Involvement of nuclear factor κB in platelet CD40 signaling

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Hachem, Ahmed; Yacoub, Daniel; Zaid, Younes; Mourad, Walid; Merhi, Yahye

2012-01-01

Highlights: ► sCD40L induces TRAF2 association to CD40 and NF-κB activation in platelets. ► IκBα phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. ► IκBα is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.

Involvement of nuclear factor {kappa}B in platelet CD40 signaling

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Hachem, Ahmed [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Yacoub, Daniel [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1 (Canada); Zaid, Younes [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Mourad, Walid [Universite de Montreal, Department of Medicine, 2900 boul. Edouard-Montpetit, Montreal, Quebec, Canada H3T 1J4 (Canada); Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1 (Canada); Merhi, Yahye, E-mail: yahye.merhi@icm-mhi.org [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Universite de Montreal, Department of Medicine, 2900 boul. Edouard-Montpetit, Montreal, Quebec, Canada H3T 1J4 (Canada)

2012-08-17

Highlights: Black-Right-Pointing-Pointer sCD40L induces TRAF2 association to CD40 and NF-{kappa}B activation in platelets. Black-Right-Pointing-Pointer I{kappa}B{alpha} phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. Black-Right-Pointing-Pointer I{kappa}B{alpha} is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-{kappa}B). Given that platelets contain NF-{kappa}B, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of I{kappa}B{alpha}, which are abolished by CD40L blockade. Inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced I{kappa}B{alpha} phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on I{kappa}B{alpha} phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-{kappa}B activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo

Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris.

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Ohkuri, Takatoshi; Murase, Eri; Sun, Shu-Lan; Sugitani, Jun; Ueda, Tadashi

2013-10-01

A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

CD4 Depletion or CD40L Blockade Results in Antigen-Specific Tolerance in a Red Blood Cell Alloimmunization Model

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Prabitha Natarajan

2017-08-01

Full Text Available Approximately 3–10% of human red blood cell (RBC transfusion recipients form alloantibodies to non-self, non-ABO blood group antigens expressed on donor RBCs, with these alloantibodies having the potential to be clinically significant in transfusion and pregnancy settings. However, the majority of transfused individuals never form detectable alloantibodies. Expanding upon observations that children initially transfused with RBCs at a young age are less likely to form alloantibodies throughout their lives, we hypothesized that “non-responders” may not only be ignorant of antigens on RBCs but instead tolerized. We investigated this question in a reductionist murine model, in which transgenic donors express the human glycophorin A (hGPA antigen in an RBC-specific manner. Although wild-type mice treated with poly IC and transfused with hGPA RBCs generated robust anti-hGPA IgG alloantibodies that led to rapid clearance of incompatible RBCs, those transfused in the absence of an adjuvant failed to become alloimmunized. Animals depleted of CD4+ cells or treated with CD40L blockade prior to initial hGPA RBC exposure, in the presence of poly IC, failed to generate detectable anti-hGPA IgG alloantibodies. These non-responders to a primary transfusion remained unable to generate anti-hGPA IgG alloantibodies upon secondary hGPA exposure and did not prematurely clear transfused hGPA RBCs even after their CD4 cells had returned or their CD40L blockade had resolved. This observed tolerance was antigen (hGPA specific, as robust IgG responses to transfused RBCs expressing a third-party antigen occurred in all studied groups. Experiments completed in an RBC alloimmunization model that allowed evaluation of antigen-specific CD4+ T-cells (HOD (hen egg lysozyme, ovalbumin, and human duffyb demonstrated that CD40L blockade prevented the expansion of ovalbumin 323-339 specific T-cells after HOD RBC transfusion and also prevented germinal center formation. Taken

Cyclosporine-resistant, Rab27a-independent Mobilization of Intracellular Preformed CD40L Mediates Antigen-specific T Cell Help In Vitro

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Koguchi, Yoshinobu; Gardell, Jennifer L.; Thauland, Timothy J.; Parker, David C.

2011-01-01

CD40L is critically important for the initiation and maintenance of adaptive immune responses. It is generally thought that CD40L expression in CD4+ T cells is regulated transcriptionally and made from new mRNA following antigen recognition. However, recent studies with two-photon microscopy revealed that the majority of cognate interactions between effector CD4+ T cells and APCs are too short for de novo synthesis of CD40L. Given that effector and memory CD4+ T cells store preformed CD40L (pCD40L) in lysosomal compartments and that pCD40L comes to the cell surface within minutes of antigenic stimulation, we and others have proposed that pCD40L might mediate T cell-dependent activation of cognate APCs during brief encounters in vivo. However, it has not been shown that this relatively small amount of pCD40L is sufficient to activate APCs, owing to the difficulty of separating the effects of pCD40L from those of de novo CD40L and other cytokines in vitro. Here we show that pCD40L surface mobilization is resistant to cyclosporine or FK506 treatment, while de novo CD40L and cytokine expression are completely inhibited. These drugs thus provide a tool to dissect the role of pCD40L in APC activation. We find that pCD40L mediates selective activation of cognate but not bystander APCs in vitro and that mobilization of pCD40L does not depend on Rab27a, which is required for mobilization of lytic granules. Therefore, effector CD4+ T cells deliver pCD40L specifically to APCs on the same time scale as the lethal hit of CTLs but with distinct molecular machinery. PMID:21677130

Efficient adenovector CD40 ligand immunotherapy of canine malignant melanoma.

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von Euler, Henrik; Sadeghi, Arian; Carlsson, Björn; Rivera, Patricio; Loskog, Angelica; Segall, Thomas; Korsgren, Olle; Tötterman, Thomas H

2008-05-01

Cutaneous canine melanomas are usually benign in contrast to human malignant melanoma. However, the canine oropharyngeal, uveal, and mucocutaneous neoplasms are aggressive and have metastatic potential. Surgery and to a lesser extent radiotherapy and chemotherapy are widely adopted treatments but are seldom curative in advanced stages. The similarities between human and canine melanoma make spontaneous canine melanoma an excellent disease model for exploring novel therapies. Herein, we report the first 2 adenovector CD40L immunogene (AdCD40L) treatments of aggressive canine malignant melanoma. Case no. 1 was an advanced stage III oral melanoma that was cured from malignant melanoma with 2 intratumor AdCD40L injections before cytoreductive surgery. After treatment, the tumor tissue was infiltrated with T lymphocytes and B lymphocytes suggesting immune activation. This dog survived 401 days after the first round of gene therapy and was free of melanoma at autopsy. Case no. 2 had a conjunctival malignant melanoma with a rapid progression. This case was treated with 6 AdCD40L injections over 60 days. One hundred and twenty days after start of gene therapy and 60 days after the last injection, the tumor had regressed dramatically, and the dog had a minimal tumor mass and no signs of progression or metastasis. Our results indicate that AdCD40L immunogene therapy is beneficial in canine malignant melanoma and could be considered for human malignant melanoma as well.

CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type 1 immunity.

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Zaccard, Colleen R; Watkins, Simon C; Kalinski, Pawel; Fecek, Ronald J; Yates, Aarika L; Salter, Russell D; Ayyavoo, Velpandi; Rinaldo, Charles R; Mailliard, Robbie B

2015-02-01

The ability of dendritic cells (DC) to mediate CD4(+) T cell help for cellular immunity is guided by instructive signals received during DC maturation, as well as the resulting pattern of DC responsiveness to the Th signal, CD40L. Furthermore, the professional transfer of antigenic information from migratory DC to lymph node-residing DC is critical for the effective induction of cellular immune responses. In this study we report that, in addition to their enhanced IL-12p70 producing capacity, human DC matured in the presence of inflammatory mediators of type 1 immunity are uniquely programmed to form networks of tunneling nanotube-like structures in response to CD40L-expressing Th cells or rCD40L. This immunologic process of DC reticulation facilitates intercellular trafficking of endosome-associated vesicles and Ag, but also pathogens such HIV-1, and is regulated by the opposing roles of IFN-γ and IL-4. The initiation of DC reticulation represents a novel helper function of CD40L and a superior mechanism of intercellular communication possessed by type 1 polarized DC, as well as a target for exploitation by pathogens to enhance direct cell-to-cell spread. Copyright © 2015 by The American Association of Immunologists, Inc.

Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.

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Harald Schwarz

Full Text Available Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU. When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml. We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

Structure-Based Design of a Soluble Prefusion-Closed HIV-1 Env Trimer with Reduced CD4 Affinity and Improved Immunogenicity

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Chuang, Gwo-Yu; Geng, Hui; Pancera, Marie; Xu, Kai; Cheng, Cheng; Acharya, Priyamvada; Chambers, Michael; Druz, Aliaksandr; Tsybovsky, Yaroslav; Wanninger, Timothy G.; Yang, Yongping; Doria-Rose, Nicole A.; Georgiev, Ivelin S.; Gorman, Jason; Joyce, M.Gordon; O; Dell, Sijy; Zhou, Tongqing; McDermott, Adrian B.; Mascola, John R.; Kwong, Peter D. (NIH); (FNL)

2017-03-08

The HIV-1 envelope (Env) trimer is a target for vaccine design as well as a conformational machine that facilitates virus entry by transitioning between prefusion-closed, CD4-bound, and coreceptor-bound conformations by transitioning into a postfusion state. Vaccine designers have sought to restrict the conformation of the HIV-1 Env trimer to its prefusion-closed state as this state is recognized by most broadly neutralizing, but not nonneutralizing, antibodies. We previously identified a disulfide bond, I201C-A433C (DS), which stabilizes Env in the vaccine-desired prefusion-closed state. When placed into the context of BG505 SOSIP.664, a soluble Env trimer mimic developed by Sanders, Moore, and colleagues, the engineered DS-SOSIP trimer showed reduced conformational triggering by CD4. Here, we further stabilize DS-SOSIP through a combination of structure-based design and 96-well-based expression and antigenic assessment. From 103 designs, we identified one, named DS-SOSIP.4mut, with four additional mutations at the interface of potentially mobile domains of the prefusion-closed structure. We also determined the crystal structures of DS-SOSIP.4mut at 4.1-Å resolution and of an additional DS-SOSIP.6mut variant at 4.3-Å resolution, and these confirmed the formation of engineered disulfide bonds. Notably, DS-SOSIP.4mut elicited a higher ratio of tier 2 autologous titers versus tier 1 V3-sensitive titers than BG505 SOSIP.664. DS-SOSIP.4mut also showed reduced recognition of CD4 and increased thermostability. The improved antigenicity, thermostability, and immunogenicity of DS-SOSIP.4mut suggest utility as an immunogen or a serologic probe; moreover, the specific four alterations identified here, M154, M300, M302, and L320 (4mut), can also be transferred to other HIV-1 Env trimers of interest to improve their properties.

IMPORTANCEOne approach to elicit broadly neutralizing antibodies against HIV-1 is to stabilize the

HIV-1 neutralizing antibodies induced by native-like envelope trimers

NARCIS (Netherlands)

Sanders, Rogier W.; van Gils, Marit J.; Derking, Ronald; Sok, Devin; Ketas, Thomas J.; Burger, Judith A.; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J.; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J.; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne C.; Julien, Jean-Philippe; Rakasz, Eva G.; Seaman, Michael S.; Guttman, Miklos; Lee, Kelly K.; Klasse, Per Johan; Labranche, Celia; Schief, William R.; Wilson, Ian A.; Overbaugh, Julie; Burton, Dennis R.; Ward, Andrew B.; Montefiori, David C.; Dean, Hansi; Moore, John P.

2015-01-01

A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505

Modulation of neuronal differentiation by CD40 isoforms

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Hou Huayu; Obregon, Demian; Lou, Deyan; Ehrhart, Jared; Fernandez, Frank; Silver, Archie; Tan Jun

2008-01-01

Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40 isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40 -/- deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express mostly CD40 type I in cerebral tissues at relatively high levels, in age and gender-matched CD40 -/- mice CD40 type I expression was almost completely absent; suggesting a predominance of the non-signal-transducible CD40 type II isoform. Younger, 1 day old wild-type mice displayed less CD40 type I, and more CD40 type II, as well as, greater expression of soluble CD40 (CD40L/CD40 signal inhibitor), compared with 1 month old mice. Neuron-like N2a cells express CD40 type I and type II isoforms while in an undifferentiated state, however once induced to differentiate, CD40 type I predominates. Further, differentiated N2a cells treated with CD40 ligand express high levels of neuron specific nuclear protein (NeuN); an effect reduced by anti-CD40 type I siRNA, but not by control (non-targeting) siRNA. Altogether these data suggest that CD40 isoforms may act in a temporal fashion to modulate neuron differentiation during brain development. Thus, modulation of neuronal CD40 isoforms and CD40 signaling may represent

Anti-tumor immunotherapy by blockade of the PD-1/PD-L1 pathway with recombinant human PD-1-IgV.

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Zhang, C; Wu, S; Xue, X; Li, M; Qin, X; Li, W; Han, W; Zhang, Y

2008-01-01

Blockade of the programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway can delay tumor growth and prolong the survival of tumor-bearing mice. The extracellular immunoglobulin (Ig) V domain of PD-1 is important for the interaction between PD-1 and PD-L1, suggesting that PD-1-IgV may be a potential target for anti-tumor immunotherapy. The extracellular sequence of human PD-1-IgV (hPD-1-IgV) was expressed in Escherichia coli and purified. The anti-tumor effect of hPD-1-IgV on tumor-bearing mice was tested. hPD-1-IgV recombinant protein could bind PD-L1 at molecular and cellular levels and enhance Cytotoxic T Lymphocyte (CTL) activity and anti-tumor effect on tumor-bearing mice in vivo. The percentage of CD4(+)CD25(+) T cells in tumor-bearing mice was decreased compared with control mice after administration of the recombinant protein. Our results suggest that inhibition of the interaction between PD-1 and PD-L1 by hPD-1-IgV may be a promising strategy for specific tumor immunotherapy.

Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

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JIANG Hua-wei

2014-09-01

Full Text Available Objective: To establish the Chinese Hamster Ovary (CHO cell lines with stable expression of soluble CD40 ligands (sCD40L. Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR, reverse transcription-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA from aspects of deoxyribose nucleic acid (DNA, messenger ribonucleic acid (mRNA and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11 were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed ＞90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1 ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02 % to (34.8±8.75%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32% to (20.7±5.24%, and the differences

Induction of IL-12 Production in Human Peripheral Monocytes by Trypanosoma cruzi Is Mediated by Glycosylphosphatidylinositol-Anchored Mucin-Like Glycoproteins and Potentiated by IFN-γ and CD40-CD40L Interactions

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Lúcia Cristina Jamli Abel

2014-01-01

Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi, is characterized by immunopathology driven by IFN-γ secreting Th1-like T cells. T. cruzi has a thick coat of mucin-like glycoproteins covering its surface, which plays an important role in parasite invasion and host immunomodulation. It has been extensively described that T. cruzi or its products—like GPI anchors isolated from GPI-anchored mucins from the trypomastigote life cycle stage (tGPI-mucins—are potent inducers of proinflammatory responses (i.e., cytokines and NO production by IFN-γ primed murine macrophages. However, little is known about whether T. cruzi or GPI-mucins exert a similar action in human cells. We therefore decided to further investigate the in vitro cytokine production profile from human mononuclear cells from uninfected donors exposed to T. cruzi as well as tGPI-mucins. We observed that both living T. cruzi trypomastigotes and tGPI-mucins are potent inducers of IL-12 by human peripheral blood monocytes and this effect depends on CD40-CD40L interaction and IFN-γ. Our findings suggest that the polarized T1-type cytokine profile seen in T. cruzi infected patients might be a long-term effect of IL-12 production induced by lifelong exposure to T. cruzi tGPI-mucins.

A novel PKD2L1 C-terminal domain critical for trimerization and channel function.

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Zheng, Wang; Hussein, Shaimaa; Yang, JungWoo; Huang, Jun; Zhang, Fan; Hernandez-Anzaldo, Samuel; Fernandez-Patron, Carlos; Cao, Ying; Zeng, Hongbo; Tang, Jingfeng; Chen, Xing-Zhen

2015-03-30

As a transient receptor potential (TRP) superfamily member, polycystic kidney disease 2-like-1 (PKD2L1) is also called TRPP3 and has similar membrane topology as voltage-gated cation channels. PKD2L1 is involved in hedgehog signaling, intestinal development, and sour tasting. PKD2L1 and PKD1L3 form heterotetramers with 3:1 stoichiometry. C-terminal coiled-coil-2 (CC2) domain (G699-W743) of PKD2L1 was reported to be important for its trimerization but independent studies showed that CC2 does not affect PKD2L1 channel function. It thus remains unclear how PKD2L1 proteins oligomerize into a functional channel. By SDS-PAGE, blue native PAGE and mutagenesis we here identified a novel C-terminal domain called C1 (K575-T622) involved in stronger homotrimerization than the non-overlapping CC2, and found that the PKD2L1 N-terminus is critical for dimerization. By electrophysiology and Xenopus oocyte expression, we found that C1, but not CC2, is critical for PKD2L1 channel function. Our co-immunoprecipitation and dynamic light scattering experiments further supported involvement of C1 in trimerization. Further, C1 acted as a blocking peptide that inhibits PKD2L1 trimerization as well as PKD2L1 and PKD2L1/PKD1L3 channel function. Thus, our study identified C1 as the first PKD2L1 domain essential for both PKD2L1 trimerization and channel function, and suggest that PKD2L1 and PKD2L1/PKD1L3 channels share the PKD2L1 trimerization process.

Gamma-ray mutagenesis studies in a new human-hamster hybrid, A(L)CD59(+/-), which has two human chromosomes 11 but is hemizygous for the CD59 gene

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Kraemer, S. M.; Vannais, D. B.; Kronenberg, A.; Ueno, A.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

2001-01-01

Kraemer, S. M., Vannais, D. B., Kronenberg, A., Ueno, A. and Waldren, C. A. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid, A(L)CD59(+/-), which has Two Human Chromosomes 11 but is Hemizygous for the CD59 Gene. Radiat. Res. 156, 10-19 (2001).We have developed a human-CHO hybrid cell line, named A(L)CD59(+/-), which has two copies of human chromosome 11 but is hemizygous for the CD59 gene and the CD59 cell surface antigen that it encodes. Our previous studies used the A(L) and A(L)C hybrids that respectively contain one or two sets of CHO chromosomes plus a single copy of human chromosome 11. The CD59 gene at 11p13.5 and the CD59 antigen encoded by it are the principal markers used in our mutagenesis studies. The hybrid A(L)CD59(+/-) contains two copies of human chromosome 11, only one of which carries the CD59 gene. The incidence of CD59 (-) mutants (formerly called S1(-)) induced by (137)Cs gamma rays is about fivefold greater in A(L)CD59(+/-) cells than in A(L) cells. Evidence is presented that this increase in mutant yield is due to the increased induction of certain classes of large chromosomal mutations that are lethal to A(L) cells but are tolerated in the A(L)CD59(+/-) hybrid. In addition, significantly more of the CD59 (-) mutants induced by (137)Cs gamma rays in A(L)CD59(+/-) cells display chromosomal instability than in A(L) cells. On the other hand, the yield of gamma-ray-induced CD59 (-) mutants in A(L)CD59(+/-) cells is half that of the A(L)C hybrid, which also tolerates very large mutations but has only one copy of human chromosome 11. We interpret the difference in mutability as evidence that repair processes involving the homologous chromosomes 11 play a role in determining mutant yields. The A(L)CD59(+/-) hybrid provides a useful new tool for quantifying mutagenesis and shedding light on mechanisms of genetic instability and mutagenesis.

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies

NARCIS (Netherlands)

Chung, Nancy P. Y.; Matthews, Katie; Kim, Helen J.; Ketas, Thomas J.; Golabek, Michael; de Los Reyes, Kevin; Korzun, Jacob; Yasmeen, Anila; Sanders, Rogier W.; Klasse, Per Johan; Wilson, Ian A.; Ward, Andrew B.; Marozsan, Andre J.; Moore, John P.; Cupo, Albert

2014-01-01

Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate

Human CD40 ligand-expressing type 3 innate lymphoid cells induce IL-10-producing immature transitional regulatory B cells.

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Komlósi, Zsolt I; Kovács, Nóra; van de Veen, Willem; Kirsch, Anna Isabella; Fahrner, Heinz Benedikt; Wawrzyniak, Marcin; Rebane, Ana; Stanic, Barbara; Palomares, Oscar; Rückert, Beate; Menz, Günter; Akdis, Mübeccel; Losonczy, György; Akdis, Cezmi A

2017-09-20

Type 3 innate lymphoid cells (ILC3s) are involved in maintenance of mucosal homeostasis; however, their role in immunoregulation has been unknown. Immature transitional regulatory B (itBreg) cells are innate-like B cells with immunosuppressive properties, and the in vivo mechanisms by which they are induced have not been fully clarified. We aimed to investigate the ILC3-B-cell interaction that probably takes place in human tonsils. ILC3s were isolated from peripheral blood and palatine tonsils, expanded, and cocultured with naive B cells. Tonsillar ILC3s and regulatory B cells were visualized with immunofluorescence histology. ILC3 frequencies were measured in tonsil tissue of allergic and nonallergic patients and in peripheral blood of allergic asthmatic patients and healthy control subjects. A mutually beneficial relationship was revealed between ILC3s and B cells: ILC3s induced IL-15 production in B cells through B cell-activating factor receptor, whereas IL-15, a potent growth factor for ILC3s, induced CD40 ligand (CD40L) expression on circulating and tonsillar ILC3s. IL-15-activated CD40L + ILC3s helped B-cell survival, proliferation, and differentiation of IL-10-secreting, PD-L1-expressing functional itBreg cells in a CD40L- and B cell-activating factor receptor-dependent manner. ILC3s and regulatory B cells were in close connection with each other in palatine tonsils. ILC3 frequency was reduced in tonsil tissue of allergic patients and in peripheral blood of allergic asthmatic patients. Human CD40L + ILC3s provide innate B-cell help and are involved in an innate immunoregulatory mechanism through induction of itBreg cell differentiation, which takes place in palatine tonsils in vivo. This mechanism, which can contribute to maintenance of immune tolerance, becomes insufficient in allergic diseases. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Sequential and Simultaneous Immunization of Rabbits with HIV-1 Envelope Glycoprotein SOSIP.664 Trimers from Clades A, B and C

NARCIS (Netherlands)

Klasse, P. J.; LaBranche, Celia C.; Ketas, Thomas J.; Ozorowski, Gabriel; Cupo, Albert; Pugach, Pavel; Ringe, Rajesh P.; Golabek, Michael; van Gils, Marit J.; Guttman, Miklos; Lee, Kelly K.; Wilson, Ian A.; Butera, Salvatore T.; Ward, Andrew B.; Montefiori, David C.; Sanders, Rogier W.; Moore, John P.

2016-01-01

We have investigated the immunogenicity in rabbits of native-like, soluble, recombinant SOSIP.664 trimers based on the env genes of four isolates of human immunodeficiency virus type 1 (HIV-1); specifically BG505 (clade A), B41 (clade B), CZA97 (clade C) and DU422 (clade C). The various trimers were

CD40-CD40 Ligand Pathway is a Major Component of Acute Neuroinflammation and Contributes to Long-term Cognitive Dysfunction after Sepsis.

Science.gov (United States)

Michels, Monique; Danieslki, Lucinéia Gainski; Vieira, Andriele; Florentino, Drielly; Dall'Igna, Dhébora; Galant, Letícia; Sonai, Beatriz; Vuolo, Francieli; Mina, Franciele; Pescador, Bruna; Dominguini, Diogo; Barichello, Tatiana; Quevedo, João; Dal-Pizzol, Felipe; Petronilho, Fabrícia

2015-03-26

Sepsis-associated encephalopathy (SAE) is associated with an increased rate of morbidity and mortality. It is not understood what the exact mechanism is for the brain dysfunction that occurs in septic patients, but brain inflammation and oxidative stress are a possible theory. Such events can occur through the alteration of molecules that perpetuate the inflammatory response. Thus, it is possible to postulate that CD40 may be involved in this process. The aim of this work is to evaluate the role of CD40-CD40L pathway activation in brain dysfunction associated with sepsis in an animal model. Microglia activation induces the upregulation of CD40-CD40L, both in vitro and in vivo. The inhibition of microglia activation decreases levels of CD40-CD40L in the brain and decreases brain inflammation, oxidative damage and blood brain barrier dysfunction. Despite this, anti-CD40 treatment does not improve mortality in this model. However, it is able to improve long-term cognitive impairment in sepsis survivors. In conclusion, there is a major involvement of the CD40-CD40L signaling pathway in long-term brain dysfunction in an animal model of sepsis.

Luteolin, a flavonoid, inhibits CD40 ligand expression by activated human basophils.

Science.gov (United States)

Hirano, Toru; Arimitsu, Junsuke; Higa, Shinji; Naka, Tetsuji; Ogata, Atsushi; Shima, Yoshihito; Fujimoto, Minoru; Yamadori, Tomoki; Ohkawara, Tomoharu; Kuwabara, Yusuke; Kawai, Mari; Kawase, Ichiro; Tanaka, Toshio

2006-01-01

We have previously shown that flavonoids such as luteolin, apigenin and fisetin inhibit interleukin 4 and interleukin 13 production. In this study, we investigated whether luteolin can suppress CD40 ligand expression by basophils. A human basophilic cell line, KU812, was stimulated with A23187 and phorbol myristate acetate (PMA) with or without various concentrations of luteolin or other flavonoids for 12 h, and CD40 ligand expression was analyzed by FACS. The effect of luteolin on CD40 ligand mRNA expression was studied by semiquantitative reverse transcription PCR analysis. In addition, CD40 ligand expression was also measured in purified basophils that had been stimulated for 12 h with A23187 plus PMA with or without various concentrations of luteolin. CD40 ligand expression by KU812 cells was enhanced noticeably in response to A23187 and even more strikingly augmented by A23187 plus PMA. The expression was significantly suppressed by 10 or 30 microM of luteolin, whereas myricetin failed to inhibit. Reverse transcription PCR analyses demonstrated that luteolin inhibited CD40 ligand mRNA expression by stimulated KU812 cells. Of the six flavonoids examined, luteolin, apigenin, fisetin and quercetin at 30 microM showed a significant inhibitory effect on CD40 ligand expression. The incubation of purified basophils with A23187 plus PMA significantly enhanced CD40 ligand expression, and the presence of luteolin again had an inhibitory effect. Luteolin inhibits CD40 ligand expression by activated basophils.

The role of CD40L, IL-10 and IL-17 in radioprotection

International Nuclear Information System (INIS)

Li Ting

2003-01-01

CD40L/CD40 interaction is central to the control of thymus-dependent humoral immunity and cell mediated immune responses. IL-17 has been shown to induce the production of IL-6 and G-CSF, which can induce proliferation and differentiation of CD34 + hematopoietic progenitors. IL-10 can interfere with up-regulation of costimulatory molecules, thus suppressing the production of costimulatory cytokines, such as IL-12. IL-10 has been implicated as an essential mediator in the induction of systemic immune suppression following ultraviolet (UV) exposure. Treating UV-irradiated mice with anti-IL-10 blocks the induction of immune suppression

TLR5 signaling enhances the proliferation of human allogeneic CD40-activated B cell induced CD4hiCD25+ regulatory T cells.

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Ping-Lung Chan

Full Text Available Although diverse functions of different toll-like receptors (TLR on human natural regulatory T cells have been demonstrated recently, the role of TLR-related signals on human induced regulatory T cells remain elusive. Previously our group developed an ex vivo high-efficient system in generating human alloantigen-specific CD4(hiCD25(+ regulatory T cells from naïve CD4(+CD25(- T cells using allogeneic CD40-activated B cells as stimulators. In this study, we investigated the role of TLR5-related signals on the generation and function of these novel CD4(hiCD25(+ regulatory T cells. It was found that induced CD4(hiCD25(+ regulatory T cells expressed an up-regulated level of TLR5 compared to their precursors. The blockade of TLR5 using anti-TLR5 antibodies during the co-culture decreased CD4(hiCD25(+ regulatory T cells proliferation by induction of S phase arrest. The S phase arrest was associated with reduced ERK1/2 phosphorylation. However, TLR5 blockade did not decrease the CTLA-4, GITR and FOXP3 expressions, and the suppressive function of CD4(hiCD25(+ regulatory T cells. In conclusion, we discovered a novel function of TLR5-related signaling in enhancing the proliferation of CD4(hiCD25(+ regulatory T cells by promoting S phase progress but not involved in the suppressive function of human CD40-activated B cell-induced CD4(hiCD25(+ regulatory T cells, suggesting a novel role of TLR5-related signals in the generation of induced regulatory T cells.

