc., they could carry several elements (toxins, drugs, fluorochroms, or even nanoparticles, etc. and be used in several diagnostic procedures, or even in therapy to destroy a specific target. The conjugation of antibodies to nanoparticles can generate a product that combines the properties of both. For example, they can combine the small size of nanoparticles and their special thermal, imaging, drug carrier, or magnetic characteristics with the abilities of antibodies, such as specific and selective recognition. The hybrid product will show versatility and specificity. In this review, we analyse both antibodies and nanoparticles, focusing especially on the recent developments for antibody-conjugated nanoparticles, offering the researcher an overview of the different applications and possibilities of these hybrid carriers.

Frequency-dependent micromechanics of cellularized biopolymer networks

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Jones, Chris; Kim, Jihan; McIntyre, David; Sun, Bo

Mechanical interactions between cells and the extracellular matrix (ECM) influence many cellular behaviors such as growth, differentiation, and migration. These are dynamic processes in which the cells actively remodel the ECM. Reconstituted collagen gel is a common model ECM for studying cell-ECM interactions in vitro because collagen is the most abundant component of mammalian ECM and gives the ECM its material stiffness. We embed micron-sized particles in collagen and use holographic optical tweezers to apply forces to the particles in multiple directions and over a range of frequencies up to 10 Hz. We calculate the local compliance and show that it is dependent on both the direction and frequency of the applied force. Performing the same measurement on many particles allows us to characterize the spatial inhomogeneity of the mechanical properties and shows that the compliance decreases at higher frequencies. Performing these measurements on cell-populated collagen gels shows that cellular remodeling of the ECM changes the mechanical properties of the collagen and we investigate whether this change is dependent on the local strain and distance from nearby cells.

Evaluation of monoclonal antibodies for the development of breast cancer immunotoxins

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Bjorn, M.J.; Ring, D.; Frankel, A.

1985-01-01

Eighty-five antibodies recognizing breast cancer-selective antigens were conjugated to ricin toxin A-chain using a disulfide linkage. The cytotoxicities of the resulting immunotoxins were determined on breast cancer cells and normal human fibroblasts. Twenty-four antibodies formed immunotoxins that were toxic to at least one breast cancer cell line at concentrations of 10 nM or less but were nontoxic to human fibroblast lines used as negative controls. Some of the breast tumor-selective immunotoxins were as toxic as a conjugate between monoclonal anti-transferrin receptor and ricin toxin A-chain (50% inhibition of cellular protein synthesis at approximately 0.1 nM). Another set of four immunotoxins were indiscriminately toxic to human breast tumor cell lines, two human fibroblast cell lines, and a human lymphoblastoid line. Several of the antibodies the toxin conjugates of which specifically killed breast cancer cell lines may be useful in cancer therapy, since they show a wide range of binding to individual breast tumors and cell lines and a limited range of binding to normal tissue types

Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column.

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Kiyoshi, Masato; Caaveiro, Jose M M; Tada, Minoru; Tamura, Hiroko; Tanaka, Toru; Terao, Yosuke; Morante, Koldo; Harazono, Akira; Hashii, Noritaka; Shibata, Hiroko; Kuroda, Daisuke; Nagatoishi, Satoru; Oe, Seigo; Ide, Teruhiko; Tsumoto, Kouhei; Ishii-Watabe, Akiko

2018-03-02

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV).

Science.gov (United States)

Satoh, Ryoichi; Kaku, Ayumi; Satomura, Megumi; Kohori, Michiyo; Noura, Kanako; Furukawa, Tomoko; Kotake, Masako; Takano, Tomomi; Hohdatsu, Tsutomu

2011-06-01

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats. Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

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Grinberg, Yehudit; Benhar, Itai

2017-01-01

Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called “human cytotoxic fusion proteins”, in which antibodies are used to target human effector proteins. Here, we present three relevant approaches for reducing the immunogenicity of antibody-targeted protein therapeutics: (1) reducing the immunogenicity of the bacterial toxin, (2) fusing human cytokines to antibodies to generate immunocytokines and (3) addressing the immunogenicity of the targeting antibodies. PMID:28574434

A Single Domain–Based Anti-Her2 Antibody Has Potent Antitumor Activities

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Xiaoqiong Wu

2018-04-01

Full Text Available Human epidermal growth factor receptor 2 (HER2 is overexpressed in approximately 20% to 30% of breast cancers and various other types of cancers, which plays a vital role in the cancer progression. Monoclonal antibodies targeting Her2 are now used in the clinic to treat Her2 overexpression cancer patients. However, relapse or resistance is frequent with the current therapies. To generate a new treatment avenue against Her2, we immunized and selected a specific anti-Her2 single domain antibody C3 for further studies. The C3-Fc antibody drove antibody-dependent cell-mediated cytotoxicity against Her2-positive tumor cells in vitro and resulted in potent antitumor growth in vivo. These data suggest that the C3-Fc antibody may provide an alternative avenue for Her2-positive cancer therapy.

Cytotoxicity and cellular uptake of ZnS:Mn nanocrystals biofunctionalized with chitosan and aminoacids

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Augustine, M.S.; Anas, A.; Das, A.V.; Sreekanth, S.; Jayalekshmi, S.

into solution and by generating free radical species. In animal experiments, QDs preferentially enter the liver and spleen follow-ing intravascular injection which undergo minimal excretion if lar-ger than 6 nm, and appear to be safe to the animal. The present... recently on toxicity studies of quantum dots (QDs) in cells and animals [49,50]. Cell culture experiments have shown that QDs undergo design- dependent intracellular localiza-tion which can cause cytotoxicity by releasing free toxic materials...

Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins.

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Liu, Liming

2015-06-01

Understanding the impact of glycosylation and keeping a close control on glycosylation of product candidates are required for both novel and biosimilar monoclonal antibodies (mAbs) and Fc-fusion protein development to ensure proper safety and efficacy profiles. Most therapeutic mAbs are of IgG class and contain a glycosylation site in the Fc region at amino acid position 297 and, in some cases, in the Fab region. For Fc-fusion proteins, glycosylation also frequently occurs in the fusion partners. Depending on the expression host, glycosylation patterns in mAb or Fc-fusions can be significantly different, thus significantly impacting the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs. Glycans that have a major impact on PK and PD of mAb or Fc-fusion proteins include mannose, sialic acids, fucose (Fuc), and galactose (Gal). Mannosylated glycans can impact the PK of the molecule, leading to reduced exposure and potentially lower efficacy. The level of sialic acid, N-acetylneuraminic acid (NANA), can also have a significant impact on the PK of Fc-fusion molecules. Core Fuc in the glycan structure reduces IgG antibody binding to IgG Fc receptor IIIa relative to IgG lacking Fuc, resulting in decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activities. Glycoengineered Chinese hamster ovary (CHO) expression systems can produce afucosylated mAbs that have increased ADCC activities. Terminal Gal in a mAb is important in the complement-dependent cytotoxicity (CDC) in that lower levels of Gal reduce CDC activity. Glycans can also have impacts on the safety of mAb. mAbs produced in murine myeloma cells such as NS0 and SP2/0 contain glycans such as Galα1-3Galβ1-4N-acetylglucosamine-R and N-glycolylneuraminic acid (NGNA) that are not naturally present in humans and can be immunogenic when used as therapeutics. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

A potent human neutralizing antibody Fc-dependently reduces established HBV infections.

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Li, Dan; He, Wenhui; Liu, Ximing; Zheng, Sanduo; Qi, Yonghe; Li, Huiyu; Mao, Fengfeng; Liu, Juan; Sun, Yinyan; Pan, Lijing; Du, Kaixin; Ye, Keqiong; Li, Wenhui; Sui, Jianhua

2017-09-26

Hepatitis B virus (HBV) infection is a major global health problem. Currently-available therapies are ineffective in curing chronic HBV infection. HBV and its satellite hepatitis D virus (HDV) infect hepatocytes via binding of the preS1 domain of its large envelope protein to sodium taurocholate cotransporting polypeptide (NTCP). Here, we developed novel human monoclonal antibodies that block the engagement of preS1 with NTCP and neutralize HBV and HDV with high potency. One antibody, 2H5-A14, functions at picomolar level and exhibited neutralization-activity-mediated prophylactic effects. It also acts therapeutically by eliciting antibody-Fc-dependent immunological effector functions that impose durable suppression of viral infection in HBV-infected mice, resulting in reductions in the levels of the small envelope antigen and viral DNA, with no emergence of escape mutants. Our results illustrate a novel antibody-Fc-dependent approach for HBV treatment and suggest 2H5-A14 as a novel clinical candidate for HBV prevention and treatment of chronic HBV infection.

Particle Size-Dependent Antibacterial Activity and Murine Cell Cytotoxicity Induced by Graphene Oxide Nanomaterials

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Lin Zhao

2016-01-01

Full Text Available Recent studies have indicated that graphene and its derivative graphene oxide (GO engage in a wide range of antibacterial activities with limited toxicity to human cells. Here, we systematically evaluate the dependence of GO toxicity on the size of the nanoparticles used in treatments: we compare the cytotoxic effects of graphene quantum dots (GQDs, <15 nm, small GOs (SGOs, 50–200 nm, and large GOs (LGOs, 0.5–3 μm. We synthesize the results of bacterial colony count assays and SEM-based observations of morphological changes to assess the antibacterial properties that these GOs bring into effect against E. coli. We also use Live/Dead assays and morphological analysis to investigate changes to mammalian (Murine macrophage-like Raw 264.7 cells induced by the presence of the various GO particle types. Our results demonstrate that LGOs, SGOs, and GQDs possess antibacterial activities and cause mammalian cell cytotoxicity at descending levels of potency. Placing our observations in the context of previous simulation results, we suggest that both the lateral size and surface area of GO particles contribute to cytotoxic effects. We hope that the size dependence elucidated here provides a useful schematic for tuning GO-cell interactions in biomedical applications.

Ovarian carcinoma glyco-antigen targeted by human IgM antibody.

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Yi Chen

Full Text Available Epithelial Ovarian Cancer (EOC cells expression of a novel carbohydrate antigen was defined using a human VH4-34 encoded IgM monoclonal antibody (mAb216. MAb216 binds to a poly N-acetyllactosamine epitope expressed on B cells and kills normal and malignant B cells in vitro and in vivo. EOC patient ascites and EOC cell lines were used to study the anti tumor effect of mAb216. Various assays were used to characterize the epitope and demonstrate antibody-mediated binding and cytotoxicity in EOC. Drug and antibody combination effects were determined by calculating the combination index values using the Chou and Talalay method. MAb216 displays direct antibody mediated cytotoxicity on a population of human EOC tumor and ascites samples and EOC cell lines, which express high amounts of poly N-acetyllactosamine epitope, carried by CD147/CD98. Eighty four percent of patient samples, including platin resistant, had a tumor population that bound the monoclonal antibody. The binding pattern of mAb216 and mechanism of cytotoxicity was similar to that seen on normal and malignant B cells with unique general membrane disruption and "pore" formation. In vitro incubation with mAb216 and cisplatin enhanced killing of OVCAR3 cell line. In EOC cell lines percent cytotoxicity correlated with percent expression of epitope. Although in vitro data shows specific EOC cytotoxicity, for possible treatment of EOC MAb216 would need to be evaluated in a clinical trial with or without chemotherapy.

Environmentally persistent free radicals amplify ultrafine particle mediated cellular oxidative stress and cytotoxicity

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Balakrishna Shrilatha

2009-04-01

Full Text Available Abstract Background Combustion generated particulate matter is deposited in the respiratory tract and pose a hazard to the lungs through their potential to cause oxidative stress and inflammation. We have previously shown that combustion of fuels and chlorinated hydrocarbons produce semiquinone-type radicals that are stabilized on particle surfaces (i.e. environmentally persistent free radicals; EPFRs. Because the composition and properties of actual combustion-generated particles are complex, heterogeneous in origin, and vary from day-to-day, we have chosen to use surrogate particle systems. In particular, we have chosen to use the radical of 2-monochlorophenol (MCP230 as the EPFR because we have previously shown that it forms a EPFR on Cu(IIO surfaces and catalyzes formation of PCDD/F. To understand the physicochemical properties responsible for the adverse pulmonary effects of combustion by-products, we have exposed human bronchial epithelial cells (BEAS-2B to MCP230 or the CuO/silica substrate. Our general hypothesis was that the EPFR-containing particle would have greater toxicity than the substrate species. Results Exposure of BEAS-2B cells to our combustion generated particle systems significantly increased reactive oxygen species (ROS generation and decreased cellular antioxidants resulting in cell death. Resveratrol treatment reversed the decline in cellular glutathione (GSH, glutathione peroxidase (GPx, and superoxide dismutase (SOD levels for both types of combustion-generated particle systems. Conclusion The enhanced cytotoxicity upon exposure to MCP230 correlated with its ability to generate more cellular oxidative stress and concurrently reduce the antioxidant defenses of the epithelial cells (i.e. reduced GSH, SOD activity, and GPx. The EPFRs in MCP230 also seem to be of greater biological concern due to their ability to induce lipid peroxidation. These results are consistent with the oxidizing nature of the CuO/silica ultrafine

Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

Science.gov (United States)

Di Cristofaro, Julie; Frassati, Coralie; Montagnie, Rolande; Basire, Agnes; Merieux, Yves; Picard, Christophe

2015-01-01

Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future.

Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

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Roit, Fabio Da; Engelberts, Patrick J.; Taylor, Ronald P.; Breij, Esther C.W.; Gritti, Giuseppe; Rambaldi, Alessandro; Introna, Martino; Parren, Paul W.H.I.; Beurskens, Frank J.; Golay, Josée

2015-01-01

The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3–3 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs. PMID:25344523

Cellular internalisation kinetics and cytotoxic properties of statistically designed and optimised neo-geometric copper nanocrystals.

Science.gov (United States)

Murugan, Karmani; Choonara, Yahya E; Kumar, Pradeep; du Toit, Lisa C; Pillay, Viness

2017-09-01

This study aimed to highlight a statistic design to precisely engineer homogenous geometric copper nanoparticles (CuNPs) for enhanced intracellular drug delivery as a function of geometrical structure. CuNPs with a dual functionality comprising geometric attributes for enhanced cell uptake and exerting cytotoxic activity on proliferating cells were synthesized as a novel drug delivery system. This paper investigated the defined concentrations of two key surfactants used in the reaction to mutually control and manipulate nano-shape and optimisation of the geometric nanosystems. A statistical experimental design comprising a full factorial model served as a refining factor to achieve homogenous geometric nanoparticles using a one-pot method for the systematic optimisation of the geometric CuNPs. Shapes of the nanoparticles were investigated to determine the result of the surfactant variation as the aim of the study and zeta potential was studied to ensure the stability of the system and establish a nanosystem of low aggregation potential. After optimisation of the nano-shapes, extensive cellular internalisation studies were conducted to elucidate the effect of geometric CuNPs on uptake rates, in addition to the vital toxicity assays to further understand the cellular effect of geometric CuNPs as a drug delivery system. In addition to geometry; volume, surface area, orientation to the cell membrane and colloidal stability is also addressed. The outcomes of the study demonstrated the success of homogenous geometric NP formation, in addition to a stable surface charge. The findings of the study can be utilized for the development of a drug delivery system for promoted cellular internalisation and effective drug delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

Size-dependent cytotoxicity and inflammatory responses of PEGylated silica-iron oxide nanocomposite size series

Science.gov (United States)

Injumpa, Wishulada; Ritprajak, Patcharee; Insin, Numpon

2017-04-01

incubation with the highest concentration of 1000 μg/mL. Although 1000 μg/mL of all sizes of the nanocomposites decreased macrophage viability, the cytotoxicity of the nanocomposites was notably less than silica. The inflammatory response of macrophage was also observed by ELISA, and we found that the size of 20 and 40 nm, but not 100 and 200 nm, obviously stimulated IL-6 production. From this study, the preparations of multifunctional superparamagnetic nanocomposites of different sizes along with the size-dependent effects on cellular toxicity and inflammatory response were demonstrated and could be applied for designing of new drug carriers.

Size-dependent cytotoxicity and inflammatory responses of PEGylated silica-iron oxide nanocomposite size series

Energy Technology Data Exchange (ETDEWEB)

Injumpa, Wishulada [Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330 (Thailand); Ritprajak, Patcharee [Department of Microbiology, and RU in Oral Microbiology and Immunology, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330 (Thailand); Insin, Numpon, E-mail: Numpon.I@chula.ac.th [Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330 (Thailand)

2017-04-01

incubation with the highest concentration of 1000 μg/mL. Although 1000 μg/mL of all sizes of the nanocomposites decreased macrophage viability, the cytotoxicity of the nanocomposites was notably less than silica. The inflammatory response of macrophage was also observed by ELISA, and we found that the size of 20 and 40 nm, but not 100 and 200 nm, obviously stimulated IL-6 production. From this study, the preparations of multifunctional superparamagnetic nanocomposites of different sizes along with the size-dependent effects on cellular toxicity and inflammatory response were demonstrated and could be applied for designing of new drug carriers. - Highlights: • Magnetic iron oxide-silica nanocomposites (MNCs) size series were synthesized. • PPEGMA-MNCs exhibited low cytotoxicity against fibroblast and macrophage lines. • The effects on the sizes of PPEGMA-coated MNCs on immune responses were observed.

Preclinical activity of the type II CD20 antibody GA101 (obinutuzumab) compared with rituximab and ofatumumab in vitro and in xenograft models.

Science.gov (United States)

Herter, Sylvia; Herting, Frank; Mundigl, Olaf; Waldhauer, Inja; Weinzierl, Tina; Fauti, Tanja; Muth, Gunter; Ziegler-Landesberger, Doris; Van Puijenbroek, Erwin; Lang, Sabine; Duong, Minh Ngoc; Reslan, Lina; Gerdes, Christian A; Friess, Thomas; Baer, Ute; Burtscher, Helmut; Weidner, Michael; Dumontet, Charles; Umana, Pablo; Niederfellner, Gerhard; Bacac, Marina; Klein, Christian

2013-10-01

We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation. ©2013 AACR.

Cell cycle phase dependent emergence of thymidylate synthase studied by monoclonal antibody (M-TS-4).

Science.gov (United States)

Shibui, S; Hoshino, T; Iwasaki, K; Nomura, K; Jastreboff, M M

1989-05-01

A method of identifying thymidylate synthase (TS) at the cellular level was developed using anti-TS monoclonal antibody (M-TS-4), a monoclonal antibody created against purified TS from a HeLa cell line. In HeLa cells and four human glioma cell lines (U-251, U-87, 343-MGA, and SF-188), TS was identified primarily in the cytoplasm. Autoradiographic and flow cytometric studies showed that TS appeared mainly in the G1 phase and subsided early in the S phase; thus, the G1 phase can be divided into TS-positive and -negative fractions. Nuclear TS was not demonstrated unequivocally with M-TS-4, and the relationship between nuclear TS and DNA synthesis could not be determined. Although the percentage of TS-positive cells was larger than the S-phase fraction measured by autoradiography after a pulse of tritiated thymidine or by the immunoperoxidase method using BUdR, the ratios were within a similar range (1.2-1.4) in all cell lines studied. Therefore, the S-phase fraction can be estimated indirectly from the percentage of TS-positive cells measured by M-TS-4. Because the emergence of TS detected by our method is cell cycle dependent, M-TS-4 may be useful for biochemical studies of TS and for cytokinetic analysis.

Successful kidney transplantation across a positive complement-dependent cytotoxicity crossmatch by using C1q assay-directed, bortezomib-assisted desensitization

Science.gov (United States)

Lee, Juhan; Park, Borae G.; Jeong, Hyang Sook; Park, Youn Hee; Kim, Sinyoung; Kim, Beom Seok; Kim, Hye Jin; Huh, Kyu Ha; Jeong, Hyeon Joo; Kim, Yu Seun

2017-01-01

Abstract Rationale: Human leukocyte antigen (HLA) is the major immunologic barrier in kidney transplantation (KT). Various desensitization protocols to overcome the HLA barrier have increased the opportunity for transplantation in sensitized patients. In addition, technological advances in solid-phase assays have permitted more comprehensive assessment of donor-specific antibodies. Although various desensitization therapies and immunologic techniques have been developed, the final transplantation decision is still based on the classic complement-dependent cytotoxicity (CDC) crossmatch (XM) technique. Some patients who fail to achieve negative XM have lost their transplant opportunities, even after receiving sufficient desensitization therapies. Patient concerns: A 57-year-old male with end-stage renal disease secondary to chronic glomerulonephritis was scheduled to have a second transplant from his son, but CDC XM was positive. Diagnoses: Initial CDC XM (Initial T-AHG 1:32) and flow-cytometry XM were positive. Anti-HLA-B59 donor specific antibody was detected by Luminex single antigen assay. Interventions: Herein, we report a successful case of KT across a positive CDC XM (T-AHG 1:8 at the time of transplantation) by using C1q assay-directed, bortezomib-assisted desensitization. After confirming a negative conversion in the C1q donor-specific antibody, we decided to perform KT accepting a positive AHG-CDC XM of 1:8 at the time of transplantation. Outcomes: The posttransplant course was uneventful and a protocol biopsy at 3 months showed no evidence of rejection. The patient had excellent graft function at 12 months posttransplant. Lessons: The results of XM test and solid-phase assay should be interpreted in the context of the individual patient. PMID:28953652

Effects of Iron-Oxide Nanoparticle Surface Chemistry on Uptake Kinetics and Cytotoxicity in CHO-K1 Cells

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Camille C. Hanot

2015-12-01

Full Text Available Superparamagnetic iron-oxide nanoparticles (SPIONs show great promise for multiple applications in biomedicine. While a number of studies have examined their safety profile, the toxicity of these particles on reproductive organs remains uncertain. The goal of this study was to evaluate the cytotoxicity of starch-coated, aminated, and PEGylated SPIONs on a cell line derived from Chinese Hamster ovaries (CHO-K1 cells. We evaluated the effect of particle diameter (50 and 100 nm and polyethylene glycol (PEG chain length (2k, 5k and 20k Da on the cytotoxicity of SPIONs by investigating cell viability using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT and sulforhodamine B (SRB assays. The kinetics and extent of SPION uptake by CHO-K1 cells was also studied, as well as the resulting generation of intracellular reactive oxygen species (ROS. Cell toxicity profiles of SPIONs correlated strongly with their cellular uptake kinetics, which was strongly dependent on surface properties of the particles. PEGylation caused a decrease in both uptake and cytotoxicity compared to aminated SPIONs. Interestingly, 2k Da PEG-modifed SPIONs displayed the lowest cellular uptake and cytotoxicity among all studied particles. These results emphasize the importance of surface coatings when engineering nanoparticles for biomedical applications.

Uniform TiO2 nanoparticles induce apoptosis in epithelial cell lines in a size-dependent manner.

Science.gov (United States)

Sun, Qingqing; Ishii, Takayuki; Kanehira, Koki; Sato, Takeshi; Taniguchi, Akiyoshi

2017-05-02

The size of titanium dioxide (TiO 2 ) nanoparticles is a vital parameter that determines their cytotoxicity. However, most reported studies have employed irregular shapes and sizes of TiO 2 nanoparticles, as it is difficult to produce nanoparticles of suitable sizes for research. We produced good model TiO 2 nanoparticles of uniform shape and size for use in studying their cytotoxicity. In this work, spherical, uniform polyethylene glycol-modified TiO 2 (TiO 2 -PEG) nanoparticles of differing sizes (100, 200, and 300 nm) were prepared using the sol-gel method. A size-dependent decrease in cell viability was observed with increasing nanoparticle size. Furthermore, apoptosis was found to be positively associated with nanoparticle size, as evidenced by an increase in caspase-3 activity with increasing nanoparticle size. Larger nanoparticles exhibited higher cellular uptake, suggesting that larger nanoparticles more strongly induce apoptosis. In addition, the cellular uptake of different sizes of nanoparticles was energy dependent, suggesting that there are size-dependent uptake pathways. We found that 100 and 200 nm (but not 300 nm) nanoparticles were taken up via clathrin-mediated endocytosis. These results utilizing uniform nanoparticles suggest that the size-dependent cytotoxicity of nanoparticles involves active cellular uptake, caspase-3 activation, and apoptosis in the epithelial cell line (NCI-H292). These findings will hopefully aid in the future design and safe use of nanoparticles.

Occurrence of Fatal and Nonfatal Adverse Outcomes after Heart Transplantation in Patients with Pretransplant Noncytotoxic HLA Antibodies

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Luciano Potena

2013-01-01

Full Text Available HLA antibodies (HLA ab in transplant candidates have been associated with poor outcome. However, clinical relevance of noncytotoxic antibodies after heart transplant (HT is controversial. By using a Luminex-based HLA screening, we retested pretransplant sera from HT recipients testing negative for cytotoxic HLA ab and for prospective crossmatch. Out of the 173 consecutive patients assayed (52±13y; 16% females; 47% ischemic etiology, 32 (18% showed pretransplant HLA ab, and 12 (7% tested positive against both class I and class II HLA. Recipients with any HLA ab had poorer survival than those without (65±9 versus 82±3%; P=0.02, accounting for a doubled independent mortality risk (P=0.04. In addition, HLA-ab detection was associated with increased prevalence of early graft failure (35 versus 15%; P=0.05 and late cellular rejection (29 versus 11%; P=0.03. Of the subgroup of 37 patients suspected for antibody mediated rejection (AMR, the 9 with pretransplant HLA ab were more likely to display pathological AMR grade 2 (P=0.04. By an inexpensive, luminex-based, HLA-screening assay, we were able to detect non-cytotoxic HLA ab predicting fatal and nonfatal adverse outcomes after heart transplant. Allocation strategies and desensitization protocols need to be developed and prospectively tested in these patients.

Luminol-dependent chemiluminescence in antibody-sensitized neutrophils stimulated with protein A-bearing staphylococci.

Science.gov (United States)

Nishihara, S; Seki, K; Ikigai, H; Masuda, S

1988-01-01

When mouse polymorphonuclear leukocytes (PMNs) sensitized with rabbit antibody to mouse Ehrlich ascites tumor cells were stimulated by Staphylococcus aureus Cowan I cells, a conspicuous luminol-dependent chemiluminescence was observed in the absence of opsonin. The profile of the chemiluminescence (CL) response evoked by staphylococcal cells from antibody-sensitized PMNs had two peaks. An initial peak, observed within 1 min after stimulation, was sharp and high and a second peak, observed about 5 min after stimulation, was low and extended. The CL response of antibody-sensitized PMNs stimulated by S. aureus Cowan I cells was dose-dependently blocked by preincubation with soluble SpA. Cells of a mutant derived from S. aureus Cowan I strain with trace amounts of cell-bound SpA failed to stimulate the antibody-sensitized PMNs to generate the CL response. The antibody-sensitized PMNs were found to phagocytize SpA-bearing S. aureus cells even in the absence of opsonic serum. These results suggest that the observation presented here might provide a useful tool for the investigation of CL response of PMNs.

Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

OpenAIRE

Grinberg, Yehudit; Benhar, Itai

2017-01-01

Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called ?human cytotoxic fusion protein...

Evaluation of Different Parameters of Humoral and Cellular Immune Responses in HIV Serodiscordant Heterosexual Couples: Humoral Response Potentially Implicated in Modulating Transmission Rates

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María Julia Ruiz

2017-12-01

Full Text Available As the HIV/AIDS pandemic still progresses, understanding the mechanisms governing viral transmission as well as protection from HIV acquisition is fundamental. In this context, cohorts of HIV serodiscordant heterosexual couples (SDC represent a unique tool. The present study was aimed to evaluate specific parameters of innate, cellular and humoral immune responses in SDC. Specifically, plasma levels of cytokines and chemokines, HIV-specific T-cell responses, gp120-specific IgG and IgA antibodies, and HIV-specific antibody-dependent cellular cytotoxicity (ADCC activity were assessed in nine HIV-exposed seronegative individuals (ESN and their corresponding HIV seropositive partners (HIV+-P, in eighteen chronically infected HIV subjects (C, nine chronically infected subjects known to be HIV transmitters (CT and ten healthy HIV− donors (HD. Very low magnitude HIV-specific cellular responses were found in two out of six ESN. Interestingly, HIV+-P had the highest ADCC magnitude, the lowest IgA levels and the highest IgG/IgA ratio, all compared to CT. Positive correlations between CD4+ T-cell counts and both IgG/IgA ratios and %ADCC killing uniquely distinguished HIV+-P. Additionally, evidence of IgA interference with ADCC responses from HIV+-P and CT is provided. These data suggest for the first time a potential role of ADCC and/or gp120-specific IgG/IgA balance in modulating heterosexual transmission. In sum, this study provides key information to understand the host factors that influence viral transmission, which should be considered in both the development of prophylactic vaccines and novel immunotherapies for HIV-1 infection.

Cellular injury evidenced by impedance technology and infrared microspectroscopy

Science.gov (United States)

le Roux, K.; Prinsloo, L. C.; Meyer, D.

2015-03-01

Fourier Transform Infrared (FTIR) spectroscopy is finding increasing biological application, for example in the analysis of diseased tissues and cells, cell cycle studies and investigating the mechanisms of action of anticancer drugs. Cancer treatment studies routinely define the types of cell-drug responses as either total cell destruction by the drug (all cells die), moderate damage (cell deterioration where some cells survive) or reversible cell cycle arrest (cytostasis). In this study the loss of viability and related chemical stress experienced by cells treated with the medicinal plant, Plectranthus ciliatus, was investigated using real time cell electronic sensing (RT-CES) technology and FTIR microspectroscopy. The use of plants as medicines is well established and ethnobotany has proven that crude extracts can serve as treatments against various ailments. The aim of this study was to determine whether FTIR microspectroscopy would successfully distinguish between different types of cellular injury induced by a potentially anticancerous plant extract. Cervical adenocarcinoma (HeLa) cells were treated with a crude extract of Pciliatus and cells monitored using RT-CES to characterize the type of cellular responses induced. Cell populations were then investigated using FTIR microspectroscopy and statistically analysed using One-way Analysis of Variance (ANOVA) and Principal Component Analysis (PCA). The plant extract and a cancer drug control (actinomycin D) induced concentration dependent cellular responses ranging from nontoxic, cytostatic or cytotoxic. Thirteen spectral peaks (915 cm-1, 933 cm-1, 989 cm-1, 1192 cm-1, 1369 cm-1, 1437 cm-1, 1450 cm-1, 1546 cm-1, 1634 cm-1, 1679 cm-1 1772 cm-1, 2874 cm-1 and 2962 cm-1) associated with cytotoxicity were significantly (p value < 0.05, one way ANOVA, Tukey test, Bonferroni) altered, while two of the bands were also indicative of early stress related responses. In PCA, poor separation between nontoxic and cytostatic

Pichia pastoris-Expressed Bivalent Virus-Like Particulate Vaccine Induces Domain III-Focused Bivalent Neutralizing Antibodies without Antibody-Dependent Enhancement in Vivo

OpenAIRE

Rahul Shukla; Ravi K. Rajpoot; Upasana Arora; Ankur Poddar; Sathyamangalam Swaminathan; Navin Khanna; Navin Khanna; Navin Khanna

2018-01-01

Dengue, a significant public health problem in several countries around the world, is caused by four different serotypes of mosquito-borne dengue viruses (DENV-1, -2, -3, and -4). Antibodies to any one DENV serotype which can protect against homotypic re-infection, do not offer heterotypic cross-protection. In fact, cross-reactive antibodies may augment heterotypic DENV infection through antibody-dependent enhancement (ADE). A recently launched live attenuated vaccine (LAV) for dengue, which ...

