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Sample records for antigen-specific polyclonal cytotoxic

  1. Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

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    Markus Munder

    Full Text Available Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i CD8(+ T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

  2. Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

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    Laurence Rolland

    1992-06-01

    Full Text Available In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

  3. Increasing a Robust Antigen-Specific Cytotoxic T Lymphocyte Response by FMDV DNA Vaccination with IL-9 Expressing Construct

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    Qiang Zou

    2010-01-01

    Full Text Available Various chemokines and cytokines as adjuvants can be used to improve efficacy of DNA vaccination. In this study, we sought to investigate if a DNA construct expressing IL-9 (designed as proV-IL9 as a molecular adjuvant enhance antigen specific immune responses elicited by the pcD-VP1 DNA vaccination. Mice immunized with pcD-VP1 combined with proV-IL9 developed a strong humoral response. In addition, the coinoculation induced significant higher level of antigen-specific cell proliferation and cytotoxic response. This agreed well with higher expression level of IFN-γ and perforin in CD8+ T cells, but not with IL-17 in these T cells. The results indicate that IL-9 induces the development of IFN-γ-producing CD8+ T cells (Tc1, but not the IL-17-producing CD8+ T cells (Tc17. Up-regulated expressions of BCL-2 and BCL-XL were exhibited in these Tc1 cells, suggesting that IL-9 may trigger antiapoptosis mechanism in these cells. Together, these results demonstrated that IL-9 used as molecular adjuvant could enhance the immunogenicity of DNA vaccination, in augmenting humoral and cellular responses and particularly promoting Tc1 activations. Thus, the IL-9 may be utilized as a potent Tc1 adjuvant for DNA vaccines.

  4. Demethylating agent decitabine induces autologous cancer testis antigen specific cytotoxic T lymphocytes in vivo

    Institute of Scientific and Technical Information of China (English)

    ZHOU Ji-hao; YAO Yu-shi; WANG Li-xin; WANG Jia; LI Yong-hui; JIANG Meng-meng; ZHOU Min-hang

    2013-01-01

    Background Cancer testis antigens (CTAs) are a novel group of tumor associated antigens.Demethylating agent decitabine was reported to be able to up-regulate CTAs through its hypomethylation mechanism,thus enhance the immunogenicity of leukemia cells.However,few researches have ever focused on the questions that whether this immunostimulatory effect of decitabine could induce autologous CTA specific cytotoxic T lymphocytes (CTLs) in vivo,and if so,whether this effect contributes to disease control.In this study,we aimed to show that decitabine could induce specific autologous CTLs against some mouse CTAs in leukemia cells in vitro and in vivo.Methods Several mouse CTAs were screened by RT-PCR.CTL specific to one of the CTAs named P1A was detected and sorted by P1A specific dimer by flow cytometry.The activity of specific CTLs was measured by real time RT-PCR.Results We firstly screened expression of some CTAs in mouse leukemia cells before and after decitabine treatment and found that decitabine treatment did up-regulate expression of many CTAs.Then we measured the CTLs' activity specific to a mouse CTA P1A in vivo and showed that this activity increased after decitabine treatment.Finally,we sorted these in vivo induced P1A specific CTLs by flow cytometry and demonstrated their cytotoxicity against decitabine treated leukemia cells.Conclusions Our study showed the autologous immune response induced by decitabine in vivo.And more importantly,we firstly proved that this response may contribute to disease control.We believe that this immunostimulatory effect is another anti-cancer mechanism of decitabine,and this special effect would inspire new applications of decitabine in the field of leukemia treatment in the future.

  5. Effective Delivery of Antigen-Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection.

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    Choi, Bongseo; Moon, Hyojin; Hong, Sung Joon; Shin, Changsik; Do, Yoonkyung; Ryu, Seongho; Kang, Sebyung

    2016-08-23

    In cancer immunotherapy, robust and efficient activation of cytotoxic CD8(+) T cell immune responses is a promising, but challenging task. Dendritic cells (DCs) are well-known professional antigen presenting cells that initiate and regulate antigen-specific cytotoxic CD8(+) T cells that kill their target cells directly as well as secrete IFN-γ, a cytokine critical in tumor rejection. Here, we employed recently established protein cage nanoparticles, encapsulin (Encap), as antigenic peptide nanocarriers by genetically incorporating the OT-1 peptide of ovalbumin (OVA) protein to the three different positions of the Encap subunit. With them, we evaluated their efficacy in activating DC-mediated antigen-specific T cell cytotoxicity and consequent melanoma tumor rejection in vivo. DCs efficiently engulfed Encap and its variants (OT-1-Encaps), which carry antigenic peptides at different positions, and properly processed them within phagosomes. Delivered OT-1 peptides were effectively presented by DCs to naïve CD8(+) T cells successfully, resulting in the proliferation of antigen-specific cytotoxic CD8(+) T cells. OT-1-Encap vaccinations in B16-OVA melanoma tumor bearing mice effectively activated OT-1 peptide specific cytotoxic CD8(+) T cells before or even after tumor generation, resulting in significant suppression of tumor growth in prophylactic as well as therapeutic treatments. A large number of cytotoxic CD8(+) T cells that actively produce both intracellular and secretory IFN-γ were observed in tumor-infiltrating lymphocytes collected from B16-OVA tumor masses originally vaccinated with OT-1-Encap-C upon tumor challenges. The approaches we describe herein may provide opportunities to develop epitope-dependent vaccination systems that stimulate and/or modulate efficient and epitope-specific cytotoxic T cell immune responses in nonpathogenic diseases.

  6. Increasing a Robust Antigen-Specific Cytotoxic T Lymphocyte Response by FMDV DNA Vaccination with IL-9 Expressing Construct

    OpenAIRE

    Qiang Zou; Bing Wu; Xiaodan He; Yizhi Zhang; Youmin Kang; Jin Jin; Hanqian Xu; Hu Liu; Bin Wang

    2010-01-01

    Various chemokines and cytokines as adjuvants can be used to improve efficacy of DNA vaccination. In this study, we sought to investigate if a DNA construct expressing IL-9 (designed as proV-IL9) as a molecular adjuvant enhance antigen specific immune responses elicited by the pcD-VP1 DNA vaccination. Mice immunized with pcD-VP1 combined with proV-IL9 developed a strong humoral response. In addition, the coinoculation induced significant higher level of antigen-specific cell proliferation and...

  7. Induction/Engineering, Detection, Selection, and Expansion of Clinical-Grade Human Antigen-Specific CD8+ Cytotoxic T Cell Clones for Adoptive Immunotherapy

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    Matjaž Jeras

    2010-01-01

    Full Text Available Adoptive transfer of effector antigen-specific immune cells is becoming a promising treatment option in allogeneic transplantation, infectious diseases, cancer, and autoimmune disorders. Within this context, the important role of CD8+ cytotoxic T cells (CTLs is objective of intensive studies directed to their in vivo and ex vivo induction, detection, selection, expansion, and therapeutic effectiveness. Additional questions that are being addressed by the scientific community are related to the establishment and maintenance of their longevity and memory state as well as to defining critical conditions underlying their transitions between discrete, but functionally different subtypes. In this article we review and comment latest approaches and techniques used for preparing large amounts of antigen-specific CTLs, suitable for clinical use.

  8. Antigen-specific B cells reactivate an effective cytotoxic T cell response against phagocytosed Salmonella through cross-presentation.

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    Jelle de Wit

    Full Text Available BACKGROUND: The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8(+ T cell response. Dendritic cells (DCs are considered to orchestrate the cytotoxic CD8(+ T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8(+ T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8(+ T cells is dependent on CD4(+ T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis. CONCLUSIONS/SIGNIFICANCE: B cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection.

  9. Antigen-specific cytotoxic T lymphocytes target airway CD103+ and CD11b+ dendritic cells to suppress allergic inflammation.

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    Daniels, N J; Hyde, E; Ghosh, S; Seo, K; Price, K M; Hoshino, K; Kaisho, T; Okada, T; Ronchese, F

    2016-01-01

    Allergic airway inflammation is driven by the recognition of inhaled allergen by T helper type 2 (Th2) cells in the airway and lung. Allergen-specific cytotoxic T lymphocytes (CTLs) can strongly reduce airway inflammation, however, the mechanism of their inhibitory activity is not fully defined. We used mouse models to show that allergen-specific CTLs reduced early cytokine production by Th2 cells in lung, and their subsequent accumulation and production of interleukin (IL)-4 and IL-13. In addition, treatment with specific CTLs also increased the proportion of caspase(+) dendritic cells (DCs) in mediastinal lymph node (MLN), and decreased the numbers of CD103(+) and CD11b(+) DCs in the lung. This decrease required expression of the cytotoxic mediator perforin in CTLs and of the appropriate MHC-antigen ligand on DCs, suggesting that direct CTL-DC contact was necessary. Lastly, lung imaging experiments revealed that in airway-challenged mice XCR1-GFP(+) DCs, corresponding to the CD103(+) DC subset, and XCR1-GFP(-) CD11c(+) cells, which include CD11b(+) DCs and alveolar macrophages, both clustered in the areas surrounding the small airways and were closely associated with allergen-specific CTLs. Thus, allergen-specific CTLs reduce allergic airway inflammation by depleting CD103(+) and CD11b(+) DC populations in the lung, and may constitute a mechanism through which allergic immune responses are regulated.

  10. Fibronectin EDA and CpG synergize to enhance antigen-specific Th1 and cytotoxic responses.

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    Julier, Ziad; de Titta, Alexandre; Grimm, Alizée J; Simeoni, Eleonora; Swartz, Melody A; Hubbell, Jeffrey A

    2016-05-01

    Subunit vaccines, employing purified protein antigens rather than intact pathogens, require the addition of adjuvants for enhanced immunogenicity with a correct balance between strong activation of the immune system and low toxicity. Here we show that the endogenous (i.e., autologous) non-toxic TLR4 agonist extra domain A type III repeat of fibronectin (FNIII EDA) can synergize with the exogenous (i.e., bacterial), toxic-at-high-dose, TLR9 agonist CpG to induce efficient cellular immune responses while keeping the dose of CpG low. The efficacy of the combined TLR agonists, even at half-doses, led to stronger dendritic cell activation, enhanced cytotoxic T lymphocyte activation as well as stronger humoral response, compared to the individual agonists given at full doses. Immune cells induced after vaccination with the co-adjuvanted formulation could mediate tumor regression in an E.G7-OVA tumor model, and eradicate circulating hepatitis B virus (HBV) in a transgenic HBV model. Together, these results show that endogenous TLR agonists, such as variants of FNIII EDA, can synergize with exogenous TLR ligands, such as CpG, and strongly enhance cellular immune responses, while improving their safety profile. PMID:27016652

  11. Tracking in vivo migration and distribution of antigen-specific cytotoxic T lymphocytes by 5,6-carboxyfluorescein diacetate succinimidyl ester staining during cancer immunotherapy

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    XU Wei-li; LI Suo-lin; WEN Ming; WEN Jun-ye; HAN Jie; ZHANG Hong-zhen; GAO Fei

    2013-01-01

    Background Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte (CTL) numbers,immunophenotype,tumor-specificity,and in vivo residence time,migration,and distribution.Therefore,tracing in vivo persistence,migration,and distribution of CTLs is important for cancer immunotherapy.Methods Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 (CCK-8) assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled melanoma antigen-specific cytotoxic T lymphocytes (CFSE-CTLs) for malignant melanoma cells in vitro were compared.Additionally,CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma,and their residence time,migration,and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue (%FI/g) in various tissues and analyzing tumor/non-tumor (T/NT) values.Anti-tumor effects of transferred CTLs and correlation between %FI/g and D-value of tumor size were analyzed.Results Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity.No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing (P=0.849) or interleukin-2 (P=0.318) and interferon-y (P=0.201)levels.Distribution of CTLs in vivo varied with time.A negative correlation between %FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed.CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion.CTLs were observed up to three weeks later in the tumor,liver,kidneys,and spleen; this was related to the abundant blood supply or the nature of immune organs.Conclusions CCK-8 assay is a novel method to select optimal CFSE staining concentrations.Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors.CFSE fluorescent markers can trace in vivo CTL persistence,migration,and distribution because of its

  12. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

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    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

    2016-03-01

    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 (Fc domain) regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

  13. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

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    Alison E Mahan

    2016-03-01

    Full Text Available 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 (Fc domain regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

  14. Regeneration of tumor antigen-specific CTLs utilizing iPS technology.

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    Maeda, Takuya; Masuda, Kyoko; Kawamoto, Hiroshi

    2016-08-01

    Tumor immunotherapy, especially tumor antigen specific T cell therapy, is currently attracting attention. However, a critical issue still awaits resolution; it is difficult to efficiently expand tumor antigen-specific T cells. To solve this problem, we are now utilizing iPS cell technology. When iPS cells are established from tumor antigen specific T cells, T cells regenerated from these iPS cells are expected to express the same TCRs as the original T cells. In line with this concept, we succeeded in regenerating tumor antigen specific cytotoxic T cells. The regenerated T cells exhibited TCR specific killing activity comparable to that of the original cells, and were able to kill leukemia cells in an antigen-specific manner. We are currently endeavoring to apply this method clinically. In the future, we intend to establish an allogeneic transfusion system, in which various tumor antigen specific T-iPS cells from a wide range of HLA haplotype homozygous donors will be lined up as a "T-iPS cell bank", with the aim of making off-the-shelf tumor immunotherapy a reality. PMID:27599425

  15. Antigen-specific active immunotherapy for ovarian cancer

    NARCIS (Netherlands)

    Leffers, N.; Daemen, T.; Helfrich, W.; Boezen, H. M.; Cohlen, B. J.; Melief, Cornelis; Nijman, H. W.

    2010-01-01

    BACKGROUND: Despite advances in chemotherapy, prognosis of ovarian cancer remains poor. Antigen-specific active immunotherapy aims to induce a tumour-antigen-specific anti-tumour immune responses as an alternative treatment for ovarian cancer. OBJECTIVES: To assess feasibility of antigen-specific ac

  16. Antigen-Specific versus Non-Antigen-Specific Immunoadsorption in ABO-Incompatible Renal Transplantation.

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    Gerold Thölking

    Full Text Available ABO-incompatible (ABOi renal transplantation (RTx from living donors is an established procedure to expand the donor pool for patients with end stage renal disease. Immunoadsorption (IA is a standard procedure for the removal of preformed antibodies against the allograft. In this study, antigen-specific and non-antigen-specific IA in ABOi RTx were compared.10 patients underwent antigen-specific IA (Glycosorb group and 13 patients non-antigen-specific IA (Immunosorba group. The effects of both procedures regarding antibody reduction, number of treatments, complications, costs, as well as the allograft function and patient survival were compared between both groups.Although the IgG levels were reduced equally by both procedures (p=0.82, the reduction of the IgM level was more effective in the Glycosorb group (p=0.0172. Patients in both groups required a median number of 6 IA before ABOi RTx. Allograft function at one year after AB0i RTx was similar in both groups (estimated glomerular filtration rate: 66 vs. 64 ml/min/1.73m² respectively, with a death-censored graft survival of 90.0% and 92.3% respectively. Complication rates did not differ between procedures. Due to the reuse of non-antigen-specific Immunosorba columns, costs were considerably lower in this group; however, the use of the Immunosorba-based IA was less time-efficient.Considering upcoming alternatives as simultaneous performance of dialysis and IA or a possible reuse of Glycosorb columns, this might become less relevant in the future.

  17. Rapid production of antigen-specific monoclonal antibodies from a variety of animals

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    Kurosawa Nobuyuki

    2012-09-01

    Full Text Available Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

  18. Human antigen-specific regulatory T cells generated by T cell receptor gene transfer.

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    Todd M Brusko

    Full Text Available BACKGROUND: Therapies directed at augmenting regulatory T cell (Treg activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff activity as determined by tumor cell growth and luciferase reporter

  19. Measuring antigen-specific immune responses: 2008 Update

    NARCIS (Netherlands)

    J.W. Gratama (Jan-Willem); F. Kern (Florian); F. Manca (Fabrizio); M. Roederer (Mario)

    2008-01-01

    textabstractOverall, the last 10 years have seen an explosion in the field of antigen-specific immune response monitoring. As summarized in this issue of Cytometry and at the MASIR conferences, these technologies have provided new insights into the basic biology of the immune system and are beginnin

  20. Dissection of T-cell antigen specificity in human melanoma

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Albæk Thrue, Charlotte; Junker, Niels;

    2012-01-01

    Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma-as...... from different fragments of resected melanoma lesions. In summary, our findings provide an initial definition of T-cell populations contributing to tumor recognition in TILs although the specificity of many tumor-reactive TILs remains undefined.......Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma......-associated antigens and applying a novel technology for high-throughput analysis of T-cell responses, we dissected the composition of melanoma-restricted T-cell responses in 63 TIL cultures. T-cell reactivity screens against 175 melanoma-associated epitopes detected 90 responses against 18 different epitopes...

  1. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

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    Maria Domina

    Full Text Available There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  2. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

    KAUST Repository

    Domina, Maria

    2014-12-04

    There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  3. Antigen-specific immune reactions to ischemic stroke

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    Xabier eUrra

    2014-09-01

    Full Text Available Brain proteins are detected in the CSF and blood of stroke patients and their concentration is related to the extent of brain damage. Antibodies against brain antigens develop after stroke, suggesting a humoral immune response to the brain injury. Furthermore, induced immune tolerance is beneficial in animal models of cerebral ischemia. The presence of circulating T cells sensitized against brain antigens, and antigen presenting cells (APCs carrying brain antigens in draining lymphoid tissue of stroke patients support the notion that stroke might induce antigen-specific immune responses. After stroke, brain proteins that are normally hidden from the periphery, inflammatory mediators, and danger signals can exit the brain through several efflux routes. They can reach the blood after leaking out of the damaged blood-brain barrier or following the drainage of interstitial fluid to the dural venous sinus, or reach the cervical lymph nodes through the nasal lymphatics following CSF drainage along the arachnoid sheaths of nerves across the nasal submucosa. The route and mode of access of brain antigens to lymphoid tissue could influence the type of response. Central and peripheral tolerance prevents autoimmunity, but the actual mechanisms of tolerance to brain antigens released into the periphery in the presence of inflammation, danger signals, and APCs, are not fully characterized. Stroke does not systematically trigger autoimmunity, but under certain circumstances, such as pronounced systemic inflammation or infection, autoreactive T cells could escape the tolerance controls. Further investigation is needed to elucidate whether antigen-specific immune events could underlie neurological complications impairing stroke outcome.

  4. Polyclonal Peptide Antisera.

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    Pihl, Tina H; Illigen, Kristin E; Houen, Gunnar

    2015-01-01

    Polyclonal antibodies are relatively easy to produce and may supplement monoclonal antibodies for some applications or even have some advantages. The choice of species for production of (peptide) antisera is based on practical considerations, including availability of immunogen (vaccine) and animals. Two major factors govern the production of antisera: the nature of adaptive immune responses, which take place over days/weeks and ethical guidelines for animal welfare. Here, simple procedures for immunization of mice, rabbits, sheep, goats, pigs, horses, and chickens are presented. PMID:26424267

  5. IL-35 inhibits HBV antigen-specific IFN-γ-producing CTLs in vitro.

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    Li, Xuefen; Tian, Li; Dong, Yuejiao; Zhu, Qiaoyun; Wang, Yiyin; Han, Wenzheng; Liu, Xia; Ni, Qin; Chen, Yu; Li, Lanjuan

    2015-09-01

    Interleukin (IL)-35 is an inhibitory cytokine consisting of IL-12A and Epstein-Barr virus-induced gene 3 (Ebi3) and is required by regulatory T-cells (Tregs) for maximal activity. During chronic hepatitis B virus (HBV) infection, Tregs have immunosuppressive effects on HBV-specific T helper (Th) cells, yet little is known about the complex regulation of Tregs and their contribution to the inadequate immune system response to the virus. In the present study, we investigated whether IL-35 is involved in HBV-related cellular immune responses. Cluster of differentiation (CD)4(+) T-cells from peripheral blood were derived from healthy volunteers, resolved HBV individuals and chronic active hepatitis B patients and stimulated with CD3/28-conjugated beads. We analysed mRNA and protein levels of IL-35 and assessed the inhibitory effect of IL-35 on HBV core antigen-specific cytotoxic T lymphocytes (CTLs), dendritic cells (DCs) and effector T-cells (Teffs). Correlation analyses between liver inflammation and HBV DNA load were conducted. Results show that chronic HBV patients harbour significantly higher levels of Ebi3 mRNA and protein in CD4(+) T-cells compared with healthy volunteers and resolved HBV individuals. IL-35 suppressed the proliferation of HBV antigen-specific CTLs and interferon (IFN)-γ production in vitro. Ex vivo, IL-35 decreased the proliferation of CD4(+)CD45RA(+) naïve T-cells, especially in CD4(+)CD25(-)CD45RA(+) naïve Teffs. IL-35 inhibited the expansion of CD11c(+) DCs. Our data indicate that IL-35 is highly expressed in chronic HBV CD4(+) T-cells and plays an important role in the inhibition of the cellular immune response in chronic HBV.

  6. Suppression of experimental autoimmune encephalomyelitis by intravenously administered polyclonal immunoglobulins.

    Science.gov (United States)

    Achiron, A; Mor, F; Margalit, R; Cohen, I R; Lider, O; Miron, S

    2000-11-01

    Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats either by active immunization with myelin basic protein (MBP) or by adoptive transfer using anti-MBP specific CD4(+)T cells. Treatment with human polyclonal immunoglobulins (IgG) effectively suppressed active EAE. Time-dependent experiments demonstrated that the effect of IgG was manifested only when treatment was given immediately after immunization; administration from day 7 after disease induction did not suppress the disease. In the adoptive transfer model of EAE, IgG had no effect in vivo. However, pretreatment in vitro of the antigen-specific T-cells with IgG inhibited their ability to mediate adoptive EAE, as it did in active EAE. Similarly, in vitro IgG pretreatment of the antigen-specific T-cells suppressed the proliferative response to MBP. Fluorescent Activated Cell Sorter (FACS) analysis demonstrated the binding of IgG to activated T-cell lines that was inhibited by soluble Fc molecules. The differential effects of IgG on active EAE and on the adoptive transfer of EAE suggest that IgG in vivo can suppress disease by acting during the early phase of the immune response which involves naive T cells. The inhibition of T-cell proliferation and adoptive transfer of EAE by incubation of T cells in vitro appears to require higher concentrations of IgG than those obtained in vivo. PMID:11040073

  7. Do natural Treg become activated to antigen specific Treg in transplantation and in autoimmunity?

    OpenAIRE

    Bruce Milne Hall; Tran, Giang T.; Nirupama eVerma; Karren ePlain; Catherine M. Robinson; Masaru eNomura; Hodgkinson, Suzanne J.

    2013-01-01

    Antigen specific Treg are often CD4+CD25+FoxP3+T cells, with a phenotype similar to natural Treg (nTreg). It is assumed that nTreg cannot develop into an antigen specific Treg as repeated culture with IL-2 and a specific antigen does not increase the capacity or potency of nTreg to promote immune tolerance or suppress in vitro. This has led to an assumption that antigen specific Treg mainly develop from CD4+CD25-FoxP3- T cells, by activation with antigen and TGF-beta in the absence of infla...

  8. Adoptive Immunotherapy for Hematological Malignancies Using T Cells Gene-Modified to Express Tumor Antigen-Specific Receptors

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujiwara

    2014-12-01

    Full Text Available Accumulating clinical evidence suggests that adoptive T-cell immunotherapy could be a promising option for control of cancer; evident examples include the graft-vs-leukemia effect mediated by donor lymphocyte infusion (DLI and therapeutic infusion of ex vivo-expanded tumor-infiltrating lymphocytes (TIL for melanoma. Currently, along with advances in synthetic immunology, gene-modified T cells retargeted to defined tumor antigens have been introduced as “cellular drugs”. As the functional properties of the adoptive immune response mediated by T lymphocytes are decisively regulated by their T-cell receptors (TCRs, transfer of genes encoding target antigen-specific receptors should enable polyclonal T cells to be uniformly redirected toward cancer cells. Clinically, anticancer adoptive immunotherapy using genetically engineered T cells has an impressive track record. Notable examples include the dramatic benefit of chimeric antigen receptor (CAR gene-modified T cells redirected towards CD19 in patients with B-cell malignancy, and the encouraging results obtained with TCR gene-modified T cells redirected towards NY-ESO-1, a cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. This article overviews the current status of this treatment option, and discusses challenging issues that still restrain the full effectiveness of this strategy, especially in the context of hematological malignancy.

  9. Characterizing complex polysera produced by antigen-specific immunization through the use of affinity-selected mimotopes.

    Directory of Open Access Journals (Sweden)

    Galina Denisova

    Full Text Available BACKGROUND: Antigen-based (as opposed to whole organism vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity. METHODOLOGY/PRINCIPAL FINDINGS: Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets. CONCLUSIONS/SIGNIFICANCE: By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines.

  10. Impaired Antigen-Specific Immune Response to Vaccines in Children with Antibody Production Defects

    OpenAIRE

    Szczawinska-Poplonyk, Aleksandra; Breborowicz, Anna; Samara, Husam; Ossowska, Lidia; Dworacki, Grzegorz

    2015-01-01

    The impaired synthesis of antigen-specific antibodies, which is indispensable for an adaptive immune response to infections, is a fundamental pathomechanism that leads to clinical manifestations in children with antibody production defects. The aim of this study was to evaluate the synthesis of antigen-specific antibodies following immunization in relation to peripheral blood B cell subsets in young children with hypogammaglobulinemia. Twenty-two children, aged from 8 to 61 months, with a def...

  11. Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Kvistborg, Pia; Frøsig, Thomas Mørch;

    2012-01-01

    Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specif......(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h....

  12. Antigen-specific memory Treg control memory responses to influenza virus infection

    OpenAIRE

    Brincks, Erik L.; Roberts, Alan D.; Cookenham, Tres; Sell, Stewart; Kohlmeier, Jacob E.; Blackman, Marcia A.; Woodland, David L

    2013-01-01

    Regulatory CD4+FoxP3+ T cells (Treg) are key regulators of inflammatory responses and control the magnitude of cellular immune responses to viral infections. However, little is known about how Treg contribute to immune regulation during memory responses to previously-encountered pathogens. Here we utilized influenza NP311-325/IAb Class II tetramers to track the antigen-specific Treg response to primary and secondary influenza virus infections. During secondary infections, antigen-specific mem...

  13. Tracking antigen-specific T-cells during clinical tolerance induction in humans.

    Directory of Open Access Journals (Sweden)

    Aamir Aslam

    Full Text Available Allergen immunotherapy presents an opportunity to define mechanisms of induction of clinical tolerance in humans. Significant progress has been made in our understanding of changes in T cell responses during immunotherapy, but existing work has largely been based on functional T cell assays. HLA-peptide-tetrameric complexes allow the tracking of antigen-specific T-cell populations based on the presence of specific T-cell receptors and when combined with functional assays allow a closer assessment of the potential roles of T-cell anergy and clonotype evolution. We sought to develop tools to facilitate tracking of antigen-specific T-cell populations during wasp-venom immunotherapy in people with wasp-venom allergy. We first defined dominant immunogenic regions within Ves v 5, a constituent of wasp venom that is known to represent a target antigen for T-cells. We next identified HLA-DRB1*1501 restricted epitopes and used HLA class II tetrameric complexes alongside cytokine responses to Ves v 5 to track T-cell responses during immunotherapy. In contrast to previous reports, we show that there was a significant initial induction of IL-4 producing antigen-specific T-cells within the first 3-5 weeks of immunotherapy which was followed by reduction of circulating effector antigen-specific T-cells despite escalation of wasp-venom dosage. However, there was sustained induction of IL-10-producing and FOXP3 positive antigen-specific T cells. We observed that these IL-10 producing cells could share a common precursor with IL-4-producing T cells specific for the same epitope. Clinical tolerance induction in humans is associated with dynamic changes in frequencies of antigen-specific T-cells, with a marked loss of IL-4-producing T-cells and the acquisition of IL-10-producing and FOXP3-positive antigen-specific CD4+ T-cells that can derive from a common shared precursor to pre-treatment effector T-cells. The development of new approaches to track antigen

  14. Suppression of polyclonal and antigen-specific murine IgG1 but not IgE responses by neutralizing interleukin-6 in vivo

    OpenAIRE

    Ommen, R.; Vredendaal, Astrid; Savelkoul, Huub

    1994-01-01

    textabstractThe crucial role of interleukin (IL)-4 in the induction of murine IgG1 and IgE responses, which are coupled through the process of sequential isotype switching, has been well documented. Whereas IL-4 is obligatory for the induction of IgE responses, it enhances IgG1 responses. In this study, using neutralizing antibodies, we provide evidence that, besides IL-4, also IL-6 is required for obtaining peak IgG1 responses. The mRNA levels of these two cytokines are coordinately expresse...

  15. Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines

    DEFF Research Database (Denmark)

    Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl

    2016-01-01

    Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is ......Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen...... in the cells even throughout division. This leads to daughter cells containing half the fluorescence of their parents. When lymphocytes are loaded with CFSE prior to ex vivo stimulation with specific antigen, the measurement of serial halving of its fluorescence by flow cytometry identifies the cells...

  16. Antigen-specific monoclonal antibodies isolated from B cells expressing constitutively active STAT5.

    Directory of Open Access Journals (Sweden)

    Ferenc A Scheeren

    Full Text Available BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5. Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells.

  17. Cryopreservation of MHC Multimers: Recommendations for Quality Assurance in Detection of Antigen Specific T Cells

    OpenAIRE

    Hadrup, Sine Reker; Maurer, Dominik; Laske, Karoline; Frøsig, Thomas Mørch; Andersen, Sofie Ramskov; Britten, Cedrik M.; Sjoerd H van der Burg; Walter, Steffen; Gouttefangeas, Cécile

    2014-01-01

    Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the pep...

  18. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kleinhenz, M.E.; Ellner, J.J.

    1985-07-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

  19. Antigen-Specific CD4+ T Cells Recognize Epitopes of Protective Antigen following Vaccination with an Anthrax Vaccine

    OpenAIRE

    Laughlin, Elsa M.; Miller, Joseph D.; James, Eddie; Fillos, Dimitri; Ibegbu, Chris C.; Mittler, Robert S.; Akondy, Rama; Kwok, William; Ahmed, Rafi; Nepom, Gerald,

    2007-01-01

    Detection of antigen-specific CD4+ T cells is facilitated by the use of fluorescently labeled soluble peptide-major histocompatibility complex (MHC) multimers which mirror the antigen specificity of T-cell receptor recognition. We have used soluble peptide-MHC class II tetramers containing peptides from the protective antigen (PA) of Bacillus anthracis to detect circulating T cells in peripheral blood of subjects vaccinated with an anthrax vaccine. PA-specific HLA class II-restricted T lympho...

  20. Physiological level Production of Antigen-Specific Human Immunoglobulin in Cloned Transchromosomic Cattle

    OpenAIRE

    Akiko Sano; Hiroaki Matsushita; Hua Wu; Jin-An Jiao; Poothappillai Kasinathan; Eddie J. Sullivan; Zhongde Wang; Yoshimi Kuroiwa

    2013-01-01

    Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human...

  1. Procedure for preparing peptide-major histocompatibility complex tetramers for direct quantification of antigen-specific cytotoxic T lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Xian-Hui He; Li-Hui Xu; Yi Liu

    2005-01-01

    AIM: To establish a simplified method for generating peptide-major histocompatibility complex (MHC) class I tetramers.METHODS: cDNAs encoding the extracellular domain of human lymphocyte antigen (HLA)-A*0201 heavy chain (A2) and β2-microglobulin (β2m) from total RNA extracted from leukocytes of HLA-A2+ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and β2m proteins were expressed in Escherichia coli strain BL21(DE3) and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain β2m and HLA-A2-restricted peptide antigens. The refolded A2monomers were biotinylated with a commercial biotinylation enzyme (BirA) and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4:1. Flow cytometry was used to confirm the expected tetramer staining of CD8+ T cells.RESULTS: Recombinant genes for HLA-A*0201 heavy chain (A2) fused to a BirA substrate peptide (A2-BSP) and mature β2m from HLA-A2+ donor leukocytes were successfully doned and highly expressed in E. coli. Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of β2m and peptides loaded with either human cytomegalovirus pp65495-503 peptide (NLVPMVATV,NLV; designated as A2-NLV) or influenza virus matrix protein Mp58-66 peptide (GILGFVFTL, GIL; designated as A2-GIL). Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplication as revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8+ T cells from a HLA-A2+ donor,but failed to bind to those from a HLA-A2- donor.CONCLUSION: The procedure is simple and efficient for generating peptide-MHC tetramers.

  2. Antigen-Specific Cytotoxic T Lymphocytes can Target Chemoresistant Side-Population Tumor Cells in Hodgkin’s Lymphoma

    OpenAIRE

    Shafer, Jessica A.; Cruz, Conrad R.; Leen, Ann M; Ku, Stephanie; LU, AN; Rousseau, Alexandra; Heslop, Helen E.; Rooney, Cliona M; Bollard, Catherine M.; Foster, Aaron E.

    2010-01-01

    Side-population (SP) analysis has been used to identify progenitor cells from normal and malignant tissues as well as revealing tumor cells with increased resistance to radiation and chemotherapy. Despite enhanced chemoresistance, tumor SP cells may still express tumor associated antigens (TAA) which may render them susceptible to elimination by the immune system. In this study, we show that both Hodgkin’s lymphoma (HL) cell lines and primary HL tumor samples contain a distinct SP phenotype. ...

  3. A Peptide Mimicking a Region in Proliferating Cell Nuclear Antigen Specific to Key Protein Interactions Is Cytotoxic to Breast Cancer

    OpenAIRE

    Smith, Shanna J.; Gu, Long; Phipps, Elizabeth A.; Lacey E Dobrolecki; Mabrey, Karla S.; Gulley, Pattie; Dillehay, Kelsey L; Dong, Zhongyun; Fields, Gregg B.; Chen, Yun-Ru; Ann, David; Hickey, Robert J.; Malkas, Linda H.

    2015-01-01

    Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and much of the regulation is a result of the inherent competition for this docking site. If this target region of PCNA is modified, the DNA replication and repair process in cancer cells is potentially altered. Exploitation of this cancer-associated region has imp...

  4. Impaired Antigen-Specific Immune Response to Vaccines in Children with Antibody Production Defects.

    Science.gov (United States)

    Szczawinska-Poplonyk, Aleksandra; Breborowicz, Anna; Samara, Husam; Ossowska, Lidia; Dworacki, Grzegorz

    2015-08-01

    The impaired synthesis of antigen-specific antibodies, which is indispensable for an adaptive immune response to infections, is a fundamental pathomechanism that leads to clinical manifestations in children with antibody production defects. The aim of this study was to evaluate the synthesis of antigen-specific antibodies following immunization in relation to peripheral blood B cell subsets in young children with hypogammaglobulinemia. Twenty-two children, aged from 8 to 61 months, with a deficiency in one or more major immunoglobulin classes participated in the study. Postvaccination antibodies against tetanus and diphtheria toxoids, the surface antigen of the hepatitis B virus, and the capsular Haemophilus influenzae type b polysaccharide antigen were assessed along with an immunophenotypic evaluation of peripheral blood B lymph cell maturation. A deficiency of antibodies against the tetanus toxoid was assessed in 73% of cases and that against the diphtheria toxoid was assessed in 68% of cases, whereas a deficiency of antibodies against the surface antigen of the hepatitis B virus was revealed in 59% of the children included in the study. A defective response to immunization with a conjugate vaccine with the Haemophilus influenzae type b polysaccharide antigen was demonstrated in 55% of hypogammaglobulinemic patients. Increased proportions of transitional B lymph cells and an accumulation of plasmablasts accompanied antibody deficiencies. The defective response to vaccine protein and polysaccharide antigens is a predominating disorder of humoral immunity in children with hypogammaglobulinemia and may result from a dysfunctional state of the cellular elements of the immune system. PMID:26018535

  5. Antigen-Specific Gene Therapy after Immunisation Reduces the Severity of Collagen-Induced Arthritis

    Directory of Open Access Journals (Sweden)

    Tove Eneljung

    2013-01-01

    Full Text Available Reestablishment of tolerance induction in rheumatoid arthritis (RA would be an optimal treatment with few, if any, side effects. However, to develop such a treatment further insights in the immunological mechanisms governing tolerance are needed. We have developed a model of antigen-specific tolerance in collagen type II (CII induced arthritis (CIA using lentivirus-based gene therapy. The immunodominant epitope of CII was inserted into a lentivirus vector to achieve expression on the MHC class II molecule and the lentiviral particles were subsequently intravenously injected at different time points during CIA. Injection of lentiviral particles in early phases of CIA, that is, at day 7 or day 26 after CII immunisation, partially prevented development of arthritis, decreased the serum levels of CII-specific IgG antibodies, and enhanced the suppressive function of CII-specific T regulatory cells. When lentiviral particles were injected during manifest arthritis, that is, at day 31 after CII immunisation, the severity of arthritis progression was ameliorated, the levels of CII-specific IgG antibodies decreased and the proportion of T regulatory cells increased. Thus, antigen-specific gene therapy is effective when administered throughout the inflammatory course of arthritis and offers a good model for investigation of the basic mechanisms during tolerance in CIA.

  6. Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination.

    Science.gov (United States)

    Qi, Qian; Cavanagh, Mary M; Le Saux, Sabine; NamKoong, Hong; Kim, Chulwoo; Turgano, Emerson; Liu, Yi; Wang, Chen; Mackey, Sally; Swan, Gary E; Dekker, Cornelia L; Olshen, Richard A; Boyd, Scott D; Weyand, Cornelia M; Tian, Lu; Goronzy, Jörg J

    2016-03-30

    Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood can escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. Although all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen-reactive TCRs, including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection that occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single-booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important readout to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection.

  7. Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination.

    Science.gov (United States)

    Qi, Qian; Cavanagh, Mary M; Le Saux, Sabine; NamKoong, Hong; Kim, Chulwoo; Turgano, Emerson; Liu, Yi; Wang, Chen; Mackey, Sally; Swan, Gary E; Dekker, Cornelia L; Olshen, Richard A; Boyd, Scott D; Weyand, Cornelia M; Tian, Lu; Goronzy, Jörg J

    2016-03-30

    Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood can escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. Although all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen-reactive TCRs, including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection that occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single-booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important readout to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection. PMID:27030598

  8. High-throughput identification of antigen-specific TCRs by TCR gene capture

    DEFF Research Database (Denmark)

    Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia;

    2013-01-01

    The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We...... have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting...... knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer....

  9. Recent Advance in Antigen-Specific Immunotherapy for Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Norimitsu Kadowaki

    2011-01-01

    Full Text Available Relapse after chemotherapy is inevitable in the majority of patients with acute myeloid leukemia (AML. Thus, it is necessary to develop novel therapies that have different antileukemic mechanisms. Recent advances in immunology and identification of promising leukemia-associated antigens open the possibilities for eradicating minimal residual diseases by antigen-specific immunotherapy after chemotherapy. Several methods have been pursued as immunotherapies for AML: peptide vaccines, granulocyte-macrophage colony-stimulating factor-secreting tumor vaccines, dendritic cell vaccines, and adoptive T cell therapy. Whereas immunogenicity and clinical outcomes are improving in these trials, severe adverse events were observed in highly avid engineered T cell therapies, indicating the importance of the balance between effectiveness and side effects in advanced immunotherapy. Such progress in inducing antitumor immune responses, together with strategies to attenuate immunosuppressive factors, will establish immunotherapy as an important armament to combat AML.

  10. Tumor associated antigen specific T-cell populations identified in ex vivo expanded TIL cultures

    DEFF Research Database (Denmark)

    Junker, Niels; Kvistborg, Pia; Køllgaard, Tania;

    2012-01-01

    Ex vivo expanded tumor infiltrating lymphocytes (TILs) from malignant melanoma (MM) and head & neck squamous cell carcinoma (HNSCC) share a similar oligoclonal composition of T effector memory cells, with HLA class I restricted lysis of tumor cell lines. In this study we show that ex vivo expanded...... TILs from MM and HNSCC demonstrate a heterogeneous composition in frequency and magnitude of tumor associated antigen specific populations by Elispot IFN¿ quantitation. TILs from MM and HNSCC shared reactivity towards NY ESO-1, cyclin B1 and Bcl-x derived peptides. Additionally we show that dominating...... the heterogeneous tumors upon adoptive transfer; increasing the probability of tumor control by minimizing immune evasion by tumor cell escape variants....

  11. Analysis of antigen specific T cells in diabetes - Lessons from pre-clinical studies and early clinical trials.

    Science.gov (United States)

    Krishnamurthy, Balasubramanian; Selck, Claudia; Chee, Jonathan; Jhala, Guarang; Kay, Thomas W H

    2016-07-01

    Antigen-specific immune tolerance promises to provide safe and effective therapies to prevent type 1 diabetes (T1D). Antigen-specific therapy requires two components: well-defined, clinically relevant autoantigens; and safe approaches to inducing tolerance in T cells specific for these antigens. Proinsulin is a critical autoantigen in both NOD mice, based on knockout mouse studies and induction of immune tolerance to proinsulin preventing disease whereas most antigens cannot, and also in human T1D based on proinsulin-specific T cells being found in the islets of affected individuals and the early appearance of insulin autoantibodies. Effective antigen-specific therapies that prevent T1D in humans have not yet been developed although doubt remains about the best molecular form of the antigen, the dose and the route of administration. Preclinical studies suggest that antigen specific therapy is most useful when administered before onset of autoimmunity but this time-window has not been tested in humans until the recent "pre-point" study. There may be a 'window of opportunity' during the neonatal period when 'vaccine' like administration of proinsulin for a short period may be sufficient to prevent diabetes. After the onset of autoimmunity, naive antigen-specific T cells have differentiated into antigen-experienced memory cells and the immune responses have spread to multiple antigens. Induction of tolerance at this stage becomes more difficult although recent studies have suggested generation of antigen-specific TR1 cells can inhibit memory T cells. Preclinical studies are required to identify additional 'help' that is required to induce tolerance to memory T cells and develop protocols for effective therapy in individuals with established autoimmunity. PMID:27083395

  12. Amiloride enhances antigen specific CTL by faciliting HBV DNA vaccine entry into cells.

    Directory of Open Access Journals (Sweden)

    Shuang Geng

    Full Text Available The induction of relatively weak immunity by DNA vaccines in humans can be largely attributed to the low efficiency of transduction of somatic cells. Although formulation with liposomes has been shown to enhance DNA transduction of cultured cells, little, if any, effect is observed on the transduction of somatic tissues and cells. To improve the rate of transduction, DNA vaccine delivery by gene gun and the recently developed electroporation techniques have been employed. We report here that to circumvent requirement for such equipment, amiloride, a drug that is prescribed for hypertension treatment, can accelerate plasmid entry into antigen presenting cells (APCs both in vitro and in vivo. The combination induced APCs more dramatically in both maturation and cytokine secretion. Amiloride enhanced development of full CD8 cytolytic function including induction of high levels of antigen specific CTL and expression of IFN-γ+perforin+granzymeB+ in CD8+ T cells. Thus, amiloride is a facilitator for DNA transduction into host cells which in turn enhances the efficiency of the immune responses.

  13. The generation and antigen-specificity of CD4+CD25+ regulatory T cells.

    Science.gov (United States)

    Taams, Leonie S; Curnow, S John; Vukmanovic-Stejic, M; Akbar, Arne N

    2006-09-01

    CD4+CD25+ regulatory T cells are essential components of the immune system. They help to maintain immune tolerance by exerting suppressive effects on cells of the adaptive and innate immune system. In the last few years there has been an abundance of papers addressing the suppressive effects of CD4+CD25+ regulatory T cells and their putative role in various experimental disease models and human diseases. Despite the enormous amounts of data on these cells a number of controversial issues still exists. CD4+CD25+ regulatory T cells were originally described as thymus-derived anergic/suppressive T cells. Recent papers however indicate that these cells might also be generated in the periphery. Due to the thymic development of CD4+CD25+ regulatory T cells it was thought that these cells were specific for self-antigens. Indeed it was shown that CD4+CD25+ regulatory T cells could be positively selected upon high affinity interaction with self-antigens. However, evidence is accumulating that these cells might also interact with non-self antigens. Finally, in the literature there is conflicting evidence regarding the role of soluble factors versus cell-contact in the mechanism of suppression. The aim of this review is to summarize the evidence supporting these opposing viewpoints and to combine them into a general model for the origin, function and antigen-specificity of CD4+CD25+ regulatory T cells. PMID:16918478

  14. Lunasin alleviates allergic airway inflammation while increases antigen-specific Tregs.

    Directory of Open Access Journals (Sweden)

    Xiaowei Yang

    Full Text Available Lunasin is a naturally occurring peptide isolated from soybeans and has been explored in cancer treatment. Lunasin inhibits NF-κB activation and thus pro-inflammatory cytokine and mediator production in macrophages. In this study we demonstrate that lunasin can effectively suppress allergic airway inflammation in two murine models of asthma. In an OVA+Alum sensitization model, intranasal lunasin treatment at the time of OVA challenges significantly reduced total cells counts in bronchoalveolar lavage (BAL fluid and eosinophilia, peribronchiolar inflammatory infiltration, goblet cell metaplasia and airway IL-4 production. In an OVA+LPS intranasal sensitization model, lunasin treatment either at the time of sensitization or challenge has similar effects in suppress allergic airway inflammation including significantly reduced total cell and eosinophil counts in BAL fluid, inflammatory gene Fizz1 expression in the lung, and IL-4 production by OVA re-stimulated cells from mediastinal lymph nodes. We further show that intranasal instillation of OVA+lunasin significantly increases OVA-specific regulatory T cell (Treg accumulation in the lung comparing to OVA only treatment. Taken together, our results suggest lunasin as an anti-inflammatory agent can be potentially used in asthma therapy or as an adjuvant to enhance the induction of antigen-specific Tregs and thus boost the efficacy of allergy immunotherapy.

  15. Antigen-specific T cell–mediated gene therapy in collagen-induced arthritis

    Science.gov (United States)

    Nakajima, Atsuo; Seroogy, Christine M.; Sandora, Matthew R.; Tarner, Ingo H.; Costa, Gina L.; Taylor-Edwards, Cariel; Bachmann, Michael H.; Contag, Christopher H.; Fathman, C. Garrison

    2001-01-01

    Autoantigen-specific T cells have tissue-specific homing properties, suggesting that these cells may be ideal vehicles for the local delivery of immunoregulatory molecules. We tested this hypothesis by using type II collagen–specific (CII-specific) CD4+ T hybridomas or primary CD4+ T cells after gene transfer, as vehicles to deliver an immunoregulatory protein for the treatment of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). CII-specific T cells or hybridomas were transduced using retroviral vectors to constitutively express the IL-12 antagonist, IL-12 p40. Transfer of engineered CD4+ T cells after immunization significantly inhibited the development of CIA, while cells transduced with vector control had no effect. The beneficial effect on CIA of IL-12 p40-transduced T cells required TCR specificity against CII, since transfer of T cells specific for another antigen producing equivalent amounts of IL-12 p40 had no effect. In vivo cell detection using bioluminescent labels and RT-PCR showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed joints in mice with CIA. These results indicate that the local delivery of IL-12 p40 by T cells inhibited CIA by suppressing autoimmune responses at the site of inflammation. Modifying antigen-specific T cells by retroviral transduction for local expression of immunoregulatory proteins thus offers a promising strategy for treating RA. PMID:11375419

  16. Preserved antiviral adaptive immunity following polyclonal antibody immunotherapy for severe murine influenza infection.

    Science.gov (United States)

    Stevens, Natalie E; Hatjopolous, Antoinette; Fraser, Cara K; Alsharifi, Mohammed; Diener, Kerrilyn R; Hayball, John D

    2016-01-01

    Passive immunotherapy may have particular benefits for the treatment of severe influenza infection in at-risk populations, however little is known of the impact of passive immunotherapy on the formation of memory responses to the virus. Ideally, passive immunotherapy should attenuate the severity of infection while still allowing the formation of adaptive responses to confer protection from future exposure. In this study, we sought to determine if administration of influenza-specific ovine polyclonal antibodies could inhibit adaptive immune responses in a murine model of lethal influenza infection. Ovine polyclonal antibodies generated against recombinant PR8 (H1N1) hemagglutinin exhibited potent prophylactic capacity and reduced lethality in an established influenza infection, particularly when administered intranasally. Surviving mice were also protected against reinfection and generated normal antibody and cytotoxic T lymphocyte responses to the virus. The longevity of ovine polyclonal antibodies was explored with a half-life of over two weeks following a single antibody administration. These findings support the development of an ovine passive polyclonal antibody therapy for treatment of severe influenza infection which does not affect the formation of subsequent acquired immunity to the virus. PMID:27380890

  17. Preserved antiviral adaptive immunity following polyclonal antibody immunotherapy for severe murine influenza infection

    Science.gov (United States)

    Stevens, Natalie E.; Hatjopolous, Antoinette; Fraser, Cara K.; Alsharifi, Mohammed; Diener, Kerrilyn R.; Hayball, John D.

    2016-01-01

    Passive immunotherapy may have particular benefits for the treatment of severe influenza infection in at-risk populations, however little is known of the impact of passive immunotherapy on the formation of memory responses to the virus. Ideally, passive immunotherapy should attenuate the severity of infection while still allowing the formation of adaptive responses to confer protection from future exposure. In this study, we sought to determine if administration of influenza-specific ovine polyclonal antibodies could inhibit adaptive immune responses in a murine model of lethal influenza infection. Ovine polyclonal antibodies generated against recombinant PR8 (H1N1) hemagglutinin exhibited potent prophylactic capacity and reduced lethality in an established influenza infection, particularly when administered intranasally. Surviving mice were also protected against reinfection and generated normal antibody and cytotoxic T lymphocyte responses to the virus. The longevity of ovine polyclonal antibodies was explored with a half-life of over two weeks following a single antibody administration. These findings support the development of an ovine passive polyclonal antibody therapy for treatment of severe influenza infection which does not affect the formation of subsequent acquired immunity to the virus. PMID:27380890

  18. Nitric Oxide Limits the Expansion of Antigen-Specific T Cells in Mice Infected with the Microfilariae of Brugia pahangi

    Science.gov (United States)

    O'Connor, Richard A.; Devaney, Eileen

    2002-01-01

    Infection of BALB/c mice with the microfilariae (Mf) of the filarial nematode Brugia pahangi results in an antigen-specific proliferative defect that is induced by high levels of NO. Using carboxyfluorescein diacetate succinimydl ester and cell surface labeling, it was possible to identify a population of antigen-specific T cells from Mf-infected BALB/c mice that expressed particularly high levels of CD4 (CD4hi). These cells proliferated in culture only when inducible NO synthase was inhibited and accounted for almost all of the antigen-specific proliferative response under those conditions. CD4hi cells also expressed high levels of CD44, consistent with their status as activated T cells. A similar population of CD4hi cells was observed in cultures from Mf-infected gamma interferon receptor knockout (IFN-γR−/−) mice. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining revealed that the CD4+ T cells from Mf-infected wild-type mice were preferentially susceptible to apoptosis compared to CD4+ T cells from IFN-γR−/− mice. These studies suggest that the expansion of antigen-specific T cells in Mf-infected mice is limited by NO. PMID:12379675

  19. Transforming growth factor-beta inhibits human antigen-specific CD4(+) T cell proliferation without modulating the cytokine response

    NARCIS (Netherlands)

    Tiemessen, MM; Kunzmann, S; Schmidt-Weber, CB; Garssen, J; Bruijnzeel-Koomen, CAFM; Knol, EF; Van Hoffen, E

    2003-01-01

    Transforming growth factor (TGF)-beta has been demonstrated to play a key role in the regulation of the immune response, mainly by its suppressive function towards cells of the immune system. In humans, the effect of TGF-beta on antigen-specific established memory T cells has not been investigated y

  20. MRI contrast demonstration of antigen-specific targeting with an iron-based ferritin construct

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Edward G., E-mail: edward_walsh@brown.edu [Brown University, Department of Neuroscience (United States); Mills, David R. [Rhode Island Hospital/Warren Alpert Medical School of Brown University, Division of Hematology and Oncology, Department of Medicine (United States); Lim, Sierin; Sana, Barindra [Nanyang Technological University, Division of Bioengineering (Singapore); Brilliant, Kate E. [Rhode Island Hospital/Warren Alpert Medical School of Brown University, Division of Hematology and Oncology, Department of Medicine (United States); Park, William K. C. [Rhode Island Hospital, Department of Diagnostic Imaging (United States)

    2013-01-15

    A genetically modified ferritin has been examined for its properties as a tumor-selective magnetic resonance imaging (MRI) contrast agent. The engineered ferritin described herein was derived from Archaeoglobus fulgidus (AfFtn-AA), which stores a significantly greater quantity of iron than wild-type ferritins. Relaxivity measurements were taken at 3 Tesla of ferritin particles uniformly distributed in an agarose gel to assess relaxivities r{sub 1} and r{sub 2}. The r{sub 1} and r{sub 2} values of the uniformly distributed modified ferritin were significantly higher (r{sub 1} = 1,290 mM{sup -1} s{sup -1} and r{sub 2} = 5,740 mM{sup -1} s{sup -1}) than values observed for wild-type ferritin (e.g., horse spleen, r{sub 1} = 0.674 mM{sup -1} s{sup -1}, r{sub 2} = 95.54 mM{sup -1} s{sup -1}). The modified iron-enriched ferritin (14.5 nm diameter) was conjugated with a monoclonal antibody (10 nm length) against rat Necl-5, a cell surface glycoprotein overexpressed by many epithelial cancers. In vitro studies showed strong reactivity of the assembled nanoconjugate to transformed Necl-5 positive rat prostate epithelial cells. Furthermore, MRI demonstrated a significant T{sub 2} contrast with negligible T{sub 1} effect when bound to cells. These findings highlight the utility of the modified ferritin construct as a novel MRI contrast agent that can be manipulated to target antigen-specific tissues.

  1. Molecular tracking of antigen-specific T cell clones in neurological immune-mediated disorders

    Science.gov (United States)

    Muraro, Paolo A.; Wandinger, Klaus-Peter; Bielekova, Bibiana; Gran, Bruno; Marques, Adriana; Utz, Ursula; McFarland, Henry F.; Jacobson, Steve; Martin, Roland

    2016-01-01

    Summary T cells recognizing self or microbial antigens may trigger or reactivate immune-mediated diseases. Monitoring the frequency of specific T cell clonotypes to assess a possible link with the course of disease has been a difficult task with currently available technology. Our goal was to track individual candidate pathogenic T cell clones, selected on the basis of previous extensive studies from patients with immune-mediated disorders of the CNS, including multiple sclerosis, HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/ TSP) and chronic Lyme neuroborreliosis. We developed and applied a highly specific and sensitive technique to track single CD4+ and CD8+ T cell clones through the detection and quantification of T cell receptor (TCR) α or β chain complementarity-determining region 3 transcripts by real-time reverse transcriptase (RT)-PCR. We examined the frequency of the candidate pathogenic T cell clones in the peripheral blood and CSF during the course of neurological disease. Using this approach, we detected variations of clonal frequencies that appeared to be related to clinical course, significant enrichment in the CSF, or both. By integrating clono-type tracking with direct visualization of antigen-specific staining, we showed that a single T cell clone contributed substantially to the overall recognition of the viral peptide/MHC complex in a patient with HAM/ TSP. T cell clonotype tracking is a powerful new technology enabling further elucidation of the dynamics of expansion of autoreactive or pathogen-specific T cells that mediate pathological or protective immune responses in neurological disorders. PMID:12477694

  2. Polyclonal Antibody Therapies for Clostridium difficile Infection

    Directory of Open Access Journals (Sweden)

    Michael R. Simon

    2014-10-01

    Full Text Available Clostridium difficile infection has emerged as a growing worldwide health problem. The colitis of Clostridium difficile infection results from the synergistic action of C. difficile secreted toxins A and B upon the colon mucosa. A human monoclonal IgG anti-toxin has demonstrated the ability in combination therapy to reduce mortality in C. difficile challenged hamsters. This antibody is currently in a clinical trial for the treatment of human Clostridium difficile infection. More than one group of investigators has considered using polyclonal bovine colostral antibodies to toxins A and B as an oral passive immunization. A significant proportion of the healthy human population possesses polyclonal antibodies to the Clostridium difficile toxins. We have demonstrated that polyclonal IgA derived from the pooled plasma of healthy donors possesses specificity to toxins A and B and can neutralize these toxins in a cell-based assay. This suggests that secretory IgA prepared from such pooled plasma IgA may be able to be used as an oral treatment for Clostridium difficile infection.

  3. Tumor Antigen Specific Activation of Primary Human T-Cells Expressing a Virally Encoded Chimeric T-Cell Receptor Specific for p185HER2

    Institute of Scientific and Technical Information of China (English)

    杨建民; MichaelSFRIEDMAN; ChristopherMREYNOLDS; MarianneTHUBEN; LeeWILKE; JenniferFULLER; 李桥; ZeligESHHAR; JamesJMULE; KevimTMCDONAGH

    2004-01-01

    We have developed and tested chimeric T-cell receptors (TCR) specific for p185HER2. In these experiments,retroviral vectors expressing the N297 or N29ξ receptors were constructed in pRET6. Amphotropic viral producer cells were established in the GALV-based PG13 packaging cell line. Ficoll purified human peripheral blood lymphocytes (PBL) were vitally transduced using an optimized protocol incorporating activation with immobilized anti-CD3/anti-CD28 monoclonal antibodies, followed by viral infection in the presence of fibronectin fragment CH296. Transduced cells were co-cultured with human tumor cell lines that overexpress (SK-OV-3) or underexpress (MCF7) p185HER2 to assay for antigen specific immune responses. Both CD4+ and CD8+ T-cells transduced with the N297 or N29ξ chTCR demonstrated HER2-specific antigen responses, as determined by release of Th1 like cytokines, and cellular cytotoxicity assays. Our results support the feasibility of adoptive immunothempy with genetically modified T-cells expressing a chTCR specific for p185HER2.

  4. Flow cytometric assessment of antigen-specific proliferation in peripheral chicken T cells by CFSE dilution

    DEFF Research Database (Denmark)

    Dalgaard, Tina; Norup, Liselotte Rothmann; Rubbenstroth, Dennis;

    2010-01-01

    peripheral mononuclear blood cells (PBMC) and to evaluate and optimize its performance in relation to detection of vaccine-induced chicken T cells specific for Newcastle disease virus (NDV). The method was based on analysis of CFSE dilution upon ex vivo recall stimulation with whole vaccine antigen. Analysis...... fetal calf serum with serum-free medium. It was rendered probable that antigen-specific cellular immunity can be assessed by this method as NDV-vaccinated chickens showed a significantly higher proliferative capacity than age-matched naïve controls. Furthermore it was shown that the recall stimulation...... lead to a proliferative response in T cells expressing αβ-type TCRs but also those expressing the γδ-type. In summary, the method was found challenging but nevertheless useful to quantify the proliferative response of chicken antigen-specific T cells. Further investigations though, are needed in order...

  5. Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates.

    Science.gov (United States)

    Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

    2014-12-18

    In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates.

  6. TIM-4, expressed by medullary macrophages, regulates respiratory tolerance by mediating phagocytosis of antigen-specific T cells

    OpenAIRE

    Albacker, Lee A; Yu, Sanhong; Bedoret, Denis; Lee, Wan-Ling; Umetsu, Sarah E.; Monahan, Sheena; Freeman, Gordon J.; Umetsu, Dale T.; DeKruyff, Rosemarie H.

    2012-01-01

    Respiratory exposure to antigen induces T cell tolerance via several overlapping mechanisms that limit the immune response. While the mechanisms involved in the development of Treg cells have received much attention, those that result in T cell deletion are largely unknown. Herein, we show that F4/80+ lymph node medullary macrophages expressing TIM-4, a phosphatidylserine receptor, remove antigen-specific T cells during respiratory tolerance, thereby reducing secondary T cell responses. Block...

  7. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

    OpenAIRE

    Kellermann, Gottfried H.; Lehmann, Paul V; Diana R. Roen; Chenggang Jin

    2013-01-01

    Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B....

  8. Fms-like tyrosine kinase-3 ligand alters antigen-specific responses to infections after severe burn injury

    OpenAIRE

    Bohannon, Julia; Fang, Geping; Cui, Weihua; Sherwood, Edward; Toliver-Kinsky, Tracy

    2009-01-01

    Burn patients are susceptible to opportunistic infections due partly to decreased immune functions, especially Th1-driven antigen-specific responses, which are regulated by dendritic cells. The dendritic cell growth factor, fms-like tyrosine kinase-3 ligand (FL), has been shown to increase resistance to P. aeruginosa, in a dendritic cell-dependent manner, in a mouse model of burn wound infection. The specific mechanisms of protection are not known. This study tested the hypothesis that FL can...

  9. Overnight resting of PBMC changes functional signatures of antigen specific T- cell responses: impact for immune monitoring within clinical trials.

    Directory of Open Access Journals (Sweden)

    Sarah Kutscher

    Full Text Available Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37 °C on the quantity and functionality of HIV-1, EBV, CMV, HBV and HCV specific CD4 and CD8 T-cell responses in a cohort of 21 individuals. We quantified total antigen specific T cells by multimer staining and used 10-color intracellular cytokine staining (ICS to determine IFNγ, TNFα, IL2 and MIP1β production. After an overnight resting significantly higher numbers of functionally active T cells were detectable by ICS for all tested antigen specificities, whereas the total number of antigen specific T cells determined by multimer staining remained unchanged. Overnight resting shifted the quality of T-cell responses towards polyfunctionality and increased antigen sensitivity of T cells. Our data suggest that the observed effect is mediated by T cells rather than by antigen presenting cells. We conclude that overnight resting of PBMC prior to ex vivo analysis of antiviral T-cell responses represents an efficient method to increase sensitivity of ICS-based methods and has a prominent impact on the functional phenotype of T cells.

  10. Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination.

    Science.gov (United States)

    Ellebedy, Ali H; Jackson, Katherine J L; Kissick, Haydn T; Nakaya, Helder I; Davis, Carl W; Roskin, Krishna M; McElroy, Anita K; Oshansky, Christine M; Elbein, Rivka; Thomas, Shine; Lyon, George M; Spiropoulou, Christina F; Mehta, Aneesh K; Thomas, Paul G; Boyd, Scott D; Ahmed, Rafi

    2016-10-01

    Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody. PMID:27525369

  11. Intranasal Vaccination Affords Localization and Persistence of Antigen-Specific CD8+ T Lymphocytes in the Female Reproductive Tract

    Directory of Open Access Journals (Sweden)

    Shailbala Singh

    2016-03-01

    Full Text Available Immunization strategies generating large numbers of antigen-specific T cells in the female reproductive tract (FRT can provide barrier protection against sexually-transmitted pathogens, such as the human immunodeficiency virus (HIV and human papillomaviruses (HPV. The kinetics and mechanisms of regulation of vaccine-induced adaptive T cell-mediated immune responses in FRT are less well defined. We present here evidence for intranasal delivery of the model antigen ovalbumin (OVA along with alpha-galactosylceramide adjuvant as a protein vaccine to induce significantly higher levels of antigen-specific effector and memory CD8+ T cells in the FRT, relative to other systemic and mucosal tissues. Antibody blocking of the CXCR3 receptor significantly reduced antigen-specific CD8+ T cells subsequent to intranasal delivery of the protein vaccine suggesting an important role for the CXCR3 chemokine-receptor signaling for T cell trafficking. Further, intranasal vaccination with an adenoviral vector expressing OVA or HIV-1 envelope was as effective as intramuscular vaccination for generating OVA- or ENV-specific immunity in the FRT. These results support the application of the needle-free intranasal route as a practical approach to delivering protein as well as DNA/virus vector-based vaccines for efficient induction of effector and memory T cell immunity in the FRT.

  12. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs

    Science.gov (United States)

    Dutta, Avijit; Huang, Ching-Tai; Lin, Chun-Yen; Chen, Tse-Ching; Lin, Yung-Chang; Chang, Chia-Shiang; He, Yueh-Chia

    2016-09-01

    Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity.

  13. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs

    Science.gov (United States)

    Dutta, Avijit; Huang, Ching-Tai; Lin, Chun-Yen; Chen, Tse-Ching; Lin, Yung-Chang; Chang, Chia-Shiang; He, Yueh-Chia

    2016-01-01

    Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity. PMID:27596047

  14. Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray.

    Directory of Open Access Journals (Sweden)

    Daniel S Chen

    2005-10-01

    Full Text Available BACKGROUND: In contrast to many animal model studies, immunotherapeutic trials in humans suffering from cancer invariably result in a broad range of outcomes, from long-lasting remissions to no discernable effect. METHODS AND FINDINGS: In order to study the T cell responses in patients undergoing a melanoma-associated peptide vaccine trial, we have developed a high-throughput method using arrays of peptide-major histocompatibility complexes (pMHC together with antibodies against secreted factors. T cells were specifically immobilized and activated by binding to particular pMHCs. The antibodies, spotted together with the pMHC, specifically capture cytokines secreted by the T cells. This technique allows rapid, simultaneous isolation and multiparametric functional characterization of antigen-specific T cells present in clinical samples. Analysis of CD8+ lymphocytes from ten melanoma patients after peptide vaccination revealed a diverse set of patient- and antigen-specific profiles of cytokine secretion, indicating surprising differences in their responsiveness. Four out of four patients who showed moderate or greater secretion of both interferon-gamma (IFNgamma and tumor necrosis factor-alpha (TNFalpha in response to a gp100 antigen remained free of melanoma recurrence, whereas only two of six patients who showed discordant secretion of IFNgamma and TNFalpha did so. CONCLUSION: Such multiparametric analysis of T cell antigen specificity and function provides a valuable tool with which to dissect the molecular underpinnings of immune responsiveness and how this information correlates with clinical outcome.