A novel recombinantly produced banana lectin isoform is a valuable tool for glycoproteomics and a potent modulator of the proliferation response in CD3(+), CD4(+), and CD8(+) populations of human PBMCs

DEFF Research Database (Denmark)

Gavrovic-Jankulovic, M; Poulsen, Knud; Brckalo, T

2008-01-01

Lectins as carbohydrate-binding proteins have been employed in various biological assays for the detection and characterization of glycan structures on glycoproteins, including clinical biomarkers in disease states. A mannose-specific banana lectin (BanLec) is unique in its specificity for internal......, the immunomodulatory potential of rBanLec and nBanLec were comparable as assessed by an inhibition assay and a human T cell proliferation assay where they induced a strong proliferation response in CD3(+), CD4(+), and CD8(+) populations of human PBMCs. This recombinant BanLec is a useful reagent for glycoproteomics...

CD40–CD40 Ligand Pathway Is a Major Component of Acute Neuroinflammation and Contributes to Long-term Cognitive Dysfunction after Sepsis

Science.gov (United States)

Michels, Monique; Danieslki, Lucinéia Gainski; Vieira, Andriele; Florentino, Drielly; Dall’Igna, Dhébora; Galant, Letícia; Sonai, Beatriz; Vuolo, Francieli; Mina, Franciele; Pescador, Bruna; Dominguini, Diogo; Barichello, Tatiana; Quevedo, João; Dal-Pizzol, Felipe; Petronilho, Fabrícia

2015-01-01

Sepsis-associated encephalopathy (SAE) is associated with an increased rate of morbidity and mortality. It is not understood what the exact mechanism is for the brain dysfunction that occurs in septic patients, but brain inflammation and oxidative stress are a possible theory. Such events can occur through the alteration of molecules that perpetuate the inflammatory response. Thus, it is possible to postulate that CD40 may be involved in this process. The aim of this work is to evaluate the role of CD40–CD40L pathway activation in brain dysfunction associated with sepsis in an animal model. Microglia activation induces the upregulation of CD40–CD40L, both in vitro and in vivo. The inhibition of microglia activation decreases levels of CD40–CD40L in the brain and decreases brain inflammation, oxidative damage and blood brain barrier dysfunction. Despite this, anti-CD40 treatment does not improve mortality in this model. However, it is able to improve long-term cognitive impairment in sepsis survivors. In conclusion, there is a major involvement of the CD40–CD40L signaling pathway in long-term brain dysfunction in an animal model of sepsis. PMID:25822797

Biophysical characterisation of GlycoPEGylated recombinant human factor VIIa

DEFF Research Database (Denmark)

Plesner, Bitten; Westh, Peter; Nielsen, Anders D.

2011-01-01

The effects of GlycoPEGylation on the structural, kinetic and thermal stability of recombinant human FVIIa were investigated using rFVIIa and linear 10 kDa and branched 40 kDa GlycoPEGylated® recombinant human FVIIa derivatives. The secondary and tertiary structure of rFVIIa measured by circular...... dichroism (CD) was maintained upon PEGylation. In contrast, the thermal and kinetic stability of rFVIIa was affected by GlycoPEGylation, as the apparent unfolding temperature Tm measured by differential scanning calorimetry (DSC) and the temperature of aggregation, Tagg, measured by light scattering (LS......) both increased with GlycoPEGylation. Both Tm and Tagg were independent of the molecular weight and the shape of the PEG chain. From the present biophysical characterisation it is concluded that after GlycoPEGylation, rFVIIa appears to be unaffected structurally (secondary and tertiary structure...

Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide

International Nuclear Information System (INIS)

Lai Yuping; Peng Yifei; Zuo Yi; Li Jun; Huang Jing; Wang Linfa; Wu Zirong

2005-01-01

Antimicrobial peptides from human skin are an important component of the innate immune response and play a key role as a first line of defense against infections. One such peptide is the recently discovered dermcidin-1L. To better understand its mechanism and to further investigate its antimicrobial spectrum, recombinant dermcidin-1L was expressed in Escherichia coli as a fusion protein and purified by affinity chromatography. The fusion protein was cleaved by factor Xa protease to produce recombinant dermcidin-1L. Antimicrobial and hemolytic assays demonstrated that dermcidin-1L displayed microbicidal activity against several opportunistic nosocomial pathogens, but no hemolytic activity against human erythrocytes even at concentrations up to 100 μM. Structural studies performed by circular dichroism spectroscopy indicated that the secondary structure of dermcidin-1L was very flexible, and both α-helix and β-sheet structures might be required for the antimicrobial activity. Our results confirmed previous findings indicating that dermcidin-1L could have promising therapeutic potentials and shed new light on the structure-function relationship of dermcidin-1L

[Cloning of human CD45 gene and its expression in Hela cells].

Science.gov (United States)

Li, Jie; Xu, Tianyu; Wu, Lulin; Zhang, Liyun; Lu, Xiao; Zuo, Daming; Chen, Zhengliang

2015-11-01

To clone human CD45 gene PTPRC and establish Hela cells overexpressing recombinant human CD45 protein. The intact cDNA encoding human CD45 amplified using RT-PCR from the total RNA extracted from peripheral blood mononuclear cells (PBMCs) of a healthy donor was cloned into pMD-18T vector. The CD45 cDNA fragment amplified from the pMD-18T-CD45 by PCR was inserted to the coding region of the PcDNA3.1-3xflag vector, and the resultant recombinant expression vector PcDNA3.1-3xflag-CD45 was transfected into Hela cells. The expression of CD45 in Hela cells was detected by flow cytometry and Western blotting, and the phosphastase activity of CD45 was quantified using an alkaline phosphatase assay kit. The cDNA fragment of about 3 900 bp was amplified from human PBMCs and cloned into pMD-18T vector. The recombinant expression vector PcDNA3.1-3xflag-CD45 was constructed, whose restriction maps and sequence were consistent with those expected. The expression of CD45 in transfected Hela cells was detected by flow cytometry and Western blotting, and the expressed recombinant CD45 protein in Hela cells showed a phosphastase activity. The cDNA of human CD45 was successfully cloned and effectively expressed in Hela cells, which provides a basis for further exploration of the functions of CD45.

In Vivo Replication and Pathogenesis of Vesicular Stomatitis Virus Recombinant M40 Containing Ebola Virus L-Domain Sequences

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Takashi Irie

2012-01-01

Full Text Available The M40 VSV recombinant was engineered to contain overlapping PTAP and PPxY L-domain motifs and flanking residues from the VP40 protein of Ebola virus. Replication of M40 in cell culture is virtually indistinguishable from that of control viruses. However, the presence of the Ebola PTAP motif in the M40 recombinant enabled this virus to interact with and recruit host Tsg101, which was packaged into M40 virions. In this brief report, we compared replication and the pathogenic profiles of M40 and the parental virus M51R in mice to determine whether the presence of the Ebola L-domains and flanking residues altered in vivo characteristics of the virus. Overall, the in vivo characteristics of M40 were similar to those of the parental M51R virus, indicating that the Ebola sequences did not alter pathogenesis of VSV in this small animal model of infection.

Radiative and interfacial recombination in CdTe heterostructures

Energy Technology Data Exchange (ETDEWEB)

Swartz, C. H., E-mail: craig.swartz@txstate.edu; Edirisooriya, M.; LeBlanc, E. G.; Noriega, O. C.; Jayathilaka, P. A. R. D.; Ogedengbe, O. S.; Hancock, B. L.; Holtz, M.; Myers, T. H. [Materials Science, Engineering, and Commercialization Program, Texas State University, 601 University Dr., San Marcos, Texas 78666 (United States); Zaunbrecher, K. N. [National Renewable Energy Laboratory, 15013 Denver West Parkway, Mississippi RSF200, Golden, Colorado 80401 (United States)

2014-12-01

Double heterostructures (DH) were produced consisting of a CdTe film between two wide band gap barriers of CdMgTe alloy. A combined method was developed to quantify radiative and non-radiative recombination rates by examining the dependence of photoluminescence (PL) on both excitation intensity and time. The measured PL characteristics, and the interface state density extracted by modeling, indicate that the radiative efficiency of CdMgTe/CdTe DHs is comparable to that of AlGaAs/GaAs DHs, with interface state densities in the low 10{sup 10 }cm{sup −2} and carrier lifetimes as long as 240 ns. The radiative recombination coefficient of CdTe is found to be near 10{sup −10} cm{sup 3}s{sup −1}. CdTe film growth on bulk CdTe substrates resulted in a homoepitaxial interface layer with a high non-radiative recombination rate.

Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome

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Maria J. Abrey Recalde

2017-10-01

Full Text Available Shiga toxin (Stx, produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS, which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L, which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions.

Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome

Science.gov (United States)

Abrey Recalde, Maria J.; Alvarez, Romina S.; Alberto, Fabiana; Mejias, Maria P.; Ramos, Maria V.; Fernandez Brando, Romina J.; Bruballa, Andrea C.; Exeni, Ramon A.; Alconcher, Laura; Ibarra, Cristina A.; Amaral, María M.; Palermo, Marina S.

2017-01-01

Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions. PMID:29068360

Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

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Rong Jin

Full Text Available Recent work has revealed an essential involvement of soluble CD40L (sCD40L in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40, i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better

Functional Analysis of CD28/B7 and CD40/CD40L Costimulation During the in vivo Type 2 Immune Response

Science.gov (United States)

1995-10-06

Fay, T.M., Masters, S.R, Laman , J.D., and Noelle, R.J. (1994b). The role of CD40 in the regulation ofhumoral and cell-mediated immunity. Immuno...1567-1575. 187 Foy, T.M., Laman , J.D., Ledbetter, J.A., Aruff’o, A., Claassen, E., and Noelle, R.J. (1994). gp39-CD40 interaction are essential for...cystine knot motif. Cell 73,421-424. Mohan, C., Shi, Y., Laman , J.D., and Datta, S.K. (1995). Interaction between CD40 and its ligand gp39 in the

Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

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Zanotto Carlo

2011-11-01

Full Text Available Abstract Background Human papilloma virus (HPV-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1 have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species. Methods A new fowlpox virus recombinant encoding HPV-L1 (FPL1 was engineered and evaluated for the correct expression of HPV-L1 in vitro, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays. Results The FPL1 recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector. Conclusion This FPL1 recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.

The Signaling Role of CD40 Ligand in Platelet Biology and in Platelet Component Transfusion

Science.gov (United States)

Aoui, Chaker; Prigent, Antoine; Sut, Caroline; Tariket, Sofiane; Hamzeh-Cognasse, Hind; Pozzetto, Bruno; Richard, Yolande; Cognasse, Fabrice; Laradi, Sandrine; Garraud, Olivier

2014-01-01

The CD40 ligand (CD40L) is a transmembrane molecule of crucial interest in cell signaling in innate and adaptive immunity. It is expressed by a variety of cells, but mainly by activated T-lymphocytes and platelets. CD40L may be cleaved into a soluble form (sCD40L) that has a cytokine-like activity. Both forms bind to several receptors, including CD40. This interaction is necessary for the antigen specific immune response. Furthermore, CD40L and sCD40L are involved in inflammation and a panoply of immune related and vascular pathologies. Soluble CD40L is primarily produced by platelets after activation, degranulation and cleavage, which may present a problem for transfusion. Soluble CD40L is involved in adverse transfusion events including transfusion related acute lung injury (TRALI). Although platelet storage designed for transfusion occurs in sterile conditions, platelets are activated and release sCD40L without known agonists. Recently, proteomic studies identified signaling pathways activated in platelet concentrates. Soluble CD40L is a good candidate for platelet activation in an auto-amplification loop. In this review, we describe the immunomodulatory role of CD40L in physiological and pathological conditions. We will focus on the main signaling pathways activated by CD40L after binding to its different receptors. PMID:25479079

Human CD180 Transmits Signals via the PIM-1L Kinase.

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Nicole Egli

Full Text Available Toll-like receptors (TLRs are important sensors of the innate immune system that recognize conserved structural motifs and activate cells via a downstream signaling cascade. The CD180/MD1 molecular complex is an unusual member of the TLR family, since it lacks the components that are normally required for signal transduction by other TLRs. Therefore the CD180/MD 1 complex has been considered of being incapable of independently initiating cellular signals. Using chemogenetic approaches we identified specifically the membrane bound long form of PIM-1 kinase, PIM-1L as the mediator of CD180-dependent signaling. A dominant negative isoform of PIM-1L, but not of other PIM kinases, inhibited signaling elicited by cross-linking of CD180, and this effect was phenocopied by PIM inhibitors. PIM-1L was directed to the cell membrane by its N-terminal extension, where it colocalized and physically associated with CD180. Triggering CD180 also induced increased phosphorylation of the anti-apoptotic protein BAD in a PIM kinase-dependent fashion. Also in primary human B cells, which are the main cells expressing CD180 in man, cross-linking of CD180 by monoclonal antibodies stimulated cell survival and proliferation that was abrogated by specific inhibitors. By associating with PIM-1L, CD180 can thus obtain autonomous signaling capabilities, and this complex is then channeling inflammatory signals into B cell survival programs. Pharmacological inhibition of PIM-1 should therefore provide novel therapeutic options in diseases that respond to innate immune stimulation with subsequently increased B cell activity, such as lupus erythematosus or myasthenia gravis.

Despite disorganized synapse structure, Th2 cells maintain directional delivery of CD40L to antigen-presenting B cells.

Science.gov (United States)

Gardell, Jennifer L; Parker, David C

2017-01-01

Upon recognition of peptide displayed on MHC molecules, Th1 and Th2 cells form distinct immunological synapse structures. Th1 cells have a bull's eye synapse structure with TCR/ MHC-peptide interactions occurring central to a ring of adhesion molecules, while Th2 cells have a multifocal synapse with small clusters of TCR/MHC interactions throughout the area of T cell/antigen-presenting cell interaction. In this study, we investigated whether this structural difference in the immunological synapse affects delivery of T cell help. The immunological synapse is thought to ensure antigen-specific delivery of cytolytic granules and killing of target cells by NK cells and cytolytic T cells. In helper T cells, it has been proposed that the immunological synapse may direct delivery of other effector molecules including cytokines. CD40 ligand (CD40L) is a membrane-bound cytokine essential for antigen-specific T cell help for B cells in the antibody response. We incubated Th1 and Th2 cells overnight with a mixture of antigen-presenting and bystander B cells, and the delivery of CD40L to B cells and subsequent B cell responses were compared. Despite distinct immunological synapse structures, Th1 and Th2 cell do not differ in their ability to deliver CD40L and T cell help in an antigen-specific fashion, or in their susceptibility to inhibition of help by a blocking anti-CD40L antibody.

Assessment of serum levels of soluble CD40L in Egyptian children and adolescents with type 1 diabetes mellitus: Relationship to microalbuminuria and glycemic control

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Kotb Abbass Metwalley

2013-01-01

Full Text Available Context: Soluble CD40 ligand (sCD40L is known to be elevated in different clinical situations including hypercholesterolemia, acute coronary syndromes, and type 2 diabetes mellitus (T2DM, Data about the relationship between type 1 diabetes mellitus (T1DM and sCD40L is limited. In addition, the potential role ofsCD40Lin the pathogenesis of vascular complications in children and adolescents with T1DM is to be clarified. Hence, the study aimed at assessment of sCD40L levels in children and adolescents with T1DM and correlation of these levels with glycemic control and microalbuminuria. Settings and Design: Cross-sectional controlled study. Materials and Methods: The study was performed in the Pediatric Endocrinology and Diabetes Unit, Assuit University Children Hospital, Assiut, Egypt. It included 70 children and adolescents with T1DM (mean age 14. 76 ± 2.21 years. Cases were further subdivided into 43 cases with normoalbuminuria and 27 cases with microalbuminuria according to presence or absence or microalbuminuria in fresh urine samples. Twentyfive healthy subjects, age- and sex-matched were included as control group (mean age = 13.62 ± 2.11 years. Studied cases were subjected to medical history, clinical examination, and laboratory assessment of fasting blood glucose (FBG, lipid profile, glycosylated hemoglobin (HbA1c, and sCD40L were performed. Results: Mean HbA1c and sCD40L were significantly higher in diabetic children (n = 70 compared to control (n = 25 (P < 0.001 for each. Mean HbA1c and sCD40L levels were significantly higher in microalbuminuric cases (n = 27 compared to normoalbuminuric cases (n = 43 (P < 0.05 and <0.01, respectively.We also observed a significant positive correlation between sCD40L levels and the age, diabetes duration, HbA1c, and urinary albumin creatinine ratio. Conclusions: The high serum sCD40L levels in children and adolescents with T1DM particularly in those with microalbminuria and its positive correlation with

Requirement for CD40 ligand, CD4(+) T cells, and B cells in an infectious mononucleosis-like syndrome

DEFF Research Database (Denmark)

Brooks, J W; Hamilton-Easton, A M; Christensen, J P

1999-01-01

(+) CD8(+) population that is found in mice with different major histocompatibility complex (MHC) haplotypes. Aspects of the CD8(+)-T-cell response are substantially modified in mice that lack B cells, CD4(+) T cells, or the CD40 ligand (CD40L). The B-cell-deficient mice show no increase in Vbeta4(+) CD8......(+) T cells. Similar abrogation of the Vbeta4(+) CD8(+) response is seen following antibody-mediated depletion of the CD4(+) subset, through the numbers of CD8(+) CD62L(lo) cells are still significantly elevated. Virus-specific CD4(+)-T-cell frequencies are minimal in the CD40L(-/-) mice, and the Vbeta4......(+) CD8(+) population remains unexpanded. Apparently B-cell-CD4(+)-T-cell interactions play a part in the gammaHV-68 induction of both splenomegaly and non-MHC-restricted Vbeta4(+) CD8(+)-T-cell expansion....

Cyclic trimer of human cystatin C, an amyloidogenic protein - molecular dynamics and experimental studies

Science.gov (United States)

Chrabåszczewska, Magdalena; Maszota-Zieleniak, Martyna; Pietralik, Zuzanna; Taube, Michał; Rodziewicz-Motowidło, Sylwia; Szymańska, Aneta; Szutkowski, Kosma; Clemens, Daniel; Grubb, Anders; Kozak, Maciej

2018-05-01

Human cystatin C (HCC) is a cysteine protease inhibitor that takes a series of oligomeric forms in solution (e.g., dimers, trimers, tetramers, decamers, dodecamers, and other higher oligomers). The best-known form of cystatin C is the dimer, which arises as a result of a domain swapping mechanism. The formation of the HCC oligomeric forms, which is most likely due to this domain swapping mechanism, is associated with the aggregation of HCC into amyloid fibrils and deposits. To investigate the structure of a specific HCC oligomer, we developed a covalently stabilized trimer of HCC. An atomic model of this HCC trimer was proposed on the basis of molecular docking and molecular dynamics simulations. The most stable model of the HCC trimer obtained from the molecular dynamics simulations is characterized by a well-preserved secondary structure. The molecular size and structural parameters of the HCC trimer in solution were also confirmed by Small Angle Neutron Scattering and Nuclear Magnetic Resonance Diffusometry.

Purification, crystallization and preliminary X-ray diffraction study of human ribosomal protein L10 core domain

International Nuclear Information System (INIS)

Nishimura, Mitsuhiro; Kaminishi, Tatsuya; Kawazoe, Masahito; Shirouzu, Mikako; Takemoto, Chie; Yokoyama, Shigeyuki; Tanaka, Akiko; Sugano, Sumio; Yoshida, Takuya; Ohkubo, Tadayasu; Kobayashi, Yuji

2007-01-01

A truncated variant of human ribosomal protien L10 was prepared and crystallized. Diffraction data were collected to 2.5 Å resolution. Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to space group P3 1 21 or P3 2 21

Interaction of calreticulin with CD40 ligand, TRAIL and Fas ligand

DEFF Research Database (Denmark)

Duus, K; Pagh, R T; Holmskov, U

2007-01-01

is utilized by many other functionally diverse molecules and in this work the interaction of calreticulin with C1q and structurally similar molecules was investigated. In addition to C1q and MBL, CD40 ligand (CD40L), tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) were...... found to bind calreticulin strongly. A low level or no binding was observed for adiponectin, tumour necrosis factor-alpha (TNF-alpha), CD30L, surfactant protein-A and -D and collagen VIII. The interaction with calreticulin required a conformational change in CD40L, TRAIL and FasL and showed the same...

Charge-carrier transport and recombination in heteroepitaxial CdTe

International Nuclear Information System (INIS)

Kuciauskas, Darius; Farrell, Stuart; Dippo, Pat; Moseley, John; Moutinho, Helio; Li, Jian V.; Allende Motz, A. M.; Kanevce, Ana; Zaunbrecher, Katherine; Gessert, Timothy A.; Levi, Dean H.; Metzger, Wyatt K.; Colegrove, Eric; Sivananthan, S.

2014-01-01

We analyze charge-carrier dynamics using time-resolved spectroscopy and varying epitaxial CdTe thickness in undoped heteroepitaxial CdTe/ZnTe/Si. By employing one-photon and nonlinear two-photon excitation, we assess surface, interface, and bulk recombination. Two-photon excitation with a focused laser beam enables characterization of recombination velocity at the buried epilayer/substrate interface, 17.5 μm from the sample surface. Measurements with a focused two-photon excitation beam also indicate a fast diffusion component, from which we estimate an electron mobility of 650 cm 2 (Vs) −1 and diffusion coefficient D of 17 cm 2  s −1 . We find limiting recombination at the epitaxial film surface (surface recombination velocity S surface  = (2.8 ± 0.3) × 10 5  cm s −1 ) and at the heteroepitaxial interface (interface recombination velocity S interface  = (4.8 ± 0.5) × 10 5  cm s −1 ). The results demonstrate that reducing surface and interface recombination velocity is critical for photovoltaic solar cells and electronic devices that employ epitaxial CdTe.

HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations.

Science.gov (United States)

Ma, Xiaochu; Lu, Maolin; Gorman, Jason; Terry, Daniel S; Hong, Xinyu; Zhou, Zhou; Zhao, Hong; Altman, Roger B; Arthos, James; Blanchard, Scott C; Kwong, Peter D; Munro, James B; Mothes, Walther

2018-03-21

HIV-1 entry into cells requires binding of the viral envelope glycoprotein (Env) to receptor CD4 and coreceptor. Imaging of individual Env molecules on native virions shows Env trimers to be dynamic, spontaneously transitioning between three distinct well-populated conformational states: a pre-triggered Env (State 1), a default intermediate (State 2) and a three-CD4-bound conformation (State 3), which can be stabilized by binding of CD4 and coreceptor-surrogate antibody 17b. Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we show the default intermediate configuration to be asymmetric, with individual protomers adopting distinct conformations. During entry, this asymmetric intermediate forms when a single CD4 molecule engages the trimer. The trimer can then transition to State 3 by binding additional CD4 molecules and coreceptor.

Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris.

Science.gov (United States)

Dagar, Vikas Kumar; Adivitiya; Devi, Nirmala; Khasa, Yogender Pal

2016-10-01

Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145 mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475 mg/L with a high volumetric productivity of 4.39 mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni-NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40 % α-helix, 12 % β-sheets with an emission maxima at 343 nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.

Involvement of mitogen-activated protein kinases and NFκB in LPS-induced CD40 expression on human monocytic cells

International Nuclear Information System (INIS)

Wu Weidong; Alexis, Neil E.; Chen Xian; Bromberg, Philip A.; Peden, David B.

2008-01-01

CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFκB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFκB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFκB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFκB activation, and CD40 expression. Moreover, blockage of MAPK and NFκB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFκB

Human surfactant protein A2 gene mutations impair dimmer/trimer assembly leading to deficiency in protein sialylation and secretion.

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Yi Song

Full Text Available Surfactant protein A2 (SP-A2 plays an essential role in surfactant metabolism and lung host defense. SP-A2 mutations in the carbohydrate recognition domain have been related to familial pulmonary fibrosis and can lead to a recombinant protein secretion deficiency in vitro. In this study, we explored the molecular mechanism of protein secretion deficiency and the subsequent biological effects in CHO-K1 cells expressing both wild-type and several different mutant forms of SP-A2. We demonstrate that the SP-A2 G231V and F198S mutants impair the formation of dimmer/trimer SP-A2 which contributes to the protein secretion defect. A deficiency in sialylation, but not N-linked glycosylation, is critical to the observed dimmer/trimer impairment-induced secretion defect. Furthermore, both mutant forms accumulate in the ER and form NP-40-insoluble aggregates. In addition, the soluble mutant SP-A2 could be partially degraded through the proteasome pathway but not the lysosome or autophagy pathway. Intriguingly, 4-phenylbutyrate acid (4-PBA, a chemical chaperone, alleviates aggregate formation and partially rescued the protein secretion of SP-A2 mutants. In conclusion, SP-A2 G231V and F198S mutants impair the dimmer/trimer assembly, which contributes to the protein sialylation and secretion deficiency. The intracellular protein mutants could be partially degraded through the proteasome pathway and also formed aggregates. The treatment of the cells with 4-PBA resulted in reduced aggregation and rescued the secretion of mutant SP-A2.

CD40 in clinical inflammation: From multiple sclerosis to atherosclerosis

NARCIS (Netherlands)

Laman, J.D.; Boer, M. de; Hart, B.A. 't

1998-01-01

The interactions of CD40 and CD40L have been known for some time to critically regulate B-cell responses with respect to proliferation, isotype switching, antibody production, and memory formation. More recent findings demonstrated that CD40 can be expressed on several other antigen-presenting cell

Serum Levels of Platelet Released CD40 Ligand Are Increased in Early Onset Occlusive Carotid Artery Disease

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József Balla

2006-01-01

Full Text Available Objective: Soluble CD40 ligand (sCD40L has been suggested as a key mediator between inflammation and atherosclerosis, and the CD40-CD40L interaction has a role in atherosclerotic lesion progression. We evaluated if platelet released serum sCD40L and sCD40 levels differ between patients with early onset occlusive carotid artery disease and age-matched controls.

Increased concentrations of soluble CD40 ligand platelet in patients with primary antiphospholipidic syndrome.

Science.gov (United States)

Galicia López, Aida; Olguín Ortega, Lourdes; Saavedra, Miguel A; Méndez Cruz, René; Jimenez Flores, Rafael; García de la Peña, Maximiliano

2013-01-01

To determine the concentrations of sCD40L in patients with PAPS, and establish its association with the number of thrombosis. We included patients with PAPS and healthy controls of the same age and sex. For analysis, patients with PAPS were divided into 2 groups: 1) patients with 1 thrombosis, and 2) patients with >1 thrombosis. Soluble CD40L concentrations were determined by ELISA method. sCD40L concentrations were significantly higher in patients with PAPS compared with the controls (9.72 ng ± 11.23 ng/ml vs. 4.69 ± 4.04 ng/ml) (P=.04) There was no association between serum levels of sCD40L and the number of thrombosis (1 thrombosis: 9.81 ± 9.87 ng/ml vs 9.63 ± 12.75 ng/ml in ≥ 1thrombosis (P=.13). In women with pregnancy and abortions, (13 patients) concentrations of sCD40L were higher than in those patients without a history of abortion (26 patients) but without statically significant difference (12.11 ± 16.46 ng/ml vs. 8.80 ± 8.61 ng/ml) (P=.33). There was no correlation between levels of sCD40L and the total number of thrombosis. Patients with PAPS have higher concentrations of sCD40L compared with healthy subjects, although this is not associated with a greater number of thrombosis. Among patients with PAPS, there is a tendency to higher concentrations of sCD40L in women with pregnancy and history of abortion. Since the platelet is the main cellular source of sCD40L, is possible that this pathway plays a pathogenic role in patients with PAPS. Copyright © 2012 Elsevier España, S.L. All rights reserved.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

Science.gov (United States)

Doublier, Sophie; Zennaro, Cristina; Musante, Luca; Spatola, Tiziana; Candiano, Giovanni; Bruschi, Maurizio; Besso, Luca; Cedrino, Massimo; Carraro, Michele; Ghiggeri, Gian Marco; Camussi, Giovanni; Lupia, Enrico

2017-01-01

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

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Sophie Doublier

Full Text Available CD40/CD40 ligand (CD40L dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs. We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS, and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS, and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

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Mattias N E Forsell

2008-10-01

Full Text Available The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3. Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4 rabbits with envelope glycoprotein (Env trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

Science.gov (United States)

Forsell, Mattias N E; Dey, Barna; Mörner, Andreas; Svehla, Krisha; O'dell, Sijy; Högerkorp, Carl-Magnus; Voss, Gerald; Thorstensson, Rigmor; Shaw, George M; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T

2008-10-03

The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

Blood Mixing Upregulates Platelet Membrane-Bound CD40 Ligand Expression in vitro Independent of Abo Compatibility.