In vitro cytotoxicity of alpha conjugates for human pancreatic cancer cell lines

International Nuclear Information System (INIS)

Qu, C.; Li, Y.; Rizvi, M.A.; Allen, B.; Samra, J.; Smith, R.

2003-01-01

Targeted Alpha therapy (TAT) can inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The aim of this study is to demonstrate the cytotoxicity of different alpha conjugates in vitro to human metastatic pancreatic cancer cell lines (CAPAN-1, CFPAN-1 and PANC-1). We are labeling the C595 and J591 (non-specific controls) monoclonal antibodies (Mabs) with 213 Bi were performed according to the standard methods in our laboratory. 213 Bi-C595 is specifically cytotoxic to CAPAN-1, CFPAN-1 and PANC-1cell lines in a concentration-dependent fashion. While non-specific alpha conjugates only killed very small fractions of pancreatic cancer cells. These alpha conjugates might be useful agents for the treatment of micro-metastases in pancreatic cancer patients with over-expression of the targeted receptors

Extracellular thiol-assisted selenium uptake dependent on the x(c)(-) cystine transporter explains the cancer-specific cytotoxicity of selenite

DEFF Research Database (Denmark)

Olm, E.; Fernandes, A. P.; Hebert, C.

2009-01-01

The selenium salt selenite (SeO32-) is cytotoxic in low to moderate concentrations, with a remarkable specificity for cancer cells resistant to conventional chemotherapy. Our data show that selenium uptake and accumulation, rather than intracellular events, are crucial to the specific selenite...... cytotoxicity observed in resistant cancer cells. We show that selenium uptake depends on extracellular reduction, and that the extracellular environment is a key factor specific to selenite cytotoxicity. The extracellular reduction is mediated by cysteine, and the efficacy is determined by the uptake...

Cytotoxicity of β-D-glucose/sucrose-coated silver nanoparticles depends on cell type, nanoparticles concentration and time of incubation

Science.gov (United States)

Vergallo, Cristian; Panzarini, Elisa; Carata, Elisabetta; Ahmadi, Meysam; Mariano, Stefania; Tenuzzo, Bernardetta Anna; Dini, Luciana

2016-06-01

The use of silver NanoParticles (AgNPs) in several consumer commercialized products, like food contact materials, medical devices and cosmetics has increased significantly, owing to their antibacterial and antifungal properties. Even though the NPs are widely diffused, due to the great variety in size, coating or shape, controversial data on their possible detrimental health effects still exist. Herein, by performing an easy and fast green method synthesis, we used β-D-glucose/sucrose to stabilize AgNPs and avoid the release of cytotoxic soluble silver ions Ag+ in the culture medium. The cytotoxic effects of these β-D-Glucose/Sucrose-Coated AgNPs (AgNPs-GS) was assessed on two cell culture models, which are human liver HepG2 and human Peripheral Blood Lymphocytes (PBLs) cells. AgNPs-GS, as determined by Transmission Electron Microscopy (TEM) analyses, had an average diameter of 30±5 nm, a spherical shape and were well-dispersed in the freshly-prepared solution. In addition, they were found spectrophotometrically stable throughout the experiment. Cytotoxicity, determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, was evaluated by using two AgNPs-GS amounts, indicated as highest (10×103 of NPs/cell) and lowest (2×103 NPs/cell) concentration for 6, 12 and 24 h. The highest concentration of AgNPs-GS was significantly cytotoxic for both HepG2 and PBLs cells at all times, when compared with the negative control; conversely, the lowest amount of AgNPs-GS was toxic only for HepG2 cells. A significant increase of Reactive Oxygen Species (ROS) levels, determined by Nitro Blue Tetrazolium (NBT) reduction assay, was observed only in PBLs after treatment with NPs, by reaching maximum levels after the incubation with the lowest amount of NPs for 24 h. Significant morphological changes, depending on NPs/cell amount, characteristic of cell toxicity, like shape, cytoplasm, and nucleus alterations, were observed in lymphocytes and Hep

Antitumor activity of anti-C-ERC/mesothelin monoclonal antibody in vivo.

Science.gov (United States)

Inami, Koichi; Abe, Masaaki; Takeda, Kazuyoshi; Hagiwara, Yoshiaki; Maeda, Masahiro; Segawa, Tatsuya; Suyama, Masafumi; Watanabe, Sumio; Hino, Okio

2010-04-01

Mesothelioma is an aggressive cancer often caused by chronic asbestos exposure, and its prognosis is very poor despite the therapies currently used. Due to the long latency period between asbestos exposure and tumor development, the worldwide incidence will increase substantially in the next decades. Thus, novel effective therapies are warranted to improve the prognosis. The ERC/mesothelin gene (MSLN) is expressed in wide variety of human cancers, including mesotheliomas, and encodes a precursor protein cleaved by proteases to generate C-ERC/mesothelin and N-ERC/mesothelin. In this study, we investigated the antitumor activity of C-ERC/mesothelin-specific mouse monoclonal antibody, 22A31, against tumors derived from a human mesothelioma cell line, ACC-MESO-4, in a xenograft experimental model using female BALB/c athymic nude mice. Treatment with 22A31 did not inhibit cell proliferation of ACC-MESO-4 in vitro; however, therapeutic treatment with 22A31 drastically inhibited tumor growth in vivo. 22A31 induced antibody-dependent cell-mediated cytotoxicity by natural killer (NK) cells, but not macrophages, in vitro. Consistently, the F(ab')(2) fragment of 22A31 did not inhibit tumor growth in vivo, nor did it induce antibody-dependent cell mediated cytotoxicity (ADCC) in vitro. Moreover, NK cell depletion diminished the antitumor effect of 22A31. Thus, 22A31 induced NK cell-mediated ADCC and exerted antitumor activity in vivo. 22A31 could have potential as a therapeutic tool to treat C-ERC/mesothelin-expressing cancers including mesothelioma.

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.

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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.

Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage.

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Jui-Hua Hsieh

Full Text Available Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2 using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo while the other evaluates cell membrane integrity (i.e., cell death, flor. Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our

Induction of human immunodeficiency virus neutralizing antibodies using fusion complexes.

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Zipeto, Donato; Matucci, Andrea; Ripamonti, Chiara; Scarlatti, Gabriella; Rossolillo, Paola; Turci, Marco; Sartoris, Silvia; Tridente, Giuseppe; Bertazzoni, Umberto

2006-05-01

Human immunodeficiency virus-1 (HIV-1) infects cells by membrane fusion that is mediated by the envelope proteins gp120/gp41 and the cellular receptors CD4 and CCR5. During this process, some conserved viral epitopes are temporarily exposed and may induce a neutralizing antibody response when fixed in the fusogenic conformation. These transient structures are conserved and may be effective antigens for use in an anti-HIV-1 vaccine. In this study we tested different conditions of preparation of fusion complexes inducing neutralizing antibodies against both R5 and X4 tropic HIV-1 strains. Cell lines expressing HIV-1 gp120/gp41 and CD4-CCR5 were prepared and conditions for producing fusion complexes were tested. Complexes produced at different temperature and fixative combinations were used to immunize mice. Results indicated that (a) fusion complexes prepared at either 21 degrees C, 30 degrees C or 37 degrees C were immunogenic and induced neutralizing antibodies against both R5 and X4 HIV-1 heterologous isolates; (b) after extensive purification of antibodies there was no cytotoxic effect; (c) complexes prepared at 37 degrees C were more immunogenic and induced higher titers of neutralizing antibodies than complexes prepared at either 21 degrees C or 30 degrees C; (d) the fixative used did not affect the titer of neutralizing antibodies except for glutaraldehyde which was ineffective; (e) the neutralizing activity was retained after CD4-CCR5 antibody removal. The production of higher titers of neutralizing antibody with fusion complexes prepared at 37 degrees C, as compared to lower temperatures, may be related to the induction of antibodies against many different conformation intermediates that subsequently act synergistically at different steps in the fusion process.

Antibody responses in allogeneic radiation chimeras

International Nuclear Information System (INIS)

Coico, R.F.

1982-01-01

The construction of long-lived allogeneic radiation chimeras, free of graft-versus-host disease, has been achieved using serologic elimination of Thy 1 + cells from donor bone marrow. Humoral immune function was not restored in these animals as evidenced by lack of primary antibody responses to a T cell-dependent antigen, namely, sheep erythrocytes (SRBC) both in vivo and in vitro. No evidence for a suppressor cell-mediated mechanism was found. Using separated chimera spleen cell populations and specific helper cell soluble mediators, the functional capabilities of chimera B cells, T cells, and macrophages were assessed. These findings suggested that the failure of chimeras to produce antibody is not the result of impaired B cell, T cell, or macrophage function, but rather, that it is due to ineffective cellular interactions. Physiologic cellular interactions depend upon the sharing of major histocompatibility complex (MHC) determinants between interacting cells. However, the self-recognition repertoire of developing T cells may be influenced by the environment which these cells differentiate such that they learn to recognize host MHC determinants as self. These findings support the interpretation that the immunologic hyporeactivity of allogeneic bone marrow chimeras reflects the role of the host environment in restricting the interactive capabilities of donor-derived cells

Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity.

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Schettini, Jorge; Kidiyoor, Amritha; Besmer, Dahlia M; Tinder, Teresa L; Roy, Lopamudra Das; Lustgarten, Joseph; Gendler, Sandra J; Mukherjee, Pinku

2012-11-01

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.

A comparison of the cytotoxic activity of eosinophils and other cells by 51chromium release and time lapse microcinematography

International Nuclear Information System (INIS)

Sanderson, C.J.; Thomas, J.A.

1978-01-01

Antibody dependent cytotoxicity of chicken erythrocytes by purified rat eosinophils, neutrophils, macrophages and K cells has been compared by 51 Cr release and time lapse microcinematography. Techniques have been developed for purifying these effector cell types. Both eosinophils and neutrophils caused rapid release of 51 Cr from erythrocytes. Time lapse observations indicated that this was the result of phagocytosis. Eosinophils showed rapid membrane movement and repeatedly engulfed and regurgitated the erythrocytes. On the other hand, neutrophils became quiescent after phagocytosing erythrocytes, and remained quiescent until the remains of the cell were expelled. Neutrophils presumably have a mechanism for the release of soluble material, as 51 Cr was released rapidly. Macrophages showed a similar quiescence after phagocytosis, but in these cells there was apparently no rapid mechanism to expel material, as there was no significant 51 Cr release over 20 h. K cells appeared to damage chicken erythrocytes more slowly than they destroyed tumour cells. Mast cells caused antibody-independent cytotoxicity which can be attributed to the release of toxic materials. None of these effector cells produced the type of lysis seen with antibody and complement. (author)

Cellular inactivation of nitric oxide induces p53-dependent ...

African Journals Online (AJOL)

Tropical Journal of Pharmaceutical Research August 2016; 15 (8): 1595-1603 ... Cellular inactivation of nitric oxide induces p53-dependent apoptosis in ... apoptosis induced by a selective iNOS inhibitor, N-[(3-aminomethyl) benzyl] acetamidine (1400W), .... and nitrate. ... Nitrite production was measured in culture media.

Cellular impedance measurement as a new tool for poxvirus titration, antibody neutralization testing and evaluation of antiviral substances

International Nuclear Information System (INIS)

Witkowski, Peter T.; Schuenadel, Livia; Wiethaus, Julia; Bourquain, Daniel R.; Kurth, Andreas; Nitsche, Andreas

2010-01-01

Research highlights: → Real-time data acquisition by RT-CES requires low operative effort. → Time to result is reduced by using RT-CES instead of conventional methods. → RT-CES enables quantification of virus titers in unknown samples. → RT-CES is a useful tool for high-throughput characterization of antiviral agents. → An RT-CES-based virus neutralization test was established. -- Abstract: Impedance-based biosensing known as real-time cell electronic sensing (RT-CES) belongs to an emerging technology for analyzing the status of cells in vitro. In the present study protocols were developed for an RT-CES-based system (xCELLigence TM , Roche Applied Science, ACEA Biosciences Inc.) to supplement conventional techniques in pox virology. First, proliferation of cells susceptible to orthopoxviruses was monitored. For virus titration cells were infected with vaccinia virus and cell status, represented by the dimensionless impedance-based cell index (CI), was monitored. A virus-dose dependent decrease in electrical impedance could be shown. Calculation of calibration curves at a suitable CI covering a dynamic range of 4 log enabled the quantification of virus titers in unknown samples. Similarly, antiviral effects could be determined as shown for anti-poxviral agents ST-246 and Cidofovir. Published values for the in vitro concentration that inhibited virus replication by 50% (IC 50 ) could be confirmed while cytotoxicity in effective concentrations was excluded in long-term incubation experiments. Finally, an RT-CES-based virus neutralization test was established. Various poxvirus-specific antibodies were examined for their neutralizing activity and a calculation mode for the neutralizing antibody titer was introduced. In summary, the presented RT-CES-based methods outmatch end-point assays by observing the cell population throughout the entire experiment while workload and time to result are reduced.

Cytotoxicity of leukocytes from normal and Shigella-susceptible (opium-treated) guinea pigs against virulent Shigella sonnei.

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Morgan, D R; DuPont, H L; Wood, L V; Kohl, S

1984-01-01

Intraepithelial lymphocytes were collected from the ileum of adult Hartley strain guinea pigs and used as effector cells in a 60-min bactericidal assay with virulent Shigella sonnei as target cells. Natural killer cytotoxicity (NKC) and antibody-dependent cellular cytotoxicity (ADCC) were measured and correlated with the resistance of the animals to infection by S. sonnei. Normal guinea pig intraepithelial lymphocytes exhibited mean NKC and ADCC values of 22.8 +/- 5.0 and 34.1 +/- 13.6, respectively. These animals were resistant to oral challenge with virulent S. sonnei. Intraepithelial lymphocytes from guinea pigs which were fasted for 4 days demonstrated NKC and ADCC values similar to those of normal animals (31.0 +/- 8.1 and 41.7 +/- 6.7, respectively). These animals also were resistant to oral challenge. Intraepithelial lymphocytes from guinea pigs which were given 1 ml of deodorized tincture of opium 2 h before cell collection demonstrated deficient NKC (4.7 +/- 4.2) and ADCC (5.3 +/- 4.9) values but remained resistant to infection by S. sonnei. When guinea pigs were fasted for 4 days and given opium, deficient NKC (2.0 +/- 2.0) and ADCC (1.3 +/- 1.3) values were demonstrated; this group of animals was susceptible to infection by S. sonnei (P less than 0.04). These experiments demonstrated that opium treatment depresses one form of gut immunity. When combined with starvation, opium treatment may increase susceptibility to infection by shigellae by modulation of immunity in addition to the effects on gut motility and bacterial flora. PMID:6384044

Monitoring the systemic human memory B cell compartment of melanoma patients for anti-tumor IgG antibodies.

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Amy E Gilbert

Full Text Available 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.

Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

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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

Calcein AM release-based cytotoxic cell assay for fish leucocytes.

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Iwanowicz, Luke R; Densmore, Christine L; Ottinger, Christopher A

2004-02-01

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (Pantibody (Pcytotoxic cell activity of approximately 18% was observed following 8 h of incubation at the 2:1 and 1:1 leucocyte to target cell ratios. Percent cytotoxic cell activity using calcein AM was similar to values reported for rainbow trout leucocytes using the 51Cr-release assay.

Anti sperm antibodies detection in infertile patients by radioimmunometry

International Nuclear Information System (INIS)

ELnabarawy, F.; Megahed, Y.M.; Tadrous, G.A.; Hamada, T.; Elbadry, A.

1992-01-01

Three different methods of testing for anti sperm antibodies were compared: complement cytotoxicity, sperm agglutination, and radiolabelled anti globulin antibody technique, for detection of anti sperm antibodies in serum and secretions (seminal plasma and cervical mucus). Sample from 120 patients with infertility were investigated by the previous three methods. The results of unexplained infertile patients revealed wide variations in figures, concerning the positivity of anti sperm antibody whether in their serum or secretions, by using the cytotoxicity or sperm agglutination tests. Using a specific radiolabelled anti globulin test, a subset of patients (44.9% in the serum of men and 50% in seminal plasma) with IgG anti sperm antibody was identified, and this antibody was present in 65.4% and 78,6% of infertile wives sera and cervical mucus, respectively. Therefore, this test has been used to identify and quantitate antibodies directed toward other human cell surfaces. It was concluded that this radiolabelled method is a clinically useful and a potentially versatile procedure that can be successfully applied to the diagnosis and management of patients with suspected immunologic infertility. 1 fig., 5 tab

Successful kidney transplantation across a positive complement-dependent cytotoxicity crossmatch by using C1q assay-directed, bortezomib-assisted desensitization: A case report.

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Lee, Juhan; Park, Borae G; Jeong, Hyang Sook; Park, Youn Hee; Kim, Sinyoung; Kim, Beom Seok; Kim, Hye Jin; Huh, Kyu Ha; Jeong, Hyeon Joo; Kim, Yu Seun

2017-09-01

Human leukocyte antigen (HLA) is the major immunologic barrier in kidney transplantation (KT). Various desensitization protocols to overcome the HLA barrier have increased the opportunity for transplantation in sensitized patients. In addition, technological advances in solid-phase assays have permitted more comprehensive assessment of donor-specific antibodies. Although various desensitization therapies and immunologic techniques have been developed, the final transplantation decision is still based on the classic complement-dependent cytotoxicity (CDC) crossmatch (XM) technique. Some patients who fail to achieve negative XM have lost their transplant opportunities, even after receiving sufficient desensitization therapies. A 57-year-old male with end-stage renal disease secondary to chronic glomerulonephritis was scheduled to have a second transplant from his son, but CDC XM was positive. Initial CDC XM (Initial T-AHG 1:32) and flow-cytometry XM were positive. Anti-HLA-B59 donor specific antibody was detected by Luminex single antigen assay. Herein, we report a successful case of KT across a positive CDC XM (T-AHG 1:8 at the time of transplantation) by using C1q assay-directed, bortezomib-assisted desensitization. After confirming a negative conversion in the C1q donor-specific antibody, we decided to perform KT accepting a positive AHG-CDC XM of 1:8 at the time of transplantation. The posttransplant course was uneventful and a protocol biopsy at 3 months showed no evidence of rejection. The patient had excellent graft function at 12 months posttransplant. The results of XM test and solid-phase assay should be interpreted in the context of the individual patient.

Effect of vitamin E on cytotoxicity, DNA single strand breaks, chromosomal aberrations, and mutation in Chinese hamster V-79 cells exposed to ultraviolet-B light

International Nuclear Information System (INIS)

Sugiyama, M.; Tsuzuki, K.; Matsumoto, K.; Ogura, R.

1992-01-01

The effect of pretreatment with vitamin E on cytotoxicity, DNA single strand breaks, and chromosomal aberrations as well as on mutation induced by ultraviolet-B light (UV-B) was investigated in Chinese hamster V-79 cells. Cellular pretreatment with non-toxic levels of 25 μM α-tocopherol succinate (vitamin E) for 24h prior to exposure resulted in a 10-fold increase in cellular levels of α-tocopherol. Using a colony-forming assay, this pretreatment decreased the cytotoxicity of UV-B light. However, alkaline elution assays demonstrated that pretreatment with vitamin E did not affect the number of DNA single strand breaks caused by UV-B light. UV-B exposure produced a dose-dependent induction of chromosomal aberrations and mutations at the HGPRT locus, and neither of these actions of UV-B was influenced by pretreatment with the vitamin. These results suggest that vitamin E protects cells from UV-B-induced cytotoxicity, possibly through its ability to scavenge free radicals. The genotoxicity induced by UV-B light may not correlate directly with the cytotoxic action of this wavelength region in sunlight. (author)

The cytotoxicity of polycationic iron oxide nanoparticles: Common endpoint assays and alternative approaches for improved understanding of cellular response mechanism

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Hoskins Clare

2012-04-01

Full Text Available Abstract Background Iron oxide magnetic nanoparticles (MNP's have an increasing number of biomedical applications. As such in vitro characterisation is essential to ensure the bio-safety of these particles. Little is known on the cellular interaction or effect on membrane integrity upon exposure to these MNPs. Here we synthesised Fe3O4 and surface coated with poly(ethylenimine (PEI and poly(ethylene glycol (PEG to achieve particles of varying surface positive charges and used them as model MNP's to evaluate the relative utility and limitations of cellular assays commonly applied for nanotoxicity assessment. An alternative approach, atomic force microscopy (AFM, was explored for the analysis of membrane structure and cell morphology upon interacting with the MNPs. The particles were tested in vitro on human SH-SY5Y, MCF-7 and U937 cell lines for reactive oxygen species (ROS production and lipid peroxidation (LPO, LDH leakage and their overall cytotoxic effect. These results were compared with AFM topography imaging carried out on fixed cell lines. Results Successful particle synthesis and coating were characterised using FTIR, PCS, TEM and ICP. The particle size from TEM was 30 nm (−16.9 mV which increased to 40 nm (+55.6 mV upon coating with PEI and subsequently 50 nm (+31.2 mV with PEG coating. Both particles showed excellent stability not only at neutral pH but also in acidic environment of pH 4.6 in the presence of sodium citrate. The higher surface charge MNP-PEI resulted in increased cytotoxic effect and ROS production on all cell lines compared with the MNP-PEI-PEG. In general the effect on the cell membrane integrity was observed only in SH-SY5Y and MCF-7 cells by MNP-PEI determined by LDH leakage and LPO production. AFM topography images showed consistently that both the highly charged MNP-PEI and the less charged MNP-PEI-PEG caused cell morphology changes possibly due to membrane disruption and cytoskeleton remodelling. Conclusions

TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells.

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Lee, Yong Chan; Chuang, Chun-Yu; Lee, Pak-Kei; Lee, Jin-Soo; Harper, Richart W; Buckpitt, Alan B; Wu, Reen; Oslund, Karen

2008-05-01

Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.

Cytotoxicity of metal and semiconductor nanoparticles indicated by cellular micromotility.

Science.gov (United States)

Tarantola, Marco; Schneider, David; Sunnick, Eva; Adam, Holger; Pierrat, Sebastien; Rosman, Christina; Breus, Vladimir; Sönnichsen, Carsten; Basché, Thomas; Wegener, Joachim; Janshoff, Andreas

2009-01-27

In the growing field of nanotechnology, there is an urgent need to sensitively determine the toxicity of nanoparticles since many technical and medical applications are based on controlled exposure to particles, that is, as contrast agents or for drug delivery. Before the in vivo implementation, in vitro cell experiments are required to achieve a detailed knowledge of toxicity and biodegradation as a function of the nanoparticles' physical and chemical properties. In this study, we show that the micromotility of animal cells as monitored by electrical cell-substrate impedance analysis (ECIS) is highly suitable to quantify in vitro cytotoxicity of semiconductor quantum dots and gold nanorods. The method is validated by conventional cytotoxicity testing and accompanied by fluorescence and dark-field microscopy to visualize changes in the cytoskeleton integrity and to determine the location of the particles within the cell.

Putrescine-dependent re-localization of TvCP39, a cysteine proteinase involved in Trichomonas vaginalis cytotoxicity.

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Bertha Isabel Carvajal-Gamez

Full Text Available Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB (an inhibitor of putrescine biosynthesis, diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced ∼ 80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization.

CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1.

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Osada, Takuya; Patel, Sandip P; Hammond, Scott A; Osada, Koya; Morse, Michael A; Lyerly, H Kim

2015-06-01

Bispecific T cell-engaging (BiTE) antibodies recruit polyclonal cytotoxic T cells (CTL) to tumors. One such antibody is carcinoembryonic antigen (CEA) BiTE that mediates T cell/tumor interaction by simultaneously binding CD3 expressed by T cells and CEA expressed by tumor cells. A widely operative mechanism for mitigating cytotoxic T cell-mediated killing is the interaction of tumor-expressed PD-L1 with T cell-expressed PD-1, which may be partly reversed by PD-1/PD-L1 blockade. We hypothesized that PD-1/PD-L1 blockade during BiTE-mediated T cell killing would enhance CTL function. Here, we determined the effects of PD-1 and PD-L1 blockade during initial T cell-mediated killing of CEA-expressing human tumor cell lines in vitro, as well as subsequent T cell-mediated killing by T lymphocytes that had participated in tumor cell killing. We observed a rapid upregulation of PD-1 expression and diminished cytolytic function of T cells after they had engaged in CEA BiTE-mediated killing of tumors. T cell cytolytic activity in vitro could be maximized by administration of anti-PD-1 or anti-PD-L1 antibodies alone or in combination if applied prior to a round of T cell killing, but T cell inhibition could not be fully reversed by this blockade once the T cells had killed tumor. In conclusion, our findings demonstrate that dual blockade of PD-1 and PD-L1 maximizes T cell killing of tumor directed by CEA BiTE in vitro, is more effective if applied early, and provides a rationale for clinical use.

Artocarpin Induces Apoptosis in Human Cutaneous Squamous Cell Carcinoma HSC-1 Cells and Its Cytotoxic Activity Is Dependent on Protein-Nutrient Concentration

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Stephen Chu-Sung Hu

2015-01-01

Full Text Available Artocarpin, a natural prenylated flavonoid, has been shown to have various biological properties. However, its effects on human cutaneous squamous cell carcinoma (SCC have not been previously investigated. We set out to determine whether artocarpin has cytotoxic effects on SCC cells and whether its pharmacological activity is dependent on protein-nutrient concentration. Our results showed that treatment of HSC-1 cells (a human cutaneous SCC cell line with artocarpin decreased cell viability and induced cell apoptosis by increasing caspase 3/7 activity. These effects were more pronounced at low fetal bovine serum (FBS concentrations. Artocarpin induced an increase in the level of phospho-p38 and a decrease in the levels of phospho-ERK, phospho-JNK, phospho-Akt, phospho-mTOR, and phospho-S6K. High FBS concentrations in the culture media inhibited and delayed the uptake of artocarpin from the extracellular compartment (culture media into the intracellular compartment, as determined by high performance liquid chromatography (HPLC analysis. In conclusion, artocarpin induces apoptosis in HSC-1 cells through modulation of MAPK and Akt/mTOR pathways. Binding of artocarpin to proteins in the FBS may inhibit cellular uptake and reduce the cytotoxic activity of artocarpin on HSC-1 cells. Therefore, artocarpin may have potential use in the future as a form of treatment for cutaneous SCC.

Long-term persistence of robust antibody and cytotoxic T cell responses in recovered patients infected with SARS coronavirus.

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Taisheng Li

2006-12-01

Full Text Available Most of the individuals infected with SARS coronavirus (SARS-CoV spontaneously recovered without clinical intervention. However, the immunological correlates associated with patients' recovery are currently unknown. In this report, we have sequentially monitored 30 recovered patients over a two-year period to characterize temporal changes in SARS-CoV-specific antibody responses as well as cytotoxic T cell (CTL responses. We have found persistence of robust antibody and CTL responses in all of the study subjects throughout the study period, with a moderate decline one year after the onset of symptoms. We have also identified two potential major CTL epitopes in N proteins based on ELISPOT analysis of pooled peptides. However, despite the potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients have not yet been restored to normal levels. In summary, our study has, for the first time, characterized the temporal and dynamic changes of humoral and CTL responses in the natural history of SARS-recovered individuals, and strongly supports the notion that high and sustainable levels of immune responses correlate strongly with the disease outcome. Our findings have direct implications for future design and development of effective therapeutic agents and vaccines against SARS-CoV infection.

Size and shape-dependent cytotoxicity profile of gold nanoparticles for biomedical applications.

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Woźniak, Anna; Malankowska, Anna; Nowaczyk, Grzegorz; Grześkowiak, Bartosz F; Tuśnio, Karol; Słomski, Ryszard; Zaleska-Medynska, Adriana; Jurga, Stefan

2017-06-01

Metallic nanoparticles, in particular gold nanoparticles (AuNPs), offer a wide spectrum of applications in biomedicine. A crucial issue is their cytotoxicity, which depends greatly on various factors, including morphology of nanoparticles. Because metallic nanoparticles have an effect on cell membrane integrity, their shape and size may affect the viability of cells, due to their different geometries as well as physical and chemical interactions with cell membranes. Variations in the size and shape of gold nanoparticles may indicate particular nanoparticle morphologies that provide strong cytotoxicity effects. Synthesis of different sized and shaped bare AuNPs was performed with spherical (~ 10 nm), nanoflowers (~ 370 nm), nanorods (~ 41 nm), nanoprisms (~ 160 nm) and nanostars (~ 240 nm) morphologies. These nanostructures were characterized and interacting with cancer (HeLa) and normal (HEK293T) cell lines and cell viability tests were performed by WST-1 tests and fluorescent live/dead cell imaging experiments. It was shown that various shapes and sizes of gold nanostructures may affect the viability of the cells. Gold nanospheres and nanorods proved to be more toxic than star, flower and prism gold nanostructures. This may be attributed to their small size and aggregation process. This is the first report concerning a comparison of cytotoxic profile in vitro with a wide spectrum of bare AuNPs morphology. The findings show their possible use in biomedical applications.

Toxicology and cellular effect of manufactured nanomaterials

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Chen, Fanqing

2014-07-22

The increasing use of nanotechnology in consumer products and medical applications underlies the importance of understanding its potential toxic effects to people and the environment. Herein are described methods and assays to predict and evaluate the cellular effects of nanomaterial exposure. Exposing cells to nanomaterials at cytotoxic doses induces cell cycle arrest and increases apoptosis/necrosis, activates genes involved in cellular transport, metabolism, cell cycle regulation, and stress response. Certain nanomaterials induce genes indicative of a strong immune and inflammatory response within skin fibroblasts. Furthermore, the described multiwall carbon nanoonions (MWCNOs) can be used as a therapeutic in the treatment of cancer due to its cytotoxicity.

A novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1.

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Hazin, John; Moldenhauer, Gerhard; Altevogt, Peter; Brady, Nathan R

2015-08-01

Monoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity. Copyright © 2015 Elsevier B.V. All rights reserved.

Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis

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Swetha Tati

2017-09-01

Full Text Available JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11. Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1 suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

Association of Angiotensin-Converting Enzyme (ACE) Gene Polymorphism with Inflammation and Cellular Cytotoxicity in Vitiligo Patients.

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Rashed, Laila; Abdel Hay, Rania; Mahmoud, Rania; Hasan, Nermeen; Zahra, Amr; Fayez, Salwa

2015-01-01

Vitiligo is a disorder with profound heterogeneity in its aetio-pathophysiology. Angiotensin converting enzyme (ACE) plays an important role in the physiology of the vasculature, blood pressure and inflammation. An insertion/deletion (I/D) polymorphism of the ACE gene was reported be associated with the development of vitiligo. Our aim was to evaluate the ACE I/D polymorphism in vitiligo patients and controls. Our second aim was to find a possible association between ACE gene polymorphism and inflammatory mediators (as interleukin (IL)-6) and/or cellular cytotoxicity induced by serum nitrite (as a breakdown product of the cytotoxic nitric oxide) in vitiligo patients. This case-control study included 74 vitiligo patients and 75 apparently healthy controls. The distribution of ACE gene I/D genotype was investigated using PCR. Serum ACE, IL-6 and nitrite were measured by colorimetric method, ELISA and Griess assay respectively. The ACE allele frequency was significantly different between vitiligo patients and healthy controls (P = 0.026). However there was no significant difference between the ACE genotyping frequency in both groups (P = 0.115). There were statistically significant higher VIDA score (P = 0.007), and serum IL-6 (P ACE, IL-6 and nitrite in vitiligo patients were statistically significantly higher than those in controls. As a conclusion, ACE gene polymorphism might grant susceptibility to develop vitiligo. Serum IL-6 and nitrite levels might have an important role in the pathogenesis of vitiligo. Targeting these two factors might have an implication in the treatment of some resistant cases.

DNA binding, antioxidant, cytotoxicity (MTT, lactate dehydrogenase, NO), and cellular uptake studies of structurally different nickel(II) thiosemicarbazone complexes: synthesis, spectroscopy, electrochemistry, and X-ray crystallography.

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Prabhakaran, R; Kalaivani, P; Huang, R; Poornima, P; Vijaya Padma, V; Dallemer, F; Natarajan, K

2013-02-01

Three new nickel(II) thiosemicarbazone complexes have been synthesized and characterized by analytical, spectral, and single-crystal X-ray diffraction studies. In complex 1, the ligand 2-hydroxy-1-naphthaldehydethiosemicarbazone coordinated as a monobasic tridentate donor, whereas in complexes 2 and 3, the ligands salicylaldehyde-4(N)-ethylthiosemicarbazone and 2-hydroxy-1-naphthaldehyde-4(N)-ethylthiosemicarbazone coordinated as a dibasic tridentate donor. The DNA binding ability of the complexes in calf thymus DNA was explored by absorption and emission titration experiments. The antioxidant property of the new complexes was evaluated to test their free-radical scavenging ability. In vitro cytotoxicity assays were performed for the new complexes in A549 and HepG2 cell lines. The new compounds overcome cisplatin resistance in the A549 cell line and they were also active in the HepG2 cell line. The cellular uptake study showed the accumulation of the complexes in tumor cells depended on the nature of the ligand attached to the nickel ion.

Biocompatibility of a fish scale-derived artificial cornea: Cytotoxicity, cellular adhesion and phenotype, and in vivo immunogenicity.

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van Essen, T H; van Zijl, L; Possemiers, T; Mulder, A A; Zwart, S J; Chou, C-H; Lin, C C; Lai, H J; Luyten, G P M; Tassignon, M J; Zakaria, N; El Ghalbzouri, A; Jager, M J

2016-03-01

To determine whether a fish scale-derived collagen matrix (FSCM) meets the basic criteria to serve as an artificial cornea, as determined with in vitro and in vivo tests. Primary corneal epithelial and stromal cells were obtained from human donor corneas and used to examine the (in)direct cytotoxicity effects of the scaffold. Cytotoxicity was assessed by an MTT assay, while cellular proliferation, corneal cell phenotype and adhesion markers were assessed using an EdU-assay and immunofluorescence. For in vivo-testing, FSCMs were implanted subcutaneously in rats. Ologen(®) Collagen Matrices were used as controls. A second implant was implanted as an immunological challenge. The FSCM was implanted in a corneal pocket of seven New Zealand White rabbits, and compared to sham surgery. The FSCM was used as a scaffold to grow corneal epithelial and stromal cells, and displayed no cytotoxicity to these cells. Corneal epithelial cells displayed their normal phenotypical markers (CK3/12 and E-cadherin), as well as cell-matrix adhesion molecules: integrin-α6 and β4, laminin 332, and hemi-desmosomes. Corneal stromal cells similarly expressed adhesion molecules (integrin-α6 and β1). A subcutaneous implant of the FSCM in rats did not induce inflammation or sensitization; the response was comparable to the response against the Ologen(®) Collagen Matrix. Implantation of the FSCM in a corneal stromal pocket in rabbits led to a transparent cornea, healthy epithelium, and, on histology, hardly any infiltrating immune cells. The FSCM allows excellent cell growth, is not immunogenic and is well-tolerated in the cornea, and thus meets the basic criteria to serve as a scaffold to reconstitute the cornea. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of cytotoxic and tumor targeting capability of (177)Lu-DOTATATE-nanoparticles: a trailblazing strategy in peptide receptor radionuclide therapy.

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Arora, Geetanjali; Dubey, Priyanka; Shukla, Jaya; Ghosh, Sourabh; Bandopadhyaya, Gurupad

2016-06-01

We propose an innovative strategy of nanoparticle-mediated-peptide receptor radionuclide therapy (PRRT) employing PLGA-nanoparticles together with anti-β-hCG antibodies that can protect kidneys from radiation damage while simultaneously enhancing its tumor targeting and cytotoxic ability for somatostatin receptor (SSR) positive tumors. PEG-coated-(177)Lu-DOTATATE-PLGA-nanoparticles (PEG-LuD-NP) were formulated and characterized. In vitro toxicity of these particles was tested on human glioblastoma cell line U87MG over a radiation dose range of 19-78 Gy, using MTT assay and flow cytometry. To further enhance cytotoxicity and test the feasibility of active tumor targeting, apoptosis-inducing anti-β-hCG monoclonal antibodies were employed in vitro, after confirming expression of β-hCG on U87MG. In vivo tumor targeting ability of these particles, in comparison to uncoated particles and un-encapsulated (177)Lu-DOTATATE, was assessed by intravenous administration in tumor-induced wistar rats. Rats were first imaged in a gamma camera followed by euthanasia for organ extraction and counting in gamma counter. The particles were spherical in shape with mean diameter of 300 nm. Highest cytotoxicity that could be achieved with PEG-LuD-NP, on radio-resistant U87MG cells, was 35.8 % due to complex cellular response triggered by ionizing radiation. Interestingly, synergistic action of antibodies and PEG-LuD-NP doubled the cytotoxicity (80 %). PEG-LuD-NP showed the highest tumor uptake (4.3 ± 0.46 % ID/g) as compared to (177)Lu-DOTATATE (3.5 ± 0.31 %) and uncoated-(177)Lu-DOTATATE-nanoparticles (3.4 ± 0.35 %) in tumor-inoculated wistar rats (p targeting SSR positive tumors for enhanced cytoxicity and reduced renal radiation dose associated with conventional PRRT. To our knowledge of literature, this is the first study to establish in vitro and in vivo efficacy profile of nanoparticles in PRRT providing a stepping-stone for undergoing and future research

Detection of tumor-specific cytotoxic drug activity in vitro using the fluorometric microculture cytotoxicity assay and primary cultures of tumor cells from patients.

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Nygren, P; Fridborg, H; Csoka, K; Sundström, C; de la Torre, M; Kristensen, J; Bergh, J; Hagberg, H; Glimelius, B; Rastad, J

1994-03-01

The semi-automated fluorometric microculture cytotoxicity assay (FMCA), based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) by viable cells, was employed for cytotoxic drug sensitivity testing of tumor cells from patients with hematological or solid tumors. In total, 390 samples from 20 diagnoses were tested with up to 12 standard cytotoxic drugs. The technical success rate for different tumor types ranged from 67 to 95%. Fluorescence was linearly related to cell number but variably steep depending on tumor type. Samples from most solid tumors thus showed higher signal-to-noise ratios than hematological samples. A wide spectrum of in vitro drug activity was obtained, with acute leukemias and non-Hodgkin's lymphomas being sensitive to almost all tested drugs, whereas renal and adrenocortical carcinomas were essentially totally resistant. Between these extremes were samples of breast and ovarian carcinomas and sarcomas. When in vitro response was compared with known clinical response patterns, a good correspondence was observed. The results indicate that the FMCA is a rapid and efficient method for in vitro measurement of tumor-specific drug activity both in hematological and in solid tumors. The assay may be suitable for new drug development and direction of phase-2 trials to suitable patients.

Hepatitis C virus NS2 protein activates cellular cyclic AMP-dependent pathways

International Nuclear Information System (INIS)

Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki

2007-01-01

Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway

Effects of daratumumab on natural killer cells and impact on clinical outcomes in relapsed or refractory multiple myeloma

DEFF Research Database (Denmark)

Casneuf, Tineke; Xu, Xu Steven; Adams, Homer C

2017-01-01

Daratumumab, a human CD38 imunoglobulin G 1κ monoclonal antibody, has demonstrated clinical activity and a manageable safety profile in monotherapy and combination therapy clinical trials in relapsed and/or refractory multiple myeloma. CD38 is expressed at high levels on myeloma cells and......, to a lesser extent, on immune effector cells, including natural killer (NK) cells, which are important for daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Here, the pharmacodynamic effects of daratumumab monotherapy on NK cells, and the effect of NK cell dynamics on daratumumab efficacy...

Feasibility of Ecological Momentary Assessment Using Cellular Telephones in Methamphetamine Dependent Subjects

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John Mendelson

2008-01-01

Full Text Available Background: Predictors of relapse to methamphetamine use are poorly understood. State variables may play an important role in relapse, but they have been difficult to measure at frequent intervals in outpatients.Methods: We conducted a feasibility study of the use of cellular telephones to collect state variable data from outpatients. Six subjects in treatment for methamphetamine dependence were called three times per weekday for approximately seven weeks. Seven questionnaires were administered that assessed craving, stress, affect and current type of location and social environment.Results: 395/606 (65% of calls attempted were completed. The mean time to complete each call was 4.9 (s.d. 1.8 minutes and the mean time to complete each item was 8.4 (s.d. 4.8 seconds. Subjects rated the acceptability of the procedures as good. All six cellular phones and battery chargers were returned undamaged.Conclusion: Cellular telephones are a feasible method for collecting state data from methamphetamine dependent outpatients.

Cellular determinants involving mitochondrial dysfunction, oxidative stress and apoptosis correlate with the synergic cytotoxicity of epigallocatechin-3-gallate and menadione in human leukemia Jurkat T cells.

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Tofolean, Ioana Teodora; Ganea, Constanta; Ionescu, Diana; Filippi, Alexandru; Garaiman, Alexandru; Goicea, Alexandru; Gaman, Mihnea-Alexandru; Dimancea, Alexandru; Baran, Irina

2016-01-01

We have investigated the growth-suppressive action of epigallocatechin-3-gallate (EGCG) on human leukemia Jurkat T cells. Results show a strong correlation between the dose-dependent reduction of clonogenic survival following acute EGCG treatments and the EGCG-induced decline of the mitochondrial level of Ca(2+). The cell killing ability of EGCG was synergistically enhanced by menadione. In addition, the cytotoxic effect of EGCG applied alone or in combination with menadione was accompanied by apoptosis induction. We also observed that in acute treatments EGCG displays strong antioxidant properties in the intracellular milieu, but concurrently triggers some oxidative stress generating mechanisms that can fully develop on a longer timescale. In parallel, EGCG dose-dependently induced mitochondrial depolarization during exposure, but this condition was subsequently reversed to a persistent hyperpolarized mitochondrial state that was dependent on the activity of respiratory Complex I. Fluorimetric measurements suggest that EGCG is a mitochondrial Complex III inhibitor and indicate that EGCG evokes a specific cellular fluorescence with emission at 400nm and two main excitation bands (at 330nm and 350nm) that may originate from a mitochondrial supercomplex containing dimeric Complex III and dimeric ATP-synthase, and therefore could provide a valuable means to characterize the functional properties of the respiratory chain. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antibody Engineering and Therapeutics

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Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul WHI; Xu, Kai Y

2014-01-01

The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates. PMID:24589717

A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer

Science.gov (United States)

Elvington, Michelle; Huang, Yuxiang; Morgan, B. Paul; Qiao, Fei; van Rooijen, Nico; Atkinson, Carl

2012-01-01

Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer. PMID:22442351

Clinical cytometry and progress in HLA antibody detection.

Science.gov (United States)

Bray, Robert A; Tarsitani, Christine; Gebel, Howard M; Lee, Jar-How

2011-01-01

For most solid organ and selected stem cell transplants, antibodies against mismatched HLA antigens can lead to early and late graft failure. In recognition of the clinical significance of these antibodies, HLA antibody identification is one of the most critical functions of histocompatibility laboratories. Early methods employed cumbersome and insensitive complement-dependent cytotoxicity assays with a visual read-out. A little over 20 years ago flow cytometry entered the realm of antibody detection with the introduction of the flow cytometric crossmatch. Cytometry's increased sensitivity and objectivity quickly earned it popularity as a preferred crossmatch method especially for sensitized recipients. Although a sensitive method, the flow crossmatch was criticized as being "too sensitive" as false positive reactions were a know drawback. In part, the shortcomings of the flow crossmatch were due to the lack of corresponding sensitive and specific HLA antibody screening assays. However, in the mid 1990s, solid phase assays, capable of utilizing standard flow cytometers, were developed. These assays used microparticles coated with purified HLA molecules. Hence, the era of solid-phase, microparticle technology for HLA antibody detection was born permitting the sensitive and specific detection of HLA antibody. It was now possible to provide better correlation between HLA antibody detection and the flow cytometric crossmatch. This flow-based technology was soon followed by adaptation to the Luminex platform permitting a mutltiplexed approach for the identification and characterization of HLA antibodies. It is hoped that these technologies will ultimately lead to the identification of parameters that best correlate with and/or predict transplant outcomes. Copyright © 2011 Elsevier Inc. All rights reserved.

Drug delivery systems--2. Site-specific drug delivery utilizing monoclonal antibodies.

Science.gov (United States)

Ranade, V V

1989-10-01

Monoclonal antibodies (MAbs) are purified antibodies produced by a single clone of cells. They are engineered to recognize and bind to a single specific antigen. Accordingly, when administered, MAbs home in on a particular circulating protein or on cells that bear the correct antigenic signature on their surfaces. It is the specificity of MAbs that has made them valuable tools for health professions. Following the discovery of Kohler and Milstein regarding the method of somatic cell hybridization, a number of investigators have successfully adopted this technique to obtain T-lymphocyte hybrid cell lines by fusion of activated T (thymus derived) lymphocytes with a T lymphoma cell line leading to an immortalization of a specific differentiated function. The hybrids thus obtained were subsequently shown to produce homogeneous effector molecules with a wide variety of immune functions such as enhancement or suppression of antibody responses, generation of helper T cells, suppressor T cells and cytotoxic T cells. Study of these regulatory molecules has been further shown to provide a greater insight into the genetic, biochemical and molecular mechanisms responsible for cellular development, and the interaction and triggering of various cell types. The successful application of hybridoma technology has now resulted into several advances in the understanding the mechanism and treatment of diseases, especially cancer and development of vaccines, promotion of organ transplantation and therapy against parasites as well. Since monoclonal antibodies could be made in unlimited supply, they have been used in genetic studies such as mRNA and gene isolation, chromosomal isolation of specific genes, immunoglobulin structure, detection of new or rare immunoglobulin gene products, structural studies of enzymes and other proteins and structural and population studies of protein polymorphisms. In some instances, the monoclonal antibodies have been found to replace conventional antisera

Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605 Reduces Asbestos-Induced Cytotoxicity in an Nrf2-Dependent and -Independent Manner

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Ralph A. Pietrofesa

2018-03-01

Full Text Available Asbestos exposure triggers inflammatory processes associated with oxidative stress and tissue damage linked to malignancy. LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models, including exposure to asbestos fibers. Nuclear factor-E2 related factor 2 (Nrf2 activation and boosting of endogenous tissue defenses were associated with the protective action of LGM2605 from asbestos-induced cellular damage. To elucidate the role of Nrf2 induction by LGM2605 in protection from asbestos-induced cellular damage, we evaluated LGM2605 in asbestos-exposed macrophages from wild-type (WT and Nrf2 disrupted (Nrf2−/− mice. Cells were pretreated with LGM2605 (50 µM and 100 µM and exposed to asbestos fibers (20 µg/cm2 and evaluated 8 h and 24 h later for inflammasome activation, secreted cytokine levels (interleukin-1β (IL-1β, interleukin-18 (IL-18, interleukin-6 (IL-6, and tumor necrosis factor alpha (TNFα, cytotoxicity and cell death, nitrosative stress, and Nrf2-regulated enzyme levels. Asbestos exposure induced robust oxidative and nitrosative stress, cell death and cytotoxicity, which were equally mitigated by LGM2605. Inflammasome activation was significantly attenuated in Nrf2−/− macrophages compared to WT, and the protective action of LGM2605 was seen only in WT cells. In conclusion, in a cell model of asbestos-induced toxicity, LGM2605 acts via protective mechanisms that may not involve Nrf2 activation.

Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605) Reduces Asbestos-Induced Cytotoxicity in an Nrf2-Dependent and -Independent Manner

Science.gov (United States)

Pietrofesa, Ralph A.; Chatterjee, Shampa; Park, Kyewon; Arguiri, Evguenia; Albelda, Steven M.; Christofidou-Solomidou, Melpo

2018-01-01

Asbestos exposure triggers inflammatory processes associated with oxidative stress and tissue damage linked to malignancy. LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models, including exposure to asbestos fibers. Nuclear factor-E2 related factor 2 (Nrf2) activation and boosting of endogenous tissue defenses were associated with the protective action of LGM2605 from asbestos-induced cellular damage. To elucidate the role of Nrf2 induction by LGM2605 in protection from asbestos-induced cellular damage, we evaluated LGM2605 in asbestos-exposed macrophages from wild-type (WT) and Nrf2 disrupted (Nrf2−/−) mice. Cells were pretreated with LGM2605 (50 µM and 100 µM) and exposed to asbestos fibers (20 µg/cm2) and evaluated 8 h and 24 h later for inflammasome activation, secreted cytokine levels (interleukin-1β (IL-1β), interleukin-18 (IL-18), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα)), cytotoxicity and cell death, nitrosative stress, and Nrf2-regulated enzyme levels. Asbestos exposure induced robust oxidative and nitrosative stress, cell death and cytotoxicity, which were equally mitigated by LGM2605. Inflammasome activation was significantly attenuated in Nrf2−/− macrophages compared to WT, and the protective action of LGM2605 was seen only in WT cells. In conclusion, in a cell model of asbestos-induced toxicity, LGM2605 acts via protective mechanisms that may not involve Nrf2 activation. PMID:29498660

A Mathematical Model for Cisplatin Cellular Pharmacodynamics

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Ardith W. El-Kareh

2003-03-01

Full Text Available A simple theoretical model for the cellular pharmacodynamics of cisplatin is presented. The model, which takes into account the kinetics of cisplatin uptake by cells and the intracellular binding of the drug, can be used to predict the dependence of survival (relative to controls on the time course of extracellular exposure. Cellular pharmacokinetic parameters are derived from uptake data for human ovarian and head and neck cancer cell lines. Survival relative to controls is assumed to depend on the peak concentration of DNA-bound intracellular platinum. Model predictions agree well with published data on cisplatin cytotoxicity for three different cancer cell lines, over a wide range of exposure times. In comparison with previously published mathematical models for anticancer drug pharmacodynamics, the present model provides a better fit to experimental data sets including long exposure times (∼100 hours. The model provides a possible explanation for the fact that cell kill correlates well with area under the extracellular concentration-time curve in some data sets, but not in others. The model may be useful for optimizing delivery schedules and for the dosing of cisplatin for cancer therapy.

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

DEFF Research Database (Denmark)

Pratt-Riccio, Lilian Rose; Bianco, Cesare; Totino, Paulo Renato Rivas

2011-01-01

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region h...

Modulation of immune response by bacterial lipopolysaccharide (LPS): cellular basis of stimulatory and inhibitory effects of LPS on the in vitro IGM antibody response to a T-dependent antigen

International Nuclear Information System (INIS)

Uchiyama, T.; Jacobs, D.M.

1978-01-01

The role of thymus-derived lymphocytes (T cells) in LPS modulation of T cell-development antibody responses has been investigated. We have assessed the effect of LPS on the primary anti-TNP response to TNP-SRBC of cultures of whole spleen cells or T cell-depleted spleen cells that were supplemented with various subpopulations of carrier-primed (SRBC) spleen cells. The TNP-PFC response was enhanced in the presence of irradiated SRBC-primed spleen cells by addition of 0.16 to 20 μg/ml LPS, but inhibition was observed when irradiation of primed cells was omitted. Enhancement but no inhibition occurred when added primed cells were first passed through a nylon wool column. LPS-mediated enhancement was dependent on a T cell in the primed population. These results suggest that LPS modulation of antibody synthesis is dependent on two populations of antigen-specific cells that have opposing effects on B cell responses to a T-dependent antigen: a helper cell that is irradiation resistant, nonadherent to nylon wool, and sensitive to anti-T cell serum, and a suppressor cell that is irradiation sensitive and adherent to nylon wool

Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection

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Miao Wang

2017-05-01

Full Text Available Dengue virus (DENV co-circulates as four serotypes (DENV1-4. Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS. Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR, a process known as antibody dependent enhancement (ADE. Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2 and DENV-2 prM monoclonal antibody (prM mAb could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo, interferon-α and γ receptor-deficient mice (AG6 were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10 and alaninea minotransferase (ALT in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo, suggested that anti-idiotypic antibodies might be a new choice to be considered to

Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection.

Science.gov (United States)

Wang, Miao; Yang, Fan; Huang, Dana; Huang, Yalan; Zhang, Xiaomin; Wang, Chao; Zhang, Shaohua; Zhang, Renli

2017-01-01

Dengue virus (DENV) co-circulates as four serotypes (DENV1-4). Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody dependent enhancement (ADE). Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM) of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2) and DENV-2 prM monoclonal antibody (prM mAb) could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs) specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo , interferon-α and γ receptor-deficient mice (AG6) were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10) and alaninea minotransferase (ALT) in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo , suggested that anti-idiotypic antibodies might be a new choice to be considered to treat

Molecular and cellular mechanisms of the age-dependency of opioid analgesia and tolerance

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Zhao Jing

2012-05-01

Full Text Available Abstract The age-dependency of opioid analgesia and tolerance has been noticed in both clinical observation and laboratory studies. Evidence shows that many molecular and cellular events that play essential roles in opioid analgesia and tolerance are actually age-dependent. For example, the expression and functions of endogenous opioid peptides, multiple types of opioid receptors, G protein subunits that couple to opioid receptors, and regulators of G protein signaling (RGS proteins change with development and age. Other signaling systems that are critical to opioid tolerance development, such as N-methyl-D-aspartic acid (NMDA receptors, also undergo age-related changes. It is plausible that the age-dependent expression and functions of molecules within and related to the opioid signaling pathways, as well as age-dependent cellular activity such as agonist-induced opioid receptor internalization and desensitization, eventually lead to significant age-dependent changes in opioid analgesia and tolerance development.

Size-dependent cytotoxicity of europium doped NaYF4 nanoparticles in endothelial cells

International Nuclear Information System (INIS)

Chen, Shizhu; Zhang, Cuimiao; Jia, Guang; Duan, Jianlei; Wang, Shuxiang; Zhang, Jinchao

2014-01-01

Lanthanide-doped sodium yttrium fluoride (NaYF 4 ) nanoparticles exhibit novel optical properties which make them be widely used in various fields. The extensive applications increase the chance of human exposure to these nanoparticles and thus raise deep concerns regarding their riskiness. In the present study, we have synthesized europium doped NaYF 4 (NaYF 4 :Eu 3+ ) nanoparticles with three diameters and used endothelial cells (ECs) as a cell model to explore the potential toxic effect. The cell viability, cytomembrane integrity, cellular uptake, intracellular localization, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis detection, caspase-3 activity and expression of inflammatory gene were studied. The results indicated that these nanoparticles could be uptaken into ECs and decrease the cell viability, induce the intracellular lactate dehydrogenase (LDH) release, increase the ROS level, and decrease the cell MMP in a size-dependent manner. Besides that, the cells were suffered to apoptosis with the caspase-3 activation, and the inflammation specific gene expressions (ICAM1 and VCAM1) were also increased. Our results suggest that the damage pathway may be related to the ROS generation and mitochondrial damage. The results provide novel evidence to elucidate their toxicity mechanisms and may be helpful for more rational applications of these compounds in the future. - Highlights: • NaYF 4 :Eu 3+ nanoparticles with three diameters have been synthesized. • NaYF 4 :Eu 3+ nanoparticles could be uptaken by endothelial cells (ECs). • NaYF 4 :Eu 3+ nanoparticles show a significant cytotoxicity on ECs. • The size of NaYF 4 :Eu 3+ nanoparticles may be important to their toxicology effect

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody

OpenAIRE

Donahue, Renee N.; Lepone, Lauren M.; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A.; Heery, Christopher R.; Gulley, James L.; Schlom, Jeffrey

2017-01-01

Background Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range...

Cellular uptake and transport of zein nanoparticles: effects of sodium caseinate.

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Luo, Yangchao; Teng, Zi; Wang, Thomas T Y; Wang, Qin

2013-08-07

Cellular evaluation of zein nanoparticles has not been studied systematically due to their poor redispersibility. Caseinate (CAS)-stabilized zein nanoparticles have been recently developed with better redispersibility in salt solutions. In this study, zein-CAS nanoparticles were prepared with different zein/CAS mass ratios. The prepared nanoparticles demonstrated good stabilities to maintain particle size (120-140 nm) in cell culture medium and HBSS buffer at 37 °C. The nanoparticles showed no cytotoxicity for Caco-2 cells for 72 h. CAS not only significantly enhanced cell uptake of zein nanoparticles in a concentration- and time-dependent manner but also remarkably improved epithelial transport through Caco-2 cell monolayer. The cell uptake of zein-CAS nanoparticles indicated an energy-dependent endocytosis process as evidenced by cell uptake under blocking conditions, that is, 4 °C, sodium azide, and colchicine. Fluorescent microscopy clearly showed the internalization of zein-CAS nanoparticles. This study may shed some light on the cellular evaluations of hydrophobic protein nanoparticles.

The chemopreventive effect of the dietary compound kaempferol on the MCF-7 human breast cancer cell line is dependent on inhibition of glucose cellular uptake.

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Azevedo, Cláudia; Correia-Branco, Ana; Araújo, João R; Guimarães, João T; Keating, Elisa; Martel, Fátima

2015-01-01

Our aim was to investigate the effect of several dietary polyphenols on glucose uptake by breast cancer cells. Uptake of (3)H-deoxy-D-glucose ((3)H-DG) by MCF-7 cells was time-dependent, saturable, and inhibited by cytochalasin B plus phloridzin. In the short-term (26 min), myricetin, chrysin, genistein, resveratrol, kaempferol, and xanthohumol (10-100 µM) inhibited (3)H-DG uptake. Kaempferol was found to be the most potent inhibitor of (3)H-DG uptake [IC50 of 4 µM (1.6-9.8)], behaving as a mixed-type inhibitor. In the long-term (24 h), kaempferol (30 µM) was also able to inhibit (3)H-DG uptake, associated with a 40% decrease in GLUT1 mRNA levels. Interestingly enough, kaempferol (100 µM) revealed antiproliferative (sulforhodamine B and (3)H-thymidine incorporation assays) and cytotoxic (extracellular lactate dehydrogenase activity determination) properties, which were mimicked by low extracellular (1 mM) glucose conditions and reversed by high extracellular (20 mM) glucose conditions. Finally, exposure of cells to kaempferol (30 µM) induced an increase in extracellular lactate levels over time (to 731 ± 32% of control after a 24 h exposure), due to inhibition of MCT1-mediated lactate cellular uptake. In conclusion, kaempferol potently inhibits glucose uptake by MCF-7 cells, apparently by decreasing GLUT1-mediated glucose uptake. The antiproliferative and cytotoxic effect of kaempferol in these cells appears to be dependent on this effect.

Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in BXPC-3 and PANC-1 human pancreatic cancer cells.

Science.gov (United States)

Wang, Lin; Zhu, Zhi-Xia; Zhang, Wen-Ying; Zhang, Wei-Min

2011-09-01

Previous studies have shown that both pemetrexed, a cytotoxic drug, and erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), inhibit the cell growth of pancreatic cancer cells. However, whether they exert a synergistic antitumor effect on pancreatic cancer cells remains unknown. The present study aimed to assess the synergistic effect of erlotinib in combination with pemetrexed using different sequential administration schedules on the proliferation of human pancreatic cancer BXPC-3 and PANC-1 cells and to probe its cellular mechanism. The EGFR and K-ras gene mutation status was examined by quantitative PCR high-resolution melting (qPCR-HRM) analysis. BXPC-3 and PANC-1 cells were incubated with pemetrexed and erlotinib using different administration schedules. MTT assay was used to determine cytotoxicity, and cell cycle distribution was determined by flow cytometry. The expression and phosphorylation of EGFR, HER3, AKT and MET were determined using Western blotting. Both pemetrexed and erlotinib inhibited the proliferation of BXPC-3 and PANC-1 cells in a dose- and time-dependent manner in vitro. Synergistic effects on cell proliferation were observed when pemetrexed was used in combination with erlotinib. The degree of the synergistic effects depended on the administration sequence, which was most obvious when erlotinib was sequentially administered at 24-h interval following pemetrexed. Cell cycle studies revealed that pemetrexed induced S arrest and erlotinib induced G(0)/G(1) arrest. The sequential administration of erlotinib following pemetrexed induced S arrest. Western blot analyses showed that pemetrexed increased and erlotinib decreased the phosphorylation of EGFR, HER3 and AKT, respectively. However, both pemetrexed and erlotinib exerted no significant effects on the phosphorylation of c-MET. The phosphorylation of EGFR, HER3 and AKT was significantly suppressed by scheduled incubation with pemetrexed followed by erlotinib

Phase I dose escalation pharmacokinetic assessment of intravenous humanized anti-MUC1 antibody AS1402 in patients with advanced breast cancer.