  15. Resistant prolactinoma: Is it monoclonal or polyclonal?

    Directory of Open Access Journals (Sweden)

    K. V. S. Hari Kumar

    2013-01-01

    Full Text Available Prolactinomas are solitary benign neoplasms and resistance to dopamine agonists occur in a small percentage of prolactinomas. Multiple pituitary adenomas are reported in less than 1% of pituitary adenomas and rarely result in resistant prolactinoma. We recently encountered an interesting patient of hyperprolactinemia with multiple pituitary microadenomas. Dopamine agonist use resulted in prolactin normalization and subsequent pregnancy resulted in drug withdrawal. Repeat evaluation after delivery showed a macroprolactinoma and dopamine agonist therapy resulted in biochemical cure without reduction in tumor size. We report the case for its presentation with multiple microadenomas progressing to macroprolactinoma suggesting polyclonal in origin.

  16. Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle.

    Directory of Open Access Journals (Sweden)

    Akiko Sano

    Full Text Available Therapeutic human polyclonal antibodies (hpAbs derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc cattle carrying a human artificial chromosome (HAC comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH and kappa-chain (hIGK germline loci (named as κHAC are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO. However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ in the pre-B cell receptor (pre-BCR complex, we partially replaced (bovinized the hIgM constant domain with the counterpart of bovine IgM (bIgM that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO, the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG can be produced from such Tc cattle.

  17. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Scott G Kitchen

    Full Text Available There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR. Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.

  18. Nematode-Derived Proteins Suppress Proliferation and Cytokine Production of Antigen-Specific T Cells via Induction of Cell Death

    Science.gov (United States)

    Hartmann, Wiebke; Brenz, Yannick; Kingsley, Manchang Tanyi; Ajonina-Ekoti, Irene; Brattig, Norbert W.; Liebau, Eva; Breloer, Minka

    2013-01-01

    In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host’s immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocercavolvulus-derived excreted/secreted proteins. Addition of two recombinant O. volvulus proteins, abundant larval transcript-2 (OvALT-2) and novel larval transcript-1 (OvNLT-1) to cell cultures of T cell receptor transgenic CD4+ and CD8+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by 3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O. volvulus protein, microfilariae surface-associated antigen (Ov103), did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O. volvulus, which interfere with the function of antigen-specific T cells in vitro. PMID:23861729

  19. Nematode-derived proteins suppress proliferation and cytokine production of antigen-specific T cells via induction of cell death.

    Directory of Open Access Journals (Sweden)

    Wiebke Hartmann

    Full Text Available In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host's immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocercavolvulus-derived excreted/secreted proteins. Addition of two recombinant O. volvulus proteins, abundant larval transcript-2 (OvALT-2 and novel larval transcript-1 (OvNLT-1 to cell cultures of T cell receptor transgenic CD4(+ and CD8(+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4(+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by (3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8(+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O. volvulus protein, microfilariae surface-associated antigen (Ov103, did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O. volvulus, which interfere with the function of antigen-specific T cells in vitro.

  20. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    Science.gov (United States)

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells. PMID:20164855

  1. Antigen-specific regulatory T cells and low dose of IL-2 in treatment of type 1 diabetes

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    Minh N. Pham

    2016-01-01

    Full Text Available Regulatory T cells (Tregs play an important role in preventing effector T-cell (Teff targeting of self-antigens that can lead to tissue destruction in autoimmune settings, including type 1 diabetes (T1D. Autoimmunity is caused in part by an imbalance between Teff and Tregs. Early attempts to treat with immunosuppressive agents have led to serious side effects, thus requiring a more targeted approach. Low-dose IL-2 (LD IL-2 can provide immuno-regulation with few side effects by preferentially acting on Tregs to drive tolerance. The concept of LD IL-2 as a therapeutic approach is supported by data in mouse models where autoimmunity is cured and further strengthened by success in human clinical studies in Hepatitis C Virus (HCV induced vasculitis, chronic graft vs host disease (GVHD and Alopecia areata (AA. Treatment will require identification of a safe therapeutic window, which is a difficult task given that patients are reported to have deficient or defective IL-2 production or signalling and have experienced mild activation of NK cells and eosinophils with LD IL-2 therapy. In T1D, a LD IL-2 clinical trial concluded that Tregs can be safely expanded in humans; however, the study was not designed to address efficacy. Antigen-specific therapies have also aimed at regulation of the autoimmune response, but have been filled with disappointment despite an extensive list of diverse islet antigens tested in humans. This approach could be enhanced through the addition of LD IL-2 to the antigenic treatment regimen to improve the frequency and function of antigen-specific Tregs, without global immunosuppression. Here we will discuss the use of LD IL-2 and islet antigen to enhance antigen-specific Tregs in T1D and focus on what is known about their immunological impact, their safety and potential efficacy, and need for better methods to identify therapeutic effectiveness.

  2. Prokaryotic Expression and Polyclonal Antibody Preparation of PRL3

    Institute of Scientific and Technical Information of China (English)

    SHEN Xing-gui; LI Wan-nan; WANG Jing; JIANG Yi-qun; LI Qing-shan; FU Xue-qi

    2007-01-01

    Phosphatase of regenerating liver 3(PRL3), which belongs to the superfamily of protein tyrosine phosphatases (PTPs), represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes.Presented here are the results of cloning, prokaryotic expression, purification, and polyclonal antibody preparation of PRL3. To obtain a specific polyclonal antibody against PRL3, the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns. Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein. The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.

  3. Vaccination with human induced pluripotent stem cells creates an antigen-specific immune response against HIV-1 gp160

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    Shinji eYoshizaki

    2011-02-01

    Full Text Available Induced pluripotent stem cells (iPSCs are artificially derived from somatic cells that have been transduced with defined reprogramming factors. A previous report has indicated the possibility of using iPSCs as an immune stimulator to generate antigen-specific immunity. In our current study, we have investigated whether human iPSCs (hiPSCs have the ability to enhance specific immune response against a human immunodeficiency virus type 1 (HIV-1 antigen in a xenogenic mouse model. Our results show that BALB/c mice immunized with hiPSCs transduced with an adenoviral vector encoding HIV-1 gp160 exhibited prominent antigen-specific cellular immune responses. We further found that pre-treatment of hiPSCs with ionizing radiation promotes the secretion of pro-inflammatory cytokines such as interleukin-1 alpha (IL-1α, IL-12 and IL-18. These cytokines might promote the activation of antigen-presenting cells and the effective induction of cellular immunity. Our present findings thus demonstrate that a hiPSCs-based vaccine has the potential to generate cellular immunity against viral antigens such as HIV-1 gp160 in a xenogenic condition.

  4. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

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    Gottfried H. Kellermann

    2013-09-01

    Full Text Available Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-g as a biomarker, we developed a new enzyme-linked immunospot method (iSpot Lyme™ to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease.

  5. CD8α− Dendritic Cells Induce Antigen-Specific T Follicular Helper Cells Generating Efficient Humoral Immune Responses

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    Changsik Shin

    2015-06-01

    Full Text Available Recent studies on T follicular helper (Tfh cells have significantly advanced our understanding of T cell-dependent B cell responses. However, little is known about the early stage of Tfh cell commitment by dendritic cells (DCs, particularly by the conventional CD8α+ and CD8α− DC subsets. We show that CD8α− DCs localized at the interfollicular zone play a pivotal role in the induction of antigen-specific Tfh cells by upregulating the expression of Icosl and Ox40l through the non-canonical NF-κB signaling pathway. Tfh cells induced by CD8α− DCs function as true B cell helpers, resulting in significantly increased humoral immune responses against various human pathogenic antigens, including Yersinia pestis LcrV, HIV Gag, and hepatitis B surface antigen. Our findings uncover a mechanistic role of CD8α− DCs in the initiation of Tfh cell differentiation and thereby provide a rationale for investigating CD8α− DCs in enhancing antigen-specific humoral immune responses for improving vaccines and therapeutics.

  6. Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration

    Science.gov (United States)

    Lopes Pinheiro, Melissa A; Kamermans, Alwin; Garcia-Vallejo, Juan J; van het Hof, Bert; Wierts, Laura; O'Toole, Tom; Boeve, Daniël; Verstege, Marleen; van der Pol, Susanne MA; van Kooyk, Yvette; de Vries, Helga E; Unger, Wendy WJ

    2016-01-01

    Trafficking of myelin-reactive CD4+ T-cells across the brain endothelium, an essential step in the pathogenesis of multiple sclerosis (MS), is suggested to be an antigen-specific process, yet which cells provide this signal is unknown. Here we provide direct evidence that under inflammatory conditions, brain endothelial cells (BECs) stimulate the migration of myelin-reactive CD4+ T-cells by acting as non-professional antigen presenting cells through the processing and presentation of myelin-derived antigens in MHC-II. Inflamed BECs internalized myelin, which was routed to endo-lysosomal compartment for processing in a time-dependent manner. Moreover, myelin/MHC-II complexes on inflamed BECs stimulated the trans-endothelial migration of myelin-reactive Th1 and Th17 2D2 cells, while control antigen loaded BECs did not stimulate T-cell migration. Furthermore, blocking the interaction between myelin/MHC-II complexes and myelin-reactive T-cells prevented T-cell transmigration. These results demonstrate that endothelial cells derived from the brain are capable of enhancing antigen-specific T cell recruitment. DOI: http://dx.doi.org/10.7554/eLife.13149.001 PMID:27336724

  7. Tumor-derived exosomes confer antigen-specific immunosuppression in a murine delayed-type hypersensitivity model.

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    Chenjie Yang

    Full Text Available Exosomes are endosome-derived small membrane vesicles that are secreted by most cell types including tumor cells. Tumor-derived exosomes usually contain tumor antigens and have been used as a source of tumor antigens to stimulate anti-tumor immune responses. However, many reports also suggest that tumor-derived exosomes can facilitate tumor immune evasion through different mechanisms, most of which are antigen-independent. In the present study we used a mouse model of delayed-type hypersensitivity (DTH and demonstrated that local administration of tumor-derived exosomes carrying the model antigen chicken ovalbumin (OVA resulted in the suppression of DTH response in an antigen-specific manner. Analysis of exosome trafficking demonstrated that following local injection, tumor-derived exosomes were internalized by CD11c+ cells and transported to the draining LN. Exosome-mediated DTH suppression is associated with increased mRNA levels of TGF-β1 and IL-4 in the draining LN. The tumor-derived exosomes examined were also found to inhibit DC maturation. Taken together, our results suggest a role for tumor-derived exosomes in inducing tumor antigen-specific immunosuppression, possibly by modulating the function of APCs.

  8. The role of Ia molecules in the activation of T lymphocytes. III. Antigen-specific, Ia-restricted, interleukin 2-producing T cell hybridomas with detectable affinity for the restricting I-A molecule

    OpenAIRE

    1983-01-01

    Antigen-specific I region-restricted, interleukin 2-producing T cell hybridomas were produced by fusing GAT-specific T cell blasts with BW5147. Two antigen-specific phenotypes were identified, one autoreactive and one nonautoreactive. All of the antigen-specific and autoreactive clones were H-2 restricted, mapping to the IA subregion by genetic analysis and monoclonal antibody inhibition. Both the antigen- specific and autoreactive stimulation are the property of a single cell and required no...

  9. Development of Anti-Isoproturon Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    LI Fang-shi; SUN Feng; LIU Xian-jin; CUI Heng-hua

    2007-01-01

    A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon,3-(4-isopropylphenyl)-1,1-dimethylurea, in food and environmental samples was developed. Two haptens named 1-(3-carboxypropyl)-3-(4-isopropylphenyl)-1-methylurca (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin(OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen,with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36:1 and 46:1, respectively, as calculated by a UV spectrophotometry.The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6×105. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L-1 and 1.0 × 105, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.07 mg mL-1.The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and

  10. Polyclonal clustering algorithm and its convergence

    Institute of Scientific and Technical Information of China (English)

    MA Li; JIAO Li-cheng; BAI Lin; CHEN Chang-guo

    2008-01-01

    Being characteristic of non-teacher learning, self-organization, memory, and noise resistance, the artificial immune system is a research focus in the field of intelligent information processing. Based on the basic principles of organism immune and clonal selection, this article presents a polyclonal clustering algorithm characteristic of self-adaptation. According to the core idea of the algorithm, various immune operators in the artificial immune system are employed in the clustering process; moreover, clustering numbers are adjusted in accordance with the affinity function. Introduction of the recombination operator can effectively enhance the diversity of the individual antibody in a generation population, so that the searching scope for solutions is enlarged and the premature phenomenon of the algorithm is avoided. Besides, introduction of the inconsistent mutation operator enhances the adaptability and optimizes the performance of local solution seeking. Meanwhile, the convergence of the algorithm is accelerated. In addition, the article also proves the convergence of the algorithm by employing the Markov chain. Results of the data simulation experiment show that the algorithm is capable of obtaining reasonable and effective cluster.

  11. Get effective polyclonal antisera in one month

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    According to the traditional immunization procedure, after the first injection of the sample A (emulsion of aimed antigen and Freund's complete adjuvant) to immunize rabbit, successive injections of the sample B (emulsion of aimed antigen and Freund's incomplete adjuvant) were followed every 2-4 weeks. In general,high titer of the corresponding polyclonal antisera will be observed after 4-5 injections of sample B in 3-4months. This report presents a simply modified procedure that was able to stimulate the antisera formation in one month and achieve enough avidity to satisfy either Western blot or immunohistochemistry analysis.It just applied an additional injection of the sample A to the rabbit at the 3rd day after the primary immunization injection. You could gain the high titer of the antisera right after the first sample B injection in one month. This method has produced the desired results in three different recombinant antigens with different molecular weight (5.9 KD-55 KD) expressed from prokaryotic or eukaryotic cells.

  12. Preparation and Characterization of Olaquindox Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    CHE,Huilian; SHI Baoxia; CHEN,Jinlan; ZHU,Xuejia; HE,Jiguo

    2009-01-01

    2-[N-(2-Hydroxyethyl)-carbamoyl]-3-methyl-oxoquinoxaline-l,4-oxides (OLA) was first mono-esterificated by succinic acid anhydride, and was then turned into a derivative with carboxyl. The synthetic product was purified by recrystallization with a yield of 52.47%. Its chemical structure was determined by NMR, IR and MS spectra. The determined structure was used to synthese OLA hapten, which had the molecular structural characteristics of OLA. The OLA hapten and bovine serum albumin (BSA) were conjugated using the activated ester method, while the hapten and ovalbumin (OVA) were coupled by the mixed anhydride method. The UV scanning and infrared spectrum results showed that the hapten and the carrier protein were successfully coupled to BSA and OVA, with the combined ratios of 3.8 : 1 and 5 : 1, respectively. OLA-BSA was used as the immunogen to immunize four New Zealand white rabbits, and the high titer anti-serum produced relatively. The titers were 1 " 6400, 1 : 1600, 1 : 12800 and 1 : 6400, respectively, determined by indirect ELISA OLA-OVA as the coating antigen. The intermediate inhibition concentration (IC50) of OLA in the indirect ELISA test was 743.3 ng/mL. The lowest detection limit (IC20) was 5.71 ng/mL. High-purity OLA polyclonal antibody has been obtained by saturated ammonium sulfate and ion-exchange chromatography.

  13. [Antigen specific immunoglobulin E to grass and weed pollens in the plasma of patients with seasonal allergic rhinitis].

    Science.gov (United States)

    Silny, W; Kuchta, D; Siatecka, D; Silny, P

    1999-01-01

    The study involved 22 patients with seasonal allergic rhinitis between 13 and 53 years of age. The level of antigen specific IgE (AS IgE) to 5 grass and 3 weed pollens was determined with the use of CAP FEIA (Pharmacia, Uppsala, Sweden). The control group consisted of 20 persons. All above AS IgE were significantly higher in the patients with seasonal allergic rhinithis than in the control group. The most commonly present hypersensitivities were to Meadow fescue (Festuca elatior), Meadow grass (Poa pratensis), Cocksfoot (Dactylis glomerata), Ribwort (Plantago lanceolata) and Mugwort (Artemisia vulgaris) allergens. The authors believe that the pathomechanism of the development of seasonal allergic rhinithis is governed to a large degree by hypersensitivity to grass and weed pollens and suggest that precise determination of AS IgE to these allergens in patients blood sera should form the basis of the construction of the vaccine used in their immunotherapy. PMID:10337158

  14. Focal radiation therapy combined with 4-1BB activation and CTLA-4 blockade yields long-term survival and a protective antigen-specific memory response in a murine glioma model.

    Directory of Open Access Journals (Sweden)

    Zineb Belcaid

    Full Text Available BACKGROUND: Glioblastoma (GBM is the most common malignant brain tumor in adults and is associated with a poor prognosis. Cytotoxic T lymphocyte antigen -4 (CTLA-4 blocking antibodies have demonstrated an ability to generate robust antitumor immune responses against a variety of solid tumors. 4-1BB (CD137 is expressed by activated T lymphocytes and served as a co-stimulatory signal, which promotes cytotoxic function. Here, we evaluate a combination immunotherapy regimen involving 4-1BB activation, CTLA-4 blockade, and focal radiation therapy in an immune-competent intracranial GBM model. METHODS: GL261-luciferace cells were stereotactically implanted in the striatum of C57BL/6 mice. Mice were treated with a triple therapy regimen consisted of 4-1BB agonist antibodies, CTLA-4 blocking antibodies, and focal radiation therapy using a small animal radiation research platform and mice were followed for survival. Numbers of brain-infiltrating lymphocytes were analyzed by FACS analysis. CD4 or CD8 depleting antibodies were administered to determine the relative contribution of T helper and cytotoxic T cells in this regimen. To evaluate the ability of this immunotherapy to generate an antigen-specific memory response, long-term survivors were re-challenged with GL261 glioma en B16 melanoma flank tumors. RESULTS: Mice treated with triple therapy had increased survival compared to mice treated with focal radiation therapy and immunotherapy with 4-1BB activation and CTLA-4 blockade. Animals treated with triple therapy exhibited at least 50% long-term tumor free survival. Treatment with triple therapy resulted in a higher density of CD4+ and CD8+ tumor infiltrating lymphocytes. Mechanistically, depletion of CD4+ T cells abrogated the antitumor efficacy of triple therapy, while depletion of CD8+ T cells had no effect on the treatment response. CONCLUSION: Combination therapy with 4-1BB activation and CTLA-4 blockade in the setting of focal radiation therapy

  15. Application of Adoptive T-Cell Therapy Using Tumor Antigen-Specific T-Cell Receptor Gene Transfer for the Treatment of Human Leukemia

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    Toshiki Ochi

    2010-01-01

    Full Text Available The last decade has seen great strides in the field of cancer immunotherapy, especially the treatment of melanoma. Beginning with the identification of cancer antigens, followed by the clinical application of anti-cancer peptide vaccination, it has now been proven that adoptive T-cell therapy (ACT using cancer antigen-specific T cells is the most effective option. Despite the apparent clinical efficacy of ACT, the timely preparation of a sufficient number of cancer antigen-specific T cells for each patient has been recognized as its biggest limitation. Currently, therefore, attention is being focused on ACT with engineered T cells produced using cancer antigen-specific T-cell receptor (TCR gene transfer. With regard to human leukemia, ACT using engineered T cells bearing the leukemia antigen-specific TCR gene still remains in its infancy. However, several reports have provided preclinical data on TCR gene transfer using Wilms' tumor gene product 1 (WT1, and also preclinical and clinical data on TCR gene transfer involving minor histocompatibility antigen, both of which have been suggested to provide additional clinical benefit. In this review, we examine the current status of anti-leukemia ACT with engineered T cells carrying the leukemia antigen-specific TCR gene, and discuss the existing barriers to progress in this area.

  16. In vitro and in vivo analyses of a genetically—restricted antigen specific factor from mixed cell cultures of macrophage,T and B lymphocytes

    Institute of Scientific and Technical Information of China (English)

    CHAURMW; LAUASK

    1990-01-01

    An immunostimulatory factor was identified to be secreted by antigen-pulsed macrophages.This factor was able to induce the generation of antigen specific T helper lymphocytes in vitro as well as in vivo.Further in vitro experiments testing for the genetic restriction of this factor indicated that it is a geneticallyrestricted antigen specific factor (ASF).The Cunningham plaque assay was used to quantify the generation of T helper lymphocytes by measuring the number of plaque forming cells after sequential incubations of antigen-qulsed macrophages with T lymphocytes,and then spleen cells,and finally the TNP-coated sheep red blood cells.

  17. Antigen-specific tolerance induced by IL-10 gene modified immature dendritic cells in experimental autoimmune myocarditis in rats

    Institute of Scientific and Technical Information of China (English)

    LI Wei-min; LI Yue; LIU Wei; GAO Cheng; ZHOU Bao-guo; YANG Shu-sen; WANG Zheng; ZHANG Rui-hong; GAN Run-tao; KONG Yi-hui

    2006-01-01

    underwent maturation induced by in vitro exposure to LPS. IL-10 gene modified iDC inhibited the antigen specific T cell responses towards cardiac myosin. IκB protein was up-regulated significantly in the IL-10 gene modified iDC group.Conclusions IL-10 gene modified iDC induced antigen-specific tolerance in EAM. The underlying mechanisms may be related to costimulatory molecules down-regulation and NF-κB pathway inhibition.

  18. Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

    Directory of Open Access Journals (Sweden)

    Karasuyama Hajime

    2011-04-01

    Full Text Available Abstract Background There have been few reports on the role of Fc receptors (FcRs and immunoglobulin G (IgG in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa. Methods In FcγRIIB deficient (KO and C57BL/6 (WT mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA. Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c+ MHC class II+ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c+ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL. Results In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c+ MHC class II+ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c+ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously. Conclusion Antigen-specific IgG ameliorates

  19. Induction of an antigen specific gut inflammatory reaction in mice and rats: a model for human Inflammatory Bowel Disease

    Directory of Open Access Journals (Sweden)

    Gerlinde Agate Platais Brasil Teixeira

    2009-06-01

    Full Text Available Food allergy is an adverse reaction that occurs in susceptible people when they eat sensitizing foods and is one of the causes of Inflammatory Bowel Disease (IBD. The effort to understand the induction process of these diseases is important as IBD is increasing worldwide, including in Brazil. The aim of this study was to develop an experimental antigen specific inflammatory process of the gut of mice and rats, using peanut seeds. Animals were immunized with peanut protein extract before their exposure to the in natura peanut seeds. Results showed that systemic immunization with peanut protein extracts rendered significantly higher antibody titers than control groups and that immunized animals submitted to a challenge diet containing peanuts presented time dependent alterations of the gut similar to celiac disease. In conclusion, results suggested that this experimental model was a convenient tool to study the evolution of alterations in chronic antigen specific gut inflammatory process.A alergia alimentar consiste em uma reação adversa que ocorre em pessoas susceptíveis quando ingerem alimentos sensibilizantes, sendo uma das causas das Doenças Inflamatórias Intestinais (IBD. O objetivo deste estudo foi desenvolver um protocolo experimental de indução de um processo inflamatório intestinal antígeno-específico em camundongos e ratos. Foi escolhida para a indução deste processo a semente de amendoim. Os animais foram imunizados com o extrato protéico previamente à exposição com a semente in natura. Nossos resultados mostram que a imunização sistêmica com extratos protéicos de amendoim ocasiona títulos significativamente maiores de anticorpos quando comparado ao grupo controle e que os animais imunizados submetidos ao desafio com a dieta contendo exclusivamente amendoim apresentam alterações intestinais tempo-dependente similares àquelas observadas na doença celíaca. Os resultados obtidos sugerem que este modelo

  20. Feasibility and diagnostic utility of antigen-specific interferon-gamma responses for rapid immunodiagnosis of tuberculosis using induced sputum.

    Directory of Open Access Journals (Sweden)

    Tamaryn J Cashmore

    Full Text Available BACKGROUND: The diagnosis of smear-negative or sputum-scarce tuberculosis (TB is problematic as culture takes several weeks and representative biological samples are difficult to obtain. RD-1 antigen-specific interferon-gamma release assays (IGRAs are sensitive and specific blood-based tests for the diagnosis of M. tuberculosis infection. The feasibility and diagnostic utility of this rapid immunodiagnostic assay, using cells from induced sputum, is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Cells isolated from induced sputum were co-cultured with ESAT-6 and CFP-10 antigens using a standardized enzyme-linked immunospot (ELISPOT assay (T-SPOT.TB in 101 consecutively recruited TB suspects or non-TB controls. An optimization phase using 28 samples was followed by a validation phase using samples from 73 participants (20 with definite or probable TB, and 48 with non-TB. Despite optimization of sputum processing 65/73 (89% of the IGRAs in the validation phase were inconclusive. 44/73 (60% tests failed due to sputum induction-related factors [sputum induction-related adverse events (n = 5, inadequate sputum volume (n = 8, non-homogenisable sputum (n = 7, and insufficient numbers of cells to perform the assay (n = 24], whilst 20/73 (27% tests failed due T-SPOT.TB assay-related factors [excessive debris precluding reading of spots in the ELISPOT well (n = 6, failure of the positive control (n = 11, or high spot count in the negative control (n = 3]. Only 8/73 (11% of the available samples could therefore be correctly categorized (7 definite or probable TB, and 1 non-TB patient. Thus, 13/20 (65% of the definite or probable TB cases remained undiagnosed. CONCLUSIONS/SIGNIFICANCE: Rapid immunodiagnosis of pulmonary TB by antigen-specific IFN-gamma ELISPOT responses, using cells from induced sputum, is possible. However, the test, in its current ELISPOT format, is not clinically useful because the majority of the assays are inconclusive.

  1. Proinsulin multi-peptide immunotherapy induces antigen-specific regulatory T cells and limits autoimmunity in a humanized model.

    Science.gov (United States)

    Gibson, V B; Nikolic, T; Pearce, V Q; Demengeot, J; Roep, B O; Peakman, M

    2015-12-01

    Peptide immunotherapy (PIT) is a targeted therapeutic approach, involving administration of disease-associated peptides, with the aim of restoring antigen-specific immunological tolerance without generalized immunosuppression. In type 1 diabetes, proinsulin is a primary antigen targeted by the autoimmune response, and is therefore a strong candidate for exploitation via PIT in this setting. To elucidate the optimal conditions for proinsulin-based PIT and explore mechanisms of action, we developed a preclinical model of proinsulin autoimmunity in a humanized HLA-DRB1*0401 transgenic HLA-DR4 Tg mouse. Once proinsulin-specific tolerance is broken, HLA-DR4 Tg mice develop autoinflammatory responses, including proinsulin-specific T cell proliferation, interferon (IFN)-γ and autoantibody production. These are preventable and quenchable by pre- and post-induction treatment, respectively, using intradermal proinsulin-PIT injections. Intradermal proinsulin-PIT enhances proliferation of regulatory [forkhead box protein 3 (FoxP3(+))CD25(high) ] CD4 T cells, including those capable of proinsulin-specific regulation, suggesting this as its main mode of action. In contrast, peptide delivered intradermally on the surface of vitamin D3-modulated (tolerogenic) dendritic cells, controls autoimmunity in association with proinsulin-specific IL-10 production, but no change in regulatory CD4 T cells. These studies define a humanized, translational model for in vivo optimization of PIT to control autoimmunity in type 1 diabetes and indicate that dominant mechanisms of action differ according to mode of peptide delivery. PMID:26206289

  2. Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells

    Science.gov (United States)

    Yang, Lili; Baltimore, David

    2005-03-01

    A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both and chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

  3. Production of antigen-specific suppressive T cell factor by radiation leukemia virus-transformed suppressor T cells

    International Nuclear Information System (INIS)

    Hen egg-white lysozyme-specific suppressor T cells induced in C57BL/6 mice have been selected by sequential passage over plates coated with goat anti-mouse Ig and HEL. These suppressor T cells, 80% I-J+, were infected in vitro with radiation leukemia virus and injected intravenously into sublethally irradiated syngeneic recipients. After 4 to 6 months, 6 out of 20 injected mice developed thymic lymphomas, which were maintained by transplantation into histocompatible hosts and subsequently established as permanent cell lines. Cells of these six thymomas were screened for the presence of Thy 1.2, Lyt 1, Lyt 2, I-J/sup b/, and Ig cell surface antigens by direct or indirect immunofluorescence. One tumor was found to express the expected phenotype of suppressor T cells. High-speed supernatants of extracts obtained from L4 cells were able to induce HEL-specific suppression in a T cell proliferative assay, demonstrating the presence of an antigen-specific suppressive T cell factor

  4. Clinical fluctuations in MuSK myasthenia gravis are related to antigen-specific IgG4 instead of IgG1

    NARCIS (Netherlands)

    Niks, E H; van Leeuwen, Y; Leite, M I; Dekker, F W; Wintzen, A R; Wirtz, P W; Vincent, A; van Tol, M J D; Jol-van der Zijde, C M; Verschuuren, J J G M

    2008-01-01

    We studied the longitudinal relation between disease severity and titers of antigen-specific IgG subclasses in sera of patients with myasthenia gravis and antibodies to Muscle Specific Kinase (MuSK MG). Six patients were included of whom 55 samples had been collected during 2.5-13.4 years. Anti-MuSK

  5. Diabetes is associated with lower tuberculosis antigen-specific interferon gamma release in Tanzanian tuberculosis patients and non-tuberculosis controls

    DEFF Research Database (Denmark)

    Faurholt-Jepsen, Daniel; Aabye, Martine Grosos; Jensen, Andreas Vestergaard;

    2014-01-01

    in diabetes patients and therefore the validity of interferon gamma release assays (IGRA) may be compromised. The aim of the present study was to assess the association between diabetes and Mycobacterium tuberculosis (Mtb) antigen-specific interferon gamma (IFN-γ) release in a TB endemic area among culture...

  6. Preparation and characterization of polyclonal antibodies against ARL-1 protein

    Institute of Scientific and Technical Information of China (English)

    Jun-Fei Jin; Liu-Di Yuan; Li Liu; Zhu-Jiang Zhao; Wei Xie

    2003-01-01

    AIM: To prepare and characterize polyclonal antibodies against aldose reductase-like (ARL-1) protein.METHODS: ARL-L gene was inserted into the E.coli expression vector pGEX-4T-1(His)6C and vector pQE-30. Recombinant ARL1 proteins named ARL-(His)6 and ARL-GST were expressed.They were purified by affinity chromatography. Sera from domestic rabbits immunized with ARL-(His)6 were purified by CNBr-activated sepharose 4B coupled ARL-GST. Polyclonal antibodies were detected by Western blotting.RESULTS: Recombinant proteins of ARL-(His)6 with molecular weight of 35.7 KD and ARL-GST with molecular weight of 60.8 KD were highly expressed. The expression levels of ARL-GST and ARL-(His)6 were 15.1% and 27.7 %among total bacteria proteins, respectively. They were soluble, predominantly in supernatant. After purification by non-denatured way, SDS-PAGE showed one band. In the course of polyclonal antibodies purification, only one elution peak could be seen. Western blotting showed positive signals in the two purified proteins and the bacteria transformed with pGEX-4T-1(His)6 C-ARL and pQE-30-ARL individually.CONCLUSION: Polyclonal antibodies are purified and highly specific against ARL-1 protein. ARL-GST and ARL-(His)6 are highly expressed and purified.

  7. Deep Sequencing of the T-cell Receptor Repertoire Demonstrates Polyclonal T-cell Infiltrates in Psoriasis [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Jamie L. Harden

    2015-08-01

    Full Text Available It is well known that infiltration of pathogenic T-cells plays an important role in psoriasis pathogenesis. However, the antigen specificity of these activated T-cells is relatively unknown. Previous studies using T-cell receptor polymerase chain reaction technology (TCR-PCR have suggested there are expanded T-cell receptor (TCR clones in psoriatic skin, suggesting a response to an unknown psoriatic antigen. Here we describe the results of high-throughput deep sequencing of the entire αβ- and γδ- TCR repertoire in normal healthy skin and psoriatic lesional and non-lesional skin. From this study, we were able to determine that there is a significant increase in the abundance of unique β- and γ- TCR sequences in psoriatic lesional skin compared to non-lesional and normal skin, and that the entire T-cell repertoire in psoriasis is polyclonal, with similar diversity to normal and non-lesional skin. Comparison of the αβ- and γδ- TCR repertoire in paired non-lesional and lesional samples showed many common clones within a patient, and these close were often equally abundant in non-lesional and lesional skin, again suggesting a diverse T-cell repertoire. Although there were similar (and low amounts of shared β-chain sequences between different patient samples, there was significantly increased sequence sharing of the γ-chain in psoriatic skin from different individuals compared to those without psoriasis. This suggests that although the T-cell response in psoriasis is highly polyclonal, particular γδ- T-cell subsets may be associated with this disease. Overall, our findings present the feasibility of this technology to determine the entire αβ- and γδ- T-cell repertoire in skin, and that psoriasis contains polyclonal and diverse αβ- and γδ- T-cell populations.

  8. Age-related impairment of humoral response to influenza is associated with changes in antigen specific T follicular helper cell responses.

    Science.gov (United States)

    Lefebvre, Julie S; Masters, April R; Hopkins, Jacob W; Haynes, Laura

    2016-01-01

    T follicular helper (TFH) cell responses are essential for generation of protective humoral immunity during influenza infection. Aging has a profound impact on CD4(+) T cell function and humoral immunity, yet the impact of aging on antigen specific TFH responses remains unclear. Influenza specific TFH cells are generated in similar numbers in young and aged animals during infection, but TFH cells from aged mice exhibit significant differences, including reduced expression of ICOS and elevated production of IL-10 and IFNγ, which potentially impairs interaction with cognate B cells. Also, more influenza specific T cells in aged mice have a regulatory phenotype, which could contribute to the impaired TFH function. Adoptive transfer studies with young T cells demonstrated that TGF-β1 in the aged environment can drive increased regulatory T cell accumulation. Aging and the aged environment thus impact antigen specific TFH cell function and formation, which contribute to reduced protective humoral responses. PMID:27109638

  9. Antibody-dependent cellular cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy trial target multiple epitopes and preferentially use the VH1 gene family.

    Science.gov (United States)

    Bonsignori, Mattia; Pollara, Justin; Moody, M Anthony; Alpert, Michael D; Chen, Xi; Hwang, Kwan-Ki; Gilbert, Peter B; Huang, Ying; Gurley, Thaddeus C; Kozink, Daniel M; Marshall, Dawn J; Whitesides, John F; Tsao, Chun-Yen; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Kim, Jerome H; Michael, Nelson L; Tomaras, Georgia D; Montefiori, David C; Lewis, George K; DeVico, Anthony; Evans, David T; Ferrari, Guido; Liao, Hua-Xin; Haynes, Barton F

    2012-11-01

    The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs. PMID:22896626

  10. Age-related impairment of humoral response to influenza is associated with changes in antigen specific T follicular helper cell responses

    OpenAIRE

    Lefebvre, Julie S; Masters, April R; Hopkins, Jacob W; Laura Haynes

    2016-01-01

    T follicular helper (TFH) cell responses are essential for generation of protective humoral immunity during influenza infection. Aging has a profound impact on CD4+ T cell function and humoral immunity, yet the impact of aging on antigen specific TFH responses remains unclear. Influenza specific TFH cells are generated in similar numbers in young and aged animals during infection, but TFH cells from aged mice exhibit significant differences, including reduced expression of ICOS and elevated p...

  11. Antagonism of Airway Tolerance by Endotoxin/LPS Through Promoting OX40L and Suppressing Antigen-specific Foxp3+ Treg

    OpenAIRE

    Duan, Wei; So, Takanori; Croft, Michael

    2008-01-01

    Respiratory exposure to allergens can lead to airway tolerance. Factors that antagonize tolerance mechanisms in the lung might result in susceptibility to diseases such as asthma. We show that inhalation of endotoxin/LPS with antigen prevented airway tolerance and abolished protection from T cell-driven asthmatic lung inflammation. Under conditions leading to tolerance, adaptive antigen-specific CD4+Foxp3+ Treg were generated following exposure to intranasal antigen and outnumbered IL-4- and ...

  12. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay

    OpenAIRE

    M Anthony Moody; Mattia Bonsignori

    2012-01-01

    The traditional enzyme-linked immunospot (ELISpot) assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities ha...

  13. A single exposure to iron oxide nanoparticles attenuates antigen-specific antibody production and T-cell reactivity in ovalbumin-sensitized BALB/c mice

    Directory of Open Access Journals (Sweden)

    Shen CC

    2011-06-01

    Full Text Available Chien-Chang Shen1, Chia-Chi Wang1, Mei-Hsiu Liao2, Tong-Rong Jan11Department and Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan; 2Division of Isotope Application, Institute of Energy Research, Taoyuan, TaiwanBackground: Superparamagnetic iron oxide nanoparticles have been used in clinical applications as a diagnostic contrasting agent. Previous studies showed that iron oxide nanoparticles deposited in the liver and spleen after systemic administration. The present study investigated the effect of iron oxide nanoparticles on antigen-specific immune responses in mice sensitized with the T cell-dependent antigen ovalbumin (OVA.Methods: BALB/c mice were intravenously administered with a single dose of iron oxide nanoparticles (10-60 mg Fe/kg 1 hour prior to OVA sensitization, and the serum antibody production and splenocyte reactivity were examined 7 days later.Results: The serum levels of OVA-specific IgG1 and IgG2a were significantly attenuated by treatment with iron oxide nanoparticles. The production of interferon-γ and interleukin-4 by splenocytes re-stimulated with OVA in culture was robustly suppressed in mice administered with iron oxide nanoparticles. The viability of OVA-stimulated splenocytes was also attenuated. In contrast, treatment with iron oxide nanoparticles did not affect the viability of splenocytes stimulated with concanavalin A, a T-cell mitogen.Conclusion: Collectively, these data indicate that systemic exposure to a single dose of iron oxide nanoparticles compromises subsequent antigen-specific immune reactions, including the serum production of antigen-specific antibodies, and the functionality of T cells.Keywords: iron oxide nanoparticle, antigen-specific, immune, ovalbumin

  14. Intranasal Vaccination Affords Localization and Persistence of Antigen-Specific CD8⁺ T Lymphocytes in the Female Reproductive Tract.

    Science.gov (United States)

    Singh, Shailbala; Schluns, Kimberly S; Yang, Guojun; Anthony, Scott M; Barry, Michael A; Sastry, K Jagannadha

    2016-03-17

    Immunization strategies generating large numbers of antigen-specific T cells in the female reproductive tract (FRT) can provide barrier protection against sexually-transmitted pathogens, such as the human immunodeficiency virus (HIV) and human papillomaviruses (HPV). The kinetics and mechanisms of regulation of vaccine-induced adaptive T cell-mediated immune responses in FRT are less well defined. We present here evidence for intranasal delivery of the model antigen ovalbumin (OVA) along with alpha-galactosylceramide adjuvant as a protein vaccine to induce significantly higher levels of antigen-specific effector and memory CD8⁺ T cells in the FRT, relative to other systemic and mucosal tissues. Antibody blocking of the CXCR3 receptor significantly reduced antigen-specific CD8⁺ T cells subsequent to intranasal delivery of the protein vaccine suggesting an important role for the CXCR3 chemokine-receptor signaling for T cell trafficking. Further, intranasal vaccination with an adenoviral vector expressing OVA or HIV-1 envelope was as effective as intramuscular vaccination for generating OVA- or ENV-specific immunity in the FRT. These results support the application of the needle-free intranasal route as a practical approach to delivering protein as well as DNA/virus vector-based vaccines for efficient induction of effector and memory T cell immunity in the FRT.

  15. Production and Purification of Rabbit's Polyclonal Antibody Against Factor VIII.

    OpenAIRE

    Sohrabi, Simin; Akbarzadeh, Azim; Norouzian, Dariush; Farhangi, Ali; Mortazavi, Mehri; Mehrabi, Mohammad Reza; Chiani, Mohsen; Saffari, Zahra; Ghassemi, Soheil

    2011-01-01

    The attempt is made to produce recombinant factor VIII but the first step in producing such product is production and purification of rabbit's polyclonal antibody against factor VIII. The second and third steps involve monoclonal antibody and recombinant factor VIII production. Factor VIII is one of the most important coagulating factor where its deficiency leads to diseases like hemophilia type A or classic. It is an inherited disease. Previously, it was obtained through fractionation of blo...

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Macrophages play an essential role in antigen-specific immune suppression mediated by T CD8⁺ cell-derived exosomes.

    Science.gov (United States)

    Nazimek, Katarzyna; Ptak, Wlodzimierz; Nowak, Bernadeta; Ptak, Maria; Askenase, Philip W; Bryniarski, Krzysztof

    2015-09-01

    Murine contact sensitivity (CS) reaction could be antigen-specifically regulated by T CD8(+) suppressor (Ts) lymphocytes releasing microRNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of Mφ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated Mφ. Additionally, exosome-pulsed, TNP-conjugated Mφ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation. PMID:25808106

  18. A recombinant adenovirus expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice.

    Science.gov (United States)

    Li, Wu; Deng, Guangcun; Li, Min; Zeng, Jin; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2014-11-01

    Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide, which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report, the generation of Ad5-CEAB, a recombinant adenovirus expressing Mtb antigens of CFP10, ESAT6, Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB, mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response, particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines, IFN-γ, TNF-α, IL-2 and IL-12. In addition, the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore, an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice, suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo, which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.

  19. Dexamethasone and Monophosphoryl Lipid A-Modulated Dendritic Cells Promote Antigen-Specific Tolerogenic Properties on Naive and Memory CD4+ T Cells

    Science.gov (United States)

    Maggi, Jaxaira; Schinnerling, Katina; Pesce, Bárbara; Hilkens, Catharien M.; Catalán, Diego; Aguillón, Juan C.

    2016-01-01

    Tolerogenic dendritic cells (DCs) are a promising tool to control T cell-mediated autoimmunity. Here, we evaluate the ability of dexamethasone-modulated and monophosphoryl lipid A (MPLA)-activated DCs [MPLA-tolerogenic DCs (tDCs)] to exert immunomodulatory effects on naive and memory CD4+ T cells in an antigen-specific manner. For this purpose, MPLA-tDCs were loaded with purified protein derivative (PPD) as antigen and co-cultured with autologous naive or memory CD4+ T cells. Lymphocytes were re-challenged with autologous PPD-pulsed mature DCs (mDCs), evaluating proliferation and cytokine production by flow cytometry. On primed-naive CD4+ T cells, the expression of regulatory T cell markers was evaluated and their suppressive ability was assessed in autologous co-cultures with CD4+ effector T cells and PPD-pulsed mDCs. We detected that memory CD4+ T cells primed by MPLA-tDCs presented reduced proliferation and proinflammatory cytokine expression in response to PPD and were refractory to subsequent stimulation. Naive CD4+ T cells were instructed by MPLA-tDCs to be hyporesponsive to antigen-specific restimulation and to suppress the induction of T helper cell type 1 and 17 responses. In conclusion, MPLA-tDCs are able to modulate antigen-specific responses of both naive and memory CD4+ T cells and might be a promising strategy to “turn off” self-reactive CD4+ effector T cells in autoimmunity.

  20. Macrophages play an essential role in antigen-specific immune suppression mediated by T CD8⁺ cell-derived exosomes.

    Science.gov (United States)

    Nazimek, Katarzyna; Ptak, Wlodzimierz; Nowak, Bernadeta; Ptak, Maria; Askenase, Philip W; Bryniarski, Krzysztof

    2015-09-01

    Murine contact sensitivity (CS) reaction could be antigen-specifically regulated by T CD8(+) suppressor (Ts) lymphocytes releasing microRNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of Mφ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated Mφ. Additionally, exosome-pulsed, TNP-conjugated Mφ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation.

  1. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    Directory of Open Access Journals (Sweden)

    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  2. Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis.

    OpenAIRE

    Appel, H; Kuon, W; Kuhne, M; P. Wu; Kuhlmann, S.; Kollnberger, S.; Thiel, A.; Bowness, P.; Sieper, J

    2004-01-01

    Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8+ T cells in HLA-B27+-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8+ T cells. The application of HLA-B27 tetramers in HLA-...

  3. Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis.

    OpenAIRE

    Appel, H; Kuon, W; Kuhne, M; P. Wu; Kuhlmann, S.; Kollnberger, S.; Thiel, A.; Bowness, P.; Sieper, J

    2004-01-01

    Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8+ T cells in HLA-B27+-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein-Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8+ T cells. The application of HLA-B27 tetramers in HLA-...

  4. An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice

    Directory of Open Access Journals (Sweden)

    Chin'ombe Nyasha

    2009-04-01

    Full Text Available Abstract Background The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli β-galactosidase α-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated. Results High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-γ and IL-4 immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-γ and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-γ produced by GFP peptide-stimulated cells was 65.2-fold above background (p Conclusion These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.

  5. Particle-mediated Intravenous Delivery of Antigen mRNA Results in Strong Antigen-specific T-cell Responses Despite the Induction of Type I Interferon

    Science.gov (United States)

    Broos, Katrijn; Van der Jeught, Kevin; Puttemans, Janik; Goyvaerts, Cleo; Heirman, Carlo; Dewitte, Heleen; Verbeke, Rein; Lentacker, Ine; Thielemans, Kris; Breckpot, Karine

    2016-01-01

    Cancer vaccines based on mRNA are extensively studied. The fragile nature of mRNA has instigated research into carriers that can protect it from ribonucleases and as such enable its systemic use. However, carrier-mediated delivery of mRNA has been linked to production of type I interferon (IFN) that was reported to compromise the effectiveness of mRNA vaccines. In this study, we evaluated a cationic lipid for encapsulation of mRNA. The nanometer-sized, negatively charged lipid mRNA particles (LMPs) efficiently transfected dendritic cells and macrophages in vitro. Furthermore, i.v. delivery of LMPs resulted in rapid expression of the mRNA-encoded protein in spleen and liver, predominantly in CD11c+ cells and to a minor extent in CD11b+ cells. Intravenous immunization of mice with LMPs containing ovalbumin, human papilloma virus E7, and tyrosinase-related protein-2 mRNA, either combined or separately, elicited strong antigen-specific T-cell responses. We further showed the production of type I IFNs upon i.v. LMP delivery. Although this decreased the expression of the mRNA-encoded protein, it supported the induction of antigen-specific T-cell responses. These data question the current notion that type I IFNs hamper particle-mediated mRNA vaccines. PMID:27327138

  6. Regulation of delayed-type hypersensitivity: VI. Antigen-specific suppressor T cells and suppressor factor for delayed-type hypersensitivity to histocompatibility antigens

    International Nuclear Information System (INIS)

    Mice develop highly significant levels of delayed-type hypersensitivity (DTH) to major and minor histocompatibility antigens when injected s.c. with lymphoid cells from X-irradiated allogeneic donors. However, when mice are inoculated i.v. with a high dose of X-irradiated allogeneic lymphoid cells, they not only fail to develop DTH to the allogeneic cells, but their ability to respond to an immunogenic challenge of the alloantigens is also significantly depressed. This suppression is adoptively transferable by antigen-specific suppressor T cells and not by immune serum. Cell surface phenotypic analysis shows that the primary suppressor cells for alloantigens are Thy-1+, Lyt-1+2-, and Ia-, whereas the secondary suppressor cells appearing after boosting injection are Thy-+, Lyt-1+2+, and Ia-. These suppressor T (Ts) cells localize in the lymphoid organs shortly after their induction and are largely absent from the spleen or lymph node 1 month later.However, ''suppressor memory'' can be recalled by an immunogenic dose of alloantigens which would normally induce DTH effector cells rather than suppressor cells in naive mice. When the suppressor cells were cultured in vitro for 48 hr, the supernatant contained suppressive activity. It appears likely that the manifestation of the suppressor cells is via soluble, antigen-specific suppressor factor(s), the production of which is dependent on viable T cells

  7. Detection of Salmonella typhimurium using polyclonal antibody immobilized magnetostrictive biosensors

    Science.gov (United States)

    Guntupalli, R.; Hu, Jing; Lakshmanan, Ramji S.; Wan, Jiehui; Huang, Shichu; Yang, Hong; Barbaree, James M.; Huang, T. S.; Chin, Bryan A.

    2006-05-01

    Novel mass-sensitive, magnetostrictive sensors have a characteristic resonant frequency that can be determined by monitoring the magnetic flux emitted by the sensor in response to an applied, time varying, magnetic field. This magnetostrictive platform has a unique advantage over conventional sensor platforms in that measurement is wireless or remote. These biosensors can thus be used in-situ for detecting pathogens and biological threat agents. In this work, we have used a magnetostrictive platform immobilized with a polyclonal antibody (the bio-molecular recognition element) to form a biosensor for the detection of Salmonella typhimurium. Upon exposure to solutions containing Salmonella typhimurium bacteria, the bacteria were bound to the sensor and the additional mass of the bound bacteria caused a shift in the sensor's resonant frequency. Responses of the sensors to different concentrations of S. typhimurium were recorded and the results correlated with those obtained from scanning electron microscopy (SEM) images of samples. Good agreement between the measured number of bound bacterial cells (attached mass) and frequency shifts were obtained. The longevity and specificity of the selected polyclonal antibody were also investigated and are reported.

  8. A whole blood monokine-based reporter assay provides a sensitive and robust measurement of the antigen-specific T cell response

    Directory of Open Access Journals (Sweden)

    Bennett Sophia C

    2011-08-01

    Full Text Available Abstract Background The ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFNγ-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpot assays cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFNγ signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood. Methods Whole blood or peripheral blood mononuclear cells (PBMCs from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9, IP-10 (CXCL10 and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot. Results Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFNγ-R signaling allows responses to be detected in as little as 25 μL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing. Conclusions A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be

  9. Back to the future: recombinant polyclonal antibody therapeutics.

    Science.gov (United States)

    Wang, Xian-Zhe; Coljee, Vincent W; Maynard, Jennifer A

    2013-11-01

    Antibody therapeutics are one of the fastest growing classes of pharmaceuticals, with an annual US market over $20 billion, developed to treat a variety of diseases including cancer, auto-immune and infectious diseases. Most are currently administered as a single molecule to treat a single disease, however there is mounting evidence that cocktails of multiple antibodies, each with a unique binding specificity and protective mechanism, may improve clinical efficacy. Here, we review progress in the development of oligoclonal combinations of antibodies to treat disease, focusing on identification of synergistic antibodies. We then discuss the application of modern antibody engineering technologies to produce highly potent antibody preparations, including oligoclonal antibody cocktails and truly recombinant polyclonal antibodies. Specific examples illustrating the synergy conferred by multiple antibodies will be provided for diseases caused by botulinum toxin, cancer and immune thrombocytopenia. The bioprocessing and regulatory options for these preparations will be discussed. PMID:24443710

  10. Preparation and Application of Polyclonal Antibody against a Recombinant Laccase

    Institute of Scientific and Technical Information of China (English)

    Yinghai Xu; Yuzhi Hong; Yazhong Xiao; Wei Fang

    2007-01-01

    A laccase gene from Trametes sp. 420 was recombinantly expressed in Pichia pastoris, producing the enzyme rLacD.Six mutant enzymes were produced by site-directed mutation at six potential glycosylation sites in the enzyme rLacD respectively. To probe the mutants with lower activities sensitively and specifically, the antiserum containing specific polyclonal antibodies were prepared by immunizing healthy male rabbits, about 4-month-old and 2 kilogram weight, using pure rLacD as an immunogen. Antibodies were collected after the fifth immunization injection. The antiserum had titres of 1:32 in double immunodiffusion test and of 1:128,000 in enzyme-linked immunosorbent assay (ELISA). The results obtained by Western blot analysis showed that the antiserum could react with rLacD and its mutants with highly specific and sensitive affinities.

  11. Polyclonal B-cell lymphocytosis with binucleated lymphocytes (PPBL

    Directory of Open Access Journals (Sweden)

    Xavier Troussard

    2008-10-01

    Full Text Available Xavier Troussard1, Edouard Cornet1, Jean-François Lesesve2, Carine Kourel3, Hossein Mossafa31Laboratoire d’Hématologie Côte de Nacre, Université Caen Basse Normandie Caen, Registre Régional des Hémopathies Malignes de Basse Normandie, France; 2Laboratoire d’Hématologie, Vandoeuvre-Les-Nancy Cedex, France; 3Département de Génétique Humaine, Laboratoire pasteur-Cerba, Cergy-Pontoise, FranceFor the Groupe Français d’Hématologie cellulaire (GFHCAbstract: Persistent polyclonal B-cell lymphocytosis (PPBL is a rare and recently described entity. The review of the literature show PPBL is diagnosed predominantly but not exclusively in women, usually smokers. PPBL is recognized by a moderate, chronic and absolute lymphocytosis (>4 × 109/l in the peripheral blood. In 10% of cases without lymphocytosis, the PPBL diagnosis has to be suggested by peripheral blood examination showing in all cases atypical binucleated lymphocytes. A polyclonal serum IgM is also associated and HLA-DR7 expression is present in most cases. Contrary to B-cell chronic lymphoproliferative disorders (B-CLPD, peripheral B cells are polyclonal with kappa and lambda light-chain expression and no clonal rearrangement of immunoglobulin heavy chain genes is usually demonstrated. The detection of an extra isochromosome for the long arm of chromosome 3 +i(3(q10 has to be considered as a specific marker of PPBL. We performed conventional cytogenetic analysis (CCA in 111 patients with typical PPBL we followed-up more than 4 years. +i(3q was detected in 34% (33/98, PCC in 8% (8/98 and both abnormalities in 31% (30/98. CCA showed neither +i(3q nor PCC in 28% (27/98. Fluorescence in situ hybridization (FISH was also performed in 84 cases and +i(3q was detected in 71% (60/84. When combining both procedures in 84 patients, +i(3q was detected in 17 patients with negative CCA and was confirmed in 43 patients with positive CCA. CCA and FISH were both negative in 24 cases. Whether

  12. Polyclonal antibody to ovomucoid determination in gamma irradiated laying eggs

    International Nuclear Information System (INIS)

    To determine allergenic food proteins, one of the most used tests is the immunoassays such as ELISA (enzyme linked immunosorbent assay), where the antibody recognizes the antigen and this connection is showed by an enzymatic system, in other words, optical density. The aim of this study was to determine the polyclonal antibody efficiency, produced in laboratory, to identify the presence the ovomucoid antigen in treated eggs by gamma irradiation for its inactivation. To evaluate the treatments, polyclonal antibody was produced in female rabbits immunized with bioconjugated ovomucoid. Was used Freund Complete Adjuvant at first immunization and PBS Buffer at four subsequently immunizations every fifteen days, plus a booster 48 hours before the blood retreated. The blood serum was tittered by PTA-ELISA (Plate trapped antigen). All procedures were according to European Norms for ethical and animal welfare. It was used, in nature, commercial laying eggs. So the samples were submitted to the gamma radiation coming from a source of Co60, type Multipurpose, under a dose rate of 19.4 and 31.8 Gy/hour, in the doses: 0 (control); 10 KGy; 20 KGy and 30 KGy, in all rates. By the ELISA.s test we can find the egg allergen ovomucoid and the radiation treatment do not showed considerable changes. So we can concluded that the antibody produced is capable of identify the ovomucoid allergenic protein and the gamma irradiation in such rates does not shows changes in that protein, therefore showed some changes in the color and visual viscosity of the egg samples. (author)

  13. Polyclonal antibody to ovomucoid determination in gamma irradiated laying eggs

    Energy Technology Data Exchange (ETDEWEB)

    Harder, Marcia N.C.; Arthur, Valter; Silva, Lucia C.A.S.; Lopes, Tatiana G.G. [Centro de Energia Nuclear na Agricultura (CENA/USP, Piracicaba, SP. Dept. de Radiobiologia e Ambiente) (Brazil)], e-mail: mnharder@cena.usp.br, e-mail: arthur@cena.usp.br, e-mail: tgglopes@cena.usp.br; Duarte, Keila M.R. [Instituto de Zootecnia (IZ . Nova Odessa), Nova Odessa, SP (Brazil)], e-mail: keila@iz.sp.gov.br; Canniatti-Brazaca, Solange G.; Savino, Vicente J.M.; Coelho, Antonio A.D. [Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP), Piracicaba, SP (Brazil)], e-mail: sgcbraza@esalq.usp.br, e-mail: vjmsavin@esalq.usp.br, e-mail: aadcoelh@esalq.usp.br

    2009-07-01

    To determine allergenic food proteins, one of the most used tests is the immunoassays such as ELISA (enzyme linked immunosorbent assay), where the antibody recognizes the antigen and this connection is showed by an enzymatic system, in other words, optical density. The aim of this study was to determine the polyclonal antibody efficiency, produced in laboratory, to identify the presence the ovomucoid antigen in treated eggs by gamma irradiation for its inactivation. To evaluate the treatments, polyclonal antibody was produced in female rabbits immunized with bioconjugated ovomucoid. Was used Freund Complete Adjuvant at first immunization and PBS Buffer at four subsequently immunizations every fifteen days, plus a booster 48 hours before the blood retreated. The blood serum was tittered by PTA-ELISA (Plate trapped antigen). All procedures were according to European Norms for ethical and animal welfare. It was used, in nature, commercial laying eggs. So the samples were submitted to the gamma radiation coming from a source of Co{sup 60}, type Multipurpose, under a dose rate of 19.4 and 31.8 Gy/hour, in the doses: 0 (control); 10 KGy; 20 KGy and 30 KGy, in all rates. By the ELISA.s test we can find the egg allergen ovomucoid and the radiation treatment do not showed considerable changes. So we can concluded that the antibody produced is capable of identify the ovomucoid allergenic protein and the gamma irradiation in such rates does not shows changes in that protein, therefore showed some changes in the color and visual viscosity of the egg samples. (author)

  14. Effect of prior dietary exposure to cow’s milk protein on antigen-specific and nonspecific cellular proliferation in mice

    DEFF Research Database (Denmark)

    Pedersen, Susanne Brix; Magyar, O.H.; Barkholt, Vibeke;

    2005-01-01

    in cell donors. Focusing on the immunostimulatory potential of cows' milk proteins and peptides, we studied the impact of prior dietary exposure to cows' milk on proliferation of murine immune cells upon ex vivo stimulation with bovine milk proteins. Nonspecific proliferation induced by beta-casein...... the importance of employing immune cells from mice unexposed to cows' milk for studies of the immunomodulating capacity of cows' milk proteins and peptides, in order to rule out the interference caused by antigen-specific immune responses. By using such cells, we here show that some beta-casein peptides possess......The impact of dietary components on the immune system is gaining increased attention in the effort to develop safe food products, some even with health-promoting potential, as well as to improve the basic understanding of the immunomodulatory potential of common food components. In such studies...

  15. Vaccination with Antigen Combined with αβ-ATP as a Vaccine Adjuvant Enhances Antigen-Specific Antibody Production via Dendritic Cell Activation.

    Science.gov (United States)

    Matsuo, Kazuhiko; Nishiuma, Satoshi; Hasegawa, Yuta; Kawabata, Fumika; Kitahata, Kosuke; Nakayama, Takashi

    2016-01-01

    Adjuvants are required to enhance antigen-specific immune responses by vaccines. Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X and P2Y receptors and triggers the activation of dendritic cells (DCs). Here we investigated the in vivo adjuvant efficacy of α,β-methylene-ATP (αβ-ATP), a non-hydrolysable form of ATP. We found that intradermal injection of ovalbumin (OVA), as a model antigen, combined with αβ-ATP, as the adjuvant, enhanced OVA-specific immune responses more than OVA alone. Additionally, DCs in the skin of mice injected with OVA and αβ-ATP had increased expression of major histocompatibility complex class II and co-stimulator molecules, CD40, CD80, and CD86, suggesting that αβ-ATP activated DC. These findings indicate that αβ-ATP functions as a potent vaccine adjuvant. PMID:27251512

  16. Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Lynette Beattie

    2010-03-01

    Full Text Available Kupffer cells (KCs represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

  17. A Novel Scheme for Production of Polyclonal Antibody against Estrogenic Bisphenols

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A polyclonal antibody against the currently concerned estrogenic bisphcnol compoundswas produced according to a new scheme. 4,4-Bis (4-hydroxyphenyl) valeric acid was used tosynthesize the complete antigen in which the characteristic bisphenol structure was exposed to thelargest extent. The produced polyclonal antibody showed high specificity and affinity forbisphenol A.

  18. Up-regulation of HLA class-I antigen expression and antigen-specific CTL response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid

    Directory of Open Access Journals (Sweden)

    Lizano-Soberón Marcela

    2006-12-01

    Full Text Available Abstract Background DNA hypermethylation and histone deacetylation are epigenetic events that contribute to the absence or downregulated expression of different components of the tumor recognition complex. These events affect the processing and presentation of antigenic peptides to CTLs by HLA class-I molecules. In this work evaluated the effect of the DNA hypomethylating agent hydralazine and the histone deacetylase inhibitor valproic acid, on the expression of HLA class-I molecules and on the antigen-specific immune recognition of cervical cancer cells. Methods Cell lines C33A (HPV-, CaSki (HPV-16+ and MS751 (HPV-18+ were treated with hydralazine and valproic acid to assess the expression of HLA class-I molecules by flow cytometry and RT-PCR. Promoter methylation of HLA class-I -A, -B and C, was also evaluated by Methylation-Specific PCR. Primary cervical tumors of four HLA-A*0201 allele patients were typed for HPV and their CTL's stimulated in vitro with the T2 cell line previously loaded with 50 μM of the HPV peptides. Cytotoxicity of stimulated CTL's was assayed against Caski and MS751 cells pre-treated with hydralazine and valproic acid. Results Valproic acid and hydralazine/valproic acid up-regulated the constitutive HLA class-I expression as evaluated by flow cytometry and RT-PCR despite constitutive promoter demethylation at these loci. Hydralazine and valproic acid in combination but no IFN-gamma hyperacetylated histone H4 as evaluated by ChiP assay. The antigenic immune recognition of CaSki and MS751 cells by CTLs specific to HPV-16/18 E6 and E7-derived epitopes, was increased by VA and H/VA and the combination of H/VA/IFN-gamma. Conclusion These results support the potential use of hydralazine and valproic acid as an adjuvant for immune intervention in cervical cancer patients whenever clinical protocols based on tumor antigen recognition is desirable, like in those cases where the application of E6 and E7 based therapeutic vaccines

  19. Proteome-wide screening reveals immunodominance in the CD8 T cell response against classical swine fever virus with antigen-specificity dependent on MHC class I haplotype expression.

    Directory of Open Access Journals (Sweden)

    Giulia Franzoni

    Full Text Available Vaccination with live attenuated classical swine fever virus (CSFV vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-γ responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-γ expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-γ induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core₂₄₁₋₂₅₅ PESRKKLEKALLAWA and NS3₁₉₀₂₋₁₉₁₂ VEYSFIFLDEY, or minimal length antigenic peptides: E2₉₉₆₋₁₀₀₃ YEPRDSYF, NS2₁₂₂₃₋₁₂₃₀ STVTGIFL and NS5A₃₀₇₀₋₃₀₇₈ RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-α in addition to IFN-γ. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS₁₂₂₃₋₁₂₃₀ STVTGIFL and NS3₁₉₀₂₋₁₉₁₂ VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed

  20. Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    Science.gov (United States)

    Papa, Michelle Premazzi; de Morais, Ana Theresa Silveira; Peçanha, Ligia Maria Torres; de Arruda, Luciana Barros

    2015-01-01

    Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV in vitro. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types in vivo might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients. PMID:26656738

  1. Production and purification of polyclonal antibody against melatonin hormone

    Directory of Open Access Journals (Sweden)

    Fooladsaz K

    1999-09-01

    Full Text Available Nowadays immunochemical techniques have played a very important and valuable role in quantitative and qualitative assays of liquid compounds of the body. Producing antibody against immunogenes is the first step to make immunochemical kits. In this study production and purification of polyclonal antibody against melatonin has been considered. This hormone which has several important functions in physiological conditions such as migraine, cirrhosis, mammary gland cancer and other diseases, is the most important pineal gland secretion. This gland is a circumventricular organ of brain and according to histological and anatomical studies, it is a high secretory organ, that secretes active biological substances like melatonin, oxytocin, serotonin and ect. In this study, melatonin has been considered as hapten and has become an immunogen by being linked to the bovine serum Albumin. Then, by the immunization of three white New Zeland rabbits that had the booster injections in regular intervals, the antibody titer was detected to be 1/2000, by using checkboard curves, and with the use of melatonin linked to penicillinase as a labeled antigen, the titer was detected 1/200. Finally an antibody with high purification rate has been obtained, which can be used in immunochemical assays like RIA, ELISA, and EIA.

  2. Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance.