Science.gov (United States)

Huang, Go-Shine; Hu, Mei-Hua; Lin, Tso-Chou; Lin, Yi-Chang; Tsai, Yi-Ting; Lin, Chih-Yuan; Ke, Hung-Yen; Zheng, Xu-Zhi; Tsai, Chien-Sung

2017-11-30

Platelets play a central role in the inflammation response via CD40 ligand (CD40L) expression, which may lead to transfusion reactions. The precise role of platelet CD40L-mediated inflammation in transfusion reactions is unclear. Therefore, we assessed the effects of in vitro blood mixing on platelet CD40L expression. In addition, we examined the effect of ABO compatibility on CD40L expression. Donor packed red blood cells were acquired from a blood bank, and recipient blood was obtained from patients undergoing cardiac surgery and prepared as washed platelets. Donor blood was mixed with suspended, washed recipient platelets to obtain a final mixing ratio of 1%, 5%, or 10% (vol/vol). The blood mixtures were divided into three groups: Group M, cross-matched blood-type mixing (n = 20); Group S, ABO type-specific uncross-matched blood (n = 20); and Group I, ABO incompatibility (not ABO type-specific blood and not process cross-matched) mixing (n = 20). The blood mixtures were used to detect platelet membrane-bound CD40L expression by flow cytometry. Blood mixing resulted in an increase in CD40L expression in Group M (P role in the induction of CD40L expression.

Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies

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Shridhar Bale

2018-05-01

Full Text Available Native flexibly linked (NFL HIV-1 envelope glycoprotein (Env trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1.8 g/l. BG505 NFL trimers purified by single-step lectin-affinity chromatography displayed a native-like closed structure, efficient recognition by trimer-preferring bNAbs, no recognition by non-neutralizing CD4 binding site-directed and V3-directed antibodies, long-term stability, and proper N-glycan processing. Following negative-selection, formulation in ISCOMATRIX adjuvant and inoculation into rabbits, the trimers rapidly elicited potent autologous tier 2 neutralizing antibodies. These antibodies targeted the N-glycan “hole” naturally present on the BG505 Env proximal to residues at positions 230, 241, and 289. The BG505 NFL trimers that did not expose V3 in vitro, elicited low-to-no tier 1 virus neutralization in vivo, indicating that they remained intact during the immunization process, not exposing V3. In addition, BG505 NFL and BG505 SOSIP trimers expressed from 293F cells, when formulated in Adjuplex adjuvant, elicited equivalent BG505 tier 2 autologous neutralizing titers. These titers were lower in potency when compared to the titers elicited by CHO-M cell derived trimers. In addition, increased neutralization of tier 1 viruses was detected. Taken together, these data indicate that both adjuvant and cell-type expression can affect the elicitation of tier 2 and tier 1 neutralizing responses in vivo.

Reduced influenza viral neutralizing activity of natural human trimers of surfactant protein D

DEFF Research Database (Denmark)

Hartshorn, Kevan L; White, Mitchell R; Tecle, Tesfaldet

2007-01-01

BACKGROUND: Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection. Common human polymorphisms of SP-D have been found in many human populations and associated with increased risk of certain infections. We recently reported that the Thr...... on the CRD of SP-D were found to have differing effects on antiviral activity. Using an mAb that did not interfere with antiviral activity of SP-D, we confirm that natural SP-D trimers had reduced ability to bind to IAV. In addition, the trimers had reduced ability to neutralize IAV as compared to natural...... indicate that a common human polymorphic form of SP-D may modulate host defense against IAV and give impetus to clinical studies correlating this genotype with risk for IAV infection in susceptible groups. We also show that mAbs directed against different areas on the carbohydrate recognition domain of SP...

Oral Salmonella: malaria circumsporozoite recombinants induce specific CD8+ cytotoxic T cells

OpenAIRE

1990-01-01

Oral immunization with an attenuated Salmonella typhimurium recombinant containing the full-length Plasmodium berghei circumsporozoite (CS) gene induces protective immunity against P. berghei sporozoite challenge in the absence of antibody. We found that this immunity was mediated through the induction of specific CD8+ T cells since in vivo elimination of CD8+ cells abrogated protection. In vitro studies revealed that this Salmonella-P. berghei CS recombinant induced class I- restricted CD8+ ...

Study of association of CD40-CD154 gene polymorphisms with disease susceptibility and cardiovascular risk in Spanish rheumatoid arthritis patients.

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Mercedes García-Bermúdez

Full Text Available Rheumatoid arthritis (RA is a chronic inflammatory disease associated with increased cardiovascular (CV mortality. Since CD40-CD154 binding has direct consequences on inflammation process initiation, we aimed to replicate previous findings related to disease susceptibility in Spanish RA population. Furthermore, as the major complication in RA disease patients is the development of CV events due to accelerated atherosclerosis, and elevated levels of CD40L/CD154 are present in patients with acute myocardial infarction, we assessed the potential association of CD40 and CD154/CD40L gene variants with CV risk in Spanish RA patients.One thousand five hundred and seventy-five patients fulfilling the 1987 ACR classification criteria for RA and 1600 matched controls were genotyped for the CD40 rs1883832, rs4810485 and rs1535045 and CD154 rs3092952 and rs3092920 gene polymorphisms, using predesigned TaqMan single nucleotide polymorphism genotyping assays. Afterwards, we investigated the influence of CD40-CD154 gene variants in the development of CV events. Also, in a subgroup of 273 patients without history of CV events, we assessed the influence of these polymorphisms in the risk of subclinical atherosclerosis determined by carotid ultrasonography.Nominally significant differences in the allele frequencies for the rs1883832 CD40 gene polymorphism between RA patients and controls were found (p=0.038. Although we did not observe a significant association of CD40-CD154 gene variants with the development of CV events, an ANCOVA model adjusted for sex, age at the time of the ultrasonography assessment, follow-up time, traditional CV risk factors and anti-cyclic citrullinated peptide antibodies disclosed a significant association (p=0.0047 between CD40 rs1535045 polymorphism and carotid intima media thickness, a surrogate marker of atherosclerosis.Data from our pilot study indicate a potential association of rs1883832 CD40 gene polymorphism with susceptibility

Recombinant heat shock protein 27 (HSP27/HSPB1) protects against cadmium-induced oxidative stress and toxicity in human cervical cancer cells.

Science.gov (United States)

Alvarez-Olmedo, Daiana G; Biaggio, Veronica S; Koumbadinga, Geremy A; Gómez, Nidia N; Shi, Chunhua; Ciocca, Daniel R; Batulan, Zarah; Fanelli, Mariel A; O'Brien, Edward R

2017-05-01

Cadmium (Cd) is a carcinogen with several well-described toxicological effects in humans, but its molecular mechanisms are still not fully understood. Overexpression of heat shock protein 27 (HSP27/HSPB1)-a multifunctional protein chaperone-has been shown to protect cells from oxidative damage and apoptosis triggered by Cd exposure. The aims of this work were to investigate the potential use of extracellular recombinant HSP27 to prevent/counteract Cd-induced cellular toxicity and to evaluate if peroxynitrite was involved in the development of Cd-induced toxicity. Here, we report that the harmful effects of Cd correlated with changes in oxidative stress markers: upregulation of reactive oxygen species, reduction in nitric oxide (NO) bioavailability, increment in lipid peroxidation, peroxynitrite (PN), and protein nitration; intracellular HSP27 was reduced. Treatments with Cd (100 μM) for 24 h or with the peroxynitrite donor, SIN-1, decreased HSP27 levels (~50%), suggesting that PN formation is responsible for the reduction of HSP27. Pre-treatments of the cells either with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a pharmacological inhibitor of NO synthase) or with recombinant HSP27 (rHSP27) attenuated the disruption of the cellular metabolism induced by Cd, increasing in a 55 and 52%, respectively, the cell viability measured by CCK-8. Cd induced necrotic cell death pathways, although apoptosis was also activated; pre-treatment with L-NAME or rHSP27 mitigated cell death. Our findings show for the first time a direct relationship between Cd-induced toxicity and PN production and a role for rHSP27 as a potential therapeutic agent that may counteract Cd toxicity.

Interleukin-9 receptor α chain mRNA formation in CD8+ T cells producing anti-human immunodeficiency virus type 1 substance(s)

International Nuclear Information System (INIS)

Hossain, M.M.; Tsuchie, H.; Detorio, M.A.; Shirono, H.; Hara, C.; Nishimoto, A.; Saji, A.; Koga, J.; Takata, N.; Maniar, J.K.; Saple, D.G.; Taniguchi, K.; Kageyama, S.; Ichimura, H.; Kurimura, T.

1998-01-01

A search for gene(s) associated with anti-human immunodeficiency virus type 1 (HIV-l) activity of CD8 + T cells was attempted using molecular cloning and the relation between the anti-HIV activity of CD8 + T cells and the interleukin-9 receptor a chain (IL-9R-α) mRNA expression from the cDNA clones obtained was examined. The anti-HIV-l activity of CD8 + T cell culture supernatants was assessed by measuring the level of HIV-l replication in a CD4 + T cell line transfected with an infectious HIV-l DNA clone. IL-9R-a mRNA was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR). Of 5 cases showing high level of anti-HIV-l activity (more than 80% suppression of HIV-l replication), the mRNA was detected in 4 cases. Of 10 cases showing low level of anti-HIV-l activity (less than 80% suppression of HIV-l replication), the mRNA was detected in one case. Soluble recombinant human IL-9 receptor (rhIL-9sR) did not suppress HIV-l replication at a concentration of 1 μg/ml. These data suggest that the IL-9R-a mRNA formation in CD8 + T cells may correlate with and play some role in the anti-HIV-l activity of CD8+ T cells from HIV-l-infected individuals. Key words: CD8+ T cells; anti-HIV-l activity; cytokines; interleukin-9 receptor (authors)

Expression by Streptomyces lividans of the Rat α Integrin CD11b A-Domain as a Secreted and Soluble Recombinant Protein

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Dorra Zouari Ayadi

2007-01-01

Full Text Available We already reported the use of a long synthetic signal peptide (LSSP to secrete the Streptomyces sp. TO1 amylase by Streptomyces lividans strain. We herein report the expression and secretion of the rat CD11b A-domain using the same LSSP and S. lividans as host strain. We have used the Escherichia coli/Streptomyces shuttle vector pIJ699 for the cloning of the A-domain DNA sequence downstream of LSSP and under the control of the constitutive ermE-up promoter of Streptomyces erythraeus. Using this construct and S. lividans as a host strain, we achieved the expression of 8 mg/L of soluble secreted recombinant form of the A-domain of the rat leukocyte β2 integrin CD11/CD18 alpha M subunit (CD11b. This secreted recombinant CD11b A-domain reacted with a function blocking antibody showing that this protein is properly folded and probably functional. These data support the capability of Streptomyces to produce heterologous recombinant proteins as soluble secreted form using the “LSSP” synthetic signal peptide.

Cre-/IoxP-Mediated Recombination between the SIL and SCL Genes Leads to a Block in T-Cell Development at the CD4-CD8- to CD4+CD8+ Transition

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Yue Cheng

2007-04-01

Full Text Available In the most common form of stem cell leukemia (SCL gene rearrangement, an interstitial deletion of 82 kb brings SCL under the control of regulatory elements that normally govern expression of the ubiquitously expressed SCL interrupting locus (SIL gene, which is located directly upstream of SCL. To investigate the effect of this fusion in a mouse model, a bacterial artificial chromosome (BAC clone containing both human SIL and SCL genes was isolated, and IoxP sites were inserted into intron 1 of both the SIL and SCL genes, corresponding to the sites at which recombination occurs in human T-cell acute lymphocytic leukemia patients. This BAC clone was used to generate transgenic SILIoxloxSCL mice. These transgenic mice were subsequently bred to Lck-Cre mice that express the Cre recombinase specifically in the thymus. The BAC transgene was recombined between the two IoxP sites in over 50% of the thymocytes from SILIoxloxSCL/Cre double-transgenic mice, bringing the SCL gene under the direct control of SIL regulatory elements. Aberrant SCL gene expression in the thymus was verified by reverse transcription- polymerase chain reaction. Using FACS analysis, we found that mice carrying both SILIoxloxSCL and Cre transgenes have increased CD4-/CD8- thymocytes compared with transgenenegative mice. In the spleen, these transgenic mice show a marked reduction in the number of mature CD4+ or CD8+ cells. These results demonstrate that conditional activation of SCL under control of SIL regulatory elements can impair normal T-cell development.

Analysis of the association between CD40 and CD40 ligand polymorphisms and systemic sclerosis

OpenAIRE

Teruel, María; Simeón Aznar, Carmen Pilar; Broen, Jasper C.; Vonk, Madelon C.; Carreira, Patricia; Camps García, María Teresa; García-Portales, Rosa; Delgado-Frías, Esmeralda; Gallego, Maria; Espinosa Garriga, Gerard; Spanish Scleroderma Group; Beretta, Lorenzo; Airó, Paolo; Lunardi, Claudio; Riemekasten, Gabriela

2012-01-01

Introduction: The aim of the present study was to investigate the possible role of CD40 and CD40 ligand (CD40LG) genes in the susceptibility and phenotype expression of systemic sclerosis (SSc). Methods: In total, 2,670 SSc patients and 3,245 healthy individuals from four European populations (Spain, Germany, The Netherlands, and Italy) were included in the study. Five single-nucleotide polymorphisms (SNPs) of CD40 (rs1883832, rs4810485, rs1535045) and CD40LG (rs3092952, rs3092920) were genot...

Lack of evidence of CD40 ligand involvement in transfusion-related acute lung injury

NARCIS (Netherlands)

Tuinman, P. R.; Gerards, M. C.; Jongsma, G.; Vlaar, A. P.; Boon, L.; Juffermans, N. P.

2011-01-01

Activated platelets have been implicated in playing a major role in transfusion-related acute lung injury (TRALI), as platelets can trigger neutrophils, resulting in vascular damage. We hypothesized that binding of platelet CD40 ligand (CD40L) to endothelial CD40 is essential in the onset of TRALI.

Dynamics of photoexcited carrier relaxation and recombination in CdTe/CdS thin films

Energy Technology Data Exchange (ETDEWEB)

Levi, D.H.; Fluegel, B.D.; Ahrenkiel, R.K. [National Renewable Energy Lab., Golden, CO (United States)] [and others

1996-05-01

Efficiency-limiting defects in photovoltaic devices are readily probed by time-resolved spectroscopy. This paper presents the first direct optical measurements of the relaxation and recombination pathways of photoexcited carriers in the CdS window layer of CdTe/CdS polycrystalline thin films. Femtosecond time-resolved pump/probe measurements indicate the possible existence of a two-phase CdS/CdSTe layer, rather than a continuously graded alloy layer at the CdTe/CdS interface. Complementary time-resolved photoluminescence (PL) measurements show that the photoexcited carriers are rapidly captured by deep-level defects. The temporal and density-dependent properties of the photoluminescence prove that the large Stokes shift of the PL relative to the band edge is due to strong phonon coupling to deep-level defects in CdS. The authors suggest that modifications in the CdS processing may enhance carrier collection efficiency in the blue spectral region.

Predictive value of elevated soluble CD40 ligand in patients undergoing primary angioplasty for ST-segment elevation myocardial infarction.

Science.gov (United States)

Pusuroglu, Hamdi; Akgul, Ozgur; Erturk, Mehmet; Uyarel, Huseyin; Bulut, Umit; Akkaya, Emre; Buturak, Ali; Surgit, Ozgur; Fuat, Ali; Cetin, Mustafa; Yldrm, Aydn

2014-11-01

The aim of this study was to evaluate the prognostic value of soluble CD40 ligand (sCD40L) in patients with ST-segment elevation myocardial infarction (STEMI) undergoing a primary percutaneous coronary intervention (PCI). The prognostic value of sCD40L has been documented in patients with acute coronary syndrome; however, its value in acute STEMI remains unclear. We prospectively enrolled 499 consecutive STEMI patients (397 men, 102 women) undergoing primary PCI. The study population was divided into tertiles on the basis of admission sCD40L values. The high sCD40L group (n=168) included patients with a value in the third tertile (≥0.947 mg/l) and the low sCD40L group (n=331) included patients with a value in the lower two tertiles (0.947 mg/l) is a powerful independent predictor of 1-year all-cause mortality (odds ratio: 3.68; 95% confidence interval: 1.54-8.77; P=0.003). The results of this study suggest that a high sCD40L level at admission is associated with increased in-hospital and 1-year all-cause mortality rates in patients with STEMI undergoing primary PCI.

Specific blockade by CD54 and MHC II of CD40-mediated signaling for B cell proliferation and survival

DEFF Research Database (Denmark)

Doyle, I S; Hollmann, C A; Crispe, I N

2001-01-01

Regulation of B lymphocyte proliferation is critical to maintenance of self-tolerance, and intercellular interactions are likely to signal such regulation. Here, we show that coligation of either the adhesion molecule ICAM-1/CD54 or MHC II with CD40 inhibited cell cycle progression and promoted...... these effects. Addition of BCR or IL-4 signals did not overcome the effect of ICAM-1 or MHC II on CD40-induced proliferation. FasL expression was not detected in B cell populations. These results show that MHC II and ICAM-1 specifically modulate CD40-mediated signaling, so inhibiting proliferation...

Two new stilbene trimers from Cynodon dactylon.

Science.gov (United States)

Li, Bi-Jun; Liu, Yao; Gu, Ai-Tong; Zhang, Qing; Chen, Lei; Wang, Shu-Mei; Wang, Feng

2017-11-01

Many naturally occurring oligostilbenes have drawn considerable attention because of their intricate structures and diverse bioactivities. Two new stilbene trimers, cystibenetrimerol A (1) and cystibenetrimerol B (2) were isolated from the dried grass of Cynodon dactylon (L.) Pers. The planar structures and stereo configurations of them were elucidated by spectroscopic and spectrometric methods. The isolation and structures elucidation of two new stilbene trimers suggested the ordinary grass belonging to the family Poaceae may be a rich source of stilbene oligomers.

Electrophysical properties of nCdS/pCdTe heterosystem

International Nuclear Information System (INIS)

Muzafarova, S.A.

2006-01-01

Full text: In this work results of research of electrophysical properties n CdS/pCdTe heterostructure are given. It is shown that at density of current 10 -8 -10 -5 A.cm -2 vol tamper characteristics in hetero system CdTe/CdS is described by thermo ionic law, and in the range 10 -4 -10 -2 A .cm -2 the current in heterostructure is limited by recombination in electronic neutral part of high-resistance of solid structure CdTe 1-x S x . Certain life time τ p and length of diffusion L p of non basic current carriers in solid structure CdTe 1-x S x , as well as superficial recombination rate v R on border of section between CdS and solid structures were considered at influence of irradiation and γ-quanta on the mechanism of current in n CdS/pCdTe heterostructure. From the analysis of dependence doze of sites the direct volt-ampere characteristics were obtained. It is shown that monotonous increase of doze of irradiation and γ-quanta leads to nonmonotonous change of micro parameters of nCdS/pCdTe heterostructure, superficial recombination rate - v R , values of both τ p and lengths of diffusion L p of non basic carriers of potential barrier - qφ B . On border of CdTe 1-x S x there is CdS-solid structure. (author)

Development of ADA Against Recombinant Human Interferon Beta in Immune Tolerant Mice Requires Rapid Recruitment of CD4(+) T Cells, Induces Formation of Germinal Centers but Lacks Susceptibility for (Most) Adjuvants

NARCIS (Netherlands)

Kijanka, Grzegorz; Sauerborn, Melody; Boon, Louis; Schellekens, Huub; Brinks, Vera

Immunological processes leading to formation of antidrug antibodies (Abs) against recombinant human proteins remain poorly understood. Animal and clinical studies revealed that immunogenicity shares both T-cell-dependent (requirement of CD4(+) T cells, isotype switching) and T-cell-independent

CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

Science.gov (United States)

Zhang, Xiaolong; Chang Li, Xian; Xiao, Xiang; Sun, Rui; Tian, Zhigang; Wei, Haiming

2013-01-01

The peripheral Foxp3+ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4+ T cells can be readily converted to Foxp3+ iTreg in vitro, and memory CD4+ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3+ T cells from various CD4+ T-cell subsets in human peripheral blood. Though naive CD4+ T cells were readily converted to Foxp3+ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3+ T cells did not express the memory marker CD45RO as do Foxp3+ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4+ T cells, defined as CD62L+ central memory T cells, could be induced by TGF-β to differentiate into Foxp3+ T cells. It is well known that Foxp3+ T cells derived from human CD4+CD25- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4+CD62L+ central memory T cell-derived Foxp3+ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4+ T cell-derived Foxp3+ T cells. But further research showed that mouse CD4+ central memory T cells also could be induced to differentiate into Foxp3+ T cells, such Foxp3+ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4+CD62L+ central memory T cells as a novel potential source of iTreg. PMID:24155942

Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome

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Caroline Y. Kuo

2018-05-01

Full Text Available X-linked hyper-immunoglobulin M (hyper-IgM syndrome (XHIM is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9 platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs, as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.

Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis cathepsin l-like protein and a synthetic peptide containing its linear B-cell epitope.

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Daniel Menezes-Souza

2015-01-01

Full Text Available The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.

Transgenic expression of soluble human CD5 enhances experimentally-induced autoimmune and anti-tumoral immune responses.

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Rafael Fenutría

Full Text Available CD5 is a lymphoid-specific transmembrane glycoprotein constitutively expressed on thymocytes and mature T and B1a lymphocytes. Current data support the view that CD5 is a negative regulator of antigen-specific receptor-mediated signaling in these cells, and that this would likely be achieved through interaction with CD5 ligand/s (CD5L of still undefined nature expressed on immune or accessory cells. To determine the functional consequence of loss of CD5/CD5L interaction in vivo, a new transgenic mouse line was generated (shCD5EμTg, expressing a circulating soluble form of human CD5 (shCD5 as a decoy to impair membrane-bound CD5 function. These shCD5EμTg mice showed an enhanced response to autologous antigens, as deduced from the presentation of more severe forms of experimentally inducible autoimmune disease (collagen-induced arthritis, CIA; and experimental autoimmune encephalitis, EAE, as well as an increased anti-tumoral response in non-orthotopic cancer models (B16 melanoma. This enhancement of the immune response was in agreement with the finding of significantly reduced proportions of spleen and lymph node Treg cells (CD4+CD25+FoxP3+, and of peritoneal IL-10-producing and CD5+ B cells, as well as an increased proportion of spleen NKT cells in shCD5EμTg mice. Similar changes in lymphocyte subpopulations were observed in wild-type mice following repeated administration of exogenous recombinant shCD5 protein. These data reveal the relevant role played by CD5/CD5L interactions on the homeostasis of some functionally relevant lymphocyte subpopulations and the modulation of immune responses to autologous antigens.

Experimental adaptation of wild-type canine distemper virus (CDV to the human entry receptor CD150.

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Maria Bieringer

Full Text Available Canine distemper virus (CDV, a close relative of measles virus (MV, is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17(red adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (10(2 pfu/ml in Vero cells expressing human CD150 (Vero-hSLAM. After three passages using these cells virus was adapted to human CD150 and replicated to high titres (10(5 pfu/ml. Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L and Gly to Glu (G71E, and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs.

Soluble CD40 ligand contributes to blood-brain barrier breakdown and central nervous system inflammation in multiple sclerosis and neuromyelitis optica spectrum disorder.

Science.gov (United States)

Masuda, Hiroki; Mori, Masahiro; Uchida, Tomohiko; Uzawa, Akiyuki; Ohtani, Ryohei; Kuwabara, Satoshi

2017-04-15

Soluble CD40 ligand (sCD40L) is reported to disrupt the blood-brain barrier (BBB). Cerebrospinal fluid (CSF) and serum sCD40L levels were measured in 29 multiple sclerosis (MS), 29 neuromyelitis optica spectrum disorder (NMOSD), and 27 disease control (DC) patients. In MS, serum sCD40L levels were higher than in DCs and positively correlated with the CSF/serum albumin ratio (Qalb). In NMOSD, CSF sCD40L levels were significantly increased compared to DCs, and were correlated to Qalb, CSF cell counts, protein concentrations, and interleukin-6 levels. sCD40L could be involved in BBB disruption in MS, whereas it may contribute to CNS inflammation in NMOSD. Copyright © 2017 Elsevier B.V. All rights reserved.

[Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

Science.gov (United States)

Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

2015-10-01

To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

Cytoplasmic Overexpression of CD95L in Esophageal Adenocarcinoma Cells Overcomes Resistance to CD95-Mediated Apoptosis

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Gregory A. Watson

2011-03-01

Full Text Available Introduction: The CD95/CD95L pathway plays a critical role in tissue homeostasis and immune system regulation; however, the function of this pathway in malignancy remains poorly understood. We hypothesized that CD95L expression in esophageal adenocarcinoma confers advantages to the neoplasm other than immune privilege. Methods: CD95L expression was characterized in immortalized squamous esophagus (HET-1A and Barrett esophagus (BAR-T cells; adenocarcinoma cell lines FLO-1, SEG-1, and BIC-1, and MDA468 (- control; and KFL cells (+ control. Analyses included reverse transcription-polymerase chain reaction, immunoblots of whole cell and secretory vesicle lysates, FACScan analysis, laser scanning confocal microscopy of native proteins and fluorescent constructs, and assessment of apoptosis and ERK1/2 pathways. Results: Cleaved, soluble CD95L is expressed at both the RNA and protein levels in these cell lines derived from esophageal adenocarcinoma and other human tissues. CD95L was neither trafficked to the cell membrane nor secreted into the media or within vesicles, rather the protein seems to be sequestered in the cytoplasm. CD95 and CD95L colocalize by immunofluorescence, but an interaction was not proven by immunoprecipitation. Overexpression of CD95L in the adenocarcinoma cell lines induced robust apoptosis and, under conditions of pan-caspase inhibition, resulted in activation of ERK signaling. Conclusions: CD95L localization in EA cells is inconsistent with the conference of immune privilege and is more consistent with a function that promotes tumor growth through alternative CD95 signaling. Reduced cell surface expression of CD95 affects cell sensitivity to extracellular apoptotic signals more significantly than alterations in downstream modulators of apoptosis.

Unfolding study of a trimeric membrane protein AcrB.

Science.gov (United States)

Ye, Cui; Wang, Zhaoshuai; Lu, Wei; Wei, Yinan

2014-07-01

The folding of a multi-domain trimeric α-helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two-state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter-subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate-limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrBΔloop , from the SDS unfolded state. The CD spectrum of the refolded AcrBΔloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild-type AcrB. © 2014 The Protein Society.