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Pegram, Mark D; Borges, Virginia F; Ibrahim, Nuhad; Fuloria, Jyotsna; Shapiro, Charles; Perez, Susan; Wang, Karen; Schaedli Stark, Franziska; Courtenay Luck, Nigel

2009-01-01

MUC1 is a cell-surface glycoprotein that establishes a molecular barrier at the epithelial surface and engages in morphogenetic signal transduction. Alterations in MUC1 glycosylation accompany the development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion, and immune surveillance. A 20-amino-acid tandem repeat that forms the core protein of MUC1 is overexpressed and aberrantly glycosylated in the majority of epithelial tumors. AS1402 (formerly R1550) is a humanized IgG1k monoclonal antibody that binds to PDTR sequences within this tandem repeat that are not exposed in normal cells. AS1402 is a potent inducer of antibody-dependent cellular cytotoxicity (ADCC), specifically against MUC1-expressing tumor cells. The objective of this study was to determine the safety, tolerability, and pharmacokinetic (PK) characteristics of AS1402 monotherapy in patients with locally advanced or metastatic MUC1-positive breast cancer that had progressed after anthracyclines- and taxane-based therapy. Patients received AS1402 over a 1- to 3-hour intravenous (i.v.) infusion at doses between 1 and 16 mg/kg, with repeated dosing every 1 to 3 weeks (based on patient-individualized PK assessment) until disease progression. Serum AS1402 levels were measured at multiple times after i.v. administration. Human anti-human antibody (HAHA) responses were measured to determine the immunogenicity of AS1402. Noncompartmental pharmacokinetic parameters were determined and were used to assess dose dependency across the dose range studied. Twenty-six patients were treated. AS1402 was generally well tolerated. Two grade 3/4 drug-related adverse events were reported, both at the 3-mg/kg dose. Neither was observed in expanded or subsequent dosing cohorts. No anti-human antibodies were detected. Plasma concentrations of AS1402 appeared to be proportional to dose within the 1- to 16-mg/kg dose range assessed, with a mean terminal half-life of 115.4 +/- 37.1 hours

Impact of cell adhesion and migration on nanoparticle uptake and cellular toxicity.

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Pitchaimani, Arunkumar; Nguyen, Tuyen Duong Thanh; Koirala, Mukund; Zhang, Yuntao; Aryal, Santosh

2017-09-01

In vitro cell-nanoparticle (NP) studies involve exposure of NPs onto the monolayer cells growing at the bottom of a culture plate, and assumed that the NPs evenly distributed for a dose-responsive effect. However, only a few proportion of the administered dose reaches the cells depending on their size, shape, surface, and density. Often the amount incubated (administered dose) is misled as a responsive dose. Herein, we proposed a cell adhesion-migration (CAM) strategy, where cells incubated with the NP coated cell culture substrate to maximize the cell-NP interaction and investigated the physiological properties of the cells. In the present study, cell adhesion and migration pattern of human breast cancer cell (MCF-7) and mouse melanoma cell (B16-F10) on cell culture substrate decorated with toxic (cetyltrimethylammonium bromide, CTAB) and biocompatible (poly (sodium 4-styrenesulphonate), PSS) gold nanoparticles (AuNPs) of different sizes (5 and 40nm) were investigated and evaluated for cellular uptake efficiency, proliferation, and toxicity. Results showed enhanced cell adhesion, migration, and nanoparticle uptake only on biocompatible PSS coated AuNP, irrespective of its size. Whereas, cytotoxic NP shows retard proliferation with reduced cellular uptake efficiency. Considering the importance of cell adhesion and migration on cellular uptake and cytotoxicity assessment of nanoparticle, CAM strategy would hold great promises in cell-NP interaction studies. Copyright © 2017. Published by Elsevier Ltd.

Cell type-dependent induction of DNA damage by 1800 MHz radiofrequency electromagnetic fields does not result in significant cellular dysfunctions.

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Shanshan Xu

Full Text Available BACKGROUND: Although IARC clarifies radiofrequency electromagnetic fields (RF-EMF as possible human carcinogen, the debate on its health impact continues due to the inconsistent results. Genotoxic effect has been considered as a golden standard to determine if an environmental factor is a carcinogen, but the currently available data for RF-EMF remain controversial. As an environmental stimulus, the effect of RF-EMF on cellular DNA may be subtle. Therefore, more sensitive method and systematic research strategy are warranted to evaluate its genotoxicity. OBJECTIVES: To determine whether RF-EMF does induce DNA damage and if the effect is cell-type dependent by adopting a more sensitive method γH2AX foci formation; and to investigate the biological consequences if RF-EMF does increase γH2AX foci formation. METHODS: Six different types of cells were intermittently exposed to GSM 1800 MHz RF-EMF at a specific absorption rate of 3.0 W/kg for 1 h or 24 h, then subjected to immunostaining with anti-γH2AX antibody. The biological consequences in γH2AX-elevated cell type were further explored with comet and TUNEL assays, flow cytometry, and cell growth assay. RESULTS: Exposure to RF-EMF for 24 h significantly induced γH2AX foci formation in Chinese hamster lung cells and Human skin fibroblasts (HSFs, but not the other cells. However, RF-EMF-elevated γH2AX foci formation in HSF cells did not result in detectable DNA fragmentation, sustainable cell cycle arrest, cell proliferation or viability change. RF-EMF exposure slightly but not significantly increased the cellular ROS level. CONCLUSIONS: RF-EMF induces DNA damage in a cell type-dependent manner, but the elevated γH2AX foci formation in HSF cells does not result in significant cellular dysfunctions.

High-affinity uranyl-specific antibodies suitable for cellular imaging

International Nuclear Information System (INIS)

Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C.

2008-01-01

Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO 2 2+ -DCP-BSA (DCP, 1, 10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO 2 2+ -DCP-casein, we selected two highly specific mAbs against uranyl-DCP (K D = 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO 2 2+ , Fe 3+ , Zn 2+ , Cu 2+ , and Ca 2+ ) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO 2 2+ equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immuno-labeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its para-topes can accommodate a wide variety of chelating groups. (authors)

Distributed Velocity-Dependent Protocol for Multihop Cellular Sensor Networks

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Deepthi Chander

2009-01-01

Full Text Available Cell phones are embedded with sensors form a Cellular Sensor Network which can be used to localize a moving event. The inherent mobility of the application and of the cell phone users warrants distributed structure-free data aggregation and on-the-fly routing. We propose a Distributed Velocity-Dependent (DVD protocol to localize a moving event using a Multihop Cellular Sensor Network (MCSN. DVD is based on a novel form of connectivity determined by the waiting time of nodes for a Random Waypoint (RWP distribution of cell phone users. This paper analyzes the time-stationary and spatial distribution of the proposed waiting time to explain the superior event localization and delay performances of DVD over the existing Randomized Waiting (RW protocol. A sensitivity analysis is also performed to compare the performance of DVD with RW and the existing Centralized approach.

Natural cytolytic activity in mice with natural or induced cellular defects. I. Differential ability of in vitro interleukin-2 addition to augment natural cytolytic function

International Nuclear Information System (INIS)

Ades, E.W.; Hinson, A.; Butler, L.D.

1986-01-01

The ability of in vitro addition of recombinant interleukin 2 (rIL-2) to differentially enhance natural cytotoxicity was assessed using cells from mice with natural and induced cellular defects. In vivo treatment with most immunosuppressive or cytoreductive agents, anti-asialo-GM1 antibody, or gamma irradiation dramatically reduced in vitro cytotoxicity against natural killer (NK) sensitive targets by direct reduction in either percentage specific lysis or lytic units per spleen. In most cases, in vitro addition of rIL-2 (at concentrations causing augmented NK function in cells from naive Balb/C mice) enhanced cytotoxic activity of cells from treatment groups to a normal value but not within the rIL-2-enhanced range of nontreated animals. Additionally, cytotoxic activity of cells from animals treated with certain drugs or gamma irradiation could be augmented by rIL-2 when measured by percentage lysis but not lytic units per spleen. In vivo treatment with cyclosporin A did not affect natural cytotoxic activity and addition of rIL-2 augmented the NK activity in a similar fashion to the profile of naive cells. In experiments using cells from beige (C57Bl/6-bg) mice which have a natural defect in NK activity against YAC-1 targets, addition of rIL-2 (at concentrations causing augmented natural cytotoxic function in cells from C57Bl/6 mice) could not effectively enhance in vitro natural cytotoxic function

Natural cytolytic activity in mice with natural or induced cellular defects. I. Differential ability of in vitro interleukin-2 addition to augment natural cytolytic function

Energy Technology Data Exchange (ETDEWEB)

Ades, E.W.; Hinson, A.; Butler, L.D.

1986-08-01

The ability of in vitro addition of recombinant interleukin 2 (rIL-2) to differentially enhance natural cytotoxicity was assessed using cells from mice with natural and induced cellular defects. In vivo treatment with most immunosuppressive or cytoreductive agents, anti-asialo-GM1 antibody, or gamma irradiation dramatically reduced in vitro cytotoxicity against natural killer (NK) sensitive targets by direct reduction in either percentage specific lysis or lytic units per spleen. In most cases, in vitro addition of rIL-2 (at concentrations causing augmented NK function in cells from naive Balb/C mice) enhanced cytotoxic activity of cells from treatment groups to a normal value but not within the rIL-2-enhanced range of nontreated animals. Additionally, cytotoxic activity of cells from animals treated with certain drugs or gamma irradiation could be augmented by rIL-2 when measured by percentage lysis but not lytic units per spleen. In vivo treatment with cyclosporin A did not affect natural cytotoxic activity and addition of rIL-2 augmented the NK activity in a similar fashion to the profile of naive cells. In experiments using cells from beige (C57Bl/6-bg) mice which have a natural defect in NK activity against YAC-1 targets, addition of rIL-2 (at concentrations causing augmented natural cytotoxic function in cells from C57Bl/6 mice) could not effectively enhance in vitro natural cytotoxic function.

Adjuvant effect of Asparagus racemosus Willd. derived saponins in antibody production, allergic response and pro-inflammatory cytokine modulation.

Science.gov (United States)

Tiwari, Nimisha; Gupta, Vivek Kumar; Pandey, Pallavi; Patel, Dinesh Kumar; Banerjee, Suchitra; Darokar, Mahendra Pandurang; Pal, Anirban

2017-02-01

The study manifests the immunoadjuvant potential of saponin rich fraction from Asparagus racemosus in terms of cellular and humoral immune response that can be exploited against microbial infections. Asparagus racemosus (AR) has been attributed as an adaptogen and rasayana in traditional medication systems for enhancing the host defence mechanism. Spectrophotometric and HPTLC analysis ensured the presence of saponins. The saponin rich fractions were tested for immunoadjuvant property in ovalbumin immunised mice for the humoral response, quantified in terms of prolonged antibody production upto a duration of 56days. Proinflammatory cytokines (IL-6 and TNF) were estimated for the cellular immune response in LPS stimulated primary murine macrophages. The safety evaluation in terms of cytotoxicity and allergic response has also been evaluated through in-vitro (MTT) and in-vivo (IgE) respectively. ARS significantly inhibited the pro-inflammatory cytokines, in LPS stimulated murine macrophages with no intrinsic cytotoxicity. The significant increase in IgG production infers the utility of ARS for prolonged humoral response. Further, the antigen specific response of IL-12 at early stage and IgE titres also suggests the generation of cellular immune response and low allergic reaction respectively, as compared to conventional adjuvants. IL-6 and TNF fluctuations in LPS stimulated and non-stimulated macrophages along with IgG and IL-12 also confirmed the Th1/Th2 modulating effect of ARS. The study indicates potential effect of ARS as an adjuvant for the stimulation of cellular immune response in addition to generating a sustained adaptive response without any adverse effects paving way for further validation with pathogenic organisms. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

In vitro preliminary cytotoxicity testing of vegetal extracts, using colorimetric methods

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Claudia Patricia Cordero Camacho

2002-01-01

Full Text Available To advance in the study of the Colombian vegetal biodiversity, considered as a potential source of pharmacologically active products, the establishment of biological activity evaluation systems is necessary, which allow the detection of active products against pathologies with high social and economical impact, such as cancer. This work describes the implementation of a preliminary in vitro methodology for the determination of potential anticancer activity in vegetal extracts, by cytotoxicity testing upon human tumor cell lines, measuring the cellular mass indirectly with the colorimetric assays of MTT (methyl tetrazolium tiazole reduction and SRB (sulforhodamine Bstaining. HT-29, MCF-7, SiHa and HEp-2 cell lines cultures were adapted, MTT concentration, cellular density and treatment period parameters for the cytotoxicity assay were selected. Cell lines sensitivity to the chemotherapeutic agent Doxorubicin HCl was determined. Colombian vegetal species extracts cytotoxicity was tested and usefulness of the assay as a tool to bioguide the search of active products was evidenced.

In vitro preliminary cytotoxicity testing of vegetal extracts, using colorimetric methods

Directory of Open Access Journals (Sweden)

Claudia Patricia Cordero Camacho

2011-12-01

Full Text Available To advance in the study of the Colombian vegetal biodiversity, considered as a potential source of pharmacologically active products, the establishment of biological activity evaluation systems is necessary, which allow the detection of active products against pathologies with high social and economical impact, such as cancer. This work describes the implementation of a preliminary in vitro methodology for the determination of potential anticancer activity in vegetal extracts, by cytotoxicity testing upon human tumor cell lines, measuring the cellular mass indirectly with the colorimetric assays of MTT (methyl tetrazolium tiazole reduction and SRB (sulforhodamine Bstaining. HT-29, MCF-7, SiHa and HEp-2 cell lines cultures were adapted, MTT concentration, cellular density and treatment period parameters for the cytotoxicity assay were selected. Cell lines sensitivity to the chemotherapeutic agent Doxorubicin HCl was determined. Colombian vegetal species extracts cytotoxicity was tested and usefulness of the assay as a tool to bioguide the search of active products was evidenced.

Association of Angiotensin-Converting Enzyme (ACE Gene Polymorphism with Inflammation and Cellular Cytotoxicity in Vitiligo Patients.

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Laila Rashed

Full Text Available Vitiligo is a disorder with profound heterogeneity in its aetio-pathophysiology. Angiotensin converting enzyme (ACE plays an important role in the physiology of the vasculature, blood pressure and inflammation. An insertion/deletion (I/D polymorphism of the ACE gene was reported be associated with the development of vitiligo.Our aim was to evaluate the ACE I/D polymorphism in vitiligo patients and controls. Our second aim was to find a possible association between ACE gene polymorphism and inflammatory mediators (as interleukin (IL-6 and/or cellular cytotoxicity induced by serum nitrite (as a breakdown product of the cytotoxic nitric oxide in vitiligo patients.This case-control study included 74 vitiligo patients and 75 apparently healthy controls. The distribution of ACE gene I/D genotype was investigated using PCR. Serum ACE, IL-6 and nitrite were measured by colorimetric method, ELISA and Griess assay respectively.The ACE allele frequency was significantly different between vitiligo patients and healthy controls (P = 0.026. However there was no significant difference between the ACE genotyping frequency in both groups (P = 0.115. There were statistically significant higher VIDA score (P = 0.007, and serum IL-6 (P < 0.001 in patients with the DD genotype when compared to other genotypes. Serum nitrite in patients with the DD genotype was significantly higher (P = 0.007 when compared to patients with II genotype. Serum levels of ACE, IL-6 and nitrite in vitiligo patients were statistically significantly higher than those in controls.As a conclusion, ACE gene polymorphism might grant susceptibility to develop vitiligo. Serum IL-6 and nitrite levels might have an important role in the pathogenesis of vitiligo. Targeting these two factors might have an implication in the treatment of some resistant cases.

A cellular uptake and cytotoxicity properties study of gallic acid-loaded mesoporous silica nanoparticles on Caco-2 cells

Science.gov (United States)

Rashidi, Ladan; Vasheghani-Farahani, Ebrahim; Soleimani, Masoud; Atashi, Amir; Rostami, Khosrow; Gangi, Fariba; Fallahpour, Masoud; Tahouri, Mohammad Taher

2014-03-01

In this study, the effects of intracellular delivery of various concentrations of gallic acid (GA) as a semistable antioxidant, gallic acid-loaded mesoporous silica nanoparticles (MSNs-GA), and cellular uptake of nanoparticles into Caco-2 cells were investigated. MSNs were synthesized and loaded with GA, then characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, N2 adsorption isotherms, X-ray diffraction, and thermal gravimetric analysis. The cytotoxicity of MSNs and MSNs-GA at low and high concentrations were studied by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry. MSNs did not show significant toxicity in various concentrations (0-500 μg/ml) on Caco-2 cells. For MSNs-GA, cell viability was reduced as a function of incubation time and different concentrations of nanoparticles. The in vitro GA release from MSNs-GA exhibited the same antitumor properties as free GA on Caco-2 cells. Flow cytometry results confirmed those obtained using MTT assay. TEM and fluorescent microscopy confirmed the internalization of MSNs by Caco-2 cells through nonspecific cellular uptake. MSNs can easily internalize into Caco-2 cells without deleterious effects on cell viability. The cell viability of Caco-2 cells was affected during MSNs-GA uptake. MSNs could be designed as suitable nanocarriers for antioxidants delivery.

Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists Antibody-Drug Conjugate Working Group position paper.

Science.gov (United States)

Gorovits, Boris; Alley, Stephen C; Bilic, Sanela; Booth, Brian; Kaur, Surinder; Oldfield, Phillip; Purushothama, Shobha; Rao, Chetana; Shord, Stacy; Siguenza, Patricia

2013-05-01

Antibody-drug conjugates (ADCs) typically consist of a cytotoxic drug covalently bound to an antibody by a linker. These conjugates have the potential to substantially improve efficacy and reduce toxicity compared with cytotoxic small-molecule drugs. Since ADCs are generally complex heterogeneous mixtures of multiple species, these novel therapeutic products present unique bioanalytical challenges. The growing number of ADCs being developed across the industry suggests the need for alignment of the bioanalytical methods or approaches used to assess the multiple species and facilitate consistent interpretation of the bioanalytical data. With limited clinical data, the current strategies that can be used to provide insight into the relationship between the multiple species and the observed clinical safety and efficacy are still evolving. Considerations of the bioanalytical strategies for ADCs based on the current industry practices that take into account the complexity and heterogeneity of ADCs are discussed.

Identification of cellular responses to low-dose radiation by antibody array in human B-lymphoblasts IM-9 cells

Energy Technology Data Exchange (ETDEWEB)

Eom, Hyeon Soo; Kim, Ji Young; Nam, Seon Young [Low-dose Radiation Research Team, Radiation Health Institute, Korea Hydro and Nuclear Power Co. LTD., Seoul (Korea, Republic of)

2017-04-15

The low-dose radiation (LDR)-induced various responses can reduce genetic mutation, enhance cell survival, and increase infection resistance (1). The antibody array for global analysis of phosphorylated proteins might be very useful to study signaling networks of LDR-induced cellular responses (2). Therefore, global analysis of phospho- proteins in cells exposed to radiation is important to understand the signaling mechanisms induced by changes of protein phosphorylation which lead to various biological effects by radiation. The aim is to explore the possibility of LDR-specific signaling for various beneficial effects and elucidate the potential signaling pathways representing LDR responses. Our results suggest that LDR did not affect cell death and that the increased proteins phosphorylation by LDR might be involved in various cellular responses for cell homeostasis. These results might be useful to further studies aimed at investigating potential regulatory markers that represent responses to LDR.

Identification of cellular responses to low-dose radiation by antibody array in human B-lymphoblasts IM-9 cells

International Nuclear Information System (INIS)

Eom, Hyeon Soo; Kim, Ji Young; Nam, Seon Young

2017-01-01

The low-dose radiation (LDR)-induced various responses can reduce genetic mutation, enhance cell survival, and increase infection resistance (1). The antibody array for global analysis of phosphorylated proteins might be very useful to study signaling networks of LDR-induced cellular responses (2). Therefore, global analysis of phospho- proteins in cells exposed to radiation is important to understand the signaling mechanisms induced by changes of protein phosphorylation which lead to various biological effects by radiation. The aim is to explore the possibility of LDR-specific signaling for various beneficial effects and elucidate the potential signaling pathways representing LDR responses. Our results suggest that LDR did not affect cell death and that the increased proteins phosphorylation by LDR might be involved in various cellular responses for cell homeostasis. These results might be useful to further studies aimed at investigating potential regulatory markers that represent responses to LDR

Heparin-independent, PF4-dependent binding of HIT antibodies to platelets: implications for HIT pathogenesis.

Science.gov (United States)

Padmanabhan, Anand; Jones, Curtis G; Bougie, Daniel W; Curtis, Brian R; McFarland, Janice G; Wang, Demin; Aster, Richard H

2015-01-01

Antibodies specific for platelet factor 4 (PF4)/heparin complexes are the hallmark of heparin-induced thrombocytopenia and thrombosis (HIT), but many antibody-positive patients have normal platelet counts. The basis for this is not fully understood, but it is believed that antibodies testing positive in the serotonin release assay (SRA) are the most likely to cause disease. We addressed this issue by characterizing PF4-dependent binding of HIT antibodies to intact platelets and found that most antibodies testing positive in the SRA, but none of those testing negative, bind to and activate platelets when PF4 is present without any requirement for heparin (P HIT antibodies recognize PF4 in a complex with heparin, only a subset of these antibodies recognize more subtle epitopes induced in PF4 when it binds to CS, the major platelet glycosaminoglycan. Antibodies having this property could explain "delayed HIT" seen in some individuals after discontinuation of heparin and the high risk for thrombosis that persists for weeks in patients recovered from HIT. © 2015 by The American Society of Hematology.

Cytotoxicity of Cerastes cerastes snake venom: Involvement of imbalanced redox status.

Science.gov (United States)

Kebir-Chelghoum, Hayet; Laraba-Djebari, Fatima

2017-09-01

Envenomation caused by Cerastes cerastes snake venom is characterized by a local and a systemic tissue damage due to myonecrosis, hemorrhage, edema and acute muscle damage. The present study aimed to evaluate the relationship between the pro/anti-oxidants status and the cytotoxicity of C. cerastes snake venom. The in vivo cytotoxicity analysis was undertaken by the injection of C. cerastes venom (48μg/20g body weight) by i.p. route, mice were then sacrificed at 3, 24 and 48h post injection, organs were collected for further analysis. In vitro cytotoxicity analysis was investigated on cultured PBMC, hepatocytes and isolated liver. The obtained results showed a significant cell infiltration characterized by a significant increase of myeloperoxidase (MPO) and eosinoperoxidase (EPO) activities. These results showed also a potent oxidative activity of C. cerastes venom characterized by increased levels of residual nitrites and lipid peroxidation associated with a significant decrease of glutathione and catalase activity in sera and tissues (heart, lungs, liver and kidneys). The in vitro cytotoxicity of C. cerastes venom on PBMC seems to be dose-dependent (IC50 of 21μg/ml/10 6 cells) and correlated with an imbalanced redox status at high doses of venom. However, in the case of cultured hepatocytes, the LDH release and oxidative stress were observed only at high doses of the venom. The obtained results of in vivo study were confirmed by the culture of isolated liver. Therefore, these results suggest that the venom induces a direct cytotoxic effect which alters the membrane integrity causing a leakage of the cellular contents. This cytotoxic effect can lead indirectly to inflammatory response and oxidative stress. These data suggest that an early anti-inflammatory and antioxidant treatment could be useful in the management of envenomed victims. Copyright © 2017. Published by Elsevier B.V.

High-affinity uranyl-specific antibodies suitable for cellular imaging

Energy Technology Data Exchange (ETDEWEB)

Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C. [CEA Valrho, DSV, IBEB, Serv Biochim et Toxicol Nucl, F-30207 Bagnols Sur Ceze (France)

2008-07-01

Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO{sub 2}{sup 2+}-DCP-BSA (DCP, 1, 10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO{sub 2}{sup 2+}-DCP-casein, we selected two highly specific mAbs against uranyl-DCP (K{sub D} = 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO{sub 2}{sup 2+}, Fe{sup 3+}, Zn{sup 2+}, Cu{sup 2+}, and Ca{sup 2+}) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO{sub 2}{sup 2+} equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immuno-labeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its para-topes can accommodate a wide variety of chelating groups. (authors)

Suppression of cytotoxic T lymphocytes by carrageenan-activated macrophage-like cells

International Nuclear Information System (INIS)

Yung, Y.P.; Cudkowicz, G.

1978-01-01

In the presence of 100 μg/ml of carrageenans (CAR), B6D2F 1 responder spleen cells failed to generate antiparent or anti-allogeneic cytotoxic T lymphocytes in vitro, but instead generated suppressor cells. Cultured CAR-treated cells added to mixtures of B6D2F 1 anti-B6 or B6D2F 1 anti-C3H cytotoxic effectors (induced in vitro) and the appropriate 51 Cr-labeled lymphoma targets reduced or abolished cytolysis (measured as 51 Cr release) depending on the ratio of suppressor to effector cells. Cultured spleen cells not exposed to CAR failed to inhibit both types of cytotoxicity. Presuppressor cells were associated with a splenic subpopulation independent of the thymus (i.e., present in spleens of athymic nude mice), were moderately adherent to Sephadex G-10 columns, but were not phagocytic or ''sticky'' to carbonyl iron particles. Activation of such cells by CAR was not prevented by in vitro exposure to 2000 rads of γ-rays before culture, nor facilitated by antigenic stimulation. The matured suppressor cells remained radioresistant and became strongly adherent to Sephadex G-10. The suppressors lacked surface Thy-1 alloantigen detectable by antibody and rabbit complement. Suppressor cell activity was not restricted by the immunologic specificity and major histocompatibility type of effectors

21 CFR 866.5100 - Antinuclear antibody immunological test system.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antinuclear antibody immunological test system....5100 Antinuclear antibody immunological test system. (a) Identification. An antinuclear antibody... the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear...

Antibody conjugate radioimmunotherapy of superficial bladder cancer

International Nuclear Information System (INIS)

Perkins, Alan; Hopper, Melanie; Murray, Andrea; Frier, Malcolm; Bishop, Mike

2002-01-01

The administration of antibody conjugates for cancer therapy is now proving to be of clinical value. We are currently undertaking a programme of clinical studies using the monoclonal antibody C 595 (gG3) which reacts with the MUC1 glycoprotein antigen that is aberrantly expressed in a high proportion of bladder tumours. Radio immuno conjugates of the C 595 antibody have been produced with high radiolabelling efficiency and immuno reactivity using Tc-99 m and In-111 for diagnostic imaging, and disease staging and the cytotoxic radionuclides Cu-67 and Re-188 for therapy of superficial bladder cancer. A Phase I/II therapeutic trail involving the intravesical administration of antibody directly into the bladder has now begun. (author)

Distributed Velocity-Dependent Protocol for Multihop Cellular Sensor Networks

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Jagyasi Bhushan

2009-01-01

Full Text Available Abstract Cell phones are embedded with sensors form a Cellular Sensor Network which can be used to localize a moving event. The inherent mobility of the application and of the cell phone users warrants distributed structure-free data aggregation and on-the-fly routing. We propose a Distributed Velocity-Dependent (DVD protocol to localize a moving event using a Multihop Cellular Sensor Network (MCSN. DVD is based on a novel form of connectivity determined by the waiting time of nodes for a Random Waypoint (RWP distribution of cell phone users. This paper analyzes the time-stationary and spatial distribution of the proposed waiting time to explain the superior event localization and delay performances of DVD over the existing Randomized Waiting (RW protocol. A sensitivity analysis is also performed to compare the performance of DVD with RW and the existing Centralized approach.

Differential cytotoxic effects of 7-dehydrocholesterol-derived oxysterols on cultured retina-derived cells: Dependence on sterol structure, cell type, and density.

Science.gov (United States)

Pfeffer, Bruce A; Xu, Libin; Porter, Ned A; Rao, Sriganesh Ramachandra; Fliesler, Steven J

2016-04-01

Tissue accumulation of 7-dehydrocholesterol (7DHC) is a hallmark of Smith-Lemli-Opitz Syndrome (SLOS), a human inborn error of the cholesterol (CHOL) synthesis pathway. Retinal 7DHC-derived oxysterol formation occurs in the AY9944-induced rat model of SLOS, which exhibits a retinal degeneration characterized by selective loss of photoreceptors and associated functional deficits, Müller cell hypertrophy, and engorgement of the retinal pigment epithelium (RPE) with phagocytic inclusions. We evaluated the relative effects of four 7DHC-derived oxysterols on three retina-derived cell types in culture, with respect to changes in cellular morphology and viability. 661W (photoreceptor-derived) cells, rMC-1 (Müller glia-derived) cells, and normal diploid monkey RPE (mRPE) cells were incubated for 24 h with dose ranges of either 7-ketocholesterol (7kCHOL), 5,9-endoperoxy-cholest-7-en-3β,6α-diol (EPCD), 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), or 4β-hydroxy-7-dehydrocholesterol (4HDHC); CHOL served as a negative control (same dose range), along with appropriate vehicle controls, while staurosporine (Stsp) was used as a positive cytotoxic control. For 661W cells, the rank order of oxysterol potency was: EPCD > 7kCHOL > DHCEO > 4HDHC ≈ CHOL. EC50 values were higher for confluent vs. subconfluent cultures. 661W cells exhibited much higher sensitivity to EPCD and 7kCHOL than either rMC-1 or mRPE cells, with the latter being the most robust when challenged, either at confluence or in sub-confluent cultures. When tested on rMC-1 and mRPE cells, EPCD was again an order of magnitude more potent than 7kCHOL in compromising cellular viability. Hence, 7DHC-derived oxysterols elicit differential cytotoxicity that is dose-, cell type-, and cell density-dependent. These results are consistent with the observed progressive, photoreceptor-specific retinal degeneration in the rat SLOS model, and support the hypothesis that 7DHC-derived oxysterols are causally linked to that

Functional Antibody Response Against V1V2 and V3 of HIV gp120 in the VAX003 and VAX004 Vaccine Trials.