    Directory of Open Access Journals (Sweden)

    Emily Xie

    Full Text Available The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

  3. Combining Exosomes Derived from Immature DCs with Donor Antigen-Specific Treg Cells Induces Tolerance in a Rat Liver Allograft Model

    Science.gov (United States)

    Ma, Ben; Yang, Jing-Yue; Song, Wen-jie; Ding, Rui; Zhang, Zhuo-chao; Ji, Hong-chen; Zhang, Xuan; Wang, Jian-lin; Yang, Xi-sheng; Tao, Kai-shan; Dou, Ke-feng; Li, Xiao

    2016-01-01

    Allograft tolerance is the ultimate goal in the field of transplantation immunology. Immature dendritic cells (imDCs) play an important role in establishing tolerance but have limitations, including potential for maturation, short lifespan in vivo and short storage times in vitro. However, exosomes (generally 30–100 nm) from imDCs (imDex) retain many source cell properties and may overcome these limitations. In previous reports, imDex prolonged the survival time of heart or intestine allografts. However, tolerance or long-term survival was not achieved unless immune suppressants were used. Regulatory T cells (Tregs) can protect allografts from immune rejection, and our previous study showed that the effects of imDex were significantly associated with Tregs. Therefore, we incorporated Tregs into the treatment protocol to further reduce or avoid suppressant use. We defined the optimal exosome dose as approximately 20 μg (per treatment before, during and after transplantation) in rat liver transplantation and the antigen-specific role of Tregs in protecting liver allografts. In the co-treatment group, recipients achieved long-term survival, and tolerance was induced. Moreover, imDex amplified Tregs, which required recipient DCs and were enhanced by IL-2. Fortunately, the expanded Tregs retained their regulatory ability and donor-specificity. Thus, imDex and donor-specific Tregs can collaboratively induce graft tolerance. PMID:27640806

  4. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay

    Directory of Open Access Journals (Sweden)

    M. Anthony Moody

    2012-03-01

    Full Text Available The traditional enzyme-linked immunospot (ELISpot assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities have so far prevented their wide utilization as well as further expansion of their multicolor capability. An attractive solution is to replace the chromogenic reaction of the traditional ELISpot assay with a fluorescent detection system (fluorospot assay. Fluorospot assays using fluorophore-conjugated secondary antibodies in conjunction with fluorescence enhancers, FITC/anti-FITC and biotin/avidin amplification systems and dedicated equipment for spot detection have been developed to enumerate T-cells secreting two or three specific cytokines and, more recently, IgG and IgA antibody-secreting cells (ASCs. We hereby report a method for a multiplex B cell fluorospot assay that utilizes quantum-dot nanocrystals as reporters without further amplification systems or need of dedicated equipment. With this method we simultaneously enumerated HIV-1 gp41 envelope glycoprotein-specific IgG and IgM antibody-secreting cells with sensitivity comparable to that of the traditional ELISpot assay.

  5. Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen's Specific Memory T Cells in Healthy Household Contacts.

    Science.gov (United States)

    Devasundaram, Santhi; Gopalan, Akilandeswari; Das, Sulochana D; Raja, Alamelu

    2016-01-01

    In vitro mimicking conditions are thought to reflect the environment experienced by Mycobacterium tuberculosis inside the host granuloma. The majority of in vitro dormancy experimental models use laboratory-adapted strains H37Rv or Erdman instead of prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from hypoxic cultures, resolved by two-dimensional electrophoresis and protein spots were characterized by mass spectrometry. In total, 49 spots were characterized as over-expressed or newly emergent between the three strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these two genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolor flow cytometry analysis showed high levels of antigen specific CD4(+) central memory T cells in the circulation of HHC compared to PTB (p hypoxia in most prevalent clinical strains would indicate possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also elucidate the probable true representative antigens involved in adaptive mechanisms. PMID:27667981

  6. Combining Exosomes Derived from Immature DCs with Donor Antigen-Specific Treg Cells Induces Tolerance in a Rat Liver Allograft Model.

    Science.gov (United States)

    Ma, Ben; Yang, Jing-Yue; Song, Wen-Jie; Ding, Rui; Zhang, Zhuo-Chao; Ji, Hong-Chen; Zhang, Xuan; Wang, Jian-Lin; Yang, Xi-Sheng; Tao, Kai-Shan; Dou, Ke-Feng; Li, Xiao

    2016-01-01

    Allograft tolerance is the ultimate goal in the field of transplantation immunology. Immature dendritic cells (imDCs) play an important role in establishing tolerance but have limitations, including potential for maturation, short lifespan in vivo and short storage times in vitro. However, exosomes (generally 30-100 nm) from imDCs (imDex) retain many source cell properties and may overcome these limitations. In previous reports, imDex prolonged the survival time of heart or intestine allografts. However, tolerance or long-term survival was not achieved unless immune suppressants were used. Regulatory T cells (Tregs) can protect allografts from immune rejection, and our previous study showed that the effects of imDex were significantly associated with Tregs. Therefore, we incorporated Tregs into the treatment protocol to further reduce or avoid suppressant use. We defined the optimal exosome dose as approximately 20 μg (per treatment before, during and after transplantation) in rat liver transplantation and the antigen-specific role of Tregs in protecting liver allografts. In the co-treatment group, recipients achieved long-term survival, and tolerance was induced. Moreover, imDex amplified Tregs, which required recipient DCs and were enhanced by IL-2. Fortunately, the expanded Tregs retained their regulatory ability and donor-specificity. Thus, imDex and donor-specific Tregs can collaboratively induce graft tolerance. PMID:27640806

  7. γδ T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen-specific interleukin-17 response.

    Science.gov (United States)

    Zeng, Xun; Wei, Yu-Ling; Huang, Jun; Newell, Evan W; Yu, Hongxiang; Kidd, Brian A; Kuhns, Michael S; Waters, Ray W; Davis, Mark M; Weaver, Casey T; Chien, Yueh-hsiu

    2012-09-21

    γδ T cells contribute uniquely to immune competence. Nevertheless, how they function remains an enigma. It is unclear what most γδ T cells recognize, what is required for them to mount an immune response, and how the γδ T cell response is integrated into host immune defense. Here, we report that a noted B cell antigen, the algae protein phycoerythrin (PE), is a murine and human γδ T cell antigen. Employing this specificity, we demonstrated that antigen recognition activated naive γδ T cells to make interleukin-17 and respond to cytokine signals that perpetuate the response. High frequencies of antigen-specific γδ T cells in naive animals and their ability to mount effector response without extensive clonal expansion allow γδ T cells to initiate a swift, substantial response. These results underscore the adaptability of lymphocyte antigen receptors and suggest an antigen-driven rapid response in protective immunity prior to the maturation of classical adaptive immunity. PMID:22960222

  8. Near-infrared labeled, ovalbumin loaded polymeric nanoparticles based on a hydrophilic polyester as model vaccine : In vivo tracking and evaluation of antigen-specific CD8+ T cell immune response

    NARCIS (Netherlands)

    Rahimian, Sima; Kleinovink, Jan Willem; Fransen, Marieke F.; Mezzanotte, Laura; Gold, Henrik; Wisse, Patrick; Overkleeft, Hermen; Amidi, Maryam; Jiskoot, Wim; Lo¨wik, Clemens W.; Ossendorp, Ferry; Hennink, Wim E.

    2015-01-01

    Particulate antigen delivery systems aimed at the induction of antigen-specific T cells form a promising approach in immunotherapy to replace pharmacokinetically unfavorable soluble antigen formulations. In this study, we developed a delivery system using the model protein antigen ovalbumin (OVA) en

  9. Passive protection by polyclonal antibodies against Bacillus anthracis infection in guinea pigs.

    OpenAIRE

    Little, S F; Ivins, B E; Fellows, P F; Friedlander, A M

    1997-01-01

    The protective effects of polyclonal antisera produced by injecting guinea pigs with protective antigen (PA), the chemical anthrax vaccine AVA, or Sterne spore vaccine, as well as those of toxin-neutralizing monoclonal antibodies (MAbs) produced against PA, lethal factor, and edema factor, were examined in animals infected with Bacillus anthracis spores. Only the anti-PA polyclonal serum significantly protected the guinea pigs from death, with 67% of infected animals surviving. Although none ...

  10. Leishmania (Viannia) braziliensis nucleoside triphosphate diphosphohydrolase (NTPDase 1): localization and in vitro inhibition of promastigotes growth by polyclonal antibodies.

    Science.gov (United States)

    Porcino, Gabriane Nascimento; Carvalho-Campos, Cristiane; Maia, Ana Carolina Ribeiro Gomes; Detoni, Michelle Lima; Faria-Pinto, Priscila; Coimbra, Elaine Soares; Marques, Marcos José; Juliano, Maria Aparecida; Juliano, Luiz; Diniz, Vanessa Álvaro; Corte-Real, Suzana; Vasconcelos, Eveline Gomes

    2012-10-01

    Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control. PMID:22921497

  11. Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

    Directory of Open Access Journals (Sweden)

    Gomez Bianca P

    2012-10-01

    Full Text Available Abstract Background Merkel cell carcinoma (MCC is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV. The MCPyV-encoded large T (LT antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT, as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the

  12. Expression of a highly antigenic and native-like folded extracellular domain of the human α1 subunit of muscle nicotinic acetylcholine receptor, suitable for use in antigen specific therapies for Myasthenia Gravis.

    Directory of Open Access Journals (Sweden)

    Athanasios Niarchos

    Full Text Available We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR in lepidopteran insect cells (i-α1-ECD and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG. Compared to the previously expressed protein in P. pastoris (y-α1-ECD, i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies.

  13. Tumor Antigen-Specific Cytotoxic T Lymphocytes and Cancer Immunotherapy--Review%肿瘤抗原特异性细胞毒T细胞和肿瘤免疫治疗

    Institute of Scientific and Technical Information of China (English)

    刘英

    2004-01-01

    肿瘤抗原特异性细胞毒T细胞(CTL)是重要的抗肿瘤细胞.本文介绍了抗原呈递及CTL识别的分子基础,并对肿瘤相关抗原(TAA)及T细胞表位的鉴定、肿瘤相关抗原肽诱导特异性CTL应答的免疫原性、肿瘤相关抗原肽疫苗治疗肿瘤的原理以及T细胞免疫应用前景做一综述.

  14. Continuous activation of the CD122/STAT-5 signaling pathway during selection of antigen-specific regulatory T cells in the murine thymus.

    Directory of Open Access Journals (Sweden)

    Jérémie D Goldstein

    Full Text Available Signaling events affecting thymic selection of un-manipulated polyclonal natural CD25(+foxp3(+ regulatory T cells (nTreg have not been established ex vivo. Here, we report a higher frequency of phosphorylated STAT-5 (pSTAT-5 in nTreg cells in the adult murine thymus and to a lesser extent in the periphery, compared to other CD4(+CD8(- subsets. In the neonatal thymus, the numbers of pSTAT-5(+ cells in CD25(+foxp3(- and nTreg cells increased in parallel, suggesting that pSTAT-5(+CD25(+foxp3(- cells might represent the precursors of foxp3(+ regulatory T cells. This "specific" pSTAT-5 expression detected in nTreg cells ex vivo was likely due to a very recent signal given by IL-2/IL-15 cytokines in vivo since (i it disappeared rapidly if cells were left unstimulated in vitro and (ii was also observed if total thymocytes were stimulated in vitro with saturating amounts of IL-2 and/or IL-15 but not IL-7. Interestingly, STAT-5 activation upon IL-2 stimulation correlated better with foxp3 and CD122 than with CD25 expression. Finally, we show that expression of an endogenous superantigen strongly affected the early Treg cell repertoire but not the proportion of pSTAT-5(+ cells within this repertoire. Our results reveal that continuous activation of the CD122/STAT-5 signaling pathway characterize regulatory lineage differentiation in the murine thymus.

  15. Production of the Polyclonal Anti-human Metallothionein 2A Antibody with Recombinant Protein Technology

    Institute of Scientific and Technical Information of China (English)

    Faiz M.M.T.MARIKAR; Qi-Ming SUN; Zi-Chun HUA

    2006-01-01

    Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the eDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli.Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in Western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays.

  16. Use of polyclonal IgG in HIV infection and AIDS

    International Nuclear Information System (INIS)

    Nuclear Medicine should have a pivitol role to play in the investigation of patients infected with the Human Immunodeficiency Virus (HIV). Unfortunately the use of scintigraphic techniques to localize infection have not become widely used in Europe. Neither 67Ga citrate or labelled leukocytes are ideal. In a search for new agents which can be used to identify the presence of infection both 99mTc and 111In labelled polyclonal immunoglobulin-C have been investigated. It was found that 99mTC labelled polyclonal immunoglobulin-G was not able to localize infection in either the chest or the abdomen. In contrast 111In labelled polyclonal immunoglobulin-G had both high sensitivity and specificity for imaging infection in HIV infected patients. If these preliminary results are confirmed immunoglobulin-G could find an important clinical application in this specialized patient group

  17. Maintenance of Serological Memory by Polyclonal Activation of Human Memory B Cells

    Science.gov (United States)

    Bernasconi, Nadia L.; Traggiai, Elisabetta; Lanzavecchia, Antonio

    2002-12-01

    Production of antibodies can last for a lifetime, through mechanisms that remain poorly understood. Here, we show that human memory B lymphocytes proliferate and differentiate into plasma cells in response to polyclonal stimuli, such as bystander T cell help and CpG DNA. Furthermore, plasma cells secreting antibodies to recall antigens are produced in vivo at levels proportional to the frequency of specific memory B cells, even several years after antigenic stimulation. Although antigen boosting leads to a transient increase in specific antibody levels, ongoing polyclonal activation of memory B cells offers a means to maintain serological memory for a human lifetime.

  18. Mobile Robot Path Planning Using Polyclonal-Based Artificial Immune Network

    Directory of Open Access Journals (Sweden)

    Lixia Deng

    2013-01-01

    Full Text Available Polyclonal based artificial immune network (PC-AIN is utilized for mobile robot path planning. Artificial immune network (AIN has been widely used in optimizing the navigation path with the strong searching ability and learning ability. However, artificial immune network exists as a problem of immature convergence which some or all individuals tend to the same extreme value in the solution space. Thus, polyclonal-based artificial immune network algorithm is proposed to solve the problem of immature convergence in complex unknown static environment. Immunity polyclonal algorithm (IPCA increases the diversity of antibodies which tend to the same extreme value and finally selects the antibody with highest concentration. Meanwhile, immunity polyclonal algorithm effectively solves the problem of local minima caused by artificial potential field during the structure of parameter in artificial immune network. Extensive experiments show that the proposed method not only solves immature convergence problem of artificial immune network but also overcomes local minima problem of artificial potential field. So, mobile robot can avoid obstacles, escape traps, and reach the goal with optimum path and faster convergence speed.

  19. Antigenic profile of human recombinant PrP: generation and chracterization of a versatile polyclonal antiserum

    NARCIS (Netherlands)

    Sachsamanoglou, M.; Paspaltzis, I.; Petrakis, S.; Verghese-Nikolakaki, S.; Panagiotidis, C.H.; Voitlander, T.; Budka, H.; Langeveld, J.P.M.; Sklaviadis, T.

    2004-01-01

    We describe the quality of a rabbit polyclonal antiserum (Sal1) that was raised against mature human recombinant prion protein (rhuPrP). Epitope mapping demonstrated that the Sal1 antiserum recognized six to eight linear antigenic sites, depending on the animal species. The versatility of the antise

  20. Suppressor T cells, distinct from "veto cells," are induced by alloantigen priming and mediate transferable suppression of cytotoxic T lymphocyte responses in vivo

    DEFF Research Database (Denmark)

    Owens, T; Crispe, I N

    1985-01-01

    Primary and secondary cytotoxic T lymphocyte responses to minor alloantigens can be suppressed by priming host mice with a high dose (10(8) cells) of alloantigenic donor spleen cells (SC). Such suppression is antigen specific and transferable into secondary hosts with T cells. One interpretation...... described here exclude veto T cell participation in transferable alloantigen-specific suppression, and demonstrate the operation of an alloantigen-specific host-derived T suppressor (Ts) cell. The origin of the Ts has been studied directly by using Thy-1-disparate BALB/c mice. The cell responsible...... for the transfer of suppression of a secondary CTL response to B10 minors was of the host Thy-1 allotype, and so originated in the host spleen and was not introduced in the priming inoculum. Secondly, antigen-specific Ts generated in CBA female mice against B10 minors could act on CTL responses to an unequivocally...

  1. Prime-boost vaccination with Bacillus Calmette Guerin and a recombinant adenovirus co-expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis induces robust antigen-specific immune responses in mice.

    Science.gov (United States)

    Li, Wu; Li, Min; Deng, Guangcun; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2015-08-01

    Tuberculosis (TB) remains to be a prevalent health issue worldwide. At present, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the singular anti-TB vaccine available for the prevention of disease in humans; however, this vaccine only provides limited protection against Mycobacterium tuberculosis (Mtb) infection. Therefore, the development of alternative vaccines and strategies for increasing the efficacy of vaccination against TB are urgently required. The present study aimed to evaluate the ability of a recombinant adenoviral vector (Ad5-CEAB) co-expressing 10-kDa culture filtrate protein, 6-kDa early-secreted antigenic target, antigen 85 (Ag85)A and Ag85B of Mtb to boost immune responses following primary vaccination with BCG in mice. The mice were first subcutaneously primed with BCG and boosted with two doses of Ad5-CEAB via an intranasal route. The immunological effects of Ad5-CEAB boosted mice primed with BCG were then evaluated using a series of immunological indexes. The results demonstrated that the prime-boost strategy induced a potent antigen-specific immune response, which was primarily characterized by an enhanced T cell response and increased production of cytokines, including interferon-γ, tumor necrosis factor-α and interleukin-2, in mice. In addition, this vaccination strategy was demonstrated to have an elevated humoral response with increased concentrations of antigen-specific bronchoalveolar lavage secretory immunoglobulin (Ig)A and serum IgG in mice compared with those primed with BCG alone. These data suggested that the regimen of subcutaneous BCG prime and mucosal Ad5-CEAB boost was a novel strategy for inducing a broad range of antigen-specific immune responses to Mtb antigens in vivo, which may provide a promising strategy for further development of adenoviral-based vaccine against Mtb infection.

  2. Polyclonality among clinical strains of non-pigmented rapidly growing mycobacteria: phenotypic and genotypic differences and their potential implications.

    Science.gov (United States)

    García-Pedrazuela, M; Frutos, J M; Muñoz-Egea, M C; Alcaide, F; Tórtola, T; Vitoria, A; Cortés, P; Esteban, J

    2015-04-01

    To investigate the potential implications (especially the implications in clinical significance and antimicrobial susceptibility) of polyclonality among rapidly growing mycobacteria, we performed random amplified polymorphic DNA analysis in 64 clinical isolates of which the clinical significance was established. Phenotypic characteristics (antimicrobial susceptibility test, colony morphology and growth rate) of each clone were studied. Polyclonality was detected in 13 of the isolates (20.3%). There was a relationship between monoclonality and clinical significance (p 0.0096). Monoclonal and polyclonal isolates showed different behaviour in antimicrobial susceptibility. There was a strong relationship between monoclonality and those species that are more pathogenic for humans, and also with clinical significance of the isolates. PMID:25596780

  3. Therapeutic potential of CD8+ cytotoxic T lymphocytes in SLE☆

    Science.gov (United States)

    Puliaeva, I.; Puliaev, R.; Via, C.S.

    2011-01-01

    Recent evidence supports the idea that following a break in tolerance, CD8 cytotoxic T lymphocytes (CTL) may be an important but unrecognized mechanism for limiting expansion of autoreactive B cells. Failure of this mechanism could allow persistence of CD4 T cell driven polyclonal B cell activation resulting in clinical lupus. Although CD8 CTL failure may occur early in disease, work in mice supports the concept that therapeutic CTL enhancement may be both practical and beneficial in lupus. Devising such therapy for humans will first require an understanding of the in vivo mechanisms critical in CTL expansion and down regulation, particularly in the lupus setting which may differ from CTL generation in other clinical settings (e.g. tumors, infections). PMID:18725326

  4. Expression of Recombinant Human FADD, Preparation of Its Polyclonal Antiserum and the Application in Immunoassays

    Institute of Scientific and Technical Information of China (English)

    Faiz MMT Marikar; Dingyuan Ma; Jianqiang Ye; Bo Tang; Weijuan Zheng; Jing Zhang; Min Lu; Zichun Hua

    2008-01-01

    The wild-type human Fas-associated death domain (FADD) protein was expressed as a His-tag fusion protein in Escherichla coli. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refoided and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD不得 protein was allowed the production of high titre polycional antiserum. This new polyclonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofluorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays.Cellular & Molecular Immunology. 2008;5(6):471-474.

  5. Phenotypic and Functional Analysis of LCMV gp33-41-Specific CD8 T Cells Elicited by Multiple Peptide Immunization in Mice Revealed the Up-regulation of PD-1 Expression on Antigen Specific CD8 T Cells

    Institute of Scientific and Technical Information of China (English)

    Yi Liu; Lihui Xu; Yiqun Jiang; Jianfang Sun; Xianhui He

    2007-01-01

    The phenotype and function of antigen-specific CD8 T cells are closely associated with the efficacy of a therapeutic vaccination. Here we showed that multiple immunizations with LCMV gp33-41 peptide (KAV) in Freund's adjuvant could induce KAV-specific CD8 T cells with low expression of CD127 and CD62L molecules. The inhibitory receptor PD-1 was also expressed on a substantial part of KAV-specific CD8 T cells, and its expression level on KAV-specific CD8 T cells in spleen and lymph nodes was much higher when compared to those in peripheral blood. Furthermore, KAV-specific CD8 T cells could specifically kill KAV-pulsed target cells in vivo but the efficiency was low. These data suggest that prime-boost vaccination schedule with peptide in Freund's adjuvant can elicit antigen-specific CD8 T cells of effector-like phenotype with partial functional exhaustion, which may only provide short-term protection against the pathogen.

  6. Near-infrared labeled, ovalbumin loaded polymeric nanoparticles based on a hydrophilic polyester as model vaccine: In vivo tracking and evaluation of antigen-specific CD8(+) T cell immune response.

    Science.gov (United States)

    Rahimian, Sima; Kleinovink, Jan Willem; Fransen, Marieke F; Mezzanotte, Laura; Gold, Henrik; Wisse, Patrick; Overkleeft, Hermen; Amidi, Maryam; Jiskoot, Wim; Löwik, Clemens W; Ossendorp, Ferry; Hennink, Wim E

    2015-01-01

    Particulate antigen delivery systems aimed at the induction of antigen-specific T cells form a promising approach in immunotherapy to replace pharmacokinetically unfavorable soluble antigen formulations. In this study, we developed a delivery system using the model protein antigen ovalbumin (OVA) encapsulated in nanoparticles based on the hydrophilic polyester poly(lactide-co-hydroxymethylglycolic acid) (pLHMGA). Spherical nanoparticles with size 300-400 nm were prepared and characterized and showed a strong ability to deliver antigen to dendritic cells for cross-presentation to antigen-specific T cells in vitro. Using near-infrared (NIR) fluorescent dyes covalently linked to both the nanoparticle and the encapsulated OVA antigen, we tracked the fate of this formulation in mice. We observed that the antigen and the nanoparticles are efficiently co-transported from the injection site to the draining lymph nodes, in a more gradual and durable manner than soluble OVA protein. OVA-loaded pLHMGA nanoparticles efficiently induced antigen cross-presentation to OVA-specific CD8+ T cells in the lymph nodes, superior to soluble OVA vaccination. Together, these data show the potential of pLHMGA nanoparticles as attractive antigen delivery vehicles.

  7. Polyclonal antibody cocktails generated using DNA vaccine technology protect in murine models of orthopoxvirus disease

    Directory of Open Access Journals (Sweden)

    Ballantyne John

    2011-09-01

    Full Text Available Abstract Background Previously we demonstrated that DNA vaccination of nonhuman primates (NHP with a small subset of vaccinia virus (VACV immunogens (L1, A27, A33, B5 protects against lethal monkeypox virus challenge. The L1 and A27 components of this vaccine target the mature virion (MV whereas A33 and B5 target the enveloped virion (EV. Results Here, we demonstrated that the antibodies produced in vaccinated NHPs were sufficient to confer protection in a murine model of lethal Orthopoxvirus infection. We further explored the concept of using DNA vaccine technology to produce immunogen-specific polyclonal antibodies that could then be combined into cocktails as potential immunoprophylactic/therapeutics. Specifically, we used DNA vaccines delivered by muscle electroporation to produce polyclonal antibodies against the L1, A27, A33, and B5 in New Zealand white rabbits. The polyclonal antibodies neutralized both MV and EV in cell culture. The ability of antibody cocktails consisting of anti-MV, anti-EV, or a combination of anti-MV/EV to protect BALB/c mice was evaluated as was the efficacy of the anti-MV/EV mixture in a mouse model of progressive vaccinia. In addition to evaluating weight loss and lethality, bioimaging technology was used to characterize the spread of the VACV infections in mice. We found that the anti-EV cocktail, but not the anti-MV cocktail, limited virus spread and lethality. Conclusions A combination of anti-MV/EV antibodies was significantly more protective than anti-EV antibodies alone. These data suggest that DNA vaccine technology could be used to produce a polyclonal antibody cocktail as a possible product to replace vaccinia immune globulin.

  8. Polyclonal B-cell response to stimulation with Escherichia coli lipopolysaccharide in dietary protein restriction.

    OpenAIRE

    Malavé, I; Pocino, M

    1982-01-01

    The polyclonal B-cell response to Escherichia coli lipopolysaccharide was studied in C57BL/6 mice maintained after weaning on either a moderate protein-restricted diet with 8% casein or a normal diet. After in vitro or in vivo stimulation with the endotoxin, autoreactive and anti-hapten antibody-producing cells were quantitated by direct plaque assay, using bromelain-treated mouse erythrocytes and trinitrophenylated sheep erythrocytes as targets. Larger numbers of plaque-forming cells were ge...

  9. PRODUCTION AND CHARACTERIZATION OF MONOCLONAL AND POLYCLONAL ANTIBODY AGAINST RECOMBINANT OUTER MEMBRANE PROTEIN

    Directory of Open Access Journals (Sweden)

    Mahdi Fasihi-Ramandi

    2014-01-01

    Full Text Available There are many studies related to immunological and molecular methods for diagnosis of Vibrio cholera (V. cholerae. However, most assays dependent on enrichment of culture of bacteria, which need more time and involves the use of costly equipment and reagents. In this study Balb/c mice were immunized with recombinant Outer Membrane Protein (rOMPw of vibrio cholerae and splenocytes of hyper immunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using rOMPw as coating antigen. The monoclonal antibodies from ascitic fluids were purified and its reaction with rOMPw was assessed by ELISA. Polyclonal antibodies were also produced by immunization of rabbits with the above mentioned antigen. The rabbit sera was affinity purified using Hi-Trap protein G column. The result showed that monoclonal antibody specific to rOMPw has been successfully generated. The monoclonal antibody reacted with recombinant OMPw in ELISA and immunonoblat method. Rabbit polyclonal antibody was also bound to rOMPw by ELISA. The results of agglutination test with whole bacteria also showed that both mouse monoclonal and rabbit polyclonal antibodies reacted with whole vibrio cholera but not other related bacteria. The purpose of this study was to check out if anti OMPw antibodies could use as diagnostic assay for detection of V. cholerae. Our results demonstrated that anti recombinant OMPw monoclonal and polyclonal antibodies are able to diagnose whole bacteria in pure culture using agglutination test but not by home made immunochromatic strip test.

  10. Promotion of remyelination by polyclonal immunoglobulin in Theiler's virus-induced demyelination and in multiple sclerosis.

    OpenAIRE

    van Engelen, B. G.; Miller, D.J.; Pavelko, K. D.; Hommes, O R; Rodriguez, M.

    1994-01-01

    Spontaneous remyelination occurs in experimental models of demyelination and in patients with multiple sclerosis, although to a limited extent. This enables the search for factors that promote remyelination. Using the Theiler's virus model of central nervous system demyelination, promotion of remyelination was observed after passive transfer of CNS-specific antiserum and transfer of CNS-specific purified immunoglobulin. Mouse polyclonal immunoglobulin from normal non-syngeneic mice, comparabl...

  11. Preparation and Purification of Polyclonal Antibodies against Mycobacterium Avium Paratuberculosis Antigens in Rabbit

    Directory of Open Access Journals (Sweden)

    Hafezeh Alizadeh

    2012-12-01

    Full Text Available Background and Objective: Johne’s disease is the chronic granulomatous enteritis of ruminants, and a major health hazard worldwide. In recent years, researchers have focused on mycobacterium avium subsp. paratuberculosis (MAP antigens in diagnostic tests. Identification of antibodies against MAP antigens is, therefore, effective for the diagnosis or preparation of vaccine. The aim of this study was to prepare and purify polyclonal antibodies against MAP antigens. Materials and Methods: A New Zealand white rabbit was immunized at a certain time period with MAP antigens and Freund’s adjuvant. After the immunization of the animal, the rabbit was bled to obtain enriched serum. Immunoglobulins were obtained via sedimentation with ammonium sulfate 35% and then IgG was purified by ion exchange (DEAE-cellulose chromatography. Serologic test was used to evaluate the interaction of antigens and antibodies. Results: Ion exchange chromatography of IgG showed one peak, and SDS_PAGE of IgG showed a single band. Serologic test was applied and clear precipitation lines were appeared up to 1:16 dilution, which indicated the high quality of the product. Conclusion: In this study, the humoral immune response was induced well by immunization with MAP antigens in a New Zealand white rabbit and polyclonal antibodies were produced in high titers. Polyclonal antibodies are relatively inexpensive and easy to produce in large quantities and can connect to the more connective sites, resulting in better sensitivity. Identification of polyclonal antibodies via immunological tests can play a significant role in studying MAP disorders.

  12. Production and Purification of Rabbit’s Polyclonal Antibody Against Factor VIII

    OpenAIRE

    Sohrabi, Simin; Akbarzadeh, Azim; Norouzian, Dariush; Farhangi, Ali; Mortazavi, Mehri; Mehrabi, Mohammad Reza; Chiani, Mohsen; Saffari, Zahra; Ghassemi, Soheil

    2011-01-01

    The attempt is made to produce recombinant factor VIII but the first step in producing such product is production and purification of rabbit’s polyclonal antibody against factor VIII. The second and third steps involve monoclonal antibody and recombinant factor VIII production. Factor VIII is one of the most important coagulating factor where its deficiency leads to diseases like hemophilia type A or classic. It is an inherited disease. Previously, it was obtained through fractionation of blo...

  13. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    OpenAIRE

    Nazila Amini; Mohadeseh Naghi Vishteh; Omid Zarei; Reza Hadavi; Negah Ahmadvand; Hodjattallah Rabbani; Mahmood Jeddi-Tehrani

    2014-01-01

    Objective(s):Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protei...

  14. Cytotoxicity of carbon nanotubes

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    With large-scale production and application at large scale, carbon nanotubes (CNTs) may cause ad-verse response to the environment and human health. Thus, study on bio-effects and safety of CNTs has attracted great attention from scientists and governments worldwide. This report briefly summa-rizes the main results from the in vitro toxicity study of CNTs. The emphasis is placed on the descrip-tion of a variety of factors affecting CNTs cytotoxicity, including species of CNTs, impurities contained, lengths of CNTs, aspect ratios, chemical modification, and assaying methods of cytotoxicity. However, experimental information obtained thus far on CNTs’ cytotoxicity is lacking in comparability, and some-times there is controversy about it. In order to assess more accurately the potential risks of CNTs to human health, we suggest that care should be taken for issues such as chemical modification and quantitative characterization of CNTs in cytotoxicity assessment. More importantly, studies on physical and chemical mechanisms of CNTs’ cytotoxicity should be strengthened; assaying methods and evaluating criteria characterized by nanotoxicology should be gradually established.

  15. Cytotoxicity of carbon nanotubes

    Institute of Scientific and Technical Information of China (English)

    ZHU Ying; LI WenXin

    2008-01-01

    With large-scale production and application at large scale, carbon nanotubes (CNTs) may cause ad-verse response to the environment and human health. Thus, study on bio-effects and safety of CNTs has attracted great attention from scientists and governments worldwide. This report briefly summa-rizes the main results from the in vitro toxicity study of CNTs. The emphasis is placed on the descrip-tion of a variety of factors affecting CNTs cytotoxicity, including species of CNTs, impurities contained,lengths of CNTs, aspect ratios, chemical modification, and assaying methods of cytotoxicity. However,experimental information obtained thus far on CNTs' cytotoxicity is lacking in comparability, and some-times there is controversy about it. In order to assess more accurately the potential risks of CNTs to human health, we suggest that care should be taken for issues such as chemical modification and quantitative characterization of CNTa in cytotoxicity assessment. More importantly, studies on physical and chemical mechanisms of CNTs' cytotoxicity should be strengthened; assaying methods and evaluating criteria characterized by nanotoxicology should be gradually established.

  16. Preparation of Polyclonal Antibody and Expression Analysis of GR in Tomato

    Science.gov (United States)

    Xie, Yuanhong; Zhu, Benzhong; Luo, Yunbo; Chen, Xiangning; Zhang, Hongxing

    The fruit ripening of Green-ripe (Gr) mutant tomato was inhibited dramatically. To determine the expression patterns of Gr in tomato, we first produced the polyclonal antibody of Gr protein. RT-PCR was used to amplify the Gr gene from green ripe tomato fruit. And the PCR product was subcloned into prokaryotic protein expression vectors pET-30a to generate recombinant plasmid. The Gr protein was induced by IPTG in BL21 (DE3) and purified by Ni-NTA agarose column. The anti-Gr serum was produced by immunizing rabbits, and the titer of the anti-Gr serum was above 5000 by ELISA analysis. Purified by the DEAE-52 ion-column, the high purification level of anti-Gr polyclonal antibody was obtained. Furthermore, RT-CPR was used in the RNA level to demonstrate that the expression of Gr gene was specialized in some cultures of tomato. For example, the expressions of Gr were higher in seed, flower and green ripe fruit than others, and the expression level were reduced by exogenous ethylene treatment in the flower and green ripe fruit. Moreover, Polyclonal antibody of Gr was used to investigate the expression pattern of Gr in protein level by the Western blotting. Our results show that the expression level of Gr in protein level was complied with the expressions in RNA. So, we suggested that the regulation of Gr was transcriptional.

  17. Immunodiagnosis of episomal Banana streak MY virus using polyclonal antibodies to an expressed putative coat protein.

    Science.gov (United States)

    Sharma, Susheel Kumar; Kumar, P Vignesh; Baranwal, Virendra Kumar

    2014-10-01

    A cryptic Badnavirus species complex, known as banana streak viruses (BSV) poses a serious threat to banana production and genetic improvement worldwide. Due to the presence of integrated BSV sequences in the banana genome, routine detection is largely based on serological and nucleo-serological diagnostic methods which require high titre specific polyclonal antiserum. Viral structural proteins like coat protein (CP) are the best target for in vitro expression, to be used as antigen for antiserum production. However, in badnaviruses precise CP sequences are not known. In this study, two putative CP coding regions (p48 and p37) of Banana streak MY virus (BSMYV) were identified in silico by comparison with caulimoviruses, retroviruses and Rice tungro bacilliform virus. The putative CP coding region (p37) was in vitro expressed in pMAL system and affinity purified. The purified fusion protein was used as antigen for raising polyclonal antiserum in rabbit. The specificity of antiserum was confirmed in Western blots, immunosorbent electron microscopy (ISEM) and antigen coated plate-enzyme linked immunosorbent assay (ACP-ELISA). The antiserum (1:2000) was successfully used in ACP-ELISA for specific detection of BSMYV infection in field and tissue culture raised banana plants. The antiserum was also utilized in immuno-capture PCR (IC-PCR) based indexing of episomal BSMYV infection. This is the first report of in silico identification of putative CP region of BSMYV, production of polyclonal antiserum against recombinant p37 and its successful use in immunodetection.

  18. Prokaryotic Expression of Glycoprotein Gene of Infectious Hnematopoietic Necrosis Virus and Polyclonal Antibody Preparation

    Institute of Scientific and Technical Information of China (English)

    Liu; Xueguang; Zheng; Huaidong; Guo; Xinshuo; Luo; Jin; Lin; Cuicui; Wang; Qiuyu

    2014-01-01

    [Objective]The aim is to perform prokaryotic expression of the glycoprotein gene of infectious hnematopoietic necrosis virus and polyclonal antibody preparation. [Methods]Glycoprotein gene( G) of infectious hematopoietic tissue( IHNV) was synthesized,cloned to prokaryotic expression system pET-30a vector,yielding the recombinant plasmid pET-30a-IHNV-G. The yielded pET-30a-IHNV-G was transformed into E. coli strain BL21( DE3) plySs. [Results] SDSPAGE and Western blot results showed that protein G successfully expressed in E. coli at 37 ℃,1 mmol /L IPTG induction for 4 h. The molecular weight of fusion G protein was 57 KD. The polyclonal antibody was prepared by immunizing mice with the product of gel purification. ELISA analysis showed that the serum titer reached 1∶10 000. [Conclusion]The expressed G protein and the serum with polyclonal antibody obtained in this study provided the theoretical basis for the development of IHNV vaccine and detection of colloidal gold test strip.

  19. Cloning, Expression and Polyclonal Antibody Preparation of the Asialoglycoprotein Receptor of Marmota Himalayan

    Institute of Scientific and Technical Information of China (English)

    YANG Yan; HUANG Huang; ZHANG Zhenghua; WANG Baoju; TIAN Yongjun; LU Mengji; YANG Dongliang

    2007-01-01

    The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E. coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

  20. Limited transplantation of antigen-expressing hematopoietic stem cells induces long-lasting cytotoxic T cell responses.

    Directory of Open Access Journals (Sweden)

    Warren L Denning

    Full Text Available Harnessing the ability of cytotoxic T lymphocytes (CTLs to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4-6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS.

  1. Development of a polyclonal competitive enzyme-linked immunosorbent assay for detection of antibodies to Ehrlichia ruminantium

    OpenAIRE

    Sumption, Keith J.; Paxton, Edith; Bell-Sakyi, Lesley

    2003-01-01

    A polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) is described for detection of antibodies to Ehrlichia (Cowdria) ruminantium by using a soluble extract of endothelial cell culture-derived E. ruminantium as the antigen and biotin-labeled polyclonal goat immunoglobulins as the competitor. For goats, the diagnostic sensitivity and specificity were both 100% with a cutoff of 80% inhibition (80 PI), with detection of antibodies for 550 days postinfection. For cattle, diagnosti...

  2. Monoclonal and polyclonal antibodies for ante- and post-mortem detection of PrPSc in sheep

    Directory of Open Access Journals (Sweden)

    Michele Dietrich Moura Costa

    2015-04-01

    Full Text Available Scrapie is a disease that affects sheep and goats and is characterized by the accumulation of an abnormal isoform (PrPSc of the cellular prion protein, PrPC, in the central nervous system (CNS and in lymphoid tissues. Detection of PrPSc in these tissues can be attempted by a variety of techniques, including immunohistochemistry (IHC and western blotting (WB, for which a wide range of monoclonal and polyclonal antibodies are commercially available. The objective of this study was to test and compare the efficacy of monoclonal antibodiesF89/160.1.5, F99/97.6.1, and P4 and polyclonal antibodies M52 and R486 in the detection of PrPSc in lymphoid and CNS tissue samples by using IHC. Positive and negative control samples of sheep brain and tonsils were provided by the Animal Health and Veterinary Laboratories Agency (AHVLA, UK. The IHC examination of CNS samples with both monoclonal and polyclonal antibodies confirmed the granular deposition of PrPSc in the neurons of the positive control tissues. However, while the monoclonal antibodies did not produce positive reactions in the negative controls, the polyclonal antibodies showed some non-specific staining. The testing of positive control tonsil samples with polyclonal and monoclonal antibodies identified positive control-specific reactions, whereas the negative control tissues were IHC-negative with all antibodies, although P4 and the polyclonal antibodies produced some background staining. In summary, although the polyclonal antibodies may be more accessible, their use is not advisable because of possible false positive reactions. The polyclonal antibody M52 was able to identify PrPC in brain and spleen samples by WB but other lymphoid tissues were negative.

  3. Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1996-01-01

    HIV is the greatest single risk factor for the development of tuberculosis. Diseases caused by M. tuberculosis and mycobacteria are the most common opportunistic infections in HIV-infected persons, which may stem from a functional defect of the CD4+ T-cell-mediated killing of macrophages harboring...... mycobacteria. Our objective was to investigate the M.tuberculosis-and M. avium-specific cytotoxic capacity of T cells from healthy, bacille Calmette-Guérin-vaccinated, HIV-seropositive individuals. Blood mononuclear cells were obtained from 10 healthy HIV-seropositive and 10 healthy seronegative persons...... stimulation and by using purified CD4+ and CD8+ cell subsets. Substantial, but reduced antigen-specific cytotoxicity was observed in patients with asymptomatic HIV infection. The immunological dysfunction leading to reduced cytotoxic activity in healthy HIV-seropositive subjects could not be explained...

  4. A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts

    Directory of Open Access Journals (Sweden)

    Bendinelli Mauro

    2009-03-01

    Full Text Available Abstract Background Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with 51chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry. Results The method consists of culturing skin fibroblasts, immortalizing them with a replication defective clone of simian virus 40, and finally transducing them with a bicistronic vector encoding the target antigen and the reporter green fluorescent protein. When used in a flow cytometry-based assay, the target cells obtained with this method proved valuable for assessing the viral envelope protein specific cytotoxic T lymphocyte activity in domestic cats acutely or chronically infected with feline immunodeficiency virus, a lentivirus similar to human immunodeficiency virus and used as animal model for AIDS studies. Conclusion Given the versatility of the bicistronic vector used, its ability to deliver multiple and large transgenes in target cells, and its extremely wide cell specificity when pseudotyped with the vesicular stomatitis virus envelope protein, the method is potentially exploitable in many animal species.

  5. 妊娠期父源抗原特异性激活调节性T细胞的研究%Study on role of paternal antigens-specific CD4+CD25+T regulatory cells during pregnancy

    Institute of Scientific and Technical Information of China (English)

    屠海燕; 寿张飞

    2016-01-01

    Objective To investigate that the pregnancy induced T regulatory (Treg) cells are generated by aiming at pa-ternal antigens or induced by in vivo hormone change. Methods The 4 mice model groups were set up:normal pregnancy(n=15), abortion trend(n=15),third party mating(n=15) and false pregnancy(n=6). The change of Treg cells level and hormone content were observed. The Treg cells originated from paternal antigen premunity mice was adoptively instilled into the pregnant mice by the antigens-specific verification for observing its protective effect on embryonic mouse. Results The in vivo progesterone,E2 and E3 levels had no statistical differences between the abortion trend group and the normal pregnancy group (P>0.05). After in-stilling Treg cells of paternal antigen specific activation or adoptive transfer paternal antigens-specific Treg cells,the abortion rate of pregnant mice was decreased,while Treg cells level was increased. Conclusion The Treg cell amplification playing the pro-tective role during pregnancy is paternal antigens specific activation ,which is not caused by the single hormone content change.%目的:探讨妊娠诱导调节性T细胞扩增与父源抗原的关系。方法建立正常妊娠(n=15)、流产倾向(n=15)、第三方交配(n=15)及假孕(n=6)共四组小鼠模型,观察调节性T细胞水平和激素含量变化。通过抗原特异性验证将父源抗原预免疫小鼠来源的调节性T细胞过继滴注到孕鼠体内,观察其对胚鼠的保护作用。结果流产倾向组与正常妊娠组孕鼠体内黄体酮、雌二醇、雌三醇水平比较,差异均无统计学意义(P>0.05)。滴注父源抗原特异性激活调节性T细胞或经父源抗原预免疫的调节性T细胞后,孕鼠流产率下降,调节性T细胞水平升高。结论妊娠期调节性T细胞扩增发挥保护作用的是父源抗原特异性激活,并非单一激素含量变化引起。

  6. Identification of new hematopoietic cell subsets with a polyclonal antibody library specific for neglected proteins.

    Directory of Open Access Journals (Sweden)

    Monica Moro

    Full Text Available The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs. We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1 the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs.

  7. Polyclonal antibody against the DPV UL46M protein can be a diagnostic candidate

    Directory of Open Access Journals (Sweden)

    Jia Renyong

    2010-04-01

    Full Text Available Abstract Background The duck plague virus (DPV UL46 protein (VP11/12 is a 739-amino acid tegument protein encoded by the UL46 gene. We analyzed the amino acid sequence of UL46 using bioinformatics tools and defined the main antigenic domains to be between nucleotides 700-2,220 in the UL46 sequence. This region was designated UL46M. The DPV UL46 and UL46M genes were both expressed in Escherichia coli Rosetta (DE3 induced by isopropy1-β-D-thiogalactopyranoside (IPTG following polymerase chain reaction (PCR amplification and subcloning into the prokaryotic expression vector pET32a(+. The recombinant proteins were purified using a Ni-NTA spin column and used to generate the polyclonal antibody against UL46 and UL46M in New Zealand white rabbits. The titer was then tested using enzyme-linked immunosorbent assay (ELISA and agar diffusion reaction, and the specificity was tested by western blot analysis. Subsequently, we established Dot-ELISA using the polyclonal antibody and applied it to DPV detection. Results In our study, the DPV UL46M fusion protein, with a relative molecular mass of 79 kDa, was expressed in E. coli Rosetta (DE3. Expression of the full UL46 gene failed, which was consistent with the results from the bioinformatic analysis. The expressed product was directly purified using Ni-NTA spin column to prepare the polyclonal antibody against UL46M. The titer of the anti-UL46M antisera was over 1:819,200 as determined by ELISA and 1:8 by agar diffusion reaction. Dot-ELISA was used to detect DPV using a 1:60 dilution of anti-UL46M IgG and a 1:5,000 dilution of horseradish peroxidase (HRP-labeled goat anti-rabbit IgG. Conclusions The anti-UL46M polyclonal antibody reported here specifically identifies DPV, and therefore, it is a promising diagnostic tool for DPV detection in animals. UL46M and the anti-UL46M antibody can be used for further clinical examination and research of DPV.

  8. Technetium-99m labeling anti-amastigote polyclonal antibodies of Leishmania amazonensis

    Energy Technology Data Exchange (ETDEWEB)

    Araujo, J.G.V.C.; Toledo, V.P.C.P.; Guimaraes, T.M.P.D.; Bernardo-Filho, M.; Simal, C.J.R.; Mota, L.G.; Diniz, S.O.F.; Cardoso, V.N. E-mail: cardosov@farmacia.ufmg.br

    2002-05-01

    Anti-amastigote polyclonal antibody (IgG) was incubated with solutions of stannous chloride and sodium borohidride. After that, 3.7 MBq of technetium-99m ({sup 99m}Tc) was added. A labeling yield of the antibody about 84% was obtained. After filtration of {sup 99m}Tc-IgG, the radiochemical purity increased from 84 to 95%. The labeling of IgG with {sup 99m}Tc did not modify the immunoreactivity of the antibody, since it was able to identify in vitro and in vivo the specific antigen of Leishmania amazonensis.

  9. Development and characterization of new polyclonal antibodies specific for three polychlorinated biphenyls

    Institute of Scientific and Technical Information of China (English)

    Han Yu Chen; Hui Sheng Zhuang; Chun Zhou

    2009-01-01

    Three polychlorinated biphenyls(PCBs)congeners and their corresponding haptens bearing four carbon length carboxylic groups that can be linked to a protein for raising antibodies were synthesized.The three resultant immunogens were fabricated and used to stimulate immune responses in rabbits to survey the characteristics of the haptens.Three of the resultant polyclonal antibodies(Pabs)were obtained.The antiserum exhibited relatively high antibody titres(1:32-64)in double agar diffusion.◎2008 Hui Sheng Zhuang.Published by Elsevier B.V.on behalf of Chinese Chemical Society.All rights reserved.

  10. Multiple myeloma in a dog with multiple concurrent infectious diseases and persistent polyclonal gammopathy.

    Science.gov (United States)

    Geigy, Caroline; Riond, Barbara; Bley, Carla Rohrer; Grest, Paula; Kircher, Patrick; Lutz, Hans

    2013-03-01

    A 12-year-old, spayed female, mixed-breed dog was presented for acute hematuria, stranguria, polyuria, and polydipsia, as well as lameness for 8 days. Previous medical history included treatment for infection with Ehrlichia canis, Anaplasma phagocytophilum, Leishmania infantum, and Dirofilaria immitis 6.5 years prior to presentation. Besides persistently increased antibody titers to E canis and A phagocytophilum, polyclonal gammopathy with a monoclonal spike and moderate hypercalcemia were observed. There was marked hematuria, and Staphylococcus aureus was cultured from urine. Two weeks after successful treatment of the urinary tract infection, radiographs showed an extensive destructive monostotic lesion of the right humerus. Cytologic examination of fine-needle aspirates of this lesion revealed a neoplastic round cell population suggestive of multiple myeloma. The dog was treated with melphalan and prednisolone for suspected multiple myeloma and doxycycline for suspected ehrlichiosis and anaplasmosis. Treatments lead to resolution of the clinical signs, hypercalcemia, and monoclonal gammopathy, and there was radiographic improvement of bone lesions; polyclonal gammopathy persisted. About one year after presentation the dog was still in clinical remission. This is a rare report of a dog with suspected multiple myeloma and a history of multiple chronic infectious diseases, suggesting that chronic infection and uncontrolled long-term stimulation of the immune system could contribute to the pathogenesis of multiple myeloma. PMID:23278475

  11. Production of Potent Fully Human Polyclonal Antibodies against Ebola Zaire Virus in Transchromosomal Cattle

    Science.gov (United States)

    Dye, John M.; Wu, Hua; Hooper, Jay W.; Khurana, Surender; Kuehne, Ana I.; Coyle, Elizabeth M.; Ortiz, Ramon A.; Fuentes, Sandra; Herbert, Andrew S.; Golding, Hana; Bakken, Russell A.; Brannan, Jennifer M.; Kwilas, Steve A.; Sullivan, Eddie J.; Luke, Thomas C.; Smith, Gale; Glenn, Gregory; Li, Wenfang; Ye, Ling; Yang, Chinglai; Compans, Richard W.; Tripp, Ralph A.; Jiao, Jin-an

    2016-01-01

    Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities. PMID:27109916

  12. Preparation of Polyclonal Antibodies Against Testis-specific Protease 50 and Characterization of Antibody Specificity

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Testis-specific protease 50 (TSP50) is a testis-specific oncogene, which is abnormally activated in most tested patients with breast cancer. This property makes it an attractive molecular marker and a promising target for the diagnosis and therapy of breast cancer. In order to obtain the protective and specific polyclonal antibodies for further research, TSP50 cDNA was amplified by RT-PCR from normal human testicular tissue, and inserted into eukaryotic expression vector pcDNA3.1. Rabbit anti-TSP50 polyclonal antibodies were prepared by means of intramuscular injection of pcDNA3.1-TSP50 into the rabbits. Titers of the anti-sera were measured by ELISA and Western blotting with the E. coli cell lysate containing the induced GST-TSP50 fusion protein as an antigen. In addition, we examined the expression of TSP50 in both breast cancer cell line MCF-7 and breast cancer tissue by immunofluorescent and immunohistochemistry analysis.

  13. Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters.

    Science.gov (United States)

    Cheung, Kevin J; Padmanaban, Veena; Silvestri, Vanesa; Schipper, Koen; Cohen, Joshua D; Fairchild, Amanda N; Gorin, Michael A; Verdone, James E; Pienta, Kenneth J; Bader, Joel S; Ewald, Andrew J

    2016-02-16

    Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14(+) cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14(+) epithelial tumor cell clusters disseminate collectively to colonize distant organs. PMID:26831077

  14. The siRNA cocktail targeting interleukin 10 receptor and transforming growth factor-β receptor on dendritic cells potentiates tumour antigen-specific CD8(+) T cell immunity.

    Science.gov (United States)

    Ahn, Y-H; Hong, S-O; Kim, J H; Noh, K H; Song, K-H; Lee, Y-H; Jeon, J-H; Kim, D-W; Seo, J H; Kim, T W

    2015-07-01

    Dendritic cells (DCs) are promising therapeutic agents in the field of cancer immunotherapy due to their intrinsic immune-priming capacity. The potency of DCs, however, is readily attenuated immediately after their administration in patients as tumours and various immune cells, including DCs, produce various immunosuppressive factors such as interleukin (IL)-10 and transforming growth factor (TGF)-β that hamper the function of DCs. In this study, we used small interfering RNA (siRNA) to silence the expression of endogenous molecules in DCs, which can sense immunosuppressive factors. Among the siRNAs targeting various immunosuppressive molecules, we observed that DCs transfected with siRNA targeting IL-10 receptor alpha (siIL-10RA) initiated the strongest antigen-specific CD8(+) T cell immune responses. The potency of siIL-10RA was enhanced further by combining it with siRNA targeting TGF-β receptor (siTGF-βR), which was the next best option during the screening of this study, or the previously selected immunoadjuvant siRNA targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or Bcl-2-like protein 11 (BIM). In the midst of sorting out the siRNA cocktails, the cocktail of siIL-10RA and siTGF-βR generated the strongest antigen-specific CD8(+) T cell immunity. Concordantly, the knock-down of both IL-10RA and TGF-βR in DCs induced the strongest anti-tumour effects in the TC-1 P0 tumour model, a cervical cancer model expressing the human papillomavirus (HPV)-16 E7 antigen, and even in the immune-resistant TC-1 (P3) tumour model that secretes more IL-10 and TGF-β than the parental tumour cells (TC-1 P0). These results provide the groundwork for future clinical development of the siRNA cocktail-mediated strategy by co-targeting immunosuppressive molecules to enhance the potency of DC-based vaccines. PMID:25753156

  15. THE USE OF THE SPECIFIC ANTI-SALMONELLA POLYCLONAL ANTIBODIES ISOLATED FROM HEN EGGS, IN SALMONELLOSIS PROPHYLAXIS

    Directory of Open Access Journals (Sweden)

    ADRIANA CRISTE

    2013-12-01

    Full Text Available The administration of increased doses of antibodies in groups experimentallyinfected with Salmonella gallinarum, in order to record the efficiency of theiradministration in salmonellosis prophylaxis was the aim of our research. When alow infection dose, 1x107 CFU Salmonella gallinarum, was used theadministration of IgY polyclonal antibodies as immunoglobulin extract, or evenyolk administration had a protective effect against germs invasion. This effect wasnot recorded when a 10 folds higher dose was administered (1x108 CFU. Theprophylactic effect of the administration of polyclonal antibodies is demonstrated.

  16. Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coli BL21,the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG),and it was analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyclonal antibody,wi...

  17. Engineering antigen-specific immunological tolerance.

    Energy Technology Data Exchange (ETDEWEB)

    Kontos, Stephan; Grimm, Alizee J.; Hubbell, Jeffrey A.

    2015-05-01

    Unwanted immunity develops in response to many protein drugs, in autoimmunity, in allergy, and in transplantation. Approaches to induce immunological tolerance aim to either prevent these responses or reverse them after they have already taken place. We present here recent developments in approaches, based on engineered peptides, proteins and biomaterials, that harness mechanisms of peripheral tolerance both prophylactically and therapeutically to induce antigenspecific immunological tolerance. These mechanisms are based on responses of B and T lymphocytes to other cells in their immune environment that result in cellular deletion or ignorance to particular antigens, or in development of active immune regulatory responses. Several of these approaches are moving toward clinical development, and some are already in early stages of clinical testing.

  18. A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

    International Nuclear Information System (INIS)

    Highlights: → Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. → An ideal artificial APCs system was successfully prepared in vivo. → Controlled release of IL-2 leads to much more T-cell expansion. → This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

  19. A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Han, Hui [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Peng, Ji-Run, E-mail: pengjr@medmail.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Chen, Peng-Cheng; Gong, Lei [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Qiao, Shi-Shi [Department of Hepatobiliary Surgery, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052 (China); Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Leng, Xi-Sheng, E-mail: lengxs2003@yahoo.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China)

    2011-08-05

    Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

  20. Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hai-hong; ZHANG Xi-zhen; ZHAO Dong-hai; SHI He-liang; YU Yong-hui; WU Yong-ge; YU Xiang-hui; KONG Wei

    2008-01-01

    Survivin,a novel member of inhibitor ofapoptosis(IAP) protein family,is aberrantly expressed in cancer but undetectable in normal,differentiated adult tissues.The cancer-specific expression of survivin,coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment.Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B.The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21,and the relative molecule mass(Mr) of expressed fusion protein was approximately 21000.The recombinantprotein was purified through Ni2~ affinity chromatography column and characterized by SDS-PAGE and Western blot.The purified recombinant protein was then injected into rabbits,and antisurvivin polyclonal antibody with a high titer was obtained.

  1. Production and Characterization of Polyclonal Antibody for the N-Methylcarbamate Insecticide Metolcarb

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qi; LI Tie-jun; ZHU Xiao-xia; XU Li-na; LIU Feng-quan; HU Bai-shi; JIANG Ying-hua; CAO Bin

    2007-01-01

    The hapten, 3-{[1-(3-(methyl)phenyloxy)-carbonyl]amino}propanoic acid (HOM), mimicking the analyte metolcarb, was synthesized and verified by mass spectrometry (MS) and 1H-nuclear magnetic resonance spectrometry (1H-NMR). Then,HOM was conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) with stoichiometric amounts of N-hydroxysuccinimide/dicyclohexylcarbodimide (NHS/DCC) using the activated ester method. Polyclonal antibodies were raised against the conjugate of HOM-BSA in rabbits. Antiserum titres were determined by noncompetitive indirect ELISA procedures and the titer of pAb01 reached 1.28 × 106. The cross-reactivities of the structurally related Nmethylcarbamate insecticides were 0.0% except for dimethacarb. These results indicate that the antibody pAb01 with strong affinity and high specificity can be used to develop a sensitive and rapid detection protocol for metolcarb residue.

  2. Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Jianke Ren; Long Wang; Guoxiang Liu; Wen Zhang; Zhejin Sheng; Zhugang Wang; Jian Fei

    2008-01-01

    Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgenic animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibodypreparation.

  3. Antigen capture ELISA system for henipaviruses using polyclonal antibodies obtained by DNA immunization.

    Science.gov (United States)

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; Barr, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2012-08-01

    A novel antigen-capture sandwich ELISA system targeting the glycoproteins of the henipaviruses Nipah virus (NiV) and Hendra virus (HeV) was developed. Utilizing purified polyclonal antibodies derived from NiV glycoprotein-encoding DNA-immunized rabbits, we established a system that can detect the native antigenic structures of the henipavirus surface glycoproteins using simplified and inexpensive methods. The lowest detection limit against live viruses was achieved for NiV Bangladesh strain, 2.5 × 10(4) TCID(50). Considering the recent emergence of genetic variants of henipaviruses and the resultant problems that arise for PCR-based detection, this system could serve as an alternative rapid diagnostic and detection assay. PMID:22585045

  4. ERAP1 functions override the intrinsic selection of specific antigens as immunodominant peptides, thereby altering the potency of antigen-specific cytolytic and effector memory T-cell responses.

    Science.gov (United States)

    Rastall, David P W; Aldhamen, Yasser A; Seregin, Sergey S; Godbehere, Sarah; Amalfitano, Andrea

    2014-12-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a critical component of the adaptive immune system that has been shown to increase or decrease the presentation of specific peptides on MHC class I molecules. Here, we have demonstrated that ERAP1 functions are not only important during the presentation of antigen-derived peptides, but these functions can also completely change which antigen-derived peptides ultimately become selected as immunodominant T-cell epitopes. Our results suggest that ERAP1 may do this by destroying epitopes that would otherwise become immunodominant in the absence of adequate ERAP1 functionality. We further establish that ERAP1-mediated influences on T-cell functions are both qualitative and quantitative, by demonstrating that loss of ERAP1 function redirects CTL killing toward a different set of antigen-derived epitopes and increases the percent of antigen-specific memory T cells elicited by antigen exposure. As a result, our studies suggest that normal ERAP1 activity can act to suppress the numbers of T effector memory cells that respond to a given antigen. This unique finding may shed light on why certain ERAP1 single nucleotide polymorphisms are associated with several autoimmune diseases, for example, by significantly altering the robustness and quality of CD8+ T-cell memory responses to antigen-derived peptides. PMID:25087231

  5. Polyclonal antibody production and expression of CREG protein in human vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Yaling HAN; Haiwei LIU; Jian KANG; Xiaozeng WANG; Ye HU; Lianyou ZHAO; Shaohua LI

    2005-01-01

    Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.

  6. Characterization of fimbriae of Actinomyces naeslundii N16 using monoclonal and polyclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bragg, S.L.

    1988-01-01

    Two populations of fimbriae, which differ both in antigenicity and biological activity, have been identified on Actinomyces viscosus T14V cells. Although A. naeslundii serotype 1 isolates possess only one of these fimbrial populations (type 2 fimbriae), there was functional evidence to suggest that A. naeslundii serotype 3 strain N16 had both types of fimbriae. The purpose of this study was to characterize the fimbriae of A. naeslundii N16 immunologically by using both monoclonal and polyclonal antibodies. Three monoclonal antibodies (MAbs) to N16 were produced; all three bound to N16 fimbriae as determined by immunoelectron microscopy. In a solid-phase radioimmunoassay MAb 3B5.A1 reacted with 100% of the A. naeslundii serotype 3 isolates tested, but it did not react with any heterologous isolates. Type 1 and type 2 fimbriae were detected in Lancefield extracts of N16 cells by crossed immunoelectrophoresis (XIEP) using rabbit antiserum against N16 whole cells. When {sup 125}I-MAb 3B5.A1 was also incorporated into the gel, autoradiography indicated that MAb 3B5.A1 was specific for type 2 fimbriae. The N16 type 2 fimbriae were purified by gel filtration and immunoaffinity chromatography on a MAb 3B5.A1 column. Fimbriae-specific polyclonal and monoclonal antibodies were used in various immunological assays to determine that (a) N16 type 1 fimbriae are not related antigenically to type 2 fimbriae, (b) each type of fimbriae has epitopes that are present on the corresponding fimbriae of certain heterologous strains, and (c) MAb 3B5.A1 recognizes a serotype-specific epitope residing on the type 2 fimbriae of A. naeslundii serotype 3 strains.

  7. Evaluation of the inflammatory activity in chronic osteomyelitis. Contribution of the scintigraphy with polyclonal antibodies

    International Nuclear Information System (INIS)

    Active chronic osteomyelitis or complicating osteomyelitis (superimposed on diseases that changes the normal bone structure fractures, post-surgery, prosthesis) can be difficult to diagnose by anatomic radiological imaging modalities, like plain radiograph and CT. These diseases frequently cause also increased bone remodeling, leading to nonspecific uptake of Tc-99m-bone scan agents and gallium-67. New radiopharmaceuticals with greater inflammation/infection avidity and specificity are being developed, including the nonspecific polyclonal immunoglobulin (IgG) labeled with technetium-99. Tc-99m-IgG may be available as a ready to use kit, with no reported side effects, low patient absorbed radiation dose and low cost. The mechanism of IgG uptake at the inflammation site has not been fully elucidated yet. Specific (receptor linking, physico-chemical immunoglobulin properties) and nonspecific mechanisms (enhanced vascular permeability and macromolecular exudate) has been suggested. IgG scintigraphy results are affected by the isotope, labeling procedure adopted and characteristics of the inflammatory focus. Nineteen patients with suspected osteomyelitis (active chronic osteomyelitis or violated bone osteomyelitis) were studied by Tc-99m-IgG scintigraphy (directly labeled polyclonal immunoglobulin, Sandoglobuilina - Sandoz). All patients also underwent standard three-phase bone scintigraphy using methylene diphosphonate (Tc-99m-MDP), gallium-67 scintigraphy and plain radiographs. Infection was found in 8 sites. Sensitivity and specificity for Tc-99m-MDP, gallium-67 and Tc 99m-IgG scintigraphy were, respectively, 88 and 36%, 75 and 73%,88 and 82%. All patients with false positive IgG scintigraphies had previous surgery. Other current scintigraphic procedures used in the diagnosis of osteomyelitis are also reviewed. (author)

  8. [Preparation and detection of anti-influenza A virus polymerase basic protein 1 polyclonal antibody].

    Science.gov (United States)

    Qin, Yujie; Zhang, Tinghong; Ye, Xin

    2016-01-01

    Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1. PMID:27363203

  9. Adoptive immunotherapy of cancer with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells

    Directory of Open Access Journals (Sweden)

    Kim Julian A

    2004-11-01

    Full Text Available Abstract T cell-mediated cancer immunotherapy is dose dependent and optimally requires participation of antigen-specific CD4+ and CD8+ T cells. Here, we isolated tumor-sensitized T cells and activated them in vitro using conditions that led to greater than 108-fold numerical hyperexpansion of either the CD4+ or CD8+ subset while retaining their capacity for in vivo therapeutic efficacy. Murine tumor-draining lymph node (TDLN cells were segregated to purify the CD62Llow subset, or the CD4+ subset thereof. Cells were then propagated through multiple cycles of anti-CD3 activation with IL-2 + IL-7 for the CD8+ subset, or IL-7 + IL-23 for the CD4+ subset. A broad repertoire of TCR Vβ families was maintained throughout hyperexpansion, which was similar to the starting population. Adoptive transfer of hyper-expanded CD8+ T cells eliminated established pulmonary metastases, in an immunologically specific fashion without the requirement for adjunct IL-2. Hyper-expanded CD4+ T cells cured established tumors in intracranial or subcutaneous sites that were not susceptible to CD8+ T cells alone. Because accessibility and antigen presentation within metastases varies according to anatomic site, maintenance of a broad repertoire of both CD4+ and CD8+ T effector cells will augment the overall systemic efficacy of adoptive immunotherapy.