Correlation between intravoxel incoherent motion magnetic resonance imaging derived metrics and serum soluble CD40 ligand level in an embolic canine stroke model

Energy Technology Data Exchange (ETDEWEB)

Xu, Xiao Quan; Wu, Chen Jiang; Lu, Shan Shan; Gao, Qian Qian; Zu, Qing Quan; Liu, Xing Long; Shi, Hai Bin; Liu, Sheng [Dept. of Radiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing (China)

2017-09-15

To determine the relationship between intravoxel incoherent motion (IVIM) imaging derived quantitative metrics and serum soluble CD40 ligand (sCD40L) level in an embolic canine stroke model. A middle cerebral artery occlusion model was established in 24 beagle dogs. Experimental dogs were divided into low- and high-sCD40L group according to serum sCD40L level at 4.5 hours after establishing the model. IVIM imaging was scanned at 4.5 hours after model establishment using 10 b values ranging from 0 to 900 s/mm{sup 2}. Quantitative metrics diffusion coefficient (D), pseudodiffusion coefficient (D{sup *}), and perfusion fraction (f) of ischemic lesions were calculated. Quantitative metrics of ischemic lesions were normalized by contralateral hemisphere using the following formula: normalized D = D{sub stroke} / D{sub contralateral}. Differences in IVIM metrics between the low- and high-sCD40L groups were compared using t test. Pearson's correlation analyses were performed to determine the relationship between IVIM metrics and serum sCD40L level. The high-sCD40L group showed significantly lower f and normalized f values than the low-sCD40L group (f, p < 0.001; normalized f, p < 0.001). There was no significant difference in D{sup *}, normalized D{sup *}, D, or normalized D value between the two groups (All p > 0.05). Both f and normalized f values were negatively correlated with serum sCD40L level (f, r = −0.789, p < 0.001; normalized f, r = −0.823, p < 0.001). However, serum sCD40L level had no significant correlation with D{sup *}, normalized D{sup *}, D, or normalized D (All p > 0.05). The f value derived from IVIM imaging was negatively correlated with serum sCD40L level. f value might serve as a potential imaging biomarker to assess the formation of microvascular thrombosis in hyperacute period of ischemic stroke.

Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

Energy Technology Data Exchange (ETDEWEB)

Ohkawa, Mayumi [Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai 980-8578 (Japan); Ohno, Yoshiya [Laboratory of Immunobiology, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe-shi, Hyogo 650-8530 (Japan); Masuko, Kazue; Takeuchi, Akiko; Suda, Kentaro; Kubo, Akihiro; Kawahara, Rieko; Okazaki, Shogo [Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502 (Japan); Tanaka, Toshiyuki [Laboratory of Immunobiology, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe-shi, Hyogo 650-8530 (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8502 (Japan); Seki, Masayuki; Enomoto, Takemi [Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai 980-8578 (Japan); Yagi, Hideki [Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502 (Japan); Hashimoto, Yoshiyuki [Tohoku University, Sendai (Japan); Masuko, Takashi, E-mail: masuko@phar.kindai.ac.jp [Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502 (Japan)

2011-03-25

Highlights: {yields} We established LAT1 amino-acid transporter-disrupted DT40 cells. {yields} LAT1-disrupted cells showed slow growth and lost the oncogenicity. {yields} siRNA and mAb inhibited human tumor growth in vitro and in vivo. {yields} LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1{sup -/-}) cell clones, derived from a heterozygous LAT1{sup +/-} clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1{sup -/-} DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1{sup -/-} cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1{sup -/-} DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1{sup -/-} DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1{sup +/-} DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

International Nuclear Information System (INIS)

Ohkawa, Mayumi; Ohno, Yoshiya; Masuko, Kazue; Takeuchi, Akiko; Suda, Kentaro; Kubo, Akihiro; Kawahara, Rieko; Okazaki, Shogo; Tanaka, Toshiyuki; Saya, Hideyuki; Seki, Masayuki; Enomoto, Takemi; Yagi, Hideki; Hashimoto, Yoshiyuki; Masuko, Takashi

2011-01-01

Highlights: → We established LAT1 amino-acid transporter-disrupted DT40 cells. → LAT1-disrupted cells showed slow growth and lost the oncogenicity. → siRNA and mAb inhibited human tumor growth in vitro and in vivo. → LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1 -/- ) cell clones, derived from a heterozygous LAT1 +/- clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1 -/- DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1 -/- cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1 -/- DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1 -/- DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1 +/- DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

Whole-gene analysis of two groups of hepatitis B virus C/D inter-genotype recombinant strains isolated in Tibet, China.

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Tiezhu Liu

Full Text Available Tibet is a highly hepatitis B virus (HBV endemic area. Two types of C/D recombinant HBV are commonly isolated in Tibet and have been previously described. In an effort to better understand the molecular characteristic of these C/D recombinant strains from Tibet, we undertook a multistage random sampling project to collect HBsAg positive samples. Molecular epidemiological and bio-informational technologies were used to analyze the characteristics of the sequences found in this study. There were 60 samples enrolled in the survey, and we obtained 19 whole-genome sequences. 19 samples were all C/D recombinant, and could be divided into two sub-types named C/D1 and C/D2 according to the differences in the location of the recombinant breakpoint. The recombination breakpoint of the 10 strains belonging to the C/D1 sub-type was located at nt750, while the 9 stains belonging to C/D2 had their recombination break point at nt1530. According to whole-genome sequence analysis, the 19 identified strains belong to genotype C, but the nucleotide distance was more than 5% between the 19 strains and sub-genotypes C1 to C15. The distance between C/D1with C2 was 5.8±2.1%, while the distance between C/D2 with C2 was 6.4±2.1%. The parental strain was most likely sub-genotype C2. C/D1 strains were all collected in the middle and northern areas of Tibet including Lhasa, Linzhi and Ali, while C/D2 was predominant in Shannan in southern Tibet. This indicates that the two recombinant genotypes are regionally distributed in Tibet. These results provide important information for the study of special HBV recombination events, gene features, virus evolution, and the control and prevention policy of HBV in Tibet.

Targeting CD28, CTLA-4 and PD-L1 costimulation differentially controls immune synapses and function of human regulatory and conventional T-cells.

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Nahzli Dilek

Full Text Available CD28, CTLA-4 and PD-L1, the three identified ligands for CD80/86, are pivotal positive and negative costimulatory molecules that, among other functions, control T cell motility and formation of immune synapse between T cells and antigen-presenting cells (APCs. What remains incompletely understood is how CD28 leads to the activation of effector T cells (Teff but inhibition of suppression by regulatory T cells (Tregs, while CTLA-4 and PD-L1 inhibit Teff function but are crucial for the suppressive function of Tregs. Using alloreactive human T cells and blocking antibodies, we show here by live cell dynamic microscopy that CD28, CTLA-4, and PD-L1 differentially control velocity, motility and immune synapse formation in activated Teff versus Tregs. Selectively antagonizing CD28 costimulation increased Treg dwell time with APCs and induced calcium mobilization which translated in increased Treg suppressive activity, in contrast with the dampening effect on Teff responses. The increase in Treg suppressive activity after CD28 blockade was also confirmed with polyclonal Tregs. Whereas CTLA-4 played a critical role in Teff by reversing TCR-induced STOP signals, it failed to affect motility in Tregs but was essential for formation of the Treg immune synapse. Furthermore, we identified a novel role for PD-L1-CD80 interactions in suppressing motility specifically in Tregs. Thus, our findings reveal that the three identified ligands of CD80/86, CD28, CTLA-4 and PD-L1, differentially control immune synapse formation and function of the human Teff and Treg cells analyzed here. Individually targeting CD28, CTLA-4 and PD-L1 might therefore represent a valuable therapeutic strategy to treat immune disorders where effector and regulatory T cell functions need to be differentially targeted.

Properties of mouse CD40: differential expression of CD40 epitopes on dendritic cells and epithelial cells

NARCIS (Netherlands)

van den Berg, T. K.; Hasbold, J.; Renardel de Lavalette, C.; Döpp, E. A.; Dijkstra, C. D.; Klaus, G. G.

1996-01-01

In this study we describe the tissue distribution of mouse CD40 using two monoclonal antibodies (mAb) against different epitopes of the molecule. In lymphoid tissues CD40 was expressed by B lymphocytes. Most B cells in typical B-cell compartments were CD40-positive, including germinal centre B

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain.

Science.gov (United States)

Richards, Mark W; O'Regan, Laura; Roth, Daniel; Montgomery, Jessica M; Straube, Anne; Fry, Andrew M; Bayliss, Richard

2015-05-01

Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

COMPARATIVE ANALYSIS OF SOLUBLE OF CD40 LIGAND LEVELS IN HEART RECIPIENTS TREATED WITH CYCLOSPORINE A AND TACROLIMUS

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O. P. Shevchenko

2012-01-01

Full Text Available Soluble form of CD40L is platelet activating factor, which is a marker of inflammation and thrombosis. Elevated levels of sCD40L before the heart transplantation are associated with the risk of early development of cardiova- scular complications. The study included 54 patients who had received heart transplants. All recipients received a triple heart immu- nosuppressive therapy, including methylprednisolone, mycophenolate mofetil and cyclosporine A (20 recipients or methylprednisolone, mycophenolate mofetil and tacrolimus (34 recipients. Patients were not differed by age, gender, etiology of heart failure before heart transplantation (p > 0,05. In the first group of transplant recipients, the relative risk of cardiovascular events with high sCD40L levels before transplantation was 3 2 (95% CI 1,4; 12,0. In the second group of recipients, respectively, 2.69 (95% CI 1,1; 8,5. SCD40L level after heart transplan- tation was significantly higher for patients receiving cyclosporine (P < 0.05. Increasing concentrations of sCD40L are associated with a higher incidence of cardiovascular complications. 

Anthocyanin prevents CD40-activated proinflammatory signaling in endothelial cells by regulating cholesterol distribution.

Science.gov (United States)

Xia, Min; Ling, Wenhua; Zhu, Huilian; Wang, Qing; Ma, Jing; Hou, Mengjun; Tang, Zhihong; Li, Lan; Ye, Qinyuan

2007-03-01

Intracellular tumor necrosis factor receptor-associated factors (TRAFs) translocation to lipid rafts is a key element in CD40-induced signaling. The purpose of this study was to investigate the influence of anthocyanin on CD40-mediated proinflammatory events in human endothelial cells and the underlying possible molecular mechanism. Treatment of endothelial cells with anthocyanin prevented from CD40-induced proinflammatory status, measured by production of IL-6, IL-8, and monocyte chemoattractant protein-1 through inhibiting CD40-induced nuclear factor-kappaB (NF-kappaB) activation. TRAF-2 played pivotal role in CD40-NF-kappaB pathway as TRAF-2 small interference RNA (siRNA) diminished CD40-induced NF-kappaB activation and inflammation. TRAF-2 overexpression increased CD40-mediated NF-kappaB activation. Moreover, TRAF-2 almost totally recruited to lipid rafts after stimulation by CD40 ligand and depletion of cholesterol diminished CD40-mediated NF-kappaB activation. Exposure to anthocyanin not only interrupted TRAF-2 recruitment to lipid rafts but also decreased cholesterol content in Triton X-100 insoluble lipid rafts. However, anthocyanin did not influence the interaction between CD40 ligand and CD40 receptor. Our findings suggest that anthocyanin protects from CD40-induced proinflammatory signaling by preventing TRAF-2 translocation to lipid rafts through regulation of cholesterol distribution, which thereby may represent a mechanism that would explain the anti-inflammatory response of anthocyanin.

CD5L Promotes M2 Macrophage Polarization through Autophagy-Mediated Upregulation of ID3

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Lucía Sanjurjo

2018-03-01

Full Text Available CD5L (CD5 molecule-like is a secreted glycoprotein that controls key mechanisms in inflammatory responses, with involvement in processes such as infection, atherosclerosis, and cancer. In macrophages, CD5L promotes an anti-inflammatory cytokine profile in response to TLR activation. In the present study, we questioned whether CD5L is able to influence human macrophage plasticity, and drive its polarization toward any specific phenotype. We compared CD5L-induced phenotypic and functional changes to those caused by IFN/LPS, IL4, and IL10 in human monocytes. Phenotypic markers were quantified by RT-qPCR and flow cytometry, and a mathematical algorithm was built for their analysis. Moreover, we compared ROS production, phagocytic capacity, and inflammatory responses to LPS. CD5L drove cells toward a polarization similar to that induced by IL10. Furthermore, IL10- and CD5L-treated macrophages showed increased LC3-II content and colocalization with acidic compartments, thereby pointing to the enhancement of autophagy-dependent processes. Accordingly, siRNA targeting ATG7 in THP1 cells blocked CD5L-induced CD163 and Mer tyrosine kinase mRNA and efferocytosis. In these cells, gene expression profiling and validation indicated the upregulation of the transcription factor ID3 by CD5L through ATG7. In agreement, ID3 silencing reversed polarization by CD5L. Our data point to a significant contribution of CD5L-mediated autophagy to the induction of ID3 and provide the first evidence that CD5L drives macrophage polarization.

High-level secretion of native recombinant human calreticulin in yeast

DEFF Research Database (Denmark)

Čiplys, Evaldas; Žitkus, Eimantas; Gold, Leslie I.

2015-01-01

, Saccharomyces cerevisiae and Pichia pastoris. RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional...... by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration...... recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium. CONCLUSIONS: Yeasts...

Meiotic recombination in human oocytes.

Directory of Open Access Journals (Sweden)

Edith Y Cheng

2009-09-01

Full Text Available Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1 in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of "vulnerable" crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.

Anti-CD40-mediated cancer immunotherapy

DEFF Research Database (Denmark)

Hassan, Sufia Butt; Sørensen, Jesper Freddie; Olsen, Barbara Nicola

2014-01-01

activation and thus enhancement of immune responses. Treatment with anti-CD40 monoclonal antibodies has been exploited in several cancer immunotherapy studies in mice and led to the development of anti-CD40 antibodies for clinical use. Here, Dacetuzumab and Lucatumumab are in the most advanced stage...... with other cancer immunotherapies, in particular interleukin (IL)-2. An in-depth analysis of this immunotherapy is provided elsewhere. In the present review, we provide an update of the most recent clinical trials with anti-CD40 antibodies. We present and discuss recent and ongoing clinical trials...... in this field, including clinical studies which combine anti-CD40 treatment with other cancer-treatments, such as Rituximab and Tremelimumab....

Anti-tumour therapeutic efficacy of OX40L in murine tumour model.

Science.gov (United States)

Ali, Selman A; Ahmad, Murrium; Lynam, June; McLean, Cornelia S; Entwisle, Claire; Loudon, Peter; Choolun, Esther; McArdle, Stephanie E B; Li, Geng; Mian, Shahid; Rees, Robert C

2004-09-09

OX40 ligand (OX40L), a member of TNF superfamily, is a co-stimulatory molecule involved in T cell activation. Systemic administration of mOX40L fusion protein significantly inhibited the growth of experimental lung metastasis and subcutaneous (s.c.) established colon (CT26) and breast (4T1) carcinomas. Vaccination with OX40L was significantly enhanced by combination treatment with intra-tumour injection of a disabled infectious single cycle-herpes simplex virus (DISC-HSV) vector encoding murine granulocyte macrophage-colony stimulating factor (mGM-CSF). Tumour rejection in response to OX40L therapy required functional CD4+ and CD8+ T cells and correlated with splenocyte cytotoxic T lymphocytes (CTLs) activity against the AH-1 gp70 peptide of the tumour associated antigen expressed by CT26 cells. These results demonstrate the potential role of the OX40L in cancer immunotherapy.

Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

International Nuclear Information System (INIS)

Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

1990-01-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

Science.gov (United States)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Distinct and overlapping effector functions of expanded human CD4+, CD8α+ and CD4-CD8α- invariant natural killer T cells.

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Vincent O'Reilly

Full Text Available CD1d-restricted invariant natural killer T (iNKT cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+, CD8α(+ and CD4(-CD8α(- double-negative (DN subsets. CD4(+ iNKT cells expanded more readily than CD8α(+ and DN iNKT cells upon mitogen stimulation. CD8α(+ and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+ cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α and Th2 (IL-4, IL-5 and IL-13 cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+ and CD8α(+ fractions, respectively. Only CD4(+ iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+, DN or CD4(+ iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated disruption of the CD40 ligand-induced activation of primary human B cells

International Nuclear Information System (INIS)

Lu Haitian; Crawford, Robert B.; Kaplan, Barbara L.F.; Kaminski, Norbert E.

2011-01-01

Suppression of the primary antibody response is particularly sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice; however, surprisingly little is known concerning the effects of TCDD on humoral immunity or B cell function in humans. Results from a limited number of previous studies, primarily employing in vitro activation models, suggested that human B cell effector function is suppressed by TCDD. The present study sought to extend these findings by investigating, in primary human B cells, the effects of TCDD on several critical stages leading to antibody secretion including activation and plasmacytic differentiation using an in vitro CD40 ligand activation model. These studies revealed important differences in the response of human and mouse B cells to TCDD, the most striking being altered expression of plasmacytic differentiation regulators, B lymphocyte-induced maturation protein 1 and paired box protein 5, in mouse but not human B cells. The activation of human B cells was profoundly impaired by TCDD, as evidenced by decreased expression of activation markers CD80, CD86, and CD69. The impaired activation correlated with decreased cell viability, which prevented the progression of human B cells toward plasmacytic differentiation. TCDD treatment also attenuated the early activation of mitogen-activated protein kinases (MAPK) and Akt signaling in human B cells. Collectively, the present study provided experimental evidence for novel mechanisms by which TCDD impairs the effector function of primary human B cells. - Highlights: → In this study primary human and mouse B cell toxicity to TCDD was compared. → TCDD altered the expression of Blimp-1 and Pax5 in mouse but not human B cells. → TCDD markedly suppressed human B cell activation as characterized by CD80, CD86 and CD69 expression. → TCDD inhibited ERK, p38, and Akt phosphorylation in human B cells.

Gold nanoparticles conjugating recombinant influenza hemagglutinin trimers and flagellin enhanced mucosal cellular immunity.

Science.gov (United States)

Wang, Chao; Zhu, Wandi; Luo, Yuan; Wang, Bao-Zhong

2018-04-09

The immunogenicity of subunit vaccines can be augmented by formulating them into nanoparticles. We conjugated recombinant trimetric influenza A/Aichi/2/68(H3N2) hemagglutinin (HA) onto functionalized gold nanoparticles (AuNPs) surfaces in a repetitive, oriented configuration. To further improve the immunogenicity, we generated Toll-like receptor 5 (TLR5) agonist flagellin (FliC)-coupled AuNPs as particulate adjuvants. Intranasal immunizations with an AuNP-HA and AuNP-FliC particle mixture elicited strong mucosal and systemic immune responses that protected hosts against lethal influenza challenges. Compared with the AuNP-HA alone group, the addition of AuNP-FliC improved mucosal B cell responses as characterized by elevated influenza specific IgA and IgG levels in nasal, tracheal, and lung washes. AuNP-HA/AuNP-FliC also stimulated antigen-specific interferon-γ (IFN-γ)-secreting CD4 + cell proliferation and induced strong effector CD8 + T cell activation. Our results indicate that intranasal co-delivery of antigen and adjuvant-displaying AuNPs enhanced vaccine efficacy by inducing potent cellular immune responses. Copyright © 2018. Published by Elsevier Inc.

Dependence of Immunoglobulin Class Switch Recombination in B Cells on Vesicular Release of ATP and CD73 Ectonucleotidase Activity

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Francesca Schena

2013-06-01

Full Text Available Immunoglobulin (Ig isotype diversification by class switch recombination (CSR is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID. Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca2+-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5′-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.

Systemic agonistic anti-CD40 treatment of tumor bearing mice modulates hepatic myeloid suppressive cells and causes immune-mediated liver damage

Science.gov (United States)

Medina-Echeverz, José; Ma, Chi; Duffy, Austin; Eggert, Tobias; Hawk, Nga; Kleiner, David E.; Korangy, Firouzeh; Greten, Tim F.

2015-01-01

Immune stimulatory monoclonal antibodies are currently evaluated as anti tumor agents. Although overall toxicity appears to be moderate, liver toxicities have been reported and are not completely understood. We studied the effect of systemic CD40 antibody treatment on myeloid cells in spleen and liver. Naïve and tumor-bearing mice were treated systemically with agonistic anti-CD40 antibody. Immune cell subsets in liver and spleen, serum transaminases and liver histologies were analyzed after antibody administration. Nox2−/−, Cd40−/− as well as bone marrow chimeric mice were used to study the mechanism by which agonistic anti-CD40 mediates its effects in vivo. Suppressor function of murine and human tumor-induced myeloid derived suppressive cells was studied upon CD40 ligation. Agonistic CD40 antibody caused liver damage within 24 hours after injection in two unrelated tumor models and mice strains. Using bone marrow chimeras we demonstrated that CD40 antibody-induced hepatitis in tumor-bearing mice was dependent on the presence of CD40-expressing hematopoietic cells. Agonistic CD40 ligation-dependent liver damage was induced by the generation of reactive oxygen species. Furthermore, agonistic CD40 antibody resulted in increased CD80 and CD40 positive liver CD11b+Gr-1+ immature myeloid cells. CD40 ligation on tumor-induced murine and human CD14+HLA-DRlow PBMC from cancer patients reduced their immune suppressor function. Collectively, agonistic CD40 antibody treatment activated tumor-induced, myeloid cells, caused myeloid dependent hepatotoxicity and ameliorated the suppressor function of murine and human MDSC. Collectively, our data suggests that CD40 may mature immunosuppressive myeloid cells and thereby cause liver damage in mice with an accumulation of tumor-induced hepatic MDSC. PMID:25637366

Plasticity and Epitope Exposure of the HIV-1 Envelope Trimer.

Science.gov (United States)

Powell, Rebecca L R; Totrov, Maxim; Itri, Vincenza; Liu, Xiaomei; Fox, Alisa; Zolla-Pazner, Susan

2017-09-01

We recently showed that mutations in the HIV-1 envelope (Env) destabilize the V3 loop, rendering neutralization-resistant viruses sensitive to V3-directed monoclonal antibodies (MAbs). Here, we investigated the propagation of this effect on other Env epitopes, with special emphasis on V2 loop exposure. Wild-type JR-FL and 19 mutant JR-FL pseudoviruses were tested for neutralization sensitivity to 21 MAbs specific for epitopes in V2, the CD4 binding site (CD4bs), and the CD4-induced (CD4i) region. Certain glycan mutants, mutations in the gp120 hydrophobic core, and mutations in residues involved in intraprotomer interactions exposed epitopes in the V2i region (which overlies the α4β7 integrin binding site) and the V3 crown, suggesting general destabilization of the distal region of the trimer apex. In contrast, other glycan mutants, mutations affecting interprotomer interactions, and mutations affecting the CD4bs exposed V3 but not V2i epitopes. These data indicate for the first time that V3 can move independently of V2, with V3 pivoting out from its "tucked" position in the trimer while apparently leaving the V2 apex intact. Notably, none of the mutations exposed V2 epitopes without also exposing V3, suggesting that movement of V2 releases V3. Most mutations increased sensitivity to CD4bs-directed MAbs without exposure of the CD4i epitope, implying these mutations facilitate the trimers' maintenance of an intermediate energy state between open and closed conformations. Taken together, these data indicate that several transient Env epitopes can be rendered more accessible to antibodies (Abs) via specific mutations, and this may facilitate the design of V1V2-targeting immunogens. IMPORTANCE Many epitopes of the HIV envelope (Env) spike are relatively inaccessible to antibodies (Abs) compared to their exposure in the open Env conformation induced by receptor binding. However, the reduced infection rate that resulted from the vaccine used in the RV144 HIV-1 vaccine

Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein

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Anna-Janina Behrens

2016-03-01

Full Text Available The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664 maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.

The clinical investigation of OX40/OX40L from lymphocytes of patients with Graves' disease after 131I therapy

International Nuclear Information System (INIS)

Shi Bimin; Du Xuan; Wang Qin

2013-01-01

Objective To investigate the clinical value and the expression of OX40/OX40L from lymphocytes of patients with Graves' disease (GD) before and after 131 I therapy.Methods The expression of OX40/OX40L on T,B lymphocytes and the percentage of lymphocyte subsets were analyzed using immunofluorescence staining and flow cytometry in 32 patients with GD before and after 131 I therapy.Twenty-five healthy subjects were considered as the control group.The data between GD patients and controls were compared with two-sample t test.The correlation between the expression of OX40/OX40L and the function of thyroid (FT3,FT4 and TRAb level) was performed with linear correlation analysis.Results Compared with the healthy subjects,the percentage of CD4+ T cells and ratio of CD4+/CD8+ were decreased ((34.36 ±6.73)% vs (45.60-±5.72)%,t=2.634; 1.24±0.26 vs 1.84±0.37,t=2.125,both P<0.05).Whereas the percentage of CD19+ B cells in patients with GD were significantly higher than that in healthy subjects ((21.42 ±3.92)% vs (15.35 ± 3.15)%,t =2.653,P<0.05).The expression of OX40 increased in CD4+ T lymphocytes ((12.92 ± 2.26) %) and OX40L expression was up-regulated in CD19+ B lymphocytes ((15.33 ± 3.75)%) of patients with GD,compared with the healthy subjects ((4.19 ±1.45) % and (8.64 ± 1.73) %,respectively,t =2.112 and 2.467,both P<0.05).The expression of OX40 in CD4+ T lymphocytes ((7.65 ± 1.64) %) and OX40L in CD19+ B lymphocytes ((11.50 ±2.72)%) were increased after 131 I therapy(t =2.795,2.393,both P<0.05).The thyroid functions of 32 GD patients were improved.The levels of FT3,FT4 and TRAb decreased significantly after 131 I treatment (from 20.79 ±6.05 to 4.53 ± 2.54,from 49.65 ± 10.12 to 13.69 ± 4.35,and from 24.78 ± 8.46 to 11.74 ±6.19,respectively; t =2.219,2.441 and 2.293,all P<0.05).The levels of FT3 and FT4 were positively correlated with the percentage of OX40 expression in CD4+ T lymphocytes (r =0.65 and 0.73,both P<0.05).TRAb was positively correlated

Estimation of CD4+ and CD8+ T-lymphocytes in human immunodeficiency virus infection and acquired immunodeficiency syndrome patients in Manipur

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Singh H

2007-01-01

Full Text Available Purpose : To estimate and stratify CD4 + and CD8 + T-lymphocyte levels in human immunodeficiency virus (HIV infected (asymptomatic and acquired immunodeficiency syndrome (AIDS patients (symptomatic and correlate the clinical features of the patients with CD4+ and CD8+ lymphocyte level. Methods : Between April 2002 and September 2003, a total of 415 HIV seropositive adult patients (297 males and 118 females attending Regional Institute of Medical Sciences (RIMS hospitals were tested for CD4+ and CD8+ T-lymphocytes by fluorescent activated cell sorter (FACS counter (Becton Dickinson. Symptomatic patients were diagnosed as per NACO clinical case definition. Results : Ranges of 0-50, 51-100, 101-200, 201-300, 301-400, 401-500 and above 500 CD4+ T-lymphocyte per microlitre were seen in 68, 52, 101, 73, 47, 31 and 43 patients respectively whereas CD8+ T-lymphocyte ranges of 0-300, 301-600, 601-900, 901-1500, 1501-2000, 2001-3500 per microlitre were seen in 29, 84, 92, 145, 40 and 25 patients respectively. One hundred and fifty patients were asymptomatic and 265 were symptomatic. CD4/CD8 ratio in asymptomatics and symptomatics were 0.13-1.69 and 0.01-0.93 respectively. Tuberculosis and candidiasis occurred in CD4+ T-lymphocyte categories between 0-400 cells per mL in symptomatics. However, cryptosporidiosis, toxoplasmosis, herpes zoster, cryptococcal meningitis, Pneumocystis carinii pneumonia, penicilliosis and cytomegalovirus retinitis were seen in patients having CD4+ T-lymphocyte less than 200 per mL. Conclusions : CD4+ T-lymphocyte was decreased in both asymptomatic and symptomatic HIV patients, The decrease was greater in symptomatics while CD8+ T-lymphocyte was increased in both except advanced stage symptomatics. CD4:CD8 ratio was reversed in both groups. Opportunistic infections correlated with different CD4+ T-lymphocyte categories.

Increasing the ex vivo antigen-specific IFN-γ production in subpopulations of T cells and NKp46+ cells by anti-CD28, anti-CD49d and recombinant IL-12 costimulation in cattle vaccinated with recombinant proteins from Mycobacterium avium subspecies paratuberculosis

DEFF Research Database (Denmark)

Thakur, Aneesh; Riber, Ulla; Davis, William C.