Science.gov (United States)

Balasubramanian, Preetha; Williams, Constance; Shapiro, Mariya B; Sinangil, Faruk; Higgins, Keith; Nádas, Arthur; Totrov, Maxim; Kong, Xiang-Peng; Fiore-Gartland, Andrew J; Haigwood, Nancy L; Zolla-Pazner, Susan; Hioe, Catarina E

2018-01-11

Immunization with HIV AIDSVAX gp120 vaccines in the phase III VAX003 and VAX004 trials did not confer protection. To understand the shortcomings in antibody (Ab) responses induced by these vaccines, we evaluated the kinetics of Ab responses to the V1V2 and V3 regions of gp120 and the induction of Ab-mediated antiviral functions during the course of 7 vaccinations over a 30.5-month period. Plasma samples from VAX003 and VAX004 vaccinees and placebo recipients were measured for ELISA-binding Abs and for virus neutralization, Ab-dependent cellular phagocytosis (ADCP), and Ab-dependent cellular cytotoxicity (ADCC). Ab responses to V1V2 and V3 peaked after 3 to 4 immunizations and declined after 5 to 7 immunizations. The deteriorating responses were most evident against epitopes in the underside of the V1V2 β-barrel and in the V3 crown. Correspondingly, vaccinees demonstrated higher neutralization against SF162 pseudovirus sensitive to anti-V1V2 and anti-V3 Abs after 3 or 4 immunizations than after 7 immunizations. Higher levels of ADCP and ADCC were also observed at early or mid-time points as compared with the final time point. Hence, VAX003 and VAX004 vaccinees generated V1V2- and V3-binding Abs and functional Abs after 3 to 4 immunizations, but subsequent boosts did not maintain these responses.

Paucity of natural killer and cytotoxic T cells in human neuromyelitis optica lesions

Science.gov (United States)

Saadoun, Samira; Bridges, Leslie R.; Verkman, A. S.; Papadopoulos, Marios C.

2013-01-01

Neuromyelitis optica is a severe inflammatory demyelinating disease of the central nervous system. Most patients with neuromyelitis optica have circulating immunoglobulin G (IgG) antibodies against the astrocytic water channel protein aquaporin-4 (AQP4), which are pathogenic. Anti-AQP4 IgG-mediated complement-dependent astrocyte toxicity is a key mechanism of central nervous system damage in neuromyelitis optica, but the role of natural killer and cytotoxic T cells is unknown. Our objective was to determine whether natural killer and cytotoxic T cells play a role in human neuromyelitis optica lesions. We immunostained four actively demyelinating lesions, obtained from patients with anti-AQP4 IgG positive neuromyelitis optica, for Granzyme B and Perforin. The inflammatory cells were perivascular neutrophils, eosinophils and macrophages, with only occasional Granzyme B+ or Perforin + cells. Greater than 95% of inflamed vessels in each lesion had no surrounding Granzyme B+ or Perforin + cells. Granzyme B+ or Perforin+ cells were abundant in human spleen (positive control). Although natural killer cells produce central nervous system damage in mice injected with anti-AQP4 IgG, our findings here indicate that natural killer-mediated and T cell-mediated cytotoxicity are probably not involved in central nervous system damage in human neuromyelitis optica. PMID:23108041

Platelets promote tumor growth and metastasis via direct interaction between Aggrus/podoplanin and CLEC-2.

Directory of Open Access Journals (Sweden)

Satoshi Takagi

Full Text Available The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus-CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus-CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet-tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma.

Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

Science.gov (United States)

Yang, Danlin; Giragossian, Craig; Castellano, Steven; Lasaro, Marcio; Xiao, Haiguang; Saraf, Himanshu; Hess Kenny, Cynthia; Rybina, Irina; Huang, Zhong-Fu; Ahlberg, Jennifer; Bigwarfe, Tammy; Myzithras, Maria; Waltz, Erica; Roberts, Simon; Kroe-Barrett, Rachel; Singh, Sanjaya

2017-10-01

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

Fc gamma receptor activation induces the tyrosine phosphorylation of both phospholipase C (PLC)-gamma 1 and PLC-gamma 2 in natural killer cells

OpenAIRE

1992-01-01

Crosslinking of the low affinity immunoglobulin G (IgG) Fc receptor (Fc gamma R type III) on natural killer (NK) cells initiates antibody- dependent cellular cytotoxicity. During this process, Fc gamma R stimulation results in the rapid activation of phospholipase C (PLC), which hydrolyzes membrane phosphoinositides, generating inositol-1,4,5- trisphosphate and sn-1,2-diacylglycerol as second messengers. We have recently reported that PLC activation after Fc gamma R stimulation can be inhibit...

Reducing ZnO nanoparticle cytotoxicity by surface modification.

Science.gov (United States)

Luo, Mingdeng; Shen, Cenchao; Feltis, Bryce N; Martin, Lisandra L; Hughes, Anthony E; Wright, Paul F A; Turney, Terence W

2014-06-07

Nanoparticulate zinc oxide (ZnO) is one of the most widely used engineered nanomaterials and its toxicology has gained considerable recent attention. A key aspect for controlling biological interactions at the nanoscale is understanding the relevant nanoparticle surface chemistry. In this study, we have determined the disposition of ZnO nanoparticles within human immune cells by measurement of total Zn, as well as the proportions of extra- and intracellular dissolved Zn as a function of dose and surface coating. From this mass balance, the intracellular soluble Zn levels showed little difference in regard to dose above a certain minimal level or to different surface coatings. PEGylation of ZnO NPs reduced their cytotoxicity as a result of decreased cellular uptake arising from a minimal protein corona. We conclude that the key role of the surface properties of ZnO NPs in controlling cytotoxicity is to regulate cellular nanoparticle uptake rather than altering either intracellular or extracellular Zn dissolution.

Inhibition of Spontaneous Breast Cancer Metastasis by Anti—Thomsen-Friedenreich Antigen Monoclonal Antibody JAA-F11

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Jamie Heimburg

2006-11-01

Full Text Available Thomsen-Friedenreich antigen (TF-Ag is expressed in many carcinomas, including those of the breast, colon, bladder, prostate. TF-Ag is important in adhesion and metastasis and as a potential immunotherapy target. We hypothesized that passive transfer of JAAF11, an anti -TF-Ag monoclonal antibody, may create a survival advantage for patients with TIF-Ag -expressing tumors by cytotoxicity, blocking of tumor cell adhesion, inhibition of metastasis. This was tested using in vitro models of tumor cell growth; cytotoxicity assays; in vitro, ex vivo, in vivo models of cancer metastasis; and, finally, in vivo effects in mice with metastatic breast cancer. Unlike some anti-TF-Ag antibodies, JAA-F11 did not enhance breast carcinoma cell growth. JAA-F11 did not induce the killing of 4T1 tumor cells through complement-dependent cytotoxicity or apoptotic mechanisms. However, JAA-F11 blocked the stages of metastasis that involve the adhesion of human breast carcinoma cells to human endothelial cells (human umbilical vein endothelial cells and human bone marrow endothelial cells 60 in in vitro static adhesion models, in a perfused ex vivo model, in murine lung vasculature in an in vivo metastatic deposit formation assay. JAA-F11 significantly extended the median survival time of animals bearing metastatic 4T1 breast tumors and caused a > 50% inhibition of lung metastasis.

Advances in therapeutic Fc engineering - modulation of IgG associated effector functions and serum half-life

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Abhishek Saxena

2016-12-01

Full Text Available Today monoclonal immunoglobulin gamma (IgG antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs are achieved through both its antigen binding fragment (Fab and crystallizable fragment (Fc. Fab can specifically recognize tumor associated antigen (TAA and thus modulate TAA-linked downstream signaling pathways that may lead to inhibition of tumor growth, induction of tumor apoptosis and differentiation. The Fc region can further improve mAbs’ efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity (ADCC, complement-dependent cytotoxicity (CDC and antibody dependent cell-mediated phagocytosis (ADCP. Moreover, Fc is the region interacting with the neonatal Fc receptor (FcRn in a pH-dependent manner that can slow down IgG’s degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anti-cancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering based mAbs under clinical trials.

Cytotoxicity of Carbon Nanotubes on J774 Macrophages Is a Purification-Dependent Effect

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Silvia Lorena Montes-Fonseca

2012-01-01

Full Text Available The cytotoxicity of the carbon nanotubes (CNTs is an important factor for the manufacture of nanovaccines. The aim of this work was to evaluate the relationship of the purification method of CNTs in cellular toxicity using macrophages (MOs from the J774 cell line. Viability test was performed with MTT assays at 24 h of exposure at concentrations of 0.06, 0.6, and 6 mg/L of unpurified (UP-CNTs or purified (P-CNTs CNTs by two different methods: (1 reflux with 3M HNO3 and (2 sonication in H2SO4/HNO3. Characterization and COOH content of CNTs was performed using scanning electron microscopy, raman spectroscopy, and titration with NaHCO3. P-CNTs1 had lengths >100 μm and 2.76% COOH content, while P-CNTs2 had lengths >1 μm and 7% COOH content. This last particle showed a lower toxic effect. The results suggest that the lenght and COOH content are important factors in the toxicity of the CNTs.

Lack of dependence of 5-fluorodeoxyuridine-mediated radiosensitization on cytotoxicity

International Nuclear Information System (INIS)

Lawrence, T.S.; Davis, M.A.; Chang, E.Y.

1995-01-01

It has been proposed that fluoropyrimidine-mediated cytotoxicity and radiosensitization are closely correlated. We have shown that HT29 human colon cancer cells transfected with the E. coli dUTPase gene are resistant to 5-fluorodeoxyuridine (FdUrd)-mediated cytotoxicity, presumably through more effective elimination of dUTP. We used these cells to assess the association between radiosensitization and cytotoxicity produced by FdUrd. The radiation sensitivities of the clones expressing elevated dUTPase activity (dutE clones) were similar to those of untransfected HT29 cells or HT29 cells which has been transfected with only the expression vector for the E. coli gene (con clones). We found that FdUrd produced similar increases in radiation sensitivity regardless of dUTPase activity. Levels of dUTPase in the dutE clones remained elevated during the entire period of FdUrd exposure, demonstrating that the lack of difference between dutE and Con clones was not a reflection of down-regulation of dUTPase activity by FdUrd, Flow cytometry showed that all clones progressed past the G 1 /S-phase boundary and into early S phase during FdUrd treatment. These data suggest that the mechanisms of FdUrd-mediated cytotoxicity and radiosensitization are not closely linked. These findings, combined with our previous investigations, are consistent with the hypothesis that radiosensitization occurs in cells which progress past the G 1 /S-phase boundary in the presence of FdUrd. 24 refs., 2 figs., 2 tabs

Antibody-dependent enhancement of HIV-1 infection in human term syncytiotrophoblast cells cultured in vitro.

Science.gov (United States)

Tóth, F D; Mosborg-Petersen, P; Kiss, J; Aboagye-Mathiesen, G; Zdravkovic, M; Hager, H; Aranyosi, J; Lampé, L; Ebbesen, P

1994-06-01

We examined if Fc receptor-mediated antibody-dependent enhancement (FcR-ADE) or complement-mediated antibody-dependent enhancement (C'-ADE) of virus infection can contribute to increasing replication of HIV-1 in human syncytiotrophoblast (ST) cells. Here we report that both FcR-ADE and C'-ADE may result in enhanced virus release from HIV-1-infected ST cells. We show that FcR-ADE of HIV-1 infection in ST cells is mediated by FcRIII and other FcR(s) belonging to undetermined Fc classes and does not require CD4 receptors, whereas C'-ADE uses both CD4 and CR2-like receptors. FcR-ADE seems to be more efficient in enhancing HIV-1 replication than C'-ADE. While FcR-ADE leads to increased internalization of HIV-1, C'-ADE does not result in enhanced endocytosis of the virus. In addition, antibodies mediating FcR-ADE are reactive with the gp120 viral envelope antigen, whereas antibodies involved in C'-ADE react with the viral transmembrane glycoprotein gp41. Data suggest that both FcR-ADE and C'-ADE may contribute to the spread of HIV-1 from mother to the fetus.

Evaluation of the cytotoxic and antimutagenic effects of biflorin, an antitumor 1,4 o-naphthoquinone isolated from Capraria biflora L.

Science.gov (United States)

Vasconcellos, Marne C; Moura, Dinara J; Rosa, Renato M; Machado, Miriana S; Guecheva, Temenouga N; Villela, Izabel; Immich, Bruna F; Montenegro, Raquel C; Fonseca, Aluísio M; Lemos, Telma L G; Moraes, Maria Elisabete A; Saffi, Jenifer; Costa-Lotufo, Letícia V; Moraes, Manoel O; Henriques, João A P

2010-10-01

Biflorin is a natural quinone isolated from Capraria biflora L. Previous studies demonstrated that biflorin inhibits in vitro and in vivo tumor cell growth and presents potent antioxidant activity. In this paper, we report concentration-dependent cytotoxic, genotoxic, antimutagenic, and protective effects of biflorin on Salmonella tiphymurium, yeast Saccharomyces cerevisiae, and V79 mammalian cells, using different approaches. In the Salmonella/microsome assay, biflorin was not mutagenic to TA97a TA98, TA100, and TA102 strains. However, biflorin was able to induce cytotoxicity in haploid S. cerevisiae cells in stationary and exponential phase growth. In diploid yeast cells, biflorin did not induce significant mutagenic and recombinogenic effects at the employed concentration range. In addition, the pre-treatment with biflorin prevented the mutagenic and recombinogenic events induced by hydrogen peroxide (H(2)O(2)) in S. cerevisiae. In V79 mammalian cells, biflorin was cytotoxic at higher concentrations. Moreover, at low concentrations biflorin pre-treatment protected against H(2)O(2)-induced oxidative damage by reducing lipid peroxidation and DNA damage as evaluated by normal and modified comet assay using DNA glycosylases. Our results suggest that biflorin cellular effects are concentration dependent. At lower concentrations, biflorin has significant antioxidant and protective effects against the cytotoxicity, genotoxicity, mutagenicity, and intracellular lipid peroxidation induced by H(2)O(2) in yeast and mammalian cells, which can be attributed to its hydroxyl radical-scavenging property. However, at higher concentrations, biflorin is cytotoxic and genotoxic.

Cytotoxicity and intracellular dissolution of nickel nanowires

KAUST Repository

Perez, Jose E.

2015-12-22

The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 µm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

Cytotoxicity and intracellular dissolution of nickel nanowires.

Science.gov (United States)

Perez, Jose E; Contreras, Maria F; Vilanova, Enrique; Felix, Laura P; Margineanu, Michael B; Luongo, Giovanni; Porter, Alexandra E; Dunlop, Iain E; Ravasi, Timothy; Kosel, Jürgen

2016-09-01

The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis, and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage, and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 μm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

Cytotoxicity and intracellular dissolution of nickel nanowires

KAUST Repository

Perez, Jose E.; Contreras, Maria F.; Vidal, Enrique Vilanova; Felix Servin, Laura P.; Margineanu, Michael B.; Luongo, Giovanni; Porter, Alexandra E.; Dunlop, Iain E.; Ravasi, Timothy; Kosel, Jü rgen

2015-01-01

The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 µm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

Vaccination with dengue virus-like particles induces humoral and cellular immune responses in mice

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Zhang Quanfu

2011-06-01

Full Text Available Abstract Background The incidence of dengue, an infectious disease caused by dengue virus (DENV, has dramatically increased around the world in recent decades and is becoming a severe public health threat. However, there is currently no specific treatment for dengue fever, and licensed vaccine against dengue is not available. Vaccination with virus-like particles (VLPs has shown considerable promise for many viral diseases, but the effect of DENV VLPs to induce specific immune responses has not been adequately investigated. Results By optimizing the expression plasmids, recombinant VLPs of four antigenically different DENV serotypes DENV1-4 were successfully produced in 293T cells. The vaccination effect of dengue VLPs in mice showed that monovalent VLPs of each serotype stimulated specific IgG responses and potent neutralizing antibodies against homotypic virus. Tetravalent VLPs efficiently enhanced specific IgG and neutralizing antibodies against all four serotypes of DENV. Moreover, vaccination with monovalent or tetravalent VLPs resulted in the induction of specific cytotoxic T cell responses. Conclusions Mammalian cell expressed dengue VLPs are capable to induce VLP-specific humoral and cellular immune responses in mice, and being a promising subunit vaccine candidate for prevention of dengue virus infection.

Characterization of non-dimer DNA lesions and cellular damages caused by ultraviolet light

International Nuclear Information System (INIS)

Nakao, Kumi

1989-01-01

To understand the mechanisms of carcinogenicity and cytotoxicity induced by ultraviolet (UV) light, non-dimer DNA damages produced by near UV light (wave-length: 290∼320 nm) were examined by alkaline elution using Chinese hamster V-79 cells. UV exposure produced a dose-dependent induction of DNA single strand breaks and DNA-protein crosslinks. However, neither of these DNA lesions were repaired within a 24 hr incubation of the cells following UV exposure. Rather the number of these lesions increased. Also, UV exposure inhibited DNA and RNA synthesis. In addition, UV induced both cytotoxicity and chromosomal aberration. Electron spin resornance (ESR) studies showed that the exposure of cells to UV light resulted in the appearance of an ESR signal at -120degC. The roles of glutathione, vitamin E and vitamin B 2 , which were celluar antioxidant, on the induction of cytotoxicity by UV exposure were also examined. Pretreatment with vitamin E reduced the cytotoxicty caused by UV, whereas neither preteatment with vitamin B 2 nor the alteration of cellular gluthaione content affected the cytotoxicity. These results suggest that non-dimer DNA damages, such as DNA single strand breaks and DNA-protein crosslinks play an important role in inducing UV-carcinogenicity and UV-cytotoxicity, and that the mechanisms of these damages may be associated with the generation of free radicals. (author)

Investigation of internalization and cytotoxicity of 125I-[Tyr3]-octreotide in NCI-H446 cell line

International Nuclear Information System (INIS)

Sun Junjie; Fan Wo; Xu Yujie; Zhang Youjiu; Zhu Ran; Hu Mingjiang

2004-01-01

Objective: To investigate the [Tyr 3 ]-octreotide (TOC) internalizing capacity of NCI-H446 cell line, and the cytotoxicity of 125 I-TOC in NCI-H446 cell line. To assess the therapeutic radiopharmaceutical potentiality of 125 I-TOC for the somatostatin receptor (SSTR) positive tumor. Methods: NCI-H446 cells were incubated together with 125 I-TOC for different periods of time, the amount of internalized 125 I-TOC and the 125 I-TOC bound on the cellular nucleus were detected with γ counter, respectively. The viability of the cells was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time points with various doses of 125 I-TOC, free 125 I and TOC. Results: 125 I-TOC was internalized into the nucleus and bound on the nucleus in a time-dependent manner. 125 I-TOC bound on the nucleus increased to the highest level at 24 h, the amount of nucleus bound 125 I-TOC at 24 h was 7 times higher than that at 0.5 h. Cytotoxicity of 125 I-TOC in SSTR positive NCI-H446 cells was also dose- and time-dependent. The supreme effect of cytotoxicity was found at 96 h with 74 kBq 125 I-TOC, the survival ratio of cells was reduced to (44.8 ± 7.2)%. Conclusions: 125 I-TOC can be internalized into SSTR positive cells mediated by SSTR. The NCI-H446 cells can be killed by Auger electron emitting from 125 I-TOC. Effect of cytotoxicity showed dose- and time-dependent

Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

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Anna Frenzel

Full Text Available Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C. Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity.

Mouse Hepatitis Virus Strain A59 and Blocking Antireceptor Monoclonal Antibody Bind to the N-Terminal Domain of Cellular Receptor

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Dveksler, Gabriela S.; Pensiero, Michael N.; Dieffenbach, Carl W.; Cardellichio, Christine B.; Basile, Alexis A.; Elia, Patrick E.; Holmes, Kathryn V.

1993-03-01

Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

Site-specific chemical modification of antibody fragments using traceless cleavable linkers.

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Bernardes, Gonçalo J L; Steiner, Martina; Hartmann, Isabelle; Neri, Dario; Casi, Giulio

2013-11-01

Antibody-drug conjugates (ADCs) are promising agents for the selective delivery of cytotoxic drugs to specific cells (for example, tumors). In this protocol, we describe two strategies for the precise modification at engineered C- or N-terminal cysteines of antibodies in IgG, diabody and small immunoprotein (SIP) formats that yield homogenous ADCs. In this protocol, cemadotin derivatives are used as model drugs, as these agents have a potent cytotoxic activity and are easy to synthesize. However, other drugs with similar functional groups could be considered. In the first approach, a cemadotin derivative containing a sulfhydryl group results in a mixed disulfide linkage. In the second approach, a cemadotin derivative containing an aldehyde group is joined via a thiazolidine linkage. The procedures outlined are robust, enabling the preparation of ADCs with a defined number of drugs per antibody in a time frame between 7 and 24 h.

Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNFα and co-treatments

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Loo, Lit-Hsin; Bougen-Zhukov, Nicola Michelle; Tan, Wei-Ling Cecilia

2017-03-01

Signaling pathways can generate different cellular responses to the same cytotoxic agents. Current quantitative models for predicting these differential responses are usually based on large numbers of intracellular gene products or signals at different levels of signaling cascades. Here, we report a study to predict cellular sensitivity to tumor necrosis factor alpha (TNFα) using high-throughput cellular imaging and machine-learning methods. We measured and compared 1170 protein phosphorylation events in a panel of human lung cancer cell lines based on different signals, subcellular regions, and time points within one hour of TNFα treatment. We found that two spatiotemporal-specific changes in an intermediate signaling protein, p90 ribosomal S6 kinase (RSK), are sufficient to predict the TNFα sensitivity of these cell lines. Our models could also predict the combined effects of TNFα and other kinase inhibitors, many of which are not known to target RSK directly. Therefore, early spatiotemporal-specific changes in intermediate signals are sufficient to represent the complex cellular responses to these perturbations. Our study provides a general framework for the development of rapid, signaling-based cytotoxicity screens that may be used to predict cellular sensitivity to a cytotoxic agent, or identify co-treatments that may sensitize or desensitize cells to the agent.

Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy

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Szlachcic A

2016-08-01

Full Text Available Anna Szlachcic, Malgorzata Zakrzewska, Michal Lobocki, Piotr Jakimowicz, Jacek Otlewski Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Abstract: Fibroblast growth factor receptors (FGFRs are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V, was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE, and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. Keywords: fibroblast growth factor 1, FGF receptor, targeted cancer therapy, cytotoxic conjugates, FGFR-dependent cancer, MMAE, auristatin

Preventive effects of fructose and N-acetyl-L-cysteine against cytotoxicity induced by the psychoactive compounds N-methyl-5-(2-aminopropyl)benzofuran and 3,4-methylenedioxy-N-methamphetamine in isolated rat hepatocytes.

Science.gov (United States)

Nakagawa, Yoshio; Suzuki, Toshinari; Inomata, Akiko

2018-02-01

Psychoactive compounds, N-methyl-5-(2-aminopropyl)benzofuran (5-MAPB) and 3,4-methylenedioxy-N-methamphetamine (MDMA), are known to be hepatotoxic in humans and/or experimental animals. As previous studies suggested that these compounds elicited cytotoxicity via mitochondrial dysfunction and/or oxidative stress in rat hepatocytes, the protective effects of fructose and N-acetyl-l-cysteine (NAC) on 5-MAPB- and MDMA-induced toxicity were studied in rat hepatocytes. These drugs caused not only concentration-dependent (0-4 mm) and time-dependent (0-3 hours) cell death accompanied by the depletion of cellular levels of adenosine triphosphate (ATP) and glutathione (reduced form; GSH) but also an increase in the oxidized form of GSH. The toxic effects of 5-MAPB were greater than those of MDMA. Pretreatment of hepatocytes with either fructose at a concentration of 10 mm or NAC at a concentration of 2.5 mm prevented 5-MAPB-/MDMA-induced cytotoxicity. In addition, the exposure of hepatocytes to 5-MAPB/MDMA caused the loss of mitochondrial membrane potential, although the preventive effect of fructose was weaker than that of NAC. These results suggest that: (1) 5-MAPB-/MDMA-induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were ameliorated, at least in part, by the addition of fructose; and (3) GSH loss via oxidative stress was prevented by NAC. Taken collectively, these results indicate that the onset of toxic effects caused by 5-MAPB/MDMA may be partially attributable to cellular energy stress as well as oxidative stress. Copyright © 2017 John Wiley & Sons, Ltd.

The human CD38 monoclonal antibody daratumumab shows anti-tumor activity and hampers leukemia-microenvironment interactions in chronic lymphocytic leukemia

Science.gov (United States)

Matas-Céspedes, Alba; Vidal-Crespo, Anna; Rodriguez, Vanina; Villamor, Neus; Delgado, Julio; Giné, Eva; Roca-Ho, Heleia; Menéndez, Pablo; Campo, Elías; López-Guillermo, Armando; Colomer, Dolors; Roué, Gaël; Wiestner, Adrian; Parren, Paul W.H.I.; Doshi, Parul; van Bueren, Jeroen Lammerts; Pérez-Galán, Patricia

2016-01-01

Purpose To establish a proof-of-concept for the efficacy of the anti-CD38 antibody daratumumab in the poor prognosis CD38+ CLL subtype. Experimental design The mechanism of action of daratumumab was assessed in CLL primary cells and cell lines using peripheral blood mononuclear cells to analyze antibody-dependent cell cytotoxicity (ADCC), murine and human macrophages to study antibody-dependent cell phagocytosis (ADCP) or human serum to analyze complement-dependent cytotoxicity (CDC). The effect of daratumumab on CLL cell migration and adhesion to extracellular matrix was characterized. Daratumumab activity was validated in two in vivo models. Results Daratumumab demonstrated efficient lysis of patient-derived CLL cells and cell lines by ADCC in vitro and ADCP both in vitro and in vivo, while exhibited negligible CDC in these cells. To demonstrate the therapeutic effect of daratumumab in CLL, we generated a disseminated CLL mouse model with the CD38+ MEC2 cell line and CLL patient derived xenografts (CLL-PDX). Daratumumab significantly prolonged overall survival of MEC2 mice, completely eliminated cells from the infiltrated organs and significantly reduced disease burden in the spleen of CLL-PDX. The effect of daratumumab on patient-derived CLL cell dissemination was demonstrated in vitro by its effect on CXCL12-induced migration and in vivo by interfering with CLL cell homing to spleen in NSG mice. Daratumumab also reduced adhesion of CLL cells to VCAM-1, accompanied by down-regulation of the matrix metalloproteinase MMP9. Conclusions These unique and substantial effects of daratumumab on CLL viability and dissemination support the investigation of its use in a clinical setting of CLL. PMID:27637890

Fast intracellular dissolution and persistent cellular uptake of silver nanoparticles in CHO-K1 cells: implication for cytotoxicity

DEFF Research Database (Denmark)

Jiang, Xiumei; Miclaus, Teodora; Wang, Liming

2015-01-01

. Subsequent cytotoxicity studies show that Ag NPs decrease cell viability and increase ROS production. Pre-incubation with N-acetyl-l-cysteine, an efficient antioxidant and Ag+ chelator, diminished the cytotoxicity caused by Ag NPs or Ag+ exposure. Our study suggests that the cytotoxicity mechanism of Ag NPs...

Cellular dynamics in the draining lymph nodes during sensitization and elicitation phases of contact hypersensitivity

DEFF Research Database (Denmark)

Larsen, Jeppe Madura; Geisler, Carsten; Nielsen, Martin Weiss

2007-01-01

BACKGROUND: The different role of various immunological effector cells in contact hypersensitivity (CHS) is receiving increased attention. During the past decade, the involvement of different cell types in CHS has been investigated by the use of antibody-induced depletion of specific subtypes...... of immunological cells and by studying knockout mice lacking one or more of these immunological cell populations. OBJECTIVES: To develop a method for studying the collective cellular dynamics of immune cells in the draining lymph nodes during CHS in intact animals. PATIENTS/METHODS: Mice were sensitized and....../or challenged with 2,4-dinitrofluorobenzene or oxazolone. Using multi-parameter flow cytometry we determined the proliferation, activation state, and absolute number of helper T cells, cytotoxic T cells, B cells, and natural killer cells in the draining lymph nodes. RESULTS: The presented method can be applied...

Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model.

Science.gov (United States)

Maisel, Daniela; Birzele, Fabian; Voss, Edgar; Nopora, Adam; Bader, Sabine; Friess, Thomas; Goller, Bernhard; Laifenfeld, Daphna; Weigand, Stefan; Runza, Valeria

2016-01-01

CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages.

Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model.

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Daniela Maisel

Full Text Available CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP of the malignant cells by macrophages.

Artifacts by marker enzyme adsorption on nanomaterials in cytotoxicity assays with tissue cultures

International Nuclear Information System (INIS)

Wohlleben, Wendel; Kolle, Susanne N; Hasenkamp, Laura-Carolin; Boeser, Alexander; Vogel, Sandra; Vacano, Bernhard von; Ravenzwaay, Ben van; Landsiedel, Robert

2011-01-01

We used precision cut lung slices (PCLS) to study the cytotoxicity of cobalt ferrite nanomaterials with and without bovine serum albumin (BSA) stabilization. Using mitochondrial activity as an indicator of cytotoxicity (WST-1 assay) increasing concentrations of cobalt ferrite nanomaterial caused increasing levels of cytotoxicity in PCLS irrespective of BSA stabilization. However, there was no increase in released lactate dehydrogenase (LDH) levels caused by BSA stabilized nanomaterial indicating concentration depended cytotoxictiy. Moreover, non-stabilized nanomaterial caused a decrease of background LDH levels in the PCLS culture supernatant confirmed by complementary methods. Direct characterization of the protein corona of extracted nanomaterial shows that the LDH decrease is due to adsorption of LDH onto the surface of the non-stabilized nanomaterial, correlated with strong agglomeration. Preincubation with serum protein blocks the adsorption of LDH and stabilizes the nanomaterial at low agglomeration. We have thus demonstrated the cytotoxicity of nanomaterials in PCLS does not correlate with disrupted membrane integrity followed by LDH release. Furthermore, we found that intracellular enzymes such as the marker enzyme LDH are able to bind onto surfaces of nanomaterial and thereby adulterate the detection of toxic effects. A replacement of BSA by LDH or a secondary LDH-on-BSA-corona were not observed, confirming earlier indications that the protein corona exchange rate are slow or vanishing on inorganic nanomaterial. Thus, the method(s) to assess nanomaterial-mediated effects have to be carefully chosen based on the cellular effect and possible nano-specific artifacts.

Depolarization-stimulated 42K+ efflux in rat aorta is calcium- and cellular volume-dependent

International Nuclear Information System (INIS)

Magliola, L.; Jones, A.W.