  10. Cytotoxicity of halogenated graphenes

    Science.gov (United States)

    Teo, Wei Zhe; Khim Chng, Elaine Lay; Sofer, Zdeněk; Pumera, Martin

    2013-12-01

    Graphene and its family of derivatives possess unique and remarkable physicochemical properties which make them valuable materials for applications in many areas like electronics, energy storage and biomedicine. In response to the possibility of its large-scale manufacturing as commercial products in the future, an investigation was conducted to determine the cytotoxicity of one particular family of graphene derivatives, the halogenated graphenes, for the first time. Halogenated graphenes were prepared through thermal exfoliation of graphite oxide in gaseous chlorine, bromine or iodine atmospheres to yield chlorine- (TRGO-Cl), bromine- (TRGO-Br) and iodine-doped graphene (TRGO-I) respectively. 24 h exposure of human lung carcinoma epithelial cells (A549) to the three halogenated graphenes and subsequent cell viability assessments using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-8) assays revealed that all the halogenated graphenes examined are rather cytotoxic at the concentrations tested (3.125 μg mL-1 to 200 μg mL-1) and the effects are dose-dependent, with TRGO-Cl reducing the cell viability to as low as 25.7% at the maximum concentration of 200 μg mL-1. Their levels of cytotoxicity can be arranged in the order of TRGO-Cl > TRGO-Br > TRGO-I, and it is suggested that the amount of halogen present in the graphene material is the determining factor for the observed trend. Control experiments were carried out to test for possible nanomaterial-induced interference as a consequence of reaction between the halogenated graphenes and the viability markers (MTT/WST-8 reagent) or binding of the formazan products under cell-free conditions. The data obtained eliminate the probability of significant influence by these interferents as the change in the normalized percentage of formazan formed is relatively small and thorough washings were performed prior to the viability assessments to reduce the amount of halogenated

  11. Tumor-targeted delivery of IL-2 by NKG2D leads to accumulation of antigen-specific CD8+ T cells in the tumor loci and enhanced anti-tumor effects.

    Directory of Open Access Journals (Sweden)

    Tae Heung Kang

    Full Text Available Interleukin-2 (IL-2 has been shown to promote tumor-specific T-cell proliferation and differentiation but systemic administration of IL-2 results in significant toxicity. Therefore, a strategy that can specifically deliver IL-2 to the tumor location may alleviate concerns of toxicity. Because NKG2D ligands have been shown to be highly expressed in many cancer cells but not in healthy cells, we reason that a chimeric protein consisting of NKG2D linked to IL-2 will lead to the specific targeting of IL-2 to the tumor location. Therefore, we created chimeric proteins consisting of NKG2D linked to Gaussia luciferase (GLuc; a marker protein or IL-2 to form NKG2D-Fc-GLuc and NKG2D-Fc-IL2, respectively. We demonstrated that NKG2D linked to GLuc was able to deliver GLuc to the tumor location in vivo. Furthermore, we showed that TC-1 tumor-bearing mice intramuscularly injected with DNA encoding NKG2D-Fc-IL2, followed by electroporation, exhibited an increased number of luciferase-expressing E7-specific CD8+ T cells at the tumor location. More importantly, treatment with the DNA construct encoding NKG2D-Fc-IL2 significantly enhanced the therapeutic anti-tumor effects generated by intradermal vaccination with therapeutic HPV DNA in tumor-bearing mice. Therefore, by linking NKG2D to IL2, we are able to specifically deliver IL-2 to the tumor location, enhancing antigen-specific T-cell immune response and controlling tumor growth. Our approach represents a platform technology to specifically deliver proteins of interest to tumor loci.

  12. Characterization of the antigen-specific CD4+ T cell response induced by prime-boost strategies with CAF01 and CpG adjuvants administered by the intranasal and subcutaneous routes

    Directory of Open Access Journals (Sweden)

    Annalisa eCiabattini

    2015-08-01

    Full Text Available The design of heterologous prime-boost vaccine combinations that optimally shape the immune response is of critical importance for the development of next generation vaccines. Here we tested different prime-boost combinations using the tuberculosis vaccine antigen H56 with CAF01 or CpG ODN 1821 adjuvants, administered by the parenteral and nasal routes. By using peptide-MHC class II tetramers, antigen-specific CD4+ T cells were tracked following primary and booster immunizations. Both parenteral priming with H56 plus CAF01 and nasal priming with H56 plus CpG elicited significant expansion of CD4+ tetramer-positive T cells in the spleen, however only parenterally primed cells responded to booster immunization. Subcutaneous priming with H56 and CAF01 followed by nasal boosting with H56 and CpG showed the greater expansion of CD4+ tetramer-positive T cells in the spleen and lungs compared to all the other homologous and heterologous prime-boost combinations. Nasal boosting exerted a recruitment of primed CD4+ T cells into lungs that was stronger in subcutaneously than nasally primed mice, in accordance with different chemokine receptor expression induced by primary immunization. These data demonstrate that subcutaneous priming is fundamental for eliciting CD4+ T cells that can be efficiently boosted by the nasal route and results in the recruitment of antigen-experienced cells into the lungs. Combination of different vaccine formulations and routes of delivery for priming and boosting is a strategic approach for improving and directing vaccine-induced immune responses.

  13. Specific cytotoxic T-cell immune responses against autoantigens recognized by chronic lymphocytic leukaemia cells.

    Science.gov (United States)

    Zaleska, Joanna; Skorka, Katarzyna; Zajac, Malgorzata; Karczmarczyk, Agnieszka; Karp, Marta; Tomczak, Waldemar; Hus, Marek; Wlasiuk, Paulina; Giannopoulos, Krzysztof

    2016-08-01

    Mounting evidence suggests that autoreactivity and inflammatory processes are involved in the pathogenesis of chronic lymphocytic leukaemia (CLL). Cytoskeletal proteins, including non-muscle myosin heavy chain IIA (MYHIIA), vimentin (VIM) and cofilin-1 (CFL1), exposed on the surface of apoptotic cells have been identified as autoantigens that are recognized by the specific B-cell receptors of the CLL cells. In 212 CLL patients analysed with quantitative reverse transcriptase-polymerase chain reaction we found CFL1 overexpression and low expression of MYH9 in comparison with healthy volunteers. We detected specific cytotoxic immune responses for peptides derived from MYHIIA in 66·7%, VIM in 87·5% and CFL1 in 62·5% CLL patients in an Enzyme-Linked ImmunoSpot assay. Low frequencies of autoreactive peptide-specific T cells were detected against MYHIIA, VIM and CFL1 in CLL patients ex vivo; most of the detected cells had an effector-memory phenotype. Our findings support the existence of cytotoxic immune responses against three autoantigens that have been identified as targets of CLL clonotypic B-cell receptors. The presence of autoreactive CD8(+) T cells against MYHIIA, VIM and CFL1 in CLL patients indicates the involvement of antigen-specific autoreactive T cells in the pathogenesis of CLL.

  14. Identification of swine influenza virus epitopes and analysis of multiple specificities expressed by cytotoxic T cell subsets

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Breum, Solvej Østergaard; Riber, Ulla;

    2014-01-01

    Background: Major histocompatibility complex (MHC) class I peptide binding and presentation are essential for antigen-specific activation of cytotoxic T lymphocytes (CTLs) and swine MHC class I molecules, also termed swine leukocyte antigens (SLA), thus play a crucial role in the process that leads...... to elimination of viruses such as swine influenza virus (SwIV). This study describes the identification of SLA-presented peptide epitopes that are targets for a swine CTL response, and further analyses multiple specificities expressed by SwIV activated CTL subsets. Findings: Four SwIV derived peptides were...... identified as T cell epitopes using fluorescent influenza: SLA tetramers. In addition, multiple CTL specificities were analyzed using peptide sequence substitutions in two of the four epitope candidates analyzed. Interestingly both conserved and substituted peptides were found to stain the CD4-CD8+ T cell...

  15. Are diamond nanoparticles cytotoxic?

    Science.gov (United States)

    Schrand, Amanda M; Huang, Houjin; Carlson, Cataleya; Schlager, John J; Omacr Sawa, Eiji; Hussain, Saber M; Dai, Liming

    2007-01-11

    Finely divided carbon particles, including charcoal, lampblack, and diamond particles, have been used for ornamental and official tattoos since ancient times. With the recent development in nanoscience and nanotechnology, carbon-based nanomaterials (e.g., fullerenes, nanotubes, nanodiamonds) attract a great deal of interest. Owing to their low chemical reactivity and unique physical properties, nanodiamonds could be useful in a variety of biological applications such as carriers for drugs, genes, or proteins; novel imaging techniques; coatings for implantable materials; and biosensors and biomedical nanorobots. Therefore, it is essential to ascertain the possible hazards of nanodiamonds to humans and other biological systems. We have, for the first time, assessed the cytotoxicity of nanodiamonds ranging in size from 2 to 10 nm. Assays of cell viability such as mitochondrial function (MTT) and luminescent ATP production showed that nanodiamonds were not toxic to a variety of cell types. Furthermore, nanodiamonds did not produce significant reactive oxygen species. Cells can grow on nanodiamond-coated substrates without morphological changes compared to controls. These results suggest that nanodiamonds could be ideal for many biological applications in a diverse range of cell types.

  16. Are diamond nanoparticles cytotoxic?

    Science.gov (United States)

    Schrand, Amanda M; Huang, Houjin; Carlson, Cataleya; Schlager, John J; Omacr Sawa, Eiji; Hussain, Saber M; Dai, Liming

    2007-01-11

    Finely divided carbon particles, including charcoal, lampblack, and diamond particles, have been used for ornamental and official tattoos since ancient times. With the recent development in nanoscience and nanotechnology, carbon-based nanomaterials (e.g., fullerenes, nanotubes, nanodiamonds) attract a great deal of interest. Owing to their low chemical reactivity and unique physical properties, nanodiamonds could be useful in a variety of biological applications such as carriers for drugs, genes, or proteins; novel imaging techniques; coatings for implantable materials; and biosensors and biomedical nanorobots. Therefore, it is essential to ascertain the possible hazards of nanodiamonds to humans and other biological systems. We have, for the first time, assessed the cytotoxicity of nanodiamonds ranging in size from 2 to 10 nm. Assays of cell viability such as mitochondrial function (MTT) and luminescent ATP production showed that nanodiamonds were not toxic to a variety of cell types. Furthermore, nanodiamonds did not produce significant reactive oxygen species. Cells can grow on nanodiamond-coated substrates without morphological changes compared to controls. These results suggest that nanodiamonds could be ideal for many biological applications in a diverse range of cell types. PMID:17201422

  17. Development of [{sup 201}Tl](III)-DTPA-human polyclonal antibody complex for inflammation detection

    Energy Technology Data Exchange (ETDEWEB)

    Jalilian, A.R.; Kamali-Dehghan, M.; Kamrani, Y.Y. [Nuclear Research Center for Agriculture and Medicine, Karaj (Iran). Cyclotron and Nuclear Medicine Dept.; Khorrami, A.; Tavakoli, M.B. [Medical Sciences Univ. of Isfahan (Iran). Medical Physics and Engineering Dept.

    2007-07-01

    Thallium-201 (T{sub 1/2}=3.04 d) in Tl{sup +} form was converted to Tl{sup 3+} cation in presence of O{sub 3} in 6 M HCl controlled by RTLC/gel electrophoresis methods and used in the labeling of human polyclonal antibody (HIgG) after conjugation with freshly prepared cyclic DTPA-dianhydride. The best results of the conjugation were obtained by the addition of 1 mL of a HIgG pharmaceutical solution (5 mg/ml, in phosphate buffer, pH=7) to a glass tube pre-coated with DTPA-dianhydride (0.01 mg) at 25 C with continuous mild stirring for 30 min. The final isotonic [{sup 201}Tl](III)-DTPA-HIgG complex was checked by radio-TLC using several solvent systems to ensure the formation of only one species followed by filtration through a 0.22 {mu} filter (specific activity= 33.7 TBq/mM, radiochemical purity >95%). Preliminary bio-distribution studies in normal and inflammation-bearing rats were performed. The target/skin and target/blood ratios were 4 and 6 after 28 h respectively, showing the selectivity of the radiopharmaceutical for the inflammatory lesions. (orig.)

  18. Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    A.J.A. Barbosa

    1991-03-01

    Full Text Available The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160, whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.

  19. Scintigraphic evaluation of chronic osteomyelitis with technetium 99 m labeled polyclonal immunoglobulin

    International Nuclear Information System (INIS)

    Active chronic osteomyelitis or complicating osteomyelitis are difficult to be diagnosed by radiological imaging modalities, such as plain radiograph and CT. They frequently cause increased bone remodeling, leading to nonspecific uptake of Tc 99 m-bone scan agents and gallium-67. New radiopharmaceuticals with greater infection avidity are being developed, including the nonspecific polyclonal immunoglobulin (IgG) labeled with technetium 99 m. Tc 99 m-IgG may be available as a ready to use kit, with no reported side effects, low patient absorbed radiation dose and low cost. Material and Methods: 23 bone segments with suspected active chronic osteomyelitis or violated bone osteomyelitis were studied by Tc 99 m IgG scintigraphy. All patients underwent standard three phase bone scintigraphy using methylene diphosphonate (Tc 99 m MDP), gallium 67 scintigraphy and plain radiographs, compared with clinical evaluation and laboratory tests values. Results: Infection was found in 8 sites. Sensitivity and specificity for Tc-99 m MDP, gallium 67 and Tc 99 m IgG scintigraphy were, respectively, 88 and 36%, 75 and 73%, 88 and 82%. Conclusion: Tc-99 m IgG may be useful in the scintigraphic evaluation of osteomyelitis. (author)

  20. Persistent Polyclonal B Cell Lymphocytosis B Cells Can Be Activated through CD40-CD154 Interaction

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    Emmanuelle Dugas-Bourdages

    2014-01-01

    Full Text Available Persistent polyclonal B cell lymphocytosis (PPBL is a rare disorder, diagnosed primarily in adult female smokers and characterized by an expansion of CD19+CD27+IgM+ memory B cells, by the presence of binucleated lymphocytes, and by a moderate elevation of serum IgM. The clinical course is usually benign, but it is not known whether or not PPBL might be part of a process leading to the emergence of a malignant proliferative disorder. In this study we sought to investigate the functional response of B cells from patients with PPBL by use of an optimal memory B cell culture model based on the CD40-CD154 interaction. We found that the proliferation of PPBL B cells was almost as important as that of B cells from normal controls, resulting in high immunoglobulin secretion with in vitro isotypic switching. We conclude that the CD40-CD154 activation pathway is functional in the memory B cell population of PPBL patients, suggesting that the disorder may be due to either a dysfunction of other cells in the microenvironment or a possible defect in another B cell activation pathway.

  1. Prokaryotic expression of soluble Arabidopsis protein AtERF1 and preparation of its polyclonal antibodies

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    ZHANG Yu

    2013-08-01

    Full Text Available AtERF1 encodes a member of the ERF subfamily B-3 of ERF/AP2 transcription factor family.It has been demonstrated almost every member of the B3 subgroup of AP2/ERF genes is involved in defense responses in Arabidopsis.Codon usage within a gene is a critical determinant of achievable protein expression level in E.coli. Gene optimization,therefore,is an effective method for synthetic genes with the aim of enhancing soluble expression,particular in heterologous hosts.In this paper,the AtERF1 protein of Arabidopsis thaliana was expressed in Escherichia coli using its optimized DNA sequence for E.coli. and yielded high level of soluble AtERF-1 protein in recombinant E.coli. The AtERF1 protein was used as an antigen to immune rabbits and obtains high titer antibodies.The immunological specificity of the polyclonal antibodies to AtERF1 was confirmed by Western blot assay.The prepared antibody in this work would facilitate the further functional investigation of AtERF1 in biochemical levels in Arabidopsis anther development.

  2. Characterization of polyclonal antibodies against nonstructural protein 9 from the porcine reproductive and respiratory syndrome virus

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    Mengmeng ZHAO,Juanjuan QIAN,Jiexiong XIE,Tiantian CUI,Songling FENG,Guoqiang WANG,Ruining WANG,Guihong ZHANG

    2016-06-01

    Full Text Available Porcine reproductive and respiratory syndrome (PRRS is considered to be one of the most important infectious diseases impacting the swine industry and is characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages. The nonstructural protein 9 gene, Nsp9, encoding the RNA-dependent RNA polymerase, is generally regarded as fairly conserved when compared to other viral proteins. Antibodies against Nsp9 will be of great importance for the diagnosis and treatment of the causal agent, PRRS virus. A study was undertaken to generate polyclonal antibodies against the immunodominant Nsp9. For this purpose, the Nsp9 was expressed in Escherichia coli and subsequently used as an antigen to immunize New Zealand rabbits. Antiserum was identified via an indirect ELISA, and then verified based on the ability to react with both naturally and artificially expressed Nsp9. Results of virus neutralization test showed that this antiserum could not neutralize the PRRSV. Nevertheless, this antiserum as a diagnostic core reagent should prove invaluable for further investigations into the mechanism of PRRS pathogenesis.

  3. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

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    Nazila Amini

    2014-06-01

    Full Text Available Objective(s:Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH (and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. Results: The antibody could recognize the immunizing peptide in ELISA. It could also recognize            β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Conclusion: Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA,immunocytochemistry and immunohistochemistry.

  4. Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein.

    Science.gov (United States)

    Choudhary, Nandlal; Roy, Avijit; Guillermo, Leon M; Picton, D D; Wei, G; Nakhla, M K; Levy, L; Brlansky, R H

    2013-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.

  5. A comprehensive immunoassay for the detection of microcystins in waters based on polyclonal antibodies

    International Nuclear Information System (INIS)

    Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria (blue-green algae), and microcystin-leucine-arginine (MC-LR) is among the most frequent and most toxic microcystin congeners. In this study, a free amino group was introduced to MC-LR at its seventh amino acid residue with 2-mercaptoethylamine, and the product aminoethyl-MC-LR was coupled to bovine serum albumin (BSA) and horseradish peroxidise (HRP) by glutaraldehyde to be complete antigen (MC-LR-BSA) and labelled hapten (MC-LR-HRP), respectively. Polyclonal antibodies against MC-LR were generated by immunization with MC-LR-BSA. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was established to detect the MCs in waters, which showed a good cross-reactivity with MC-LR, MC-RR, MC-YR, MC-LF, MC-LW and nodularin, and have a detection limit for MC-LR 0.12 μg L-1, the 50% inhibition concentration (IC50) for MC-LR was 0.63 ± 0.06 μg L-1 and the quantitative detection range was from 0.17 to 2.32 μg L-1, the analysis result of water samples showed good recovery and reliability. So the comprehensive and reliable dc-ELISA will well potentially suit for sensitive analysis for total MCs in drinking as well as resource water samples

  6. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

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    Tana A. Omokoko

    2016-01-01

    Full Text Available Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay’s ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay’s combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.

  7. Monoclonal and Polyclonal Antibodies Specific for Foot and Mouth Disease Virus Type A and Type O VP1

    OpenAIRE

    CHO, JIN GU; Jo, Yeong Joon; Sung, Jong-Hyuk; Hong, Jang-Kwan; Hwang, Ji-Hyeon; Park, Jong-Hyeon; Lee, Kwang-Nyeong; Park, Sang Gyu

    2012-01-01

    The foot and mouth disease virus (FMDV) is an RNA virus composed of single stranded positive sense RNA. FMDV has been known to infect cloven-hoofed animals, including pigs, cattle, and sheep. FMDV is rapidly spreading outward to neighboring regions, often leading to a high mortality rate. Thus, early diagnosis of FMDV is critical to suppress propagation of FMDV and minimize economic losses. In this study, we report the generation and characterization of polyclonal and six monoclonal antibodie...

  8. Production of Polyclonal Antibody against Interleukin-33 and Assessment of Its Distribution in Murine Liver and Lung

    OpenAIRE

    Xiaojin Liu; Yan Wu; Mingcai Li; Sui Chen; Yanchun Zhou

    2009-01-01

    Interleukin (IL)-33 is the latest member of IL-1 cytokine family. In this study, the cloning, expression, purification, and polyclonal antibody preparation of mouse IL-33 were described. The coding region of IL-33 mature protein was cloned into the prokaryotic expression vector pET-44. The recombinant protein, IL-33 containing a hexahistidine tag in the C-terminal, was expressed in Escherichia coli. The expressed soluble protein was purified by immobilized metal-ion affinity chromatography us...

  9. Characterization of Rabbit Polyclonal Sera against Recombinant Shiga Toxin and its Subunits for Detection of Stx-Producing E. coli

    OpenAIRE

    Mana Oloomi; Saeid Bouzari

    2011-01-01

    Shiga toxin (Stx) is the principal virulence factor of Shigatoxigenic Escherichia coli (STEC), a food-born pathogen associated disease with uncomplicated diarrhea or the hemolytic-uremic syndrome. In this study, rabbit polyclonal anti recombinant A, B subunits of Shiga toxin and holotoxin antisera were raised and employed for immunological purpose. By immunoblotting, these antisera recognized respective subunit and the holotoxin antiserum recognized both subunits, equally. The raised antisera...

  10. Polyacrylamide gel electrophoresis of RNA compared with polyclonal- and monoclonal-antibody-based enzyme immunoassays for rotavirus.

    OpenAIRE

    Pacini, D L; Brady, M T; Budde, C T; Connell, M J; Hamparian, V V; Hughes, J. H.

    1988-01-01

    Polyacrylamide gel electrophoresis (PAGE) of rotaviral RNA, a sensitive and highly specific test for detecting rotavirus in stool, was compared with two commercially available enzyme immunoassays (EIAs), monoclonal (Pathfinder) and polyclonal (Rotazyme II). Stool samples from 204 children with nosocomial diarrhea were tested for rotavirus by both EIAs and by PAGE of RNA extracted from raw stools or 10% stool suspensions. Samples which tested positive by either EIA but were negative by PAGE we...

  11. Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

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    Sofía Duque-Beltrán

    2002-12-01

    Full Text Available The present study developed and standardized an enzime-linked immunosorbent assay (ELISA to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate. One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%; specificity, 95% (95% CI: 88.6-97.6%; positive predictive value, 91% (95% CI: 81.4-95.9%; and negative predictive value, 100% (95% CI: 96.1-100%. This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

  12. Production, purification and characterization of polyclonal antibody against the truncated gK of the duck enteritis virus

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    Zhang Shunchuan

    2010-09-01

    Full Text Available Abstract Duck virus enteritis (DVE is an acute, contagious herpesvirus infection of ducks, geese, and swans, which has produced significant economic losses in domestic and wild waterfowl. With the purpose of decreasing economic losses in the commercial duck industry, studying the unknown glycoprotein K (gK of DEV may be a new method for preferably preventing and curing this disease. So this is the first time to product and purify the rabbit anti-tgK polyclonal antibody. Through the western blot and ELISA assay, the truncated glycoprotein K (tgK has good antigenicity, also the antibody possesses high specificity and affinity. Meanwhile the rabbit anti-tgK polyclonal antibody has the potential to produce subunit vaccines and the functions of neutralizing DEV and anti-DEV infection because of its neutralization titer. Indirect immunofluorescent microscopy using the purified rabbit anti-tgK polyclonal antibody as diagnostic antibody was susceptive to detect a small quantity of antigen in tissues or cells. This approach also provides effective experimental technology for epidemiological investigation and retrospective diagnose of the preservative paraffin blocks.

  13. DETECTION OF ROTAVIRUS WITH A NEW POLYCLONAL ANTIBODY ENZYME IMMUNOASSAY (ROTAZYME 2) AND A COMMERCIAL LATEX AGGLUTINATION TEXT (ROTALEX): COMPARISON WITH A MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

    Science.gov (United States)

    A total of 176 human fecal specimens were examined for the presence of rotavirus using four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, Chicago, IL (Rotazyme I); a modification of...

  14. Purification of full-length human Pregnane and Xenobiotic Receptor: polyclonal antibody preparation for immunological characterization

    Institute of Scientific and Technical Information of China (English)

    Mallampati SARADHI; Biji KRISHNA; Gauranga MUKHOPADHYAY; Rakesh K TYAGI

    2005-01-01

    Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body's homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. Thefull-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21 (DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.

  15. Prokaryotic overexpression of TEV-rhGH and characterization of its polyclonal antibody.

    Science.gov (United States)

    Murad, Hossam; Ali, Bouthina; Makeya, Rima; Abbady, Abdul Qader

    2014-05-25

    Recombinant protein technology represents one of the best solutions to achieve rapid, efficient, and cost-effective protein expression and purification of therapeutic proteins. Growth hormone (GH) is an excellent example of these proteins used in the therapy of hormone deficiencies. In this work, a plasmid, pRSET-TEV-rhGH, has been constructed to overexpress recombinant human GH (rhGH) by cloning its gene downstream of an N-terminal 6 × His-tagged polypeptide (43 aa) in the T7 promoter-plasmid pRSET. This polypeptide was cleavable by means of the integrated recognition site for the tobaccos etch virus (TEV) protease, resulting in an rhGH protein at an exact length and sequence. After IPTG induction, this plasmid effectively expressed TEV-rhGH protein (27 kDa) in the cytoplasm of Escherichia coli, which accumulated in the form of inclusion bodies. The 6 × His-tagged protein, with a yield of ~150 mg/L of culture, was purified from the cell extract using metal affinity chromatography, as shown after SDS-PAGE blue staining, and was confirmed by immunoblotting using specific commercial monoclonal antibodies. In order to detect TEV-rhGH, in ELISA and immunoblotting, specific polyclonal antibody, with high titer (~10⁻⁵ fold dilution), was produced in a rabbit and purified using affinity chromatography. Preliminary tests have proved that TEV-rhGH protein and its specific purified IgG antibody could provide valuable tools for rhGH productive and diagnostic purposes. PMID:24534464

  16. Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, A.; Chen, W.F.; Scollay, R.; Shortman, K. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

    1982-08-13

    A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic /sup 111/In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8-9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2/sup +/ T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects.

  17. Measurement of lymphatic function with technetium-99m-labelled polyclonal immunoglobulin

    International Nuclear Information System (INIS)

    A reliable method for measuring lymph flow in physiological units would be valuable, especially in conditions in which it is uncertain whether lymph flow is increased or decreased. The requirements of a radiopharmaceutical for such measurement include stable radionuclide labelling and rapid access to lymphatic vessels following tissue injection but no access to blood vessels. A soluble macromolecule is likely to come closest to meeting these requirements. Technetium-99m- labelled human polyclonal immunoglobulin (HIG) was therefore investigated firstly in comparison with 99mTc-labelled human serum albumin (HSA) in patients undergoing routine lymphoscintigraphy and secondly with respect to injection site in a group of volunteers with post-mastectomy oedema (PMO). Subcutaneous injection of 99mTc-HIG into the web space of a distal extremity gave images in which lymphatic vessels were more clearly defined compared with images obtained after injection of 99mTc-HSA. Lymph nodes were also more clearly defined, suggesting specific retention of HIG, possibly through Fc-mediated binding. Peripheral blood sampling showed a delayed arrival in blood of radioactivity after 99mTc-HIG compared with 99mTc-HSA, although ultimately, the blood recovery of 99mTc-HIG was significantly higher (P 99mTc-HSA. Clearance rates of radioactivity from the injection site were not sinificantly different, however, between the two agents. In patients with PMO, web space injection of 99mTc-HIG gave excellent images of normal lymphatic vessels, of lymph nodes and of abnormal lymph drainage such as dermal backflow in swollen arms. In contrast, neither lymphatic vessels nor lymph nodes were visualised after injection into the skin of the dorsum of the distal forearm. Although there was no difference in clearance rates from the injection sites between normal and swollen arms with either agent in PMO, clearance was significantly faster following injection into the web space (0.11% per minute for normal and

  18. Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle.

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    Hiroaki Matsushita

    Full Text Available Large-scale production of fully human IgG (hIgG or human polyclonal antibodies (hpAbs by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.

  19. Cytotoxic glucosphingolipid from Celtis Africana

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    Shagufta Perveen

    2015-01-01

    Full Text Available Background: Literature survey proved the use of the powdered sun-dried bark and roots of Celtis africana for the treatment of cancer in South Africa. Objective: The aim of this study was to do further isolation work on the ethyl acetate fraction and to investigate the cytotoxic activities of the various fractions and isolated compound. Materials and Methods: Cytotoxicity of petroleum ether, chloroform, ethyl acetate, n-butanol fractions and compound 1 were tested on mouse lymphoma cell line L5178Y using the microculture tetrazolium assay. Results: One new glucosphingolipid 1 was isolated from the aerial parts of C. africana. The structure of the new compound was determined by extensive analysis by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry. The ethyl acetate fraction and compound 1 showed strong cytotoxic activity with an EC 50 value of 8.3 μg/mL and 7.8 μg/mL, respectively, compared with Kahalalide F positive control (6.3 μg/mL. Conclusion: This is the first report of the occurrence of a cytotoxic glucosphingolipid in family Ulmaceae.

  20. Polyclonal B cell differentiation and loss of gastrointestinal tract germinal centers in the earliest stages of HIV-1 infection.

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    Marc C Levesque

    2009-07-01

    Full Text Available The antibody response to HIV-1 does not appear in the plasma until approximately 2-5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1-specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4(+ T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI included not only HIV-1-specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1-induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer's patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis.Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1-induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors' Summary.

  1. Prokaryotic Expression and Purification of Human TLE1 N-terminal Q Domain Fragment and Production of its Polyclonal Antibody

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    Su WANG

    2010-11-01

    Full Text Available Background and objective TLE1 is an important protein in regulating Wnt, Notch and EGFR signaling pathways. The TLE1 N-terminal Q domain regulates the pathways by mediating its oligomerization and interaction with LEF1. The aim of this study is to construct the human TLE1 N-terminal Q domain fragment in prokaryotic expression system, express and purify protein TLE1 N-terminal Q domain and prepare its polyclonal antibody. Methods The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA, was cloned into the prokaryotic expression vector pGEX-4T-1 containing Glutathione S-transferase (GST. Vector pGEX-4T1-TLE1-Q was transformed into E.coli BL21 condon plus. The GST-TLE1-Q(1-136 fusion protein was induced by IPTG, digested by Thrombin, purified with glutathione-sepharose beads and FPLC, identified by SDS-PAGE. Then rabbits were immunized with the purified protein TLE1-Q(1-136 for obtaining the antiserum. The titers and specificity of antibodies were measured by ELISA and Western blot. Results The PCR identification and the sequencing of recombinant plasmid demonstrated that vector pGEX-4T1-TLE1-Q was successfully constructed. The SDS-PAGE shows target protein (14 000 Da is the interest protein TLE1-Q(1-136. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired, with an antibody titer of 1:20 000. Conclusion Expression vector pGEX-4T1-TLE1-Q is correctly constructed. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired. These work established the foundation for further biological study between TLE1 and lung cancers.

  2. Preparation and Preliminary Application of MAdCAM-1 Polyclonal Antibody in Dairy Cows with Subclinical Mastitis.

    Science.gov (United States)

    Xu, Chuang; Chen, Yuanyuan; Chang, Qiaocheng; Xia, Cheng; Yang, Wei; Zhang, Hongyou

    2015-08-01

    MAdCAM-1 plays an important role in mediating immune response and inflammation. This study aimed to express and purify a fusion protein of MAdCAM-1 in prokaryotic cells and to prepare rat anti-bovine MAdCAM-1 polyclonal antibodies. Prokaryotic expression vector pGEX-4T-1-MAdCAM-1 and pET-28a-MAdCAM-1 were constructed, respectively. The above plasmids were transformed into BL21 Escherichia coli strain. These recombinant strains were induced by IPTG and identified by Western blot analysis and SDS-PAGE. Wistar rats were immunized with recombinant protein (pET-28a-MAdCAM-1) emulsified with Freund's adjuvant, and antibody titers were measured by indirect ELISA. Antibody titers reached the highest value (1:128,000) after the third immunization. Western blot showed that rat anti-bovine MAdCAM-1 polyclonal antibody can not only recognize recombinant MAdCAM-1 protein expressed in E. coli but also recognizes natural MAdCAM-1 protein extracted from bovine tissues. However, commercial anti-mouse MAdCAM-1 monoclonal antibodies did not recognize the recombinant MAdCAM-1 protein or natural protein, which indicated no cross-reactivity between bovine MAdCAM-1 and mouse MAdCAM-1. Real-time fluorescence quantitative polymerase chain reaction and Western blot analysis showed that MAdCAM-1 expression was limited in mammary lymphoid nodes of subclinical mastitis in dairy cows. We speculate that MAdCAM-1 expression is inconsistent in different periods of the dairy cows. The successful preparation of rat anti-bovine MAdCAM-1 polyclonal antibody and its preliminary application in dairy cows provide the foundation for further study of the mechanism of anti-inflammation of MAdCAM-1 in dairy cows with subclinical mastitis. PMID:26301930

  3. Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection

    OpenAIRE

    Dowall, Stuart David; Callan, Jo; Zeltina, Antra; Al-Abdulla, Ibrahim; Strecker, Thomas; Sarah K Fehling; Krähling, Verena; Bosworth, Andrew; Rayner, Emma; Taylor, Irene; Charlton, Sue; Landon, John; Cameron, Ian; Hewson, Roger; Nasidi, Abdulsalami

    2015-01-01

    The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOT...

  4. In vivo evaluation of the cross-genotype neutralizing activity of polyclonal antibodies against hepatitis C virus

    DEFF Research Database (Denmark)

    Meuleman, Philip; Bukh, Jens; Verhoye, Lieven;

    2011-01-01

    1977 (H77). In this study we investigated whether polyclonal antibodies isolated from Patient H in 2006 (H06), which display high cross-genotype neutralizing activity in both the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems, were also able to prevent HCV infection of different...... genotypes (gt) in vivo. Following passive immunization with H06-antibodies, chimeric mice were challenged with the consensus strains H77C (gt1a), ED43 (gt4a), or HK6a (gt6a). In accordance with previous results, H06-antibodies prevented infection of chimeric mice with the autologous virus. However, the...

  5. Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production

    OpenAIRE

    Hiroaki Matsushita; Akiko Sano; Hua Wu; Jin-An Jiao; Poothappillai Kasinathan; Eddie J. Sullivan; Zhongde Wang; Yoshimi Kuroiwa

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM−/− , bIGHM...

  6. Triple Immunoglobulin Gene Knockout Transchromosomic (Tc) Cattle: Bovine Lambda Cluster Deletion and its Effect on Fully Human Polyclonal Antibody Production

    OpenAIRE

    Matsushita, H.; Sano, A.; Wu, H.; J. Jiao; Kasinathan, P.; Sullivan, E. J.; Wang, Zhongde; Kuroiwa, K

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML...

  7. Expression, purification of herpes simplex virus type 1 US11 Protein, and production of US11 polyclonal antibody

    OpenAIRE

    Yao Shanglong; Wuyunerdeni,; Huang Yizhong

    2011-01-01

    Abstract Background The US11 protein of herpes simplex virus type 1 (HSV-1) is a small, highly basic phosphoprotein expressed at late times during infection. To date, the function of US11 protein in cell culture and animal models is poorly understood. To further investigate the function of the US11 protein, this study was undertaken to express the US11 protein and raise a polyclonal antibody. Results The US11 gene was cloned into the prokaryotic expression vector pET-32a (+) to express His-ta...

  8. Saponins as cytotoxic agents: a review

    OpenAIRE

    Podolak, Irma; Galanty, Agnieszka; Sobolewska, Danuta

    2010-01-01

    Saponins are natural glycosides which possess a wide range of pharmacological properties including cytotoxic activity. In this review, the recent studies (2005–2009) concerning the cytotoxic activity of saponins have been summarized. The correlations between the structure and the cytotoxicity of both steroid and triterpenoid saponins have been described as well as the most common mechanisms of action.

  9. Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    A.J.A. Barbosa

    1991-03-01

    Full Text Available The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160, whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.O método daperoxidase-antiperoxidase foi utilizado para estudar as propriedades imunocitoquimicas de Leishmanias e de amastigotas do Trypanosoma cruzi, in situ, após os tecidos terem sido submetidos a diferentes tipos de fixação. Anti-soros foram obtidos de coelhos cronicamente infectados com três cepas de T. cruzi ou imunizados com L. mexicana ámazonensis e L. braziliensis guyanensis e aplicados nos cortes histológicos de 5 µm de espessura. Os antígenos de T. cruzi foram corados muito bem pelos três soros anti-T. cruzi e pelos dois soros anti-Leishmania com diluições entre 1:1.000 e 1:2.000. Diferentemente, os antígenos dç Leishmania foram revelados pelos soros anti- Leishmania somente em baixas diluições, ou seja, entre 1:60 e 1:160 enquanto que os soros

  10. Development of an Indirect Competitive ELISA Based on Polyclonal Antibody for the Detection of Diethylstilbestrol in Water Samples

    Institute of Scientific and Technical Information of China (English)

    WANG,Wen-Jun; LING,Yun; XU,Ting; GAO,Hong-Bin; SHENG,Wei; LI,Ji

    2007-01-01

    An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on polyclonal antibody for the estrogen diethylstilbestrol (DES) was developed. With this aim, two different haptens mono-O-3-carboxypropyldiethylstilbestrol (DES-CP) and mono-O-carboxymethyldiethylstilbestrol (DES-CM) with carboxylic group that preserve the molecular structure character of diethylstilbestrol were synthesized. The haptens were conjugated with the carrier proteins bovine serum albumin (BSA) by mixed-anhydride method for immunogen and conjugated with ovalbumin (OVA) by active ester method for coating antigen. Polyclonal antibodies for diethylstilbestrol were raised by immunizing mice with immune antigen DES-CP-BSA. Under optimized system, the lowest limit of detection (LLD) of diethylstilbestrol was 0.01 ng/mL, and IC50= 1.02 ng/mL. Its analogs were tested and no obvious cross-reactivity was found to anti-diethylstilbestrol antibody. DES-fortified water samples were determined by simple dilution to diminish the matrix effect. The comparison between the amount of DES estimated by ELISA and the amount added indicates good agreement for all water samples tested, with mean recovery values ranging from 86% to 120.2%.

  11. Cytotoxic geranylflavonoids from Bonannia graeca

    OpenAIRE

    Rosselli, Sergio; Bruno, Maurizio; Maggio, Antonella; Raccuglia, Rosa Angela; Safder, Muhammad; Lai, Chin-Yu; Bastow, Kenneth F.; Lee, Kuo-Hsiung

    2011-01-01

    The analysis of the aerial parts of Bonannia graeca led to the isolation and characterization of two new polar geranylated flavonoids (6 and 7). The structure elucidation was performed by extensive spectroscopic methods (1D and 2D NMR) and comparison with literature data. All natural flavonoids isolated from B. graeca (1–7) and some synthetic derivatives (8–11) were tested for cytotoxic activity against four human tumor cell lines. Preliminary structure-activity relationship correlations are ...

  12. HLA-G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to investigate whether the non-classi-cal HLA-G classⅠmolecule protects the porcine endothelial cells (PECs) from the lysis mediated by human immune cells in pig to human discordant xenotransplantation, we have cloned HLA-G cDNA from a human placenta by RT-PCR. Mammalian expression vector, pEFG-neo, was constructed by insertion of HLA-G cDNA in pEF-neo. We obtained efficiently expressed PECs by stable transfection. Cytotoxicity assay showed that overexpression of HLA-G on PECs was sufficient to inhibit human NK-92 cell lysis. The level of lysis was equal to or less than that of the lysis of human umbilical vein endothelial cells mediated by human NK-92 cells. It also indicated that HLA-G inhibited the lysis of PECs mediated by xeno-antigen specific T lymphocytes. The reduction of lysis ranged between 59.1% and 88.9%. These findings suggest that the transgenic approach to overexpress HLA-G is believed to be a new immunotherapy in overcoming the immune rejections in xenotransplantion, including delayed xenograft rejection and cell-mediated rejection.

  13. Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity.

    Science.gov (United States)

    Quast, Isaak; Keller, Christian W; Maurer, Michael A; Giddens, John P; Tackenberg, Björn; Wang, Lai-Xi; Münz, Christian; Nimmerjahn, Falk; Dalakas, Marinos C; Lünemann, Jan D

    2015-11-01

    IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.

  14. Comparative cytotoxicity of periodontal bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, R.H.; Hammond, B.F.

    1988-11-01

    The direct cytotoxicity of sonic extracts (SE) from nine periodontal bacteria for human gingival fibroblasts (HGF) was compared. Equivalent dosages (in terms of protein concentration) of SE were used to challenge HGF cultures. The cytotoxic potential of each SE was assessed by its ability to (1) inhibit HGF proliferation, as measured by direct cell counts; (2) inhibit 3H-thymidine incorporation in HGF cultures; or (3) cause morphological alterations of the cells in challenged cultures. The highest concentration (500 micrograms SE protein/ml) of any of the SEs used to challenge the cells was found to be markedly inhibitory to the HGFs by all three of the criteria of cytotoxicity. At the lowest dosage tested (50 micrograms SE protein/ml); only SE from Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Fusobacterium nucleatum caused a significant effect (greater than 90% inhibition or overt morphological abnormalities) in the HGFs as determined by any of the criteria employed. SE from Capnocytophaga sputigena, Eikenella corrodens, or Wolinella recta also inhibited cell proliferation and thymidine incorporation at this dosage; however, the degree of inhibition (5-50%) was consistently, clearly less than that of the first group of three organisms named above. The SE of the three other organisms tested (Actinomyces odontolyticus, Bacteroides intermedius, and Streptococcus sanguis) had little or no effect (0-10% inhibition) at this concentration. The data suggest that the outcome of the interaction between bacterial components and normal resident cells of the periodontium is, at least in part, a function of the bacterial species.

  15. Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+CD28+ T lymphocyte responses

    Institute of Scientific and Technical Information of China (English)

    Xia Yu; Yuan Fang; Xi Li; Nuo Zhou; Yong-Xiang Zhao; Xiao-Ling Lu; Jian He; Sodaly Mongkhoune; Yi Peng; Yuan Xie; Jing Su; Su-Fang Zhou; Xiao-Xun Xie; Guo-Rong Luo

    2013-01-01

    To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8+CD28+ cytotoxic T lymphocyte (CTL) responses. Methods: Cell-sized Dynabeads® M-450 Epoxy beads coated with H-2Kb:Ig-TRP2180-188 and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8+CD28+CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs. Results: Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8+CD28+ CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN-γ under the stimulation of H-2Kb:Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells. Conclusions: The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8+CD28+ CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.

  16. Comparison of the C-mediating killing activity and C-activating properties of mouse monoclonal and polyclonal antibodies against Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    T. L. Kipnis

    1992-01-01

    Full Text Available A Mouse polyclonal antiserum against Trypanosoma cruzi or its IgG and IgM fractions and five monoclonal antibodies (two IgM, two IgG1 and one IgG2a recognize and combine with membrane components of trypomastigote forms of the parasite as revealed by immunofluorescence. Although all these antibodies sensitize trypomastigotes and prepare them to activate the complement (C system, as measured by consumption of total C, C4, B and C3, only the polyclonal antiserum or its IgG, IgM and Fabμ fragments were able to induce trypanosome lysis by the alternative C pathway.

  17. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    Science.gov (United States)

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function. PMID:26205082

  18. Cytotoxic quassinoids from Simaba cedron.

    Science.gov (United States)

    Ozeki, A; Hitotsuyanagi, Y; Hashimoto, E; Itokawa, H; Takeya, K; de Mello Alves, S

    1998-06-26

    Four new quassinoids, cedronolactones A-D (1-4), together with nine known compounds, simalikalactone D (5), chaparrinone (6), chaparrin (7), glaucarubolone (8), glaucarubol (9), samaderine Z (10), guanepolide (11), ailanquassin A (12), and polyandrol (13), were isolated from the wood of Simaba cedron. The chemical structures of 1-4 were elucidated on the basis of their chemical and spectral properties. Cedronolactone A (1) was shown to exhibit a significant in vitro cytotoxicity (IC50 0.0074 microg/mL) against P-388 cells.

  19. 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 cytotoxicit......, produced from redox cycling of the catechol structure with molecular oxygen, is responsible in part for the cytotoxicity of this subgroup of flavonoids....

  20. Production and Application of Polyclonal Antibodies Against Recombinant Capsid Protein of Extra Small Virus of Macrobrachium rosenbergii.

    Science.gov (United States)

    Neethi, V; Sivakumar, N; Kumar, Kundan; Rajendran, K V; Makesh, M

    2012-12-01

    Macrobrachium rosenbergii nodavirus along with a satellite virus, extra small virus (XSV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. Infected M. rosenbergii postlarvae were collected from a hatchery in Kakinada, Andhra Pradesh. The gene coding the capsid protein of XSV was cloned in a bacterial expression vector pRSET A and the recombinant protein was expressed in Escherichia coli BL21(DE3)pLysS cells. The recombinant protein was purified by Nickel affinity chromatography. Polyclonal antibodies were produced in mice against the recombinant protein and the antibodies reacted specifically with the recombinant protein and XSV in WTD-infected tissues. This is the first report of detection of XSV using antibodies against recombinant capsid protein.

  1. Purification of Herpes Simplex Virus Type 1 for Production of High Titer Polyclonal Antibody against the Virus

    Directory of Open Access Journals (Sweden)

    Z Meshkat

    2008-12-01

    Full Text Available ABSTRACT: Introduction & Objective: Herpes simplex virus type 1 infection is one of the most prevalent viral infections worldwide. Different methods are being investigated for the virus’ detection, prevention and therapy. The aim of the present study was to purify the virus and to produce a high titer polyclonal antibody against the virus. Materials & Methods: This experimental study was done in the Virology Department of Tarbiat Modares University from 2001 to 2002. Virus purification was done using serial dilution and plaque purification protocols. A single plaque was chosen and propagated, and the virus titer was determined. In inoculated animals, the titer of produced antibody against the virus was measured by virus neutralization test. Results: Using virus neutralization test, it was found that the high level of antibody has been raised in animals against the virus. Conclusion: Considering the preparation of high titer antibody against the virus, the produced antibody can be used for the development and optimization of different diagnostic methods.

  2. Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    CHEN Yang; YANG Su-juan; FU Yao; WANG Jia-peng; ZHAO Zhi-zhuang; FU Xue-qi

    2008-01-01

    A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. Polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPa.

  3. Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection

    Science.gov (United States)

    Dowall, Stuart David; Callan, Jo; Zeltina, Antra; Al-Abdulla, Ibrahim; Strecker, Thomas; Fehling, Sarah K.; Krähling, Verena; Bosworth, Andrew; Rayner, Emma; Taylor, Irene; Charlton, Sue; Landon, John; Cameron, Ian; Hewson, Roger; Nasidi, Abdulsalami; Bowden, Thomas A.; Carroll, Miles W.

    2016-01-01

    The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOTAb, was prepared from the antisera and used for an in vivo guinea pig study. When EBOTAb was delivered 6 hours after challenge, all animals survived without experiencing fever or other clinical manifestations. In a second series of guinea pig studies, the administration of EBOTAb dosing was delayed for 48 or 72 hours after challenge, resulting in 100% and 75% survival, respectively. These studies illustrate the usefulness of EBOTAb in protecting against EBOV-induced disease. PMID:26715676

  4. Cytotoxicity of Ustilago maydis isolated from maize

    Directory of Open Access Journals (Sweden)

    Magdalena Twarużek

    2013-03-01

    Full Text Available The main pathogen of maize are fungi of the genus Fusarium. Besides phytopathogenic Fusarium, Ustilago maydis is another fungal genus affecting maize yields, causing lesions, known as smut. The objective of the study was evaluation of the cytotoxicity of Ustilago maydis isolated from maize. Nine Ustilago maydis strains were selected to a detailed evaluation of their cytotoxicity using a 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT test. Ustilago maydis strains showed medium and high cytotoxicity compared to control. High levels of cytotoxicity of Ustilago maydis may be indicative of their toxigenic potential.

  5. The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation

    Directory of Open Access Journals (Sweden)

    Mei Liu

    2013-04-01

    Full Text Available The enolase2 gene is usually expressed in mature neurons and also named neuron specific enolase (NSE. In the present study, we first obtained the NSE gene cDNA sequence by using the RACE method based on the expressed sequence tag (EST fragment from the cDNA library of Gekko japonicus and identified one transcript of about 2.2 kb in central nervous system of Gekko japonicus by Northern blotting. The open reading frame of NSE is 1305 bp, which encodes a 435 amino-acid protein. We further investigated the multi-tissue expression pattern of NSE by RT-PCR and found that the expression of NSE mRNA was very high in brain, spinal cord and low in heart, while it was not detectable in other tissues. The real-time quantitative PCR was used to investigate the time-dependent change in the expression of the NSE mRNA level after gecko spinal cord transection and found it significantly increased at one day, reaching its highest level three days post-injury and then decreasing at the seventh day of the experiment. The recombinant plasmid of pET-32a-NSE was constructed and induced to express His fused NSE protein. The purified NSE protein was used to immunize rabbits to generate polyclonal antisera. The titer of the antiserum was more than 1:65536 determined by ELISA. Western blotting showed that the prepared antibody could specifically recognize the recombinant and endogenous NSE protein. The result of immunohistochemistry revealed that positive signals were present in neurons of the brain and the spinal cord. This study provided the tools of cDNA and polyclonal antibody for studying NSE function in Gekko japonicus.

  6. Generation of genic diversity among Streptococcus pneumoniae strains via horizontal gene transfer during a chronic polyclonal pediatric infection.

    Directory of Open Access Journals (Sweden)

    N Luisa Hiller

    Full Text Available Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci.

  7. Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. In sugar cane (Saccharum spp. Using differente polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    Lúcia Gracinda da Silva-Froufe

    2009-12-01

    Full Text Available The species Gluconacetobacterdiazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 10(5 cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.

  8. Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    Lan LI; Yi-shu YANG; Ze-lin LI; Yi ZENG

    2008-01-01

    To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 Vif was amplified by PCR from a plasmid of HIV-1 NL4.3 cDNA, and the APOBEC3G gene was obtained by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E. coli cells, the accuracy of inserted gene and specificity of proteins were detected by the two enzyme digestion method, SDS-PAGE, and Western blotting. Rabbits were then immunized by Vif or APOBEC3G protein and serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assays were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of the anti-APOBEC3G antibodies was 1:102400. Thus the antibodies could detect the antigen expression in the cells, demonstrating that fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved.

  9. Tumor specific cytotoxicity of glucosylceramide.

    Science.gov (United States)

    Oku, Hirosuke; Wongtangtintharn, Sawitree; Iwasaki, Hironori; Inafuku, Masashi; Shimatani, Masayuki; Toda, Takayoshi

    2007-11-01

    To develop a new taxon of anti-cancer agent with lower side effect, this study described a tumor selective cytotoxicity of glucosylceramide extracted from malt feed of beer brewing waste. Interpretation of (13)C- and (1)H-NMR spectra identified the chemical structure of major component of glucosylceramide as 1-O-beta-D: -glucopyranosyl-2(2'-hydroxyeicosanoylamino)-4,11-octadecadiene-1,3-diol. Selective cytotoxicity was studied with three pairs of normal and cancer cells: liver, skin and lung. The glucosylceramide selectively lowered the relative viability of cancer cells. Of the pairs, the selectivity was most pronounced with the liver cells, and, for this reason, further experiment was conducted with this pair of normal (CS-HC) and cancer cells (HepG2) to get more insight into the selective toxicity. The glucosylceramide significantly increased the cell population at G(2)/M phase in HepG2 cells, and also increased the numbers of apoptotic (sub-G(0)/G(1)) cells, but to much lesser extent compared with the increase in G(2)/M phase. Treatment of HepG2 cells with this agent selectively disrupted the mitochondrial membrane integrity without activation of caspase pathway to induce apoptosis. These findings suggested that the glucosylceramide specifically suppressed the growth of cancer cells by inhibiting cell renewal capacity rather than induction of apoptosis. The underlying mechanism for the selectivity remains to be answered in the forthcoming study.

  10. Bioluminescence imaging of leukemia cell lines in vitro and in mouse xenografts: effects of monoclonal and polyclonal cell populations on intensity and kinetics of photon emission

    Directory of Open Access Journals (Sweden)

    Christoph Sandra

    2013-01-01

    Full Text Available Abstract Background We investigated the utility of bioluminescence imaging (BLI using firefly luciferase in monoclonal and polyclonal populations of leukemia cells in vitro and in vivo. Methods Monoclonal and polyclonal human lymphoid and myeloid leukemia cell lines transduced with firefly luciferase were used for BLI. Results Kinetics and dynamics of bioluminescence signal were cell line dependent. Luciferase expression decreased significantly over time in polyclonal leukemia cells in vitro. Transplantation of polyclonal luciferase-tagged cells in mice resulted in inconsistent signal intensity. After selection of monoclonal cell populations, luciferase activity was stable, equal kinetic and dynamic of bioluminescence intensity and strong correlation between cell number and light emission in vitro were observed. We obtained an equal development of leukemia burden detected by luciferase activity in NOD-scid-gamma mice after transplantation of monoclonal populations. Conclusion The use of monoclonal leukemia cells selected for stable and equal luciferase activity is recommended for experiments in vitro and xenograft mouse models. The findings are highly significant for bioluminescence imaging focused on pre-clinical drug development.

  11. High-density fecal Enterococcus faecium colonization in hospitalized patients is associated with the presence of the polyclonal subcluster CC17

    NARCIS (Netherlands)

    Ruiz-Garbajosa, P.; de Regt, M.; Bonten, M.; Baquero, F.; Coque, T. M.; Canton, R.; Harmsen, H. J. M.; Willems, Rob J. L.

    2012-01-01

    Enterococcus faecium belonging to the polyclonal subcluster CC17, with a typical ampicillin-resistant E. faecium (AREfm) phenotype, have become prevalent among nosocomial infections around the world. High-density intestinal AREfm colonization could be one of the factors contributing to the successfu

  12. Production and characterization of a broad-specificity polyclonal antibody for O,O-diethyl organophosphorus pesticides and a quantitative structure-activity relationship study of antibody recognition

    Science.gov (United States)

    Polyclonal antibody (PAb) with broad-specificity for O,O-diethyl organophosphorus pesticides (OPs) against a generic hapten, 4-(diethoxyphosphoro thioyloxy) benzoic acid, was produced. The obtained PAb showed high sensitivity to seven commonly used O,O-diethyl OPs in a competitive indirect enzyme-l...

  13. Polyclonal Mycobacterium avium infections in patients with AIDS: variations in antimicrobial susceptibilities of different strains of M. avium isolated from the same patient.

    Science.gov (United States)

    von Reyn, C F; Jacobs, N J; Arbeit, R D; Maslow, J N; Niemczyk, S

    1995-01-01

    Broth microdilution MICs were determined for pairs of strains isolated from five AIDS patients with polyclonal Mycobacterium avium infection. Four (80%) of the five patients were infected simultaneously with strains having different antimicrobial susceptibility patterns. These findings have implications for the interpretation of susceptibility data in M. avium prophylaxis and treatment trials. PMID:7790424

  14. Polyclonal Mycobacterium avium infections in patients with AIDS: variations in antimicrobial susceptibilities of different strains of M. avium isolated from the same patient.

    OpenAIRE

    von Reyn, C F; Jacobs, N J; Arbeit, R D; Maslow, J.N.; Niemczyk, S

    1995-01-01

    Broth microdilution MICs were determined for pairs of strains isolated from five AIDS patients with polyclonal Mycobacterium avium infection. Four (80%) of the five patients were infected simultaneously with strains having different antimicrobial susceptibility patterns. These findings have implications for the interpretation of susceptibility data in M. avium prophylaxis and treatment trials.

  15. Structure-cytotoxicity relationships for dietary flavonoids

    DEFF Research Database (Denmark)

    Breinholt, V.; Dragsted, L.O.

    1998-01-01

    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...

  16. Clonogenic cytotoxicity testing by microdrop encapsulation

    Science.gov (United States)

    Goguen, Blane

    1993-05-01

    Cytotoxicity is defined as the in situ measurement of cellular function in response to a toxic substance. Cytotoxicity assays are widely used in drug screening and development research to minimize the number of compounds on which expensive mammalian animal model toxicology studies are performed.

  17. DNA vaccine-generated duck polyclonal antibodies as a postexposure prophylactic to prevent hantavirus pulmonary syndrome (HPS.

    Directory of Open Access Journals (Sweden)

    Rebecca Brocato

    Full Text Available Andes virus (ANDV is the predominant cause of hantavirus pulmonary syndrome (HPS in South America and the only hantavirus known to be transmitted person-to-person. There are no vaccines, prophylactics, or therapeutics to prevent or treat this highly pathogenic disease (case-fatality 35-40%. Infection of Syrian hamsters with ANDV results in a disease that closely mimics human HPS in incubation time, symptoms of respiratory distress, and disease pathology. Here, we evaluated the feasibility of two postexposure prophylaxis strategies in the ANDV/hamster lethal disease model. First, we evaluated a natural product, human polyclonal antibody, obtained as fresh frozen plasma (FFP from a HPS survivor. Second, we used DNA vaccine technology to manufacture a polyclonal immunoglobulin-based product that could be purified from the eggs of vaccinated ducks (Anas platyrhynchos. The natural "despeciation" of the duck IgY (i.e., Fc removed results in an immunoglobulin predicted to be minimally reactogenic in humans. Administration of ≥ 5,000 neutralizing antibody units (NAU/kg of FFP-protected hamsters from lethal disease when given up to 8 days after intranasal ANDV challenge. IgY/IgYΔFc antibodies purified from the eggs of DNA-vaccinated ducks effectively neutralized ANDV in vitro as measured by plaque reduction neutralization tests (PRNT. Administration of 12,000 NAU/kg of duck egg-derived IgY/IgYΔFc protected hamsters when administered up to 8 days after intranasal challenge and 5 days after intramuscular challenge. These experiments demonstrate that convalescent FFP shows promise as a postexposure HPS prophylactic. Moreover, these data demonstrate the feasibility of using DNA vaccine technology coupled with the duck/egg system to manufacture a product that could supplement or replace FFP. The DNA vaccine-duck/egg system can be scaled as needed and obviates the necessity of using limited blood products obtained from a small number of HPS survivors. This

  18. Cytotoxic Effects of Bangladeshi Medicinal Plant Extracts

    Directory of Open Access Journals (Sweden)

    Shaikh J. Uddin

    2011-01-01

    Full Text Available To investigate the cytotoxic effect of some Bangladeshi medicinal plant extracts, 16 Bangladeshi medicinal plants were successively extracted with n-hexane, dichloromethane, methanol and water. The methanolic and aqueous extracts were screened for cytotoxic activity against healthy mouse fibroblasts (NIH3T3 and three human cancer-cell lines (gastric: AGS; colon: HT-29; and breast: MDA-MB-435S using the MTT assay. Two methanolic extracts (Hygrophila auriculata and Hibiscus tiliaceous and one aqueous extract (Limnophila indica showed no toxicity against healthy mouse fibroblasts, but selective cytotoxicity against breast cancer cells (IC50 1.1–1.6 mg mL−1. Seven methanolic extracts from L. indica, Clerodendron inerme, Cynometra ramiflora, Xylocarpus moluccensis, Argemone mexicana, Ammannia baccifera and Acrostichum aureum and four aqueous extracts from Hygrophila auriculata, Bruguiera gymnorrhiza, X. moluccensis and Aegiceras corniculatum showed low toxicity (IC50 > 2.5 mg mL−1 against mouse fibroblasts but selective cytotoxicity (IC50 0.2–2.3 mg mL−1 against different cancer cell lines. The methanolic extract of Blumea lacera showed the highest cytotoxicity (IC50 0.01–0.08 mg mL−1 against all tested cell lines among all extracts tested in this study. For some of the plants their traditional use as anticancer treatments correlates with the cytotoxic results, whereas for others so far unknown cytotoxic activities were identified.

  19. Oxaliplatin regulates expression of stress ligands in ovarian cancer cells and modulates their susceptibility to natural killer cell-mediated cytotoxicity.

    Science.gov (United States)

    Siew, Yin-Yin; Neo, Soek-Ying; Yew, Hui-Chuing; Lim, Shun-Wei; Ng, Yi-Cheng; Lew, Si-Min; Seetoh, Wei-Guang; Seow, See-Voon; Koh, Hwee-Ling

    2015-12-01

    Selected cytotoxic chemicals can provoke the immune system to recognize and destroy malignant tumors. Most of the studies on immunogenic cell death are focused on the signals that operate on a series of receptors expressed by dendritic cells to induce tumor antigen-specific T-cell responses. Here, we explored the effects of oxaliplatin, an immunogenic cell death inducer, on the induction of stress ligands and promotion of natural killer (NK) cell-mediated cytotoxicity in human ovarian cancer cells. The results indicated that treatment of tumor cells with oxaliplatin induced the production of type I interferons and chemokines and enhanced the expression of major histocompatibility complex class I-related chains (MIC) A/B, UL16-binding protein (ULBP)-3, CD155 and TNF-related apoptosis-inducing ligand (TRAIL)-R1/R2. Furthermore, oxaliplatin but not cisplatin treatment enhanced susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. In addition, activated NK cells completely abrogated the growth of cancer cells that were pretreated with oxaliplatin. However, cancer cells pretreated with the same concentration of oxaliplatin alone were capable of potentiating regrowth over a period of time. These results suggest an advantage in combining oxaliplatin and NK cell-based therapy in the treatment of ovarian cancer. Further investigation on such potential combination therapy is warranted.

  20. Expression, purification and polyclonal antibody generation of p23,an Hsp90 cochaperone, in the amphioxus Branchiostoma belcheri

    Institute of Scientific and Technical Information of China (English)

    ZHAO Bosheng; ZHANG Shicui; PANG Qiuxiang; LIU Zhenhui; LIANG Yujun

    2006-01-01

    The cDNA of amphioxus p23, a highly conserved co-chaperone for Hsp90, was cloned into a bacterial expression vector pGEX-6P-1 and the GST-tagged fusion protein was produced in Eschherichia coli cells. The recombinant p23 was purified by affinity purification, and its molecular mass was estimated to be approximately 22 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminus of purified p23 was sequenced, and the resulting amino acid sequence matches exactly the predicted residues deduced from the amphioxus p23 gene. Besides, polyclonal antibodies against the recombinant p23 were generated, and these antibodies not only recognized specifically the fusion protein GST-p23 from induced E. coli cells, purified GST-p23 and p23 protein, but also reacted with the total protein extracted from the adult amphioxus and formed a single positive band. These results pave the way for identifying its tissue and subcellular localization, and may open the door to clarifying its structure and mechanisms of biological role.

  1. Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli.

    Science.gov (United States)

    Shapira, R; Paster, N; Menasherov, M; Eyal, O; Mett, A; Meiron, T; Kuttin, E; Salomon, R

    1997-03-01

    Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods. PMID:9055416

  2. Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.

    2015-12-01

    Full Text Available Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims.

  3. Cloning of neuromedin B and its receptor in the rabbit and generating a polyclonal antibody to the neuromedin B protein.

    Science.gov (United States)

    Guo, Ting-Ting; Su, Juan; Ma, Zhi-Yu; Ma, Jun-Xiao; Jin, Meng-Meng; Li, Xiang; Lei, Zhi-Hai

    2015-06-10

    Neuromedin B (NMB) is a highly conserved bombesin-related neuropeptide found in mammals. Neuromedin B (NMB) executes its effect by binding to the cell surface receptor, neuromedin B receptor (NMBR). In this study, we cloned the rabbit NMB and NMBR genes. The similarity and phylogenetic analyses of NMB and NMBR gene sequences were performed. The expression of NMB and NMBR mRNA in the rabbit was investigated using real-time RT-PCR. Our bioinformatic analysis demonstrated that the cloned rabbit NMB precursor cDNA encoded Gly-His-Phe-Met-NH2 amino acids at the C-terminus, and that its receptor possessed typical transmembrane features. The NMB mRNA was highly expressed in the CNS, while the NMBR mRNA was widely expressed in many tissues, with the highest expression in the gastrointestinal tract. The studies on the NMB distribution and function are limited by the lack of a specific antibody to this neuropeptide. In this paper, polyclonal NMB antibody was generated in mice. Western blotting analysis revealed that the prepared antibody could specifically recognize the recombinant and the endogenous NMB proteins. Immunohistochemistry analysis indicated that the NMB protein was localized in the cytoplasm of the pituitary cells. The existence of NMB protein in the hypothalamic-pituitary-gonadal axis suggests that NMB might function in rabbit reproduction. PMID:25796599

  4. POLYCLONAL OUTBREAK OF BLOODSTREAM INFECTIONS CAUSED BY Burkholderia cepacia COMPLEX IN HEMATOLOGY AND BONE MARROW TRANSPLANT OUTPATIENT UNITS

    Directory of Open Access Journals (Sweden)

    Icaro Boszczowski

    2014-01-01

    Full Text Available Aim: The objective was to describe an outbreak of bloodstream infections by Burkholderia cepacia complex (Bcc in bone marrow transplant and hematology outpatients. Methods: On February 15, 2008 a Bcc outbreak was suspected. 24 cases were identified. Demographic and clinical data were evaluated. Environment and healthcare workers' (HCW hands were cultured. Species were determined and typed. Reinforcement of hand hygiene, central venous catheter (CVC care, infusion therapy, and maintenance of laminar flow cabinet were undertaken. 16 different HCWs had cared for the CVCs. Multi-dose heparin and saline were prepared on counter common to both units. Findings: 14 patients had B. multivorans (one patient had also B. cenopacia, six non-multivorans Bcc and one did not belong to Bcc. Clone A B. multivorans occurred in 12 patients (from Hematology; in 10 their CVC had been used on February 11/12. Environmental and HCW cultures were negative. All patients were treated with meropenem, and ceftazidime lock-therapy. Eight patients (30% were hospitalized. No deaths occurred. After control measures (multidose vial for single patient; CVC lock with ceftazidime; cleaning of laminar flow cabinet; hand hygiene improvement; use of cabinet to store prepared medication, no new cases occurred. Conclusions: This polyclonal outbreak may be explained by a common source containing multiple species of Bcc, maybe the laminar flow cabinet common to both units. There may have been contamination by B. multivorans (clone A of multi-dose vials.