2013-01-01

-γ secretion by CD4, CD8, γδ T cells and NK cells. Age matched male jersey calves, experimentally infected with Mycobacterium avium subsp. paratuberculosis (MAP), were vaccinated with a cocktail of recombinant MAP proteins or left unvaccinated. Vaccine induced ex vivo recall responses were measured through Ag......T cells, which encounter specific antigen (Ag), require additional signals to mount a functional immune response. Here, we demonstrate activation of signal 2, by anti-CD28 mAb (aCD28) and other costimulatory molecules (aCD49d, aCD5), and signal 3, by recombinant IL-12, enhance Ag-specific IFN...

B-cell activation with CD40L or CpG measures the function of B-cell subsets and identifies specific defects in immunodeficient patients.

Science.gov (United States)

Marasco, Emiliano; Farroni, Chiara; Cascioli, Simona; Marcellini, Valentina; Scarsella, Marco; Giorda, Ezio; Piano Mortari, Eva; Leonardi, Lucia; Scarselli, Alessia; Valentini, Diletta; Cancrini, Caterina; Duse, Marzia; Grimsholm, Ola; Carsetti, Rita

2017-01-01

Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B-cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B-cell functions in infancy and throughout childhood. We show that T-independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T-dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system. © 2016 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CD40 expression in Wehi-164 cell line.

Science.gov (United States)

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Moazzeni, Seyed Mohammad

2010-07-01

CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body's defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein expression of CD40 but no surface expression. These results suggest that the Wehi-164 cell line down regulates expression of CD40 on the surface for evasion of immune system.

Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function.

Science.gov (United States)

Patel, Kashyap R; Roberts, Jacob T; Subedi, Ganesh P; Barb, Adam W

2018-03-09

CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates ( N -glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N -glycans (23%). These proportions indicated restricted N -glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N -glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ϵ receptor γ-chain in HEK293F cells was expected to produce N -glycoforms similar to NK cell-derived CD16a but yielded N -glycoforms different from NK cell-derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N -glycan composition affected antibody binding: CD16a with oligomannose N -glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N -glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein's structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N -glycan composition and antibody-binding affinity. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1

NARCIS (Netherlands)

Sanders, Rogier W.; Vesanen, Mika; Schuelke, Norbert; Master, Aditi; Schiffner, Linnea; Kalyanaraman, Roopa; Paluch, Maciej; Berkhout, Ben; Maddon, Paul J.; Olson, William C.; Lu, Min; Moore, John P.

2002-01-01

The envelope glycoprotein (Env) complex of human immunodeficiency virus type I has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41

BIP induces mice CD19(hi) regulatory B cells producing IL-10 and highly expressing PD-L1, FasL.

Science.gov (United States)

Tang, Youfa; Jiang, Qing; Ou, Yanghui; Zhang, Fan; Qing, Kai; Sun, Yuanli; Lu, Wenjie; Zhu, Huifen; Gong, Feili; Lei, Ping; Shen, Guanxin

2016-01-01

Many studies have shown that B cells possess a regulatory function in mouse models of autoimmune diseases. Regulatory B cells can modulate immune response through many types of molecular mechanisms, including the production of IL-10 and the expression of PD-1 Ligand and Fas Ligand, but the microenvironmental factors and mechanisms that induce regulatory B cells have not been fully identified. BIP (binding immunoglobulin protein), a member of the heat shock protein 70 family, is a type of evolutionarily highly conserved protein. In this article, we have found that IL-10(+), PD-L1(hi) and FasL(hi) B cells are discrete cell populations, but enriched in CD19(hi) cells. BIP can induce IL-10-producing splenic B cells, IL-10 secretion and B cells highly expressing PD-L1 and FasL. CD40 signaling acts in synergy with BIP to induce regulatory B cells. BIP increased surface CD19 molecule expression intensity and IL-10(+), PD-L1(hi) and FasL(hi) B cells induced by BIP share the CD19(hi) phenotype. Furthermore, B cells treated with BIP and anti-CD40 can lead to suppression of T cell proliferation and the effect is partially IL-10-dependent and mainly BIP-induced. Taken together, our findings identify a novel function of BIP in the induction of regulatory B cells and add a new reason for the therapy of autoimmune disorders or other inflammatory conditions. Copyright © 2015. Published by Elsevier Ltd.

Effect of treatment with human apolipoprotein A-I on atherosclerosis in uremic apolipoprotein-E deficient mice

DEFF Research Database (Denmark)

Pedersen, Tanja Xenia; Bro, Susanne; Andersen, Mikkel H

2009-01-01

OBJECTIVE: Uremia markedly increases the risk of atherosclerosis. Thus, effective anti-atherogenic treatments are needed for uremic patients. This study examined effects of non-lipidated recombinant human apoA-I (h-apoA-I) and a recombinant trimeric apoA-I molecule (TripA-I) on lipid metabolism a...

Blockade of OX40/OX40 ligand to decrease cytokine messenger RNA expression in acute renal allograft rejection in vitro.

Science.gov (United States)

Wang, Y-L; Li, G; Fu, Y-X; Wang, H; Shen, Z-Y

2013-01-01

The aim of this study was to investigate cytokine messenger RNA (mRNA) expression by peripheral blood mononuclear cells (PBMCs) from renal recipients experiencing acute rejection by blocking OX40-OX40L interactions with recombinant human OX40-Fc fusion protein (rhOX40Fc) in vitro. PBMCs were isolated from 20 recipients experiencing acute rejection episodes (rejection group) and 20 recipients with stable graft function (stable group). Levels of Th1 (interferon [IFN]-γ) and Th2 (interleukin [IL]-4) mRNA expressions by PBMCs were measured using real-time reverse transcriptase-polymerase chain reactions. IFN-γ mRNA expression levels were significantly higher in the rejection than the stable group (P rejection group, rhOX40Fc reduced significantly the expression of IFN-γ and IL-4 mRNA by anti-CD3-monoclonal antibody stimulated PBMCs (P type cytokines. Copyright © 2013 Elsevier Inc. All rights reserved.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

Science.gov (United States)

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Impact of extended defects on recombination in CdTe heterostructures grown by molecular beam epitaxy

Energy Technology Data Exchange (ETDEWEB)

Zaunbrecher, Katherine N. [Department of Physics, Colorado State University, Fort Collins, Colorado 80523 (United States); National Renewable Energy Laboratory, Golden, Colorado 80401 (United States); Kuciauskas, Darius; Dippo, Pat; Barnes, Teresa M. [National Renewable Energy Laboratory, Golden, Colorado 80401 (United States); Swartz, Craig H.; Edirisooriya, Madhavie; Ogedengbe, Olanrewaju S.; Sohal, Sandeep; Hancock, Bobby L.; LeBlanc, Elizabeth G.; Jayathilaka, Pathiraja A. R. D.; Myers, Thomas H. [Materials Science, Engineering and Commercialization Program, Texas State University, San Marcos, Texas 78666 (United States)

2016-08-29

Heterostructures with CdTe and CdTe{sub 1-x}Se{sub x} (x ∼ 0.01) absorbers between two wider-band-gap Cd{sub 1-x}Mg{sub x}Te barriers (x ∼ 0.25–0.3) were grown by molecular beam epitaxy to study carrier generation and recombination in bulk materials with passivated interfaces. Using a combination of confocal photoluminescence (PL), time-resolved PL, and low-temperature PL emission spectroscopy, two extended defect types were identified and the impact of these defects on charge-carrier recombination was analyzed. The dominant defects identified by confocal PL were dislocations in samples grown on (211)B CdTe substrates and crystallographic twinning-related defects in samples on (100)-oriented InSb substrates. Low-temperature PL shows that twin-related defects have a zero-phonon energy of 1.460 eV and a Huang-Rhys factor of 1.50, while dislocation-dominated samples have a 1.473-eV zero-phonon energy and a Huang-Rhys factor of 1.22. The charge carrier diffusion length near both types of defects is ∼6 μm, suggesting that recombination is limited by diffusion dynamics. For heterostructures with a low concentration of extended defects, the bulk lifetime was determined to be 2.2 μs with an interface recombination velocity of 160 cm/s and an estimated radiative lifetime of 91 μs.

Multimeric and trimeric subunit SP-D are interconvertible structures with distinct ligand interaction

DEFF Research Database (Denmark)

Sørensen, Grith Lykke; Hoegh, Silje V; Leth-Larsen, Rikke

2009-01-01

-D compared to Met11 SP-D. Multimerization has proven important for enhancement of microbial phagocytosis. In the present study defined multimeric forms of Met11Thr SP-D were isolated from human amniotic fluid. Implementation of ManNAc-affinity chromatography allowed high recovery of natural trimeric SP......-D multimers. Trimeric SP-D subunits also showed greater binding to endogenous lipoproteins: LDL, oxLDL, and HDL, than multimeric SP-D. In conclusion, purified trimeric and multimeric SP-D represent separate and only partly interconvertible molecular populations with distinct biochemical properties....

CD8αα expression marks terminally differentiated human CD8+ T cells expanded in chronic viral infection

Directory of Open Access Journals (Sweden)

Lucy Jane Walker

2013-08-01

Full Text Available The T cell co-receptor CD8αβ enhances T cell sensitivity to antigen, however studies indicate CD8αα has the converse effect and acts as a co-repressor. Using a combination of Thymic Leukaemia antigen (TL tetramer, which directly binds CD8αα, anti-CD161 and anti-Vα7.2 antibodies we have been able for the first time to clearly define CD8αα expression on human CD8 T cells subsets. In healthy controls CD8αα is most highly expressed by CD161 bright (CD161++ mucosal associated invariant T (MAIT cells, with CD8αα expression highly restricted to the TCR Vα7.2+ cells of this subset. We also identified CD8αα-expressing populations within the CD161 mid (CD161+ and negative (CD161- non-MAIT CD8 T cell subsets and show TL-tetramer binding to correlate with expression of CD8β at low levels in the context of maintained CD8α expression (CD8α+CD8βlow. In addition, we found CD161-CD8α+CD8βlow populations to be significantly expanded in the peripheral blood of HIV-1 and hepatitis B (mean of 47% and 40% of CD161- T cells respectively infected individuals. Such CD8αα expressing T cells are an effector-memory population (CD45RA-, CCR7-, CD62L- that express markers of activation and maturation (HLA-DR+, CD28-, CD27-, CD57+ and are functionally distinct, expressing greater levels of TNF-α and IFN-γ on stimulation and perforin at rest than their CD8α+CD8βhigh counterparts. Antigen-specific T cells in HLA-B*4201+HIV-1 infected patients are found within both the CD161-CD8α+CD8βhigh and CD161-CD8α+CD8βlow populations. Overall we have clearly defined CD8αα expressing human T cell subsets using the TL-tetramer, and have demonstrated CD161-CD8α+CD8βlow populations, highly expanded in disease settings, to co-express CD8αβ and CD8αα. Co-expression of CD8αα on CD8αβ T cells may impact on their overall function in-vivo and contribute to the distinctive phenotype of highly differentiated populations in HBV and HIV-1 infection.

Evaluation of the impact of chitosan/DNA nanoparticles on the differentiation of human naive CD4+ T cells

Science.gov (United States)

Liu, Lanxia; Bai, Yuanyuan; Zhu, Dunwan; Song, Liping; Wang, Hai; Dong, Xia; Zhang, Hailing; Leng, Xigang

2011-06-01

Chitosan (CS) is one of the most widely studied polymers in non-viral gene delivery since it is a cationic polysaccharide that forms nanoparticles with DNA and hence protects the DNA against digestion by DNase. However, the impact of CS/DNA nanoparticle on the immune system still remains poorly understood. Previous investigations did not found CS/DNA nanoparticles had any significant impact on the function of human and murine macrophages. To date, little is known about the interaction between CS/DNA nanoparticles and naive CD4+ T cells. This study was designed to investigate whether CS/DNA nanoparticles affect the initial differentiation direction of human naive CD4+ T cells. The indirect impact of CS/DNA nanoparticles on naive CD4+ T cell differentiation was investigated by incubating the nanoparticles with human macrophage THP-1 cells in one chamber of a transwell co-incubation system, with the enriched human naive CD4+ T cells being placed in the other chamber of the transwell. The nanoparticles were also co-incubated with the naive CD4+ T cells to explore their direct impact on naive CD4+ T cell differentiation by measuring the release of IL-4 and IFN-γ from the cells. It was demonstrated that CS/DNA nanoparticles induced slightly elevated production of IL-12 by THP-1 cells, possibly owing to the presence of CpG motifs in the plasmid. However, this macrophage stimulating activity was much less significant as compared with lipopolysaccharide and did not impact on the differentiation of the naive CD4+ T cells. It was also demonstrated that, when directly exposed to the naive CD4+ T cells, the nanoparticles induced neither the activation of the naive CD4+ T cells in the absence of recombinant cytokines (recombinant human IL-4 or IFN-γ) that induce naive CD4+ T cell polarization, nor any changes in the differentiation direction of naive CD4+ T cells in the presence of the corresponding cytokines.

Evaluation of the impact of chitosan/DNA nanoparticles on the differentiation of human naive CD4+ T cells

International Nuclear Information System (INIS)

Liu Lanxia; Bai Yuanyuan; Zhu Dunwan; Song Liping; Wang Hai; Dong Xia; Zhang Hailing; Leng Xigang

2011-01-01

Chitosan (CS) is one of the most widely studied polymers in non-viral gene delivery since it is a cationic polysaccharide that forms nanoparticles with DNA and hence protects the DNA against digestion by DNase. However, the impact of CS/DNA nanoparticle on the immune system still remains poorly understood. Previous investigations did not found CS/DNA nanoparticles had any significant impact on the function of human and murine macrophages. To date, little is known about the interaction between CS/DNA nanoparticles and naive CD4 + T cells. This study was designed to investigate whether CS/DNA nanoparticles affect the initial differentiation direction of human naive CD4 + T cells. The indirect impact of CS/DNA nanoparticles on naive CD4 + T cell differentiation was investigated by incubating the nanoparticles with human macrophage THP-1 cells in one chamber of a transwell co-incubation system, with the enriched human naive CD4 + T cells being placed in the other chamber of the transwell. The nanoparticles were also co-incubated with the naive CD4 + T cells to explore their direct impact on naive CD4 + T cell differentiation by measuring the release of IL-4 and IFN-γ from the cells. It was demonstrated that CS/DNA nanoparticles induced slightly elevated production of IL-12 by THP-1 cells, possibly owing to the presence of CpG motifs in the plasmid. However, this macrophage stimulating activity was much less significant as compared with lipopolysaccharide and did not impact on the differentiation of the naive CD4 + T cells. It was also demonstrated that, when directly exposed to the naive CD4 + T cells, the nanoparticles induced neither the activation of the naive CD4 + T cells in the absence of recombinant cytokines (recombinant human IL-4 or IFN-γ) that induce naive CD4 + T cell polarization, nor any changes in the differentiation direction of naive CD4 + T cells in the presence of the corresponding cytokines.

CD30 ligand is frequently expressed in human hematopoietic malignancies of myeloid and lymphoid origin.

Science.gov (United States)

Gattei, V; Degan, M; Gloghini, A; De Iuliis, A; Improta, S; Rossi, F M; Aldinucci, D; Perin, V; Serraino, D; Babare, R; Zagonel, V; Gruss, H J; Carbone, A; Pinto, A

1997-03-15

CD30 ligand (CD30L) is a type-II membrane glycoprotein capable of transducing signals leading to either cell death or proliferation through its specific counterstructure CD30. Although several lines of evidence indicate that CD30L plays a key role as a paracrine- or autocrine-acting surface molecule in the deregulated cytokine cascade of Hodgkin's disease, little is known regarding its distribution and biologic significance in other human hematopoietic malignancies. By analyzing tumor cells from 181 patients with RNA studies and immunostaining by the anti-CD30L monoclonal antibody M80, we were able to show that human hematopoietic malignancies of different lineage and maturation stage display a frequent and broad expression of the ligand. CD30L mRNA and surface protein were detected in 60% of acute myeloid leukemias (AMLs), 54% of B-lineage acute lymphoblastic leukemias (ALLs), and in a consistent fraction (68%) of B-cell lymphoproliferative disorders. In this latter group, hairy cell leukemia and high-grade B-cell non-Hodgkin's lymphoma (B-NHL) expressed a higher surface density of CD30L as compared with B-cell chronic lymphocytic leukemia and low-grade B-NHL. Purified plasmacells from a fraction of multiple myeloma patients also displayed CD30L mRNA and protein. A more restricted expression of CD30L was found in T-cell tumors that was mainly confined to neoplasms with an activated peripheral T-cell phenotype, such as T-cell prolymphocytic leukemia, peripheral T-NHL, and adult T-cell leukemia/lymphoma. In contrast, none of the T-lineage ALLs analyzed expressed the ligand. In AML, a high cellular density of CD30L was detected in French-American-British M3, M4, and M5 phenotypes, which are directly associated with the presence on tumor cells of certain surface structures, including the p55 interleukin-2 receptor alpha-chain, the alpha(M) (CD11b) chain of beta2 integrins, and the intercellular adhesion molecule-1 (CD54). Analysis of normal hematopoietic cells

Enhanced photovoltaic performance of quantum dot-sensitized solar cells with a progressive reduction of recombination using Cu-doped CdS quantum dots

International Nuclear Information System (INIS)

Muthalif, Mohammed Panthakkal Abdul; Lee, Young-Seok; Sunesh, Chozhidakath Damodharan; Kim, Hee-Je; Choe, Youngson

2017-01-01

Highlights: • Cu-doped CdS QDs were deposited on TiO_2 by SILAR method. • Cu-doped CdS electrodes contributes reduction of charge recombination and longer electron lifetime. • A promising power conversion efficiency of 3% is obtained for the Cu-doped CdS Quantum dot sensitized solar cell. - Abstract: In this article, we have systematically probed the effect of Cu-doping in CdS quantum dots (QDs) to enhance the photovoltaic performance of the quantum dot-sensitized solar cells (QDSSCs). The Cu-doped CdS photoanodes were prepared by successive ionic layer adsorption and reaction (SILAR) method and the corresponding cell devices were fabricated using CuS counter electrodes with a polysulfide electrolyte. The photovoltaic performance results demonstrate that 3 mM Cu-doped CdS QDs based QDSSCs exhibit the efficiency (η) of 3% including J_S_C = 9.40 mA cm"−"2, V_O_C = 0.637 V, FF = 0.501, which are higher than those with bare CdS (η = 2.05%, J_S_C = 7.12 mA cm"−"2, V_O_C = 0.588 V, FF = 0.489). The structural, topographical and optical properties of the thin films have been studied with the help of X-ray diffraction pattern (XRD), atomic force microscopy (AFM) and UV–vis spectrophotometer. Electrochemical impedance spectroscopy (EIS) and open circuit voltage decay (OCVD) measurements indicate that Cu-dopant can inhibit the charge recombination at the photoanode/electrolyte interface and extend the lifetime of electrons. These results reveal that incorporation of copper metal in CdS QDs is a simple and effective method to improve the photovoltaic properties of QDSSCs.

Enhanced photovoltaic performance of quantum dot-sensitized solar cells with a progressive reduction of recombination using Cu-doped CdS quantum dots

Energy Technology Data Exchange (ETDEWEB)

Muthalif, Mohammed Panthakkal Abdul [Department of Polymer Science and Chemical Engineering, Pusan National University, Geumjeong-Ku, Jangjeong-Dong, Busan 609-735 (Korea, Republic of); Lee, Young-Seok [School of Electrical Engineering, Pusan National University, Geumjeong-Ku, Jangjeong-Dong, Busan 609-735 (Korea, Republic of); Sunesh, Chozhidakath Damodharan [Department of Polymer Science and Chemical Engineering, Pusan National University, Geumjeong-Ku, Jangjeong-Dong, Busan 609-735 (Korea, Republic of); Kim, Hee-Je [School of Electrical Engineering, Pusan National University, Geumjeong-Ku, Jangjeong-Dong, Busan 609-735 (Korea, Republic of); Choe, Youngson, E-mail: choe@pusan.ac.kr [Department of Polymer Science and Chemical Engineering, Pusan National University, Geumjeong-Ku, Jangjeong-Dong, Busan 609-735 (Korea, Republic of)

2017-02-28

Highlights: • Cu-doped CdS QDs were deposited on TiO{sub 2} by SILAR method. • Cu-doped CdS electrodes contributes reduction of charge recombination and longer electron lifetime. • A promising power conversion efficiency of 3% is obtained for the Cu-doped CdS Quantum dot sensitized solar cell. - Abstract: In this article, we have systematically probed the effect of Cu-doping in CdS quantum dots (QDs) to enhance the photovoltaic performance of the quantum dot-sensitized solar cells (QDSSCs). The Cu-doped CdS photoanodes were prepared by successive ionic layer adsorption and reaction (SILAR) method and the corresponding cell devices were fabricated using CuS counter electrodes with a polysulfide electrolyte. The photovoltaic performance results demonstrate that 3 mM Cu-doped CdS QDs based QDSSCs exhibit the efficiency (η) of 3% including J{sub SC} = 9.40 mA cm{sup −2}, V{sub OC} = 0.637 V, FF = 0.501, which are higher than those with bare CdS (η = 2.05%, J{sub SC} = 7.12 mA cm{sup −2}, V{sub OC} = 0.588 V, FF = 0.489). The structural, topographical and optical properties of the thin films have been studied with the help of X-ray diffraction pattern (XRD), atomic force microscopy (AFM) and UV–vis spectrophotometer. Electrochemical impedance spectroscopy (EIS) and open circuit voltage decay (OCVD) measurements indicate that Cu-dopant can inhibit the charge recombination at the photoanode/electrolyte interface and extend the lifetime of electrons. These results reveal that incorporation of copper metal in CdS QDs is a simple and effective method to improve the photovoltaic properties of QDSSCs.

Soluble human CD4 elicits an antibody response in rhesus monkeys that inhibits simian immunodeficiency virus replication

International Nuclear Information System (INIS)

Watanabe, Mamoru; Chen, Zheng W.; Tsubota, Hiroshi; Lord, C.I.; Levine, C.G.; Letvin, N.L.

1991-01-01

Rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIV mac ) demonstrate significant virologic and clinical improvement as a result of treatment with human recombinant soluble CD4 (rsCD4). The authors show that human rsCD4 does not efficiently inhibit SIV mac replication in bone marrow macrophages of rhesus monkeys and does not significantly augment bone marrow hematopoietic colony formation in vitro. However, plasma of human rsCD4-treated rhesus monkeys does exhibit significant anti-SIV mac activity in vitro. Plasma of these animals efficiently blocks SIV mac replicaton in peripheral blood lymphocytes and bone marrow macrophages. It also increases granulocyte/macrophage colony formation in vitro by bone marrow cells of SIV mac -infected monkeys. This plasma and the IgG fraction of plasma from a rhesus monkey immunized with human rsCD4 in adjuvant demonstrate reactivity with a soluble form of the rhesus monkey CD4 molecule, exhibit binding to CD4 + but not CD8 + concanavalin A-activated rhesus monkey peripheral blood lymphocytes, and precipitate the CD4 molecule from surface-labeled activated rhesus monkey peripheral blood lymphocytes. Moreover, anti-viral activity is demonstrable in the IgG fraction of plasma from a human rsCD4-immunized monkey. These studies raise the possibility that a modified human CD4 molecule serving as an immunogen might elicit an antibody response that could potentially induce a beneficial therapeutic response in human immunodeficiency virus-infected individuals

Facile synthesis of N-acetyl-L-cysteine capped CdHgSe quantum dots and selective determination of hemoglobin.

Science.gov (United States)

Wang, Qingqing; Zhan, Guoqing; Li, Chunya

2014-01-03

Using N-acetyl-L-cysteine (NAC) as a stabilizer, well water-dispersed, high-quality and stable CdHgSe quantum dots were facilely synthesized via a simple aqueous phase method. The as-prepared NAC capped CdHgSe quantum dots were thoroughly characterized by fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, energy dispersive X-ray spectroscopy and transmission electron microscopy. A novel method for the selective determination of hemoglobin (Hb) was developed based on fluorescence quenching of the NAC capped CdHgSe quantum dots. A number of key factors including pH value of phosphate buffer solution, quantum dots concentration, the adding sequence of reagents and reaction time that influence the analytical performance of the NAC capped CdHgSe quantum dots in Hb determination were investigated. Under the optimal experimental conditions, the change of fluorescence intensity (ΔI) was linearly proportional to the concentration of Hb in the range of 4.0×10(-9)-4.4×10(-7) mol L(-1) with a detection limit of 2.0×10(-9) mol L(-1). The developed method has been successfully employed to determine Hb in human urine samples. Copyright © 2013. Published by Elsevier B.V.

Single crystalline multi-petal Cd nanoleaves prepared by thermal reduction of CdO

International Nuclear Information System (INIS)

Khan, Waheed S.; Cao, Chuanbao; Aslam, Imran; Ali, Zulfiqar; Butt, Faheem K.; Mahmood, Tariq; Nabi, Ghulam; Ihsan, Ayesha; Usman, Zahid; Rehman, Asma

2013-01-01

Highlights: ► Cd nanoleaves are obtained on abraded Cu substrate by thermal reduction of CdO. ► Vapour solid (VS) growth mechanism governs the formation of Cd nanoleaves (CdNLs). ► PL spectrum for CdNLs exhibits a strong ultraviolet (UV) emission band at 353 nm. ► UV band is attributed to interband radiative recombination under Xe illumination. -- Abstract: Multi-petal cadmium metal nanoleaves with 30–40 nm thickness were fabricated on abraded copper substrate by simple thermal reduction of cadmium oxide (CdO) powder at 1050 °C inside horizontal tube furnace (HTF) under nitrogen gas flow. The structural, compositional and morphological characterizations of the as-prepared cadmium nanoleaves (CdNLs) were performed by X-ray diffraction, energy dispersive X-ray spectroscopy, scanning electron microscopy, high resolution transmission electron microscopy and selected area electron diffraction. Non-catalytic vapour–solid (VS) process based growth mechanism governing the formation of CdNLs has been proposed and discussed briefly. Photoluminescence (PL) spectrum for CdNLs measured at room temperature exhibited a single prominent emission band at 353 nm which may either be ascribed to surface oxidation effects or interband radiative recombination under Xe light illumination.

Single crystalline multi-petal Cd nanoleaves prepared by thermal reduction of CdO

Energy Technology Data Exchange (ETDEWEB)

Khan, Waheed S. [Research Centre of Materials Science, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 (China); National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box No. 577, Jhang Road, Faisalabad (Pakistan); Cao, Chuanbao, E-mail: cbcao@bit.edu.cn [Research Centre of Materials Science, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 (China); Aslam, Imran; Ali, Zulfiqar; Butt, Faheem K.; Mahmood, Tariq; Nabi, Ghulam [Research Centre of Materials Science, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 (China); Ihsan, Ayesha [National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box No. 577, Jhang Road, Faisalabad (Pakistan); Usman, Zahid [Research Centre of Materials Science, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 (China); Rehman, Asma [National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box No. 577, Jhang Road, Faisalabad (Pakistan)

2013-02-15

Highlights: ► Cd nanoleaves are obtained on abraded Cu substrate by thermal reduction of CdO. ► Vapour solid (VS) growth mechanism governs the formation of Cd nanoleaves (CdNLs). ► PL spectrum for CdNLs exhibits a strong ultraviolet (UV) emission band at 353 nm. ► UV band is attributed to interband radiative recombination under Xe illumination. -- Abstract: Multi-petal cadmium metal nanoleaves with 30–40 nm thickness were fabricated on abraded copper substrate by simple thermal reduction of cadmium oxide (CdO) powder at 1050 °C inside horizontal tube furnace (HTF) under nitrogen gas flow. The structural, compositional and morphological characterizations of the as-prepared cadmium nanoleaves (CdNLs) were performed by X-ray diffraction, energy dispersive X-ray spectroscopy, scanning electron microscopy, high resolution transmission electron microscopy and selected area electron diffraction. Non-catalytic vapour–solid (VS) process based growth mechanism governing the formation of CdNLs has been proposed and discussed briefly. Photoluminescence (PL) spectrum for CdNLs measured at room temperature exhibited a single prominent emission band at 353 nm which may either be ascribed to surface oxidation effects or interband radiative recombination under Xe light illumination.

CD40 expression in Wehi-164 cell line

OpenAIRE

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Moazzeni, Seyed Mohammad

2010-01-01

CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body’s defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein ex...