1987-01-01

The purpose of this study was to investigate the factors controlling membrane permeability to potassium of smooth muscle cells from rat aorta stimulated by depolarization. The increase 42 K+ efflux (change in the rate constant) induced by depolarization (application of high concentrations of potassium chloride) was inhibited significantly by the calcium antagonists diltiazem and nisoldipine. Parallel inhibitory effects on contraction were observed. Diltiazem also inhibited potassium-stimulated 36 Cl- efflux. The addition of 25-150 mM KCl to normal physiologic solution stimulated 42 K+ efflux in a concentration-dependent manner. Diltiazem suppressed potassium-stimulated 42 K+ efflux approximately 90% at 25 mM KCl and approximately 40% at 150 mM KCl. The ability of nisoldipine to inhibit 42 K+ efflux also diminished as the potassium chloride concentration was elevated. The component of efflux that was resistant to calcium antagonists probably resulted from a decrease in the electrochemical gradient for potassium. Cellular water did not change during potassium addition. Substitution of 80 and 150 mM KCl for sodium chloride produced cellular swelling and enhanced potassium-stimulated 42 K+ efflux compared with potassium chloride addition. The addition of sucrose to prevent cellular swelling reduced efflux response to potassium substitution toward that of potassium addition. A hypoosmolar physiologic solution produced an increase in the 42 K+ efflux and a contracture that were both prevented by the addition of sucrose. We concluded that the depolarization-mediated 42 K+ efflux has three components: one is calcium dependent; a second is dependent on cellular volume; and a third is resistant to inhibition by calcium antagonists

Chlorpromazine inhibits tumour necrosis factor synthesis and cytotoxicity in vitro.

Science.gov (United States)

Zinetti, M; Galli, G; Demitri, M T; Fantuzzi, G; Minto, M; Ghezzi, P; Alzani, R; Cozzi, E; Fratelli, M

1995-11-01

Chlorpromazine (CPZ) has been previously shown to protect against endotoxin [lipopolysaccharide (LPS)] lethality and inhibit the release of tumour necrosis factor in vivo. We investigated at the cellular level whether this was due to direct inhibition of tumour necrosis factor-alpha (TNF-alpha) synthesis, using LPS-stimulated THP-1 human monocytic leukemia cells. We also studied the effect of CPZ on human TNF-alpha action by assessing TNF-alpha cytotoxicity on mouse fibrosarcoma L929 cells. CPZ (1-100 microM) inhibited TNF-alpha production in THP-1 cells in a dose dependent manner by a maximum of 80%. This effect was comparable to that of two well-known inhibitory drugs, dexamethasone and cyclicAMP. Inhibition was also evident at the mRNA level. On the other hand CPZ (10-25 microM) also inhibited TNF-alpha activity: in fact it reduced the cytotoxicity of TNF-alpha on L929 cells (EC50 was increased four times) and could provide protection even as a post-treatment. CPZ inhibited TNF-induced apoptosis in L929 cells, as detected by analysis of nuclear morphology. However, since we showed that apoptosis was very limited, and was not the main mode of cell death in our conditions, this could not explain the overall protection. Since CPZ did not interfere with either the oligomerization state of TNF-alpha or its receptor binding, our data suggest that it reduced cytotoxicity by inhibiting some steps in the TNF-alpha signalling pathways.

Zinc-Dependent Protection of Tobacco and Rice Cells From Aluminum-Induced Superoxide-Mediated Cytotoxicity

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Lin, Cun; Hara, Ayaka; Comparini, Diego; Bouteau, François; Kawano, Tomonori

2015-01-01

Al3+ toxicity in growing plants is considered as one of the major factors limiting the production of crops on acidic soils worldwide. In the last 15 years, it has been proposed that Al3+ toxicity are mediated with distortion of the cellular signaling mechanisms such as calcium signaling pathways, and production of cytotoxic reactive oxygen species (ROS) causing oxidative damages. On the other hand, zinc is normally present in plants at high concentrations and its deficiency is one of the most widespread micronutrient deficiencies in plants. Earlier studies suggested that lack of zinc often results in ROS-mediated oxidative damage to plant cells. Previously, inhibitory action of Zn2+ against lanthanide-induced superoxide generation in tobacco cells have been reported, suggesting that Zn2+ interferes with the cation-induced ROS production via stimulation of NADPH oxidase. In the present study, the effect of Zn2+ on Al3+-induced superoxide generation in the cell suspension cultures of tobacco (Nicotiana tabacum L., cell-line, BY-2) and rice (Oryza sativa L., cv. Nipponbare), was examined. The Zn2+-dependent inhibition of the Al3+-induced oxidative burst was observed in both model cells selected from the monocots and dicots (rice and tobacco), suggesting that this phenomenon (Al3+/Zn2+ interaction) can be preserved in higher plants. Subsequently induced cell death in tobacco cells was analyzed by lethal cell staining with Evans blue. Obtained results indicated that presence of Zn2+ at physiological concentrations can protect the cells by preventing the Al3+-induced superoxide generation and cell death. Furthermore, the regulation of the Ca2+ signaling, i.e., change in the cytosolic Ca2+ ion concentration, and the cross-talks among the elements which participate in the pathway were further explored. PMID:26648960

Characterisation of anifrolumab, a fully human anti-interferon receptor antagonist antibody for the treatment of systemic lupus erythematosus

Science.gov (United States)

Rajan, Bhargavi; Zerrouki, Kamelia; Karnell, Jodi L; Sagar, Divya; Vainshtein, Inna; Farmer, Erika; Rosenthal, Kimberly; Morehouse, Chris; de los Reyes, Melissa; Schifferli, Kevin; Liang, Meina; Sanjuan, Miguel A; Sims, Gary P; Kolbeck, Roland

2018-01-01

Objective We investigated the mechanistic and pharmacological properties of anifrolumab, a fully human, effector-null, anti-type I interferon (IFN) alpha receptor 1 (IFNAR1) monoclonal antibody in development for SLE. Methods IFNAR1 surface expression and internalisation on human monocytes before and after exposure to anifrolumab were assessed using confocal microscopy and flow cytometry. The effects of anifrolumab on type I IFN pathway activation were assessed using signal transducer and activator of transcription 1 (STAT1) phosphorylation, IFN-stimulated response element–luciferase reporter cell assays and type I IFN gene signature induction. The ability of anifrolumab to inhibit plasmacytoid dendritic cell (pDC) function and plasma cell differentiation was assessed by flow cytometry and ELISA. Effector-null properties of anifrolumab were assessed in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays with B cells. Results Anifrolumab reduced cell surface IFNAR1 by eliciting IFNAR1 internalisation. Anifrolumab blocked type I IFN-dependent STAT1 phosphorylation and IFN-dependent signalling induced by recombinant and pDC-derived type I IFNs and serum of patients with SLE. Anifrolumab suppressed type I IFN production by blocking the type I IFN autoamplification loop and inhibited proinflammatory cytokine induction and the upregulation of costimulatory molecules on stimulated pDCs. Blockade of IFNAR1 suppressed plasma cell differentiation in pDC/B cell co-cultures. Anifrolumab did not exhibit CDC or ADCC activity. Conclusions Anifrolumab potently inhibits type I IFN-dependent signalling, including the type I IFN autoamplification loop, and is a promising therapeutic for patients with SLE and other diseases that exhibit chronic dysfunctional type I IFN signalling. PMID:29644082

Cytotoxicity and Genotoxicity of Cypermethrin in Hepatocarcinoma Cells: A Dose- and Time-Dependent Study.

Science.gov (United States)

AlKahtane, Abdullah A; Alarifi, Saud; Al-Qahtani, Ahmed A; Ali, Daoud; Alomar, Suliman Y; Aleissia, Mohammed S; Alkahtani, Saad

2018-01-01

Most of the agricultural workers are potentially exposed to pesticides through different routes. Inhalation exposures may result in numerous diseases that can adversely affect an individual's health and capacity to perform at work. The aim of this study was to determine the cytotoxic potential of cypermethrin pesticide on cultured human hepatocarcinoma (HepG2) cells. The HepG2 cells were exposed to cypermethrin (0, 5, 15, 40 ng/mL) for 24 and 48 hours. We observed that cypermethrin caused cell death of HepG2 cells using 3-(4, 5-dimethylthiozolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase tests. Furthermore, cypermethrin reduced HepG2 cells viability in a time and dose dependent basis, that was probably mediated through the induction of reactive oxygen species (ROS) and apoptosis. An increase in ROS generation with a concomitant increase in expression of the proapoptotic protein Bcl-2 and cytochrome c and decrease in the antiapoptosis protein Bax suggested that a mitochondria-mediated pathway was involved in cypermethrin-induced apoptosis. These findings provide insights into the underlying mechanisms involved in cytotoxicity of cypermethrin in HepG2 cells.

Replicating Rather than Nonreplicating Adenovirus-Human Immunodeficiency Virus Recombinant Vaccines Are Better at Eliciting Potent Cellular Immunity and Priming High-Titer Antibodies

OpenAIRE

Peng, Bo; Wang, Liqun Rejean; Gómez-Román, Victor Raúl; Davis-Warren, Alberta; Montefiori, David C.; Kalyanaraman, V. S.; Venzon, David; Zhao, Jun; Kan, Elaine; Rowell, Thomas J.; Murthy, Krishna K.; Srivastava, Indresh; Barnett, Susan W.; Robert-Guroff, Marjorie

2005-01-01

A major challenge in combating the human immunodeficiency virus (HIV) epidemic is the development of vaccines capable of inducing potent, persistent cellular immunity and broadly reactive neutralizing antibody responses to HIV type 1 (HIV-1). We report here the results of a preclinical trial using the chimpanzee model to investigate a combination vaccine strategy involving sequential priming immunizations with different serotypes of adenovirus (Ad)/HIV-1MNenv/rev recombinants and boosting wit...

Shedding of Ebola Virus Surface Glycoprotein Is a Mechanism of Self-regulation of Cellular Cytotoxicity and Has a Direct Effect on Virus Infectivity.

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Dolnik, Olga; Volchkova, Valentina A; Escudero-Perez, Beatriz; Lawrence, Philip; Klenk, Hans-Dieter; Volchkov, Viktor E

2015-10-01

The surface glycoprotein (GP) is responsible for Ebola virus (EBOV) attachment and membrane fusion during virus entry. Surface expression of highly glycosylated GP causes marked cytotoxicity via masking of a wide range of cellular surface molecules, including integrins. Considerable amounts of surface GP are shed from virus-infected cells in a soluble truncated form by tumor necrosis factor α-converting enzyme. In this study, the role of GP shedding was investigated using a reverse genetics approach by comparing recombinant viruses possessing amino acid substitutions at the GP shedding site. Virus with an L635V substitution showed a substantial decrease in shedding, whereas a D637V substitution resulted in a striking increase in the release of shed GP. Variations in shedding efficacy correlated with observed differences in the amounts of shed GP in the medium, GP present in virus-infected cells, and GP present on virions. An increase in shedding appeared to be associated with a reduction in viral cytotoxicity, and, vice versa, the virus that shed less was more cytotoxic. An increase in shedding also resulted in a reduction in viral infectivity, whereas a decrease in shedding efficacy enhanced viral growth characteristics in vitro. Differences in shedding efficacy and, as a result, differences in the amount of mature GP available for incorporation into budding virions did not equate to differences in overall release of viral particles. Likewise, data suggest that the resulting differences in the amount of mature GP on the cell surface led to variations in the GP content of released particles and, as a consequence, in infectivity. In conclusion, fine-tuning of the levels of EBOV GP expressed at the surface of virus-infected cells via GP shedding plays an important role in EBOV replication by orchestrating the balance between optimal virion GP content and cytotoxicity caused by GP. © The Author 2015. Published by Oxford University Press on behalf of the Infectious

Activating Transcription Factor 3 regulates in part the enhanced tumour cell cytotoxicity of the histone deacetylase inhibitor M344 and cisplatin in combination

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St Germain Carly

2010-09-01

Full Text Available Abstract Background Activating Transcription Factor (ATF 3 is a key regulator of the cellular integrated stress response whose expression has also been correlated with pro-apoptotic activities in tumour cell models. Combination treatments with chemotherapeutic drugs, such as cisplatin, and histone deacetylase (HDAC inhibitors have been demonstrated to enhance tumour cell cytotoxicity. We recently demonstrated a role for ATF3 in regulating cisplatin-induced apoptosis and others have shown that HDAC inhibition can also induce cellular stress. In this study, we evaluated the role of ATF3 in regulating the co-operative cytotoxicity of cisplatin in combination with an HDAC inhibitor. Results The HDAC inhibitor M344 induced ATF3 expression at the protein and mRNA level in a panel of human derived cancer cell lines as determined by Western blot and quantitative RT-PCR analyses. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatments also enhanced the cytotoxic effects of cisplatin in these cancer cell lines. The mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways and dependent on ATF4, a known regulator of ATF3 expression. ATF4 heterozygote (+/- and knock out (-/- mouse embryonic fibroblast (MEF as well as chromatin immunoprecipitation (ChIP assays were utilized in determining the mechanistic induction of ATF3 by M344. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. Conclusion This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

The Influences of Cell Type and ZnO Nanoparticle Size on Immune Cell Cytotoxicity and Cytokine Induction

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Thurber Aaron

2009-01-01

Full Text Available Abstract Nanotechnology represents a new and enabling platform that promises to provide a range of innovative technologies for biological applications. ZnO nanoparticles of controlled size were synthesized, and their cytotoxicity toward different human immune cells evaluated. A differential cytotoxic response between human immune cell subsets was observed, with lymphocytes being the most resistant and monocytes being the most susceptible to ZnO nanoparticle-induced toxicity. Significant differences were also observed between previously activated memory lymphocytes and naive lymphocytes, indicating a relationship between cell-cycle potential and nanoparticle susceptibility. Mechanisms of toxicity involve the generation of reactive oxygen species, with monocytes displaying the highest levels, and the degree of cytotoxicity dependent on the extent of nanoparticle interactions with cellular membranes. An inverse relationship between nanoparticle size and cytotoxicity, as well as nanoparticle size and reactive oxygen species production was observed. In addition, ZnO nanoparticles induce the production of the proinflammatory cytokines, IFN-γ, TNF-α, and IL-12, at concentrations below those causing appreciable cell death. Collectively, these results underscore the need for careful evaluation of ZnO nanoparticle effects across a spectrum of relevant cell types when considering their use for potential new nanotechnology-based biological applications.

The impact of pretransplant donor-specific antibodies on graft outcome in renal transplantation: a six-year follow-up study

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Elias David-Neto

2012-01-01

Full Text Available OBJECTIVE: The significance of pretransplant, donor-specific antibodies on long-term patient outcomes is a subject of debate. This study evaluated the impact and the presence or absence of donor-specific antibodies after kidney transplantation on short- and long-term graft outcomes. METHODS: We analyzed the frequency and dynamics of pretransplant donor-specific antibodies following renal transplantation from a randomized trial that was conducted from 2002 to 2004 and correlated these findings with patient outcomes through 2009. Transplants were performed against a complement-dependent T- and B-negative crossmatch. Pre- and posttransplant sera were available from 94 of the 118 patients (80%. Antibodies were detected using a solid-phase (LuminexH, single-bead assay, and all tests were performed simultaneously. RESULTS: Sixteen patients exhibited pretransplant donor-specific antibodies, but only 3 of these patients (19% developed antibody-mediated rejection and 2 of them experienced early graft losses. Excluding these 2 losses, 6 of 14 patients exhibited donor-specific antibodies at the final follow-up exam, whereas 8 of these patients (57% exhibited complete clearance of the donor-specific antibodies. Five other patients developed ''de novo'' posttransplant donor-specific antibodies. Death-censored graft survival was similar in patients with pretransplant donor-specific and non-donor-specific antibodies after a mean follow-up period of 70 months. CONCLUSION: Pretransplant donor-specific antibodies with a negative complement-dependent cytotoxicity crossmatch are associated with a risk for the development of antibody-mediated rejection, although survival rates are similar when patients transpose the first months after receiving the graft. Our data also suggest that early posttransplant donor-specific antibody monitoring should increase knowledge of antibody dynamics and their impact on long-term graft outcome.

Discovery Of Human Antibodies Against Spitting Cobra Toxins

DEFF Research Database (Denmark)

Bojsen-Møller, Laura; Lohse, Brian; Harrison, Robert

Current snakebite envenoming treatment options consist of animal-derived antisera and are associated with severe adverse reactions due to the heterologous nature of the animal-derived antibodies present in these antisera, and the presence of therapeutically irrelevant antibodies. The African...... spitting cobras are among the most medically important snakes in sub-Saharan regions due to the severity of the clinical outcomes caused by their cytotoxic venom, which is derived from cytotoxins of the 3FTx toxin family and PLA2. Here we report the results of our progress in identifying human antibodies...... targeting relevant toxins from the venom of the black necked spitting cobra (Naja nigricolis)....

Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis.

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Yu, Xiaowen; Yang, Yu-Ping; Dikici, Emre; Deo, Sapna K; Daunert, Sylvia

2017-06-12

The emergence of novel binding proteins or antibody mimetics capable of binding to ligand analytes in a manner analogous to that of the antigen-antibody interaction has spurred increased interest in the biotechnology and bioanalytical communities. The goal is to produce antibody mimetics designed to outperform antibodies with regard to binding affinities, cellular and tumor penetration, large-scale production, and temperature and pH stability. The generation of antibody mimetics with tailored characteristics involves the identification of a naturally occurring protein scaffold as a template that binds to a desired ligand. This scaffold is then engineered to create a superior binder by first creating a library that is then subjected to a series of selection steps. Antibody mimetics have been successfully used in the development of binding assays for the detection of analytes in biological samples, as well as in separation methods, cancer therapy, targeted drug delivery, and in vivo imaging. This review describes recent advances in the field of antibody mimetics and their applications in bioanalytical chemistry, specifically in diagnostics and other analytical methods.

Mathematical modeling of antibody drug conjugates with the target and tubulin dynamics to predict AUC.

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Byun, Jong Hyuk; Jung, Il Hyo

2018-04-14

Antibody drug conjugates (ADCs)are one of the most recently developed chemotherapeutics to treat some types of tumor cells. They consist of monoclonal antibodies (mAbs), linkers, and potent cytotoxic drugs. Unlike common chemotherapies, ADCs combine selectively with a target at the surface of the tumor cell, and a potent cytotoxic drug (payload) effectively prevents microtubule polymerization. In this work, we construct an ADC model that considers both the target of antibodies and the receptor (tubulin) of the cytotoxic payloads. The model is simulated with brentuximab vedotin, one of ADCs, and used to investigate the pharmacokinetic (PK) characteristics of ADCs in vivo. It also predicts area under the curve (AUC) of ADCs and the payloads by identifying the half-life. The results show that dynamical behaviors fairly coincide with the observed data and half-life and capture AUC. Thus, the model can be used for estimating some parameters, fitting experimental observations, predicting AUC, and exploring various dynamical behaviors of the target and the receptor. Copyright © 2018 Elsevier Ltd. All rights reserved.

Luminescent cyclometalated iridium(III) polypyridine indole complexes--synthesis, photophysics, electrochemistry, protein-binding properties, cytotoxicity, and cellular uptake.

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Lau, Jason Shing-Yip; Lee, Pui-Kei; Tsang, Keith Hing-Kit; Ng, Cyrus Ho-Cheong; Lam, Yun-Wah; Cheng, Shuk-Han; Lo, Kenneth Kam-Wing

2009-01-19

A series of luminescent cyclometalated iridium(III) polypyridine indole complexes, [Ir(N--C)(2)(N--N)](PF(6)) (HN--C = 2-phenylpyridine (Hppy), N--N = 4-((2-(indol-3-yl)ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-ind) (1a), N--N = 4-((5-((2-(indol-3-yl)ethyl)aminocarbonyl)pentyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-C6-ind) (1b); HN--C = 7,8-benzoquinoline (Hbzq), N--N = bpy-ind (2a), N--N = bpy-C6-ind (2b); and HN--C = 2-phenylquinoline (Hpq), N--N = bpy-ind (3a), N--N = bpy-C6-ind (3b)), have been synthesized, characterized, and their photophysical and electrochemical properties and lipophilicity investigated. Photoexcitation of the complexes in fluid solutions at 298 K and in alcohol glass at 77 K resulted in intense and long-lived luminescence (lambda(em) = 540-616 nm, tau(o) = 0.13-5.15 mus). The emission of the complexes has been assigned to a triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi(Ir) --> pi*(N--N)) excited state, probably with some mixing of triplet intraligand ((3)IL) (pi --> pi*) (pq) character for complexes 3a,b. Electrochemical measurements revealed that all the complexes showed an irreversible indole oxidation wave at ca. +1.1 V versus SCE, a quasi-reversible iridium(IV/III) couple at ca. +1.3 V, and a reversible diimine reduction couple at ca. -1.3 V. The interactions of these complexes with an indole-binding protein, bovine serum albumin (BSA), have been studied by emission titrations, and the K(a) values are on the order of 10(4) M(-1). Additionally, the cytotoxicity of the complexes toward human cervix epithelioid carcinoma (HeLa) cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) values of the complexes ranged from 1.1 to 6.3 microM, which are significantly smaller than that of cisplatin (30.7 microM) under the same experimental conditions. Furthermore, the cellular uptake of the complexes has been investigated by flow cytometry and laser

Ca2+-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

International Nuclear Information System (INIS)

Stenovec, Matjaz; Kreft, Marko; Grilc, Sonja; Potokar, Maja; Kreft, Mateja Erdani; Pangrsic, Tina; Zorec, Robert

2007-01-01

Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca 2+ -dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca 2+ level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca 2+ -triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles

Interactions between ibrutinib and anti-CD20 antibodies; competing effects on the outcome of combination therapy

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Skarzynski, Martin; Niemann, Carsten U; Lee, Yuh Shan; Martyr, Sabrina; Maric, Irina; Salem, Dalia; Stetler-Stevenson, Maryalice; Marti, Gerald E; Calvo, Katherine R; Yuan, Constance; Valdez, Janet; Soto, Susan; Farooqui, Mohammed Z.H.; Herman, Sarah E.M.; Wiestner, Adrian

2015-01-01

Purpose Clinical trials of ibrutinib combined with anti-CD20 monoclonal antibodies (mAbs) for chronic lymphocytic leukemia (CLL) report encouraging results. Paradoxically, in pre-clinical studies in vitro ibrutinib was reported to decrease CD20 expression and inhibits cellular effector mechanisms. We therefore set out to investigate effects of in vivo ibrutinib treatment that could explain this paradox. Experimental Design Patients received single agent ibrutinib (420mg daily) on an investigator-initiated phase 2 trial. Serial blood samples were collected pre-treatment and during treatment for ex vivo functional assays to examine the effects on CLL cell susceptibility to anti-CD20 mAbs. Results We demonstrate that CD20 expression on ibrutinib was rapidly and persistently down-regulated (median reduction 74%, day 28, Pibrutinib were less susceptible to anti-CD20 mAb-mediated complement-dependent cytotoxicity than pre-treatment cells (median reduction 75%, Pibrutinib, providing a likely mechanism for the preserved C3d opsonization. Additionally, ibrutinib significantly inhibited trogocytosis, a major contributor to antigen loss and tumor escape during mAb therapy. Conclusions Our data indicate that ibrutinib promotes both positive and negative interactions with anti-CD20 mAbs, suggesting that successfully harnessing maximal anti-tumor effects of such combinations requires further investigation. PMID:26283682

Interactions between Ibrutinib and Anti-CD20 Antibodies: Competing Effects on the Outcome of Combination Therapy.

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Skarzynski, Martin; Niemann, Carsten U; Lee, Yuh Shan; Martyr, Sabrina; Maric, Irina; Salem, Dalia; Stetler-Stevenson, Maryalice; Marti, Gerald E; Calvo, Katherine R; Yuan, Constance; Valdez, Janet; Soto, Susan; Farooqui, Mohammed Z H; Herman, Sarah E M; Wiestner, Adrian

2016-01-01

Clinical trials of ibrutinib combined with anti-CD20 monoclonal antibodies (mAb) for chronic lymphocytic leukemia (CLL) report encouraging results. Paradoxically, in preclinical studies, in vitro ibrutinib was reported to decrease CD20 expression and inhibit cellular effector mechanisms. We therefore set out to investigate effects of in vivo ibrutinib treatment that could explain this paradox. Patients received single-agent ibrutinib (420 mg daily) on an investigator-initiated phase II trial. Serial blood samples were collected pretreatment and during treatment for ex vivo functional assays to examine the effects on CLL cell susceptibility to anti-CD20 mAbs. We demonstrate that CD20 expression on ibrutinib was rapidly and persistently downregulated (median reduction 74%, day 28, P ibrutinib were less susceptible to anti-CD20 mAb-mediated complement-dependent cytotoxicity than pretreatment cells (median reduction 75%, P ibrutinib, providing a likely mechanism for the preserved C3d opsonization. In addition, ibrutinib significantly inhibited trogocytosis, a major contributor to antigen loss and tumor escape during mAb therapy. Our data indicate that ibrutinib promotes both positive and negative interactions with anti-CD20 mAbs, suggesting that successfully harnessing maximal antitumor effects of such combinations requires further investigation. ©2015 American Association for Cancer Research.

Identification of stable cytotoxic factors in the gas phase extract of cigarette smoke and pharmacological characterization of their cytotoxicity.

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Noya, Yoichi; Seki, Koh-Ichi; Asano, Hiroshi; Mai, Yosuke; Horinouchi, Takahiro; Higashi, Tsunehito; Terada, Koji; Hatate, Chizuru; Hoshi, Akimasa; Nepal, Prabha; Horiguchi, Mika; Kuge, Yuji; Miwa, Soichi

2013-12-06

Smoking is a major risk factor for atherosclerotic vascular diseases, but the mechanism for its genesis is unknown. We have recently shown that the gas phase of cigarette smoke (nicotine- and tar-free cigarette smoke extract; CSE) likely to reach the systemic circulation contains stable substances which cause cytotoxicity like plasma membrane damage and cell death in cultured cells, and also that the plasma membrane damage is caused through sequential activation of protein kinase C (PKC) and NADPH oxidase (NOX) and the resulting generation of reactive oxygen species (PKC/NOX-dependent mechanism), whereas cell death is caused through PKC/NOX-dependent and -independent mechanisms. To identify these stable substances, the CSE was prepared by passing the main-stream smoke of 10 cigarettes through a Cambridge glass fiber filter, trapping of the smoke in a vessel cooled at -80°C, and subsequent dissolution in 10ml of water. The CSE was fractionated into nine fractions using reversed-phase HPLC, and each fraction was screened for cytotoxicity in cultured cells, using propidium iodide uptake assay for cell membrane damage and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction assay for cell viability. The cytotoxicity was positive in two of the nine fractions (Fr2 and Fr5). After extraction of the active fractions into dichloromethane, GC/MS analysis identified 2-cyclopenten-1-one (CPO) in Fr5 but none in Fr2. After derivatization of the active fractions with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride, GC/MS analysis identified acrolein, acetone and propionaldehyde in Fr2, and methyl vinyl ketone (MVK) in Fr5. After 4-h incubation, authentic acrolein and MVK induced concentration-dependent cytotoxicity with EC50 values of 75.9±8.2 and 47.0±8.0μM (mean±SEM; n=3), respectively, whereas acetone, propionaldehyde and CPO were without effect. However, after 24-h incubation, CPO induced concentration-dependent

Considerations for the nonclinical safety evaluation of antibody drug conjugates for oncology.

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Roberts, Stanley A; Andrews, Paul A; Blanset, Diann; Flagella, Kelly M; Gorovits, Boris; Lynch, Carmel M; Martin, Pauline L; Kramer-Stickland, Kimberly; Thibault, Stephane; Warner, Garvin

2013-12-01

Antibody drug conjugates (ADCs) include monoclonal antibodies that are linked to cytotoxic small molecules. A number of these agents are currently being developed as anti-cancer agents designed to improve the therapeutic index of the cytotoxin (i.e., cytotoxic small molecule or cytotoxic agent) by specifically delivering it to tumor cells. This paper presents primary considerations for the nonclinical safety evaluation of ADCs and includes strategies for the evaluation of the entire ADC or the various individual components (i.e., antibody, linker or the cytotoxin). Considerations are presented on how to design a nonclinical safety assessment program to identify the on- and off-target toxicities to enable first-in-human (FIH) studies. Specific discussions are also included that provide details as to the need and how to conduct the studies for evaluating ADCs in genetic toxicology, tissue cross-reactivity, safety pharmacology, carcinogenicity, developmental and reproductive toxicology, biotransformation, toxicokinetic monitoring, bioanalytical assays, immunogenicity testing, test article stability and the selection of the FIH dose. Given the complexity of these molecules and our evolving understanding of their properties, there is no single all-encompassing nonclinical strategy. Instead, each ADC should be evaluated on a case-by-case scientifically-based approach that is consistent with ICH and animal research guidelines. Copyright © 2013 Elsevier Inc. All rights reserved.

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid.

Science.gov (United States)

Feuser, Paulo Emilio; Arévalo, Juan Marcelo Carpio; Junior, Enio Lima; Rossi, Gustavo Rodrigues; da Silva Trindade, Edvaldo; Rocha, Maria Eliane Merlin; Jacques, Amanda Virtuoso; Ricci-Júnior, Eduardo; Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H Hermes

2016-12-01

Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Results showed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of the polymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0-1 h) followed by a slow and sustained release, indicating a biphasic release system. Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggests that this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.

Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

International Nuclear Information System (INIS)

Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

1988-01-01

A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125 I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10 -8 to 1 x 10 -6 M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca 2+ , Mg 2+ , or Mn 2+ partially inhibited binding of 125 I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved γ-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins

Treatment of a solid tumor using engineered drug-resistant immunocompetent cells and cytotoxic chemotherapy.

Science.gov (United States)

Dasgupta, Anindya; Shields, Jordan E; Spencer, H Trent

2012-07-01

Multimodal therapy approaches, such as combining chemotherapy agents with cellular immunotherapy, suffers from potential drug-mediated toxicity to immune effector cells. Overcoming such toxic effects of anticancer cellular products is a potential critical barrier to the development of combined therapeutic approaches. We are evaluating an anticancer strategy that focuses on overcoming such a barrier by genetically engineering drug-resistant variants of immunocompetent cells, thereby allowing for the coadministration of cellular therapy with cytotoxic chemotherapy, a method we refer to as drug-resistant immunotherapy (DRI). The strategy relies on the use of cDNA sequences that confer drug resistance and recombinant lentiviral vectors to transfer nucleic acid sequences into immunocompetent cells. In the present study, we evaluated a DRI-based strategy that incorporates the immunocompetent cell line NK-92, which has intrinsic antitumor properties, genetically engineered to be resistant to both temozolomide and trimetrexate. These immune effector cells efficiently lysed neuroblastoma cell lines, which we show are also sensitive to both chemotherapy agents. The antitumor efficacy of the DRI strategy was demonstrated in vivo, whereby neuroblastoma-bearing NOD/SCID/γ-chain knockout (NSG) mice treated with dual drug-resistant NK-92 cell therapy followed by dual cytotoxic chemotherapy showed tumor regression and significantly enhanced survival compared with animals receiving either nonengineered cell-based therapy and chemotherapy, immunotherapy alone, or chemotherapy alone. These data show there is a benefit to using drug-resistant cellular therapy when combined with cytotoxic chemotherapy approaches.