  5. Development of Lateral-flow Immunoassay for WSSV with Polyclonal Antibodies Raised against Recombinant VP (19+28) Fusion Protein

    Institute of Scientific and Technical Information of China (English)

    Qing-yu CHENG; Xiao-lin MENG; Jin-ping XU; Wei LU; Jian WANG

    2007-01-01

    We have developed a sensitive and rapid lateral-flow immunoassay (LFIA) for WSSV,using colloidal gold as an indicator.The fusion protein,VP (19+28),was expressed in E.coli,purified and used to prepare polyclonal antibodies.The purified anti-VP (19+28) IgG were conjugated with colloidal gold.Unconjugated anti-VP (19+28) IgG and goat anti-rabbit IgG were immobilized on nitrocellulose membranes.After assembly,three groups (5 individual animals in each group) of shrimp samples were tested which included healthy,moribund and dead shrimps.For each group,three different tissues (body juices,gills and hepatopancreas) were tested at the same time.In parallel,all the samples were also analyzed using PCR for comparison.Out of 45 samples tested,30 were detected as positive while 15 were classified as negative.The results of LFIA correlate with those obtained by the PCR analysis,indicating that these two detection methods have the same efficacy in the limited number of samples tested in this preliminary study.

  6. Expansion of polyclonal B-cell precursors in bone marrow from children treated for acute lymphoblastic leukemia.

    Science.gov (United States)

    Duval, M; Fenneteau, O; Cave, H; Gobillot, C; Rohrlich, P; Guidal, C; Lescoeur, B; Legac, S; Schlegel, N; Sterkers, G; Vilmer, E

    1997-06-01

    In a series of 12 patients (mean age: 3 years at diagnosis) receiving chemotherapy for acute lymphoblastic leukemia, bone marrow examinations performed during hematopoietic recovery following treatment-induced agranulocytosis or completion of maintenance treatment showed at least 15% of non malignant immature cells which were sometimes hardly distinguishable from leukemic cells. No comparable data was observed in patients treated with G-CSF. The cytological features of these cells as well as their immunophenotyping were defined. Results showed that the majority of cells expressed HLA-DR, CD19, CD10 and cytoplasmic IgM but not the CD34 markers. This predominant and homogeneous pre-B cell population which likely represents the expansion of a minor population detectable in normal bone marrow is phenotypically indistinguishable from leukemic cells. The pattern of IgH gene rearrangements studied by PCR amplification of the CDRIII region showed that these cells were polyclonal. Except in one patient, minimal residual disease was not detected using probes specific for IgH or TCR gene rearrangement of the malignant clone. In children during the hematopoietic recovery after chemotherapy, immature marrow cells in great numbers, even with an highly homogeneous immunophenotype identical to the malignant clone's, are not sufficient for the diagnosis of relapse.

  7. Immunization with antigenic peptides complexed with β-glucan induces potent cytotoxic T-lymphocyte activity in combination with CpG-ODNs.

    Science.gov (United States)

    Mochizuki, Shinichi; Morishita, Hiromi; Kobiyama, Kouji; Aoshi, Taiki; Ishii, Ken J; Sakurai, Kazuo

    2015-12-28

    The induction of antigen-specific immune responses requires immunization with not only antigens, but also adjuvants. CpG oligonucleotides (CpG-ODNs) are well-known ligands for Toll-like receptor 9 and a potent adjuvant that induces both Th1-type humoral and cellular immune responses including cytotoxic T-lymphocyte responses. We previously demonstrated that β-glucan schizophyllan (SPG) can form complexes with CpG-ODNs with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses by co-administration of antigenic proteins, namely ovalbumin (OVA). Immunization with antigenic peptides, OVA257-264, did not induce these antigen-specific immune responses even in combination with CpG-dA/SPG, indicating that peptides require a carrier to antigen presenting cells. In this study, we prepared conjugates comprising OVA257-264 and dA40, and made complexes with SPG. Immunization with OVA257-264-dA/SPG induced peptide-specific immune responses in combination with CpG-dA regardless of complexation with SPG both in vitro and in vivo. When splenocytes from immunized mice were incubated with E.G7-OVA tumor model cells presenting OVA peptides, the number of cells drastically decreased after 24h. Furthermore, mice pre-immunized with OVA257-264-dA/SPG and CpG-ODNs exhibited a long delay in tumor growth after tumor inoculation. Therefore, these peptide-dA/SPG and CpG-dA/SPG complexes could be used as a potent vaccine for the treatment of cancers and infectious diseases. PMID:26562685

  8. B Cells Are Critical to T-cell—Mediated Antitumor Immunity Induced by a Combined Immune-Stimulatory/Conditionally Cytotoxic Therapy for Glioblastoma

    Directory of Open Access Journals (Sweden)

    Marianela Candolfi

    2011-10-01

    Full Text Available We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT mice, it completely failed in B-cell-deficient Igh6-/- mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6-/- mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1, in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells’ production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs. Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression.

  9. Hypoxia-inducible miR-210 regulates the susceptibility of tumor cells to lysis by cytotoxic T cells.

    Science.gov (United States)

    Noman, Muhammad Zaeem; Buart, Stéphanie; Romero, Pedro; Ketari, Sami; Janji, Bassam; Mari, Bernard; Mami-Chouaib, Fathia; Chouaib, Salem

    2012-09-15

    Hypoxia in the tumor microenvironment plays a central role in the evolution of immune escape mechanisms by tumor cells. In this study, we report the definition of miR-210 as a miRNA regulated by hypoxia in lung cancer and melanoma, documenting its involvement in blunting the susceptibility of tumor cells to lysis by antigen-specific cytotoxic T lymphocytes (CTL). miR-210 was induced in hypoxic zones of human tumor tissues. Its attenuation in hypoxic cells significantly restored susceptibility to autologous CTL-mediated lysis, independent of tumor cell recognition and CTL reactivity. A comprehensive approach using transcriptome analysis, argonaute protein immunoprecipitation, and luciferase reporter assay revealed that the genes PTPN1, HOXA1, and TP53I11 were miR-210 target genes regulated in hypoxic cells. In support of their primary importance in mediating the immunosuppressive effects of miR-210, coordinate silencing of PTPN1, HOXA1, and TP53I11 dramatically decreased tumor cell susceptibility to CTL-mediated lysis. Our findings show how miR-210 induction links hypoxia to immune escape from CTL-mediated lysis, by providing a mechanistic understanding of how this miRNA mediates immunosuppression in oxygen-deprived regions of tumors where cancer stem-like cells and metastatic cellular behaviors are known to evolve. PMID:22962263

  10. Antibody-Dependent Cell-Mediated Cytotoxicity to Hemagglutinin of Influenza A Viruses After Influenza Vaccination in Humans

    Science.gov (United States)

    Zhong, Weimin; Liu, Feng; Wilson, Jason R.; Holiday, Crystal; Li, Zhu-Nan; Bai, Yaohui; Tzeng, Wen-Pin; Stevens, James; York, Ian A.; Levine, Min Z.

    2016-01-01

    Background. Detection of neutralizing antibodies (nAbs) to influenza A virus hemagglutinin (HA) antigens by conventional serological assays is currently the main immune correlate of protection for influenza vaccines However, current prepandemic avian influenza vaccines are poorly immunogenic in inducing nAbs despite considerable protection conferred. Recent studies show that Ab-dependent cell-mediated cytotoxicity (ADCC) to HA antigens are readily detectable in the sera of healthy individuals and patients with influenza infection. Methods. Virus neutralization and ADCC activities of serum samples from individuals who received either seasonal or a stock-piled H5N1 avian influenza vaccine were evaluated by hemagglutination inhibition assay, microneutralization assay, and an improved ADCC natural killer (NK) cell activation assay. Results. Immunization with inactivated seasonal influenza vaccine led to strong expansion of both nAbs and ADCC-mediating antibodies (adccAbs) to H3 antigen of the vaccine virus in 24 postvaccination human sera. In sharp contrast, 18 individuals vaccinated with the adjuvanted H5N1 avian influenza vaccine mounted H5-specific antibodies with strong ADCC activities despite moderate virus neutralization capacity. Strength of HA-specific ADCC activities is largely associated with the titers of HA-binding antibodies and not with the fine antigenic specificity of anti-HA nAbs. Conclusions. Detection of both nAbs and adccAbs may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines.

  11. Production of anti-Gordonia amarae mycolic acid polyclonal antibody for detection of mycolic acid-containing bacteria in activated sludge foam.

    Science.gov (United States)

    Iwahori, K; Miyata, N; Takata, N; Morisada, S; Mochizuki, T

    2001-01-01

    Mycolic acid-containing actinomycetes (mycolata) are considered the causative agents of foaming of activated sludge and scum formation in activated sludge treatment plants. In this study, the production of anti-Gordonia amarae mycolic acid polyclonal antibodies was investigated. Rabbits were immunized with a conjugate of keyhole limpet hemocyanin and mycolic acids of G. amarae, which contained 48 to 56 carbon atoms (average, 52.0). Enzyme-linked immunosorbent assay (ELISA) demonstrated that the polyclonal antibodies could recognize cells of G. amarae ranging from 0.1 to 10 microg. The antibodies also reacted with other tested mycolata strains belonging to the genera Nocardia, Rhodococcus, Dietzia, Mycobacterium and Tsukamurella. However, reactivities against other gram-positive and gram-negative bacteria not containing mycolic acid were negligible or much lower. The results indicate that the anti-G. amarae mycolic acid antibodies show a reactivity selective for a group of mycolata involved in the foaming of activated sludge. PMID:16233121

  12. Combined TLR2/4-activated dendritic/tumor cell fusions induce augmented cytotoxic T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Shigeo Koido

    Full Text Available Induction of antitumor immunity by dendritic cell (DC-tumor fusion cells (DC/tumor can be modulated by their activation status. In this study, to address optimal status of DC/tumor to induce efficient antigen-specific cytotoxic T lymphocytes (CTLs, we have created various types of DC/tumor: 1 un-activated DC/tumor; 2 penicillin-killed Streptococcus pyogenes (OK-432; TLR4 agonist-activated DC/tumor; 3 protein-bound polysaccharides isolated from Coriolus versicolor (PSK; TLR2 agonist-activated DC/tumor; and 4 Combined OK-432- and PSK-activated DC/tumor. Moreover, we assessed the effects of TGF-β1 derived from DC/tumor on the induction of MUC1-specific CTLs. Combined TLR2- and TLR4-activated DC/tumor overcame immune-suppressive effect of TGF-β1 in comparison to those single activated or un-activated DC/tumor as demonstrated by: 1 up-regulation of MHC class II and CD86 expression on DC/tumor; 2 increased fusion efficiency; 3 increased production of fusions derived IL-12p70; 4 activation of CD4(+ and CD8(+ T cells that produce high levels of IFN-γ; 5 augmented induction of CTL activity specific for MUC1; and 6 superior efficacy in inhibiting CD4(+CD25(+Foxp3(+ T cell generation. However, DC/tumor-derived TGF-β1 reduced the efficacy of DC/tumor vaccine in vitro. Incorporating combined TLRs-activation and TGF-β1-blockade of DC/tumor may enhance the effectiveness of DC/tumor-based cancer vaccines and have the potential applicability to the field of adoptive immunotherapy.

  13. Cell Death Mechanisms Induced by Cytotoxic Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Ch(a)vez-Gal(a)n L; Arenas-Del Angel MC; Zenteno E; Ch(a)vez R; Lascurain R

    2009-01-01

    One of the functions of the immune system is to recognize and destroy abnormal or infected cells to maintain homeostasis. This is accomplished by cytotoxic lymphocytes. Cytotoxicity is a highly organized multifactor process. Here, we reviewed the apoptosis pathways induced by the two main cytotoxic lymphocyte subsets, natural killer (NK) cells and CD8+T cells. In base to recent experimental evidence, we reviewed NK receptors involved in recognition of target-cell, as well as lytic molecules such as perforin, granzymes-A and -B, and granulysin. In addition, we reviewed the Fas-FasL intercellular linkage mediated pathway, and briefly the cross-linking of tumor necrosis factor (TNF) and TNF receptor pathway. We discussed three models of possible molecular interaction between lyric molecules from effector cytotoxic cells and target-cell membrane to induction of apoptosis.

  14. Cytotoxic Activity of Selected Nigerian Plants

    OpenAIRE

    Sowemimo, A; M. Venter; Baatjies, L; Koekemoer, T

    2009-01-01

    Cancer is one of the most prominent human diseases which has stimulated scientific and commercial interest in the discovery of new anticancer agents from natural sources. The current study investigates the cytotoxic activity of ethanolic extracts of sixteen Nigerian plants used locally for the treatment of cancer using the MTT assay on the HeLa cell line. Sapium ellipticum leaves showed activity comparable to the reference compound Cisplatin and greater cytotoxic activity than Combretum panic...

  15. Cytotoxic T lymphocyte antigen-4 (CTLA-4 A49G polymorphism and autoimmune blood diseases

    Directory of Open Access Journals (Sweden)

    Faruk Aktürk

    2010-06-01

    Full Text Available Objective: The cytotoxic T lymphocyte associated antigen-4 (CTLA-4 is expressed on T lymphocytes, and inhibits the T-cell responses. In animal models, it has been shown that complete CTLA-4 deficiency was lethal due to massive infiltration of tissues by polyclonally proliferating lymphocytes. CTLA-4 A49G polymorphism, which has been suggested to reduce the inhibitory function of the CTLA-4 molecule, was found to be associated with various autoimmune diseases in recent studies. Material and Methods: In this study, we evaluated the frequency of CTLA-4 A49G polymorphism in 46 patients with autoimmune hemolytic anemia (AIHA, 62 patients with immune thrombocytopenic purpura (ITP, and 150 healthy individuals. Results: Allele frequencies and genotype distributions were similar in both ITP and AIHA patients compared to healthy individuals. In subgroup analysis, however, we found that in chronic lymphocytic leukemia (CLL patients with AIHA (n=4, all patients had CTLA-4 A49G polymorphism (3 had AG, 1 had GG. There was no significant statistical association between G allele and systemic lupus erythematosus (SLE or AIHA.Conclusion: These data suggest that CTLA-4 A49G polymorphism does not contribute to the pathogenesis of lymphoproliferative diseases itself, nor does it increase the risk of autoimmune complications in patients with lymphoproliferative disease.

  16. Human antibody responses to the polyclonal Dryvax vaccine for smallpox prevention can be distinguished from responses to the monoclonal replacement vaccine ACAM2000.

    Science.gov (United States)

    Pugh, Christine; Keasey, Sarah; Korman, Lawrence; Pittman, Phillip R; Ulrich, Robert G

    2014-06-01

    Dryvax (Wyeth Laboratories, Inc., Marietta, PA) is representative of the vaccinia virus preparations that were previously used for preventing smallpox. While Dryvax was highly effective, the national supply stocks were depleted, and there were manufacturing concerns regarding sterility and the clonal heterogeneity of the vaccine. ACAM2000 (Acambis, Inc./Sanofi-Pasteur Biologics Co., Cambridge, MA), a single-plaque-purified vaccinia virus derivative of Dryvax, recently replaced the polyclonal smallpox vaccine for use in the United States. A substantial amount of sequence heterogeneity exists within the polyclonal proteome of Dryvax, including proteins that are missing from ACAM2000. Reasoning that a detailed comparison of antibody responses to the polyclonal and monoclonal vaccines may be useful for identifying unique properties of each antibody response, we utilized a protein microarray comprised of approximately 94% of the vaccinia poxvirus proteome (245 proteins) to measure protein-specific antibody responses of 71 individuals receiving a single vaccination with ACAM2000 or Dryvax. We observed robust antibody responses to 21 poxvirus proteins in vaccinated individuals, including 11 proteins that distinguished Dryvax responses from ACAM2000. Analysis of protein sequences from Dryvax clones revealed amino acid level differences in these 11 antigenic proteins and suggested that sequence variation and clonal heterogeneity may contribute to the observed differences between Dryvax and ACAM2000 antibody responses.

  17. Investigation of a panel of monoclonal antibodies and polyclonal sera against anthrax toxins resulted in identification of an anti-lethal factor antibody with disease-enhancing characteristics.

    Science.gov (United States)

    Kulshreshtha, Parul; Tiwari, Ashutosh; Priyanka; Joon, Shikha; Sinha, Subrata; Bhatnagar, Rakesh

    2015-12-01

    Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization.

  18. Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli.

    Science.gov (United States)

    Sharma, Prachi; Sharma, Susheel; Singh, Jasvir; Saha, Swati; Baranwal, V K

    2016-04-01

    Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production. PMID:26850143

  19. A freeze dried kit formulation for the preparation of 99m Tc labelled human polyclonal IgG for the detection of infection and inflammation

    International Nuclear Information System (INIS)

    A freeze dried kit formulation for the preparation of 99mTc-labelled human polyclonal IgG for the detection of infection and inflammation employing direct methods for protein labeling was developed. The method comprises reduction of intrinsic disulphide bridges within the antibody molecule by the use of the reductant 2-mercaptoethanol. Following a subsequent purification, the resulting reduced antibody is labeled via Sn2+ reduction of pertechnetate in the presence of a weak competing ligand ethane-1-hydroxy-1,1-diphosphonate (EHDP). High labeling efficiencies (>90%) were obtained with high in vitro stability and without effect upon antibody immunoreactivity. Methods of analysis were also established permitting identification of radiochemical impurities which may be present in radiopharmaceutical solution. 99m Tc-polyclonal IgG prepared by the kit method was evaluated for scintigraphic localization of inflammatory lesions and abscesses in rabbits. Data demonstrated that the 99m Tc-polyclonal IgG after kit-reconstitution shows excellent stability and is an effective imaging agent of infection and/or inflammation. (Author)

  20. Produção de anticorpos policlonais anti-ricina Production of polyclonal anti-ricin antibodies

    Directory of Open Access Journals (Sweden)

    Roselayne Ferro Furtado

    2011-02-01

    Full Text Available A ricina é uma proteína bastante tóxica presente nas sementes de mamona que impossibilita o uso da torta de mamona "in natura", como ração. A torta de mamona destoxificada necessita ainda de métodos de análise que garantam a ausência de traços dessa proteína. Objetivou-se, neste trabalho, produzir e avaliar a sensibilidade e especificidade de anticorpos policlonais anti-ricina, para serem empregados como possíveis componentes de métodos sorológicos na detecção de ricina em torta de mamona destoxificada. Foram avaliadas três doses da proteína: 400, 180 e 100 µg cada uma dividida em duas aplicações em coelhos. A primeira dose foi injetada no animal no início do experimento e a segunda após 21 dias. O método de ELISA indicou que as duas doses menores (100 e 180 µg induziram respostas imunológicas primária e secundária com produção de anticorpos específicos. Enquanto a dose maior (400 µg de ricina apresentou uma resposta primária com elevação dos títulos de anticorpos, seguida de uma supressão da resposta. Esse perfil é sugestivo de tolerância imunológica. Pela técnica de Western blotting verificou-se que os anticorpos policlonais produzidos são bastante específicos para a ricina, no entanto, por detectarem ricina na forma nativa e desnaturada não são recomendados para o monitoramento de ricina em torta de mamona destoxificada por tratamento térmico.Ricin is a very toxic protein found in castor bean plants, making it impossible to use natural castor cake as animal food. The detoxificated castor cake needs to be analyzed by methods that ensure the absence of traces of this protein. This work had the objective to produce and to evaluate the sensitivity and specificity of anti-ricin polyclonal antibodies, to be employed as component of sorologic methods as the ELISA in the detection of ricin in detoxificated castor cake. Three doses of protein, 400, 180 and 100 µg were evaluated each one injected twice into

  1. A feasible enzyme-linked immunosorbent assay system using monoclonal and polyclonal antibodies against glucosyltransferase-B from Streptococcus mutans.

    Science.gov (United States)

    Shinozaki-Kuwahara, Noriko; Hashizume-Takizawa, Tomomi; Hirasawa, Masatomo; Takada, Kazuko

    2012-06-01

    Streptococcus mutans has been considered the principal etiological agent of dental caries in humans. S. mutans can secrete three kinds of glucosyltransferases (GTFs). One of these, GTF-B, which synthesizes water-insoluble glucans from sucrose, has been considered to be one of the most important factors of cariogenic dental plaque formation. Therefore, determination of whether GTF-B is present in plaque and saliva samples may contribute to the evaluation of individual virulence potential (caries risk). The aim of this study was to develop a feasible enzyme-linked immunosorbent assay (ELISA) for the routine quantification of GTF-B in plaque-derived cultures and clinical samples, and to apply this assay to an epidemiological study. To determine the presence of GTF-B in plaque samples, a sandwich-ELISA was devised, consisting of mouse monoclonal and rabbit polyclonal antibodies against GTF-B and a horseradish peroxidase-conjugated anti-rabbit antibody. The developed ELISA allowed for quantification of the amounts of purified GTF-B with satisfactory sensitivity and specificity; this method was not affected by other components such as plaque and saliva. Plaque samples from healthy volunteers were examined using this ELISA method and microbial analysis to apply the assay to an epidemiological study. A correlation was observed between the amount of extracted GTF-B and S. mutans levels as determined by ELISA and cultivated with Mitis Salivarius Bacitracin agar plates derived from plaque samples, although there were some exceptions. In this regard, this ELISA system has the advantage of estimating both the individual numbers of S. mutans and the productivity of GTF-B, namely, the cariogenic potential of S. mutans simultaneously. These results indicate that this ELISA method is a useful tool for the diagnosis of caries risk.

  2. Effects of the Sheep Polyclonal Antibodies Against the Porcine Adipocyte Plasma Membrane Proteins on Porcine Carcass Composition and Meat Quality

    Institute of Scientific and Technical Information of China (English)

    GAO Shi-zheng; HU Hong-mei; LIU Ling-yun; ZHANG Xi; LIU Yong-gang; GE Chang-rong

    2007-01-01

    To detect the effects of the polyclonal antibodies raised in sheep against porcine adipocyte plasma membranes on the porcine carcass composition and meat quality, 30 pigs assigned into 6 treatment groups were given intraperitoneal injections of sheep antipig adipocyte plasma membrane immunoglobulin (ASIg) or sheep nonimmune serum immunoglobulin (NSIg). At the end of the experiment, the pigs were slaughtered at 90 kg body weight, and carcasses and meat quality were evaluated. The results showed that when pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, 20 mg purified ASIg twice at 15 and 60 kg body weight, or 20 mg purified ASIg at 60 kg body weight, respectively, their lean meat percentage, fat meat percentage, backfat thickness, loin eye area leaf fat weight, caul fat weight, heart weight, liver weight, and kidney weight were significantly affected. However, the kidney weight, lung weight, dressing percentage,and spleen weight did not remarkably change. Our results indicated that pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, and 20 mg ASIg twice at 15 and 60 kg body weight, have significantly different drip loss rate,cooked meat ratio, tenderness, storage loss rate, muscle fiber diameter, moisture content, dry matter content, crude protein content, and crude fat content from the control group that received 20 mg NSIg at 15 kg body weight. However, meat pH,meat color value, meat marbling score, inosinate, and myohemoglobin were not significantly affected. Our results indicated ASIg could not significantly affect the content of most muscular amino acids and intramuscular fatty acids.

  3. Development of a class-specific polyclonal antibody-based indirect competitive ELISA for detecting fluoroquinolone residues in milk

    Institute of Scientific and Technical Information of China (English)

    Guo-ying FAN; Ruo-song YANG; Jin-qing JIANG; Xin-yao CHANG; Jun-jie CHEN; Yong-hua QI; Shi-xiu WU; Xue-feng YANG

    2012-01-01

    Modified 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method was employed to synthesize the artificial antigen of norfloxacin (NOR),and New Zealand rabbits were used to produce anti-NOR polyclonal antibody (pAb).Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay (icELISA)standard curve was established.This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml,with the half maximal inhibitory concentration (IC50) and limit of detection (LOD) values of 2.7 ng/ml and 0.06 ng/ml,respectively.The produced pAb exhibited high cross-reactivity to fluoroquinolones (FQs) tested,and the IC50 values to enoxacin,ciprofloxacin,and pefloxacin were 3.1,3.4,and 4.1 ng/ml,respectively.It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10% and 30%.When spiked in milk at 5,20,and 50 ng/ml,the recoveries for NOR,enoxacin,ciprofloxacin,and pefloxacin ranged 90.5%-98.0%,84.0%-95.2%,94.0%-106.0%,and 89.5%-100.0%,respectively.The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.

  4. An automated packed protein G micro-pipette tip assay for rapid quantification of polyclonal antibodies in ovine serum.

    Science.gov (United States)

    Chhatre, Sunil; Francis, Richard; Bracewell, Daniel G; Titchener-Hooker, Nigel J

    2010-11-15

    The demands on the biopharmaceutical sector to expedite process development have instigated the deployment of micro-biochemical engineering techniques to acquire manufacturing insight with extremely small sample volumes. In conjunction with automated liquid handlers, this permits the simultaneous evaluation of multiple operating conditions and reduces manual intervention. For these benefits to be sustained, novel ways are now required to accelerate analysis and so prevent this becoming a throughput bottleneck. For example, although Protein G HPLC is used to quantify antibody titres in bioprocess feedstocks, it can be time-consuming owing to the serial nature of its application. Although commercial options are available that can process many samples simultaneously, these require separate, potentially expensive instruments. A more integrated approach is desirable wherein the assay is implemented directly on a robot. This article describes a high-throughput alternative to antibody HPLC analysis which uses an eight-channel liquid handler to control pipette tips packed with 40 μL of Protein G affinity matrix. The linearity, range, limit of detection, specificity and precision of the method were established, with results showing that antibody was detected reliably and specifically between 0.10 and 1.00 mg/mL. Subsequently, the technique was used to quantify the antibody titre in ovine serum, which is used as feed material by BTG PLC for manufacturing FDA-approved polyclonal bio-therapeutics. The mean concentration determined by the tips was comparable to that found by HPLC, but the tip method delivered its results in less than 40% of the time and with the potential for further, substantial time-savings possible by using higher capacity robots.

  5. Amplification and polyclonal antibody preparation of M13 phage display library%M13噬菌体肽库扩增及兔抗血清制备

    Institute of Scientific and Technical Information of China (English)

    任晓峰; 马晓微

    2013-01-01

    为制备M13噬菌体多克隆抗体,将噬菌体十二肽原始文库进行大量扩增,作为免疫原制备兔抗M13的多克隆抗体血清并进行间接ELISA鉴定.结果表明,该多抗效价达1∶1 048 576,说明此多抗可与扩增噬菌体文库发生很好的抗原抗体反应.将制备的噬菌体M13多克隆抗体与商业化M13抗体水平比较,制备多抗与商业化M13抗体效果相当.本研究成功制备兔抗M13噬菌体多克隆抗体,为深入研究噬菌体展示技术提供了材料.%In order to generate polyclonal antibodies of the M13 Phage,one M13 of Ph..D.-12TM Phage Display Peptide Library was amplified and used to immunize a rabbit to generate polyclonal antibody.Indirect ELISA analysis showed that the titer of the polyclonal antibody was approximately 1∶1 048 576,showing that the anti-M13 antibody had a high titer.Compared this polyclonal antibody with a Rabbit polyclonal antibody (Rb pAb) to M13 Bacteriophage Coat Proteins (commercialization),the binding activity of the produced polyclonal antibody to the identical target was as good as that of the Rb pAb to M13 Bacteriophage Coat Proteins.In the study,polyclonal antibody to this M13 of phage library was successfully generated and such phage polyclonal antibody is important material for functional analysis with phage.

  6. The cytotoxicity study of praziquantel enantiomers

    Directory of Open Access Journals (Sweden)

    Sun Q

    2016-06-01

    Full Text Available Qian Sun, Ruifeng Mao, Dongling Wang, Changyan Hu, Yang Zheng, Dequn Sun Department of Pharmacy, Marine College, Shandong University, Weihai, People’s Republic of China Abstract: Praziquantel (PZQ is prescribed as a racemic mixture (racemic-PZQ, rac-PZQ, which is composed of (R-PZQ and (S-PZQ. In this work, the cytotoxicity of rac-PZQ and its two enantiomers (R-PZQ and (S-PZQ on eight cell lines (L-02, HepG2, prf-plc-5, SH-SY5Y, HUVEC, A549, HCT-15, Raw264.7 was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays. The morphology of apoptotic cells was studied by fluorescence microscope using Hoechst 33342 staining, and the cytotoxicity of the compounds was also tested by lactate dehydrogenase assay. Results revealed that (R-PZQ had negligible cytotoxicity against L-02, SH-SY5Y, HUVEC, A549, HCT-15, and Raw264.7 cells but selectively inhibited tumor cell lines (prf-plc-5 and HepG2. However, in contrast to (R-PZQ, the (S-isomer showed higher cytotoxicity against L-02 cells and lower inhibition on prf-plc-5 and HepG2 cells. Besides, (R-PZQ showed lower cytotoxicity on SH-SY5Y cells than (S-PZQ. Meanwhile, (R-PZQ at <80 µM concentration could promote proliferation of macrophage cells (Raw264.7. Our research revealed that (R-PZQ has lower cytotoxicity than (S-PZQ and has similar cytotoxicity with rac-PZQ. (S-PZQ is the principal enantiomer to cause side effects on human definitive hosts. These findings gave the reasonable reasons for World Health Organization to produce (R-PZQ as a replacement for rac-PZQ for the treatment of schistosomiasis. Keywords: isomer, MTT, selectivity, (R-PZQ

  7. The cytotoxicity study of praziquantel enantiomers

    Science.gov (United States)

    Sun, Qian; Mao, Ruifeng; Wang, Dongling; Hu, Changyan; Zheng, Yang; Sun, Dequn

    2016-01-01

    Praziquantel (PZQ) is prescribed as a racemic mixture (racemic-PZQ, rac-PZQ), which is composed of (R)-PZQ and (S)-PZQ. In this work, the cytotoxicity of rac-PZQ and its two enantiomers (R)-PZQ and (S)-PZQ on eight cell lines (L-02, HepG2, prf-plc-5, SH-SY5Y, HUVEC, A549, HCT-15, Raw264.7) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide and lactate dehydrogenase assays. The morphology of apoptotic cells was studied by fluorescence microscope using Hoechst 33342 staining, and the cytotoxicity of the compounds was also tested by lactate dehydrogenase assay. Results revealed that (R)-PZQ had negligible cytotoxicity against L-02, SH-SY5Y, HUVEC, A549, HCT-15, and Raw264.7 cells but selectively inhibited tumor cell lines (prf-plc-5 and HepG2). However, in contrast to (R)-PZQ, the (S)-isomer showed higher cytotoxicity against L-02 cells and lower inhibition on prf-plc-5 and HepG2 cells. Besides, (R)-PZQ showed lower cytotoxicity on SH-SY5Y cells than (S)-PZQ. Meanwhile, (R)-PZQ at <80 μM concentration could promote proliferation of macrophage cells (Raw264.7). Our research revealed that (R)-PZQ has lower cytotoxicity than (S)-PZQ and has similar cytotoxicity with rac-PZQ. (S)-PZQ is the principal enantiomer to cause side effects on human definitive hosts. These findings gave the reasonable reasons for World Health Organization to produce (R)-PZQ as a replacement for rac-PZQ for the treatment of schistosomiasis. PMID:27445457

  8. Epitope hunting in rheumatoid arthritis : towards antigen specific immunotherapy

    NARCIS (Netherlands)

    de Jong, H.

    2013-01-01

    Current treatment options in rheumatoid arthritis aim to dampen the immune response a-specifically. In the last decennia new strategies have emerged that have fewer side effects due to more specificity by focussing on those cells of the immune system that deal with regulation. Epitope specific immun

  9. Circadian control of antigen-specific T cell responses

    Directory of Open Access Journals (Sweden)

    Nobis CC

    2016-09-01

    Full Text Available Chloé C Nobis,1–3 Nathalie Labrecque,2–4 Nicolas Cermakian1,5–8 1Douglas Mental Health University Institute, 2Maisonneuve-Rosemont Hospital Research Centre, 3Department of Microbiology, Infectious Diseases and Immunology, 4Department of Medicine, University of Montreal, 5Department of Psychiatry, 6Department of Microbiology and Immunology, 7Department of Neurology and Neurosurgery, 8Department of Physiology, McGill University, Montreal, QC, Canada Abstract: The immune system is composed of two arms, the innate and the adaptive immunity. While the innate response constitutes the first line of defense and is not specific for a particular pathogen, the adaptive response is highly specific and allows for long-term memory of the pathogen encounter. T lymphocytes (or T cells are central players in the adaptive immune response. Various aspects of T cell functions vary according to the time of day. Circadian clocks located in most tissues and cell types generate 24-hour rhythms of various physiological processes. These clocks are based on a set of clock genes, and this timing mechanism controls rhythmically the expression of numerous other genes. Clock genes are expressed in cells of the immune system, including T cells. In this review, we provide an overview of the circadian control of the adaptive immune response, with emphasis on T cells, including their development, trafficking, response to antigen, and effector functions. Keywords: circadian clock, adaptive immune response, T lymphocyte, antigen, cytokine, proliferation

  10. The antigen specific composition of melanoma tumor infiltrating lymphocytes?

    DEFF Research Database (Denmark)

    Hadrup, Sine Reker

    2012-01-01

    Large numbers of tumor associated antigens has been characterized, but only a minor fraction of these are recognized by tumor infiltrating lymphocytes of melanoma, although these have shown the ability to recognize tumor and provide tumor regression upon adoptive transfer. Thus the peptide...... recognition of the majority of the CD8 tumor infiltrating lymphocytes remains to be identified....

  11. Synthetic peptides with antigenic specificity for bacterial toxins.

    Science.gov (United States)

    Sela, M; Arnon, R; Jacob, C O

    1986-01-01

    The attachment of a diphtheria toxin-specific synthetic antigenic determinant and a synthetic adjuvant to a synthetic polymeric carrier led to production of a totally synthetic macromolecule which provoked protective antibodies against diphtheria when administered in aqueous solution. When peptides related to the B subunit of cholera toxin were synthesized and attached to tetanus toxoid, antibodies produced against the conjugate reacted in some but not all cases with intact cholera toxin and (especially with peptide CTP 3, residues 50-64) neutralized toxin reactivity, as tested by permeability in rabbit skin, fluid accumulation in ligated small intestinal loops and adenylate cyclase activation. Polymerization of the peptide without any external carrier, or conjugation with the dipalmityl lysine group, had as good an effect in enhancing the immune response as its attachment to tetanus toxoid. Prior exposure to the carrier suppressed the immune response to the epitope attached to it, whereas prior exposure to the synthetic peptide had a good priming effect when the intact toxin was given; when two different peptides were attached to the same carrier, both were expressed. Antisera against peptide CTP 3 were highly cross-reactive with the heat-labile toxin of Escherichia coli and neutralized it to the same extent as cholera toxin, which is not surprising in view of the great homology between the two proteins. A synthetic oligonucleotide coding for CTP 3 has been used to express the peptide in a form suitable for immunization. It led to a priming effect against the intact cholera toxin. PMID:2426052

  12. Antigenic specificity of serum antibodies in mice fed soy protein

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Bruun, S.W.; Frøkiær, Hanne

    2003-01-01

    Background: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about...

  13. 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.

  14. Isoflavanones from Desmodium oxyphyllum and their cytotoxicity.

    Science.gov (United States)

    Li, Yan-Ping; Li, Yin-Ke; Du, Gang; Yang, Hai-Yin; Gao, Xue-Mei; Hu, Qiu-Fen

    2014-01-01

    Two new isoflavanones, (3R)-7-hydroxy-4'-methoxy-5-methoxycarbonyl-isoflavanone (1) and (3R)-8-hydroxy-4'-methoxy-7-methoxycarbonyl-isoflavanone (2), together with seven known isoflavanones (3-9) were isolated from Desmodium oxyphyllum of the Leguminosae family. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. Compound 1 showed good cytotoxicity against NB4 and SHSY5Y cell lines with IC50 values of 3.1 and 2.5 μM; compound 2 exhibited cytotoxicity against PC3 cell lines with a IC50 value of 3.6 μM; compound 4 showed cytotoxicity against A549 and SHSY5Y cell lines with IC50 values of 3.6 and 2.8 μM; and compound 5 displayed cytotoxicity against NB4, SHSY5Y, and MCF7 cell lines with IC50 values of 2.6, 3.8, and 2.8 μM, respectively. Other compounds also showed moderate cytotoxicity for some tested cell lines with IC50 values between 5.4 and 8.8 μM. PMID:24749537

  15. 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. PMID:26692167

  16. [Synthesis and cytotoxicity of allobetulin derivatives].

    Science.gov (United States)

    Kazakova, O B; Smirnova, I E; Khusnutdinova, E F; Zhukova, O S; Fetisova, L V; Apryshko, G N; Medvedeva, N I; Iamansarov, E Iu; Baĭkova, I P; Nguen, Thanh Tra; Thu, Do Thi H

    2014-01-01

    The synthesis and screening of antitumor activity in vitro (cytotoxicity) of various oxygen, nitrogen, sulfur and platinum-containing derivatives of allobetulin, including different arrangements of the double bonds in the A and B rings, penta- and hexacyclic ring A, 21-acetyl-20,28-epoxy-18α,19βH-ursane-isomeric cycle E, was carry out. (3R,5R)-19β,28-Epoxy-4,5-seco-18α-olean-3(5)-ozonide and 2,3-indolo-21β-acetyl-20β,28-epoxy-18α, H-19β-ursane showed significant cytotoxic activity against melanoma MeWo and Leukemia SR cells, appropriately. (3S,5S)-Diastereomer of the first compound showed no cytotoxicity. PMID:25895356

  17. Cytotoxic Compounds Isolated from Murraya tetramera Huang

    Directory of Open Access Journals (Sweden)

    Chun-Xue You

    2014-08-01

    Full Text Available A new compound and seven known compounds were isolated from Murraya tetramera Huang for the first time, and they were identified with NMR and MS spectral analysis. It was confirmed that the new compound was 10-methoxy-7-methyl-2H-benzo[g]chromen-2-one (3 and the others were β-eudesmol (1, trans-3β-(1-hydroxy-1-methylethyl-8aβ-methyl-5-methylenedecalin-2-one (2, 5,7-dimethoxy-8-[(Z-3'-methyl-butan-1',3'-dienyl]coumarin (4, 7-geranyloxy-6-methoxycoumarin (5, 5,7-dimethoxy-8-(3-methyl-2-oxo-butylcoumarin (6, murrangatin acetate (7 and toddalenone (8. Furthermore, the cytotoxic activity against human lung adenocarcinoma (A549, human hepatocellular carcinoma cells (SMMC-7721, human bladder tumor cells (EJ, human cervical carcinoma cells (HeLa, and human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1 was evaluated for all compounds. It was found that five of them displayed various degrees of cytotoxicity against different testing targets. Compound 1 showed significant cytotoxic activity against the five cell lines (A549, SMMC-7721, EJ, Hela and BALL-1. Compounds 2 and 5 showed significant cytotoxicity against three cell lines (A549, SMMC-7721 and BALL-1. Compound 4 showed significant cytotoxicity against three cell lines (A549, EJ and BALL-1. However, compound 3 only showed fair cytotoxicity against the BALL-1 cell line. The structure-active relationships were investigated as well. These active compounds might be potential lead compounds for the treatment of cancer.

  18. Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production.

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsushita

    Full Text Available Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs in transchromosomic (Tc cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC comprising the entire unrearranged human immunoglobulin (Ig heavy-chain (hIGH, kappa-chain (hIGK, and lambda-chain (hIGL germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML1-/-; double knockouts or DKO. However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK and lambda-chain (bIGL genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ. To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J and constant (C gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5 by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM-/-, bIGHML1-/- and bIGL-/-; TKO Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ.

  19. Cytotoxic Terpene Quinones from Marine Sponges

    Directory of Open Access Journals (Sweden)

    Marina Gordaliza

    2010-11-01

    Full Text Available The 1,4-benzoquinone moiety is a common structural feature in a large number of compounds that have received considerable attention owing to their broad spectrum of biological activities. The cytotoxic and antiproliferative properties of many natural sesquiterpene quinones and hydroquinones from sponges of the order Dictyoceratida, such as avarol, avarone, illimaquinone, nakijiquinone and bolinaquinone, offer promising opportunities for the development of new antitumor agents. The present review summarizes the structure and cytotoxicity of natural terpenequinones/hydroquinones and their bioactive analogues and derivatives.

  20. Cytotoxicity of Two Triterpenoids from Nigella glandulifera

    Directory of Open Access Journals (Sweden)

    Erxi Wu

    2006-09-01

    Full Text Available During an investigation of antitumor substances from Nigella glandulifera Freyn et Sint. (Ranunculaceae the cytotoxicity of two oleanane triterpene saponins isolated from the seeds of this species, kalopanaxsaponins A and I, was evaluated against HepG2, drug resistant HepG2 (R-HepG2 (two hepatocyte cell lines and primary cultured normal mouse hepatocytes. Evident cytotoxic activities were observed. Morphological observations and cell cycle analysis suggest that these compounds inhibit the proliferation of hepatoma by inducing apoptosis and consequently kalopanaxsaponins A and I may be potential therapeutic agents for the treatment of parental and drug resistant hepatoma.

  1. Cytotoxic effect of endodontic irrigants in vitro

    OpenAIRE

    Bajrami, Donika; Hoxha, Veton; Gorduysus, Omer; Muftuoglu, Sevda; Zeybek, Naciye Dilara; Küçükkaya, Selen

    2014-01-01

    Background Cytotoxicity of root canal irrigants is important due to their close contact with host tissues. The aim of this study was to assess the cytotoxic effect of NaOCl 3%, Chx 2%, and MTAD on rat periodontal ligament fibroblasts, at 0.1 and 100 μl/mL, using WST-1 colorimetric method. Material/Method Rat ligamental fibroblasts were exposed to the irrigants and their viability was assessed after 1, 24, 48, and 72 h. The measurements were determined using WST-1 assay, using a micro ELISA re...

  2. Incipient cytotoxicity: A time-independent measure of cytotoxic potency in vitro.

    Science.gov (United States)

    Gülden, Michael; Kähler, Daria; Seibert, Hasso

    2015-09-01

    Time is an important determinant of toxicity but largely ignored in in vitro toxicity assays where exposure times chosen are rather arbitrary. To investigate the impact of time on the cytotoxic potency of chemicals in vitro, the concentration dependent cytotoxic action of selected chemicals (surfactants, metals, oxidative stressors, a mitochondrial poison) was determined after various exposure times (1-72 h) in cultures of Balb/c 3T3 cells. Time affected the cytotoxic potency as well as the cytotoxic efficacy. The median cytotoxic concentrations, EC50, decreased and in most cases approached an "incipient" value, EC50,∞, within 72 h. Cytotoxicity due to mitochondrial insult occurred after a threshold time which was dependent on the medium glucose concentration. Within the chemicals studied the extent of potency change with time ranged from 3- to >1000-fold and the "time to incipient cytotoxicity", tic, from 4 to >72 h. Hence, also the relative cytotoxic potencies depend on exposure time. Ignoring this may lead to severe bias in toxicological hazard and risk assessment. Therefore it is recommended to determine the incipient cytotoxic potency of chemical compounds, represented by, e.g., the incipient median effect (EC50,∞), no effect (NEC∞) or lowest effect concentrations (LEC∞) instead of measures obtained after arbitrary exposure times. If this is not possible, the 72 h-potency measurements appear to be useful surrogates. These time-independent incipient potency values can be reasonably compared between substances, endpoints, cells and biological test systems and may serve to define points of departure for quantitative in vitro-in vivo extrapolations.

  3. Polyclonal antibody against an insect excitatory toxin BmKIT from Buthus Martensii karsch and detection of BmKIT expressed in transgenic cotton

    Institute of Scientific and Technical Information of China (English)

    Hao Chanjuan; Xu Chenggang; Zhang Zhiyun; Liang Aihua

    2008-01-01

    An insect excitatory toxin gene from Buthus martensii Karsch (BmKIT) was cloned into the expression vector, pET-28a. BmKIT was expressed as inclusion bodies in Escherichia coli BL21 (DE3) host cells. The authenticity of in vitro expressed protein was confirmed by Western blot. The inclusion body protein band in SDS-PAGE was excised and the protein, BmKIT, was extracted. Polyclonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed BmKIT and was used to quantify its presence in transgenic cotton.

  4. Suppression of in vivo polyclonal IgE responses by monoclonal antibody to the lymphokine B-cell stimulatory factor 1.

    OpenAIRE

    Finkelman, F D; Katona, I M; Urban, J F; Snapper, C M; Ohara, J; Paul, W E

    1986-01-01

    The lymphokine B-cell stimulatory factor 1 (BSF-1) has been shown to greatly enhance the differentiation of lipopolysaccharide-activated B cells into IgG1- and IgE-secreting cells in vitro. To determine whether in vivo IgG1 and IgE antibody responses are BSF-1 dependent, the ability of a monoclonal rat IgG1 anti-BSF-1 antibody, 11B11, to affect polyclonal IgG1 and IgE production in mice infected with the nematode parasite Nippostrongylus brasiliensis or injected with a purified goat antibody ...

  5. Polyclonal anti-idiotypic antibodies mimicking the small cell lung carcinoma antigen cluster-5A interact with a panel of antibodies and induce specific immune response in animals.

    OpenAIRE

    Zwicky, C.; Stahel, R A; Jaksche, H.; Waibel, R.; Lehmann, H. P.; Loibner, H

    1991-01-01

    Polyclonal anti-idiotypic antibodies (ab2) were generated by immunising goats with the murine IgG2a monoclonal antibody SWA20 which recognises the SCLC antigen cluster-5A, a tumour-associated sialoglycoprotein. Ab2 was shown to bind specifically to antibody SWA20, but not to isotype matched control antibodies. Pre-incubation with ab2 completely inhibited target cell binding of antibody SWA20 and of four other antibodies to cluster-5A antigen, while no effect was seen with antibodies to cluste...

  6. Natural deep eutectic solvents: cytotoxic profile.

    Science.gov (United States)

    Hayyan, Maan; Mbous, Yves Paul; Looi, Chung Yeng; Wong, Won Fen; Hayyan, Adeeb; Salleh, Zulhaziman; Mohd-Ali, Ozair

    2016-01-01

    The purpose of this study was to investigate the cytotoxic profiles of different ternary natural deep eutectic solvents (NADESs) containing water. For this purpose, five different NADESs were prepared using choline chloride as a salt, alongside five hydrogen bond donors (HBD) namely glucose, fructose, sucrose, glycerol, and malonic acid. Water was added as a tertiary component during the eutectics preparation, except for the malonic acid-based mixture. Coincidentally, the latter was found to be more toxic than any of the water-based NADESs. A trend was observed between the cellular requirements of cancer cells, the viscosity of the NADESs, and their cytotoxicity. This study also highlights the first time application of the conductor-like screening model for real solvent (COSMO-RS) software for the analysis of the cytotoxic mechanism of NADESs. COSMO-RS simulation of the interactions between NADESs and cellular membranes' phospholipids suggested that NADESs strongly interacted with cell surfaces and that their accumulation and aggregation possibly defined their cytotoxicity. This reinforced the idea that careful selection of NADESs components is necessary, as it becomes evident that organic acids as HBD highly contribute to the increasing toxicity of these neoteric mixtures. Nevertheless, NADESs in general seem to possess relatively less acute toxicity profiles than their DESs parents. This opens the door for future large scale utilization of these mixtures. PMID:27386357

  7. Cytotoxicity Potentials of Eleven Bangladeshi Medicinal Plants

    Directory of Open Access Journals (Sweden)

    Amina Khatun

    2014-01-01

    Full Text Available Various forms of cancer are rising all over the world, requiring newer therapy. The quest of anticancer drugs both from natural and synthetic sources is the demand of time. In this study, fourteen extracts of different parts of eleven Bangladeshi medicinal plants which have been traditionally used for the treatment of different types of carcinoma, tumor, leprosy, and diseases associated with cancer were evaluated for their cytotoxicity for the first time. Extraction was conceded using methanol. Phytochemical groups like reducing sugars, tannins, saponins, steroids, gums, flavonoids, and alkaloids were tested using standard chromogenic reagents. Plants were evaluated for cytotoxicity by brine shrimp lethality bioassay using Artemia salina comparing with standard anticancer drug vincristine sulphate. All the extracts showed potent to moderate cytotoxicity ranging from LC50 2 to 115 µg/mL. The highest toxicity was shown by Hygrophila spinosa seeds (LC50=2.93 µg/mL and the lowest by Litsea glutinosa leaves (LC50=114.71 µg/mL in comparison with standard vincristine sulphate (LC50=2.04 µg/mL. Among the plants, the plants traditionally used in different cancer and microbial treatments showed highest cytotoxicity. The results support their ethnomedicinal uses and require advanced investigation to elucidate responsible compounds as well as their mode of action.

  8. Two Cytotoxic Eremophilanolides from Senecio tsoongianus

    Institute of Scientific and Technical Information of China (English)

    Qi Jun ZHANG; Hui DOU; Qun Xiong ZHENG; Chang Xin ZHOU; Zhao Jun XU; Hua PENG; Yu ZHAO

    2005-01-01

    Two new eremophilanolides, tsoongianolide E and F, were isolated from Senecio tsoongianus. Their structures were elucidated on the basis of NMR and MS spectra. Both of these two compounds showed in vitro cytotoxicity to cultured KB and A-549 cancer cell lines.

  9. Cytotoxicity potentials of eleven Bangladeshi medicinal plants.

    Science.gov (United States)

    Khatun, Amina; Rahman, Mahmudur; Haque, Tania; Rahman, Md Mahfizur; Akter, Mahfuja; Akter, Subarna; Jhumur, Afrin

    2014-01-01

    Various forms of cancer are rising all over the world, requiring newer therapy. The quest of anticancer drugs both from natural and synthetic sources is the demand of time. In this study, fourteen extracts of different parts of eleven Bangladeshi medicinal plants which have been traditionally used for the treatment of different types of carcinoma, tumor, leprosy, and diseases associated with cancer were evaluated for their cytotoxicity for the first time. Extraction was conceded using methanol. Phytochemical groups like reducing sugars, tannins, saponins, steroids, gums, flavonoids, and alkaloids were tested using standard chromogenic reagents. Plants were evaluated for cytotoxicity by brine shrimp lethality bioassay using Artemia salina comparing with standard anticancer drug vincristine sulphate. All the extracts showed potent to moderate cytotoxicity ranging from LC50 2 to 115 µg/mL. The highest toxicity was shown by Hygrophila spinosa seeds (LC50 = 2.93 µg/mL) and the lowest by Litsea glutinosa leaves (LC50 = 114.71 µg/mL) in comparison with standard vincristine sulphate (LC50 = 2.04 µg/mL). Among the plants, the plants traditionally used in different cancer and microbial treatments showed highest cytotoxicity. The results support their ethnomedicinal uses and require advanced investigation to elucidate responsible compounds as well as their mode of action. PMID:25431796

  10. Titanocene Difluorides with Improved Cytotoxic Activity

    OpenAIRE

    Eger, Silvia; Immel, Timo; Claffey, James; Müller-Bunz, Helge; Tacke, Matthias; Groth, Ulrich; Huhn, Thomas

    2010-01-01

    Titanocene difluorides can be obtained by halide metathesis of the respective titanocene dichlorides with trimethyltin fluoride (Me3SnF), giving access to a new class of cytotoxic active substances. Furthermore, an improved method for the synthesis of diaryl-substituted titanocene dichlorides is presented.

  11. Cytotoxicity of the rhizome of medicinal plants

    Institute of Scientific and Technical Information of China (English)

    Shakhawoat Hossain; Golam Kader; Farjana Nikkon; Tanzima Yeasmin

    2012-01-01

    Objective:To investigate the cytotoxicity of the crude ethanol extract of the rhizome of Zingiber zerumbet (Z. zerumbet) (L) Smith. and Curcuma zedoaria (C. zedoaria) Rosc. against Artemia salina Leach. Methods:Fresh rhizomes of Z. zerumbet (L) Smith. and C. zedoaria Rosc. were extracted separately in cold with ethanol (2.5 L) and after concentration a brownish syrupy suspension of ethanol extracts of Z. zerumbet (L) Smith. and C. zedoaria Rosc. was obtained. The cytotoxic effect of the crude ethanol extracts of both plants was determined by brine shrimp lethality bioassay. Results: Crude ethanol extracts of the rhizome of Z. zerumbet (L) Smith. showed the highest cytotoxicity (LC50 was 1.24μg/mL) against brine shrimp nauplii as compared with C. zedoaria Rosc. (LC50 was 33.593μg/mL) after 24 h of exposure. Conclusions:It can be concluded that the rhizome of Z. zerumbet (L) Smith. and C. zedoaria Rosc. can be used as a source of cytotoxic agent.

  12. Measuring mucosal damage induced by cytotoxic therapy.

    NARCIS (Netherlands)

    Blijlevens, N.M.A.; Land, B. van 't; Donnelly, J.P.; Rabet, L. M'; Pauw, B.E. de

    2004-01-01

    We scored oral mucositis and gut toxicity and measured sugar permeability testing among 56 recipients of a haematopoietic stem cell transplant (HSCT) given myeloablative conditioning with idarubicin, cyclophosphamide and TBI, and a group of 18 patients given cytotoxic chemotherapy for newly diagnose

  13. SYNTHESIS AND CYTOTOXICITY OF NOVEL LIGNANS

    NARCIS (Netherlands)

    Middel, O; Woerdenbag, H.J.; van Uden, W.; van Oeveren, A.; Jansen, J.F.G.A.; Feringa, B.L.; Konings, A.WT; Pras, N.; Kellogg, R.M

    1995-01-01

    In this study the syntheses of 11 novel lignans are described. Their cytotoxicities are studied in GLC(4), a human small cell lung carcinoma cell line, using the microculture tetrazolium (MTT) assay. Ten of these compounds were substituted with a menthyloxy group on the 5-position of the lactone. Th

  14. Biodistribution and radioimmunoimage of iodine-125 labeled anti-human ADAM15 polyclonal antibody in nude mice bearing human gastric carcinoma xenografts

    International Nuclear Information System (INIS)

    Objective: To investigate the biodistribution of Iodine-125 labeled anti-human ADAM15 polyclonal antibody in nude mice bearing human gastric carcinoma xenografts. Methods: The anti-human ADAM15 polyclonal antibody was labeled with I-125 using Chloramine-T method. The labeling efficiency and radiochemical purity of 125I-anti-ADAM15 antibody were measured. The SPECT planar imaging of nude mice bearing gastric carcinoma xenografts were performed at 1, 4, 8, 24, and 48 h post-injection and the biodistribution of 125I-anti-ADAM15 antibody was measured at 48 h after injection. Results: The labeling efficiency of 125I-anti-ADAM15 antibody was (75.16±9.43)% and its radiochemical purity was (99.44±0.21)% . Tumors could be cleanly visualized in SPECT planar images, and the radioactivity ratio of tumor to non-tumor tissue was 3.84±0.43 at 48 h post-injection. Conclusion: 125I-anti-ADAM15 antibody can target the gastric carcinoma in vivo, and provide good radioimmunoimages. (authors)

  15. CD4 T cell epitope specificity determines follicular versus non-follicular helper differentiation in the polyclonal response to influenza infection or vaccination

    Science.gov (United States)

    Knowlden, Zackery A. G.; Sant, Andrea J.

    2016-01-01

    Follicular helper T cells (Tfh) are essential for B cell production of high-affinity, class-switched antibodies. Much interest in Tfh development focuses on the priming environment of CD4 T cells. Here we explored the role that peptide specificity plays in the partitioning of the polyclonal CD4 T cell repertoire between Tfh and NonTfh lineages during the response to influenza. Surprisingly, we found that CD4 T cells specific for different epitopes exhibited distinct tendencies to segregate into Tfh or NonTfh. To alter the microenvironment and abundance, viral antigens were introduced as purified recombinant proteins in adjuvant as native proteins. Also, the most prototypical epitopes were expressed in a completely foreign protein. In many cases, the epitope-specific response patterns of Tfh vs. NonTfh persisted. The functional TcR avidity of only a subset of epitope-specific cells correlated with the tendency to drive a Tfh response. Thus, we conclude that in a polyclonal CD4 T cell repertoire, features of TcR-peptide:MHC class II complex have a strong deterministic influence on the ability of CD4 T cells to become a Tfh or a NonTfh. Our data is most consistent with at least 2 checkpoints of Tfh selection that include both TcR affinity and B cell presentation. PMID:27329272

  16. Multilineage polyclonal engraftment of Cal-1 gene-modified cells and in vivo selection after SHIV infection in a nonhuman primate model of AIDS.

    Science.gov (United States)

    Peterson, Christopher W; Haworth, Kevin G; Burke, Bryan P; Polacino, Patricia; Norman, Krystin K; Adair, Jennifer E; Hu, Shiu-Lok; Bartlett, Jeffrey S; Symonds, Geoff P; Kiem, Hans-Peter

    2016-01-01

    We have focused on gene therapy approaches to induce functional cure/remission of HIV-1 infection. Here, we evaluated the safety and efficacy of the clinical grade anti-HIV lentiviral vector, Cal-1, in pigtailed macaques (Macaca nemestrina). Cal-1 animals exhibit robust levels of gene marking in myeloid and lymphoid lineages without measurable adverse events, suggesting that Cal-1 transduction and autologous transplantation of hematopoietic stem cells are safe, and lead to long-term, multilineage engraftment following myeloablative conditioning. Ex vivo, CD4+ cells from transplanted animals undergo positive selection in the presence of simian/human immunodeficiency virus (SHIV). In vivo, Cal-1 gene-marked cells are evident in the peripheral blood and in HIV-relevant tissue sites such as the gastrointestinal tract. Positive selection for gene-marked cells is observed in blood and tissues following SHIV challenge, leading to maintenance of peripheral blood CD4+ T-cell counts in a normal range. Analysis of Cal-1 lentivirus integration sites confirms polyclonal engraftment of gene-marked cells. Following infection, a polyclonal, SHIV-resistant clonal repertoire is established. These findings offer strong preclinical evidence for safety and efficacy of Cal-1, present a new method for tracking protected cells over the course of virus-mediated selective pressure in vivo, and reveal previously unobserved dynamics of virus-dependent T-cell selection. PMID:26958575

  17. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Energy Technology Data Exchange (ETDEWEB)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  18. CD4 T cell epitope specificity determines follicular versus non-follicular helper differentiation in the polyclonal response to influenza infection or vaccination.

    Science.gov (United States)

    Knowlden, Zackery A G; Sant, Andrea J

    2016-01-01

    Follicular helper T cells (Tfh) are essential for B cell production of high-affinity, class-switched antibodies. Much interest in Tfh development focuses on the priming environment of CD4 T cells. Here we explored the role that peptide specificity plays in the partitioning of the polyclonal CD4 T cell repertoire between Tfh and NonTfh lineages during the response to influenza. Surprisingly, we found that CD4 T cells specific for different epitopes exhibited distinct tendencies to segregate into Tfh or NonTfh. To alter the microenvironment and abundance, viral antigens were introduced as purified recombinant proteins in adjuvant as native proteins. Also, the most prototypical epitopes were expressed in a completely foreign protein. In many cases, the epitope-specific response patterns of Tfh vs. NonTfh persisted. The functional TcR avidity of only a subset of epitope-specific cells correlated with the tendency to drive a Tfh response. Thus, we conclude that in a polyclonal CD4 T cell repertoire, features of TcR-peptide:MHC class II complex have a strong deterministic influence on the ability of CD4 T cells to become a Tfh or a NonTfh. Our data is most consistent with at least 2 checkpoints of Tfh selection that include both TcR affinity and B cell presentation. PMID:27329272

  19. Radiolabeled, nonspecific, polyclonal human immunoglobulin in the detection of focal inflammation by scintigraphy: Comparison with gallium-67 citrate and technetium-99m-labeled albumin

    International Nuclear Information System (INIS)

    The accumulation of nonspecific polyclonal human immunoglobulin (IgG) radiolabeled with 125I or 111In was compared to that of [67Ga]citrate and [99mTc]albumin in rats with deep thigh inflammation due to Escherichia coli infection. Serial scintigrams were acquired at 1, 3, 24, and in some cases, 48 hr after injection. As early as 3 hr postinjection, [111In]IgG showed greater accumulation at the lesion than [99mTc]HSA (p less than 0.01). Both [125I]IgG and [111In]IgG showed greater accumulation than [67Ga]citrate (p less than 0.01). At 24 hr, IgG image definition increased, while HSA image definition decreased, and the intensity of accumulation of both IgG preparations was greater than that of [67Ga]citrate or [99mTc]HSA (p less than 0.01). At all imaging times, [67Ga]citrate accumulation was surprisingly low. In inflammation produced by Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Candida albicans, or turpentine, [111In]IgG accumulation was similar to the results obtained with Escherichia coli. These studies suggest that focal sites of inflammation can be detected with radiolabeled nonspecific human polyclonal IgG

  20. Technetium-99m-human polyclonal IgG radiolabeled via the hydrazino nicotinamide derivative for imaging focal sites of infection in rats

    International Nuclear Information System (INIS)

    The biologic behavior of human polyclonal immunoglobulin (IgG) radiolabeled with technetium-99m (99mTc) by a novel method, via a nicotinyl hydrazine derivative, was evaluated in rats. Technetium-99m- and indium-111-IgG were co-administered to normal rats and biodistribution was determined at 2, 6, and 16 hr. The inflammation imaging properties of the two reagents were compared in rats with deep-thigh infection due to Escherichia coli. Blood clearance of both antibody preparations was well described by a bi-exponential function: (99mTc-IgG: t1/2 = 3.82 +/- 0.89 and 57.52 +/- 1.70 hr. 111In-IgG: 3.93 +/- 0.117 and 40.71 +/- 1.26 hr). Biodistributions in the solid organs were similar, however, small but statistically significant differences were detected: 99mTc-IgG greater than 111In-IgG in lung, liver, and spleen; 99mTc-IgG less than 111In-IgG in kidney and skeletal muscle (p less than 0.01). At all three imaging times, target-to-background ratio and percent residual activity for the two compounds were remarkably similar. These studies establish that human polyclonal IgG labeled with 99mTc via a nicotinyl hydrazine modified intermediate is equivalent to 111In-IgG for imaging focal sites of infection in experimental animals

  1. Preparation and Application of Polyclonal Antibody with a Peptide EDS1%EDS1多肽抗体的制备和应用

    Institute of Scientific and Technical Information of China (English)

    陈卓; 宋宝安; 刘开兴; 于丹丹; 刘家驹; 王贞超; 李向阳; 毕亮; 胡德禹; 杨松

    2011-01-01

    EDS1 is an important protein with regulation function at SA signal transduction pathway at plant host. In order to acquire polyclonal antibody of high titer and specificity against enhanced disease susceptibility 1 (EDS1), according to the primary structure of information about EDS1 in NCBI GenBank, 3 polypeptides with sequence-specific was obtained by using bioinformatics software contained Blastn,Blastx and Expasy. One polypeptide was synthetized by fmoc solid phase synthesis methods, and determined theirs purity and molecular weight by using HPLC and GC-MS, purity value reaching at 89.37% and molecular weight being at 1978.33. The polypeptide was coupled to keyhole limpet hemocyanin (KLH) to form a complex of Pep-KLH by us EDC. Anti-sera were acquired by immunizing rabbit with Pep-KLH emulsified by Complete Freund' s adjuvant (CFA) and Incomplete Freund' s adjuvant (IFA), and polyclonal antibody was purified by affinity chromatography. The titer and specificity of anti-sera and polyclonal antibody were determined by Indirect-ELISA and Western blotting. The results shown that anti-sera and polyclonal antibody reacted with Pep-KLH and detected a specific band of 70 kD, and the size was agreed with the predicted molecular mass. The EDS1 polyclonal antibody revealed high sensitivity and specificity.%EDS1蛋白是植物寄主SA信号转导通路中具有重要调控功能的蛋白,为获得效价高和选择性强的EDS1抗体,根据NCBI GenBank中报道的EDS1蛋白一级结构信息,采用Blastn、Blastx和ExPASy等生物信息学软件进行序列同源性分析,获得三段序列特异性较高的多肽,并从中优选一段序列特异性多肽,采用9-氟甲氧羰基固相合成法获得序列特异性最好的多肽,采用HPLC和GC-MS测定合成多肽的浓度和分子量,试验表明目的多肽纯度达89.37%,目的多肽分子量为1978.33.采用碳化二亚胺法将多肽与KLH进行偶联获得免疫原-Pep-KLH,并将其免疫新西兰大白兔以获

  2. Cytotoxic constituents of ethyl acetate fraction from Dianthus superbus.

    Science.gov (United States)

    Ding, Chengli; Zhang, Wu; Li, Jie; Lei, Jiachuan; Yu, Jianqing

    2013-01-01

    The ethyl acetate fraction (EE-DS) from Dianthus superbus was found to possess the cytotoxic activity against cancer cells in previous study. To investigate cytotoxic constituents, the bioassay-guided isolation of compounds from EE-DS was performed. Two dianthramides (1 and 2), three flavonoids (3-5), two coumarins (6 and 7) and three other compounds (8-10) were obtained. Structures of isolated compounds were identified by spectroscopic analysis. Cytotoxicity of the compounds against HepG2 cells was evaluated. Compound 1 showed the strongest cytotoxicity, compounds 10, 4, 3 and 5 had moderate cytotoxicity.