The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3.

Science.gov (United States)

Sirena, Dominique; Lilienfeld, Benjamin; Eisenhut, Markus; Kälin, Stefan; Boucke, Karin; Beerli, Roger R; Vogt, Lorenz; Ruedl, Christiane; Bachmann, Martin F; Greber, Urs F; Hemmi, Silvio

2004-05-01

Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.

CD40 Ligand Deficient C57BL/6 Mouse Is a Potential Surrogate Model of Human X-Linked Hyper IgM (X-HIGM Syndrome for Characterizing Immune Responses against Pathogens

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Catalina Lopez-Saucedo

2015-01-01

Full Text Available Individuals with X-HIGM syndrome fail to express functional CD40 ligand; consequently they cannot mount effective protective antibody responses against pathogenic bacteria. We evaluated, compared, and characterized the humoral immune response of wild type (WT and C57-CD40L deficient (C57-CD40L−/− mice infected with Citrobacter rodentium. Basal serum isotype levels were similar for IgM and IgG3 among mice, while total IgG and IgG2b concentrations were significantly lower in C57-CD40L−/− mice compared with WT. Essentially IgG1 and IgG2c levels were detectable only in WT mice. C57-CD40L−/− animals, orally inoculated with 2×109 CFU, presented several clinical manifestations since the second week of infection and eventually died. In contrast at this time point no clinical manifestations were observed among C57-CD40L−/− mice infected with 1×107 CFU. Infection was subclinical in WT mice inoculated with either bacterial dose. The serum samples from infected mice (1×107 CFU, collected at day 14 after infection, had similar C. rodentium-specific IgM titres. Although C57-CD40L−/− animals had lower IgG and IgG2b titres than WT mice, C57-CD40L−/− mice sera displayed complement-mediated bactericidal activity against C. rodentium. C. rodentium-infected C57-CD40L−/− mice are capable of producing antibodies that are protective. C57-CD40L−/− mouse is a useful surrogate model of X-HIGM syndrome for studying immune responses elicited against pathogens.

Evaluation of the impact of chitosan/DNA nanoparticles on the differentiation of human naive CD4{sup +} T cells

Energy Technology Data Exchange (ETDEWEB)

Liu Lanxia; Bai Yuanyuan; Zhu Dunwan; Song Liping; Wang Hai; Dong Xia; Zhang Hailing; Leng Xigang, E-mail: lengxg@bme.org.cn [Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials, Lab of Bioengineering, Institute of Biomedical Engineering (China)

2011-06-15

Chitosan (CS) is one of the most widely studied polymers in non-viral gene delivery since it is a cationic polysaccharide that forms nanoparticles with DNA and hence protects the DNA against digestion by DNase. However, the impact of CS/DNA nanoparticle on the immune system still remains poorly understood. Previous investigations did not found CS/DNA nanoparticles had any significant impact on the function of human and murine macrophages. To date, little is known about the interaction between CS/DNA nanoparticles and naive CD4{sup +} T cells. This study was designed to investigate whether CS/DNA nanoparticles affect the initial differentiation direction of human naive CD4{sup +} T cells. The indirect impact of CS/DNA nanoparticles on naive CD4{sup +} T cell differentiation was investigated by incubating the nanoparticles with human macrophage THP-1 cells in one chamber of a transwell co-incubation system, with the enriched human naive CD4{sup +} T cells being placed in the other chamber of the transwell. The nanoparticles were also co-incubated with the naive CD4{sup +} T cells to explore their direct impact on naive CD4{sup +} T cell differentiation by measuring the release of IL-4 and IFN-{gamma} from the cells. It was demonstrated that CS/DNA nanoparticles induced slightly elevated production of IL-12 by THP-1 cells, possibly owing to the presence of CpG motifs in the plasmid. However, this macrophage stimulating activity was much less significant as compared with lipopolysaccharide and did not impact on the differentiation of the naive CD4{sup +} T cells. It was also demonstrated that, when directly exposed to the naive CD4{sup +} T cells, the nanoparticles induced neither the activation of the naive CD4{sup +} T cells in the absence of recombinant cytokines (recombinant human IL-4 or IFN-{gamma}) that induce naive CD4{sup +} T cell polarization, nor any changes in the differentiation direction of naive CD4{sup +} T cells in the presence of the corresponding

Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults.

Science.gov (United States)

Szpakowski, Piotr; Biet, Franck; Locht, Camille; Paszkiewicz, Małgorzata; Rudnicka, Wiesława; Druszczyńska, Magdalena; Allain, Fabrice; Fol, Marek; Pestel, Joël; Kowalewicz-Kulbat, Magdalena

2015-01-01

Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18) and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4(+) T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.

Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults

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Piotr Szpakowski

2015-01-01

Full Text Available Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG, the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18 and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4+ T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.

EBV promotes human CD8 NKT cell development.

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Yuling He

2010-05-01

Full Text Available The reports on the origin of human CD8(+ Valpha24(+ T-cell receptor (TCR natural killer T (NKT cells are controversial. The underlying mechanism that controls human CD4 versus CD8 NKT cell development is not well-characterized. In the present study, we have studied total 177 eligible patients and subjects including 128 healthy latent Epstein-Barr-virus(EBV-infected subjects, 17 newly-onset acute infectious mononucleosis patients, 16 newly-diagnosed EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. We have established human-thymus/liver-SCID chimera, reaggregated thymic organ culture, and fetal thymic organ culture. We here show that the average frequency of total and CD8(+ NKT cells in PBMCs from 128 healthy latent EBV-infected subjects is significantly higher than in 17 acute EBV infectious mononucleosis patients, 16 EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. However, the frequency of total and CD8(+ NKT cells is remarkably increased in the acute EBV infectious mononucleosis patients at year 1 post-onset. EBV-challenge promotes CD8(+ NKT cell development in the thymus of human-thymus/liver-SCID chimeras. The frequency of total (3% of thymic cells and CD8(+ NKT cells ( approximately 25% of NKT cells is significantly increased in EBV-challenged chimeras, compared to those in the unchallenged chimeras (<0.01% of thymic cells, CD8(+ NKT cells undetectable, respectively. The EBV-induced increase in thymic NKT cells is also reflected in the periphery, where there is an increase in total and CD8(+ NKT cells in liver and peripheral blood in EBV-challenged chimeras. EBV-induced thymic CD8(+ NKT cells display an activated memory phenotype (CD69(+CD45RO(hiCD161(+CD62L(lo. After EBV-challenge, a proportion of NKT precursors diverges from DP thymocytes, develops and differentiates into mature CD8(+ NKT cells in thymus in EBV-challenged human-thymus/liver-SCID chimeras or

Immunoglobulin class-switch recombination deficiencies.

Science.gov (United States)

Durandy, Anne; Kracker, Sven

2012-07-30

Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches.

Increased T cell expression of CD154 (CD40-ligand) in multiple sclerosis

DEFF Research Database (Denmark)

Jensen, J; Krakauer, M; Sellebjerg, F

2001-01-01

CD154 (CD40-ligand, gp39), expressed on activated T cells, is crucial in T cell-dependent immune responses and may be involved in the pathogenesis of multiple sclerosis (MS). We studied cerebro-spinal fluid and peripheral blood T cell expression of CD154 in MS by flow cytometry. Patients with sec......CD154 (CD40-ligand, gp39), expressed on activated T cells, is crucial in T cell-dependent immune responses and may be involved in the pathogenesis of multiple sclerosis (MS). We studied cerebro-spinal fluid and peripheral blood T cell expression of CD154 in MS by flow cytometry. Patients...

Cascading electron and hole transfer dynamics in a CdS/CdTe core-shell sensitized with bromo-pyrogallol red (Br-PGR): slow charge recombination in type II regime.

Science.gov (United States)

Maity, Partha; Debnath, Tushar; Chopra, Uday; Ghosh, Hirendra Nath

2015-02-14

Ultrafast cascading hole and electron transfer dynamics have been demonstrated in a CdS/CdTe type II core-shell sensitized with Br-PGR using transient absorption spectroscopy and the charge recombination dynamics have been compared with those of CdS/Br-PGR composite materials. Steady state optical absorption studies suggest that Br-PGR forms strong charge transfer (CT) complexes with both the CdS QD and CdS/CdTe core-shell. Hole transfer from the photo-excited QD and QD core-shell to Br-PGR was confirmed by both steady state and time-resolved emission spectroscopy. Charge separation was also confirmed by detecting electrons in the conduction band of the QD and the cation radical of Br-PGR as measured from femtosecond transient absorption spectroscopy. Charge separation in the CdS/Br-PGR composite materials was found to take place in three different pathways, by transferring the photo-excited hole of CdS to Br-PGR, electron injection from the photo-excited Br-PGR to the CdS QD, and direct electron transfer from the HOMO of Br-PGR to the conduction band of the CdS QD. However, in the CdS/CdTe/Br-PGR system hole transfer from the photo-excited CdS to Br-PGR and electron injection from the photo-excited Br-PGR to CdS take place after cascading through the CdTe shell QD. Charge separation also takes place via direct electron transfer from the Br-PGR HOMO to the conduction band of CdS/CdTe. Charge recombination (CR) dynamics between the electron in the conduction band of the CdS QD and the Br-PGR cation radical were determined by monitoring the bleach recovery kinetics. The CR dynamics were found to be much slower in the CdS/CdTe/Br-PGR system than in the CdS/Br-PGR system. The formation of the strong CT complex and the separation of charges cascading through the CdTe shell help to slow down charge recombination in the type II regime.

Upregulation of Fas-Fas-L (CD95/CD95L)-mediated epithelial apoptosis--a putative role in pouchitis?

LENUS (Irish Health Repository)

Coffey, J C

2012-02-03

INTRODUCTION: Ileal pouch-anal anastomosis (IPAA) remains the gold standard for patients with refractory ulcerative colitis. Pouchitis causes considerable morbidity in 40% of patients with IPAA. This study examined the role of increased epithelial apoptosis in the etiology of pouchitis. METHODS: Following ethical approval pouch biopsies taken from patients with a history of pouchitis were compared with age-matched controls from patients who were pouchitis free. Apoptosis was detected immunohistochemically using a monoclonal antibody (M30) and terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin end labeling (TUNEL). Villous atrophy was assessed histologically and correlated with levels of apoptosis. Epithelial Fas-ligand (L) was also assessed immunohistochemically. RESULTS: A significant increase in TUNEL staining was seen at the epithelial but not at the lamina propria level for known pouchitis patients versus controls (0.091 vs 0.035; P < 0.01). Similarly, epithelial M30 immunoreactivity (0.225 vs 0.082; P < 0.05) and villous atrophy (0.035 vs 0.10; P < 0.05) were significantly increased in pouches with previous pouchitis when compared with normal pouches. Upregulation of Fas-L expression was characteristic of this epithelium. Mononuclear cells were strongly positive for Fas-L. Increased epithelial levels of apoptosis correlated with increased levels of villous atrophy. CONCLUSIONS: Our data suggest a role for elevated Fas-Fas-L (CD95-CD95L)-mediated epithelial apoptosis in the etiology of pouchitis. Increased levels of villous atrophy may result from increased apoptosis and thereby predispose to infection by otherwise apathogenic organisms.

Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential

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Yu Y

2017-03-01

all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdcem26Il2rgem26Nju (NCG mice transplanted with CEM cells without any obvious decrease in body weight. Further studies on NCG mice model with patient-derived T-ALL cells, dhuVHH6-PE38 treatment, significantly prolonged mice survival with ~40% survival improvement. However, it was also noticed that although dhuVHH6-PE-LR showed strong antitumor effect in vitro, its in vivo antitumor efficacy was disappointing. Conclusion: We have successfully constructed a targeted CD7 molecule-modified nanobody (CD7 molecule-improved nanobody immunotoxin dhuVHH6-PE38 and demonstrated its potential for treating CD7-positive malignant tumors, especially T-cell acute lymphoblastic leukemia. Keywords: CD7, humanized nanobody, T-cell acute lymphoblastic leukemia, patient-derived xenograft model, recombinant immunotoxins, Pseudomonas exotoxin A

DNA fragmentation and cytotoxicity by recombinant human tumor necrosis factor in L929 fibroblast cells

International Nuclear Information System (INIS)

Kosaka, T.; Kuwabara, M.; Koide, F.

1992-01-01

Induction of cell DNA fragmentation by treatment of recombinant human Tumor Necrosis Factor alpha (rhTNF alpha) was examined by using mouse L929 cells derived from mouse fibroblast cells. The amount of DNA fragments derived from rhTNF alpha-treated cells, detected by alkaline elution technique, was smaller than that derived from X-irradiated cells. The rhTNF alpha caused the DNA fragmentation depending on its incubation time and concentration. The DNA damage caused by rhTNF alpha treatment correlated with its cytotoxicity. This result suggested that the DNA fragmentation is one of causes of cell death. The treatment with proteinase K of DNA obtained from rhTNF alpha-treated cells did not increase the amount of DNA fragmentation, which indicates that rhTNF alpha causes DNA-fragmentation but not DNA-protein cross-linking

Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA.

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Matthew G Cottingham

2008-02-01

Full Text Available The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS99 12415-20. The genomic BAC clone was 'rescued' back to infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA, now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full-length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8(+ T cell immunogenicity in BALB/c mice, but deletion of B15R enhanced specific CD8(+ T cell responses to one of two endogenous viral epitopes (from the E2 and F2 proteins, in accordance with published work (Staib et al., 2005, J. Gen. Virol.86, 1997-2006. In addition, we found a higher frequency of triple-positive IFN-gamma, TNF-alpha and IL-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, a recombinant vaccine capable of inducing CD8(+ T cells against an epitope from Plasmodium berghei was created using GalK counterselection to insert an antigen expression cassette lacking a tandem marker gene into the traditional thymidine kinase locus of MVA-BAC. MVA continues to feature prominently in clinical trials of recombinant vaccines against diseases such as HIV-AIDS, malaria and tuberculosis. Here we demonstrate in proof-of-concept experiments that MVA-BAC recombineering is a viable route to more rapid and efficient generation of new candidate mutant and recombinant

Energy relaxation and transfer in excitonic trimer

International Nuclear Information System (INIS)

Herman, Pavel; Barvik, Ivan; Urbanec, Martin

2004-01-01

Two models describing exciton relaxation and transfer (the Redfield model in the secular approximation and Capek's model) are compared for a simple example - a symmetric trimer coupled to a phonon bath. Energy transfer within the trimer occurs via resonance interactions and coupling between the trimer and the bath occurs via modulation of the monomer energies by phonons. Two initial conditions are adopted: (1) one of higher eigenstates of the trimer is initially occupied and (2) one local site of the trimer is initially occupied. The diagonal exciton density matrix elements in the representation of eigenstates are found to be the same for both models, but this is not so for the off-diagonal density matrix elements. Only if the off-diagonal density matrix elements vanish initially (initial condition (1)), they then vanish at arbitrary times in both models. If the initial excitation is local, the off-diagonal matrix elements essentially differ

Bayesian inference of shared recombination hotspots between humans and chimpanzees.

Science.gov (United States)

Wang, Ying; Rannala, Bruce

2014-12-01

Recombination generates variation and facilitates evolution. Recombination (or lack thereof) also contributes to human genetic disease. Methods for mapping genes influencing complex genetic diseases via association rely on linkage disequilibrium (LD) in human populations, which is influenced by rates of recombination across the genome. Comparative population genomic analyses of recombination using related primate species can identify factors influencing rates of recombination in humans. Such studies can indicate how variable hotspots for recombination may be both among individuals (or populations) and over evolutionary timescales. Previous studies have suggested that locations of recombination hotspots are not conserved between humans and chimpanzees. We made use of the data sets from recent resequencing projects and applied a Bayesian method for identifying hotspots and estimating recombination rates. We also reanalyzed SNP data sets for regions with known hotspots in humans using samples from the human and chimpanzee. The Bayes factors (BF) of shared recombination hotspots between human and chimpanzee across regions were obtained. Based on the analysis of the aligned regions of human chromosome 21, locations where the two species show evidence of shared recombination hotspots (with high BFs) were identified. Interestingly, previous comparative studies of human and chimpanzee that focused on the known human recombination hotspots within the β-globin and HLA regions did not find overlapping of hotspots. Our results show high BFs of shared hotspots at locations within both regions, and the estimated locations of shared hotspots overlap with the locations of human recombination hotspots obtained from sperm-typing studies. Copyright © 2014 by the Genetics Society of America.

Effect of uv-irradiation on genetic recombination of Simian virus 40 mutants

International Nuclear Information System (INIS)

Gentil, A.; Margot, A.; Sarasin, A.

1983-01-01

Genetic recombination in monkey kidney cells has been studied using Simian virus 40 (SV40) as a molecular probe. Control or uv-irradiated cells have been co-infected with two thermosensitive mutants of SV40, tsA58 and tsA30. Recombination between the two viral genomes gives rise to a wild type virus phenotype, able to grow at the restrictive temperature of 41 0 C, which was taken as a measure of the recombination activity of the host cells. Results show that recombination takes place at a low frequency when viruses are not uv-irradiated. Irradiation of one or both viruses increases drastically recombination frequency. Pretreatment of the host cells with uv-light or mitomycin C 24 hours before being infected does not increase recombination frequency measured in our experimental conditions. 23 references, 5 tables

Physalis peruviana extract induces apoptosis in human Hep G2 cells through CD95/CD95L system and the mitochondrial signaling transduction pathway.

Science.gov (United States)

Wu, Shu-Jing; Ng, Lean-Teik; Lin, Doung-Liang; Huang, Shan-Ney; Wang, Shyh-Shyan; Lin, Chun-Ching

2004-11-25

Physalis species is a popular folk medicine used for treating cancer, leukemia, hepatitis and other diseases. Studies have shown that the ethanol extract of Physalis peruviana (EEPP) inhibits growth and induces apoptotic death of human Hep G2 cells in culture, whereas proliferation of the mouse BALB/C normal liver cells was not affected. In this study, we performed detailed studies to define the molecular mechanism of EEPP-induced apoptosis in Hep G2 cells. The results further confirmed that EEPP inhibited cell proliferation in a dose- and time-dependent manner. At 50 microg/ml, EEPP significantly increased the accumulation of the sub-G1 peak (hypoploid) and the portion of apoptotic annexin V positive cells. EEPP was found to trigger apoptosis through the release of cytochrome c, Smac/DIABLO and Omi/HtrA2 from mitochondria to cytosol and consequently resulted in caspase-3 activation. Pre-treatment with a general caspase inhibitor (z-VAD-fmk) prevented cytochrome c release. After 48 h of EEPP treatment, the apoptosis of Hep G2 cells was found to associate with an elevated p53, and CD95 and CD95L proteins expression. Furthermore, a marked down-regulation of the expression of the Bcl-2, Bcl-XL and XIAP, and up-regulation of the Bax and Bad proteins were noted. Taken together, the present results suggest that EEPP-induced Hep G2 cell apoptosis was possibly mediated through the CD95/CD95L system and the mitochondrial signaling transduction pathway.

A comparative immunogenicity study in rabbits of disulfide-stabilized, proteolytically cleaved, soluble trimeric human immunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140 and monomeric gp120

International Nuclear Information System (INIS)

Beddows, Simon; Franti, Michael; Dey, Antu K.; Kirschner, Marc; Iyer, Sai Prasad N.; Fisch, Danielle C.; Ketas, Thomas; Yuste, Eloisa; Desrosiers, Ronald C.; Klasse, Per Johan; Maddon, Paul J.; Olson, William C.; Moore, John P.

2007-01-01

The human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein (Env) complex, a homotrimer containing gp120 surface glycoprotein and gp41 transmembrane glycoprotein subunits, mediates the binding and fusion of the virus with susceptible target cells. The Env complex is the target for neutralizing antibodies (NAbs) and is the basis for vaccines intended to induce NAbs. Early generation vaccines based on monomeric gp120 subunits did not confer protection from infection; one alternative approach is therefore to make and evaluate soluble forms of the trimeric Env complex. We have directly compared the immunogenicity in rabbits of two forms of soluble trimeric Env and monomeric gp120 based on the sequence of HIV-1 JR-FL . Both protein-only and DNA-prime, protein-boost immunization formats were evaluated, DNA-priming having little or no influence on the outcome. One form of trimeric Env was made by disrupting the gp120-gp41 cleavage site by mutagenesis (gp140 UNC ), the other contains an intramolecular disulfide bond to stabilize the cleaved gp120 and gp41 moieties (SOSIP.R6 gp140). Among the three immunogens, SOSIP.R6 gp140 most frequently elicited neutralizing antibodies against the homologous, neutralization-resistant strain, HIV-1 JR-FL . All three proteins induced NAbs against more sensitive strains, but the breadth of activity against heterologous primary isolates was limited. When antibodies able to neutralize HIV-1 JR-FL were detected, antigen depletion studies showed they were not directed at the V3 region but were targeted at other, undefined gp120 and also non-gp120 epitopes

Increasing antiviral activity of surfactant protein d trimers by introducing residues from bovine serum collectins: dissociation of mannan-binding and antiviral activity

DEFF Research Database (Denmark)

Hartshorn, K L; White, M R; Smith, K

2010-01-01

Collectins contribute to host defence through interactions with glycoconjugates on pathogen surfaces. We have prepared recombinant trimeric neck and carbohydrate recognition domains (NCRD) of collectins, and we now show that the NCRD of bovine conglutinin and CL-46 (like that of CL-43) have greater...

Nanodiamond enhances immune responses in mice against recombinant HA/H7N9 protein.

Science.gov (United States)

Pham, Ngoc Bich; Ho, Thuong Thi; Nguyen, Giang Thu; Le, Thuy Thi; Le, Ngoc Thu; Chang, Huan-Cheng; Pham, Minh Dinh; Conrad, Udo; Chu, Ha Hoang

2017-10-05

The continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles. In this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization). These results

Anti-inflammatory and anti-thrombotic intervention strategies using atorvastatin, clopidogrel and knock-down of CD40L do not modify radiation-induced atherosclerosis in ApoE null mice

NARCIS (Netherlands)

Hoving, Saske; Heeneman, Sylvia; Gijbels, Marion J. J.; te Poele, Johannes A. M.; Pol, Jeffrey F. C.; Gabriels, Karen; Russell, Nicola S.; Daemen, Mat J. A. P.; Stewart, Fiona A.

2011-01-01

We previously showed that irradiating the carotid arteries of ApoE(-/-) mice accelerated the development of macrophage-rich, inflammatory and thrombotic atherosclerotic lesions. In this study we investigated the potential of anti-inflammatory (atorvastatin, CD40L knockout) and anti-thrombotic

Long-lived trimers in a quasi-two-dimensional Fermi system

Science.gov (United States)

Laird, Emma K.; Kirk, Thomas; Parish, Meera M.; Levinsen, Jesper

2018-04-01

We consider the problem of three distinguishable fermions confined to a quasi-two-dimensional (quasi-2D) geometry, where there is a strong harmonic potential in one direction. We go beyond previous theoretical work and investigate the three-body bound states (trimers) for the case where the two-body short-range interactions between fermions are unequal. Using the scattering parameters from experiments on ultracold 6Li atoms, we calculate the trimer spectrum throughout the crossover from two to three dimensions. We find that the deepest Efimov trimer in the 6Li system is unaffected by realistic quasi-2D confinements, while the first excited trimer smoothly evolves from a three-dimensional-like Efimov trimer to an extended 2D-like trimer as the attractive interactions are decreased. We furthermore compute the excited trimer wave function and quantify the stability of the trimer against decay into a dimer and an atom by determining the probability that three fermions approach each other at short distances. Our results indicate that the lifetime of the trimer can be enhanced by at least an order of magnitude in the quasi-2D geometry, thus opening the door to realizing long-lived trimers in three-component Fermi gases.

Effects of anti-CD40 mAb on inducing malignant B cells proliferation arrest and apoptosis and its mechanism

International Nuclear Information System (INIS)

Tang Lin; Zhuang Yumei; Zhou Zhaohua; Yu Gehua; Pan Jianzhong; Zhang Xueguang

2002-01-01

Objective: To study the expression of CD 40 molecule and the biological effects mediated by CD 40 molecules on malignant B cells. Methods: Agonistic anti-human CD 40 monoclonal antibody (clone 5C11) was added to cell culture system. Cell counting, PI staining, Annexin-V staining and flow cytometric analysis were used to study the behavior of malignant B cell lines after treatment with mAb clone 5C11. Results: 5C11 induced homotypic aggregation and proliferation arrest and mediated apoptosis in multiple myeloma cell line XG2 that expressed CD 40 strongly; 5C11 induced B lymphoma cell line Daudi homotypic aggregation and proliferation arrest and apoptosis, the apoptosis of XG2 and Daudi by CD40 activation was not mediated by TNF. Conclusion: Agonistic anti-CD 40 mAb 5C11 can inhibit the proliferation of malignant B cells by inducing them to die apoplectically

Production of recombinant proteins GST L1, E6 and E7 tag HPV 16 ...

African Journals Online (AJOL)

In the present work recombinant proteins were produced for used in LUMINEX in order to undergo serological study of Tunisian female population. HPV types 16 L1, E6 and E7 sequences fused to their 3'-end to a sequence encoding the terminal undecapeptide of the SV40 large T-antigen (tag) were isolated from plasmids ...

Immunogenicity of recombinant Lactobacillus plantarum NC8 expressing goose parvovirus VP2 gene in BALB/c mice.

Science.gov (United States)

Liu, Yu-Ying; Yang, Wen-Tao; Shi, Shao-Hua; Li, Ya-Jie; Zhao, Liang; Shi, Chun-Wei; Zhou, Fang-Yu; Jiang, Yan-Long; Hu, Jing-Tao; Gu, Wei; Yang, Gui-Lian; Wang, Chun-Feng

2017-06-30

Goose parvovirus (GPV) continues to be a threat to goose farms and has significant economic effects on the production of geese. Current commercially available vaccines only rarely prevent GPV infection. In our study, Lactobacillus (L.) plantarum NC8 was selected as a vector to express the VP2 gene of GPV, and recombinant L. plantarum pSIP409-VP2/NC8 was successfully constructed. The molecular weight of the expressed recombinant protein was approximately 70 kDa. Mice were immunized with a 2 × 10 9 colony-forming unit/200 μL dose of the recombinant L. plantarum strain, and the ratios and numbers of CD11c + , CD3 + CD4 + , CD3 + CD8 + , and interferon gamma- and tumor necrosis factor alpha-expressing spleen lymphocytes in the pSIP409-VP2/NC8 group were higher than those in the control groups. In addition, we assessed the capacity of L. plantarum SIP409-VP2/NC8 to induce secretory IgA production. We conclude that administered pSIP409-VP2/NC8 leads to relatively extensive cellular responses. This study provides information on GPV infection and offers a clear framework of options available for GPV control strategies.

Immunotoxicity assessment of rice-derived recombinant human serum albumin using human peripheral blood mononuclear cells.

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Kai Fu

Full Text Available Human serum albumin (HSA is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA before a First-in-human (FIH trial, we compared OsrHSA and plasma-derived HSA (pHSA, evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs. The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4(+ and CD8(+ T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ, tumor necrosis factor-alpha (TNF-α, interleukin (IL-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development.

Creation of Hybrid Nanorods From Sequences of Natural Trimeric Fibrous Proteins Using the Fibritin Trimerization Motif

Science.gov (United States)

Papanikolopoulou, Katerina; van Raaij, Mark J.; Mitraki, Anna

Stable, artificial fibrous proteins that can be functionalized open new avenues in fields such as bionanomaterials design and fiber engineering. An important source of inspiration for the creation of such proteins are natural fibrous proteins such as collagen, elastin, insect silks, and fibers from phages and viruses. The fibrous parts of this last class of proteins usually adopt trimeric, β-stranded structural folds and are appended to globular, receptor-binding domains. It has been recently shown that the globular domains are essential for correct folding and trimerization and can be successfully substituted by a very small (27-amino acid) trimerization motif from phage T4 fibritin. The hybrid proteins are correctly folded nanorods that can withstand extreme conditions. When the fibrous part derives from the adenovirus fiber shaft, different tissue-targeting specificities can be engineered into the hybrid proteins, which therefore can be used as gene therapy vectors. The integration of such stable nanorods in devices is also a big challenge in the field of biomechanical design. The fibritin foldon domain is a versatile trimerization motif and can be combined with a variety of fibrous motifs, such as coiled-coil, collagenous, and triple β-stranded motifs, provided the appropriate linkers are used. The combination of different motifs within the same fibrous molecule to create stable rods with multiple functions can even be envisioned. We provide a comprehensive overview of the experimental procedures used for designing, creating, and characterizing hybrid fibrous nanorods using the fibritin trimerization motif.