Anti·red cell activity of lymphocytotoxic antibodies: and in vitro and in ...

African Journals Online (AJOL)

The need to obtain non-toxic antilymphocyre sera (ALS) led to the in vitro and in vivo evaluarion of its crossreactivity for red cells. The findings showed thar the antibodies coating the erythrocytes in vitro are idenrical with the antibodies that sensitize lymphocytes by the cytotoxicity rest. It would appear that rhe observed ...

Dose-Dependent Cytotoxic Effects of Boldine in HepG-2 Cells—Telomerase Inhibition and Apoptosis Induction

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Sakineh Kazemi Noureini

2015-02-01

Full Text Available Plant metabolites are valuable sources of novel therapeutic compounds. In an anti-telomerase screening study of plant secondary metabolites, the aporphine alkaloid boldine (1,10-dimethoxy-2,9-dihydroxyaporphine exhibited a dose and time dependent cytotoxicity against hepatocarcinoma HepG-2 cells. Here we focus on the modes and mechanisms of the growth-limiting effects of this compound. Telomerase activity and expression level of some related genes were estimated by real-time PCR. Modes of cell death also were examined by microscopic inspection, staining methods and by evaluating the expression level of some critically relevant genes. The growth inhibition was correlated with down-regulation of the catalytic subunit of telomerase (hTERT gene (p < 0.01 and the corresponding reduction of telomerase activity in sub-cytotoxic concentrations of boldine (p < 0.002. However, various modes of cell death were stimulated, depending on the concentration of boldine. Very low concentrations of boldine over a few passages resulted in an accumulation of senescent cells so that HepG-2 cells lost their immortality. Moreover, boldine induced apoptosis concomitantly with increasing the expression of bax/bcl2 (p < 0.02 and p21 (p < 0.01 genes. Boldine might thus be an interesting candidate as a potential natural compound that suppresses telomerase activity in non-toxic concentrations.

Radioimmunoimaging of experimental gliomas using radiolabelled monoclonal antibodies

International Nuclear Information System (INIS)

Glaessner, H.

1986-01-01

The biodistribution and tumour uptake of radiolabelled (131 I) glioma-seeking monoclonal antibodies (14 AC1) and their F(ab') 2 fragments were investigated in nude mice having received glioma transplants. Radioimmunoimaging by external scintigraphy at 48 and 96 hours pointed to a superior tumour localisation by the fragments that was clearly related to the dose. Wholebody determinations of the biokinetic behaviour led to the following results: Faster clearance anc more ready elimination from the blood pool for the fragments, preferential uptake in the tumour; intact antibodies; binding in the liver, spleen and lungs. The study confirmed the value of fragments of monoclonal antibodies in the diagnosis of tumours and pointed to the possibility of using intact monoclonal antibodies as carriers of radioisotopes and cytotoxic drugs within the scope of therapeutic programmes. (TRV) [de

A cytotoxic serine proteinase isolated from mouse submandibular gland.

Science.gov (United States)

Shimamura, T; Nagumo, N; Ikigai, H; Murakami, K; Okubo, S; Toda, M; Ohnishi, R; Tomita, M

1989-08-01

We have isolated a novel cytotoxic factor from the submandibular glands of male BALB/c mice by Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. The cytotoxic factor is a serine proteinase, which belongs to the mouse glandular kallikrein (mGK) family, with an Mr of approximately 27,000. The purified serine proteinase showed cytotoxic activity against mouse thymocytes in a dose-dependent manner, and a serine proteinase inhibitor, diisopropyl fluorophosphate, blocked its cytotoxic activity.

Immune Antibody Libraries: Manipulating The Diverse Immune Repertoire for Antibody Discovery.

Science.gov (United States)

Lim, Theam Soon; Chan, Soo Khim

2016-01-01

Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries. Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection. This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases. We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Submicron and nano formulations of titanium dioxide and zinc oxide stimulate unique cellular toxicological responses in the green microalga Chlamydomonas reinhardtii

Energy Technology Data Exchange (ETDEWEB)

Gunawan, Cindy, E-mail: c.gunawan@unsw.edu.au [ARC Centre of Excellence for Functional Nanomaterials, School of Chemical Engineering, The University of New South Wales, Sydney, NSW (Australia); Sirimanoonphan, Aunchisa [ARC Centre of Excellence for Functional Nanomaterials, School of Chemical Engineering, The University of New South Wales, Sydney, NSW (Australia); Teoh, Wey Yang [Clean Energy and Nanotechnology (CLEAN) Laboratory, School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Region (Hong Kong); Marquis, Christopher P., E-mail: c.marquis@unsw.edu.au [School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW (Australia); Amal, Rose [ARC Centre of Excellence for Functional Nanomaterials, School of Chemical Engineering, The University of New South Wales, Sydney, NSW (Australia)

2013-09-15

Highlights: • Uptake of TiO{sub 2} solids by C. reinhardtii generates ROS as an early stress response. • Submicron and nanoTiO{sub 2} exhibit benign effect on cell proliferation. • Uptake of ZnO solids and leached zinc by C. reinhardtii inhibit the alga growth. • No cellular oxidative stress is detected with submicron and nano ZnO exposure. • The toxicity of particles is not necessarily mediated by cellular oxidative stress. -- Abstract: The work investigates the eco-cytoxicity of submicron and nano TiO{sub 2} and ZnO, arising from the unique interactions of freshwater microalga Chlamydomonas reinhardtii to soluble and undissolved components of the metal oxides. In a freshwater medium, submicron and nano TiO{sub 2} exist as suspended aggregates with no-observable leaching. Submicron and nano ZnO undergo comparable concentration-dependent fractional leaching, and exist as dissolved zinc and aggregates of undissolved ZnO. Cellular internalisation of solid TiO{sub 2} stimulates cellular ROS generation as an early stress response. The cellular redox imbalance was observed for both submicron and nano TiO{sub 2} exposure, despite exhibiting benign effects on the alga proliferation (8-day EC50 > 100 mg TiO{sub 2}/L). Parallel exposure of C. reinhardtii to submicron and nano ZnO saw cellular uptake of both the leached zinc and solid ZnO and resulting in inhibition of the alga growth (8-day EC50 ≥ 0.01 mg ZnO/L). Despite the sensitivity, no zinc-induced cellular ROS generation was detected, even at 100 mg ZnO/L exposure. Taken together, the observations confront the generally accepted paradigm of cellular oxidative stress-mediated cytotoxicity of particles. The knowledge of speciation of particles and the corresponding stimulation of unique cellular responses and cytotoxicity is vital for assessment of the environmental implications of these materials.

Submicron and nano formulations of titanium dioxide and zinc oxide stimulate unique cellular toxicological responses in the green microalga Chlamydomonas reinhardtii

International Nuclear Information System (INIS)

Gunawan, Cindy; Sirimanoonphan, Aunchisa; Teoh, Wey Yang; Marquis, Christopher P.; Amal, Rose

2013-01-01

Highlights: • Uptake of TiO 2 solids by C. reinhardtii generates ROS as an early stress response. • Submicron and nanoTiO 2 exhibit benign effect on cell proliferation. • Uptake of ZnO solids and leached zinc by C. reinhardtii inhibit the alga growth. • No cellular oxidative stress is detected with submicron and nano ZnO exposure. • The toxicity of particles is not necessarily mediated by cellular oxidative stress. -- Abstract: The work investigates the eco-cytoxicity of submicron and nano TiO 2 and ZnO, arising from the unique interactions of freshwater microalga Chlamydomonas reinhardtii to soluble and undissolved components of the metal oxides. In a freshwater medium, submicron and nano TiO 2 exist as suspended aggregates with no-observable leaching. Submicron and nano ZnO undergo comparable concentration-dependent fractional leaching, and exist as dissolved zinc and aggregates of undissolved ZnO. Cellular internalisation of solid TiO 2 stimulates cellular ROS generation as an early stress response. The cellular redox imbalance was observed for both submicron and nano TiO 2 exposure, despite exhibiting benign effects on the alga proliferation (8-day EC50 > 100 mg TiO 2 /L). Parallel exposure of C. reinhardtii to submicron and nano ZnO saw cellular uptake of both the leached zinc and solid ZnO and resulting in inhibition of the alga growth (8-day EC50 ≥ 0.01 mg ZnO/L). Despite the sensitivity, no zinc-induced cellular ROS generation was detected, even at 100 mg ZnO/L exposure. Taken together, the observations confront the generally accepted paradigm of cellular oxidative stress-mediated cytotoxicity of particles. The knowledge of speciation of particles and the corresponding stimulation of unique cellular responses and cytotoxicity is vital for assessment of the environmental implications of these materials

B7h-expressing dendritic cells and plasma B cells mediate distinct outcomes of ICOS costimulation in T cell-dependent antibody responses

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Larimore Kevin

2012-06-01

Full Text Available Abstract Background The ICOS-B7h costimulatory receptor-ligand pair is required for germinal center formation, the production of isotype-switched antibodies, and antibody affinity maturation in response to T cell-dependent antigens. However, the potentially distinct roles of regulated B7h expression on B cells and dendritic cells in T cell-dependent antibody responses have not been defined. Results We generated transgenic mice with lineage-restricted B7h expression to assess the cell-type specific roles of B7h expression on B cells and dendritic cells in regulating T cell-dependent antibody responses. Our results show that endogenous B7h expression is reduced on B cells after activation in vitro and is also reduced in vivo on antibody-secreting plasma B cells in comparison to both naïve and germinal center B cells from which they are derived. Increasing the level of B7h expression on activated and plasma B cells in B-B7hTg mice led to an increase in the number of antibody-secreting plasma cells generated after immunization and a corresponding increase in the concentration of antigen-specific high affinity serum IgG antibodies of all isotypes, without affecting the number of responding germinal center B cells. In contrast, ICOS costimulation mediated by dendritic cells in DC-B7hTg mice contributed to germinal center formation and selectively increased IgG2a production without affecting the overall magnitude of antibody responses. Conclusions Using transgenic mice with lineage-restricted B7h expression, we have revealed distinct roles of ICOS costimulation mediated by dendritic cells and B cells in the regulation of T cell-dependent antibody responses.

Anticancer agent CHS-828 inhibits cellular synthesis of NAD

DEFF Research Database (Denmark)

Olesen, U.H.; Christensen, M.K.; Bjorkling, F.

2008-01-01

Malignant cells display increased demands for energy production and DNA repair. Nicotinamide adenine dinucleotide (NAD) is required for both processes and is also continuously degraded by cellular enzymes. Nicotinamide phosphoribosyltransferase (Nampt) is a crucial factor in the resynthesis of NAD......, and thus in cancer cell survival. Here, we establish the cytotoxic mechanism of action of the small molecule inhibitor CHS-828 to result from impaired synthesis of NAD. Initially, we detected cross-resistance in cells between CHS-828 and a known inhibitor of Nampt, FK866, a compound of a structurally...... different class. We then showed that nicotinamide protects against CHS-828-mediated cytotoxicity. Finally, we observed that treatment with CHS-828 depletes cellular NAD levels in sensitive cancer cells. In conclusion, these results strongly suggest that, like FK866, CHS-828 kills cancer cells by depleting...

Analysis of cytotoxic effects of nickel on human blood lymphocytes.

Science.gov (United States)

Zarei, Mohammad Hadi; Hosseini Shirazi, Seyed Farshad; Aghvami, Marjan; Salimi, Ahmad; Pourahmad, Jalal

2018-02-01

Nickel compounds possess many applications in different industrial processes. Human beings are exposed to nickel commonly through occupational exposure and food. Although a few studies so far have investigated the effects of nickel compounds on human lymphocytes, the complete mechanism of cytotoxicity of this metal on human lymphocytes is yet to be determined. The intention of this paper was to determine the cytotoxicity mechanism of water soluble NiCl 2 toward human lymphocytes using the accelerated cytotoxicity mechanisms screening (ACMS) technique. Human lymphocytes were isolated from the blood of healthy subjects based on Ficoll-Paque PLUS standard method. For the assessment of cell viability, lymphocytes were incubated with 0.05-1 mM NiCl 2 for 12 h. Determination of mechanistic parameters was performed 2, 4 and 6 h after treatment of cells with ½ EC50 12h , EC50 12h and 2EC50 12h of NiCl 2 . Our results demonstrate that cytotoxicity of NiCl 2 on human lymphocytes is associated with increased ROS formation, mitochondrial membrane potential collapse, glutathione depletion, lysosomal membrane damage, cellular proteolysis and activation of caspase-3 before cytotoxicity ensued.

Radiolabelled cellular blood elements

International Nuclear Information System (INIS)

Sinzinger, H.

1990-01-01

This book reports on radiolabelled cellular blood elements, covering new advances made during the past several years, in particular the use of Tc-99 as a tracer for blood elements. Coverage extends to several radiolabelled monoclonal antibodies that are specific for blood components and may label blood elements in vivo

Cytotoxic effects of psychotropic benzofuran derivatives, N-methyl-5-(2-aminopropyl)benzofuran and its N-demethylated derivative, on isolated rat hepatocytes.

Science.gov (United States)

Nakagawa, Yoshio; Suzuki, Toshinari; Tada, Yukie; Inomata, Akiko

2017-03-01

The novel psychoactive compounds derived from amphetamine have been illegally abused as recreational drugs, some of which are known to be hepatotoxic in humans and experimental animals. The cytotoxic effects and mechanisms of 5-(2-aminopropyl)benzofuran (5-APB) and N-methyl-5-(2-aminopropyl)benzofuran (5-MAPB), both of which are benzofuran analogues of amphetamine, and 3,4-methylenedioxy-N-methamphetamine (MDMA) were studied in freshly isolated rat hepatocytes. 5-MAPB caused not only concentration-dependent (0-4.0 mm) and time-dependent (0-3 h) cell death accompanied by the depletion of cellular ATP and reduced glutathione and protein thiol levels, but also accumulation of oxidized glutathione. Of the other analogues examined at a concentration of 4 mm, 5-MAPB/5-APB-induced cytotoxicity with the production of reactive oxygen species and loss of mitochondrial membrane potential was greater than that induced by MDMA. In isolated rat liver mitochondria, the benzofurans resulted in a greater increase in the rate of state 4 oxygen consumption than did MDMA, with a decrease in the rate of state 3 oxygen consumption. Furthermore, the benzofurans caused more of a rapid mitochondrial swelling dependent on the mitochondrial permeability transition than MDMA. 5-MAPB at a weakly toxic level (1 mm) was metabolized slowly: levels of 5-MAPB and 5-APB were approximately 0.9 mm and 50 μm, respectively, after 3 h incubation. Taken collectively, these results indicate that mitochondria are target organelles for the benzofuran analogues and MDMA, which elicit cytotoxicity through mitochondrial failure, and the onset of cytotoxicity may depend on the initial and/or residual concentrations of 5-MAPB rather than on those of its metabolite 5-APB. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Development of ELISA-detected anti-HLA antibodies precedes the development of bronchiolitis obliterans syndrome and correlates with progressive decline in pulmonary function after lung transplantation.

Science.gov (United States)

Jaramillo, A; Smith, M A; Phelan, D; Sundaresan, S; Trulock, E P; Lynch, J P; Cooper, J D; Patterson, G A; Mohanakumar, T

1999-04-27

Development of anti-HLA antibodies after lung transplantation (LT) is thought to play an important role in the etiology of bronchiolitis obliterans syndrome (BOS). However, a cause-effect relationship between anti-HLA antibodies and BOS has not been established. This study was conducted to determine the temporal relationship between the development of anti-HLA antibodies and BOS after LT, and to determine the antigenic specificity of the antibodies developed in BOS patients. Sera from 15 BOS+ LT patients and 12 BOS- LT patients were obtained before LT and collected again at 6, 12, 24, 36, and 48 months after LT. Anti-HLA antibodies were detected by the PRA-STAT ELISA system and by complement-dependent cytotoxicity assays. Anti-HLA reactivity was further characterized by flow cytometry and absorption/elution with human platelets. When analyzed by ELISA, 10 of 15 BOS+ patients developed anti-HLA antibodies, whereas 0 of 12 BOS- patients developed anti-HLA antibodies (PELISA after LT can provide an early identification of an important subset of LT patients with an increased risk of developing BOS.

A malaria vaccine that elicits in humans antibodies able to kill Plasmodium falciparum.

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Pierre Druilhe

2005-11-01

Full Text Available Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant.Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum-infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation.This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory.

Selective lymphoid irradiation: III. Prolongation of cardiac xenografts and allografts in presensitized rats

International Nuclear Information System (INIS)

Hardy, M.A.; Oluwole, S.; Fawwaz, R.; Satake, K.; Nowygrod, R.; Reemtsma, K.

1982-01-01

Selective lymphoid irradiation (SLI) with palladium-109-hematoporphyrin (Pd-H) combined with antilymphocyte globulin (ALG) induces either donor-specific permanent rat heart allograft acceptance or significant allograft prolongation depending on the degree of donor-recipient matching. The purpose of this study was to determine if SLI combined with ALG can affect ACI heart allograft survival in Lewis recipients presensitized to ACI, and of hamster heart xenografts of Lewis rats. SLI combined with ALG delays allograft and xenograft rejection in the presence of induced or preformed antidonor antibodies, and converts primarily a humoral rejection into a cellular rejection by mechanisms as yet uncertain. Such peritransplant treatment had significant effect on the levels of antidonor complement-dependent cytotoxic antibody titers but did not correlate directly with graft survival. Histological analysis of rejected hearts in all groups demonstrated primarily a humoral hyperacute rejection in control animals and in recipients treated with ALG alone, while peritransplant treatment with Pd-H and ALG resulted not only in prolonged graft survival but histologically, primarily a cellular rejection of the graft

New-onset mediastinal and central nervous system sarcoidosis in a patient with metastatic melanoma undergoing CTLA4 monoclonal antibody treatment.

LENUS (Irish Health Repository)

Murphy, Kevin P

2014-01-01

Ipilimumab, a cytotoxic monoclonal antibody that inhibits cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), has been established as an effective therapy in the management of advanced melanoma. Immune-mediated adverse events are a common side effect.

Cytotoxicity and Effects on Cell Viability of Nickel Nanowires

KAUST Repository

Rodriguez, Jose E.

2013-05-01

Recently, magnetic nanoparticles are finding an increased use in biomedical applications and research. Nanobeads are widely used for cell separation, biosensing and cancer therapy, among others. Due to their properties, nanowires (NWs) are gaining ground for similar applications and, as with all biomaterials, their cytotoxicity is an important factor to be considered before conducting biological studies with them. In this work, the cytotoxic effects of nickel NWs (Ni NWs) were investigated in terms of cell viability and damage to the cellular membrane. Ni NWs with an average diameter of 30-34 nm were prepared by electrodeposition in nanoporous alumina templates. The templates were obtained by a two-step anodization process with oxalic acid on an aluminum substrate. Characterization of NWs was done using X-Ray diffraction (XRD) and energy dispersive X-Ray analysis (EDAX), whereas their morphology was observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell viability studies were carried out on human colorectal carcinoma cells HCT 116 by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell proliferation colorimetric assay, whereas the lactate dehydrogenase (LDH) homogenous membrane fluorimetric assay was used to measure the degree of cell membrane rupture. The density of cell seeding was calculated to obtain a specific cell number and confluency before treatment with NWs. Optical readings of the cell-reduced MTT products were measured at 570 nm, whereas fluorescent LDH membrane leakage was recorded with an excitation wavelength of 525 nm and an emission wavelength of 580 - 640 nm. The effects of NW length, cell exposure time, as well as NW:cell ratio, were evaluated through both cytotoxic assays. The results show that cell viability due to Ni NWs is affected depending on both exposure time and NW number. On the other hand, membrane rupture and leakage was only significant at later exposure times. Both

Pichia pastoris-Expressed Bivalent Virus-Like Particulate Vaccine Induces Domain III-Focused Bivalent Neutralizing Antibodies without Antibody-Dependent Enhancement in Vivo

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Rahul Shukla

2018-01-01

Full Text Available Dengue, a significant public health problem in several countries around the world, is caused by four different serotypes of mosquito-borne dengue viruses (DENV-1, -2, -3, and -4. Antibodies to any one DENV serotype which can protect against homotypic re-infection, do not offer heterotypic cross-protection. In fact, cross-reactive antibodies may augment heterotypic DENV infection through antibody-dependent enhancement (ADE. A recently launched live attenuated vaccine (LAV for dengue, which consists of a mixture of four chimeric yellow-fever/dengue vaccine viruses, may be linked to the induction of disease-enhancing antibodies. This is likely related to viral interference among the replicating viral strains, resulting in an unbalanced immune response, as well as to the fact that the LAV encodes prM, a DENV protein documented to elicit ADE-mediating antibodies. This makes it imperative to explore the feasibility of alternate ADE risk-free vaccine candidates. Our quest for a non-replicating vaccine centered on the DENV envelope (E protein which mediates virus entry into the host cell and serves as an important target of the immune response. Serotype-specific neutralizing epitopes and the host receptor recognition function map to E domain III (EDIII. Recently, we found that Pichia pastoris-expressed DENV E protein, of all four serotypes, self-assembled into virus-like particles (VLPs in the absence of prM. Significantly, these VLPs displayed EDIII and elicited EDIII-focused DENV-neutralizing antibodies in mice. We now report the creation and characterization of a novel non-replicating recombinant particulate vaccine candidate, produced by co-expressing the E proteins of DENV-1 and DENV-2 in P. pastoris. The two E proteins co-assembled into bivalent mosaic VLPs (mVLPs designated as mE1E2bv VLPs. The mVLP, which preserved the serotype-specific antigenic integrity of its two component proteins, elicited predominantly EDIII-focused homotypic virus

Assay dependence of Brucella antibody prevalence in a declining Alaskan harbor seal (Phoca vitulina population

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Hueffer Karsten

2013-01-01

Full Text Available Abstract Background Brucella is a group of bacteria that causes brucellosis, which can affect population health and reproductive success in many marine mammals. We investigated the serological prevalence of antibodies against Brucella bacteria in a declining harbor seal population in Glacier Bay National Park, Alaska. Results Prevalence ranged from 16 to 74 percent for those tests detecting antibodies, indicating that harbor seals in Glacier Bay have been exposed to Brucella bacteria. However, the actual level of serological prevalence could not be determined because results were strongly assay-dependent. Conclusions This study reinforces the need to carefully consider assay choice when comparing different studies on the prevalence of anti–Brucella antibodies in pinnipeds and further highlights the need for species- or taxon-specific assay validation for both pathogen and host species.

Dynamics and mechanisms of quantum dot nanoparticle cellular uptake

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Telford William G

2010-06-01

Full Text Available Abstract Background The rapid growth of the nanotechnology industry and the wide application of various nanomaterials have raised concerns over their impact on the environment and human health. Yet little is known about the mechanism of cellular uptake and cytotoxicity of nanoparticles. An array of nanomaterials has recently been introduced into cancer research promising for remarkable improvements in diagnosis and treatment of the disease. Among them, quantum dots (QDs distinguish themselves in offering many intrinsic photophysical properties that are desirable for targeted imaging and drug delivery. Results We explored the kinetics and mechanism of cellular uptake of QDs with different surface coatings in two human mammary cells. Using fluorescence microscopy and laser scanning cytometry (LSC, we found that both MCF-7 and MCF-10A cells internalized large amount of QD655-COOH, but the percentage of endocytosing cells is slightly higher in MCF-7 cell line than in MCF-10A cell line. Live cell fluorescent imaging showed that QD cellular uptake increases with time over 40 h of incubation. Staining cells with dyes specific to various intracellular organelles indicated that QDs were localized in lysosomes. Transmission electron microscopy (TEM images suggested a potential pathway for QD cellular uptake mechanism involving three major stages: endocytosis, sequestration in early endosomes, and translocation to later endosomes or lysosomes. No cytotoxicity was observed in cells incubated with 0.8 nM of QDs for a period of 72 h. Conclusions The findings presented here provide information on the mechanism of QD endocytosis that could be exploited to reduce non-specific targeting, thereby improving specific targeting of QDs in cancer diagnosis and treatment applications. These findings are also important in understanding the cytotoxicity of nanomaterials and in emphasizing the importance of strict environmental control of nanoparticles.

PDADMAC flocculation of Chinese hamster ovary cells: enabling a centrifuge-less harvest process for monoclonal antibodies.

Science.gov (United States)

McNerney, Thomas; Thomas, Anne; Senczuk, Anna; Petty, Krista; Zhao, Xiaoyang; Piper, Rob; Carvalho, Juliane; Hammond, Matthew; Sawant, Satin; Bussiere, Jeanine

2015-01-01

High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.

Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models

International Nuclear Information System (INIS)

Liu, Jing; Hu, Rui; Liu, Jianwei; Zhang, Butian; Wang, Yucheng; Liu, Xin; Law, Wing-Cheung; Liu, Liwei; Ye, Ling; Yong, Ken-Tye

2015-01-01

The toxicity of quantum dots (QDs) has been extensively studied over the past decade. Some common factors that originate the QD toxicity include releasing of heavy metal ions from degraded QDs and the generation of reactive oxygen species on the QD surface. In addition to these factors, we should also carefully examine other potential QD toxicity causes that will play crucial roles in impacting the overall biological system. In this contribution, we have performed cytotoxicity assessment of four types of QD formulations in two different human cancer cell models. The four types of QD formulations, namely, mercaptopropionic acid modified CdSe/CdS/ZnS QDs (CdSe-MPA), PEGylated phospholipid encapsulated CdSe/CdS/ZnS QDs (CdSe-Phos), PEGylated phospholipid encapsulated InP/ZnS QDs (InP-Phos) and Pluronic F127 encapsulated CdTe/ZnS QDs (CdTe-F127), are representatives for the commonly used QD formulations in biomedical applications. Both the core materials and the surface modifications have been taken into consideration as the key factors for the cytotoxicity assessment. Through side-by-side comparison and careful evaluations, we have found that the toxicity of QDs does not solely depend on a single factor in initiating the toxicity in biological system but rather it depends on a combination of elements from the particle formulations. More importantly, our toxicity assessment shows different cytotoxicity trend for all the prepared formulations tested on gastric adenocarcinoma (BGC-823) and neuroblastoma (SH-SY5Y) cell lines. We have further proposed that the cellular uptake of these nanocrystals plays an important role in determining the final faith of the toxicity impact of the formulation. The result here suggests that the toxicity of QDs is rather complex and it cannot be generalized under a few assumptions reported previously. We suggest that one have to evaluate the QD toxicity on a case to case basis and this indicates that standard procedures and comprehensive

Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models

Energy Technology Data Exchange (ETDEWEB)

Liu, Jing [Institute of Gerontology and Geriatrics & Beijing Key Lab of Aging and Geriatrics, Chinese PLA General Hospital, Beijing 100853 (China); Hu, Rui [School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Liu, Jianwei [Institute of Gerontology and Geriatrics & Beijing Key Lab of Aging and Geriatrics, Chinese PLA General Hospital, Beijing 100853 (China); Zhang, Butian; Wang, Yucheng [School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Liu, Xin [Lawrence Berkeley National Laboratory, Berkeley, CA (United States); Law, Wing-Cheung [Department of Industrial and System Engineering, The Hang Kong Polytechnic University, Hung Hom (Hong Kong); Liu, Liwei [School of Science, Changchun University of Science and Technology, Changchun 130022 (China); Ye, Ling, E-mail: lye_301@163.com [Institute of Gerontology and Geriatrics & Beijing Key Lab of Aging and Geriatrics, Chinese PLA General Hospital, Beijing 100853 (China); Yong, Ken-Tye, E-mail: ktyong@ntu.edu.sg [School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore)

2015-12-01

The toxicity of quantum dots (QDs) has been extensively studied over the past decade. Some common factors that originate the QD toxicity include releasing of heavy metal ions from degraded QDs and the generation of reactive oxygen species on the QD surface. In addition to these factors, we should also carefully examine other potential QD toxicity causes that will play crucial roles in impacting the overall biological system. In this contribution, we have performed cytotoxicity assessment of four types of QD formulations in two different human cancer cell models. The four types of QD formulations, namely, mercaptopropionic acid modified CdSe/CdS/ZnS QDs (CdSe-MPA), PEGylated phospholipid encapsulated CdSe/CdS/ZnS QDs (CdSe-Phos), PEGylated phospholipid encapsulated InP/ZnS QDs (InP-Phos) and Pluronic F127 encapsulated CdTe/ZnS QDs (CdTe-F127), are representatives for the commonly used QD formulations in biomedical applications. Both the core materials and the surface modifications have been taken into consideration as the key factors for the cytotoxicity assessment. Through side-by-side comparison and careful evaluations, we have found that the toxicity of QDs does not solely depend on a single factor in initiating the toxicity in biological system but rather it depends on a combination of elements from the particle formulations. More importantly, our toxicity assessment shows different cytotoxicity trend for all the prepared formulations tested on gastric adenocarcinoma (BGC-823) and neuroblastoma (SH-SY5Y) cell lines. We have further proposed that the cellular uptake of these nanocrystals plays an important role in determining the final faith of the toxicity impact of the formulation. The result here suggests that the toxicity of QDs is rather complex and it cannot be generalized under a few assumptions reported previously. We suggest that one have to evaluate the QD toxicity on a case to case basis and this indicates that standard procedures and comprehensive

Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by Mycobacterium ulcerans.

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Jean-Pierre Dangy

2016-06-01

Full Text Available Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone.Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone.The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target.