  3. African horse sickness virus serotype 4 antigens, VP1-1, VP2-2, VP4, VP7 and NS3, induce cytotoxic T cell responses in vitro.

    Science.gov (United States)

    Faber, F E; van Kleef, M; Tshilwane, S I; Pretorius, A

    2016-07-15

    It was shown in a previous study that proliferating CD8+ T cells could be detected in immune horse peripheral blood mononuclear cells (PBMC) when stimulated with African horse sickness virus serotype 4 (AHSV4). In this study the cytotoxicity of CD8+ T cells were tested by using the fluorescent antigen-transfected target cells-cytotoxic T lymphocytes (FATT-CTL) assay, for both the virus and its individual proteins expressed in Escherichia coli. This CTL assay measures the killing of viral protein expressing cells. AHSV proteins were successfully expressed in E. coli using the pET102/D-TOPO expression vector and the effector cells were stimulated with these recombinant proteins or with live viable virulent AHSV4. The AHSV genes were amplified and cloned into the pIRES-hrGFP II (pGFPempty) vector and these plasmid vectors encoding antigen-green fluorescent protein (GFP) fusion proteins were used to nucleofect PBMC, the target cells. The elimination of antigen-GFP expressing cells by CTL was quantified by flowcytometry. VP1-1, VP2-2, VP4, VP7 and NS3, antigen-specific CD8+ T cells resulted in cell lysis suggesting that CTL may play a role in the immune response induced against the AHSV4 vaccine strain. PMID:27063332

  4. Polyclonal Recipient nTregs Are Superior to Donor or Third-Party Tregs in the Induction of Transplantation Tolerance

    Directory of Open Access Journals (Sweden)

    Nina Pilat

    2015-01-01

    Full Text Available Induction of donor-specific tolerance is still considered as the “Holy Grail” in transplantation medicine. The mixed chimerism approach is virtually the only tolerance approach that was successfully translated into the clinical setting. We have previously reported successful induction of chimerism and tolerance using cell therapy with recipient T regulatory cells (Tregs to avoid cytotoxic recipient treatment. Treg therapy is limited by the availability of cells as large-scale expansion is time-consuming and associated with the risk of contamination with effector cells. Using a costimulation-blockade based bone marrow (BM transplantation (BMT model with Treg therapy instead of cytoreductive recipient treatment we aimed to determine the most potent Treg population for clinical translation. Here we show that CD4+CD25+ in vitro activated nTregs are superior to TGFβ induced iTregs in promoting the induction of chimerism and tolerance. Therapy with nTregs (but not iTregs led to multilineage chimerism and donor-specific tolerance in mice receiving as few as 0.5 × 106 cells. Moreover, we show that only recipient Tregs, but not donor or third-party Tregs, had a beneficial effect on BM engraftment at the tested doses. Thus, recipient-type nTregs significantly improve chimerism and tolerance and might be the most potent Treg population for translation into the clinical setting.

  5. Improved cytotoxicity testing of magnesium materials

    International Nuclear Information System (INIS)

    Metallic magnesium (Mg) and its alloys are highly suitable for medical applications as biocompatible and biodegradable implant materials. Magnesium has mechanical properties similar to bone, stimulates bone regeneration, is an essential non-toxic element for the human body and degrades completely within the body environment. In consequence, magnesium is a promising candidate as implant material for orthopaedic applications. Protocols using the guideline of current ISO standards should be carefully evaluated when applying them for the characterization of the cytotoxic potential of degradable magnesium materials. For as-cast material we recommend using 10 times more extraction medium than recommended by the ISO standards to obtain reasonable results for reliable cytotoxicity rankings of degradable materials in vitro. In addition primary isolated human osteoblasts or mesenchymal stem cells should be used to test magnesium materials.

  6. Molecular Mechanisms of Lymphocyte-Mediated Cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Zusen Fan; Qixiang Zhang

    2005-01-01

    Granule-mediated cytotoxicity is the major mechanism for lymphocytes to kill viruses, intracellular bacteria and tumors. The cytotoxic granules move to the immunological synapse by exocytosis after recognition of a killer cell.The contents of the granules are delivered into target cells with the help of perforin by endocytosis. A group of serine protease granzymes cleave their critical substrates to initiate DNA damage and cell death. The most abundant granzymes are granzyme A and B. They induce cell death through alternate and nonoverlapping pathways. The substrates and functions of the majority of the orphan granzymes have not yet been identified. It is possible that the diversity of granzymes provides fail-safe mechanisms for killing viruses and tumor cells.

  7. Improved cytotoxicity testing of magnesium materials

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Janine, E-mail: janine.fischer@hzg.de [Helmholtz-Zentrum Geesthacht, Institute of Materials Research, Department for Structural Research on Macromolecules, Max-Planck Str. 1, D - 21502 Geesthacht (Germany); Proefrock, Daniel [Helmholtz-Zentrum Geesthacht, Institute for Coastal Research, Department for Marine Bioanalytical Chemistry, Max-Planck Str. 1, D - 21502 Geesthacht (Germany); Hort, Norbert [Helmholtz-Zentrum Geesthacht, Institute of Materials Research, Department for Magnesium Processing, Max-Planck Str. 1, D - 21502 Geesthacht (Germany); Willumeit, Regine; Feyerabend, Frank [Helmholtz-Zentrum Geesthacht, Institute of Materials Research, Department for Structural Research on Macromolecules, Max-Planck Str. 1, D - 21502 Geesthacht (Germany)

    2011-06-25

    Metallic magnesium (Mg) and its alloys are highly suitable for medical applications as biocompatible and biodegradable implant materials. Magnesium has mechanical properties similar to bone, stimulates bone regeneration, is an essential non-toxic element for the human body and degrades completely within the body environment. In consequence, magnesium is a promising candidate as implant material for orthopaedic applications. Protocols using the guideline of current ISO standards should be carefully evaluated when applying them for the characterization of the cytotoxic potential of degradable magnesium materials. For as-cast material we recommend using 10 times more extraction medium than recommended by the ISO standards to obtain reasonable results for reliable cytotoxicity rankings of degradable materials in vitro. In addition primary isolated human osteoblasts or mesenchymal stem cells should be used to test magnesium materials.

  8. Cytotoxic diterpenoids from Rabdosia lophanthoides var. gerardianus.

    Science.gov (United States)

    Lin, Chao-Zhan; Zhao, Wei; Feng, Xiu-Li; Liu, Fang-Le; Zhu, Chen-Chen

    2016-03-01

    Two new abietane diterpenoids, Gerardianin B (1) and Gerardianin C (2), one new lignan glycoside, Gerardianin D (3) and one new lupane-type triterpenoid, Gerardianol A (4), together with seven known abietane diterpenoids were isolated from the aerial parts of Rabdosia lophanthoides var. gerardianus. Their structures were determined by 1D and 2D NMR spectroscopic data. The cytotoxic activities of the nine diterpenoids were evaluated on human cancer cell lines. Compounds 6-11 exhibited significant cytotoxic activities against HepG2 cell lines with IC50 from 4.68 to 9.43μM and HCF-8 cell lines with IC50 from 9.12 to 13.53μM. PMID:26608401

  9. Mycoplasma pneumoniae induces cytotoxic activity in guinea pig bronchoalveolar cells

    International Nuclear Information System (INIS)

    Precultured guinea pig alveolar macrophages (AM) and freshly harvested alveolar cells (FHAC) activated by interaction with Mycoplasma pneumoniae were cytotoxic for xenogeneic 75selenomethionine-labeled tumor target cells. Phagocytosis of whole opsonized or nonopsonized M. pneumoniae cells was more effective in eliciting cytotoxicity than uptake of sonicated microorganisms. The addition of living mycoplasma cells to the assay system enhanced the cytotoxic effect considerably. Target cells were significantly more susceptible to the cytotoxic action of phagocytes if they were coated with mycoplasma antigen or cocultured together with M. pneumoniae. The activation of the phagocytes could be inhibited by 2-deoxy-D-glucose but not by antimicrobial substances suppressing mycoplasma protein synthesis. It was accompanied by 51Cr release without detectable signs of cell damage. The supernatants of activated cells were cytotoxic for approximately 24 h. Inhibition, release, and cytotoxic activity indicate the necessity of an intact metabolism of the effector cells and suggest a secretion of cytotoxic substances

  10. Cytotoxic Terpene Quinones from Marine Sponges

    OpenAIRE

    Marina Gordaliza

    2010-01-01

    The 1,4-benzoquinone moiety is a common structural feature in a large number of compounds that have received considerable attention owing to their broad spectrum of biological activities. The cytotoxic and antiproliferative properties of many natural sesquiterpene quinones and hydroquinones from sponges of the order Dictyoceratida, such as avarol, avarone, illimaquinone, nakijiquinone and bolinaquinone, offer promising opportunities for the development of new antitumor agents. The present rev...

  11. Novel cytotoxic annonaceous acetogenins from Annona muricata.

    Science.gov (United States)

    Chang, F R; Wu, Y C

    2001-07-01

    Seven new annonaceous acetogenins, muricins A-G (1-7), as well as five known compounds, a mixture of muricatetrocin A (8) and muricatetrocin B (9), longifolicin (10), corossolin (11), and corossolone (12), were isolated from the seeds of Annona muricata. The structures of all isolates were elucidated and characterized by spectral and chemical methods. These acetogenins showed significantly selective in vitro cytotoxicities toward the human hepatoma cell lines Hep G(2) and 2,2,15.

  12. Four new cytotoxic xanthones from Garcinia nujiangensis.

    Science.gov (United States)

    Tang, Zhong-Yan; Xia, Zheng-Xiang; Qiao, Shi-Ping; Jiang, Chao; Shen, Guo-Rong; Cai, Mei-Xiang; Tang, Xiao-Yan

    2015-04-01

    Bioassay-guided fractionation of the acetone extract of the twigs of Garcinia nujiangensis resulted in the isolation of four new prenylated xanthones, nujiangexanthones C-F (1-4), and ten known related analogues. The structures of compounds 1-4 were elucidated by interpretation of their spectroscopic data. The compounds isolated were evaluated for their cytotoxic effects against three cancer cell lines, the test substances demonstrated selectivity toward the cancer cells. PMID:25727735

  13. Cytotoxic Polyisoprenyl Benzophenonoids from Garcinia subelliptica

    OpenAIRE

    Zhang, Li-Jie; Chiou, Chun-Tang; Cheng, Jing-Jy; Huang, Hui-Chi; Yang Kuo, Li-Ming; Liao, Chia-Chin; Bastow, Kenneth F.; Lee, Kuo-Hsiung; Kuo, Yao-Haur

    2010-01-01

    Six new polyisoprenyl benzophenonoids, (±)-garcinialiptone A (1, 2), garcinialiptone B (3), (−)-cycloxanthochymol (4), garcinialiptone C (5), and garcinialiptone D (6), along with three known compounds, xanthochymol (7), isoxanthochymol (8), and cycloxanthochymol (9), were isolated from the fruits of Garcinia subelliptica. The structures of 1–6 were elucidated by spectroscopic analysis. Biological evaluation showed that all compounds 1–9 exhibited cytotoxic activity against a small panel of h...

  14. Cytotoxicity Potentials of Eleven Bangladeshi Medicinal Plants

    OpenAIRE

    Amina Khatun; Mahmudur Rahman; Tania Haque; Md. Mahfizur Rahman; Mahfuja Akter; Subarna Akter; Afrin Jhumur

    2014-01-01

    Various forms of cancer are rising all over the world, requiring newer therapy. The quest of anticancer drugs both from natural and synthetic sources is the demand of time. In this study, fourteen extracts of different parts of eleven Bangladeshi medicinal plants which have been traditionally used for the treatment of different types of carcinoma, tumor, leprosy, and diseases associated with cancer were evaluated for their cytotoxicity for the first time. Extraction was conceded using methano...

  15. Mutagenic and cytotoxic activities of benfuracarb insecticide.

    Science.gov (United States)

    Eren, Yasin; Erdoğmuş, Sevim Feyza; Akyıl, Dilek; Özkara, Arzu

    2016-08-01

    Benfuracarb is a carbamate insecticide used to control insect pests in vegetables and it has anti-acetylcholinesterase activity lower than other carbamates. Cytotoxic effects of benfuracarb were evaluated by using root growth inhibition (EC50), mitotic index (MI), and mitotic phase determinations on the root meristem cells of Allium cepa and mutagenic effects were determined in Salmonella typhymurium Ames test by TA98 and TA100 strains with and without metabolic activation. In Allium test, 1 % DMSO was used as negative control group and 10 ppm MMS was used as positive control group. 75 ppm concentration of benfuracarb was found as EC50. In MI and mitotic phases determination study, 37.5, 75 and 150 ppm doses of benfuracarb were used. Dose-dependent cytotoxic activity was found by root growth inhibition and MI studies. It was identified that mitotic inhibition activity of benfuracarb was higher than 10 ppm MMS. In Ames test, mutagenic activity was not observed and over 200 µg/plate of benfuracarb was determined as cytotoxic to S. typhymurium strains. Benfuracarb can be called as "mitotic inhibitor" but not called as mutagen. PMID:25381170

  16. Initial cytotoxicity of novel titanium alloys.

    Science.gov (United States)

    Koike, M; Lockwood, P E; Wataha, J C; Okabe, T

    2007-11-01

    We assessed the biological response to several novel titanium alloys that have promising physical properties for biomedical applications. Four commercial titanium alloys [Super-TIX(R) 800, Super-TIX(R) 51AF, TIMETAL(R) 21SRx, and Ti-6Al-4V (ASTM grade 5)] and three experimental titanium alloys [Ti-13Cr-3Cu, Ti-1.5Si and Ti-1.5Si-5Cu] were tested. Specimens (n = 6; 5.0 x 5.0 x 3.0 mm(3)) were cast in a centrifugal casting machine using a MgO-based investment and polished to 600 grit, removing 250 mum from each surface. Commercially pure titanium (CP Ti: ASTM grade 2) and Teflon (polytetrafluoroethylene) were used as positive controls. The specimens were cleaned and disinfected, and then each cleaned specimen was placed in direct contact with Balb/c 3T3 fibroblasts for 72 h. The cytotoxicity [succinic dehydrogenase (SDH) activity] of the extracts was assessed using the MTT method. Cytotoxicity of the metals tested was not statistically different compared to the CP Ti and Teflon controls (p > 0.05). These novel titanium alloys pose cytotoxic risks no greater than many other commonly used alloys, including commercially pure titanium. The promising short-term biocompatibility of these Ti alloys is probably due to their excellent corrosion resistance under static conditions, even in biological environments. PMID:17385227

  17. Iron oxide nanoparticle enhancement of radiation cytotoxicity

    Science.gov (United States)

    Mazur, Courtney M.; Tate, Jennifer A.; Strawbridge, Rendall R.; Gladstone, David J.; Hoopes, P. Jack

    2013-02-01

    Iron oxide nanoparticles (IONPs) have been investigated as a promising means for inducing tumor cell-specific hyperthermia. Although the ability to generate and use nanoparticles that are biocompatible, tumor specific, and have the ability to produce adequate cytotoxic heat is very promising, significant preclinical and clinical development will be required for clinical efficacy. At this time it appears using IONP-induced hyperthermia as an adjunct to conventional cancer therapeutics, rather than as an independent treatment, will provide the initial IONP clinical treatment. Due to their high-Z characteristics, another option is to use intracellular IONPs to enhance radiation therapy without excitation with AMF (production of heat). To test this concept IONPs were added to cell culture media at a concentration of 0.2 mg Fe/mL and incubated with murine breast adenocarcinoma (MTG-B) cells for either 48 or 72 hours. Extracellular iron was then removed and all cells were irradiated at 4 Gy. Although samples incubated with IONPs for 48 hrs did not demonstrate enhanced post-irradiation cytotoxicity as compared to the non-IONP-containing cells, cells incubated with IONPs for 72 hours, which contained 40% more Fe than 48 hr incubated cells, showed a 25% decrease in clonogenic survival compared to their non-IONP-containing counterparts. These results suggest that a critical concentration of intracellular IONPs is necessary for enhancing radiation cytotoxicity.

  18. Initial cytotoxicity of novel titanium alloys.

    Science.gov (United States)

    Koike, M; Lockwood, P E; Wataha, J C; Okabe, T

    2007-11-01

    We assessed the biological response to several novel titanium alloys that have promising physical properties for biomedical applications. Four commercial titanium alloys [Super-TIX(R) 800, Super-TIX(R) 51AF, TIMETAL(R) 21SRx, and Ti-6Al-4V (ASTM grade 5)] and three experimental titanium alloys [Ti-13Cr-3Cu, Ti-1.5Si and Ti-1.5Si-5Cu] were tested. Specimens (n = 6; 5.0 x 5.0 x 3.0 mm(3)) were cast in a centrifugal casting machine using a MgO-based investment and polished to 600 grit, removing 250 mum from each surface. Commercially pure titanium (CP Ti: ASTM grade 2) and Teflon (polytetrafluoroethylene) were used as positive controls. The specimens were cleaned and disinfected, and then each cleaned specimen was placed in direct contact with Balb/c 3T3 fibroblasts for 72 h. The cytotoxicity [succinic dehydrogenase (SDH) activity] of the extracts was assessed using the MTT method. Cytotoxicity of the metals tested was not statistically different compared to the CP Ti and Teflon controls (p > 0.05). These novel titanium alloys pose cytotoxic risks no greater than many other commonly used alloys, including commercially pure titanium. The promising short-term biocompatibility of these Ti alloys is probably due to their excellent corrosion resistance under static conditions, even in biological environments.

  19. Antibody-Dependent Cell-Mediated Cytotoxicity to Hemagglutinin of Influenza A Viruses After Influenza Vaccination in Humans.

    Science.gov (United States)

    Zhong, Weimin; Liu, Feng; Wilson, Jason R; Holiday, Crystal; Li, Zhu-Nan; Bai, Yaohui; Tzeng, Wen-Pin; Stevens, James; York, Ian A; Levine, Min Z

    2016-04-01

    Background.  Detection of neutralizing antibodies (nAbs) to influenza A virus hemagglutinin (HA) antigens by conventional serological assays is currently the main immune correlate of protection for influenza vaccines However, current prepandemic avian influenza vaccines are poorly immunogenic in inducing nAbs despite considerable protection conferred. Recent studies show that Ab-dependent cell-mediated cytotoxicity (ADCC) to HA antigens are readily detectable in the sera of healthy individuals and patients with influenza infection. Methods.  Virus neutralization and ADCC activities of serum samples from individuals who received either seasonal or a stock-piled H5N1 avian influenza vaccine were evaluated by hemagglutination inhibition assay, microneutralization assay, and an improved ADCC natural killer (NK) cell activation assay. Results.  Immunization with inactivated seasonal influenza vaccine led to strong expansion of both nAbs and ADCC-mediating antibodies (adccAbs) to H3 antigen of the vaccine virus in 24 postvaccination human sera. In sharp contrast, 18 individuals vaccinated with the adjuvanted H5N1 avian influenza vaccine mounted H5-specific antibodies with strong ADCC activities despite moderate virus neutralization capacity. Strength of HA-specific ADCC activities is largely associated with the titers of HA-binding antibodies and not with the fine antigenic specificity of anti-HA nAbs. Conclusions.  Detection of both nAbs and adccAbs may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines. PMID:27419174

  20. Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity.

    Science.gov (United States)

    Sutton, Vivien R; Brennan, Amelia J; Ellis, Sarah; Danne, Jill; Thia, Kevin; Jenkins, Misty R; Voskoboinik, Ilia; Pejler, Gunnar; Johnstone, Ricky W; Andrews, Daniel M; Trapani, Joseph A

    2016-03-01

    The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means. PMID:26756195

  1. Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity.

    Science.gov (United States)

    Sutton, Vivien R; Brennan, Amelia J; Ellis, Sarah; Danne, Jill; Thia, Kevin; Jenkins, Misty R; Voskoboinik, Ilia; Pejler, Gunnar; Johnstone, Ricky W; Andrews, Daniel M; Trapani, Joseph A

    2016-03-01

    The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.

  2. Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain

    DEFF Research Database (Denmark)

    Vanwolleghem, Thomas; Bukh, Jens; Meuleman, Philip;

    2008-01-01

    G can prevent a de novo HCV infection in vivo and contribute to the control of viremia in infected individuals. We addressed this question with homologous in vivo protection studies in human liver-urokinase-type plasminogen activator (uPA)(+/+) severe combined immune deficient (SCID) mice. Chimeric mice...... were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV...... infection was attenuated, as evidenced by altered viral kinetics in comparison with five control IgG-treated animals. Plasma samples obtained from the mice at viral challenge neutralized H77C-HCVpp at dilutions as high as 1/400. Infection was completely prevented when, before administration to naïve...

  3. Killing of Brucella antigen-sensitized macrophages by T lymphocytes in bovine brucellosis.

    Science.gov (United States)

    Wyckoff, John H; Potts, Richard D

    2007-12-15

    The present study was an investigation into the role of T lymphocytes in the killing of antigen-sensitized macrophages (M Phi) in bovine brucellosis. Following confirmation of bovine T lymphocyte cell lines derived from Brucella abortus Strain 19 vaccinated steers as antigen-specific in proliferation studies using various antigens, we adapted an apoptosis assay for evaluation of cytotoxicity by these bovine T cells against autologous monocyte-derived macrophages (MDM Phi) as target cells. Various B. abortus antigen preparations were tested including whole gamma-irradiated B. abortus bacteria (gamma BA), a soluble cytosolic protein fraction and a membrane-associated protein fraction. Both polyclonal and cloned T lymphocyte cell lines exhibited cytotoxicity against MDM Phi targets in an antigen-specific fashion. Polyclonal and cloned T lymphocyte cell lines demonstrated cytotoxic responses to varying degrees against B. abortus antigens regardless of whether the antigen used was whole nonviable bacteria, a soluble protein extract or a membrane-associated fraction of extracted bacteria. To further develop correlation of these responses to an in vivo host defense mechanism, cytotoxicity was evaluated using target cells that had been infected with live B. abortus S19 or B. abortus Strain 2308. Cytotoxic responses were also demonstrated consistently against infected targets with either strain of B. abortus although in most cases, cytotoxicity was higher against target cells sensitized with gamma BA compared to those infected with live bacteria. Cloned T lymphocyte cell lines were all CD4+, CD8(-) cells indicating that the observed cytotoxic responses were most likely due to an inflammatory Th1 response and may represent an important host defense mechanism induced by vaccination with live attenuated strains of B. abortus in cattle. PMID:17904229

  4. The spread of Mycoplasma pneumoniae is polyclonal in both an endemic setting in France and in an epidemic setting in Israel.

    Directory of Open Access Journals (Sweden)

    Sabine Pereyre

    Full Text Available Mycoplasma pneumoniae infections occur both endemically and epidemically, and macrolide resistance has been spreading for 10 years worldwide. A substantial increased incidence of M. pneumoniae infections has been reported in several countries since 2010. Whether this increased incidence is attributed to different or to the same M. pneumoniae genotype is unknown. We have developed a multilocus variable-number tandem-repeat (VNTR analysis (MLVA for the molecular typing of M. pneumoniae isolates. In this study, the MLVA typing method was modified and validated to be applicable directly to respiratory tract specimens without culture. This method was applied to 34 M. pneumoniae-positive specimens received at the Bordeaux Hospital, France, between 2007 and 2010 in an endemic setting, and to 63 M. pneumoniae-positive specimens collected during an epidemic surge of M. pneumoniae infections in 2010 in Jerusalem, Israel. The M. pneumoniae endemic spread was shown to be polyclonal in France, with 15 MLVA types identified. Strikingly, the Israeli epidemic surge was also a multi-clonal phenomenon, with 18 circulating MLVA types. The macrolide resistance-associated substitution, A2058G, was found in 22% of the Israeli patients. Macrolide-resistant M. pneumoniae belonged to four MLVA types, the MLVA type Z being the most frequent one. An association between the MLVA type Z and macrolide resistance might exist since macrolide resistance was present or generated during the course of illness in all patients infected with this MLVA type. In conclusion, the discriminatory power of the MLVA showed that the spread of M. pneumoniae strains in France in an endemic setting was polyclonal as well as the surge of M. pneumoniae infections in Israel in 2010.

  5. Preparation of the Polyclonal Antibody against Ochratoxin A%抗赭曲霉毒素A多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    高璟瑜; 杨扬; 张红星; 刘艳华; 畅晓辉; 李丹

    2011-01-01

    为了制备赭曲霉毒素A多克隆抗体,采用戊二醛法合成赭曲霉毒素A-BSA人工抗原,通过紫外扫描法鉴定半抗原与载体蛋白偶联效果.合成的人工抗原用弗氏佐剂乳化后免疫Balb/c小白鼠,4次免疫后眼球下缘采血,分离血清.试验结果表明,免疫后产生了抗赭曲霉毒素A-BSA的特异性多克隆抗体,抗体效价为1:8100,赭曲霉毒素A的最低检出限为9ng/mL.抗赭曲霉毒素A多克隆抗体的制备为开发赭曲霉毒素A的ELISA酶联免疫试剂盒奠定了基础.%To prepare polyclonal antibody against ochratoxin A, artificial antigen of ochratoxin A-BSA were coupled by glutaraldehyde method. The couple effection between semiantigen and carrier protein was identified through ultraviolet spectrum scanning method. The synthetical antigen ochratoxin A-BSA was mixed with the same amount adjuvant and then immunized mouse. After the fourth immunize, the mouse blood was collected from heart. and blood sera was seDarated. The eXDeriment results showed that the SDecifiC Dolvclonal antibody against ochratoxin A was generated after immunity. The titer of anti-sera was 1∶8100. The detection limit of ochratoxin A was 9 ng/mL.Preparation of the polyclonal antibody against ochratoxin A established the groundwork for further research of ochratoxin A immunoassay kit.

  6. Evaluation of the inflammatory activity in chronic osteomyelitis. Contribution of the scintigraphy with polyclonal antibodies; Avaliacao de atividade inflamatoria em osteomielite cronica. Contribuicao da cintilografia com anticorpos policlonais

    Energy Technology Data Exchange (ETDEWEB)

    Sapienza, Marcelo Tatit

    1996-07-01

    Active chronic osteomyelitis or complicating osteomyelitis (superimposed on diseases that changes the normal bone structure fractures, post-surgery, prosthesis) can be difficult to diagnose by anatomic radiological imaging modalities, like plain radiograph and CT. These diseases frequently cause also increased bone remodeling, leading to nonspecific uptake of Tc-99m-bone scan agents and gallium-67. New radiopharmaceuticals with greater inflammation/infection avidity and specificity are being developed, including the nonspecific polyclonal immunoglobulin (IgG) labeled with technetium-99. Tc-99m-IgG may be available as a ready to use kit, with no reported side effects, low patient absorbed radiation dose and low cost. The mechanism of IgG uptake at the inflammation site has not been fully elucidated yet. Specific (receptor linking, physico-chemical immunoglobulin properties) and nonspecific mechanisms (enhanced vascular permeability and macromolecular exudate) has been suggested. IgG scintigraphy results are affected by the isotope, labeling procedure adopted and characteristics of the inflammatory focus. Nineteen patients with suspected osteomyelitis (active chronic osteomyelitis or violated bone osteomyelitis) were studied by Tc-99m-IgG scintigraphy (directly labeled polyclonal immunoglobulin, Sandoglobuilina - Sandoz). All patients also underwent standard three-phase bone scintigraphy using methylene diphosphonate (Tc-99m-MDP), gallium-67 scintigraphy and plain radiographs. Infection was found in 8 sites. Sensitivity and specificity for Tc-99m-MDP, gallium-67 and Tc 99m-IgG scintigraphy were, respectively, 88 and 36%, 75 and 73%,88 and 82%. All patients with false positive IgG scintigraphies had previous surgery. Other current scintigraphic procedures used in the diagnosis of osteomyelitis are also reviewed. (author)

  7. Preparation of Polyclonal Antibody against Crystalline Inclusion Protein of Entomopathogenic Bacterium%昆虫病原细菌Cip蛋白多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    游娟; 黄建林; 曹莉; 韩日畴

    2013-01-01

    Entomopathogenic Photorhabdus bacteria produce two types of intracellular crystalline inclusion proteins,CipA and CipB,as the nutrient resource for the symbiotic nematode.Based on constructed high-level prokaryotic expression system,recombinant Cip proteins were purified with a warm extract method and used as the antigens to immune New Zealand rabbits,respectively.The polyclonal antibodies with the titers of 1:6400 were raised and monitored by indirect enzyme-linked immunosorbent assay (ELISA).Western blot assay confirmed that the antibodies could specifically recognize the inclusion proteins in Photorhabdus bacteria.Preparation of Cip polyclonal antibodies will be helpful to develop the immunological assay method of these proteins.%昆虫病原发光杆菌属(Photorhabdus)细菌产生2种胞内晶体蛋白CipA和CipB,为其共生线虫的生长发育提供营养.基于Cip蛋白已在大肠杆茵原核表达系统中得到稳定且高水平表达,以纯化的重组Cip蛋白为抗原,免疫新西兰大耳兔制备多克隆抗体,ELISA测定2种蛋白抗血清效价,均可达到1∶6400.Western blot分析显示,制备的抗体均可特异识别发光杆菌的胞内晶体蛋白.Cip蛋白多克隆抗体的制备为建立该类蛋白的免疫学检测方法奠定了基础.

  8. Production of polyclonal antibody against Tehran strain influenza virus (A/H1N1/2009 hemagglutinin conserved domain (HA2: brief report

    Directory of Open Access Journals (Sweden)

    Somayeh Zamani

    2015-10-01

    Full Text Available Background: The influenza virus is one of the most important factors for higher morbidity and mortality in the world. Recently, researchers have been focused on influenza conserved antigenic proteins such as hemagglutinin stalk domain (HA2 for vaccine production and serological studies. The HA2 plays a major role in the fusion of the virus with host cells membrane. The immunity system enables to produce antibody against HA2. The aim of this study is polyclonal antibody production against influenza HA2. Methods: This study was done in the Influenza Research Lab, Pasteur Institute of Iran, Tehran for one year from September 2013 to October 2014. In the present study, recombinant HA2 protein was produced in prokaryotic system and purified using Nickel affinity chromatography. The purified HA2 was mixed with Freund’s adjuvant (complete and incomplete and injected into two New Zealand white rabbits by intramuscularly and subcutaneously routes. Immunization was continued for several months with two weeks interval. Before each immunization, blood was drawn by venous puncture from the rabbit ear. Function of rabbit's sera was evaluated using radial immunodiffusion (RID in both forms, Single RID (SRID and Double RID (DRID. Finally, antiserum activity against HA2 was evaluated using western blotting as serological assay. Results: Sedimentary line and zone was observed in RID assays (SRID and DRID represent interaction between HA2 protein and anti- HA2 antibody. As well as, western blotting results was positive for HA2 protein. Therefore, these results showed that polyclonal antibody produced against HA2 protein can identify HA2 protein antigenic sites. Conclusion: These findings show that humoral immune responses have properly been stimulated in rabbits and these antibodies can identify HA2 protein and may be suitable for other serological methods.

  9. Polyclonal infections due to Mycobacterium avium complex in patients with AIDS detected by pulsed-field gel electrophoresis of sequential clinical isolates.

    Science.gov (United States)

    Slutsky, A M; Arbeit, R D; Barber, T W; Rich, J; von Reyn, C F; Pieciak, W; Barlow, M A; Maslow, J N

    1994-01-01

    Invasive infection with organisms of the Mycobacterium avium complex (MAC) is common among patients with advanced human immunodeficiency virus infection. In previous studies, we analyzed multiple individual colonies of MAC isolated from specimens obtained at the same time and observed that 14 to 20% of patients are simultaneously infected with more than one strain. In this study, we examined sequential isolates from 12 patients with AIDS who had two or more MAC isolates available from clinical specimens collected more than 1 week apart; the intervals between the first and last specimens ranged from 8 to 192 (median, 46) days. For each isolate, restriction digests of genomic DNA were analyzed by pulsed-field gel electrophoresis; DNA was prepared by using a protocol, described here in detail, which had been optimized for conditions of bacterial growth and lysis. The pulsed-field gel electrophoresis analysis identified four patients (33%) infected with two different MAC strains. Both M. avium and M. intracellulare were cultured from blood specimens from two patients. In each of the four patients, the second strain was identified from a culture taken within 14 days of the initial study isolate, and in three of these patients, the first strain was detected again in a subsequent culture. These observations suggest that the presence of two different strains among isolates from sequential cultures may reflect ongoing polyclonal infection. We conclude that polyclonal infection with MAC is common among patients with AIDS. The identification of such infections may be critical in the development of effective treatments. Images PMID:7929773

  10. Cytotoxic activities of several geranyl-substituted flavanones.

    Science.gov (United States)

    Smejkal, Karel; Svacinová, Jana; Slapetová, Tereza; Schneiderová, Kristýna; Dall'acqua, Stefano; Innocenti, Gabbriella; Závalová, Veronika; Kollár, Peter; Chudík, Stanislav; Marek, Radek; Julínek, Ondrej; Urbanová, Marie; Kartal, Murat; Csöllei, Marek; Dolezal, Karel

    2010-04-23

    Nine geranylated flavanones isolated from the fruits of Paulownia tomentosa (4-12) and two from the roots of Morus alba (13 and 14) were examined for cytotoxicity to selected human cancer cell lines and normal human fibroblasts. Cytotoxicity was determined in vitro using a calcein AM cytotoxicity assay. Cytotoxicity for the THP-1 monocytic leukemia cell line was tested using erythrosin B cell staining. The geranylated compounds tested were compared with the known simple flavanone standards taxifolin (1), naringenin (2), and hesperetin (3) and with the standard anticancer drugs olomoucine II, diaziquone, and oxaliplatin and the antineoplastic compound camptothecin, and showed different levels of cytotoxicity. The effects of structural changes on cytotoxic activity, including geranyl substitution of the flavanone skeleton and the oxidation pattern of ring B of the flavanones, are discussed. PMID:20192247

  11. Cytotoxic phorbol esters of Croton tiglium.

    Science.gov (United States)

    Zhang, Xiao-Long; Wang, Lun; Li, Fu; Yu, Kai; Wang, Ming-Kui

    2013-05-24

    Chemical investigation of the seeds of Croton tiglium afforded eight new phorbol diesters (three phorbol diesters, 1-3, and five 4-deoxy-4α-phorbol diesters, 4-8), together with 11 known phorbol diesters (nine phorbol diesters, 9-17, and two 4-deoxy-4α-phorbol diesters, 18 and 19). The structures of compounds 1-8 were determined by spectroscopic data information and chemical degradation experiments. The cytotoxic activities of the phorbol diesters were evaluated against the SNU387 hepatic tumor cell line, and compound 3 exhibited the most potent activity (IC50 1.2 μM). PMID:23701597

  12. New Cytotoxic Tigliane Diterpenoids from Croton caudatus.

    Science.gov (United States)

    Chen, Ying-Ying; Yang, Kun-Xian; Yang, Xing-Wei; Khan, Afsar; Liu, Lu; Wang, Bei; Zhao, Yun-Li; Liu, Ya-Ping; Li, Yan; Luo, Xiao-Dong

    2016-05-01

    Three new tigliane-type diterpenoids were isolated from the methanolic extract of the twigs and leaves of Croton caudatus, trivially named crotusins A-C (1-3). The structures of compounds 1-3 were elucidated on the basis of extensive spectral methods. These new compounds were highly oxygenated and heavily substituted. Cytotoxic activity against five human tumor cell lines was assessed for compounds 1-3 of which compound 3 showed significant inhibitory activity with IC50 values ranging from 0.49 to 4.19 µM against these cells, while crotusins A and B exhibited moderate activity. PMID:27002392

  13. Cytotoxic phorbol esters of Croton tiglium.

    Science.gov (United States)

    Zhang, Xiao-Long; Wang, Lun; Li, Fu; Yu, Kai; Wang, Ming-Kui

    2013-05-24

    Chemical investigation of the seeds of Croton tiglium afforded eight new phorbol diesters (three phorbol diesters, 1-3, and five 4-deoxy-4α-phorbol diesters, 4-8), together with 11 known phorbol diesters (nine phorbol diesters, 9-17, and two 4-deoxy-4α-phorbol diesters, 18 and 19). The structures of compounds 1-8 were determined by spectroscopic data information and chemical degradation experiments. The cytotoxic activities of the phorbol diesters were evaluated against the SNU387 hepatic tumor cell line, and compound 3 exhibited the most potent activity (IC50 1.2 μM).

  14. Mitigation of quantum dot cytotoxicity by microencapsulation.

    Directory of Open Access Journals (Sweden)

    Amelia Romoser

    Full Text Available When CdSe/ZnS-polyethyleneimine (PEI quantum dots (QDs are microencapsulated in polymeric microcapsules, human fibroblasts are protected from acute cytotoxic effects. Differences in cellular morphology, uptake, and viability were assessed after treatment with either microencapsulated or unencapsulated dots. Specifically, QDs contained in microcapsules terminated with polyethylene glycol (PEG mitigate contact with and uptake by cells, thus providing a tool to retain particle luminescence for applications such as extracellular sensing and imaging. The microcapsule serves as the "first line of defense" for containing the QDs. This enables the individual QD coating to be designed primarily to enhance the function of the biosensor.

  15. Identification of HLA-A*2402-restricted HCMV immediate early-1 (IE-1 epitopes as targets for CD8+ HCMV-specific cytotoxic T lymphocytes

    Directory of Open Access Journals (Sweden)

    Lee Sang-Guk

    2009-08-01

    Full Text Available Abstract Background To identify novel HLA-A*2402-restricted human cytomegalovirus (HCMV immediate early-1 (IE-1 epitopes for adoptive immunotherapy, we explored 120 overlapping 15-amino acid spanning IE-1. Methods These peptides were screened by measuring the frequency of polyclonal CD8+ T cells producing intracellular interferon-γ (IFN-γ using flow cytometry and the epitopes were validated with a HCMV-infected target Cr release cytotoxicity assay. Results Initial screening was performed with 12 mini-pools of 10 consecutive peptides made from 120 overlapping peptides15-amino acids in length that spanned IE-1. When peripheral blood mononuclear cells (PBMCs from HLA-A*2402 HCMV-seropositive donors were sensitized with each of the 12 mini-pools, mini-pools 1 and 2 induced the highest frequency of CD8+ cytotoxic T lymphocytes (CTLs producing IFN-γ. When PBMCs were stimulated with each of the twenty peptides belonging to mini-pools 1 and 2, peptides IE-11–15MESSAKRKMDPDNPD and IE-15–19AKRKMDPDNPDEGPS induced the greatest quantities of IFN-γ production and cytotoxicity of HLA-matched HCMV-infected fibroblasts. To determine the exact HLA-A*2402-restricted epitopes within the two IE-1 proteins, we synthesized a total of twenty-one overlapping 9- or 10 amino acid peptides spanning IE-11–15 and IE-15–19. Peptide IE-13–12SSAKRKMDPD induced the greatest quantities of IFN-γ production and target cell killing by CD8+ CTLs. Conclusion HCMV IE-13–12SSAKRKMDPD is a HLA-A*2402-restricted HCMV IE-1 epitope that can serve as a common target for CD8+ HCMV-specific CTLs.

  16. Cytotoxic agents are detrimental to bone formed by distraction osteogenesis

    OpenAIRE

    Monsell, Fergal P.; Barnes, James Ralph; Bellemore, M. C.; Biston, L.; Goodship, Allen

    2013-01-01

    Distraction osteogenesis can be used to replace segmental bone loss when treating malignant bone tumors in children and adolescents. These patients often receive cytotoxic chemotherapy as part of their treatment regimen. The effect of cytotoxic drugs on the cellular processes during distraction osteogenesis and the structural and mechanical properties of regenerate bone is unknown. We therefore used a rabbit model of distraction osteogenesis to determine that cytotoxic agents had a detrimenta...

  17. NOVEL ANDROGRAPHOLIDE DERIVATIVES AND THEIR IN VITRO CYTOTOXIC ACTIVITY

    OpenAIRE

    Venkat R. P; Virohit Patil; Ravindra Patil

    2014-01-01

    A new series of sulfonyl-type of andrographolide derivatives were synthesized from andrographolide, the cytotoxic constituent of the plant Andrographis paniculata. The derived analogs (4a-4g) were evaluated for their cytotoxic activity against human small lung cancer (NCI-H187), leukemia K562, breast cancer (MCF-7/ADR) and lung adenocarcinoma (A549) cell lines. Most of the analogues show significant cytotoxic activity against tested cell lines. The methyl sulfonyl derivative 4a had higher act...

  18. Cytotoxicity and genotoxicity of biogenic silver nanoparticles

    Science.gov (United States)

    Lima, R.; Feitosa, L. O.; Ballottin, D.; Marcato, P. D.; Tasic, L.; Durán, N.

    2013-04-01

    Biogenic silver nanoparticles with 40.3 ± 3.5 nm size and negative surface charge (- 40 mV) were prepared with Fusarium oxysporum. The cytotoxicity of 3T3 cell and human lymphocyte were studied by a TaliTM image-based cytometer and the genotoxicity through Allium cepa and comet assay. The results of BioAg-w (washed) and BioAg-nw (unwashed) biogenic silver nanoparticles showed cytotoxicity exceeding 50 μg/mL with no significant differences of response in 5 and 10 μg/mL regarding viability. Results of genotoxicity at concentrations 5.0 and 10.0 ug/mL show some response, but at concentrations 0.5 and 1.0 μg/mL the washed and unwashed silver nanoparticles did not present any effect. This in an important result since in tests with different bacteria species and strains, including resistant, MIC (minimal inhibitory concentration) had good answers at concentrations less than 1.9 μg/mL. This work concludes that biogenic silver nanoparticles may be a promising option for antimicrobial use in the range where no cyto or genotoxic effect were observed. Furthermore, human cells were found to have a greater resistance to the toxic effects of silver nanoparticles in comparison with other cells.

  19. Cytotoxic effects of curcumin in osteosarcoma cells

    Directory of Open Access Journals (Sweden)

    Moran JM

    2014-11-01

    Full Text Available JM Moran, FJ Rodriguez-Velasco, R Roncero-Martin, V Vera, JD Pedrera-Zamorano Metabolic Bone Diseases Research Group, University of Extremadura, Cáceres, SpainWe read with interest the results of the study from Chang et al1 in the International Journal of Nanomedicine. This article shows that curcumin (diferuloymethane, a phenolic compound extracted from the natural plant Curcuma longa L., exerts higher cytotoxicity in osteosarcoma MG-63 cells than in healthy human osteoblasts. Based on the dosages provided by the authors it is hypothesized that with the appropriate drug carriers, curcumin could be delivered to bone tumors and selectively kill osteosarcoma cells without harming healthy osteoblasts. This hypothesis is based in the data provided that claims the dose needed to exert a significant cytotoxicity on osteosarcoma cells was much lower than that needed to exert the same effect on healthy human osteoblasts. The topic of this study is of importance for both cancer and metabolic bone disease fields, we appreciate the methodology of the study, nevertheless we believe that the hypothesis formulated by the authors and their conclusion deserves further comment.View original paper by Chang and colleagues.

  20. Pyruvate anions neutralize peritoneal dialysate cytotoxicity.

    Science.gov (United States)

    Mahiout, A; Brunkhorst, R

    1995-01-01

    A new peritoneal dialysate containing pyruvate anions was developed in order to avoid cytotoxic effect of conventional lactate-based dialysate. The dialysate has a final pH of 5.4 to 5.6 and is composed of 1.36-3.86% glucose-monohydrate; 132 mmol/l sodium; 1.75 mmol/l calcium; 0.75 mmol/l magnesium; 102 mmol/l chloride and 35 mmol/l pyruvate. For cytotoxicity testing peritoneal macrophages, and mesothelial cells (MC) were exposed to conventional lactate dialysate, and pyruvate dialysate. We investigated the O2- generation and cytokine synthesis after endotoxin stimulation in peritoneal macrophages and the proliferation of mesothelial cells of cultured human MC. After exposure to lactate dialysate O2- generation and cytokine synthesis in peritoneal macrophages and proliferation of mesothelial cells were inhibited when compared to solution containing pyruvate and the control solution. After preincubation with 3.86% glucose containing solutions, all negative effects became even more pronounced in the lactate group whereas after pre-exposure to pyruvate containing solution the toxic effects were absent. These results suggest that the acute toxic effects of commercially available peritoneal dialysates can be avoided by the use of sodium pyruvate instead of sodium lactate.

  1. Cytotoxic activities of flavonoids from Centaurea scoparia.

    Science.gov (United States)

    Ahmed, Sayed A; Kamel, Emadeldin M

    2014-01-01

    Phytochemical studies on the ethanolic extract of the aerial parts of Centaurea scoparia led to the isolation of two new flavonoids, 3',4'-dihydroxy-(3'',4''-dihydro-3''-hydroxy-4''-acetoxy)-2'',2''-dimethylpyrano-(5'',6'':7,8)-flavone-3-O- β -D-glucopyranoside (1) and 3,3',4'-trihydroxy-(3'',4''-dihydro-3'',4''-dihydroxy)-2'',2''-dimethylpyrano-(5'',6'':7,8)-flavone (2), along with eight known flavonoids isolated for the first time from this plant, cynaroside (3), Apigetrin (4), centaureidin (5), oroxylin A (6), 5,7-dihydroxy-3',4',5'-trimethoxyflavone (7), atalantoflavone (8), 5-hydroxy-3',4',8-trimethoxy-2'',2''-dimethylpyrano (5'',6'':6,7)-flavone (9), and 3',4',5,8-tetramethoxy-2'',2''-dimethylpyrano (5'',6'':6,7)-flavone (10). The structures of the isolated compounds were elucidated by means of spectroscopic tools including 1D and 2D NMR, UV, IR, and mass spectroscopy. Cytotoxic activities of the isolated compounds were evaluated against human cervical carcinoma HeLa, human hepatocellular carcinoma HepG2, and human breast carcinoma MCF-7. Compound 2 was the most potent cytotoxic agent against HeLa cells with an IC50 0.079  μM. PMID:25114960

  2. Cytotoxicity study of plant Aloe vera (Linn

    Directory of Open Access Journals (Sweden)

    Atul N Chandu

    2012-01-01

    Full Text Available Background: The objective of this study has been to evaluate the in-vitro antitumor activity of Aloe vera extract of in cultured B16F10 melanoma cell line by measuring cell viability using "Trypan blue exclusion assay" method. Aim: To find out such kind of anticancer drug which is a cheap, safe, less toxic, and more potent drug compared to chemotherapy drug. Materials and Methods: In-vitro antitumor activity cell culture1, drug treatment (standard and test extract and Trypan blue exclusion assay growth and viability test 1 were used. Treatment of Aloe vera extract against B16F10 melanoma cell line, in all concentration range, showed decrease in percent cell viability, as compared to that of negative when examined by "Trypan blue exclusion assay". Results: In overall variation of test samples, Aloe vera extract showed its best activity in the concentration of 300 μg/ml, which was approximately equal to the activity of standard drug doxorubicin. Evaluation of in-vitro antitumor activity revealed that Aloe vera extract exhibits good cytotoxic activity. The best cytotoxic activity by Aloe vera was shown at 200 μg/ml concentration. Conclusion: The study of cytoprotection against normal cells by micronucleus assay has shown that the herbal extracts have less toxic effects to the normal blood lymphocytes, as compared to that of standard anticancer drug.

  3. Preparation of polyclonal antiserum against recombinant NSP2 protein of PRRSV HH08 strain and study on biological functions of the polyclonal antiserum%猪繁殖与呼吸综合征病毒HH08株NSP2蛋白多克隆抗体的制备及其生物学功能的研究

    Institute of Scientific and Technical Information of China (English)

    马玲; 李广兴; 洪琴; 任玉东; 任晓峰

    2013-01-01

    利用RT-PCR和SOE PCR技术扩增得到猪繁殖与呼吸综合征病毒(PRRSV) HH08株NSP2全长基因,经抗原性和亲水性分析,将NSP2部分序列成功亚克隆于pET-30a(+)和PVAX1栽体中.将阳性重组质粒pET30a-NSP2转化E.coli Rosetta(DE3)感受态细胞,经诱导表达获得107 ku的重组NSP2蛋白.Western-blot检测表明,重组蛋白能够与PRRSV参考阳性血清反应.以纯化的重组NSP2蛋白免疫新西兰白兔制备多克隆抗体,其ELISA效价达到1∶1015以上;Western-blot试验表明,其具有良好的反应性和特异性.间接免疫荧光试验显示,用抗NSP2多克隆抗体可以检测到PVAX-NSP2转染BHK 21细胞所表达的NSP2蛋白,且与经典型和高致病型PRRSV毒株均有很好的特异性反应.病毒感染抑制试验表明,多抗血清对PRRSV经典和高致病性毒株的抑制率可达到68%和53%.上述研究结果为PRRSV检测及NSP2蛋白功能的深入研究奠定了基础.%In this study,the complete PRRSV HH08 NSP2 gene was cloned and the partial gene sub-cloned into prokaryotic expression vector pET-30a( + ) and eukaryotic expression vector PVAX1 after hy-drophilicity plot and antigenic index analysis. E. coli Rosetta (DE3) was transformed with the recombinant plasmid pET30a-NSP2. The recombinant NSP2 protein that molecular weight is 107 ku was expressed. It could be recognized by specific PRRSV antisera in western blot. Then the purified recombinant NSP2 protein as antigen was used to immunize rabbit for preparation of anti-NSP2 polyclonal antibody. An indirect ELISA assays showed that the titer of anti-NSP2 polyclonal antibody was 1 : 1015 , and it had highly reactivity and specialty in Western-blot. Also.IFA test demonstrated that this polyclonal antibody could react with the BHK-21 cells which can express PRRSV NSP2 protein and the Marc-145 cells infected with PRRSV. Both attenuated and highly pathogenic PRRSV strains could be inhibited by the anti-NSP2 polyclonal antibody and the

  4. Suppression of the antigen-specific T cell immune response by co-immunization with the HBV DNA vaccine and recombinant HbsAg%重组质粒与重组蛋白共免疫诱导HBsAg特异性T细胞免疫抑制

    Institute of Scientific and Technical Information of China (English)

    杜小刚; 王军朋; 康有敏; 王肖; 赵干; 王宾

    2009-01-01

    [Objective] To explore a new therapeutic strategy against acute hepatitis B and fulminant hepatitis B, we studied effect of co-immunization with HBV DNA and HBsAg on the T cell proliferation reaction. [Methods] We immunized the BALB/ c mice with HBV DNA vaccine (pcDS2) plus HBsAg by intramuscular injection. The immunization was performed on week 0, 2 and 4. The anti-HBs(IgC)antibody titer, T lymphocyte proliferation reaction , and the expression of IL-10 and Foxp3 in CD3 + T cell were detected on week 6. [Results] The anti-HBs IgC titer induced by pcDS2 plus HBsAg group was higher than that induced by pcDS2, or HBsAg alone. Compared to mice immunized with pcDS2, or HBsAg alone, the stimulated index (SI) of T cell proliferation induced by the pcDS2 plus HBsAg group tested by MTT methods decreased. Besides, the immune suppression of T cell proliferation response induced by co-immunization group was further confirmed by flow cytometry. Finally, the expression of IL-10 and Foxp3 in CD3+ T cell was up-regulated in the co-immunization group significantly. [Conclusion] The co-immunization of HBV DNA vaccine and HBsAg can induce the humoral immune response, but cannot induce antigen specific T cell proliferation reaction. Besides, the immune suppression induced by co-immunization may be correlated with the expression of IL-10 and Foxp3.%[目的]为了探索治疗急性乙型肝炎和爆发性乙型肝的新策略,本研究将HBV DNA疫苗和相应抗原的蛋白质分子联合免疫小鼠,旨在探讨联合免疫对小鼠抗原特异性T细胞增殖反应的影响.[方法]我们将HBV DNA疫苗(pcDS2)和相应抗原蛋白质分子(HBsAg)联合免疫BALB/c小鼠;分别在第0、2和4周进行免疫,在第6周用ELISA方法检测抗-HBs IgG效价,MTr和流式细胞仪检测T细胞增殖反应及流式细胞仪检测细胞因子表达水平.[结果]pcDs2和HBsAg联合免疫组小鼠的抗-HBs水平显著提高;免疫小鼠的T细胞体外经HBsAg刺激后,联

  5. Lymphocyte cytotoxicity of colchicine in Hiroshima atomic bomb survivors

    International Nuclear Information System (INIS)

    The cytotoxicity of colchicine for the peripheral blood lymphocytes of 151 Hiroshima atomic bomb survivors and their controls was determined. No radiation effect was found, but modest age-related changes for the initial number of vialble cells and for the cytotoxicity of colchicine were observed. (author)

  6. In vitro macrophage cytotoxicity of five calcium silicates.

    OpenAIRE

    Skaug, V; Davies, R.; Gylseth, B

    1984-01-01

    Five calcium silicate minerals (two naturally occurring and three synthetic compounds) with defined morphology and chemical composition were compared for their cytotoxic and lysosomal enzyme releasing effects on unstimulated mouse peritoneal macrophages in vitro. One synthetic material, a fibrous tobermorite, was cytotoxic towards the cells, and two naturally occurring wollastonites induced selective release of beta-glucuronidase from the cells.

  7. Evaluation Of Potential Cytotoxic Effects Of Herbal Extracts

    Directory of Open Access Journals (Sweden)

    Radovanovic Ana

    2015-12-01

    Full Text Available Herbal medicines have played an important role in treating different diseases since ancient times. Bioactive components of medicinal plants are a good starting point for discovering new drugs such as chemotherapeutics. Currently, there are four classes of plant-derived chemotherapeutic drugs used in clinical practice. However, to discover new potential cytotoxic molecules, the research effort on herbal extracts has not diminished. The aim of this review was to evaluate the chemical constituents of plants that possess cytotoxicity, the signalling pathways responsible for this effect, and the influence of solvent polarity on potential cytotoxic effect and to present the cytotoxic activity of selected herbal extracts. The polyphenolic, anthraquinon, diterpneoid, triterpenoid, flavonoid, betulinic acid and berberine content contributes to cytotoxicity of herbal extracts. The inhibitory effect on cancer cells viability could be a consequence of the non-apoptotic processes, such as cell cycle arrestment, and the apoptotic process in tumour cells through different signalling pathways. The influence of solvent polarity on potential cytotoxic effect of herbal extracts should not be ignored. In general, the best cytotoxic activity was found in nonpolar and moderately polar herbal extracts. The herbal extract with IC50 below 30 μg/ml could be considered a very strong cytotoxic agent. Considering that many antitumor drugs have been discovered from natural products, further research on plants and plant-derived chemicals may result in the discovery of potent anticancer agents.

  8. Study of the cytotoxicity of CCVD carbon nanotubes

    OpenAIRE

    Flahaut, Emmanuel; Durrieu, Marie-Christine; Remy-Zolghadri, Murielle; Bareille, Reine; Baquey, Charles

    2006-01-01

    The cytotoxicity of different samples of carbon nanotubes synthesised by catalytic chemical vapour deposition was investigated towards human umbilical vein endothelial cells, using two cytotoxicity standard tests (neutral red assay for the cell viability and MTT assay—tetrazolinium salt—for the cell metabolic activity). No toxicity was found for any sample, although a slight dilution effect may exist for two of them.

  9. Fluid dynamics of cytotoxic safety cabinets.

    Science.gov (United States)

    Braconnier, R; Bonthoux, F

    2010-03-01

    This study investigated the specific fluid dynamics characteristics of cytotoxic safety cabinets (CSC), particularly those used in cancer drug reconstitution operations. Measurements taken on site were used to derive characteristic data for these cabinets. An in-depth laboratory investigation of airflows inside another CSC was also conducted. Anemometric values recorded on these two installations enabled the experimental validation of computational fluid dynamics methods applied to CSC. The digital flow simulations conducted provide a better understanding of the detailed flow structure inside a CSC and made it possible to study the influence of different operating parameters on the air velocity distribution inside the cabinet front opening: recycled air temperature, product protection airflow rate, suction openings spatial distribution, air compensation mode and draughts, operator arm penetration, and operator presence in front of the cabinet. PMID:20007340

  10. Cytotoxic prenylated flavonoids from Macaranga indica.

    Science.gov (United States)

    Yang, Da-Song; Peng, Wei-Bing; Yang, Yong-Ping; Liu, Ke-Chun; Li, Xiao-Li; Xiao, Wei-Lie

    2015-06-01

    Three new prenylated flavonoids, macarindicins A-C (1-3), as well as seven known compounds (4-10) were isolated from the twigs of Macaranga indica. Their structures were elucidated on the basis of extensive spectroscopic interpretation. Compounds 2 and 3 enriched the diversity of prenyl moiety in genus Macaranga especially in the aspect of various lengths of prenyl chain. All the known compounds were isolated from M. indica for the first time and this plant was found to contain large number of ellagic acid. Compounds 1-10 were tested for their cytotoxicity against four cancer cell lines (MCF-7, Hep G2, Hela and P388) and showed IC50 values in the range of 2.61-20.35 μg/mL. PMID:25861749

  11. New Cytotoxic Saponins from Lysimachia davurica Ledeb.

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To investigate the saponins from whole plants of Lysimachia davurica Ledeb., two new saponins named davuricoside I (compound 1) and E (compound 2) were isolated. Their chemical structures were elucidated as 3β,16α, 28, 29-tetrihydroxy-olean-12-en-3-O-β-D-glucopyranosyl-(1-→2)-β-D-glucuronopyranoside (compound 1)and 3β,16α, 29-trihydroxy-13, 28-epoxy-oleanane-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranoside (compound 2) on the basis of their one- and two-dimensional nuclear magnetic resonance and mass spectrometry data, and chemical methods. Compound 1 showed significant cytotoxic activity against human A2780 cells.

  12. Cytotoxic activity and apoptosis induction by gaillardin.

    Science.gov (United States)

    Moghadam, Maryam Hamzeloo; Naghibi, Farzaneh; Atoofi, Azadeh; Rezaie, Mitra Asgharian; Irani, Mahboobeh; Mosaddegh, Mahmoud

    2013-01-01

    Cytotoxic activity of gaillardin, a sesquiterpene lactone isolated from Inula oculus-christi L. (Asteraceae), was assessed in the human breast adenocarcinoma cell line MCF-7, human hepatocellular carcinoma cell line HepG-2, human non-small cell lung carcinoma cell line A-549, and human colon adenocarcinoma cell line HT-29, resulting in IC50 values of 6.37, 6.20, 4.76, and 1.81 microg/mL, respectively, in the microculture tetrazolium-formazan MTT assay. In vitro apoptosis-inducing properties of gaillardin were also evaluated in MCF-7 cells with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. The results suggest gaillardin as a candidate for further studies in cancer therapy PMID:23819305

  13. Generation of cytotoxic T lymphocytes specific for B-cell acute lymphoblastic leukemia family-shared peptides derived from immunoglobulin heavy chain framework region

    Institute of Scientific and Technical Information of China (English)

    LIU Ying; ZHU Ping; HU Ya-mei

    2007-01-01

    Background Immunoglobulin heavy chain variable region (IgHV) is a well-characterized tumor antigen for B-cell malignancies. It can function as a target for T cell-mediated immune response. Clinical trials of IgHV protein vaccines against lymphoma have demonstrated induction of tumor-specific cytotoxic T lymphocyte (CTL) responses. However,complementary determining regions-based individual vaccines have disadvantages for wide clinical application. Although a recent study demonstrated that immunogenic peptides are derived from framework regions (FR) shared among patients with B-cell lymphoma, how to choose the appropriate peptides for each patient is still unsolved. The aim of this study was to investigate whether immunoglobulin heavy chain FR-derived peptides shared in each IgHV family are potential CTL epitopes presented by B-cell acute lymphoblastic leukemia (B-ALL). Such CTL epitopes might be beneficial to shifting vaccination strategies against B-ALL from individual specificity to family specificity.Methods Seven IgHV gene families were amplified respectively by PCR and sequenced directly from 71 childhood B-ALL cases. Bioinformatics was applied in analyzing characteristics of sequences available and predicting HLA-A*0201-restricted CTL epitopes for each IgHV family. An antigen-specific T cell expansion system was used to generate peptide-specific CTLs. The cytotoxicity of CTLs against B-ALL cells was assessed in the lactate dehydrogenase release assay.Results Complete IgHV rearrangements were identified in all of the 71 B-ALL cases. All of 40 sequences available showed ≥98% homology with the nearest germline IgHV genes, indicating IgHV genes in B-ALL of germline nature.Twelve nonapeptides of high HLA-A*0201-binding scores were obtained from 26 productive IgHV protein sequences. Ten (83%) of the peptides were located in FR1 and FR3 shared among the corresponding IgHV family. CTLs specific for the peptide QLVQSGAEV located in FR1 (3-11) shared among the IgHV1

  14. Recombinant mammaglobin A adenovirus-infected dendritic cells induce mammaglobin A-specific CD8+ cytotoxic T lymphocytes against breast cancer cells in vitro.

    Directory of Open Access Journals (Sweden)

    Huixia Cui

    Full Text Available Mammaglobin A (MGBA is a novel breast cancer-associated antigen almost exclusively over-expressed in primary and metastatic human breast cancers, making it a potential therapeutic target for breast cancer. The development of dendritic cell (DC-induced tumor antigen specific CD8(+ cytotoxic T lymphocytes (CTLs may hold promise in cancer immunotherapy. In this study we constructed recombinant replication-defective adenoviral (Ad vectors encoding MGBA and evaluated their ability to trigger anti-tumor immunity in vitro. DCs were isolated from the human peripheral blood monocyte cells (PBMCs of two HLA-A33(+ healthy female volunteers, and infected with adenovirus carrying MGBA cDNA (Ad-MGBA. After that, the Ad-MGBA-infected DCs were used to stimulate CD8(+ CTLs in vitro and the latter was used for co-culture with breast cancer cell lines. The data revealed that infection with Ad-MGBA improved DC maturation and up-regulated the expression of co-stimulatory molecules and the secretion of interleukin-12 (IL-12, but down-regulated interleukin-10 (IL-10 secretion from DCs. Ad-MGBA-infected DC-stimulated CD8(+CTLs displayed the highest cytotoxicity towards HLA-A33(+/MGBA(+ breast cancer MDA-MB-415 cells compared with other CD8(+CTL populations, and compared with the cytotoxicity towards HLA-A33(-/MGBA(+ breast cancer HBL-100 cells and HLA-A33(-/MGBA(- breast cancer MDA-MB 231 cells. In addition, Ad-MGBA-infected DC-stimulated CD8(+ CTLs showed a high level of IFNγ secretion when stimulated with HLA-A33(+/MGBA(+ breast cancer MDA-MB-415 cells, but not when stimulated with HLA-A33(-/MGBA(+ HBL-100 and HLA-A33(-/MGBA(-MDA-MB-231 cells. In addition, killing of CD8(+CTLs against breast cancer was in a major histocompability complex (MHC-limited pattern. Finally, the data also determined the importance of TNF-α in activating DCs and T cells. These data together suggest that MGBA recombinant adenovirus-infected DCs could induce specific anti-tumor immunity

  15. Synthesis and cytotoxic activities of semisynthetic zearalenone analogues.

    Science.gov (United States)

    Tadpetch, Kwanruthai; Kaewmee, Benyapa; Chantakaew, Kittisak; Kantee, Kawalee; Rukachaisirikul, Vatcharin; Phongpaichit, Souwalak

    2016-08-01

    Zearalenone is a β-resorcylic acid macrolide with various biological activities. Herein we report the synthesis and cytotoxic activities of 34 zearalenone analogues against human oral epidermoid carcinoma (KB) and human breast adenocarcinoma (MCF-7) cells as well as noncancerous Vero cells. Some zearalenone analogues showed moderately enhanced cytotoxic activities against the two cancer cell lines. We have discovered the potential lead compounds with diminished or no cytotoxicity to Vero cells. Preliminary structure-activity relationship studies revealed that the double bond at the 1' and 2' positions of zearalenone core was crucial for cytotoxic activities on both cell lines. In addition, for zearalenol analogues, the unprotected hydroxyl group at C-2 and an alkoxy substituent at C-4 played key roles on cytotoxic effects of both cell lines.