CD137 is induced by the CD40 signal on chronic lymphocytic leukemia B cells and transduces the survival signal via NF-κB activation.

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Yukana Nakaima

Full Text Available CD137 is a member of the tumor necrosis factor receptor family that is expressed on activated T cells. This molecule provides a co-stimulatory signal that enhances the survival, and differentiation of cells, and has a crucial role in the development of CD8 cytotoxic T cells and anti-tumor immunity. Here we report that CD137 expression is also induced on normal or malignant human B cells by CD40 ligation by its ligand CD154. This CD137 induction was more prominent in chronic lymphocytic leukemia (CLL cells than in other types of B cells. CD137 stimulation on B cells by its ligand induced the nuclear translocation of p52 (a non-canonical NF-κB factor. In agreement with this finding, expression of the survival factor BCL-XL was upregulated. Consequently, the CD137 signal augmented the survival of CD154-stimulated CLL B cells in vitro. This unexpected induction of CD137 on B cells by CD40 signal may influence the clinical course of CLL.

Differential requirements of CD4(+) T-cell signals for effector cytotoxic T-lymphocyte (CTL) priming and functional memory CTL development at higher CD8(+) T-cell precursor frequency.

Science.gov (United States)

Umeshappa, Channakeshava S; Nanjundappa, Roopa H; Xie, Yufeng; Freywald, Andrew; Xu, Qingyong; Xiang, Jim

2013-04-01

Increased CD8(+) T-cell precursor frequency (PF) precludes the requirement of CD4(+) helper T (Th) cells for primary CD8(+) cytotoxic T-lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) -pulsed dendritic cells (DC(OVA)) derived from C57BL/6, CD40 knockout (CD40(-/-)) or CD40 ligand knockout (CD40L(-/-)) mice were used to immunize C57BL/6, Ia(b-/-), CD40(-/-) or CD40L(-/-) mice, whose PF was previously increased with transfer of 1 × 10(6) CD8(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTI, OTI(CD40(-/-)) or OTI(CD40L(-/-)) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4(+) T-cell help and Th-provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA-expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4(+) T-cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4(+) T-cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4(+) T-cell functions. © 2012 Blackwell Publishing Ltd.

Optimization, purification and characterization of recombinant L ...

African Journals Online (AJOL)

We studied optimal L-asparaginase sequence from GenBank accession number X12746 encoding for Lasparaginase from Erwinia chrysanthemi NCPPB1125. The expression level of recombinant Lasparaginase was determined as 78% of the total proteins. The purified L-asparaginase had a molecular mass of 37 kDa with ...

Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae

International Nuclear Information System (INIS)

Gazdag, Emerich Mihai; Cirstea, Ion Cristian; Breitling, Reinhard; Lukeš, Julius; Blankenfeldt, Wulf; Alexandrov, Kirill

2010-01-01

The structures of two new crystal forms of human Cu/Zn superoxide dismutase produced in the eukaryotic expression host L. tarentolae are reported. The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30 mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5 mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research

Anti-inflammatory and anti-thrombotic intervention strategies using atorvastatin, clopidogrel and knock-down of CD40L do not modify radiation-induced atherosclerosis in ApoE null mice

Energy Technology Data Exchange (ETDEWEB)

Hoving, Saske [Division of Experimental Therapy, Netherlands Cancer Institute, Amsterdam (Netherlands); Heeneman, Sylvia [Department of Pathology, Cardiovascular Research Institute Maastricht (Netherlands); Gijbels, Marion J.J. [Department of Pathology, Cardiovascular Research Institute Maastricht (Netherlands); Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (Netherlands); Poele, Johannes A.M. te [Division of Experimental Therapy, Netherlands Cancer Institute, Amsterdam (Netherlands); Pol, Jeffrey F.C.; Gabriels, Karen [Department of Pathology, Cardiovascular Research Institute Maastricht (Netherlands); Russell, Nicola S [Division of Radiotherapy, Netherlands Cancer Institute, Amsterdam (Netherlands); Daemen, Mat J.A.P. [Department of Pathology, Cardiovascular Research Institute Maastricht (Netherlands); Department of Pathology, AMC, Amsterdam (Netherlands); Stewart, Fiona A., E-mail: f.stewart@nki.nl [Division of Experimental Therapy, Netherlands Cancer Institute, Amsterdam (Netherlands)

2011-10-15

Background and purpose: We previously showed that irradiating the carotid arteries of ApoE{sup -/-} mice accelerated the development of macrophage-rich, inflammatory and thrombotic atherosclerotic lesions. In this study we investigated the potential of anti-inflammatory (atorvastatin, CD40L knockout) and anti-thrombotic (clopidogrel) intervention strategies to inhibit radiation-induced atherosclerosis. Material and methods: ApoE{sup -/-} mice were given 0 or 14 Gy to the neck and the carotid arteries were harvested at 4 or 28 weeks after irradiation. Atorvastatin (15 mg/kg/day) or clopidogrel (20 mg/kg/day) was given in the chow; control groups received regular chow. Clopidogrel inhibited platelet aggregation by 50%. CD40L{sup -/-}/ApoE{sup -/-} and ApoE{sup -/-} littermates were also given 0 or 14 Gy to the neck and the carotid arteries were harvested after 30 weeks. Results: Clopidogrel decreased MCP-1 expression in the carotid artery at 4 weeks after irradiation. Expression of VCAM-1, ICAM-1, thrombomodulin, tissue factor and eNOS was unchanged in atorvastatin and clopidogrel-treated mice. Neither drug inhibited either age-related or radiation-induced atherosclerosis. Furthermore, loss of the inflammatory mediator CD40L did not influence the development of age-related and radiation-induced atherosclerosis. Conclusions: The effects of radiation-induced atherosclerosis could not be circumvented by these specific anti-inflammatory and anti-coagulant therapies. This suggests that more effective drug combinations may be required to overcome the radiation stimulus, or that other underlying mechanistic pathways are involved compared to age-related atherosclerosis.

Anti-inflammatory and anti-thrombotic intervention strategies using atorvastatin, clopidogrel and knock-down of CD40L do not modify radiation-induced atherosclerosis in ApoE null mice

International Nuclear Information System (INIS)

Hoving, Saske; Heeneman, Sylvia; Gijbels, Marion J.J.; Poele, Johannes A.M. te; Pol, Jeffrey F.C.; Gabriels, Karen; Russell, Nicola S.; Daemen, Mat J.A.P.; Stewart, Fiona A.

2011-01-01

Background and purpose: We previously showed that irradiating the carotid arteries of ApoE −/− mice accelerated the development of macrophage-rich, inflammatory and thrombotic atherosclerotic lesions. In this study we investigated the potential of anti-inflammatory (atorvastatin, CD40L knockout) and anti-thrombotic (clopidogrel) intervention strategies to inhibit radiation-induced atherosclerosis. Material and methods: ApoE −/− mice were given 0 or 14 Gy to the neck and the carotid arteries were harvested at 4 or 28 weeks after irradiation. Atorvastatin (15 mg/kg/day) or clopidogrel (20 mg/kg/day) was given in the chow; control groups received regular chow. Clopidogrel inhibited platelet aggregation by 50%. CD40L −/− /ApoE −/− and ApoE −/− littermates were also given 0 or 14 Gy to the neck and the carotid arteries were harvested after 30 weeks. Results: Clopidogrel decreased MCP-1 expression in the carotid artery at 4 weeks after irradiation. Expression of VCAM-1, ICAM-1, thrombomodulin, tissue factor and eNOS was unchanged in atorvastatin and clopidogrel-treated mice. Neither drug inhibited either age-related or radiation-induced atherosclerosis. Furthermore, loss of the inflammatory mediator CD40L did not influence the development of age-related and radiation-induced atherosclerosis. Conclusions: The effects of radiation-induced atherosclerosis could not be circumvented by these specific anti-inflammatory and anti-coagulant therapies. This suggests that more effective drug combinations may be required to overcome the radiation stimulus, or that other underlying mechanistic pathways are involved compared to age-related atherosclerosis.

Requirement of 8-mercaptoguanosine as a costimulus for IL-4-dependent μ to γ1 class switch recombination in CD38-activated B cells

International Nuclear Information System (INIS)

Tsukamoto, Yumiko; Uehara, Shoji; Mizoguchi, Chieko; Sato, Atsushi; Horikawa, Keisuke; Takatsu, Kiyoshi

2005-01-01

Mature B-2 cells expressing surface IgM and IgD proliferate upon stimulation by CD38, CD40 or lipopolysaccharide (LPS) and differentiate into IgG1-producing plasma cells in the presence of cytokines. The process of class switch recombination (CSR) from IgM to other isotypes is highly regulated by cytokines and activation-induced cytidine deaminase (AID). Blimp-1 and XBP-1 play an essential role in the terminal differentiation of switched B-2 cells to Ig-producing plasma cells. IL-5 induces AID and Blimp-1 expression in CD38- and CD40-activated B-2 cells, leading to μ to γ1 CSR at DNA level and IgG1 production. IL-4, a well-known IgG1-inducing factor, does not induce μ to γ1 CSR in CD38-activated B-2 cells or Blimp-1, while IL-4 induces μ to γ1 CSR, XBP-1 expression, and IgG1 production expression in CD40-activated B-2 cells. Interestingly, the addition of 8-mercaptoguanosine (8-SGuo) with IL-4 to the culture of CD38-activated B cells can induce μ to γ1 CSR, Blimp-1 expression, and IgG1 production. Intriguingly, 8-SGuo by itself induces AID expression in CD38-activated B cells. However, it does not induce μ to γ1 CSR. These results imply that the mode of B-cell activation for extracellular stimulation affects the outcome of cytokine stimulation with respect to the efficiency and direction of CSR, and the requirements of the transcriptional regulator and the generation of antibody-secreting cells. Furthermore, our data suggest the requirement of additional molecules in addition to AID for CSR

Bioavailability of Cd in 110 polluted topsoils to recombinant bioluminescent sensor bacteria. Effect of soil particulate matter

Energy Technology Data Exchange (ETDEWEB)

Ivask, Angela; Pollumaa, Lee; Kahru, Anne [National Inst. of Chemical Physics and Biophysics, Lab. of Molecular Genetics, Tallin (Estonia); Dubourguier, Henri-Charles [National Inst. of Chemical Physics and Biophysics, Lab. of Molecular Genetics, Tallin (Estonia); Estonian Univ. of Life Sciences, Tartu (Estonia); Inst. Superieur d' Agriculture, Lille (France)

2011-02-15

In this study, bioavailability and water extractability of Cd in a panel of 110 natural aged heavy metal-polluted soils from northern France containing up to 20.1 mg of Cd per kilogramme was evaluated. Materials and methods Particulate matter was removed by differential centrifugation of soil-water suspensions (liquid to solid ratio 10) resulting in soil-water extracts containing different size of particles. Chemical analysis of Cd and analysis of bioavailable Cd with recombinant bioluminescent Cd-sensing bacteria were applied in parallel to these fractionated soil solutions. Results and Discussion Extractability of Cd from soil to the aqueous phase was low-only 0.13% of the soil total Cd as a mean; however, Cd-sensing recombinant luminescent bacteria Bacillus subtilis incubated in soil-water suspensions for 2 h showed that in the conditions of contact exposure, the bioavailable fraction of Cd increased about 30-fold being 3.74% of the soil total Cd as a mean value. The total Cd content of soils was not a good predictor of either bioavailable or water-extracted fraction of Cd, but these fractions were rather determined by the combination of soil total Cd and physico-chemical properties-texture and organic matter content. Analysis of two selected ''model'' soils with Cd sensor bacteria showed that about 90% of the bioavailable Cd was associated with larger soil particles that were removed from the soil suspensions by centrifugation at 4,500 x g, and even settling of the soil suspensions for 2 h removed already 65% of bioavailable Cd. Conclusions Thus, our results indicate a potential for remarkably higher environmental hazard for soil-associated heavy metals than just aqueous exposure. (orig.)

Inhibition of the recombinant human endostatin adenavirus on experimental choroidal neovascularization in rats

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Li-Juan Chen

2017-06-01

Full Text Available AIM: To investigate the inhibition of the recombinant human endostatin adenavirus(Ad-Eson the experimental choroidal neovascularization(CNVmodels by intravitreous injection. METHODS: Experimental CNV models were induced by semiconductor laser in 30 male Brown Norway(BNrats and randomly divided into 3 groups with 10 rats in each group. At 21d after photocoagulation, the single administration group were given intravitreous injection with Ad-Es 0.01mL; the repeated administration group were given intravitreous injection with Ad-Es 0.01mL and a repeated injection 7d later; the saline control group were given intravitreous injection with saline 0.01mL. At 7d after final administration, the leakage of fundus fluorescein angiography(FFAwas observed. Various CNV areas were measured by using laser confocal microscopy of choroidal flatmount method. Pathology and ultrastructure were observed with light microscopy, the expressions of CD105 were measured by immunohistochemistry. RESULTS: The leakage of CNV of the administration group abviously decreased as compared with those in the saline group, the leakage of repeated administration group decreased compared with that of single administration group(PPCONCLUSION: Ad-Es can effectively inhibit semiconductor laser induced CNV in BN rats, and the inhibition effect of repeated administration group is better than that of single administration group. It may be a useful new method in the treatment of CNV.

Recombinant human bone morphogenetic protein induces bone formation

International Nuclear Information System (INIS)

Wang, E.A.; Rosen, V.; D'Alessandro, J.S.; Bauduy, M.; Cordes, P.; Harada, T.; Israel, D.I.; Hewick, R.M.; Kerns, K.M.; LaPan, P.; Luxenberg, D.P.; McQuaid, D.; Moutsatsos, I.K.; Nove, J.; Wozney, J.M.

1990-01-01

The authors have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 μg of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans

Structure of Simian Immunodeficiency Virus Envelope Spikes Bound with CD4 and Monoclonal Antibody 36D5.

Science.gov (United States)

Hu, Guiqing; Liu, Jun; Roux, Kenneth H; Taylor, Kenneth A

2017-08-15

The human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) envelope spike (Env) mediates viral entry into host cells. The V3 loop of the gp120 component of the Env trimer contributes to the coreceptor binding site and is a target for neutralizing antibodies. We used cryo-electron tomography to visualize the binding of CD4 and the V3 loop monoclonal antibody (MAb) 36D5 to gp120 of the SIV Env trimer. Our results show that 36D5 binds gp120 at the base of the V3 loop and suggest that the antibody exerts its neutralization effect by blocking the coreceptor binding site. The antibody does this without altering the dynamics of the spike motion between closed and open states when CD4 is bound. The interaction between 36D5 and SIV gp120 is similar to the interaction between some broadly neutralizing anti-V3 loop antibodies and HIV-1 gp120. Two conformations of gp120 bound with CD4 are revealed, suggesting an intrinsic dynamic nature of the liganded Env trimer. CD4 binding substantially increases the binding of 36D5 to gp120 in the intact Env trimer, consistent with CD4-induced changes in the conformation of gp120 and the antibody binding site. Binding by MAb 36D5 does not substantially alter the proportions of the two CD4-bound conformations. The position of MAb 36D5 at the V3 base changes little between conformations, indicating that the V3 base serves as a pivot point during the transition between these two states. IMPORTANCE Glycoprotein spikes on the surfaces of SIV and HIV are the sole targets available to the immune system for antibody neutralization. Spikes evade the immune system by a combination of a thick layer of polysaccharide on the surface (the glycan shield) and movement between spike domains that masks the epitope conformation. Using SIV virions whose spikes were "decorated" with the primary cellular receptor (CD4) and an antibody (36D5) at part of the coreceptor binding site, we visualized multiple conformations trapped by the

Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate.

Science.gov (United States)

Visciano, Maria Luisa; Tagliamonte, Maria; Stewart-Jones, Guillaume; Heyndrickx, Leo; Vanham, Guido; Jansson, Marianne; Fomsgaard, Anders; Grevstad, Berit; Ramaswamy, Meghna; Buonaguro, Franco M; Tornesello, Maria Lina; Biswas, Priscilla; Scarlatti, Gabriella; Buonaguro, Luigi

2013-07-08

Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018). Antibodies elicited in immunized rabbits show a broad binding pattern to HIV envelopes of different clades. An epitope mapping analysis reveals that, on average, the binding is mostly focused on the C1, C2, V3, V5 and C5 regions. Immune sera show neutralization activity to Tier 1 isolates of different clades, demonstrating cross clade neutralizing activity which needs to be further broadened by possible structural modifications of the clade A gp14094UG018. Our results provide a rationale for the design and evaluation of immunogens and the clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV vaccine with broad activity.

Distribution of 131I-labeled recombinant human erythropoietin in maternal and fetal organs following intravenous administration in pregnant rats

International Nuclear Information System (INIS)

Yilmaz, O.; Lambrecht, F.Y.; Durkan, K.; Gokmen, N.; Erbayraktar, S.

2007-01-01

The aim of the present study was to demonstrate the possible transplacental transmission of 131 I labeled recombinant human erythropoietin ( 131 I-rh-EPO) in pregnant rats and its distribution through maternal and fetal organs. Six Wistar Albino Rats in their pregnancy of 18 days were used 131 I labeled recombinant human erythropoietin (specific activity = 2.4 μCi/IU) was injected into the tail vein of rats. After 30 minutes labeled erythropoietin infusion maternal stomach, kidney, lung, liver, brain and heart as well as fetus were removed. Then, the same organs were removed from each fetus. Measuring weight of maternal and fetal organs as well as placenta were followed by radioactivity count via Cd(Te) detector. 131 I labeled recombinant human erythropoietin was found to be able to pass rat placenta and its distribution order in fetal organs was similar to those of maternal organs. Besides, as measurements were performed closer to cornu uteri, uptakes were decreasing in every fetus and its corresponding placenta. (author)

Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential.

Science.gov (United States)

Yu, Yuan; Li, Jialu; Zhu, Xuejun; Tang, Xiaowen; Bao, Yangyi; Sun, Xiang; Huang, Yuhui; Tian, Fang; Liu, Xiaomei; Yang, Lin

2017-01-01

Nanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies]), are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6) as well as further truncated the Pseudomonas exotoxin A (PE)-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II. Three new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdc em26 Il2rg em26 Nju (NCG) mice

Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

Directory of Open Access Journals (Sweden)

Bruno Gottstein

2014-06-01

Full Text Available Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2 and cathepsin L-1 (recCL1, were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES from adult stage liver flukes was assessed by receiver operator characteristic (ROC analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20, patients with other parasitic infections (n=87 and patients with malignancies (n=121. The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy employing the threshold (cut-off to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

Monocyte CD64 or CD89 targeting by surfactant protein D/anti-Fc receptor mediates bacterial uptake.

NARCIS (Netherlands)

Tacken, P.J.; Batenburg, J.J.

2006-01-01

We recently showed that a chimeric protein, consisting of a recombinant fragment of human surfactant protein D (rfSP-D) coupled to a Fab' fragment directed against the human Fcalpha receptor (CD89), effectively targets pathogens recognized by SP-D to human neutrophils. The present study evaluates

Genetically Modified Lactococcus lactis for Delivery of Human Interleukin-10 to Dendritic Cells

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Inge L. Huibregtse

2012-01-01

Full Text Available Interleukin-10 (IL-10 plays an indispensable role in mucosal tolerance by programming dendritic cells (DCs to induce suppressor Th-cells. We have tested the modulating effect of L. lactis secreting human IL-10 (L.  lactisIL-10 on DC function in vitro. Monocyte-derived DC incubated with L.  lactisIL-10 induced effector Th-cells that markedly suppressed the proliferation of allogenic Th-cells as compared to L. lactis. This suppressive effect was only seen when DC showed increased CD83 and CD86 expression. Furthermore, enhanced production of IL-10 was measured in both L.  lactisIL-10-derived DC and Th-cells compared to L. lactis-derived DC and Th-cells. Neutralizing IL-10 during DC-Th-cell interaction and coculturing L.  lactisIL-10-derived suppressor Th-cells with allogenic Th-cells in a transwell system prevented the induction of suppressor Th-cells. Only 130 pg/mL of bacterial-derived IL-10 and 40 times more exogenously added recombinant human IL-10 were needed during DC priming for the generation of suppressor Th-cells. The spatially restricted delivery of IL-10 by food-grade bacteria is a promising strategy to induce suppressor Th-cells in vivo and to treat inflammatory diseases.

Human CD134 (OX40) expressed on T cells plays a key role for human herpesvirus 6B replication after allogeneic hematopoietic stem cell transplantation.

Science.gov (United States)

Nagamata, Satoshi; Nagasaka, Miwako; Kawabata, Akiko; Kishimoto, Kenji; Hasegawa, Daiichiro; Kosaka, Yoshiyuki; Mori, Takeshi; Morioka, Ichiro; Nishimura, Noriyuki; Iijima, Kazumoto; Yamada, Hideto; Kawamoto, Shinichiro; Yakushijin, Kimikazu; Matsuoka, Hiroshi; Mori, Yasuko

2018-05-01

CD134 (OX40), which is a cellular receptor for human herpesvirus-6B (HHV-6B) and expresses on activated T cells, may play a key role for HHV-6B replication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Therefore, we examined the CD134 expression on T cells and HHV-6B replication after allo-HSCT, and analyzed the correlation between them. Twenty-three patients after allo-HSCT were enrolled. The percentages of CD134-positive cells within the CD4 + and CD8 + cell populations were measured by flow cytometry, and the viral copy number of HHV-6B was simultaneously quantified by real-time PCR. The correlation between CD134 and HHV-6B viral load was then statistically analyzed. HHV-6B reactivation occurred in 11 of 23 patients (47.8%). CD134 expression was seen on T cells and was coincident with the time of peak viral load. The percentage of CD134-positive cells decreased significantly when HHV-6B DNA disappeared (p = .005 in CD4 + T cells, p = .02 in CD8 + T cells). In the 4 patients who underwent umbilical cord blood transplantation (UCBT), the viral load varied with the percentage of CD134-positive cells. In the comparison between the HHV-6B reactivation group and non-reactivation group, maximum percentages of CD134-positive cells among CD4 + T cells in reactivation group were significantly higher than those in non-reactivation group (p = .04). This is the first study to show that a correlation of CD134 expression on T cells with HHV-6B replication after allo-HSCT, especially in UCBT. The results possibly indicate that CD134 on T cells plays a key role for HHV-6B replication after allo-HSCT. Copyright © 2018 Elsevier B.V. All rights reserved.

Loss of CD34 expression in aging human choriocapillaris endothelial cells.

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Elliott H Sohn

Full Text Available Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD. In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40, age-matched controls (> age 60 without AMD, or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization. Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10(-6. Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse or a trend toward decreased CD34 (human in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.

Reactive glia promote development of CD103+ CD69+ CD8+ T-cells through programmed cell death-ligand 1 (PD-L1).

Science.gov (United States)

Prasad, Sujata; Hu, Shuxian; Sheng, Wen S; Chauhan, Priyanka; Lokensgard, James R

2018-06-01

Previous work from our laboratory has demonstrated in vivo persistence of CD103 + CD69 + brain resident memory CD8 + T-cells (bT RM ) following viral infection, and that the PD-1: PD-L1 pathway promotes development of these T RM cells within the brain. Although glial cells express low basal levels of PD-L1, its expression is upregulated upon IFN-γ-treatment, and they have been shown to modulate antiviral T-cell effector responses through the PD-1: PD-L1 pathway. We performed flow cytometric analysis of cells from co-cultures of mixed glia and CD8 + T-cells obtained from wild type mice to investigate the role of glial cells in the development of bT RM . In this study, we show that interactions between reactive glia and anti-CD3 Ab-stimulated CD8 + T-cells promote development of CD103 + CD69 + CD8 + T-cells through engagement of the PD-1: PD-L1 pathway. These studies used co-cultures of primary murine glial cells obtained from WT animals along with CD8 + T-cells obtained from either WT or PD-1 KO mice. We found that αCD3 Ab-stimulated CD8 + T-cells from WT animals increased expression of CD103 and CD69 when co-cultured with primary murine glial cells. In contrast, significantly reduced expression of CD103 and CD69 was observed using CD8 + T-cells from PD-1 KO mice. We also observed that reactive glia promoted high levels of CD127, a marker of memory precursor effector cells (MPEC), on CD69 + CD8 + T-cells, which promotes development of T RM cells. Interestingly, results obtained using T-cells from PD-1 KO animals showed significantly reduced expression of CD127 on CD69 + CD8 + cells. Additionally, blocking of glial PD-L1 resulted in decreased expression of CD103, along with reduced CD127 on CD69 + CD8 + T-cells. Taken together, these results demonstrate a role for activated glia in promoting development of bT RM through the PD-1: PD-L1 pathway. © 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

Flow Cytometry Assessment of In Vitro Generated CD138+ Human Plasma Cells

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Rayelle Itoua Maïga

2014-01-01

Full Text Available The in vitro CD40-CD154 interaction promotes human B lymphocytes differentiation into plasma cells. Currently, CD138 is the hallmark marker enabling the detection of human plasma cells, both in vitro and in vivo; its presence can be monitored by flow cytometry using a specific antibody. We have developed a culture system allowing for the differentiation of memory B lymphocytes. In order to detect the newly formed plasma cells, we have compared their staining using five anti-CD138 monoclonal antibodies (mAbs. As a reference, we also tested human cell lines, peripheral blood mononuclear cells, and bone marrow samples. The five anti-CD138 mAbs stained RPMI-8226 cells (>98% with variable stain index (SI. The highest SI was obtained with B-A38 mAb while the lowest SI was obtained with DL-101 and 1D4 mAbs. However, the anti-CD138 mAbs were not showing equivalent CD138+ cells frequencies within the generated plasma cells. B-A38, B-B4, and MI-15 were similar (15–25% while DL-101 mAb stained a higher proportion of CD138-positive cells (38–42%. DL-101 and B-A38 mAbs stained similar populations in bone marrow samples but differed in their capacity to bind to CD138high and CD138lo cell lines. In conclusion, such cellular fluctuations suggest heterogeneity in human plasma cell populations and/or in CD138 molecules.

Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

Science.gov (United States)

Ho, Hsu-Tso; Fan, Li; Nowicka-Sans, Beata; McAuliffe, Brian; Li, Chang-Ben; Yamanaka, Gregory; Zhou, Nannan; Fang, Hua; Dicker, Ira; Dalterio, Richard; Gong, Yi-Fei; Wang, Tao; Yin, Zhiwei; Ueda, Yasutsugu; Matiskella, John; Kadow, John; Clapham, Paul; Robinson, James; Colonno, Richard; Lin, Pin-Fang

2006-04-01

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

IL-4 and IL-13 mediated down-regulation of CD8 expression levels can dampen anti-viral CD8⁺ T cell avidity following HIV-1 recombinant pox viral vaccination.

Science.gov (United States)

Wijesundara, Danushka K; Jackson, Ronald J; Tscharke, David C; Ranasinghe, Charani

2013-09-23

We have shown that mucosal HIV-1 recombinant pox viral vaccination can induce high, avidity HIV-specific CD8(+) T cells with reduced interleukin (IL)-4 and IL-13 expression compared to, systemic vaccine delivery. In the current study how these cytokines act to regulate anti-viral CD8(+) T, cell avidity following HIV-1 recombinant pox viral prime-boost vaccination was investigated. Out of a panel of T cell avidity markers tested, only CD8 expression levels were found to be enhanced on, KdGag197-205 (HIV)-specific CD8(+) T cells obtained from IL-13(-/-), IL-4(-/-) and signal transducer and, activator of transcription of 6 (STAT6)(-/-) mice compared to wild-type (WT) controls following, vaccination. Elevated CD8 expression levels in this instance also correlated with polyfunctionality, (interferon (IFN)-γ, tumour necorsis factor (TNF)-α and IL-2 production) and the avidity of HIVspecific CD8(+) T cells. Furthermore, mucosal vaccination and vaccination with the novel adjuvanted IL-13 inhibitor (i.e. IL-13Rα2) vaccines significantly enhanced CD8 expression levels on HIV-specific CD8(+), T cells, which correlated with avidity. Using anti-CD8 antibodies that blocked CD8 availability on CD8(+), T cells, it was established that CD8 played an important role in increasing HIV-specific CD8(+) T cell avidity and polyfunctionality in IL-4(-/-), IL-13(-/-) and STAT6(-/-) mice compared to WT controls, following vaccination. Collectively, our data demonstrate that IL-4 and IL-13 dampen CD8 expression levels on anti-viral CD8(+) T cells, which can down-regulate anti-viral CD8(+) T cell avidity and, polyfunctionality following HIV-1 recombinant pox viral vaccination. These findings can be exploited to, design more efficacious vaccines not only against HIV-1, but many chronic infections where high, avidity CD8(+) T cells help protection. Copyright © 2013 Elsevier Ltd. All rights reserved.