Targeted treatment for chronic lymphocytic leukemia: clinical potential of obinutuzumab

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Smolej L

2014-12-01

Full Text Available Lukáš Smolej 4th Department of Internal Medicine – Hematology, University Hospital Hradec Králové and Charles University in Prague, Faculty of Medicine in Hradec Králové, Hradec Králové, Czech Republic Abstract: Introduction of targeted agents revolutionized the treatment of chronic lymphocytic leukemia (CLL in the past decade. Addition of chimeric monoclonal anti-CD20 antibody rituximab to chemotherapy significantly improved efficacy including overall survival (OS in untreated fit patients; humanized anti-CD52 antibody alemtuzumab and fully human anti-CD20 antibody ofatumumab lead to improvement in refractory disease. Novel small molecule inhibitors such as ibrutinib and idelalisib demonstrated excellent activity and were very recently licensed in relapsed/refractory CLL. Obinutuzumab (GA101 is the newest monoclonal antibody approved for the treatment of CLL. This novel, glycoengineered, type II humanized anti-CD20 antibody is characterized by enhanced antibody-dependent cellular cytotoxicity and direct induction of cell death compared to type I antibodies. Combination of obinutuzumab and chlorambucil yielded significantly better OS in comparison to chlorambucil monotherapy in untreated comorbid patients. These results led to approval of obinuzutumab for the treatment of CLL. Numerous clinical trials combining obinutuzumab with other cytotoxic drugs and novel small molecules are currently under way. This review focuses on the role of obinutuzumab in the treatment of CLL. Keywords: chronic lymphocytic leukemia, anti-CD20 antibodies, chlorambucil, rituximab, ofatumumab, obinutuzumab, overall survival

Immune responses to epstein-barr virus in atomic bomb survivors: Study of precursor frequency of cytotoxic lymphocytes and titer levels of anti-Epstein-Barr virus-related antibodies

Energy Technology Data Exchange (ETDEWEB)

Kusunoki, Yoichiro; Kyoizumi, Seishi; Saito, Mayumi; Ozaki, Kyoko; Hirai, Yuko; Akiyama, Mitoshi (Radiation Effects Research Foundation, Hiroshima (Japan)); Fukuda, Yasuko (Radiation Effects Research Foundation, Hiroshima (Japan) Children' s Hospital Medical Center of Northern California, Oakland, CA (United States)); Huang, Hua (Radiation Effects Research Foundation, Hiroshima (Japan) Univ. of Wisconsin, Madison, WI (United States))

1994-04-01

Precursor frequencies of cytotoxic lymphocytes to autologous Epstein-Barr virus-transformed B cells and serum titers of anti-Epstein-Barr virus-related antibodies were measured in 68 atomic bomb survivors to clarify the immune mechanism controlling Epstein-Barr virus infection. The precursor frequency was negatively correlated with the titer of anti-early antigen lgG, which is probably produced at the stage of viral reactivation. A positive correlation between the precursor frequency and titer of anti-Epstein-Barr virus-associated nuclear antigen antibody was also observed, indicating that the precursor frequency reflects the degree of in vivo destruction by T cells of the virus-infected cells. These results suggest that T-cell memory specific to Epstein-Barr virus keeps the virus under control and that the precursor frequency assay is useful for the evaluation of immune responses to Epstein-Barr virus. However, no significant effect of atomic bomb radiation on the precursor frequency was observed in the present study, probably due to the limited number of participants. 24 refs., 4 figs., 2 tabs.

Immune responses to epstein-barr virus in atomic bomb survivors: Study of precursor frequency of cytotoxic lymphocytes and titer levels of anti-Epstein-Barr virus-related antibodies

International Nuclear Information System (INIS)

Kusunoki, Yoichiro; Kyoizumi, Seishi; Saito, Mayumi; Ozaki, Kyoko; Hirai, Yuko; Akiyama, Mitoshi; Fukuda, Yasuko; Huang, Hua

1994-01-01

Precursor frequencies of cytotoxic lymphocytes to autologous Epstein-Barr virus-transformed B cells and serum titers of anti-Epstein-Barr virus-related antibodies were measured in 68 atomic bomb survivors to clarify the immune mechanism controlling Epstein-Barr virus infection. The precursor frequency was negatively correlated with the titer of anti-early antigen lgG, which is probably produced at the stage of viral reactivation. A positive correlation between the precursor frequency and titer of anti-Epstein-Barr virus-associated nuclear antigen antibody was also observed, indicating that the precursor frequency reflects the degree of in vivo destruction by T cells of the virus-infected cells. These results suggest that T-cell memory specific to Epstein-Barr virus keeps the virus under control and that the precursor frequency assay is useful for the evaluation of immune responses to Epstein-Barr virus. However, no significant effect of atomic bomb radiation on the precursor frequency was observed in the present study, probably due to the limited number of participants. 24 refs., 4 figs., 2 tabs

Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells.

Science.gov (United States)

Yadav, Umesh C S; Ramana, K V; Srivastava, Satish K

2013-12-01

Aldose reductase (AR), a glucose-metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30 µM) relative to glucose. Acrolein, a major endogenous lipid peroxidation product as well as a component of environmental pollutants and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders, but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells (SAECs). Exposure of SAECs to varying concentrations of acrolein caused cell death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low-dose (5-10 µM) but not the high-dose (>10 µM) acrolein-induced SAEC death. AR inhibition protected SAECs from low-dose (5 µM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail moment, and annexin V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of the proapoptotic proteins Bax and Bad from the cytosol to the mitochondria and that of Bcl2 and BclXL from the mitochondria to the cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 and 2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38MAPK, and c-Jun were transiently activated in airway epithelial cells by acrolein in a concentration- and time-dependent fashion, which was significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells. Copyright © 2013 Elsevier Inc. All rights

Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells

Science.gov (United States)

Yadav, Umesh CS; Ramana, KV; Srivastava, SK

2013-01-01

Aldose reductase (AR), a glucose metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30μM) than glucose. Acrolein, a major endogenous lipid peroxidation product as well as component of environmental pollutant and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells SAECs. Exposure of SAECs to varying concentrations of acrolein caused cell-death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low (5 to 10 μM) but not high (>10 μM) concentrations of acrolein-induced SAECs cell death. AR inhibition protected SAECs from low dose (5 μM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail-moment, and annexin-V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of pro-apoptotic proteins Bax and Bad from cytosol to the mitochondria, and that of Bcl2 and BclXL from mitochondria to cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinases 1 and 2 (ERK1/2), stress-activated protein kinases/c-jun NH2-terminal kinases (SAPK/JNK) and p38MAPK, and c-jun were transiently activated in airway epithelial cells by acrolein in a concentration and time-dependent fashion, which were significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells. PMID:23770200

Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses

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Iwai Ohbayashi

2018-01-01

Full Text Available The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized pathway of this stress response involves p53 and MDM2 as key players. p53 is a crucial transcription factor that functions in response to not only nucleolar stress but also other cellular stresses such as DNA damage stress. These cellular stresses release p53 from the inhibition by MDM2, an E3 ubiquitin ligase targeting p53, in various ways, which leads to p53-dependent activation of a set of genes. In plants, genetic impairments of ribosome biogenesis factors or ribosome components have been shown to cause characteristic phenotypes, including a narrow and pointed leaf shape, implying a common signaling pathway connecting ribosomal perturbations and certain aspects of growth and development. Unlike animals, however, plants have neither p53 nor MDM2 family proteins. Then the question arises whether plant cells have a nucleolar stress response pathway. In recent years, it has been reported that several members of the plant-specific transcription factor family NAC play critical roles in the pathways responsive to various cellular stresses. In this mini review, we outline the plant cellular stress response pathways involving NAC transcription factors with reference to the p53-MDM2-dependent pathways of animal cells, and discuss the possible involvement of a plant-unique, NAC-mediated pathway in the nucleolar stress response in plants.

Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses.

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Ohbayashi, Iwai; Sugiyama, Munetaka

2017-01-01

The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized pathway of this stress response involves p53 and MDM2 as key players. p53 is a crucial transcription factor that functions in response to not only nucleolar stress but also other cellular stresses such as DNA damage stress. These cellular stresses release p53 from the inhibition by MDM2, an E3 ubiquitin ligase targeting p53, in various ways, which leads to p53-dependent activation of a set of genes. In plants, genetic impairments of ribosome biogenesis factors or ribosome components have been shown to cause characteristic phenotypes, including a narrow and pointed leaf shape, implying a common signaling pathway connecting ribosomal perturbations and certain aspects of growth and development. Unlike animals, however, plants have neither p53 nor MDM2 family proteins. Then the question arises whether plant cells have a nucleolar stress response pathway. In recent years, it has been reported that several members of the plant-specific transcription factor family NAC play critical roles in the pathways responsive to various cellular stresses. In this mini review, we outline the plant cellular stress response pathways involving NAC transcription factors with reference to the p53-MDM2-dependent pathways of animal cells, and discuss the possible involvement of a plant-unique, NAC-mediated pathway in the nucleolar stress response in plants.

Cytotoxicity and inhibitory properties against topoisomerase II of doxorubicin and its formamidine derivatives.

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Kik, Krzysztof; Studzian, Kazimierz; Wasowska-Łukawska, Małgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

2009-01-01

This work was undertaken to compare cytotoxicity, DNA damaging properties and effect on DNA cleavage by topoisomerase II of the anthracycline drug doxorubicin (DOX) and its two derivatives with a formamidino group containing a cyclic amine moiety such as morpholine (DOXM) or hexamethyleneimine (DOXH). The tetrazolium dye colorimetric assay was used to determine the cytotoxic activity of anthracyclines toward L1210 leukemia cells. DNA damage was measured by alkaline elution technique. The effect of anthracyclines on DNA cleavage was studied in a cell-free system containing supercoiled pBR322 DNA and purified human topoisomerase II. The cytotoxicity data and the results of studies on the mechanism of DNA break formation by anthracyclines at the cellular level and in the cell-free system showed that the presence of the formamidino group in the doxorubicin molecule reduced its ability to stimulate DNA cleavage by DNA topoisomerase II. DNA topoisomerase II is not a primary cellular target for DOXM or DOXH. An advantageous feature of formamidinoanthracyclines is their mechanism of cytotoxic action which is not related to the inhibition of DNA topoisomerase II. Therefore this class of anthracyclines seems to be a good source for selection of an anticancer drug directed toward cancer cells with the developed multidrug resistance attributed to the presence of altered DNA topoisomerase II.

Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells.

Science.gov (United States)

Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible.

Cellular basis of the immunohematologic defects observed in short-term semiallogeneic B6C3F1→C3H chimeras: evidence for host-versus-graft reaction initiated by radioresistant T cells

International Nuclear Information System (INIS)

Aizawa, S.; Sado, T.; Kamisaku, H.; Kubo, E.

1980-01-01

Lethally irradiated C3Hf mice reconstituted with a relatively low dose (2 x 10 6 ) of B6C3F 1 bone marrow cells (B6C3F 1 →C3Hf chimeras) frequently manifest immunohematologic deficiencies during the first month following injection of bone marrow cells. They show slow recovery of antibody-forming potential to sheep red blood cells (SRBC) as compared to that observed in syngeneic (C3Hf→C3Hf or B6C3F 1 →B6C3F 1 ) chimeras. They also show a deficiency of B-cell activity as assessed by antibody response to SRBC following further reconstitution with B6C3F 1 -derived thymus cells 1 week after injection of bone marrow cells. A significant fraction of B6C3F 1 →C3Hf chimeras was shown to manifest a sudden loss of cellularity of spleens during the second week following injection of bone marrow cells even though cellularity was restored to the normal level within 1 week. The splenic mononuclear cells recovered from such chimeras almost invariably showed strong cytotoxicity against target cells expressing donor-type specific H-2 antigens (H-2/sup b/) when assesed by 51 Cr-release assay in vitro. The effector cells responsible for the observed anti-donor specific cytotoxicity were shown to be residual host-derived T cells. These results indicate strongly that residual host T cells could develop anti-donor specific cytotoxicity even after exposure to a supralethal dose (1050 R) of radiation and that the immunohematologic disturbances observed in shortterm F 1 to parent bone marrow chimeras (B6C3F 1 →C3Hf) were due to host-versus-graft reaction (HVGR) initiated by residual host T cells. The implication of these findings on the radiobiological nature of the residual T cells and the persistence of potentially anti-donor reactive T-cell clones in long-surviving allogeneic bone marrow chimeras was discussed

Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids.

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Lawley, P D; Brookes, P

1968-09-01

1. A quantitative study was made of the relationship between survival of colony-forming ability in Escherichia coli strains B/r and B(s-1) and the extents of alkylation of cellular DNA, RNA and protein after treatment with mono- or di-functional sulphur mustards, methyl methanesulphonate or iodoacetamide. 2. The mustards and methyl methanesulphonate react with nucleic acids in the cells, in the same way as found previously from chemical studies in vitro, and with proteins. Iodoacetamide reacts only with protein, principally with the thiol groups of cysteine residues. 3. The extents of alkylation of cellular constituents required to prevent cell division vary widely according to the strain of bacteria and the nature of the alkylating agent. 4. The extents of alkylation of the sensitive and resistant strains at a given dose of alkylating agent do not differ significantly. 5. Removal of alkyl groups from DNA of cells of the resistant strains B/r and 15T(-) after alkylation with difunctional sulphur mustard was demonstrated; the product di(guanin-7-ylethyl) sulphide, characteristic of di- as opposed to mono-functional alkylation, was selectively removed; the time-scale of this effect suggests an enzymic rather than a chemical mechanism. 6. The sensitive strain B(s-1) removed alkyl groups from DNA in this way only at very low extents of alkylation. When sensitized to mustard action by treatment with iodoacetamide, acriflavine or caffeine, the extent of alkylation of cellular DNA corresponding to a mean lethal dose was decreased to approximately 3 molecules of di(guanin-7-ylethyl) sulphide in the genome of this strain. 7. Relatively large numbers of monofunctional alkylations per genome can be withstood by this sensitive strain. Iodoacetamide had the weakest cytotoxic action of the agents investigated; methyl methanesulphonate was significantly weaker in effect than the monofunctional sulphur mustard, which was in turn weaker than the difunctional sulphur mustard. 8

Cytotoxic drug sensitivity testing of tumor cells from patients with ovarian carcinoma using the fluorometric microculture cytotoxicity assay (FMCA).

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Csoka, K; Larsson, R; Tholander, B; Gerdin, E; de la Torre, M; Nygren, P

1994-08-01

The automated fluorometric microculture cytotoxicity assay (FMCA) is based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein by viable cells after a 72-hr culture period in microtiter plates. The FMCA was adopted for chemosensitivity testing of tumor cells from patients with ovarian carcinoma. Thirty-seven samples of solid tumors and malignant effusions were obtained from 35 patients at diagnosis or relapse. Tumor cells from solid samples and effusions were prepared by enzymatic digestion and centrifugation, respectively, followed by Percoll or Ficoll purification. The fluorescence was proportional to the number of cells/well and considerably higher in tumor cells than in contaminating normal cells. The effect of up to 19 cytotoxic drugs was successfully assessed in 70% of the samples and there was a good correlation between drug sensitivity data reported by the FMCA and the DiSC assay performed in parallel. The overall drug sensitivity pattern in vitro corresponded well to the clinical experience. The effect of cisplatin varied considerably between patients and resistance was found also in cases not previously exposed to cytotoxic drugs. The FMCA is a rapid and simple method that seems to report clinically relevant cytotoxic drug sensitivity data in ovarian carcinomas. In the future, this method may contribute to optimizing chemotherapy by assisting in individualized drug selection and new drug development.

A phase I study of PRO131921, a novel anti-CD20 monoclonal antibody in patients with relapsed/refractory CD20+ indolent NHL: correlation between clinical responses and AUC pharmacokinetics.

Science.gov (United States)

Casulo, Carla; Vose, Julie M; Ho, William Y; Kahl, Brad; Brunvand, Mark; Goy, Andre; Kasamon, Yvette; Cheson, Bruce; Friedberg, Jonathan W

2014-09-01

PRO131921 is a third-generation, humanized anti-CD20 monoclonal antibody with increased antibody-dependent cytotoxicity and complement-dependent cytotoxicity compared to rituximab. In this phase I study, PRO131921 was administered as a single agent to patients with CD20+, relapsed or refractory, indolent non-Hodgkin lymphoma (NHL) who had been treated with a prior rituximab-containing regimen. The primary aim of this study was safety and tolerability of PRO131921. The secondary aim of the study, and focus of this report, was to determine the pharmacokinetics (PK) profile of PRO131921 and establish a correlation between drug exposure and clinical efficacy. Patients were treated with PRO131921 by intravenous infusion weekly for 4 weeks and the dose was escalated based on safety in a 3+3 design. Twenty-four patients were treated with PRO131921 at doses from 25mg/m(2) to 800 mg/m(2). Analysis of PK data demonstrated a correlation between higher normalized drug exposure (normalized AUC) and tumor shrinkage (p = .0035). Also, normalized AUC levels were higher among responders and subjects displaying tumor shrinkage versus subjects progressing or showing no regression (p = 0.030). In conclusion, PRO131921 demonstrated clinical activity in rituximab-relapsed and refractory indolent NHL patients. The observation that higher normalized AUC may be associated with improved clinical responses has potential implications in future trials of monoclonal antibody-based therapies, and emphasizes the importance of early PK studies to optimize antibody efficacy. Copyright © 2014 Elsevier Inc. All rights reserved.

92R Monoclonal Antibody Inhibits Human CCR9+ Leukemia Cells Growth in NSG Mice Xenografts.

Science.gov (United States)

Somovilla-Crespo, Beatriz; Martín Monzón, Maria Teresa; Vela, Maria; Corraliza-Gorjón, Isabel; Santamaria, Silvia; Garcia-Sanz, Jose A; Kremer, Leonor

2018-01-01

CCR9 is as an interesting target for the treatment of human CCR9 + -T cell acute lymphoblastic leukemia, since its expression is limited to immature cells in the thymus, infiltrating leukocytes in the small intestine and a small fraction of mature circulating T lymphocytes. 92R, a new mouse mAb (IgG2a isotype), was raised using the A-isoform of hCCR9 as immunogen. Its initial characterization demonstrates that binds with high affinity to the CCR9 N-terminal domain, competing with the previously described 91R mAb for receptor binding. 92R inhibits human CCR9 + tumor growth in T and B-cell deficient Rag2 -/- mice. In vitro assays suggested complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity as possible in vivo mechanisms of action. Unexpectedly, 92R strongly inhibited tumor growth also in a model with compromised NK and complement activities, suggesting that other mechanisms, including phagocytosis or apoptosis, might also be playing a role on 92R-mediated tumor elimination. Taken together, these data contribute to strengthen the hypothesis of the immune system's opportunistic nature.

Cellular redox homeostasis in endothelial cells treated with nonmodified and Fenton-modified nanodiamond powders.

Science.gov (United States)

Solarska-Ściuk, K; Gajewska, A; Skolimowski, J; Gajek, A; Bartosz, G

2014-01-01

Diamond nanoparticles find numerous applications in pharmacy, medicine, cosmetics, and biotechnology. However, possible adverse cellular effects of diamond nanoparticle cells have been reported, which may limit their use. The aim of this study was to compare the effect of nonmodified diamond nanoparticles (D) and diamond nanoparticles modified by the Fenton reaction (D+OH) on human umbilical cord endothelial cells (HUVEC-ST). We found that both D and D+OH show time- and concentration-dependent cytotoxicity, inducing apoptosis and necrosis of HUVEC-ST. Interaction with D and D+OH also induced changes in the production of reactive oxygen and nitrogen species and changes in the level of glutathione and activities of antioxidant enzymes in the cells. These data demonstrate that diamond nanoparticles may induce oxidative stress in human endothelial cells, which contributes to their cytotoxic effects seen at higher concentrations of D and D+OH. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment

Directory of Open Access Journals (Sweden)

Tetsuya Okuda

2017-02-01

Full Text Available Protein modification by O-linked N-acetylglucosamine (O-GlcNAcylation is one of the post transcriptional modifications occurring on cellular proteins. This paper provides a data set relating to the O-GlcNAcylation of cellular proteins detected by RL2 and CTD110.6 antibodies, which are commonly used for detection of protein O-GlcNAcylation, in 2-deoxy-d-glucose (2DG-treated human teratocarcinoma NCCIT cells in support of the research article entitled “A novel, promoter-based, target-specific assay identifies 2-deoxy-d-glucose as an inhibitor of globotriaosylceramide biosynthesis” (Okuda et al., 2009 [1]. The main article described a suppressive effect of 2DG on an Sp1 target gene in NCCIT cells and discussed the relationship between the effect of 2DG and O-GlcNAcylation status of Sp1. The data in this paper complements this relationship by Western blotting and clearly showed that the 2DG treatment increased O-GlcNAcylation of cellular proteins in NCCIT cells, whereas the RL2 and CTD110.6 epitopes were detected in a different manner. The RL2 epitope was detected on Sp1 during 2DG treatment, and the level was transiently increased at 24 h. In contrast, the CTD110.6 epitope became detectable on Sp1 over 72 h after 2DG treatment, and then the other proteins containing CTD110.6 epitopes also appeared in the cell lysates and the anti-Sp1 antibody precipitates.

Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane.

Science.gov (United States)

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin; Guo, Danjing; Xu, Yuning; Wu, Liming; Zheng, Shusen

2016-08-15

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge. Copyright © 2016 Elsevier Inc. All rights reserved.

Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane

International Nuclear Information System (INIS)

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin; Guo, Danjing; Xu, Yuning; Wu, Liming; Zheng, Shusen

2016-01-01

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge.

Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane

Energy Technology Data Exchange (ETDEWEB)

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin [Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, 310003 Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health and Key Laboratory of Organ Transplantation of Zhejiang Province, The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003 (China); Guo, Danjing; Xu, Yuning [Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health and Key Laboratory of Organ Transplantation of Zhejiang Province, The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003 (China); Wu, Liming, E-mail: wlm@zju.edu.cn [Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, 310003 Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health and Key Laboratory of Organ Transplantation of Zhejiang Province, The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003 (China); Zheng, Shusen, E-mail: shusenzheng@zju.edu.cn [Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, 310003 Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health and Key Laboratory of Organ Transplantation of Zhejiang Province, The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003 (China)

2016-08-15

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge.

Next Generation Antibody Therapeutics Using Bispecific Antibody Technology.

Science.gov (United States)

Igawa, Tomoyuki

2017-01-01

Nearly fifty monoclonal antibodies have been approved to date, and the market for monoclonal antibodies is expected to continue to grow. Since global competition in the field of antibody therapeutics is intense, we need to establish novel antibody engineering technologies to provide true benefit for patients, with differentiated product values. Bispecific antibodies are among the next generation of antibody therapeutics that can bind to two different target antigens by the two arms of immunoglobulin G (IgG) molecule, and are thus believed to be applicable to various therapeutic needs. Until recently, large scale manufacturing of human IgG bispecific antibody was impossible. We have established a technology, named asymmetric re-engineering technology (ART)-Ig, to enable large scale manufacturing of bispecific antibodies. Three examples of next generation antibody therapeutics using ART-Ig technology are described. Recent updates on bispecific antibodies against factor IXa and factor X for the treatment of hemophilia A, bispecific antibodies against a tumor specific antigen and T cell surface marker CD3 for cancer immunotherapy, and bispecific antibodies against two different epitopes of soluble antigen with pH-dependent binding property for the elimination of soluble antigen from plasma are also described.

Cytotoxicity, Intestinal Transport, and Bioavailability of Dispersible Iron and Zinc Supplements

Directory of Open Access Journals (Sweden)

Jae-Min Oh

2017-04-01

Full Text Available Iron or zinc deficiency is one of the most important nutritional disorders which causes health problem. However, food fortification with minerals often induces unacceptable organoleptic changes during preparation process and storage, has low bioavailability and solubility, and is expensive. Nanotechnology surface modification to obtain novel characteristics can be a useful tool to overcome these problems. In this study, the efficacy and potential toxicity of dispersible Fe or Zn supplement coated in dextrin and glycerides (SunActive FeTM and SunActive ZnTM were evaluated in terms of cytotoxicity, intestinal transport, and bioavailability, as compared with each counterpart without coating, ferric pyrophosphate (FePP and zinc oxide (ZnO nanoparticles (NPs, respectively. The results demonstrate that the cytotoxicity of FePP was not significantly affected by surface modification (SunActive FeTM, while SunActive ZnTM was more cytotoxic than ZnO-NPs. Cellular uptake and intestinal transport efficiency of SunActive FeTM were significantly higher than those of its counterpart material, which was in good agreement with enhanced oral absorption efficacy after a single-dose oral administration to rats. These results seem to be related to dissolution, particle dispersibility, and coating stability of materials depending on suspending media. Both SunActiveTM products and their counterpart materials were determined to be primarily transported by microfold (M cells through the intestinal epithelium. It was, therefore, concluded that surface modification of food fortification will be a useful strategy to enhance oral absorption efficiency at safe levels.

Modification of the cytotoxic activity of mitomycin C

International Nuclear Information System (INIS)

Marshall, R.S.; Rauth, A.M.

1985-01-01

Utilizing a system in which oxygen levels could be altered and monitored during acute drug exposures, the authors have begun to characterize the cellular and molecular damage produced by MMC in CHO cells. The cytotoxic activity of MMC decreases sharply from 0 to 0.1% oxygen in solution, while from 0.1 to 20.0% there is little change. DNA crosslinking in cells was examined under these conditions by alkaline elution and found to be directly correlated with cell killing. While hypoxia increased crosslinking, significant levels were still observed under aerobic conditions. A cell-free assay for alkylation confirmed that overall levels increase in the absence of oxygen; however, negligible alkylation was observed under aerobic conditions. It was also noted that ascorbic acid present in the exposure medium (0.284 mM) increased the aerobic cytotoxicity without altering the hypoxic cytotoxicity. The present data suggest that MMC can be activated to an alkylating species by two mechanisms, one oxygen sensitive and one oxygen insensitive and that these two mechanisms may be independently modified

DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

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Adam J L Cook

2007-04-01

Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

Rationale for combination of therapeutic antibodies targeting tumor cells and immune checkpoint receptors: Harnessing innate and adaptive immunity through IgG1 isotype immune effector stimulation.

Science.gov (United States)

Ferris, Robert L; Lenz, Heinz-Josef; Trotta, Anna Maria; García-Foncillas, Jesús; Schulten, Jeltje; Audhuy, François; Merlano, Marco; Milano, Gerard

2018-02-01

Immunoglobulin (Ig) G1 antibodies stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). Cetuximab, an IgG1 isotype monoclonal antibody, is a standard-of-care treatment for locally advanced and recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN) and metastatic colorectal cancer (CRC). Here we review evidence regarding the clinical relevance of cetuximab-mediated ADCC and other immune functions and provide a biological rationale concerning why this property positions cetuximab as an ideal partner for immune checkpoint inhibitors (ICIs) and other emerging immunotherapies. We performed a nonsystematic review of available preclinical and clinical data involving cetuximab-mediated immune activity and combination approaches of cetuximab with other immunotherapies, including ICIs, in SCCHN and CRC. Indeed, cetuximab mediates ADCC activity in the intratumoral space and primes adaptive and innate cellular immunity. However, counterregulatory mechanisms may lead to immunosuppressive feedback loops. Accordingly, there is a strong rationale for combining ICIs with cetuximab for the treatment of advanced tumors, as targeting CTLA-4, PD-1, and PD-L1 can ostensibly overcome these immunosuppressive counter-mechanisms in the tumor microenvironment. Moreover, combining ICIs (or other immunotherapies) with cetuximab is a promising strategy for boosting immune response and enhancing response rates and durability of response. Cetuximab immune activity-including, but not limited to, ADCC-provides a strong rationale for its combination with ICIs or other immunotherapies to synergistically and fully mobilize the adaptive and innate immunity against tumor cells. Ongoing prospective studies will evaluate the clinical effect of these combination regimens and their immune effect in CRC and SCCHN and in other indications. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Industrial grade 2D molybdenum disulphide (MoS2): an in vitro exploration of the impact on cellular uptake, cytotoxicity, and inflammation

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Moore, Caroline; Movia, Dania; Smith, Ronan J.; Hanlon, Damien; Lebre, Filipa; Lavelle, Ed C.; Byrne, Hugh J.; Coleman, Jonathan N.; Volkov, Yuri; McIntyre, Jennifer

2017-06-01

The recent surge in graphene research, since its liquid phase monolayer isolation and characterization in 2004, has led to advancements which are accelerating the exploration of alternative 2D materials such as molybdenum disulphide (MoS2), whose unique physico-chemical properties can be exploited in applications ranging from cutting edge electronic devices to nanomedicine. However, to assess any potential impact on human health and the environment, the need to understand the bio-interaction of MoS2 at a cellular and sub-cellular level is critical. Notably, it is important to assess such potential impacts of materials which are produced by large scale production techniques, rather than research grade materials. The aim of this study was to explore cytotoxicity, cellular uptake and inflammatory responses in established cell-lines that mimic different potential exposure routes (inhalation, A549; ingestion, AGS; monocyte, THP-1) following incubation with MoS2 flakes of varying sizes (50 nm, 117 nm and 177 nm), produced by liquid phase exfoliation. Using high content screening (HCS) and Live/Dead assays, it was established that 1 µg ml-1 (for the three different MoS2 sizes) did not induce toxic effects on any of the cell-lines. Confocal microscopy images revealed a normal cellular morphology in all cases. Transmission electron microscopy (TEM) confirmed the uptake of all MoS2 nanomaterials in all the cell-lines, the MoS2 ultimately locating in single membrane vesicles. At such sub-lethal doses, inflammatory responses are observed, however, associated, at least partially, with the presence of lipopolysaccharide endotoxin in nanomaterial suspensions and surfactant samples. Therefore, the inflammatory response of the cells to the MoS2 or endotoxin contamination was interrogated using a 10-plex ELISA which illustrates cytokine production. The experiments carried out using wild-type and endotoxin hyporesponsive bone marrow derived dendritic cells confirmed that the

Pingyangmycin and Bleomycin Share the Same Cytotoxicity Pathway

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Yanli He

2016-06-01

Full Text Available Pingyangmycin is an anticancer drug known as bleomycin A5 (A5, discovered in the Pingyang County of Zhejiang Province of China. Bleomycin (BLM is a mixture of mainly two compounds (A2 and B2, which is on the World Health Organization’s list of essential medicines. Both BLM and A5 are hydrophilic molecules that depend on transporters or endocytosis receptors to get inside of cells. Once inside, the anticancer activities rely on their abilities to produce DNA breaks, thus leading to cell death. Interestingly, the half maximal inhibitory concentration (IC50 of BLMs in different cancer cell lines varies from nM to μM ranges. Different cellular uptake, DNA repair rate, and/or increased drug detoxification might be some of the reasons; however, the molecules and signaling pathways responsible for these processes are largely unknown. In the current study, we purified the A2 and B2 from the BLM and tested the cytotoxicities and the molecular mechanisms of each individual compound or in combination with six different cell lines, including a Chinese hamster ovary (CHO cell line defective in glycosaminoglycan biosynthesis. Our data suggested that glycosaminoglycans might be involved in the cellular uptake of BLMs. Moreover, both BLM and A5 shared similar signaling pathways and are involved in cell cycle and apoptosis in different cancer cell lines.