  16. Spectratyping analysis of the islet-reactive T cell repertoire in diabetic NOD Igμnull mice after polyclonal B cell reconstitution

    Directory of Open Access Journals (Sweden)

    Sercarz Eli E

    2011-07-01

    Full Text Available Abstract Background Non Obese Diabetic mice lacking B cells (NOD.Igμnull mice do not develop diabetes despite their susceptible background. Upon reconstitution of B cells using a chimera approach, animals start developing diabetes at 20 weeks of age. Methods We have used the spectratyping technique to follow the T cell receptor (TCR V beta repertoire of NOD.Igμnull mice following B cell reconstitution. This technique provides an unbiased approach to understand the kinetics of TCR expansion. We have also analyzed the TCR repertoire of reconstituted animals receiving cyclophosphamide treatment and following tissue transplants to identify common aggressive clonotypes. Results We found that B cell reconstitution of NOD.Igμnull mice induces a polyclonal TCR repertoire in the pancreas 10 weeks later, gradually diversifying to encompass most BV families. Interestingly, these clonotypic BV expansions are mainly confined to the pancreas and are absent from pancreatic lymph nodes or spleens. Cyclophosphamide-induced diabetes at 10 weeks post-B cell reconstitution reorganized the predominant TCR repertoires by removing potential regulatory clonotypes (BV1, BV8 and BV11 and increasing the frequency of others (BV4, BV5S2, BV9, BV16-20. These same clonotypes are more frequently present in neonatal pancreatic transplants under the kidney capsule of B-cell reconstituted diabetic NOD.Igμnull mice, suggesting their higher invasiveness. Phenotypic analysis of the pancreas-infiltrating lymphocytes during diabetes onset in B cell reconstituted animals show a predominance of CD19+ B cells with a B:T lymphocyte ratio of 4:1. In contrast, in other lymphoid organs (pancreatic lymph nodes and spleens analyzed by FACS, the B:T ratio was 1:1. Lymphocytes infiltrating the pancreas secrete large amounts of IL-6 and are of Th1 phenotype after CD3-CD28 stimulation in vitro. Conclusions Diabetes in NOD.Igμnull mice appears to be caused by a polyclonal repertoire of T cell

  17. Produção e purificação de anticorpos policlonais para Salmonella Enteritidis (Enterobacteriaceae Production and purification of polyclonal antibodies for Salmonella Enteritidis (Enterobacteriaceae

    Directory of Open Access Journals (Sweden)

    Mario Augusto Ono

    2002-04-01

    Full Text Available O objetivo deste trabalho foi produzir e purificar anticorpos policlonais específicos para Salmonella Enteritidis (Enterobacteriaceae. O anti-soro foi produzido em coelhos, empregando-se flagelina purificada. O título e a especificidade foram determinados através do ensaio imunoenzimático - ELISA e a purificação por cromatografia de afinidade com sepharose Proteína A. As suspensões bacterianas foram cultivadas em cinco diferentes meios de cultura (infusão de cérebro coração - BHI, caldo tripticase soja, caldo lactosado, caldo nutriente - CN e água peptonada. Observou-se que dependendo do meio o título do anti-soro pode variar e os melhores resultados foram obtidos com BHI e CN. O anti-soro foi específico para Salmonella Enteritidis, apresentando porcentagens de reações cruzadas com Salmonella Typhimurium, Salmonella Infantis e Salmonella Newport de 16,0, 11,9 e 6,4%, respectivamente. Menores porcentagens foram obtidas com outras enterobactérias testadas. Esses resultados indicam a possibilidade da utilização desses anticorpos na padronização de ensaios imunológicos para a detecção de Salmonella EnteritidisThe purpose of this study was to produce and to purify specific polyclonal antibodies for Salmonella Enteritidis (Enterobacteriaceae. The anti-serum was raised in rabbits using purified flagelin. Anti-serum titer and specificity were determined by an immunoassay - ELISA and its purification was performed by sepharose protein A affinity chromatography. The bacteria suspensions were cultivated in five different media (brain heart infusion - BHI, tripticase soy broth, nutrient broth - NB, peptone water. Results have showed that anti-serum titers varied depending on which media type was used and BHI and NB media yielded the most significant results. The anti-serum produced was specific for Salmonella Enteritidis. Its cross-reactivity with Salmonella Thyphimurium, Salmonella Infantis and Salmonella Newport were 16.0, 11

  18. 斑马鱼基因ATF4多克隆抗体的制备%Preparation of Polyclonal Antibody of ATF4 in Zebrafish

    Institute of Scientific and Technical Information of China (English)

    李发祥; 杨荣; 陈婷芳; 唐雄卓; 赵碧峰; 江志钢; 张义; 李永青; 吴秀山

    2012-01-01

    ATF4 , a member of the ATF/CREB transcription factor family which contains a bZIP domain, plays a important role in the embryonic development, cell proliferation and differentiation. Therefore, it is advisable to prepare an antibody for the heart development research in Zerbrafish. In this research, a ATF4-specific, hydrophilic nucleotide sequence (about 1 017 bp) was selected by using bioinformatics methods, and the sequence was amplified by PCR, the PCR product was inserted into the prokaryotic expression vector pET-28a and then transformed into Rosetta cells. After the sequence result of the recombinant plasmid pET-28a-ATF4 is verified to be right, the fusion protein was induced to express in Rosetta cells by IPTG. The mouse was immunized by injecting appropriate amount of fusion protein, and the ATF4 polyclonal antibody was obtained. The antibody' s titer and specification was identified. Our results showed that the ATF4 polyclonal antibody was practical and available for western-blotting analysis and immunohisto-chemistry.%ATF4是含有bZIP结构域的ATF/CREB转录因子家族成员,对胚胎的发育以及细胞的增殖、分化有重要的调节作用.制备ATF4的多克隆抗体对于研究其在斑马鱼心脏发育过程中的作用有重要的意义.研究首先通过生物信息学方法,选择ATF4基因中特异性强、具亲水性的一段核苷酸序列(1 017 bp),通过PCR扩增,将片段重组到原核表达载体pET-28a,然后转化人Rosetta菌株中.经测序鉴定正确后,用IPTG诱导表达融合蛋白,以该融合蛋白免疫小鼠,获得ATF4多克隆抗鼠血清.对该多抗血清抗体进行验证,具有很好特异性和较高效价,可以用作Western-blotting、免疫印迹等试验分析.

  19. Cytotoxic Activities of Flavonoids from Centaurea scoparia

    Directory of Open Access Journals (Sweden)

    Sayed A. Ahmed

    2014-01-01

    Full Text Available Phytochemical studies on the ethanolic extract of the aerial parts of Centaurea scoparia led to the isolation of two new flavonoids, 3′,4′-dihydroxy-(3′′,4′′-dihydro-3′′-hydroxy-4′′-acetoxy-2′′,2′′-dimethylpyrano-(5′′,6′′:7,8-flavone-3-O-β-D-glucopyranoside (1 and 3,3′,4′-trihydroxy-(3′′,4′′-dihydro-3′′,4′′-dihydroxy-2′′,2′′-dimethylpyrano-(5′′,6′′:7,8-flavone (2, along with eight known flavonoids isolated for the first time from this plant, cynaroside (3, Apigetrin (4, centaureidin (5, oroxylin A (6, 5,7-dihydroxy-3′,4′,5′-trimethoxyflavone (7, atalantoflavone (8, 5-hydroxy-3′,4′,8-trimethoxy-2′′,2′′-dimethylpyrano (5′′,6′′:6,7-flavone (9, and 3′,4′,5,8-tetramethoxy-2′′,2′′-dimethylpyrano (5′′,6′′:6,7-flavone (10. The structures of the isolated compounds were elucidated by means of spectroscopic tools including 1D and 2D NMR, UV, IR, and mass spectroscopy. Cytotoxic activities of the isolated compounds were evaluated against human cervical carcinoma HeLa, human hepatocellular carcinoma HepG2, and human breast carcinoma MCF-7. Compound 2 was the most potent cytotoxic agent against HeLa cells with an IC50 0.079 μM.

  20. Serum-free culture of H pylori intensifies cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Hiroyuki Ohno; Akiyuki Murano

    2007-01-01

    AIM: To perform a long culture passage of H pylori without serum, taking into account its cytotoxicity and the presence of the probable new cytotoxic factor.METHODS: One sample of H pylori 60190 (ATCC49503) was grown on Brain Heart Infusion (BHI) agar containing 0.5% 2,6-di-O-methyl-β-cyclodextrin without any serum, being passaged 70-100 times every 3-4 d for approximately 2 h, while another sample of H pylori contained 70 mL/L fetal calf serum without 2,6-di-Omethyl-β-cyclodextrin. Their supernatant and extract after 16 h in culture were evaluated for changes in cell morphology and for cell viability using HeLa cells. Furthermore, the characteristics of the probable cytotoxic factor in the extract were examined on partial purification studies and its cytotoxicity was evaluated in various human cells.RESULTS: The supernatant and the extract of the bacterium grown on serum-free medium had strong cytotoxicity compared with those grown on serumcontaining medium. They irreversibly damaged HeLa cells without vacuolation that was altogether different from that of the bacterium when grown with serum.Their cytotoxicity was easily measured by cell viability assay. The probable cytotoxic factor partially purified and detected by chromatography had characteristics difference from that of vacuolating toxin and a broad cytotoxicity toward various cell lines.CONCLUSION: Serum-free long culture method of H pylori makes its supematant and its extract cytotoxic enough to be easily measured by cell viability assay. The probable cytotoxic factor has a unique characteristic and might be a new cytotoxin.

  1. Cytotoxic T lymphocytes mediate chronic inflammation of the nasal mucosa of patients with atypical allergic rhinitis

    Directory of Open Access Journals (Sweden)

    Shuqi Qiu

    2011-01-01

    Full Text Available Background : The prevalence of chronic rhinitis is increasing rapidly; its pathogenesis is to be further understood; immune inflammation is one of the possible causative factors. Antigen specific CD8+ T cells play a critical role in the induction of chronic inflammation. Aims : This study aimed to investigate the role of antigen specific CD8+ T cells in the pathogenesis of chronic atypical allergic rhinitis. Material and Methods : Nasal mucosal epithelial surface scratching samples were obtained from patients with chronic obstruction atypical allergic rhinitis. Exosomes were purified from the scratching samples and examined by immune gold electron microscopy. The effect of exosomes on modulating dendritic cell′s properties, the effect of exosome-pulsed dendritic cells on naïve T cell differentiation and the antigen specific CD8+ T cell activation were observed by cell culture models. Results : Exosomes purified from patients with chronic atypical allergic rhinitis carried microbial products, Staphylococcal enterotoxin B (SEB, and airborne antigen, Derp1. Dendritic cells pulsed by SEB/Derp1-carrying exosomes showed high levels of CD80, CD86 and the major histocompatibility class I (MHCI. Exosome-pulsed dendritic cells could induce the naïve CD3+ T cells to differentiate into CD8+ T cells. Upon the exposure to a specific antigen, the CD8+ T cells released granzyme B and perforin; more than 30% antigen specific CD8+ T cells proliferated. Conclusions : Antigen specific CD8+ T cells play an important role in the pathogenesis of chronic obstruction atypical allergic rhinitis.

  2. Preparation and identification of the fragment of rabbit's polyclonal antibodies against human ouabain%抗哇巴因多克隆抗体F(ab)2 片段的研制及鉴定

    Institute of Scientific and Technical Information of China (English)

    张明娟; 吕卓人; 袁育康; 贺浪冲; 王颢

    2000-01-01

    Objective Anti-ouabain polyclonal antibody was digested with pepsin to prepare F fragment in order to find the base of reshaped antibody. Methods Anti-ouabain polyclonal antibody was prepared by immunizing rabbits. Then anti-ouabain polyclonal antibody was digested under different conditions. The reactants were analyzed and purified by High Performance Size Ex-clude Chromatography (HPSEC). The immune activities were detected and identified by Enzyme Linked Immunosorbent Assay (ELISA). Results The proper reactive conditions for preparation of anti-ouabain polyclonal antibody F(ab)2 fragment with pepsin were established. The eligible dose of pepsin, by which 100mg anti-onabain polyclonal antibody was digested better, was 2mg. The eligible digesuve time was 18h. The value of pH of reactive system was 3.0. Conclusion The active F fragment of anti-ouabain polyclonal antibody can be obtained with pepsin. The method is simple and feasible.%目的用胃蛋白酶酶解抗哇巴因多克隆抗体,制备出片段,为改型抗体的研究提 供依据。方法免疫家兔制备出抗哇巴因多克隆抗体,并用胃蛋白酶分别在不同的酶解条件下消化抗体,继之用高效液相排阻色谱法(HPSEC)对其进行分析、纯化,采用酶联免疫吸附法(ELISA)检测、鉴定其活性。结果用胃蛋白酶2mg/100mg抗哇巴因多克隆抗体,消化18h,反应体系的pH3.0时,可获得片段。结论用胃蛋白酶酶解抗哇巴因多克隆抗体是研制出有活性的片段的有效方法。

  3. Development of polyclonal antibody-based indirect enzyme-linked immunosorbent assay for the detection of Alicyclobacillus strains in apple juice.

    Science.gov (United States)

    Wang, Zhouli; Yue, Tianli; Yuan, Yahong; Cai, Rui; Guo, Caixia; Wang, Xin; Niu, Chen

    2012-11-01

    A sort of specific polyclonal anti-Alicyclobacillus antibody was generated by immunizing New Zealand white rabbits, and a sensitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for Alicyclobacillus detection in apple juice. A set of experimental parameters such as concentration of antigen, dilutions of the antibody and goat anti-rabbit IgG-horseradish peroxidase conjugate, selection of the blocking reagent, incubation time, and temperature was optimized. The cross-reactivity of the antibody was evaluated by ELISA and the result was consistent with Western blot analysis. The detection limit of the ELISA was about 10(5) colony forming units (CFU)/mL in apple juice samples. Samples were detected by ELISA and conventional culture method, and the ELISA results gave a good agreement with the results obtained by plating on Alicyclobacillus acidoterrestris medium agar. ELISA takes a total detection time of 6 to 7 h, which is less than the time of conventional techniques requiring more than 24 to 48 h. These results indicated that the established ELISA was a potential useful analytical method for detection of Alicyclobacillus in apple juice.

  4. Preparation of polyclonal antibodies for nateglinide (NTG) and development of a sensitive chemiluminescent immunoassay to detect NTG in tablets and serum.

    Science.gov (United States)

    Zheng, Lei; Wang, Jing; Zhang, Jie; Song, Zhaorui; Dong, Yaqing; Wang, Yufen; Tong, Zhongsheng; Deng, Chuan; Yin, Yongmei; Meng, Meng; Xi, Rimo

    2016-01-01

    In this study, we prepared polyclonal antibodies against anti-diabetic drug nateglinide (NTG), and established a sensitive chemiluminescent immunoassay (CLIA) to detect NTG in tablets and serum. Two kinds of immunogens were synthesized using ethylcarbodiimide (EDC)/hydroxysuccinimide (NHS) and carbonyldiimidazole (CDI)/4-dimethylaminopyridine (DMAP) as coupling reagents respectively. When activated by EDC/NHS, more molecules of NTG coupled with carrier protein in immunogens. A horseradish peroxidase (HRP)-luminol-H2O2 system with p-iodophenol enhancement was applied in the CLIA analysis. The antibodies in EDC/NHS group showed higher titer, sensitivity and wider detection linear range than those in CDI/DMAP group, and were chosen for next studies. The developed CLIA assay exhibited good selectivity towards NTG among structually similar analogs. The method could detect as low as 0.35 ng mL(-1) NTG in buffer, 2.1 ng mL(-1) NTG in serum and 0.84 ng mL(-1) NTG in tablets. The CLIA method provided consistent results with HPLC method (r=0.9986) in determination of NTG from 5.0 to 400 µg mL(-1). The CLIA method could detect 78 samples in one assay, and the samples need only dilution in pretreatment. As a summary, this research offers a sensitive assay for high-throughout screening of NTG in formulation control and pharmacokinetic studies. PMID:26695294

  5. Immune escape mutants of Highly Pathogenic Avian Influenza H5N1 selected using polyclonal sera: identification of key amino acids in the HA protein.

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    Ioannis Sitaras

    Full Text Available Evolution of Avian Influenza (AI viruses--especially of the Highly Pathogenic Avian Influenza (HPAI H5N1 subtype--is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1, using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number.

  6. Pengembangan Antibodi Poliklonal dari Stadium Oosista, Sporosista, dan Sporozoit Eimeria tenella (THE DEVELOPMENT OF POLYCLONAL ANTOBODY FROM EIMERIA TENELLA OOCYST, SPOROCYST, AND SPOROZOITE STADIUM

    Directory of Open Access Journals (Sweden)

    Galuh Tresnani

    2013-09-01

    Full Text Available The research on developing diagnostic method, vaccine, and drugs for coccidiosis has been focused onthe finding of the immunogenic molecule in Eimeria. The identification of this agent will need the antibodywhich can recognize the biomolecule in the antigen. Antibody that has been developed for this purposeshould be analyzed first, and one of the simple methods for analyzing this antibody is through dot blotanalysis. The objective of this research was to analyze the polyclonal antibody which developed from theoocyst, sporocyst, and sporozoite of  E. tenella using dot blot analysis. The antigen for this polyclonalantibody was made from each of the E. tenella stadium by sonication. Fifteen mice, divided into 3 groups,were then injected subcutaneously with each antigen. The sera from these mice were then collected, analyzedby using ELISA and then it will be used for the dot blot analysis. The research result showed that thepolyclonal antibody which has been developed in mice from each antigen can react with the antigen itself.From this result it can be concluded that the developing of this antibody is successful and it can be used forfurther research in immunoproteomic.

  7. Control of polyclonal immunoglobulin production from human lymphocytes by leukotrienes; leukotriene B4 induces an OKT8(+), radiosensitive suppressor cell from resting, human OKT8(-) T cells

    International Nuclear Information System (INIS)

    We report that leukotriene B4 (LTB4), a 5-lipoxygenase metabolite of arachidonic acid, is a potent suppressor of polyclonal Ig production in pokeweed mitogen (PWM)-stimulated cultures of human peripheral blood lymphocytes, while LTC4 and LTD4 have little activity in this system. Preincubation of T cells with LTB4 in nanomolar to picomolar concentrations rendered these cells suppressive of Ig production in subsequent PWM-stimulated cultures of fresh, autologous B + T cells. This LTB4-induced suppressor cell was radiosensitive, and its generation could be blocked by cyclohexamide but not by mitomycin C. The LTB4-induced suppressor cell was OKT8(+), while the precursor for the cell could be OKT8(-). The incubation of OKT8(-) T cells with LTB4 for 18 h resulted in the appearance of the OKT8(+) on 10-20% of the cells, and this could be blocked by cyclohexamide but not by mitomycin C. Thus, LTB4 in very low concentrations induces a radiosensitive OKT8(+) suppressor cell from OKT8(-) cells. In this regard, LTB4 is three to six orders of magnitude more potent than any endogenous hormonal inducer of suppressor cells previously described. Glucocorticosteroids, which block suppressor cell induction in many systems, may act by inhibiting endogenous production of LTB4

  8. A prospective comparison of sup 99m Tc-labeled polyclonal human immunoglobulin and sup 111 In granulocytes for localization of inflammatory bowel disease

    Energy Technology Data Exchange (ETDEWEB)

    Arndt, J.W.; Sluys Veer, A. van der; Blok, D.; Griffioen, G.; Verspaget, H.W.; Salvador Pena, A.; Lamers, C.B.H.W.; Pauwels, E.K.J. (Leiden Univ. Hospital (Netherlands). Div. of Nuclear Medicine, Dept. of Diagnostic Radiology, and Dept. of Gastroenterology)

    1992-03-01

    There is a need for an easily prepared radiopharmaceutical agent for the detection of inflammation and infection. In a group of 14 patients with inflammatory bowel disease (IBD), the detection of actively involved intestinal segments by nonspecific human polyclonal immunoglobulin (IgG) labeled with {sup 99m}Tc was compared with that of {sup 111}In granulocytes. To determine the specificity of {sup 99m}Tc-IgG scintigraphy, 8 control patients without clinical indications of intestinal inflammation were examined. {sup 99m}Tc-IgG was found in the left colon in 8 and in the right colon in 7 of the 8 controls 4 hours after the injection. At that time of scintigraphy only 4 IBD patients exhibited a more intensive accumulation at the site of the intestinal segments with active disease. In contrast, in a randomized comparison with {sup 111}In granulocytes scintigraphy was positive in 11 patients with the latter technique. Moreover, fewer diseased segments were seen in the 4 patients with positive {sup 99m}Tc-IgG scintigraphy (6 versus 12 with {sup 111}In granulocytes). In view of the low sensitivity and specificity, it is concluded that {sup 99m}Tc-IgG is not suitable for the scintigraphy staging of IBD patients. (orig.).

  9. Preparation and Application of Polyclonal Antibody to Butachlor%抗丁草胺多克隆抗体的制备及应用

    Institute of Scientific and Technical Information of China (English)

    郭永恩; 陈家华; 王能东; 张秀; 郭振泉; 金声

    2002-01-01

    Polyclonal antibody to butachlor was prepared from rabbits immunized with butach lor-BSA conjugate,and a competitive ELISA was developed for the detection of th e butachlor (a major component in a herbicide) within the range of 1×10-9 ~100×10-9mg/mL without cross reactions with the structurally similar c hloroac etanilide herbicides,alachlor and acetochlor.This method could also be used to m onitor the butachlor concentration of water samples from rice field.%以牛血清白蛋白-丁草胺免疫抗原免疫家兔获得了抗丁草胺多克隆抗体,并建立了竞争酶联免疫吸附检测丁草胺(除草剂中的一种主要成分)分析方法.该方法的检测范围在1×10-9~100×10-9mg/mL之间,且和化学结构相似的甲草胺和乙草胺无交叉反应.该方法也可用于检测稻田水样中丁草胺的含量.

  10. A convenient model of severe, high incidence autoimmune gastritis caused by polyclonal effector T cells and without perturbation of regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Eric Tu

    Full Text Available Autoimmune gastritis results from the breakdown of T cell tolerance to the gastric H(+/K(+ ATPase. The gastric H(+/K(+ ATPase is responsible for the acidification of gastric juice and consists of an α subunit (H/Kα and a β subunit (H/Kβ. Here we show that CD4(+ T cells from H/Kα-deficient mice (H/Kα(-/- are highly pathogenic and autoimmune gastritis can be induced in sublethally irradiated wildtype mice by adoptive transfer of unfractionated CD4(+ T cells from H/Kα(-/- mice. All recipient mice consistently developed the most severe form of autoimmune gastritis 8 weeks after the transfer, featuring hypertrophy of the gastric mucosa, complete depletion of the parietal and zymogenic cells, and presence of autoantibodies to H(+/K(+ ATPase in the serum. Furthermore, we demonstrated that the disease significantly affected stomach weight and stomach pH of recipient mice. Depletion of parietal cells in this disease model required the presence of both H/Kα and H/Kβ since transfer of H/Kα(-/- CD4(+ T cells did not result in depletion of parietal cells in H/Kα(-/- or H/Kβ(-/- recipient mice. The consistency of disease severity, the use of polyclonal T cells and a specific T cell response to the gastric autoantigen make this an ideal disease model for the study of many aspects of organ-specific autoimmunity including prevention and treatment of the disease.

  11. Biodistribution and γ imaging of 125I-labeled goat anti-human IgG polyclonal antibody in nude mice bearing human colon cancer xenografts

    International Nuclear Information System (INIS)

    The possibility of IgG secreted from tumor cells as a target for radioimmunoimaging and targeted therapy of cancers were investigated. Goat anti-human IgG polyclonal anti- body (GAHG) was radioiodinated using Iodogen method, and the in vitro stability and pharmacokinetics were evaluated. The biodistribution and γ imaging of 125I-GAHG were performed in nude mice bearing HT-29 human colon cancer xenografts. 125I-GAHG showed good in vitro stability, and its blood clearance was defined as a two-compartment model, with T1/2α and T1/2β were 1.19 h and 43.99 h, respectively. The tumor uptake of 125I-GAHG was higher than that of 125I-labeled normal goat IgG control (125I-GIgG). 125I-GAHG showed good tumor retention when injecting via intra-tumor. In the biodistribution study, the highest tumor uptake of 125I-GAHG was 6.71±2.19%ID/g at 72 h postinjection and the T/NT increased along with the postinjection time. The results show that 125I-GAHG have good tumor-specific uptake which may provide a novel idea for radioimmunoimaging and targeted therapy of cancers. (authors)

  12. Interleukin-2 activation of cytotoxic cells in postmastectomy seroma.

    Science.gov (United States)

    Gercel-Taylor, C; Hoffman, J P; Taylor, D D; Owens, K J; Eisenberg, B L

    1996-02-15

    Lymphocytes were isolated from breast seroma fluids and used to study the mechanism of activation of cytotoxic lymphocytes and possible role of immunological potentiation following surgery in breast cancer patients. Single or serial samples were obtained from patients who had undergone mastectomy or lumpectomy with axillary node dissection. Lymphocytes were activated with rIL-2 (interleukin-2) and their cytotoxic activity was studied against Daudi and K562 cells and against a breast tumor line (SKBr-3). All of the patients (21/21) responded to IL-2 stimulation by significant activation of cytotoxic activity. The unstimulated cytotoxic activity of these cells against NK targets was low with less than 10% specific release in cytotoxicity assays. In simultaneous experiments, autologous seroma fluid was included during activation of lymphocytes to study possible regulatory molecules that may be present. In 17/21 patients, the presence of their seroma fluid, during the activation period, enhanced or did not effect the cytotoxic potential of their lymphocytes; inhibition was observed when seroma fluids from 4/21 patients were included. Analysis of the cytotoxic population derived from combined IL-2 and seroma treatments indicates the presence of cells with increased expression of CD56, and CD2, as well as in some cases CD16 expression. Cytotoxic lymphocytes derived from IL-2 and seroma treatments appeared to be more effective killers. Modulation of CD2 expression with seroma alone appeared to result in the generation of this highly cytotoxic population. This study demonstrates the role of CD2 expression in the effectiveness of LAK cell killing and also potential benefit of an immunotherapeutic approach to the postoperative treatment of carcinoma of the breast.

  13. Antileishmanial activity and cytotoxicity of Brazilian plants.

    Science.gov (United States)

    Ribeiro, Tatiana G; Chávez-Fumagalli, Miguel A; Valadares, Diogo G; Franca, Juçara R; Lage, Paula S; Duarte, Mariana C; Andrade, Pedro H R; Martins, Vivian T; Costa, Lourena E; Arruda, Ana L A; Faraco, André A G; Coelho, Eduardo A F; Castilho, Rachel O

    2014-08-01

    Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance has increased the importance of discovering new therapeutic products. The present study aimed to investigate the in vitro leishmanicidal activity from 16 different Brazilian medicinal plants. Stationary-phase promastigotes of Leishmania amazonensis and murine macrophages were exposed to 44 plant extracts or fractions for 48 h at 37°C, in order to evaluate their antileishmanial activity and cytotoxicity, respectively. The most potent extracts against L. amazonensis were the hexanic extract of Dipteryx alata (IC50 of 0.08 μg/mL), the hexanic extract of Syzygium cumini (IC50 of 31.64 μg/mL), the ethanolic and hexanic extracts of leaves of Hymenaea courbaril (IC50 of 44.10 μg/mL and 35.84 μg/mL, respectively), the ethanolic extract of H. stignocarpa (IC50 of 4.69 μg/mL), the ethanolic extract of Jacaranda caroba (IC50 of 13.22 μg/mL), and the ethanolic extract of J. cuspidifolia leaves (IC50 of 10.96 μg/mL). Extracts of D. alata and J. cuspidifolia presented higher selectivity index, with high leishmanicidal activity and low cytotoxicity in the mammalian cells. The capacity in treated infected macrophages using the extracts and/or fractions of D. alata and J. cuspidifolia was also analyzed, and reductions of 95.80%, 98.31%, and 97.16%, respectively, in the parasite burden, were observed. No nitric oxide (NO) production could be observed in the treated macrophages, after stimulation with the extracts and/or fractions of D. alata and J. cuspidifolia, suggesting that the biological activity could be due to mechanisms other than macrophage activation mediated by NO production. Based on phytochemistry studies, the classes of compounds that could contribute to the observed activities are also discussed. In conclusion, the data presented in this study indicated that traditional medicinal plant extracts present effective antileishmanial activity. Future studies could focus on

  14. Stability and cytotoxicity of crystallin amyloid nanofibrils

    Science.gov (United States)

    Kaur, Manmeet; Healy, Jackie; Vasudevamurthy, Madhusudan; Lassé, Moritz; Puskar, Ljiljana; Tobin, Mark J.; Valery, Celine; Gerrard, Juliet A.; Sasso, Luigi

    2014-10-01

    Previous work has identified crystallin proteins extracted from fish eye lenses as a cheap and readily available source for the self-assembly of amyloid nanofibrils. However, before exploring potential applications, the biophysical aspects and safety of this bionanomaterial need to be assessed so as to ensure that it can be effectively and safely used. In this study, crude crystallin amyloid fibrils are shown to be stable across a wide pH range, in a number of industrially relevant solvents, at both low and high temperatures, and in the presence of proteases. Crystallin nanofibrils were compared to well characterised insulin and whey protein fibrils using Thioflavin T assays and TEM imaging. Cell cytotoxicity assays suggest no adverse impact of both mature and fragmented crystallin fibrils on cell viability of Hec-1a endometrial cells. An IR microspectroscopy study supports long-term structural integrity of crystallin nanofibrils.Previous work has identified crystallin proteins extracted from fish eye lenses as a cheap and readily available source for the self-assembly of amyloid nanofibrils. However, before exploring potential applications, the biophysical aspects and safety of this bionanomaterial need to be assessed so as to ensure that it can be effectively and safely used. In this study, crude crystallin amyloid fibrils are shown to be stable across a wide pH range, in a number of industrially relevant solvents, at both low and high temperatures, and in the presence of proteases. Crystallin nanofibrils were compared to well characterised insulin and whey protein fibrils using Thioflavin T assays and TEM imaging. Cell cytotoxicity assays suggest no adverse impact of both mature and fragmented crystallin fibrils on cell viability of Hec-1a endometrial cells. An IR microspectroscopy study supports long-term structural integrity of crystallin nanofibrils. Electronic supplementary information (ESI) available: ThT fluorescence graphs of buffers and solvents used for

  15. Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1

    Directory of Open Access Journals (Sweden)

    Savinska L. O.

    2015-08-01

    Full Text Available Aim. Generation of polyclonal antibodies specific to the ribosomal protein S6 kinase isoform – p85S6K1 and directed to the N-terminal (1–23 aa extension of p85S6K1. Methods. Animal immunization with synthetic (1–23 aa peptide, ELISA, Western blot, Immunoprecipitation, immunofluorescent analysis. Results. Polyclonal antibodies have been generated, which specifically recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofluorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension – the identification of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms.

  16. Design and synthesis of novel cytotoxic podophyllotoxin derivatives

    Institute of Scientific and Technical Information of China (English)

    Wen Li Xi; Qian Cai; Yan Bo Tang; Hua Sun; Zhi Yan Xiao

    2010-01-01

    In order to investigate the effect of different C4 linkage moieties on the cytotoxicity of podophyllotoxin derivatives, novel 4-N-and 4-C-substituted 4'-O-demethylepipodophyllotoxin derivatives were designed and synthesized. All the compounds were tested against A549 and MCF-7 tumor cells in vitro, and six compounds showed significant cytotoxicity. The most active compound 9f was superior to GL-331, and exhibited potent cytotoxicity with IC50 value at 10-7 mol/L level.

  17. Comparative cytotoxicity of quantum dot and gold conjugates

    Science.gov (United States)

    Nadeau, Jay; Kumar, Anil; Dumas, Eve-Marie

    2009-02-01

    Nanoparticle toxicity is of interest for the design of anti-bacterial and anti-cancer agents, and also for development of regulations for their safe handling and disposal. However, the same reactivity that makes nanoparticles show unique optical properties also makes them interact with standard cytotoxicity agents, causing false positive and/or false negative results. We discuss which cytotoxicity assays are most likely to work with nanoparticles and which are to be avoided, and present some results on comparative cytotoxicity of different types of conjugates.

  18. NOVEL ANDROGRAPHOLIDE DERIVATIVES AND THEIR IN VITRO CYTOTOXIC ACTIVITY

    Directory of Open Access Journals (Sweden)

    Venkat R. P

    2014-05-01

    Full Text Available A new series of sulfonyl-type of andrographolide derivatives were synthesized from andrographolide, the cytotoxic constituent of the plant Andrographis paniculata. The derived analogs (4a-4g were evaluated for their cytotoxic activity against human small lung cancer (NCI-H187, leukemia K562, breast cancer (MCF-7/ADR and lung adenocarcinoma (A549 cell lines. Most of the analogues show significant cytotoxic activity against tested cell lines. The methyl sulfonyl derivative 4a had higher activity than parent compound andrographolide 1, and reduced activity than standard drug cisplatin against tested cell lines.

  19. Synthesis and In Vitro Cytotoxic Activity of Chromenopyridones

    Directory of Open Access Journals (Sweden)

    Balwinder Singh

    2013-01-01

    Full Text Available Novel substituted chromenopyridones (3a–j and 6a–d were synthesized and evaluated in vitro for the cytotoxic activity against various human cancer cell lines such as prostate (PC-3, breast (MCF-7, CNS (IMR-32, cervix (Hela, and liver (Hep-G2. preliminary cytotoxic screening showed that all the compounds possess a good to moderate inhibitory activity against various cancer cell lines. Particularly, compound 6b bearing allyl moiety displayed a significant cytotoxic potential in comparison to standard drugs.

  20. Nanomaterial Induced Immune Responses and Cytotoxicity.

    Science.gov (United States)

    Ali, Ashraf; Suhail, Mohd; Mathew, Shilu; Shah, Muhammad Ali; Harakeh, Steve M; Ahmad, Sultan; Kazmi, Zulqarnain; Alhamdan, Mohammed Abdul Rahman; Chaudhary, Adeel; Damanhouri, Ghazi Abdullah; Qadri, Ishtiaq

    2016-01-01

    Nanomaterials are utilized in a wide array of end user products such as pharmaceuticals, electronics, clothes and cosmetic products. Due to its size (< 100 nm), nanoparticles have the propensity to enter through the airway and skin, making its path perilous with the potential to cause damages of varying severity. Once within the body, these particles have unconstrained access to different tissues and organs including the brain, liver, and kidney. As a result, nanomaterials may cause the perturbation of the immune system eliciting an inflammatory response and cytotoxicity. This potential role is dependent on many factors such as the characteristics of the nanomaterials, presence or absence of diseases, and genetic predisposition. Cobalt and nickel nanoparticles, for example, were shown to have inflammogenic properties, while silver nanoparticles were shown to reduce allergic inflammation. Just as asbestos fibers, carbon nanotubes were shown to cause lungs damage. Some nanomaterials were shown, based on animal studies, to result in cell damage, leading to the formation of pre-cancerous lesions. This review highlights the impact of nanomaterials on immune system and its effect on human health with toxicity consideration. It recommends the development of suitable animal models to study the toxicity and bio-clearance of nanomaterials and propose safety guidelines.

  1. Nanomaterial Induced Immune Responses and Cytotoxicity.

    Science.gov (United States)

    Ali, Ashraf; Suhail, Mohd; Mathew, Shilu; Shah, Muhammad Ali; Harakeh, Steve M; Ahmad, Sultan; Kazmi, Zulqarnain; Alhamdan, Mohammed Abdul Rahman; Chaudhary, Adeel; Damanhouri, Ghazi Abdullah; Qadri, Ishtiaq

    2016-01-01

    Nanomaterials are utilized in a wide array of end user products such as pharmaceuticals, electronics, clothes and cosmetic products. Due to its size (< 100 nm), nanoparticles have the propensity to enter through the airway and skin, making its path perilous with the potential to cause damages of varying severity. Once within the body, these particles have unconstrained access to different tissues and organs including the brain, liver, and kidney. As a result, nanomaterials may cause the perturbation of the immune system eliciting an inflammatory response and cytotoxicity. This potential role is dependent on many factors such as the characteristics of the nanomaterials, presence or absence of diseases, and genetic predisposition. Cobalt and nickel nanoparticles, for example, were shown to have inflammogenic properties, while silver nanoparticles were shown to reduce allergic inflammation. Just as asbestos fibers, carbon nanotubes were shown to cause lungs damage. Some nanomaterials were shown, based on animal studies, to result in cell damage, leading to the formation of pre-cancerous lesions. This review highlights the impact of nanomaterials on immune system and its effect on human health with toxicity consideration. It recommends the development of suitable animal models to study the toxicity and bio-clearance of nanomaterials and propose safety guidelines. PMID:27398432

  2. Cytotoxicity and ion release of alloy nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Hahn, Anne [Laser Zentrum Hannover e.V (Germany); Fuhlrott, Jutta; Loos, Anneke [Hannover Medical School, Biovertraeglichkeitslabor BioMedimplant (Germany); Barcikowski, Stephan, E-mail: stephan.barcikowski@uni-due.de [Laser Zentrum Hannover e.V (Germany)

    2012-01-15

    It is well-known that nanoparticles could cause toxic effects in cells. Alloy nanoparticles with yet unknown health risk may be released from cardiovascular implants made of Nickel-Titanium or Cobalt-Chromium due to abrasion or production failure. We show the bio-response of human primary endothelial and smooth muscle cells exposed to different concentrations of metal and alloy nanoparticles. Nanoparticles having primary particle sizes in the range of 5-250 nm were generated using laser ablation in three different solutions avoiding artificial chemical additives, and giving access to formulations containing nanoparticles only stabilized by biological ligands. Endothelial cells are found to be more sensitive to nanoparticle exposure than smooth muscle cells. Cobalt and Nickel nanoparticles caused the highest cytotoxicity. In contrast, Titanium, Nickel-Iron, and Nickel-Titanium nanoparticles had almost no influence on cells below a nanoparticle concentration of 10 {mu}M. Nanoparticles in cysteine dissolved almost completely, whereas less ions are released when nanoparticles were stabilized in water or citrate solution. Nanoparticles stabilized by cysteine caused less inhibitory effects on cells suggesting cysteine to form metal complexes with bioactive ions in media.

  3. [Natural cytotoxic function and ankylosing spondylitis].

    Science.gov (United States)

    Wendling, D; Racadot, E; Guidet, M

    1989-10-01

    Natural Killer (NK) cell function was evaluated in 28 ankylosing spondylitis (AS) patients (21 B27 positive), at one hand by the use of a Leu7 (HNK-1) monoclonal antibody, at the other hand by spontaneous cytotoxicity against a K562 cell line (preincubated with a fluorogenic substrate) evaluated by a flow cytometric assay. There is no difference in NK cell activity neither between AS patients and controls nor between B27 positive and negative AS. The study brings no evidence for a NK function control by the B27 gene. There is no correlation between NK activity and each of the other parameters investigated (ESR, B2m, IgA, Leu7, T4/T8). At the opposite, NK activity is significantly decreased (p = 0,006) in AS patients under NSAIDs treatment compared with non treated patients. NSAIDs rather enhance NK activity, the decrease observed in the present study could be due to evolution of the disease itself, and may be a pathogenetic factor contributing to remaining of bacterial antigens according to the current hypothesis of the disease's aetiology.

  4. Cytotoxicity effects of amiodarone on cultured cells.

    Science.gov (United States)

    Golli-Bennour, Emna El; Bouslimi, Amel; Zouaoui, Olfa; Nouira, Safa; Achour, Abdellatif; Bacha, Hassen

    2012-07-01

    Amiodarone is a potent anti-arrhythmic drug used for the treatment of cardiac arrhythmias. Although, the effects of amiodarone are well characterized on post-ischemic heart and cardiomyocytes, its toxicity on extra-cardiac tissues is still poorly understood. To this aim, we have monitored the cytotoxicity effects of this drug on three cultured cell lines including hepatocytes (HepG2), epithelial cells (EAhy 926) and renal cells (Vero). We have investigated the effects of amiodarone on (i) cell viabilities, (ii) heat shock protein expressions (Hsp 70) as a parameter of protective and adaptive response and (iii) oxidative damage.Our results clearly showed that amiodarone inhibits cell proliferation, induces an over-expression of Hsp 70 and generates significant amount of reactive oxygen species as measured by lipid peroxidation occurrence. However, toxicity of amiodarone was significantly higher in renal and epithelial cells than in hepatocytes. Vitamin E supplement restores the major part of cell mortalities induced by amiodarone showing that oxidative damage is the predominant toxic effect of the drug.Except its toxicity for the cardiac system, our findings demonstrated that amiodarone can target other tissues. Therefore, kidneys present a high sensibility to this drug which may limit its use with subjects suffering from renal disorders.

  5. Prokaryotic Expression of Rubber Elongation Factor Gene and Preparation of Its Polyclonal Antibody%橡胶延长因子基因的原核表达及其多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    陈章权; 吴坤鑫; 梁晓东; 黄震; 陆田田

    2008-01-01

    [Objective] The aim of this study was to prepare the recombination protein of rubber elongation factor and its polyclonal antibodies. [Method] The encoding gene of rubber elongation factor (REF) was amplified by RT-PCR, and cloned into the prokaryotic expression vector pDEST17 to transform into Escherichia coil BI2I-AI. The recombinant protein induced by L-Arabinose was purified by the affinity chromatography. As the immunogen, the recombination protein was used to immunize mice for preparing polyclonal antibodies, while ELISA and Western blot hybridization were used to detect the titers and specificity. [Result] The purified recombination protein of REF with high expression was used to immunize house mice for preparing polyclonal antibodies with high titer and specificity. The western blot hybridization showed that the antibody could recognize the natural REF from latex. [Conclusion] The recombination protein of REF was successfully obtained and the mouse anti REF antibody with high titer and specificity was prepared, which lays a basis for further studies on biological functions of rubber elongation factor and other membrane proteins in rubber particles.

  6. The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri.

    Science.gov (United States)

    Sohn, Hae-Jin; Kim, Jong-Hyun; Shin, Myeong-Heon; Song, Kyoung-Ju; Shin, Ho-Joon

    2010-03-01

    Naegleria fowleri destroys target cells by trogocytosis, a phagocytosis mechanism, and a process of piecemeal ingestion of target cells by food-cups. Phagocytosis is an actin-dependent process that involves polymerization of monomeric G-actin into filamentous F-actin. However, despite the numerous studies concerning phagocytosis, its role in the N. fowleri food-cup formation related with trogocytosis has been poorly reported. In this study, we cloned and characterized an Nf-actin gene to elucidate the role of Nf-actin gene in N. fowleri pathogenesis. The Nf-actin gene is composed of 1,128-bp and produced a 54.1-kDa recombinant protein (Nf-actin). The sequence identity was 82% with nonpathogenic Naegleria gruberi but has no sequence identity with other mammals or human actin gene. Anti-Nf-actin polyclonal antibody was produced in BALB/c mice immunized with recombinant Nf-actin. The Nf-actin was localized on the cytoplasm, pseudopodia, and especially, food-cup structure (amoebastome) in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri co-cultured with Chinese hamster ovary cells, Nf-actin was observed to localize around on phagocytic food-cups. We also observed that N. fowleri treated with cytochalasin D as actin polymerization inhibitor or transfected with antisense oligomer of Nf-actin gene had shown the reduced ability of food-cup formation and in vitro cytotoxicity. Finally, it suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri.

  7. Cytotoxic diterpenoids from Jatropha curcas cv. nigroviensrugosus CY Yang Roots.

    Science.gov (United States)

    Liu, JieQing; Yang, YuanFeng; Xia, JianJun; Li, XuYang; Li, ZhongRong; Zhou, Lin; Qiu, MingHua

    2015-09-01

    An investigation of phytochemicals from the roots of Jatropha curcas cv. nigroviensrugosus resulted in the isolation of twenty diterpenoids, including lathyranlactone, an unusual diterpenoid lactone possessing a 5/13/3 tricyclic skeleton, jatrocurcasenones A-E and jatrophodiones B-E, as well as 10 known analogues. All isolates were evaluated for cytotoxicity against the HL-60, SMMC-772, A-549, MCF-7 and SW480 human tumor cell lines using the MTS viability assay. Four of the known analogues showed cytotoxic activity in these cell lines, with IC50 values ranging from 2.0 to 23.0 μM. Moreover, the assessment of their cytotoxic structure-activity relationships showed the epoxy ring between C-5 and C-6 and the hydroxyl group at C-2 were the key functionalities for cytotoxicity. PMID:26209936

  8. Antibacterial and cytotoxic compounds from the bark of Cananga odorata.

    Science.gov (United States)

    Rahman, M Mukhlesur; Lopa, Simin S; Sadik, Golam; Harun-Or-Rashid; Islam, Robiul; Khondkar, Proma; Alam, A H M Khurshid; Rashid, Mohammad A

    2005-12-01

    O-Methylmoschatoline, liriodenine and 3,4-dihydroxybenzoic acid isolated from the barks of Cananga odorata showed antibacterial activities against a number of Gram (+) and Gram (-) bacteria. The compounds also showed antifungal and cytotoxic activities. PMID:16242266

  9. Cytotoxic effect of Plantago spp. on cancer cell lines.

    Science.gov (United States)

    Gálvez, Marina; Martín-Cordero, Carmen; López-Lázaro, Miguel; Cortés, Felipe; Ayuso, María Jesús

    2003-10-01

    Methanolic extracts from seven Plantago species used in traditional medicine for the treatment of cancer, were evaluated for cytotoxic activity against three human cancer cell lines recommended by the National Cancer Institute (NCI, USA). The results showed that Plantago species exhibited cytotoxic activity, showing a certain degree of selectivity against the tested cells in culture. Since the flavonoids are able to strongly inhibit the proliferation of human cancer cell lines, we have identified luteolin-7-O-beta-glucoside as major flavonoid present in most of the Plantago species. Also, we have evaluated this compound and its aglycon, luteolin, for their cytotoxic and DNA topoisomerase I poisons activities. These results could justify the traditional use of the Plantago species and topoisomerase-mediated DNA damage might be a possible mechanism by which flavonoids of Plantago exert their cytotoxicity potential. PMID:12963131

  10. Antimicrobial and cytotoxic activities of Abroma augusta Lnn. leaves extract

    Institute of Scientific and Technical Information of China (English)

    FK Saikot; Alam Khan; MF Hasan

    2012-01-01

    Objective: To evaluate the antimicrobial and cytotoxic activity of acetone extract of leaves ofAbroma augusta. Methods: Disc diffusion method was used to demonstrate antibacterial and antifungal activities. Cytotoxicity was determined against brine shrimp nauplii. In addition, minimum inhibitory concentration (MIC) was determined using serial dilution technique to determine antibacterial potency. Results: The extract showed significant antibacterial activities against three gram-positive (Bacillus subtilis, Bacillus megaterium and Staphylococcus aureus) and four gram-negative (Escherichia coli, Shigella dysenteriae, Shigella sonnei and Salmonella typhi) bacteria. The antifungal activity was found strong against five fungi (Aspergillus flavus, Aspergillus niger, Candida albicans, Rhizopus oryzae and Aspergillus fumigatus). In cytotoxicity determination, LC50 of the extract against brine shrimp nauplii was 7.06μg/ml. Conclusions: The Abroma leaves extract may be consider as a potent antimicrobial and cytotoxic agent for further advance research.

  11. Antioxidant and cytotoxic activity of Acanthus ilicifolius flower

    Institute of Scientific and Technical Information of China (English)

    Muhamad; Firdaus; Asep; Awaludin; Prihanto; Rahmi; Nurdiani

    2013-01-01

    Objective:To investigate the antioxidant and cytotoxic activity of the flower of Acanthus ilicifolius(A.ilicifolius).Methods:Antioxidant activity was determined as antiradical efficiency with diphenyl picrylhydrazil(DPPH)method and cytotoxic assay was undertaken using brine shrimp lethal toxicity test.Results:A.ilicifolius flower contained terpenoid,phenolic compounds,and alkaloid.The methanol extract of A.ilicifolius flower showed the highest antiradical efficiency(AE=1.41×10-3)against DPPH radicals and the highest cytotoxicity(LC50=22μg/mL)against brine shrimp nauplii.Conclusions:It is suggested that active compounds of A.ilicifolius flower solved in methanol play a role to inhibit free radical activity and kill Artemia salina nauplii.The substances can be considered as potential antioxidant and cytotoxic agents as well as imminent candidate for cancer therapy.

  12. Antioxidant and cytotoxic activity of Acanthus ilicifolius flower

    Institute of Scientific and Technical Information of China (English)

    Muhamad Firdaus; Asep Awaludin Prihanto; Rahmi Nurdiani

    2013-01-01

    Objective: To investigate the antioxidant and cytotoxic activity of the flower of Acanthus ilicifolius (A. ilicifolius). Methods: Antioxidant activity was determined as antiradical efficiency with diphenyl picrylhydrazil (DPPH) method and cytotoxic assay was undertaken using brine shrimp lethal toxicity test. Results: A. ilicifolius flower contained terpenoid, phenolic compounds, and alkaloid. The methanol extract of A. ilicifolius flower showed the highest antiradical efficiency (AE=1.41í10-3) against DPPH radicals and the highest cytotoxicity (LC50=22 μg/mL) against brine shrimp nauplii. Conclusions: It is suggested that active compounds of A. ilicifolius flower solved in methanol play a role to inhibit free radical activity and kill Artemia salina nauplii. The substances can be considered as potential antioxidant and cytotoxic agents as well as imminent candidate for cancer therapy.

  13. Cytotoxic and immunogenic mechanisms of recombinant oncolytic poliovirus.

    Science.gov (United States)

    Brown, Michael C; Gromeier, Matthias

    2015-08-01

    An oncolytic virus (OV) based on poliovirus (PV), the highly attenuated polio/rhinovirus recombinant PVSRIPO, may deliver targeted inflammatory cancer cell killing; a principle that is showing promise in clinical trials for recurrent glioblastoma (GBM). The two decisive factors in PVSRIPO anti-tumor efficacy are selective cytotoxicity and its in situ immunogenic imprint. While our work is focused on what constitutes PVSRIPO cancer cytotoxicity, we are also studying how this engenders host immune responses that are vital to tumor regression. We hypothesize that PVSRIPO cytotoxicity and immunogenicity are inextricably linked in essential, complimentary roles that define the anti-neoplastic response. Herein we delineate mechanisms we unraveled to decipher the basis for PVSRIPO cytotoxicity and its immunotherapeutic potential. PMID:26083317

  14. Cytotoxic sesquiterpenes from the endophytic fungus Pseudolagarobasidium acaciicola.

    Science.gov (United States)

    Wibowo, Mario; Prachyawarakorn, Vilailak; Aree, Thammarat; Mahidol, Chulabhorn; Ruchirawat, Somsak; Kittakoop, Prasat

    2016-02-01

    Twenty previously unknown compounds and two known metabolites, merulin A and merulin D, were isolated from the endophytic fungus Pseudolagarobasidium acaciicola, which was isolated from a mangrove tree, Bruguiera gymnorrhiza. Structures of the 20 compounds were elucidated by analysis of spectroscopic data. The absolute configuration of seven of these compounds was addressed by a single crystal X-ray analysis using CuKα radiation and an estimate of the Flack parameter. Three compounds also possessed a tricyclic ring system. Terpene endoperoxides isolated exhibited cytotoxic activity, while those without an endoperoxide moiety did not show activity. The endoperoxide moiety of sesquiterpenes has significant impact on cytotoxic activity, and thus is an important functionality for cytotoxicity. One terpene endoperoxide displayed potent cytotoxic activity (IC50 0.28μM), and selectively exhibited activity against the HL-60 cell line. PMID:26701647

  15. 降钙素原的克隆表达及多克隆抗体制备%Cloning,expression and the polyclonal antibody preparation of procalcitonin

    Institute of Scientific and Technical Information of China (English)

    王春芳; 周新; 谢焱; 郑璇

    2011-01-01

    目的 克隆表达与纯化降钙素原(PCT),制备多克隆抗体,为其单抗制备及在临床上的应用研究提供有用的实验基础.方法 培养人甲状腺髓伴癌细胞株(TT细胞)提取总RNA进行RT-PCR获取PCT的全长基因,构建pET-PCT重组质粒,转化至DE3经IPTG诱导表达出融合蛋白,纯化并进行10%聚丙烯酰胺凝胶电泳(SDS-PAGE)、质谱测序,融合蛋白免疫兔制备多抗,采用ELISA和Western-blot进行鉴定.结果 成功获得PCT全长基因,10%SDS-PAGE电泳和质谱测序证明表达的融合蛋白为PCT,成功制备了PCT多抗.结论 成功获得PCT的全长基因,表达出融合蛋白并制备多抗,为研究PCT的病理、生理作用及功能奠定基础,为PCT的单抗制备及在临床应用研究提供了有用的实验材料.%Objective To construct Pet-PCT recombinant plasmid which contained the full length sequence of human PCT gene, express fusion protein,and produce polyclonal antibody production of PCT to serve as a useful experimental basis for preparing the procalcitonin monoclonal antibody and clinical application. Methods Full length Mrna of PCT was obtained from TT cells through RT-PCR,and inserted into Pet-32a( + ) expression vector,which was used to transfect E. Coli DE3 cells. Transfected DE3 cells were induced by IPTG to produce fusion PCT protein. The fusion protein with His tag was purified by Ni-NTA affinity chro-matography column, and verified by multiple techniques such as 10% sodium dodecy sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ,and MALDI-TOF mass spectrometry. The purified protein was used to immunize New Zealand rabbit to produce polyclonal antibody, which was verified by ELISA and Western blot. Results Recombinant vectors containing full length PCT Mrna insert were constructed,and were used to transfect the E. Coli DE3 cells for producing recombinant PCT protein. SDS-PAGE and MALDI-TOF mass spectrometry results showed the characteristics of the recombinant protein

  16. Helper cell-independent cytotoxic clones in man

    OpenAIRE

    1982-01-01

    We report here a class of helper cell-independent cytotoxic T cell (HITc) clones in man that can proliferate in response to antigenic stimulation as well as mediate cytotoxicity. HITc appear to be rare among clones derived from primary in vitro allosensitized culture, but constitute the majority of clones derived from cells sensitized to autologous Epstein-Barr virus-transformed lymphoblastoid cell lines. The implications of the derivation and function of HITc clones are discussed.

  17. DESIGN, SYNTHESIS AND CYTOTOXIC ACTIVITY OF SOME NOVEL FLAVONES

    OpenAIRE

    Palani Venkatesh et al.

    2012-01-01

    A series of flavones (5-15) were synthesized from resorcinol via Baker-Venkataraman transformation, characterized and evaluated for their cytotoxicity against human cervical cell line (HeLa) by MTT assay method. The result of preliminary MTT cytotoxicity studies shown that the compounds 5, 9 and 10 were found to be most potent against HeLa cells; IC50 values were of 0.22, 0.88 and 1.26 µM respectively.

  18. DESIGN, SYNTHESIS AND CYTOTOXIC ACTIVITY OF SOME NOVEL FLAVONES

    Directory of Open Access Journals (Sweden)

    Palani Venkatesh et al.

    2012-02-01

    Full Text Available A series of flavones (5-15 were synthesized from resorcinol via Baker-Venkataraman transformation, characterized and evaluated for their cytotoxicity against human cervical cell line (HeLa by MTT assay method. The result of preliminary MTT cytotoxicity studies shown that the compounds 5, 9 and 10 were found to be most potent against HeLa cells; IC50 values were of 0.22, 0.88 and 1.26 µM respectively.

  19. Cytotoxicity micropollutant removal in a crossflow membrane bioreactor

    OpenAIRE

    Delgado, Luis Fernando; Faucet-Marquis, Virginie; Pfohl-Leszkowicz, Annie; Dorandeu, Christophe; Marion, Bénédicte; Schetrite, Sylvie; Albasi, Claire

    2011-01-01

    The application of membrane bioreactor (MBR) technology was investigated with the aim of evaluating its potential for cytostatic drug and cytotoxicity bioremoval. The toxicity removal was assessed from biomarker test. CP removal of up to 80% was achieved under the operating conditions studied (HRT of 48 h and a SRT of 50 days). The increase of TMP was associated with an increase of supernatant toxicity as if fouling led to retention of the toxicity. Peaks of supernatant cytotoxicity were corr...

  20. Enhanced cytotoxicity of antineoplastic agents following prolonged exposure to misonidazole.

    OpenAIRE

    Roizin-Towle, L. A.; Hall, E. J.

    1981-01-01

    Chinese hamster V79 cells cultured in vitro were used to investigated the cytotoxicity of various anti-cancer drugs subsequent to a prolonged treatment of the cells with Misonidazole (MISO). The sensitivity of the cells to Bleomycin (BLM), Melphalan or cis-Platinum (cis-DDP) was significantly increased by prior incubation with MISO under hypoxic conditions. When cysteamine, a radical scavenger, was present during the pretreatment with MISO, this enhancement of cytotoxicity was greatly reduced...

  1. Cytotoxicity and Antineoplastic Activities of Alkylamines and Their Borane Derivatives

    OpenAIRE

    Hall, Iris H.; Tse, Elaine Y.; Muhammad, Rosallah A.

    1996-01-01

    The alkylamines and their related boron derivatives demonstrated potent cytotoxicity against the growth of murine and human tissue cultured cells. These agents did not necessarily require the boron atom to possess potent cytotoxic action in certain tumor lines. Their ability to suppress tumor cell growth was based on their inhibition of DNA and protein syntheses. DNA synthesis was reduced because purine synthesis was blocked at the enzyme site of IMP dehydrogenase by the agents. In addition r...

  2. Two New Cytotoxic Sesquiterpenoids from Eupatorium lindleyanum DC.

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Two new sesquiterpenoid lactones, namely eupalinilides K and L, were isolated from Eupatorium lindleyanum DC. Their structures were determined by spectral methods, including 1D- and 2D-NMR spectra. Cytotoxic evaluation showed that eupalinilide L exhibited potent cytotoxicity against P-388 and A-549 tumor cell lines with IC50values of 0.17 and 2.60 μ mol/L, respectively.

  3. CYTOTOXIC AND IMMUNOCHEMICAL PROPERTIES OF VISCUMIN ENCAPSULATED IN POLYLACTIDE MICROPARTICLES

    OpenAIRE

    Kolotova, E.; Egorova, S.; Ramonova, A.; Bogorodski, S.; Popov, V.; Agapov, I.; Kirpichnikov, M.

    2012-01-01

    Biodegradable polylactide microparticles with encapsulated cytotoxic protein viscumin were obtained via the ultrasound-assisted supercritical fluid technique. The size of the microparticles was 10–50 µM, as shown by electron microscopy. The time course of viscumin release from microparticles was studied using an immunoenzyme test system with anti-viscumin monoclonal antibodies. It was found that 99.91% of the cytotoxic protein was incorporated into polymer microparticles. Only 0.08% of the in...

  4. Evaluation of Northern Iran Mentha pulegium L. cytotoxicity

    OpenAIRE

    Shirazi, Farshad H.; Neda Ahmadi; Mohammad Kamalinejad

    2004-01-01

    In vitro tests could be a valuable tool for the evaluation of medicinal plants’ cytotoxicity. One of the most frequently used Iranian traditional plants is Mentha Pulegium from Labiatae family. In the present study, essential oil and the methanolic extract of Mentha pulegium, were analyzed for cytotoxicity on human ovary adenocarcinoma SK-OV-3, human malignant cervix carcinoma Hela, and human lung carcinoma A549 cell lines. Two different assays of clonogenic and neutral red (NR) were used for...

  5. Cytotoxic activity of Thai medicinal plants for cancer treatment

    OpenAIRE

    Chawaboon Dechsukum; Pranee Ratanasuwan; Niwat Keawpradub; Chatchai Wattanapiromsakul; Arunporn Itharat; Athima Saetung

    2005-01-01

    Twelve Thai medicinal plants as the ingredients of a Southern Thai traditional formula for cancer treatment were selected to test cytotoxicity activity against two types of human cancer cell lines ; large cell lung carcinoma (CORL-23) and prostate cancer cell lines (PC3) and one type of normal human cell line, fibroblast cells (10FS). SRB assay was used to test cytotoxic activity against all the cell types. Two of the extracts (water and ethanolic extracts) procedures used were similar to tho...

  6. Polyclonal non multiresistant methicillin resistant Staphylococcus aureus isolates from clinical cases of infection occurring in Palermo, Italy, during a one-year surveillance period

    Directory of Open Access Journals (Sweden)

    Mammina Caterina

    2012-06-01

    Full Text Available Abstract Background The evolving epidemiology of methicillin resistant Staphylococcus aureus (MRSA is characterized by the emergence of infections caused by non multiresistant MRSA carrying staphylococcal chromosomal cassette (SCCmec IV or V in the healthcare settings. A molecular epidemiological analysis of non multiresistant MRSA isolates from four acute general hospitals was performed in Palermo, Italy, during a one year period. Methods For the purpose of the study, MRSA isolates were defined as non multiresistant when they were susceptible to at least three classes of non β-lactam antibiotics. Seventy-five isolates were submitted to antimicrobial susceptibility testing, multilocus sequence typing (MLST and polymerase chain reaction (PCR for SCCmec, accessory gene regulator (agr groups, arginine catabolic mobile element (ACME and Panton Valentine leukocidin (PVL toxin genes. For epidemiological typing, Multiple-Locus Variable-Number Tandem Repeat Fingerprinting (MLVF was performed on all isolates and pulsed field gel electrophoresis (PFGE on ST8 isolates. Results Non multiresistant MRSA isolates were isolated from all hospitals. Resistances to ciprofloxacin, macrolides and tetracycline were the most prevalent. MLST attributed 46 isolates with ST22, 13 with ST8, eight with ST1, three with ST50 and three with ST398. SCCmec type IV was found in all isolates. PVL was detected in one ST22 isolate. All isolates tested negative for the ACME element. MLVF identified 31 different patterns, some subtype clusters ranging in size between two and 22 isolates. The closely related PFGE patterns of the ST8 isolates differed from USA300. Conclusions A polyclonal circulation of non multiresistant MRSA along with blurring of boundaries between healthcare associated (HA-MRSA and community associated (CA-MRSA appear to be occurring in our epidemiological setting. A better understanding of spread of MRSA with the support of molecular typing can provide invaluable

  7. Expression and Characterization of the Extracellular Domain of Human HER2 from Escherichia Coli, and Production of Polyclonal Antibodies Against the Recombinant Proteins.

    Science.gov (United States)

    Sun, Yong; Feng, Xue; Qu, Jiao; Han, Wenqi; Liu, Zi; Li, Xu; Zou, Ming; Zhen, Yuhong; Zhu, Jie

    2015-06-01

    Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family. In this study, the whole extracellular domain gene of HER2 was amplified by RT-PCR from human breast cancer cell line SK-BR-3. The genes of membrane-distal region (A) and membrane proximal region (B) of HER2 extracellular domain were amplified from the cloned template, and then inserted into the expression vector pET-28a and pET-30a, respectively. The recombinant expression vectors were transformed into Escherichia coli BL21 (DE3) cells and induced by isopropyl-b-D-thiogalactopyranoside (IPTG) for expression of proteins His-A and His-B. The expressed proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The optimization of culture conditions led us to accomplish the recombinant protein induction with 1.0 mM IPTG at 37 °C for 8 h, and both proteins were expressed in the insoluble form. Both proteins were purified under the denaturing condition using Ni-NTA sepharose column. Balb/c mice were immunized with the purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by ELISA testing and had good specificity by western blot detection. The HER2 ECD proteins His-A and His-B could be expressed in E. coli and were suitable for production of high titer antibodies against HER2 ECD. PMID:25906688

  8. Generation of polyclonal antibody with high avidity to rosuvastatin and its use in development of highly sensitive ELISA for determination of rosuvastatin in plasma

    Directory of Open Access Journals (Sweden)

    Al-Malaq Hamoud A

    2011-07-01

    Full Text Available Abstract In this study, a polyclonal antibody with high avidity and specificity to the potent hypocholesterolaemic agent rosuvastatin (ROS has been prepared and used in the development of highly sensitive enzyme-linked immunosorbent assay (ELISA for determination of ROS in plasma. ROS was coupled to keyhole limpt hemocyanin (KLH and bovine serum albumin (BSA using carbodiimide reagent. ROS-KLH conjugate was used for immunization of female 8-weeks old New Zealand white rabbits. The immune response of the rabbits was monitored by direct ELISA using ROS-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and avidity to ROS was scarified and its sera were collected. The IgG fraction was isolated and purified by avidity chromatography on protein A column. The purified antibody showed high avidity to ROS; IC50 = 0.4 ng/ml. The specificity of the antibody for ROS was evaluated by indirect ELISA using various competitors from the ROS-structural analogues and the therapeutic agents used with ROS in a combination therapy. The proposed ELISA involved a competitive binding reaction between ROS, in plasma sample, and the immobilized ROS-BSA for the binding sites on a limited amount of the anti-ROS antibody. The bound anti-ROS antibody was quantified with horseradish peroxidase-labeled second anti-rabbit IgG antibody (HRP-IgG and 3,3',5,5'-tetramethylbenzidine (TMB as a substrate for the peroxidase enzyme. The concentration of ROS in the sample was quantified by its ability to inhibit the binding of the anti-ROS antibody to the immobilized ROS-BSA and subsequently the color intensity in the assay wells. The assay enabled the determination of ROS in plasma at concentrations as low as 40 pg/ml.

  9. Comparison of biotinylated monoclonal and polyclonal antibodies in an evaluation of a direct rapid immunohistochemical test for the routine diagnosis of rabies in southern Africa.

    Directory of Open Access Journals (Sweden)

    Andre Coetzer

    2014-09-01

    Full Text Available The major etiological agent of rabies, rabies virus (RABV, accounts for tens of thousands of human deaths per annum. The majority of these deaths are associated with rabies cycles in dogs in resource-limited countries of Africa and Asia. Although routine rabies diagnosis plays an integral role in disease surveillance and management, the application of the currently recommended direct fluorescent antibody (DFA test in countries on the African and Asian continents remains quite limited. A novel diagnostic assay, the direct rapid immunohistochemical test (dRIT, has been reported to have a diagnostic sensitivity and specificity equal to that of the DFA test while offering advantages in cost, time and interpretation. Prior studies used the dRIT utilized monoclonal antibody (MAb cocktails. The objective of this study was to test the hypothesis that a biotinylated polyclonal antibody (PAb preparation, applied in the dRIT protocol, would yield equal or improved results compared to the use of dRIT with MAbs. We also wanted to compare the PAb dRIT with the DFA test, utilizing the same PAb preparation with a fluorescent label. The PAb dRIT had a diagnostic sensitivity and specificity of 100%, which was shown to be marginally higher than the diagnostic efficacy observed for the PAb DFA test. The classical dRIT, relying on two-biotinylated MAbs, was applied to the same panel of samples and a reduced diagnostic sensitivity (83.50% and 90.78% respectively was observed. Antigenic typing of the false negative samples indicated all of these to be mongoose RABV variants. Our results provided evidence that a dRIT with alternative antibody preparations, conjugated to a biotin moiety, has a diagnostic efficacy equal to that of a DFA relying on the same antibody and that the antibody preparation should be optimized for virus variants specific to the geographical area of focus.

  10. A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by Escherichia coli in human and environmental samples.

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    Xiaohua He

    Full Text Available BACKGROUND: Shiga toxin-producing Escherichia coli (STEC are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains. METHODS AND FINDINGS: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity. CONCLUSIONS: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

  11. Cytotoxicity of commonly used luting cements -An in vitro study.

    Science.gov (United States)

    Trumpaite-Vanagiene, Rita; Bukelskiene, Virginija; Aleksejuniene, Jolanta; Puriene, Alina; Baltriukiene, Daiva; Rutkunas, Vygandas

    2015-01-01

    The study aimed to 1) evaluate the cytotoxicity of luting cements: Hoffmann's Zinc Phosphate (Hoffmann's ZP), GC Fuji Plus Resin Modified Glass Ionomer (Fuji Plus RMGI) and 3M ESPE RelyX Unicem Resin Cement (RelyX Unicem RC) and 2) test if pre-washing reduces the cements' cytotoxicity. In vitro human gingival fibroblast (HGF) culture model was chosen. The cytotoxicity was evaluated by MTT test, the cell viability -by staining the cells with AO/EB dye mixture. The means±SD of Cell Survival Ratio (CSR%) were compared among different cement types under two testing conditions, with or without cement pre-washing. The CSR%s were compared by ANOVA and linear multiple regression (LMR). Hoffmann's ZPC was less cytotoxic, while Fuji Plus RMGIC and RelyX Unicem RC were more cytotoxic (ANOVA, pcement and cement pre-washing jointly explained 90% of cell survival (LMR, pcements such as Hoffmann's ZP, Fuji Plus RMGI and RelyX Unicem RC may have a cytotoxic potential. PMID:25904168

  12. Phytochemical and Cytotoxic Investigations of Curcuma mangga Rhizomes

    Directory of Open Access Journals (Sweden)

    Sri Nurestri A. Malek

    2011-05-01

    Full Text Available Investigations on the cytotoxic effects of the crude methanol and fractionated extracts (hexane, ethyl acetate C. mangga against six human cancer cell lines, namely the hormone-dependent breast cell line (MCF-7, nasopharyngeal epidermoid cell line (KB, lung cell line (A549, cervical cell line (Ca Ski, colon cell lines (HCT 116 and HT-29, and one non-cancer human fibroblast cell line (MRC-5 were conducted using an in-vitro neutral red cytotoxicity assay. The crude methanol and fractionated extracts (hexane and ethyl acetate displayed good cytotoxic effects against MCF-7, KB, A549, Ca Ski and HT-29 cell lines, but exerted no damage on the MRC-5 line. Chemical investigation from the hexane and ethyl acetate fractions resulted in the isolation of seven pure compounds, namely (E-labda-8(17,12-dien-15,16-dial (1, (E-15,16-bisnor-labda-8(17,11-dien-13-on (2, zerumin A (3, β-sitosterol, curcumin, demethoxycurcumin and bis-demethoxycurcumin. Compounds 1 and 3 exhibited high cytotoxic effects against all six selected cancer cell lines, while compounds 2 showed no anti-proliferative activity on the tested cell lines. Compound 1 also demonstrated strong cytotoxicity against the normal cell line MRC-5. This paper reports for the first time the cytotoxic activities of C. mangga extracts on KB, A549, Ca Ski, HT-29 and MRC-5, and the occurrence of compound 2 and 3 in C. mangga.

  13. Insights on cytotoxic cells of the colonial ascidian Botryllus schlosseri

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    N Franchi

    2015-03-01

    Full Text Available Morula cells (MCs represent the most abundant circulating hemocyte of the compound ascidian Botryllus schlosseri. They are cytotoxic cells involved in the rejection reaction between contacting, genetically incompatible colonies. Upon the recognition of foreign substances, they degranulate and release their content, which contribute to the cell death along the contact borders. A major role in MC-related cytotoxicity is exerted by the enzyme phenoloxidase (PO that converts polyphenol substrata to quinones which, then, polymerize to form melanins. During this reaction, reactive oxygen species are formed which are the cause of MC-related cytotoxicity. Here, we carried out new analyses to investigate further the nature of MC content and its role in cytotoxicity. Results confirm that PO is located inside MC vacuoles together with arylsulfatase, iron and polyphenols/quinones, the latter probably representing ready-to-use cytotoxic molecules, deriving from the oxidation of DOPA-containing proteins. In addition, small DOPA-containing peptides, called tunichromes, are also present inside MCs. MC degranulation and PO-mediated cytotoxicity are prevented by secretion inhibitors and by H89 and calphostin C. The observation that PO activity is always detectable in MCs in the absence of protease treatment, and its inhibition by sulfites and sulfates, suggest a non-classical pathway of PO modulation in botryllid ascidians.

  14. Antioxidants protect keratinocytes against M. ulcerans mycolactone cytotoxicity.

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    Alvar Grönberg

    Full Text Available BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS. We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+, completely prevented mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.

  15. Molecular cytotoxic mechanisms of chlorpromazine in isolated rat hepatocytes.

    Science.gov (United States)

    MacAllister, Stephanie L; Young, Cheryl; Guzdek, Anna; Zhidkov, Nickholas; O'Brien, Peter J

    2013-01-01

    Chlorpromazine (CPZ), a member of the largest class of first-generation antipsychotic agents, is known to cause hepatotoxicity in the form of cholestasis and hepatocellular necrosis in some patients. The mechanism of CPZ hepatotoxicity is unclear, but is thought to result from reactive metabolite formation. The goal of this research was to assess potential cytotoxic mechanisms of CPZ using the accelerated cytotoxicity mechanism screening (ACMS) technique with freshly isolated rat hepatocytes. This study identified CPZ cytotoxicity and inhibition of mitochondrial membrane potential (MMP) to be concentration-dependent. Furthermore, inhibition of cytochrome P450s (CYPs), including CYP2D1 and 1A2, delayed CPZ cytotoxicity, suggesting a role for CYP activation of CPZ to a toxic metabolite(s) in this model. Metabolism studies also demonstrated glucuronide and glutathione (GSH) requirement for CPZ detoxification in hepatocytes. Inactivating the 2-electron reduction pathway, NAD(P)H quinone oxidoreductase (NQO1), caused a significant increase in hepatocyte susceptibility to CPZ, indicating quinoneimine contribution to CPZ cytotoxicity. Nontoxic concentrations of peroxidase/H(2)O(2) (inflammatory model) increased cytotoxicity in CPZ-treated hepatocytes and caused additional mitochondrial toxicity. Inflammation further depleted GSH and increased oxidized glutathione (GSSG) levels. Results suggest activation of CPZ to reactive metabolites by 2 pathways in hepatocytes: (i) a CYP-catalyzed quinoneimine pathway, and (ii) a peroxidase-catalyzed oxidation of CPZ to CPZ radicals.

  16. Long-term cytotoxic effects of contemporary root canal sealers

    Directory of Open Access Journals (Sweden)

    Emmanuel Joao Nogueira Leal da SILVA

    2013-03-01

    Full Text Available Objectives The aim of the present study was to investigate the effects of root canal sealers on the cytotoxicity of 3T3 fibroblasts during a period of 5 weeks. Material and Methods Fibroblasts (3T3, 1×105 cells per well were incubated with elutes of fresh specimens from eight root canal sealers (AH Plus, Epiphany, Endomethasone N, EndoREZ, MTA Fillapex, Pulp Canal Sealer EWT, RoekoSeal and Sealapex and with elutes of the same specimens for 5 succeeding weeks after immersing in simulated body fluid. The cytotoxicity of all root canal sealers was determined using the MTT assay. Data were analyzed using ANOVA and Tukey's test. Results RoekoSeal was the only sealer that did not show any cytotoxic effects (p<0.05. All the other tested sealers exhibited severe toxicity initially (week 0. MTA Fillapex remained moderately cytotoxic after the end of experimental period. Toxicity of the other tested sealers decreased gradually over time. The evaluated root canal sealers presented varying degrees of cytotoxicity, mainly in fresh mode. Conclusions RoekoSeal had no cytotoxic effect both freshly mixed and in the other tested time points. MTA Fillapex was associated with significantly less cell viability when compared to the other tested root canal sealers.

  17. Neurobiological changes by cytotoxic agents in mice.

    Science.gov (United States)

    Seigers, R; Loos, M; Van Tellingen, O; Boogerd, W; Smit, A B; Schagen, S B

    2016-02-15

    Cognitive deficit is a frequently reported side-effect of adjuvant chemotherapy. A large number of animal studies has been performed to examine the neurobiological mechanisms underlying this phenomenon, however, definite conclusions from these studies are restricted due to differences in experimental set-up. We systematically investigated the effects of 6 cytotoxic agents on various neurobiological parameters. C57Bl/6J mice were treated with cyclophosphamide, docetaxel, doxorubicin, 5-fluorouracil, methotrexate, or topotecan. The animals were sacrificed 3 or 15 weeks after treatment and the effect on neurogenesis, blood vessel density, and neuroinflammation was analyzed using immunohistochemistry. None of the cytostatic agents tested affected neurogenesis (cell survival or cell proliferation). Blood vessel density was increased in the hippocampus and prefrontal cortex 3 weeks after treatment with docetaxel and doxorubicin compared with control animals. A decrease in the number of microglial cells was observed in the prefrontal cortex after treatment with cyclophosphamide, docetaxel, 5-FU, and topotecan compared with control mice. The observed decrease in microglia cells is indicative of inflammation that occurred after treatment. Overall, the magnitude of the effects was relatively modest. Therefore, we conducted a similar study with topotecan in Abcg2;Abcb1a/b knock out and wildtype FVB mice. Animals were sacrificed 3 weeks after treatment and no notable effect was seen in hippocampal cell differentiation (DCX), microglia activation, or blood vessel density. Perhaps the FVB strain is more resistant to the neurotoxic effects of topotecan which makes this not the correct model to study the mechanism of chemotherapy-induced cognitive impairment. PMID:26602283

  18. Cytotoxic activity of Thai medicinal plants for cancer treatment

    Directory of Open Access Journals (Sweden)

    Chawaboon Dechsukum

    2005-08-01

    Full Text Available Twelve Thai medicinal plants as the ingredients of a Southern Thai traditional formula for cancer treatment were selected to test cytotoxicity activity against two types of human cancer cell lines ; large cell lung carcinoma (CORL-23 and prostate cancer cell lines (PC3 and one type of normal human cell line, fibroblast cells (10FS. SRB assay was used to test cytotoxic activity against all the cell types. Two of the extracts (water and ethanolic extracts procedures used were similar to those practised by Thai traditional doctors. One concentration (50 μg/ml of two different extracts was tested first against cell lines and the active plant extracts were diluted and tested for calculating IC50. The ethanolic extracts of six plants (Bridelia ovata, Curcuma zedoaria, Derris scandens, Dioscorea membranacea, Nardostachys jatamansi and Rhinacanthus nasutus showed cytotoxic activity (IC50< 30 μg/ml against lung and prostate cancer cell lines. Dioscorea membranacea roots showed the highest cytotoxic activity against lung cancer cell lines ( IC50= 4.6 μg/ml but it exhibited low cytotoxic activity against prostate cancer cell lines (IC50= 17.55 μg/ml and less cytotoxic activity against normal cell lines (IC50= 66.05 μg/ml. Curcuma zedoaria showed cytotoxic activity against COR L-23 and PC3 but less cytotoxic activity against 10FS (IC50 = 6.05, 17.84 and 55.50 μg/ml respectively Rhinacanthus nasutus root extract showed the highest cytotoxic activity against PC3 ( IC50 = 2.01 μg/ml and this extract also showed high activity against COR L-23 and 10FS (IC50=5.05 and 10.95 μg/ml respectively. The water extract of all plants exhibited no activity against all types of human cells. Two ethanolic plant extracts (Dioscorea membranacea and Curcuma zedoaria which showed specific activity against lung cancer cell lines and less cytotoxic activity against normal cells should be further investigated for active compounds against lung cancer cell.

  19. Protective effects of rilmenidine and AGN 192403 on oxidative cytotoxicity and mitochondrial inhibitor-induced cytotoxicity in astrocytes.

    Science.gov (United States)

    Choi, Dong-Hee; Kim, Dong-Hoon; Park, Yun-Gyu; Chun, Boe-Gwun; Choi, Sang-Hyun

    2002-11-15

    Oxidative stress and mitochondrial dysfunction are important aspects of pathogenesis, particularly in the brain, which is highly dependent on oxygen, and the protection of astrocytes is essential for neuroprotection. In this context, imidazoline drugs have been reported to be neuroprotective. Our recent study showed that imidazoline drugs, including guanabenz, inhibit the naphthazarin-induced oxidative cytotoxicity associated with lysosomal destabilization. We now report on a study into the protective effects of rilmenidine and AGN 192403, which have affinity for imidazoline-1 receptors, on the cytotoxicity induced by naphthazarin and inhibitors of mitochondrial respiration in astrocytes. Cytotoxicity was measured grossly by LDH release and by measuring changes in lysosomal membrane stability and features of mitochondrial membrane permeabilization. Naphthazarin-induced cytotoxicity was evidenced by the ordered development of lysosomal acridine orange relocation, decrease in mitochondrial potential, cytochrome c release, and caspase-9 activation, and was inhibited by guanabenz, rilmenidine, and AGN 192403. Antimycin A and rotenone induced mitochondrial dysfunction primarily, and their cytotoxicities were inhibited only by AGN 192403. Rilmenidine and guanabenz may have a lysosomal stabilizing effect, which underlies their protective effects. AGN 192403 might affect the mitochondrial cell death cascades, and had a novel protective effect on the cytotoxicity associated with mitochondrial dysfunction.

  20. The tumor targeted superantigen ABR-217620 selectively engages TRBV7-9 and exploits TCR-pMHC affinity mimicry in mediating T cell cytotoxicity.

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    Gunnar Hedlund

    Full Text Available The T lymphocytes are the most important effector cells in immunotherapy of cancer. The conceptual objective for developing the tumor targeted superantigen (TTS ABR-217620 (naptumomab estafenatox, 5T4Fab-SEA/E-120, now in phase 3 studies for advanced renal cell cancer, was to selectively coat tumor cells with cytotoxic T lymphocytes (CTL target structures functionally similar to natural CTL pMHC target molecules. Here we present data showing that the molecular basis for the anti-tumor activity by ABR-217620 resides in the distinct interaction between the T cell receptor β variable (TRBV 7-9 and the engineered superantigen (Sag SEA/E-120 in the fusion protein bound to the 5T4 antigen on tumor cells. Multimeric but not monomeric ABR-217620 selectively stains TRBV7-9 expressing T lymphocytes from human peripheral blood similar to antigen specific staining of T cells with pMHC tetramers. SEA/E-120 selectively activates TRBV7-9 expressing T lymphocytes resulting in expansion of the subset. ABR-217620 selectively triggers TRBV7-9 expressing cytotoxic T lymphocytes to kill 5T4 positive tumor cells. Furthermore, ABR-217620 activates TRBV7-9 expressing T cell line cells in the presence of cell- and bead-bound 5T4 tumor antigen. Surface plasmon resonance analysis revealed that ABR-217620 binds to 5T4 with high affinity, to TRBV7-9 with low affinity and to MHC class II with very low affinity. The T lymphocyte engagement by ABR-217620 is constituted by displaying high affinity binding to the tumor cells (KD approximately 1 nM and with the mimicry of natural productive immune TCR-pMHC contact using affinities of around 1 µM. This difference in kinetics between the two components of the ABR-217620 fusion protein will bias the binding towards the 5T4 target antigen, efficiently activating T-cells via SEA/E-120 only when presented by the tumor cells.

  1. CD85/LIR-1/ILT2 and CD152 (cytotoxic T lymphocyte antigen 4) inhibitory molecules down-regulate the cytolytic activity of human CD4+ T-cell clones specific for Mycobacterium tuberculosis.

    Science.gov (United States)

    Merlo, A; Saverino, D; Tenca, C; Grossi, C E; Bruno, S; Ciccone, E

    2001-10-01

    Antigen-specific cytolytic CD4+ T lymphocytes control Mycobacterium tuberculosis infection by secreting cytokines and by killing macrophages that have phagocytosed the pathogen. However, lysis of the latter cells promotes microbial dissemination, and other macrophages engulf the released bacteria. Subsequently, CD4+ T-cell-mediated killing of macrophages goes on, and this persistent process may hamper control of infection, unless regulatory mechanisms maintain a subtle balance between lysis of macrophages by cytolytic CD4+ cells and activation of cytolytic CD4+ cells by infected macrophages. We asked whether inhibitory molecules expressed by CD4+ cytolytic T lymphocytes could play a role in such a balance. To this end, human CD4+ T-cell clones specific for M. tuberculosis were produced that displayed an autologous major histocompatibility complex class II-restricted lytic ability against purified protein derivative (PPD)-pulsed antigen-presenting cells. All T-cell clones expressed CD152 (cytotoxic T-lymphocyte antigen 4 [CTLA-4]) and CD85/leukocyte immunoglobulin-like receptor 1 (LIR-1)/immunoglobulin-like transcript 2 (ILT2) inhibitory receptors, but not CD94 and the killer inhibitory receptor (or killer immunoglobulin-like receptor [KIR]) p58.2. CD3-mediated activation of the clones was inhibited in a redirected killing assay in which CD152 and CD85/LIR-1/ILT2 were cross-linked. Specific antigen-mediated proliferation of the clones was also sharply reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked by specific monoclonal antibody (MAb) followed by goat anti-mouse antiserum. In contrast, blockade of the receptors by specific MAb only increased their proliferation. Production of interleukin 2 (IL-2) and gamma interferon (IFN-gamma) by the T-cell clones was also strongly reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked. The lytic activity of the T-cell clones against PPD-pulsed autologous monocytes or Epstein-Barr virus-activated B cells was increased

  2. New approach to immunochemical determinations for triclopyr and 3,5,6-trichloro-2-pyridinol by using a bifunctional hapten, and evaluation of polyclonal antiserum.

    Science.gov (United States)

    Watanabe, Eiki; Hoshino, Ryoko; Kanzaki, Yukiko; Tokumoto, Hiroshi; Kubo, Hiroaki; Nakazawa, Hiroyuki

    2002-06-19

    The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed

  3. Human Polyclonal Antibodies Produced through DNA Vaccination of Transchromosomal Cattle Provide Mice with Post-Exposure Protection against Lethal Zaire and Sudan Ebolaviruses.

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    Callie E Bounds

    Full Text Available DNA vaccination of transchromosomal bovines (TcBs with DNA vaccines expressing the codon-optimized (co glycoprotein (GP genes of Ebola virus (EBOV and Sudan virus (SUDV produce fully human polyclonal antibodies (pAbs that recognize both viruses and demonstrate robust neutralizing activity. Each TcB was vaccinated by intramuscular electroporation (IM-EP a total of four times and at each administration received 10 mg of the EBOV-GPco DNA vaccine and 10 mg of the SUDV-GPco DNA vaccine at two sites on the left and right sides, respectively. After two vaccinations, robust antibody responses (titers > 1000 were detected by ELISA against whole irradiated EBOV or SUDV and recombinant EBOV-GP or SUDV-GP (rGP antigens, with higher titers observed for the rGP antigens. Strong, virus neutralizing antibody responses (titers >1000 were detected after three vaccinations when measured by vesicular stomatitis virus-based pseudovirion neutralization assay (PsVNA. Maximal neutralizing antibody responses were identified by traditional plaque reduction neutralization tests (PRNT after four vaccinations. Neutralizing activity of human immunoglobulins (IgG purified from TcB plasma collected after three vaccinations and injected intraperitoneally (IP into mice at a 100 mg/kg dose was detected in the serum by PsVNA up to 14 days after administration. Passive transfer by IP injection of the purified IgG (100 mg/kg to groups of BALB/c mice one day after IP challenge with mouse adapted (ma EBOV resulted in 80% protection while all mice treated with non-specific pAbs succumbed. Similarly, interferon receptor 1 knockout (IFNAR(-/- mice receiving the purified IgG (100 mg/kg by IP injection one day after IP challenge with wild type SUDV resulted in 89% survival. These results are the first to demonstrate that filovirus GP DNA vaccines administered to TcBs by IM-EP can elicit neutralizing antibodies that provide post-exposure protection. Additionally, these data describe

  4. Cytotoxic activity and phytochemical analysis of Arum palaestinum Boiss

    Institute of Scientific and Technical Information of China (English)

    Mai Mohammed Farid; Sameh Reda Hussein; Lamiaa Fawzy Ibrahim; Mohammed Ali El Desouky; Amr Mohammed Elsayed; Ahmad Ali El Oqlah; Mahmoud Mohammed Saker

    2015-01-01

    Objective:To evaluate the in vitro cytotoxic activity of the fractionated extract as well as isolated compounds of Arum palaestinum Boiss. (A. palaestinum) (black calla lily), and to identify the volatile components which may be responsible for the potential antitumor activity. Methods: A. palaestinum was collected from its natural habitats and subjected to phytochemical analysis for separation of pure compounds. In vitro cytotoxic activity was investigated against four human carcinoma cell lines Hep2, HeLa, HepG2 and MCF7 for the fractionated extract and isolated compounds. While, the diethyl ether fraction was subjected to GC–MS analysis as it exhibited the most potent cytotoxic effect to evaluate the active constituents responsible for the cytotoxic activities. Results:Four flavonoid compounds were isolated (luteolin, chrysoeriol, isoorientin, isovitexin) from the diethyl ether and ethyl acetate. The extracts and the pure isolated compounds showed a significant high antiproliferative activity against all investigated cell lines. The GC–MS analysis revealed the separation and identification of 15 compounds representing 95.01%of the extract and belonging to different groups of chemical compounds. Conclusions:The present study is considered to be the first report on the cytotoxic activities carried out on different selected fractions and pure compounds of A. palaestinum to provide evidences for its strong antitumor activities. In addition, chrysoeriol and isovitexin compounds were isolated for the first time from the studied taxa.

  5. Hormonal activity, cytotoxicity and developmental toxicity of UV filters.

    Science.gov (United States)

    Balázs, Adrienn; Krifaton, Csilla; Orosz, Ivett; Szoboszlay, Sándor; Kovács, Róbert; Csenki, Zsolt; Urbányi, Béla; Kriszt, Balázs

    2016-09-01

    Ultraviolet (UV) filters are commonly used compounds in personal care products and polymer based materials, as they can absorb solar energy in the UVA and UVB spectrum. However, they are able to bind to hormone receptors and have several and different types of hormonal activities determined by in vitro assays. One of the aims of this work was to measure the hormonal and cytotoxic activities of four frequently used UV filters using bioluminescence based yeast test organisms. Using Saccharomyces cerevisiae BLYES and BLYAS strains allowed the rapid and reliable detection of agonist and antagonist hormonal activities, whereas BLYR strain served to measure cytotoxicity. Results confirmed that all tested UV filters show multiple hormonal activities. Cytotoxicity is detected only in the case of benzophenone-3. Research data on the toxic effects of benzophenone-3, especially on aquatic organisms are scarce, so further investigations were carried out regarding its cytotoxic and teratogenic effects on bacteria and zebrafish (Danio rerio) embryos, respectively. Results revealed the cytotoxicity of benzophenone-3 not only to yeasts but to bacteria, as well as its ability to influence zebrafish embryo hatching and development. PMID:27208882

  6. CRTAM determines the CD4+ cytotoxic T lymphocyte lineage.

    Science.gov (United States)

    Takeuchi, Arata; Badr, Mohamed El Sherif Gadelhaq; Miyauchi, Kosuke; Ishihara, Chitose; Onishi, Reiko; Guo, Zijin; Sasaki, Yoshiteru; Ike, Hiroshi; Takumi, Akiko; Tsuji, Noriko M; Murakami, Yoshinori; Katakai, Tomoya; Kubo, Masato; Saito, Takashi

    2016-01-11

    Naive T cells differentiate into various effector T cells, including CD4(+) helper T cell subsets and CD8(+) cytotoxic T cells (CTL). Although cytotoxic CD4(+) T cells (CD4 +: CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4(+) T cells that express class I-restricted T cell-associated molecule (CRTAM) upon activation possesses the characteristics of both CD4(+) and CD8(+) T cells. CRTAM(+) CD4(+) T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM(+) T cells are the precursor of CD4(+)CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4(+)CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM(+) T cells traffic to mucosal tissues and inflammatory sites and developed into CD4(+)CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4(+)CTL through the induction of Eomes and CTL-related gene.

  7. Cytotoxic activity and phytochemical analysis of Arum palaestinum Boiss.

    Institute of Scientific and Technical Information of China (English)

    Mai; Mohammed; Farid; Sameh; Reda; Hussein; Lamiaa; Fawzy; Ibrahim; Mohammed; Ali; El; Desouky; Amr; Mohammed; Elsayed; Ahmad; Ali; El; Oqlah; Mahmoud; Mohammed; Saker

    2015-01-01

    Objective: To evaluate the in vitro cytotoxic activity of the fractionated extract as well as isolated compounds of Arum palaestinum Boiss.(A. palaestinum)(black calla lily), and to identify the volatile components which may be responsible for the potential antitumor activity.Methods: A. palaestinum was collected from its natural habitats and subjected to phytochemical analysis for separation of pure compounds. In vitro cytotoxic activity was investigated against four human carcinoma cell lines Hep2, He La, Hep G2 and MCF7 for the fractionated extract and isolated compounds. While, the diethyl ether fraction was subjected to GC–MS analysis as it exhibited the most potent cytotoxic effect to evaluate the active constituents responsible for the cytotoxic activities.Results: Four flavonoid compounds were isolated(luteolin, chrysoeriol, isoorientin,isovitexin) from the diethyl ether and ethyl acetate. The extracts and the pure isolated compounds showed a significant high antiproliferative activity against all investigated cell lines. The GC–MS analysis revealed the separation and identification of 15 compounds representing 95.01% of the extract and belonging to different groups of chemical compounds.Conclusions: The present study is considered to be the first report on the cytotoxic activities carried out on different selected fractions and pure compounds of A. palaestinum to provide evidences for its strong antitumor activities. In addition,chrysoeriol and isovitexin compounds were isolated for the first time from the studied taxa.

  8. Phytochemical and Cytotoxic Investigations of Alpinia mutica Rhizomes

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    Kae Shin Sim

    2011-01-01

    Full Text Available The methanol and fractionated extracts (hexane, ethyl acetate and water of Alpinia mutica (Zingiberaceae rhizomes were investigated for their cytotoxic effect against six human carcinoma cell lines, namely KB, MCF7, A549, Caski, HCT116, HT29 and non-human fibroblast cell line (MRC 5 using an in vitro cytotoxicity assay. The ethyl acetate extract possessed high inhibitory effect against KB, MCF7 and Caski cells (IC50 values of 9.4, 19.7 and 19.8 µg/mL, respectively. Flavokawin B (1, 5,6-dehydrokawain (2, pinostrobin chalcone (3 and alpinetin (4, isolated from the active ethyl acetate extract were also evaluated for their cytotoxic activity. Of these, pinostrobin chalcone (3 and alpinetin (4 were isolated from this plant for the first time. Pinostrobin chalcone (3 displayed very remarkable cytotoxic activity against the tested human cancer cells, such as KB, MCF7 and Caski cells (IC50 values of 6.2, 7.3 and 7.7 µg/mL, respectively. This is the first report of the cytotoxic activity of Alpinia mutica.

  9. Phytochemical and cytotoxic investigations of Alpinia mutica rhizomes.

    Science.gov (United States)

    Malek, Sri Nurestri Abdul; Phang, Chung Weng; Ibrahim, Halijah; Norhanom, Abdul Wahab; Sim, Kae Shin

    2011-01-01

    The methanol and fractionated extracts (hexane, ethyl acetate and water) of Alpinia mutica (Zingiberaceae) rhizomes were investigated for their cytotoxic effect against six human carcinoma cell lines, namely KB, MCF7, A549, Caski, HCT116, HT29 and non-human fibroblast cell line (MRC 5) using an in vitro cytotoxicity assay. The ethyl acetate extract possessed high inhibitory effect against KB, MCF7 and Caski cells (IC₅₀ values of 9.4, 19.7 and 19.8 µg/mL, respectively). Flavokawin B (1), 5,6-dehydrokawain (2), pinostrobin chalcone (3) and alpinetin (4), isolated from the active ethyl acetate extract were also evaluated for their cytotoxic activity. Of these, pinostrobin chalcone (3) and alpinetin (4) were isolated from this plant for the first time. Pinostrobin chalcone (3) displayed very remarkable cytotoxic activity against the tested human cancer cells, such as KB, MCF7 and Caski cells (IC₅₀ values of 6.2, 7.3 and 7.7 µg/mL, respectively). This is the first report of the cytotoxic activity of Alpinia mutica. PMID:21240148

  10. Evaluation of Northern Iran Mentha pulegium L. cytotoxicity

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    Farshad H. Shirazi

    2004-08-01

    Full Text Available In vitro tests could be a valuable tool for the evaluation of medicinal plants’ cytotoxicity. One of the most frequently used Iranian traditional plants is Mentha Pulegium from Labiatae family. In the present study, essential oil and the methanolic extract of Mentha pulegium, were analyzed for cytotoxicity on human ovary adenocarcinoma SK-OV-3, human malignant cervix carcinoma Hela, and human lung carcinoma A549 cell lines. Two different assays of clonogenic and neutral red (NR were used for evaluation of cytotoxicity. Although the methanolic extract of Mentha pulegium did not show any cytotoxic effects, the essential oil of this plant proved to be a potent cytotoxic agent on the above three cell lines. According to the clonogenic assay, LD50s of the essential oil on SK-OV-3, Hela and A549 cell lines are 14.10, 59.10 and 18.76 µg/ml, respectively. Our findings suggest that Mentha pulegium essential oil might be considered as a potentially toxic agent on human cancer cell lines, and a possible candidate for human cancer chemotherapy. However, further biological tests on the efficacy and side effects of this plant are necessary before its use in human

  11. New cytotoxic 19-norbufadienolide and bufogargarizin isolated from Chan Su.

    Science.gov (United States)

    Chen, Hu; Meng, Yu-Hui; Guo, De-An; Liu, Xuan; Liu, Jun-Hua; Hu, Li-Hong

    2015-07-01

    One new 19-norbufadienolide (1) and one new bufogargarizin (2), together with twelve known bufadienolides (3-14, resp.) were isolated from Chan Su, a traditional Chinese medicine that is used in the treatment of cancer. Their structures were elucidated on the basis of detailed spectroscopic analysis and comparison of corresponding data that is previously reported. The cytotoxic activities of the isolated compounds were evaluated on HeLa and A549 cell lines. Though 1 and 2 showed weak cytotoxic activities on both cell lines, compounds 4 and 5 showed lower IC50 values than bufalin, the most widely studied bufadienolide in Chan Su. Furthermore, four 3-ester derivatives (15-18) of compound 4 were synthesized and their cytotoxic activities were also evaluated. Analysis of the structure-activity relationship indicated that bufadienolides with aldehyde group at C-10 or α-hydroxyl group at C-11 exhibit stronger cytotoxic activities on both cell lines. The cytotoxic activity of arenobufagin-3-ester derivative 17 was 4-fold higher than compound 4. PMID:25987318

  12. Expression, Purification of E.coli NrfA Protein and Preparation of Polyclonal Antibody Against NrfA%大肠杆菌NrfA蛋白表达、纯化及多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    何婷婷; 龚钢明; 高然

    2012-01-01

    [ Objective] This study aimed to clone the E. Coli NrfA gene and construct the pET-28a( + )-NrfA prokaryotic expression vector for preparation of polyclonal antibody against E. Coli NrfA. [ Method] E. Coli NrfA gene was cloned from the E. Coli genome DNA by PCR and inserted into the vector pET-28a( + )to construct prokaryotic expression vector pET-28a( + )-NrfA. E. Coli NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by immunizing rabbit with routine method. The specificity and titer of polyclonal antibody were confirmed by ELISA and Western Blotting. [ Result] The constructed prokaryotic expression vector pET-28a( + )-NrfA was induced by IPTG, the recombinant NrfA protein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained after immunization and purification was about 1 =204 900. Western Blotting analysis indicated that the obtained polyclonal antibody against E. Coli NrfA protein had high titer and high specificity. [Conclusion] E. Coli NrfA gene was cloned and the prokaryotic expression vector pET-28a( + )-NrfA was constructed successfully, and the polyclonal antibody with high titer and high specificity was prepared, which had laid the foundation for the study of NrfA in different strains of bacteria.%[目的]克隆大肠杆菌NrfA基因,构建pET-28a(+ )-NrfA表达载体,制备相应的多克隆抗体并对其进行鉴定.[方法]以大肠杆菌基因组DNA为模板,PCR扩增得到NrfA基因编码区,构建pET-28a(+ )-NrfA表达载体;经IPTG诱导表达并纯化重组蛋白;再免疫新西兰雄兔,制备多克隆抗体;用ELISA方法检测抗体的效价,Western Blotting检测抗体的特异性.[结果]构建的表达载体pET-28a(+)-NrfA在大肠杆菌中诱导后可高效表达NrfA蛋白;免疫获得的多克隆抗体用ELISA检测,其效价为1∶204 900;经Western Blotting分析,抗体的特异性较好.[结论]成功克隆大肠杆菌的NrfA基

  13. Identification of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH as a binding protein for a 68-kDa Bacillus thuringiensis parasporal protein cytotoxic against leukaemic cells

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    Nadarajah Vishna

    2010-11-01

    Full Text Available Abstract Background Bacillus thuringiensis (Bt, an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth. Recent findings of selective human cancer cell-killing activity in non-insecticidal Bt isolates resulted in a new category of Bt parasporal protein called parasporin. However, little is known about the receptor molecules that bind parasporins and the mechanism of anti-cancer activity. A Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit preferential cytotoxic activity for human leukaemic T cells (CEM-SS but is non-cytotoxic to normal T cells or other cancer cell lines such as human cervical cancer (HeLa, human breast cancer (MCF-7 and colon cancer (HT-29 suggesting properties similar to parasporin. In this study we aim to identify the binding protein for Bt18 in human leukaemic T cells. Methods Bt18 parasporal protein was separated using Mono Q anion exchange column attached to a HPLC system and antibody was raised against the purified 68-kDa parasporal protein. Receptor binding assay was used to detect the binding protein for Bt18 parasporal protein in CEM-SS cells and the identified protein was sent for N-terminal sequencing. NCBI protein BLAST was used to analyse the protein sequence. Double immunofluorescence staining techniques was applied to localise Bt18 and binding protein on CEM-SS cell. Results Anion exchange separation of Bt18 parasporal protein yielded a 68-kDa parasporal protein with specific cytotoxic activity. Polyclonal IgG (anti-Bt18 for the 68-kDa parasporal protein was successfully raised and purified. Receptor binding assay showed that Bt18 parasporal protein bound to a 36-kDa protein from the CEM-SS cells lysate. N-terminal amino acid sequence of the 36-kDa protein was GKVKVGVNGFGRIGG. NCBI protein BLAST revealed that the binding protein was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH. Double immunofluorescence staining showed

  14. Cytotoxic Constituents from the Stems of Clausena lansium (Lour. Skeels

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    Shu Shan Du

    2013-09-01

    Full Text Available Six compounds were isolated from the stems of Clausena lansium (Lour. Skeels by repeated sillica gel column chromatography. Their chemical structures were elucidated on the basic of physicochemical and spectroscopic data. Among them, 8-geranyloxypsolaren (3 and 2-methoxy-1-(3-methyl-buten-1-yl-9H-carbazole-3-carbaldehyde (6 were isolated for the first time from this plant. These compounds were screened for cytotoxicity in human cervical cancer (Hela, leukemia (K562, lung cancer (A549, non-small lung carcinoma (H1299 and liver cancer (SMMC-7721. Within the series of cytotoxic tests, compounds 4–6 displayed potent cytotoxic activity against H1299 and SMMC-7721, with the IC50 values of 6.19 to 26.84 μg/mL.

  15. Phytochemistry, cytotoxicity and antiviral activity of Eleusine indica (sambau)

    Science.gov (United States)

    Iberahim, Rashidah; Yaacob, Wan Ahmad; Ibrahim, Nazlina

    2015-09-01

    Goose grass also known as Eleusine indica (EI) is a local medicinal plant that displays antioxidant, antimicrobial and anticancer activities. The present study is to determine the phytochemical constituents, cytotoxicity and antiviral activities for both crude extract and fraction obtained from the plant. The crude extract contained more secondary metabolites compared to the hexane fraction as gauged using standard phytochemical tests. Cytotoxicity screening against Vero cells using MTT assay showed that the CC50 values for crude extract and hexane fraction were 2.07 and 5.62 mg/ml respectively. The antiviral activity towards Herpes Simplex Virus type 1 (HSV-1) was determined using plaque reduction assay. The selective indices (SI = CC50 / EC50) for both methanol extract and hexane fraction were 12.2 and 6.2 respectively. These results demonstrate that the extract prepared from E. indica possesses phytochemical compound that was non cytotoxic to the cell with potential antiviral activity.

  16. Evaluation of in vitro cytotoxic effect of Trichosanthes dioica root

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    Sanjib Bhattacharya

    2010-01-01

    Full Text Available Background: Trichosanthes dioica Roxb. (Cucurbitaceae, called pointed gourd in English is a dioecious climber grown in India and used traditionally for various medicinal purposes. Methods: Present study was aimed to evaluate in vitro cytotoxic effect of dichloromethane (DCTD, methanol (METD, and aqueous (AQTD extracts of T. dioica root using Allium cepa root meristems by keeping them in different concentrations of each test extract under specific experimental conditions followed by determination of root growth inhibition (root length and number and mitotic index. Results: All the extracts significantly demonstrated concentration-dependent inhibition of root length and number and reduction in mitotic index, indicating antimitotic activity demonstrating cytotoxicity and genotoxicity. DCTD was found to be the most potent (EC 50 : 2.8 mg/ml, followed by METD and AQTD. Conclusion: The present study therefore, establishes promising in vitro cytotoxic and genotoxic property of T. dioica root against the test system.

  17. Synthesis and cytotoxic activity of novel curcumin analogues

    Institute of Scientific and Technical Information of China (English)

    Qin Zhang; Yao Fu; Hao Wei Wang; Tao Gong; Yong Qin; Zhi Rong Zhang

    2008-01-01

    Five novel curcumin analogues bearing different substituents at 4-position of phenyl group were synthesized. Their structures were confirmed by NMR and HRMS spectrum. Their cytotoxic activities against six tumor cell lines were tested by the standard MTT assay in vitro. The results indicated that four analogues (1A-1C, 1E) with solubilizing moieties showed selective potent cytotoxicity against HepG2, HeLa and CT26 cell lines, and analogue 1A and 1C exhibited more potent cytotoxicity than curcumin against CT26 cell line. It was suggested that introduction of appropriate substituents to 4-position of phenyl group might be a potential option for structural modification of curcumin.

  18. Antioxidant and cytotoxic agent from the rhizomes of Kaempferia pandurata

    Science.gov (United States)

    Tanjung, Mulyadi; Tjahjandarie, Tjitjik Srie; Sentosa, Mulya Hadi

    2013-01-01

    Objective To determine antioxidant and cytotoxic activity of two flavanones, pinocembrin (1) and pinostrobin (2) from the rhizomes of Kaempferia pandurata. The chemical structures of both compounds were determined based on spectroscopic data, including UV, IR, MS and NMR spectra. Methods The antioxidant activities of pinocembrin (1) and pinostrobin (2) were assayed by using 2,2-diphenyl-1-picrylhydrazyl. Cytotoxic assay was done by using brine shrimp lethality test, and cytotoxic properties was tested against murine leukemia P-388 cells. Results Compounds 1-2 were evaluated for their antioxidant properties against DPPH, showing their IC50 were 5 816 and 6 268 µmol/L; brine shrimp lethality test: LC50 23.3 and 60.5 µg/mL; murine leukemia P-388: IC50 176.3 and 218.5 µmol/L. Conclutions The results indicated that pinocembrin (1) was slightly more active than pinostrobin (2).

  19. EVALUATION OF GENOTOXICITY AND CYTOTOXICITY OF TINOSPORA CORDIFOLIA (THUNB.

    Directory of Open Access Journals (Sweden)

    AMIT KUMAR

    2013-01-01

    Full Text Available The communication deals with the genotoxic and cytotoxic effects of commonly used medicinal plant, Tinosporacordifolia (Thunb.. Genotoxicity was studied using the Allium cepa L. root tip model using three dose concentrations(10, 20 and 30 mg/mL for 24, 48 and 72 h. T-test was performed to validate the significance of data. Datarevealed dose dependent alterations in the Mitotic Index (M.I.. The M.I. first showed a significant increase andthen reduced as the concentration of extract was raised. The extract increased the rate of growth in the A. ceparoot meristem cells. Moreover, no spindle disturbances, or micronuclei formation was observed indicating thatthe stem extract did not exhibit prominent genotoxic activity. Cytotoxicity of T. cordifolia extract was evaluatedusing RBCs. The extract showed significant dose dependent cytotoxic activity (p=2.98. Considering the aboveresults, the use of aqueous stem extract of Tinospora cordifolia can be considered safe at low dose.

  20. Antioxidant Activities and Cytotoxicity of Zingiber zerumbet (L. Smith Rhizome

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    Anish Nag

    2013-09-01

    Full Text Available Zingiber zerumbet (L. Smith, popularly known as shampoo ginger, is one of the most commonly used ingredients in Indo-Malaysian traditional medicines. The antioxidant and cytotoxic activities of the ethanolic extract of Z. zerumbet rhizome (ZZ was investigated. 2,2-di(4-tert-octylphenyl-1- picryl-hydrazyl (DPPH and hydroxyl radical scavenging assays showed significant radical scavenging activities of ZZ. The extract was rich in polyphenol and flavonoids. Cytotoxicity was assessed in vitro by trypan blue exclusion test. Human peripheral blood lymphocyte cells were incubated in different concentrations of ZZ (0, 15, 30, 60, 120, 300 and 600 µg/ml for 3 h at 37 °C. The rhizome extract was found to be cytotoxic at concentrations higher for human consumption. In addition, HPLC analysis revealed ZZ as a rich source of kaempferol. Based on the results of the present investigation the rhizome may be used safely as a therapeutic antioxidant.

  1. Synthesis and cytotoxicity evaluation of 4'-amino-4'-dehydroxyloleandrin derivatives.

    Science.gov (United States)

    Chen, Hu; Lei, Min; Ma, Biao; Liu, Miao; Guo, Dean; Liu, Xuan; Hu, Lihong

    2016-09-01

    A series of C4'-substituted oleandrin analogues were designed, synthesized and evaluated for their cytotoxicity towards human cervical carcinoma cell line (HeLa). The structure-activity relationships (SARs) of these compounds were summarized in this paper, and 4'-α-amino-4'-dehydroxyloleandrin 4a (IC50=21.7nM) and 4'-β-amino-4'-dehydroxyloleandrin 4b (IC50=10.9nM) exhibited stronger cytotoxicity compared with oleandrin (IC50=33.3nM). Furthermore, the cytotoxicity of these two compounds towards another five human cancer cell lines (NCI-H266, A549, Jurkat, HL-60 and PC-3) was also evaluated and the IC50 values of β-amino derivative 4b were approximately 2-3 folds lower than that of oleandrin. PMID:27431773

  2. IgG-Mediated Immune Suppression to Erythrocytes by Polyclonal Antibodies Can Occur in the Absence of Activating or Inhibitory Fcγ Receptors in a Full Mouse Model.

    Science.gov (United States)

    Bernardo, Lidice; Yu, Honghui; Amash, Alaa; Zimring, James C; Lazarus, Alan H

    2015-09-01

    Polyclonal anti-D has been used to prevent RhD-negative mothers from becoming immunized against RhD positive fetal erythrocytes, and this mechanism has been referred as Ab or IgG-mediated immune suppression (AMIS). Although anti-D has been highly successful, the inhibitory mechanisms remain poorly understood. Two major theories behind AMIS involve the binding of IgG to activating or inhibitory FcγR, which can induce either erythrocyte clearance or immune inhibition, respectively. In this work, we explored the absolute role of activating and inhibitory FcγR in the AMIS mechanism using the HOD mouse model of RBC immunization. HOD mice contain a RBC-specific recombinant protein composed of hen egg lysozyme (HEL), OVA and human transmembrane Duffy Ag, and erythrocytes from HOD mice can stimulate an immune response to HEL. To assess the contribution of activating and inhibitory FcγR to AMIS, C57BL/6 versus FcRγ-chain(-/-) or FcγRIIB(-/-) mice were used as recipients of HOD-RBC alone or together with anti-HEL Abs (i.e., AMIS) and the resulting immune response to HEL evaluated. We show that anti-HEL polyclonal Abs induce the same degree of AMIS effect in mice lacking these IgG binding receptors as compared with wild-type mice. In agreement with this, F(ab')2 fragments of the AMIS Ab also significantly reduced the Ab response to the HOD cells. In conclusion, successful inhibition of in vivo Ab responses to HOD-RBC by polyclonal IgG can occur independently of activating or inhibitory FcγR involvement. These results may have implications for the understanding of RhD prophylaxis. PMID:26188060

  3. Prokaryotic Expression and Polyclonal Antibody Preparation of SDG711 C-terminal from Rice%水稻SDG711蛋白C末端原核表达及多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    张志刚; 李超; 林欣欣; 巫光宏; 杜平州; 王玉琪

    2013-01-01

    [目的]对水稻SDG711蛋白C末端进行原核表达,并制备其多克隆抗体.[方法]选取水稻SDG711蛋白抗原决定簇较密集的C末端进行原核表达,通过构建原核表达载体pET28a-711C,转化E.coli BL21(DE3)感受态细胞,IPTG诱导表达融合蛋白后进行纯化,再以纯化的融合蛋白为抗原免疫新西兰白兔,制备多克隆抗体,并对其进行Western-blot分析.[结果]试验制备的多克隆抗体能有效地检测抗原的表达.[结论]该研究为进一步深入研究SDG711蛋白的功能奠定了基础.%[Objective] This study aimed to conduct prokaryotic expression of rice SDG711 C-terminal and prepare its polyclonal antibody. [ Method] C-terminal of rice SDG711 containing relatively intensive antigen determinants was selected for prokaryotic expression, prokaryotic expression vector pET28a-711C was constructed and the recombinant plasmid was transformed into Escherkhia coli BL21 (DE3) competent cells. The recombinant fusion protein was induced by IPTG and purified to immunize a New Zealand white rabbit as the antigen, and polyclonal antibody was obtained for Western-blot analysis. [Result] The polyclonal antibody prepared could efficiently detect the antigen expression. [Conclusion] This study laid the foundation for further investigating the functions of SDG711 protein.

  4. Cytotoxic activity screening of Bangladeshi medicinal plant extracts.

    Science.gov (United States)

    Akter, Raushanara; Uddin, Shaikh J; Grice, I Darren; Tiralongo, Evelin

    2014-01-01

    The cytotoxic activity of 23 crude methanol extracts from 19 Bangladeshi medicinal plants was investigated against healthy mouse fibroblasts (NIH3T3), healthy monkey kidney (VERO) and four human cancer cell lines (gastric, AGS; colon, HT-29; and breast, MCF-7 and MDA-MB-231) using MTT assay. High cytotoxicity across all cell lines tested was exhibited by Aegiceras corniculatum (fruit) and Hymenodictyon excelsum (bark) extracts (IC50 values ranging from 0.0005 to 0.9980 and 0.08 to 0.44 mg/mL, respectively). Fourteen extracts from 11 plant species, namely Clitoria ternatea (flower and leaf), Dillenia indica (leaf), Diospyros peregrina (leaf), Dipterocarpus turbinatus (bark and leaf), Ecbolium viride (leaf), Glinus oppositifolius (whole plant), Gnaphalium luteoalbum (leaf), Jasminum sambac (leaf), Lannea coromandelica (bark and leaf), Mussaenda glabrata (leaf) and Saraca asoca (leaf), were also significantly cytotoxic (IC50 ternatea (flower and leaf), Caesalpinia pulcherrima (leaf), E. viride (leaf) and G. oppositifolius (whole plant) showed cytotoxicity only against both of the breast cancer cell lines (MCF-7 and MDA-MB-231). In contrast, C. ternatea (flower and leaf) exhibited high cytotoxic activity against MDA-MB-231 (IC50 values of 0.11 and 0.49 mg/mL, respectively), whereas E. viride and G. oppositifolius whole plant extracts exhibited high activity against MCF-7 cells (IC50 values of 0.06 and 0.15 mg/mL, respectively). The cytotoxic activity test results for 9 of the plant species correlate with their traditional use as anticancer agents, thus making them interesting sources for further drug development. PMID:23846168

  5. Cytotoxicity of selenium nanoparticles in rat dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Ramos JF

    2012-07-01

    Full Text Available Joseph F Ramos,1 Thomas J Webster21School of Engineering, Center of Biomedical Engineering, 2School of Engineering and Department of Orthopedics, Brown University, Providence, RI, USABackground: Ventilator-associated pneumonia is a deadly nosocomial infection caused by contaminated endotracheal tubes. It has been shown that polyvinyl chloride (PVC, the endotracheal tube substrate coated with elemental selenium nanoparticles reduces bacterial adherence and proliferation on PVC by over 99%. However, it is not known if selenium nanoparticles elicit a cytotoxic effect in vitro. The purpose of this study was to investigate the cytotoxic effects of PVC coated with selenium nanoparticles on fibroblasts, which are mammalian cells central to endotracheal tube intubation.Methods: Different concentrations of selenium nanoparticles were precipitated onto the PVC surface by reduction of selenium salts using glutathione. Characterization of PVC coated with selenium nanoparticles was done by scanning electron microscopy, energy dispersive x-ray, and contact angle measurements. For the cytotoxicity experiments, fibroblasts were seeded at a density of 5000 cm2 onto PVC coated with three different concentrations of selenium nanoparticles (high, medium, low and incubated for 4 hours (adhesion as well as for 24 hours and 72 hours (proliferation. The half-maximal inhibitory concentration (IC50 value was determined after 72 hours using an ultrahigh concentration. MTT assays were used to assess cell viability at the indicated time points.Results: The three concentrations of selenium nanoparticles did not elicit a cytotoxic effect after 72 hours (P < 0.01, n = 3. It was found that the IC50 value was at the ultrahigh concentration of selenium nanoparticles. The nanoparticulate elemental selenium concentration previously shown to decrease the function of bacteria was shown not to cause a cytotoxic effect on fibroblasts in vitro.Conclusion: These findings demonstrate

  6. 登革病毒4型E蛋白的表达与多克隆抗体的制备%Expression of the envelope protein of Dengue virus type 4 and preparation of the polyclonal antibody

    Institute of Scientific and Technical Information of China (English)

    李竹石; 俞永新; 李玉华; 林华; 杨健; 杨会强; 曾献武; 赵宇; 刘俐; 刘莉娜; 康庄

    2012-01-01

    目的 在原核细胞中表达登革病毒4型E蛋白及E蛋白Ⅲ区,分别以两种蛋白免疫家兔,获得可检测登革病毒的多克隆抗体.方法 将登革病毒4型E蛋白与E蛋白Ⅲ区编码序列克隆到pET-32a(+)质粒中,分别构建两种蛋白的表达载体,以IPTG诱导其在Rosetta细胞中的大量表达,并进行SDS-PAGE检测.分别以两种蛋白免疫家兔,制备出针对E蛋白与E蛋白Ⅲ区的多克隆抗体,并对其进行Western blot鉴定.结果 登革病毒4型E蛋白与E蛋白Ⅲ区在Rosetta细胞中以包涵体的形式大量表达;利用所制备的多克隆抗体对登革病毒进行检测,出现了预期的条带.结论 本研究制备获得的多克隆抗体可用于登革病毒的检测,为登革病毒相关研究奠定了基础.%Objective To express full length (DENV4E) and domain III (DENV4EIII) of the envelope protein of Dengue virus type 4 in procaryotic cells respectively, and to prepare the polyclonal antibody against dengue virus by immunizing rabbits with the expressed proteins. Methods The sequences encoding DENV4E and DENV4EIII proteins were cloned into pET-32a ( + ) plasmids respectively to construct expression vectors. Then DENV4E or DENV4EIII proteins were expressed in Rosetta cells by IPTG induction and detected by SDS-PAGE. Polyclonal antibody against DENV4E or DENV4EIII proteins was obtained by immunizing rabbits with the expressed proteins and was identified by Western blot. Results DENV4E and DENV4EIII proteins were both successfully expressed in Rosetta cells mainly in the form of inclusion body. The prepared polyclonal antibody formed bands as expected when reacting with the dengue virus. Conclusions The polyclonal antibody is prepared and may be used in the detection of dengue virus.

  7. Produção e caracterização de anticorpos policlonais contra Xanthomonas campestris pv. viticola Production and characterization of polyclonal antibodies against Xanthomonas campestris pv. viticola

    OpenAIRE

    João Sebastião de Paula Araujo; Fábio Bueno dos Reis Junior; Geraldo Baeta Cruz; Bruno Cardoso de Oliveira; Charles Frederick Robbs; Raul de Lucena Duarte Ribeiro; José Carlos Polidoro

    2005-01-01

    O objetivo deste trabalho foi a produção de anticorpos policlonais contra Xanthomonas campestris pv. viticola e sua caracterização pelo método Elisa indireto. Os resultados apontaram a qualidade dos anticorpos policlonais produzidos, os quais mostraram-se altamente reativos e específicos para o patovar com potencial para ser empregado no diagnóstico da doença e em programas de certificação.The objective of this work was to produce polyclonal antibodies against Xanthomonas campestris pv. vitic...

  8. Epstein-Barr virus/complement fragment C3d receptor (CR2) reacts with p53, a cellular antioncogene-encoded membrane phosphoprotein: detection by polyclonal anti-idiotypic anti-CR2 antibodies.

    OpenAIRE

    Barel, M; Fiandino, A; Lyamani, F; Frade, R

    1989-01-01

    Epstein-Barr virus and the C3d fragment of the third component of complement are specific extracellular ligands for complement receptor type 2 (CR2). However, intracellular proteins that react specifically with CR2 and are involved in post-membrane signals remain unknown. We recently prepared polyclonal anti-idiotypic anti-CR2 antibodies (Ab2) by using the highly purified CR2 molecule as original immunogen. We showed that Ab2 contained anti-idiotypic specificities that mimicked extracellular ...

  9. Preparation and Identification of the Syntenin1 Polyclonal Antibody, a Cancer Metastasis Related Gene%癌症转移相关基因Syntenin1多克隆抗体的制备与鉴定

    Institute of Scientific and Technical Information of China (English)

    夏孝强; 陈米娜; 李芳华; 蔡佩玲

    2009-01-01

    Objective To express the GST fusion protein, GST-Synteninl in E. colt, and to prepare the polyclonal antibody of Synteninl. Methods CDS fragment of Synteninl was obtained by RT-PCR from normal mouse brain and subcloned into pGEX-4T-2 to generate pGEX-4T-2-Synteninl recombinant. The confirmed recombinant was transformed into the BL21 competent cells and induced with IPTG. The recombinant fusion protein was purified with immobilized Glutathione Sepharose and confirmed by SDS-PAGE. The purified fusion protein was mixed with the Freund's adjuvant, and then injected into New Zealand white rabbits by hypodermic injection. The polyclonal antibody titer and specification were identified by Western blot. Results Synteninl polyclonal antibody-bind Syteninl protein specifically and the antiserum tiger reached to 1:20 000. Conclusion The Synteninl polyclonal antibody with high titer and high specificity was prepared successfully. This will be very helpful for the further study on Synteninl function and molecule mechanism of cancer metastasis.%目的 获得原核表达GST-Syntenin1融合蛋白,制备高效价的抗Syntenin1多克隆抗体.方法 RT-PCR扩增Syntenin1 cDNA开放阅读框(CDS)序列,亚克隆到编码GST的pGEX-4T-2原核表达载体上,将编码Syntenin1的pGEX-4T-2-Syntenin1重组质粒化学转化BL21(DE3)感受态细胞,IPTG诱导GST-Syntenin1融合蛋白表达,对融合蛋白亲和纯化.SDS-PAGE鉴定后,将纯化的融合蛋白辅以弗氏佐剂,免疫新西兰大白兔制备多克隆抗体,并用Western blot检测抗体效价及特异性.结果 成功制备GST-Syntenin1融合蛋白及多克隆抗体.Western blot结果表明Syntenin1兔多克隆抗体效价可达到1∶20 000,且与Syntenin1蛋白特异结合.结论 成功制备Syntenin1多克隆抗体,为进一步研究Syntenin1基因的功能奠定了基础.

  10. Cytotoxic effects of 4-octylphenol on fish hepatocytes.

    Science.gov (United States)

    Kaptaner, Burak

    2016-08-01

    The present study was conducted to determine cytotoxic effects of 4-octylphenol (4-OP) on primary cultured hepatocytes of pearl mullet (Alburnus tarichi). Lactate dehydrogenase (LDH) release, malondialdehyde (MDA) level, antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST)] and glutathione (GSH) content were measured after 24-h exposure to 4-OP. 4-OP caused dose- and time-dependent increases in LDH release. Significant induction of MDA level and decrease in GSH content were found. SOD and GPx activities were decreased while GST activity was increased. These findings suggest that 4-OP leads to cytotoxicity by depressing antioxidant defenses in fish hepatocytes. PMID:26415925

  11. Evaluation of the In Vitro Cytotoxicity of Crosslinked Biomaterials

    OpenAIRE

    Wang, Martha O.; Etheridge, Julie M.; Thompson, Joshua A.; Vorwald, Charlotte E.; Dean, David; Fisher, John P.

    2013-01-01

    This study evaluated the in vitro cytotoxicity of poly(propylene fumarate) (PPF). PPF is an aliphatic biodegradable polymer that has been well characterized for use in bone tissue engineering scaffolds. Four different cell types, human mesenchymal stem cells (hMSC), fibroblasts (L929), pre-osteoblasts (MC3T3), and canine mesenchymal stem cells (cMSC), were used to evaluate the cytotoxicity of PPF. These cell types represent the tissues that PPF would interact with in vivo as a bone tissue sca...

  12. New Cytotoxic 24-Homoscalarane Sesterterpenoids from the Sponge Ircinia felix

    Directory of Open Access Journals (Sweden)

    Ya-Yuan Lai

    2015-09-01

    Full Text Available Two new 24-homoscalarane sesterterpenoids, felixins F (1 and G (2, were isolated from the sponge Ircinia felix. The structures of new homoscalaranes 1 and 2 were elucidated by extensive spectroscopic methods, particularly with one-dimensional (1D and two-dimensional (2D NMR, and, by comparison, the spectral data with those of known analogues. The cytotoxicity of 1 and 2 against the proliferation of a limited panel of tumor cell lines was evaluated and 1 was found to show cytotoxicity toward the leukemia K562, MOLT-4, and SUP-T1 cells (IC50 ≤ 5.0 μM.

  13. Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance

    DEFF Research Database (Denmark)

    Tenzer, Stefan; Wee, Edmund; Burgevin, Anne;

    2009-01-01

    Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group-associated ......Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group...

  14. Natural killer cell cytotoxicity assay with time-resolved fluorimetry

    Institute of Scientific and Technical Information of China (English)

    李建中; 章竹君; 金伯泉; 田方

    1996-01-01

    A new time-resolved fluorimetric method for the measurement of natural killer (NK) cell cytotoxicity has been developed by labelling the target cell K562 with a new synthesized fluorescence marker KLUK. The method has advantages of higher sensitivity, time-saving, good reproducibility and has no radioactivity problems. A satisfactory result is obtained by comparing it with 51Cr release method. It demonstrates that the new marker provides an alternative to currently used radioactive markers for the assessment of in vitro cellular cytotoxicity.

  15. Phytochemical and Cytotoxic Investigations of Alpinia mutica Rhizomes

    OpenAIRE

    Kae Shin Sim; Norhanom Abdul Wahab; Halijah Ibrahim; Sri Nurestri Abdul Malek; Chung Weng Phang

    2011-01-01

    The methanol and fractionated extracts (hexane, ethyl acetate and water) of Alpinia mutica (Zingiberaceae) rhizomes were investigated for their cytotoxic effect against six human carcinoma cell lines, namely KB, MCF7, A549, Caski, HCT116, HT29 and non-human fibroblast cell line (MRC 5) using an in vitro cytotoxicity assay. The ethyl acetate extract possessed high inhibitory effect against KB, MCF7 and Caski cells (IC50 values of 9.4, 19.7 and 19.8 µg/mL, respectively). Flavokawin B (1), 5,6-d...

  16. New Cytotoxic 24-Homoscalarane Sesterterpenoids from the Sponge Ircinia felix.

    Science.gov (United States)

    Lai, Ya-Yuan; Chen, Li-Chai; Wu, Chug-Fung; Lu, Mei-Chin; Wen, Zhi-Hong; Wu, Tung-Ying; Fang, Lee-Shing; Wang, Li-Hsueh; Wu, Yang-Chang; Sung, Ping-Jyun

    2015-09-11

    Two new 24-homoscalarane sesterterpenoids, felixins F (1) and G (2), were isolated from the sponge Ircinia felix. The structures of new homoscalaranes 1 and 2 were elucidated by extensive spectroscopic methods, particularly with one-dimensional (1D) and two-dimensional (2D) NMR, and, by comparison, the spectral data with those of known analogues. The cytotoxicity of 1 and 2 against the proliferation of a limited panel of tumor cell lines was evaluated and 1 was found to show cytotoxicity toward the leukemia K562, MOLT-4, and SUP-T1 cells (IC50 ≤ 5.0 μM).

  17. Chemical constituents of Lecythis pisonis and cytotoxic activity

    Directory of Open Access Journals (Sweden)

    Jocélia P. C. Oliveira

    2012-10-01

    Full Text Available The phytochemical investigation of the ethanol extract from leaves of Lecythis pisonis Cambess., Lecythidaceae, resulted in the isolation of seven triterpenes: α- and β-amyrin, uvaol and erythrodiol, ursolic and oleanolic acids and 3β-friedelinol, as well as a mixture of sitosterol and stigmasterol steroids and a diterpene (E-phytol. The structures of these compounds were identified by¹H and 13C NMR spectral analysis and compared with literature data. The mixture of triterpenes ursolic and oleanolic acids isolated from the active ethereal fraction showed moderate cytotoxic activity. This paper describes for the first time the phytochemical and cytotoxic study of Lecythis pisonis' leaves.

  18. Cytotoxic studies of paclitaxel (Taxol) in human tumour cell lines.

    OpenAIRE

    Liebmann, J. E.; Cook, J. A.; Lipschultz, C.; Teague, D.; Fisher, J; Mitchell, J B

    1993-01-01

    The cytotoxicity of paclitaxel against eight human tumour cell lines has been studied with in vitro clonogenic assays. The fraction of surviving cells fell sharply after exposure for 24 h to paclitaxel concentrations ranging from 2 to 20 nM; the paclitaxel IC50 was found to range between 2.5 and 7.5 nM. Increasing the paclitaxel concentration above 50 nM, however, resulted in no additional cytotoxicity after a 24 h drug exposure. Cells incubated in very high concentrations of paclitaxel (10,0...

  19. Synthesis of new bioisosteric hemiasterlin analogues with extremely high cytotoxicity

    OpenAIRE

    The, Chinh Pham; Thi, Tuyet Anh Dang; Hoang, Thi Phuong; Ngo, Quoc Anh; Doan, Duy Tien; Thi, Thu Ha Nguyen; Thi, Tham Pham; Thi, Thu Ha Vu; Jean, Mickael; Van De Weghe, Pierre; Van Nguyen, Tuyen

    2014-01-01

    International audience In this article, we report a convenient and efficient method for the synthesis of new simplified derivativesof hemiasterlin in which the α,α-dimethylbenzylic moiety A is replaced by α,β-unsaturated aryl groupsas Michael acceptor. Most of these derivatives have a strong cytotoxic activity on three human tumorcell lines (KB, Hep-G2 and MCF7). Analogs 17b and 17f showed a high cytotoxicity against KB andHep-G2 cancer cell lines comparable to paclitaxel and ellipticine.

  20. The athymic nude rat. Immunobiological characteristics with special reference to establishment of non-antigen-specific T-cell reactivity and induction of antigen-specific immunity

    DEFF Research Database (Denmark)

    Hougen, H P

    1991-01-01

    white pulp, and the interfollicular areas of Peyer's patches, are severely cell-depleted in the athymic nude rat. The lower lymphocyte content of these organs is not reflected in the lymphocyte counts of peripheral blood, but the lymphocyte counts of thoracic duct lymph are much lower than those found...... the thymic aplasia. Normal B-lymphocyte function has been found in both in vitro and in vivo tests, whereas the T-cell function is virtually absent both in vitro and in vivo. Non-MHC-restricted cells with killer activity like NK cells are present in the nude rat, and these cells function normally both...