Anti-tumor effect of a recombinant plasmid expressing human interleukin-12: an initial research

International Nuclear Information System (INIS)

Zheng Chuansheng; Xia Xiangwen; Feng Gansheng; Li Xin; Liang Huimin; Liang Bin

2010-01-01

Objective: To study the anti-tumor effect of a recombinant plasmid expressing human interleukin-12 (pEGFP-CI I L- 12) in vivo and in vitro. Methods: We transduct the recombinant gene (pEGFP-CI I L-12) to liver cancer cell HepG 2 in vitro, and detect reproductive activity of the cell using MTT and the activity of expressing vascular endothelial growth factor(VEGF) using semiquantitative PCR. And then, we deliver the gene to rabbit liver tumor tissue intraarterial and combine with chemoembolization to observe the anti- tumor effect to VX 2 tumor in vivo. Results: There are no statistical difference compared With control group in activity of reproductive and expressing VEGF in vitro. In vivo, tumor growth rate significantly reduce in gene therapy combined with chemoembolization group. Conclusion: Recombinant gene (pEGFP-Cl I L-12) exhibit significant anti-tumor effect in vivo but not in vitro, perhaps the anti-tumor effect is associated with an indirect pathway instead of a direct pathway. (authors)

Inhibition of PAF-induced expression of CD11b and shedding of L-selectin on human neutrophils and eosinophils by the type IV selective PDE inhibitor, rolipram

NARCIS (Netherlands)

Dijkhuizen, B; deMonchy, JGR; Dubois, AEJ; Gerritsen, J; Kauffman, HF

We quantitatively determined whether the selective phosphodiesterase (PDE) inhibitor, rolipram, inhibits changes in the adhesion molecules CD11b and L-selectin on platelet-activating factor (PAF)-stimulated human neutrophils and eosinophils in vitro. Incubations were performed in human whole blood

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease.

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Mareike Florek

Full Text Available The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs in murine models of allogeneic hematopoietic cell transplantation (HCT has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L, which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.

Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

Science.gov (United States)

2016-08-01

RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI 1. INTRODUCTION 1.1 Background Vaccinia virus (VACV) is the active component of the...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in...PURIFICATION AND CHARACTERIZATION OF RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI ECBC-TR-1370

Affinity purification of recombinant human plasminogen activator ...

African Journals Online (AJOL)

Affinity purification of recombinant human plasminogen activator from ... Screening antibody was performed using rhPA milk in an ELISA-elution assay. ... useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

L-shell dielectronic recombination for 0-like ions

International Nuclear Information System (INIS)

Omar, G.; Semedal, R.; Shahin, F.; El-Sherbini, T.H.

2007-01-01

In electron-Ion (e/I) collisions, a free electron may be captured by a positively charged ion having bound electrons with a simultaneous, excitation. This radiationless capture, most probably, creates resonance (d) states. These d- states may stabilize by emission of radiation (x-rays). This two-step process is known as Dielectronic recombination (D R). At high incident-electron energy, D R dominates over the radiative recombination (R R) and three- body recombination (Tbr) processes. Thus, D R is one of the most effective recombination for ionisation balance in solar corona and artificial plasma . In addition, the D R rates are needed for the development of nuclear fusion plasma. Thus, D R is still an interesting process m both experimental and theoretical research. Previously we have done the D R rates for 0-like AL 5+ , Cl 9+ Ti 14+ and Zn 22+ ions with K-shell excitation. In this work, the D R rates are calculated for the same ions, but with L-shell excitation. It is found that, the peak values of the D R rates for L-shell excitation are 1000 times larger than that for K-shell excitation. This means that, D R process is the most efficient mechanism for outer-shell excitations. It is found also that, the Dr rates for L-shell excitation peak at Kt = 6 Ry, 22 Ry for AL 5+ and Ti 14+ ions respectively. These Kt values are much smaller than that for for K-shell excitation

eCD4-Ig promotes ADCC activity of sera from HIV-1-infected patients.

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Meredith E Davis-Gardner

2017-12-01

Full Text Available Antibody-dependent cell-mediated cytotoxity (ADCC can eliminate HIV-1 infected cells, and may help reduce the reservoir of latent virus in infected patients. Sera of HIV-1 positive individuals include a number of antibodies that recognize epitopes usually occluded on HIV-1 envelope glycoprotein (Env trimers. We have recently described eCD4-Ig, a potent and exceptionally broad inhibitor of HIV-1 entry that can be used to protect rhesus macaques from multiple high-dose challenges with simian-human immunodeficiency virus AD8 (SHIV-AD8. Here we show that eCD4-Ig bearing an IgG1 Fc domain (eCD4-IgG1 can mediate efficient ADCC activity against HIV-1 isolates with differing tropisms, and that it does so at least 10-fold more efficiently than CD4-Ig, even when more CD4-Ig molecules bound cell surface-expressed Env. An ADCC-inactive IgG2 form of eCD4-Ig (eCD4-IgG2 exposes V3-loop and CD4-induced epitopes on cell-expressed trimers, and renders HIV-1-infected cells susceptible to ADCC mediated by antibodies of these classes. Moreover, eCD4-IgG2, but not IgG2 forms of the broadly neutralizing antibodies VRC01 and 10-1074, enhances the ADCC activities of serum antibodies from patients by 100-fold, and significantly enhanced killing of two latently infected T-cell lines reactivated by vorinostat or TNFα. Thus eCD4-Ig is qualitatively different from CD4-Ig or neutralizing antibodies in its ability to mediate ADCC, and it may be uniquely useful in treating HIV-1 infection or reducing the reservoir of latently infected cells.

Efimov trimers in a harmonic potential and universality in three-body recombination

NARCIS (Netherlands)

Kokkelmans, S.J.J.M.F.; Portegies, J.W.; Gross, N.; Shotan, Z.; Khaykovich, L.

2009-01-01

We report on experimental evidence of universality in ultracold 7Li atoms’three-body recombination loss in the vicinity of a Feshbach resonance [1]. We observe a recombination minimum and an Efimov resonance in regions of positive and negative scattering lengths. Both observed features lie deeply

CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

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Shweta Jain

Full Text Available Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID, signifying class switch recombination (CSR. Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

Science.gov (United States)

Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

2011-01-01

Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

Recombinant human granulocyte colony-stimulating factor increases circulating CD34-postive cells in patients with AIDS

DEFF Research Database (Denmark)

Nielsen, S D; Dam-Larsen, S; Nielsen, C

1997-01-01

G-CSF for 3-5 consecutive days. Within 5 days of initiation of G-CSF therapy, an increase in the absolute neutrophil count (ANC) was seen in all patients. There was a median increase in ANC from 0.4 to 3.4 x 10(9)/l. In addition, G-CSF treatment significantly increased the absolute number of CD34...

Studies on mechanism of treatment of granulocyte colony-stimulating factor, recombinant human interleukin-11 and recombinant human interleukin-2 on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

International Nuclear Information System (INIS)

Li Ming; Ou Hongling; Xing Shuang; Huang Haixiao; Xiong Guolin; Xie Ling; Zhao Yanfang; Zhao Zhenhu; Wang Ning; Wang Jinxiang; Miao Jingcheng; Zhu Nankang; Luo Qingliang; Cong Yuwen; Zhang Xueguang

2010-01-01

Objective: To investigate the mechanism of treatment of granulocyte colony-stimulating factor (rhG-CSF), recombinant human interleukin-11 (rhIL-11) and recombinant human interleukin-2 (rhIL-2) on hematopoietic injuries induced by 4.5 Gy 60 Co γ-ray irradiation in beagles, and to provide experimental evidence for the clinical treatment of extremely severe myeloid acute radiation sickness (ARS). Methods: Sixteen beagle dogs were given 4.5 Gy 60 Co γ-ray total body irradiation (TBI), then randomly assigned into irradiation control group, supportive care group or cytokines + supportive care (abbreviated as cytokines) group. In addition to supportive care, rhG-CSF, rhIL-11 and rhIL-2 were administered subcutaneously to treat dogs in cytokines group. The percentage of CD34 + cells, cell cycle and apoptosis of nucleated cells in peripheral blood were examined by Flow cytometry. Results: After 4.5 Gy 60 Co γ-ray irradiation, the CD34 + cells in peripheral blood declined obviously (61.3% and 52.1% of baseline for irradiation control and supportive care group separately). The cell proportion of nucleated cells in G 0 /G 1 phase was increased notably notably (99.27% and 99.49% respectively). The rate of apoptosis (26.93% and 21.29% separately) and necrosis (3.27% and 4.14%, respectively) of nucleated cells were elevated significantly when compared with values before irradiation (P 0 /G 1 phase blockage of nucleated cells became more serious (99.71%). The rate of apoptosis (5.66%) and necrosis (1.60%) of nucleated cells were significantly lower than that of irradiation control and supportive care groups 1 d after exposure. Conclusions: Cytokines maybe mobilize CD34 + cells in bone marrow to peripheral blood, indce cell block at G 0 /G 1 phase and reduce apoptosis, and eventually cure hematopoietic injuries induced by irradiation. (authors)

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

Science.gov (United States)

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

2017-01-01

Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range of human tumor cells, and clinical studies have demonstrated anti-tumor activity versus a range of human cancers. This study was designed to investigate the effect on immune cell subsets in the peripheral blood of cancer patients prior to and following multiple administrations of avelumab. One hundred twenty-three distinct immune cell subsets in the peripheral blood of cancer patients ( n  = 28) in a phase I trial were analyzed by flow cytometry prior to and following one, three, and nine cycles of avelumab. Changes in soluble (s) CD27 and sCD40L in plasma were also evaluated. In vitro studies were also performed to determine if avelumab would mediate ADCC of PBMC. No statistically significant changes in any of the 123 immune cell subsets analyzed were observed at any dose level, or number of doses, of avelumab. Increases in the ratio of sCD27:sCD40L were observed, suggesting potential immune activation. Controlled in vitro studies also showed lysis of tumor cells by avelumab versus no lysis of PBMC from five donors. These studies demonstrate the lack of any significant effect on multiple immune cell subsets, even those expressing PD-L1, following multiple cycles of avelumab. These results complement prior studies showing anti-tumor effects of avelumab and comparable levels of adverse events with avelumab versus other anti-PD-1/PD-L1 MAbs. These studies provide the rationale to further exploit the potential ADCC mechanism of action of avelumab as well as other human IgG1 checkpoint

Vaccination with recombinant L7/L12-truncated Omp31 protein induces protection against Brucella infection in BALB/c mice.

Science.gov (United States)

Golshani, Maryam; Rafati, Sima; Dashti, Amir; Gholami, Elham; Siadat, Seyed Davar; Oloomi, Mana; Jafari, Anis; Bouzari, Saeid

2015-06-01

Brucellosis is the most common bacterial zoonotic disease worldwide and no vaccine is available for the prevention of human brucellosis. In humans, brucellosis is mostly caused by Brucella melitensis and Brucella abortus. The Outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens. In the present study, we evaluated the humoral and cellular immune responses induced by a fusion protein designed based on the Truncated form of Omp31 (TOmp31) and L7-L12 antigens. Vaccination of BALB/c mice with the recombinant fusion protein (rL7/L12-TOmp31) provided the significant protection level against B. melitensis and B. abortus challenge. Moreover, rL7/L12-TOmp31 elicited a strong specific IgG response (higher IgG2a titers) and significant IFN-γ/IL2 production and T-cell proliferation was also observed. The T helper1 (Th1) oriented response persisted for 12 weeks after the first immunization. The rL7/L12-TOmp31 could be a new potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nature of radiative recombination processes in layered semiconductor PbCdI{sub 2} nanostructural scintillation material

Energy Technology Data Exchange (ETDEWEB)

Bukivskii, A.P. [Institute of Physics of the National Academy of Sciences of Ukraine, 03028 Kyiv (Ukraine); Gnatenko, Yu.P., E-mail: yuriygnatenko@ukr.net [Institute of Physics of the National Academy of Sciences of Ukraine, 03028 Kyiv (Ukraine); Piryatinskii, Yu.P. [Institute of Physics of the National Academy of Sciences of Ukraine, 03028 Kyiv (Ukraine); Gamernyk, R.V. [Lviv National University, 8 Kyryl o and Mefodiy Str., 29005 Lviv (Ukraine)

2017-05-15

We report on the efficient photoluminescence (PL) and radioluminescence (RL) of the PbI{sub 2} nanoclusters (NCLs), which are naturally formed in the nanostructured Pb{sub 1-X}Cd{sub x}I{sub 2} alloys (X=0.70). Here, we carried out the studies of the nature of radiative recombination processes in the NCLs of various sizes by measuring PL temperature evolution. Our results indicate that at low temperatures the PL is mainly caused by exciton emission and recombination of donor-acceptor pairs, generated in volume of large NCLs. The broad bands, which are associated with the deep intrinsic surface states, including self-trapped excitons (STEs), are dominant in the PL spectra at higher temperature (>100 K). Our work shows that the nature of emission, associated with RL bands is analogous to that for PL bands. It was shown that the investigated nanostructured material is strongly radiation-resistant. Thus, the Pb{sub 1-X}Cd{sub X}I{sub 2} alloys can be considered as new effective layered semiconductor nanostructured materials which can be suitable for the elaboration of perspective semiconductor scintillators. These nanomaterials have promising prospects for applications in new generations of devices for biomedical diagnostics and industrial imaging applications. - Highlights: •The intense PL and RL of nanostructural PbCdI{sub 2} alloys were observed. •The nature of recombination processes of the nanoscintillators was established. •The low temperature PL is caused by exciton and donor-acceptor pairs recombination. •The broad PL bands are due to the deep intrinsic states formed on the NCLs surface. •The PL associated with STEs for NCLs of different sizes was analyzed in detail. •It was shown that the nature of PL and RL spectra is same.

The expression analysis of ICOS-L on activated T cells and immature dendritic cells as well as malignant B cells and Grave's-disease-derived thyroid tissues by two novel mAbs against human ICOS-L.

Science.gov (United States)

Wang, F; Zhu, W; Liu, T; Sun, Z; Ju, S; Ju, S; Yu, G; Xie, W; Deng, Z; Lu, B; Zhang, X

2007-01-01

ICOS-L, a newly identified member of B7 superfamily, plays an important role in immune responses. In this article, we report on two novel mouse anti-human ICOS-L monoclonal antibodies (mAbs) named as 11C4 and 12B11, whose specificities were verified by methods of flow cytometry, western blotting, and epitope competition assay. The two mAbs bound to distinct ICOS-L epitopes on B cells. Interestingly, mAb 11C4 could well recognize ICOS-L molecule on activated T cells and Jurkat cell lines, which is different from commercial anti-ICOS-L mAb (clone number MIH12) and the other mAb 12B11. In addition, we found that the expression of ICOS-L molecule was only detected on the surface of immature monocyte-derived dendritic cells (Mo-DCs) and was sharply decreased after induction of mature Mo-DCs activated by tumor necrosis factor-alpha or CD40. Furthermore, we showed that 11C4 could effectively suppress the maturation of Mo-DCs in vitro as evidenced by the low expression of CD80, CD86, CD83, and human leukocyte antigen-DR, which suggested that ICOS-L may be involved in the maturation of Mo-DCs. Using immunohistochemistry staining with mAb 11C4, the expression of ICOS-L was found in B lymphoma tissues and thyroid tissues from the Grave's disease but not in thyroid adenoma and normal thyroid tissues.

Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support.

Science.gov (United States)

Schellenberg, Matthew J; Petrovich, Robert M; Malone, Christine C; Williams, R Scott

2018-03-25

Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence-based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression. Published 2018. This article is a US Government work and is in the public domain in the USA.

CD8+CD122+CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity.

Science.gov (United States)

Akane, Kazuyuki; Kojima, Seiji; Mak, Tak W; Shiku, Hiroshi; Suzuki, Haruhiko

2016-03-01

The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8(+)CD122(+) cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8(+)CD122(-) population, were previously shown to include cells with regulatory activity and could be separated into CD49d(low) cells and CD49d(high) cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122(+) cells. Regulatory activity was observed in CD8(+)CD122(+)CD49d(low) but not in CD8(+)CD122(+)CD49d(high) cells, indicating that the regulatory cells in the CD8(+)CD122(+) population could be narrowed down to CD49d(low) cells. CD8(+)CD122(-) cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122(+) Tregs. CD122(+) Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8(+)CD122(-) cells, indicating that the regulation by CD122(+) Tregs is Fas/FasL-dependent. CD122(+) Tregs taken from IL-10-deficient mice could regulate CD8(+)CD122(-) cells as equally as wild-type CD122(+) Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122(+) Tregs in vitro. CD122(+) Tregs also regulated CD4(+) cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122(+) Tregs that kill activated T cells to maintain immune homeostasis.

Enhanced photovoltaic performance of quantum dot-sensitized solar cells with a progressive reduction of recombination using Cu-doped CdS quantum dots

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Muthalif, Mohammed Panthakkal Abdul; Lee, Young-Seok; Sunesh, Chozhidakath Damodharan; Kim, Hee-Je; Choe, Youngson

2017-02-01

In this article, we have systematically probed the effect of Cu-doping in CdS quantum dots (QDs) to enhance the photovoltaic performance of the quantum dot-sensitized solar cells (QDSSCs). The Cu-doped CdS photoanodes were prepared by successive ionic layer adsorption and reaction (SILAR) method and the corresponding cell devices were fabricated using CuS counter electrodes with a polysulfide electrolyte. The photovoltaic performance results demonstrate that 3 mM Cu-doped CdS QDs based QDSSCs exhibit the efficiency (η) of 3% including JSC = 9.40 mA cm-2, VOC = 0.637 V, FF = 0.501, which are higher than those with bare CdS (η = 2.05%, JSC = 7.12 mA cm-2, VOC = 0.588 V, FF = 0.489). The structural, topographical and optical properties of the thin films have been studied with the help of X-ray diffraction pattern (XRD), atomic force microscopy (AFM) and UV-vis spectrophotometer. Electrochemical impedance spectroscopy (EIS) and open circuit voltage decay (OCVD) measurements indicate that Cu-dopant can inhibit the charge recombination at the photoanode/electrolyte interface and extend the lifetime of electrons. These results reveal that incorporation of copper metal in CdS QDs is a simple and effective method to improve the photovoltaic properties of QDSSCs.

High PD-L1/CD86 MFI ratio and IL-10 secretion characterize human regulatory dendritic cells generated for clinical testing in organ transplantation.

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Zahorchak, Alan F; Macedo, Camila; Hamm, David E; Butterfield, Lisa H; Metes, Diana M; Thomson, Angus W

2018-01-01

Human regulatory dendritic cells (DCreg) were generated from CD14 immunobead-purified or elutriated monocytes in the presence of vitamin D3 and IL-10. They exhibited similar, low levels of costimulatory CD80 and CD86, but comparatively high levels of co-inhibitory programed death ligand-1 (PD-L1) and IL-10 production compared to control immature DC (iDC). Following Toll-like receptor 4 ligation, unlike control iDC, DCreg resisted phenotypic and functional maturation and further upregulated PD-L1:CD86 expression. Whereas LPS-stimulated control iDC (mature DC; matDC) secreted pro-inflammatory tumor necrosis factor but no IL-10, the converse was observed for LPS-stimulated DCreg. DCreg weakly stimulated naïve and memory allogeneic CD4 + and CD8 + T cell proliferation and IFNγ, IL-17A and perforin/granzyme B production in MLR. Their stimulatory function was enhanced however, by blocking PD-1 ligation. High-throughput T cell receptor (TCR) sequencing revealed that, among circulating T cell subsets, memory CD8 + T cells contained the most alloreactive TCR clonotypes and that, while matDC expanded these alloreactive memory CD8 TCR clonotypes, DCreg induced more attenuated responses. These findings demonstrate the feasibility of generating highly-purified GMP-grade DCreg for systemic infusion, their influence on the alloreactive T cell response, and a key mechanistic role of the PD1 pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Repressive mutations restore function-loss caused by the disruption of trimerization in Escherichia coli multidrug transporter AcrB

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Zhaoshuai eWang

2015-01-01

Full Text Available AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBP223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrBP223G. Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the trimer affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding.

A Novel de Novo Mutation in the CD40 Ligand Gene in a Patient With a Mild X-Linked Hyper-IgM Phenotype Initially Diagnosed as CVID: New Aspects of Old Diseases

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Tábata T. França

2018-05-01

Full Text Available Mutations in the CD40 ligand (CD40L gene (CD40LG lead to X-linked hyper-IgM syndrome (X-HIGM, which is a primary immunodeficiency (PID characterized by decreased serum levels of IgG and IgA and normal or elevated IgM levels. Although most X-HIGM patients become symptomatic during the first or second year of life, during which they exhibit recurrent infections, some patients exhibit mild phenotypes, which are usually associated with hypomorphic mutations that do not abrogate protein expression or function. Here, we describe a 28-year-old man who initially presented with recurrent infections since the age of 7 years, when he exhibited meningitis caused by Cryptococcus neoformans. The patient had no family history of immunodeficiency, and based on clinical and laboratory presentation, he was initially diagnosed with common variable immunodeficiency (CVID. In subsequent years, he displayed several sporadic episodes of infection, including pneumonia, pharyngotonsillitis, acute otitis media, rhinosinusitis, fungal dermatosis, and intestinal helminthiasis. The evaluation of CD40L expression on the surface of activated CD3+CD4+ T cells from the patient showed decreased expression of CD40L. Genetic analysis revealed a novel de novo mutation consisting of a 6-nucleotide insertion in exon 1 of CD40LG, which confirmed the diagnosis of X-HIGM. In this report, we describe a novel mutation in the CD40L gene and highlight the complexities of PID diagnosis in light of atypical phenotypes and hypomorphic mutations as well as the importance of the differential diagnosis of PIDs.

[Regulatory B cells activated by CpG-ODN combined with anti-CD40 monoclonal antibody inhibit CD4(+)T cell proliferation].

Science.gov (United States)

Wang, Keng; Tao, Lei; Su, Jianbing; Zhang, Yueyang; Zou, Binhua; Wang, Yiyuan; Li, Xiaojuan

2016-09-01

Objective To observe the immunosuppressive function of regulatory B cells (Bregs) in vitro after activated by CpG oligodeoxynucleotide (CpG-ODN) and anti-CD40 mAb. Methods Mice splenic CD5(+)CD1d(high)B cells and CD5(-)CD1d(low)B cells were sorted by flow cytometry. These B cells were first stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours, and then co-cultured with purified CD4(+)T cells. The interleukin 10 (IL-10) expression in the activated Bregs and other B cell subset, as well as the proliferation and interferon γ (IFN-γ) expression in the CD4(+) T cells activated by anti-CD3 mAb plus anti-CD28 mAb were determined by flow cytometry. Results CD5(+)CD1d(high) B cells activated by CpG-ODN plus anti-CD40 mAb blocked the up-regulated CD4(+)T proliferation and significantly reduced the IFN-γ level. At the same time, activated CD5(-)CD1d(low)B cells showed no inhibitory effect on CD4(+)T cells. Further study revealed that IL-10 expression in the CD5(+)CD1d(high) B cells were much higher than that in the CD5(-)CD1d(low)B cells after stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours. Conclusion CD5(+)CD1d(high) B cells activated by CpG-ODN combined with anti-CD40 mAb have immune inhibitory effects on CD4(+)T cell activation in vitro , which possibly due to IL-10 secretion.

Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B

Energy Technology Data Exchange (ETDEWEB)

Backovic, Marija [Northwestern Univ., Evanston, IL (United States); Longnecker, Richard [Northwestern Univ., Chicago, IL (United States); Jardetzky, Theodore S [Northwestern Univ., Evanston, IL (United States)

2009-03-16

Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which forms 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.

Antioxidant and membrane effects of procyanidin dimers and trimers isolated from peanut and cocoa.

Science.gov (United States)

Verstraeten, Sandra V; Hammerstone, John F; Keen, Carl L; Fraga, César G; Oteiza, Patricia I

2005-06-15

The antioxidant and membrane effects of dimer (Dim) and trimer (Trim) procyanidins isolated from cocoa (Theobroma cacao) (B- and C-bonded) and peanut (Arachis hypogea L.) skin (A-bonded) were evaluated in phosphatidyl choline liposomes. When liposomes were oxidized with a steady source of oxidants, the above dimers and trimers inhibited to a similar extent lipid oxidation in a concentration (0.33-5 microM)-dependent manner. With respect to membrane effects, Dim A1, Dim B, Trim A, and Trim C increased (Dim A1 = Dim B and Trim A = Trim C), while Dim A2 decreased, membrane surface potential. All of the procyanidins tested decreased membrane fluidity as determined by fluorescent probes at the water-lipid interface, an effect that extended into the hydrophobic region of the bilayer. Both dimers and trimers protected the lipid bilayer from disruption by Triton X-100. The magnitude of the protection was Dim A1 > Dim A2 > Dim B and Trim C > Trim A. Thus, dimers and trimers can interact with membrane phospholipids, presumably with their polar headgroup. As a consequence of this interaction, they can provide protection against the attack of oxidants and other molecules that challenge the integrity of the bilayer.

Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

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Briana Jill Williams

Full Text Available Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs with prostate specific membrane antigen (PSMA have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells. To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ. Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: Effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes

International Nuclear Information System (INIS)

Nair, M.P.N.; Pottathil, R.; Heimer, E.P.; Schwartz, S.A.

1988-01-01

Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by [ 3 H]thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3 + lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3 - lymphocytes. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection

Time-resolved photoluminescence study of CdSe/CdMnS/CdS core/multi-shell nanoplatelets

International Nuclear Information System (INIS)

Murphy, J. R.; Delikanli, S.; Demir, H. V.; Scrace, T.; Zhang, P.; Norden, T.; Petrou, A.; Thomay, T.; Cartwright, A. N.

2016-01-01

We used photoluminescence spectroscopy to resolve two emission features in CdSe/CdMnS/CdS and CdSe/CdS core/multi-shell nanoplatelet heterostructures. The photoluminescence from the magnetic sample has a positive circular polarization with a maximum centered at the position of the lower energy feature. The higher energy feature has a corresponding signature in the absorption spectrum; this is not the case for the low-energy feature. We have also studied the temporal evolution of these features using a pulsed-excitation/time-resolved photoluminescence technique to investigate their corresponding recombination channels. A model was used to analyze the temporal dynamics of the photoluminescence which yielded two distinct timescales associated with these recombination channels. The above results indicate that the low-energy feature is associated with recombination of electrons with holes localized at the core/shell interfaces; the high-energy feature, on the other hand, is excitonic in nature with the holes confined within the CdSe cores.

Time-resolved photoluminescence study of CdSe/CdMnS/CdS core/multi-shell nanoplatelets

Energy Technology Data Exchange (ETDEWEB)

Murphy, J. R. [Department of Electrical Engineering, State University of New York, University at Buffalo, Buffalo, New York 14260 (United States); Department of Physics, State University of New York, University at Buffalo, Buffalo, New York 14260 (United States); Delikanli, S.; Demir, H. V., E-mail: volkan@bilkent.edu.tr [LUMINOUS Center of Excellence for Semiconductor Lighting and Displays, School of Electrical and Electronic Engineering, School of Physical and Materials Sciences, Nanyang Technological University, Singapore 639798 (Singapore); Department of Electrical and Electronics Engineering, Department of Physics, UNAM−Institute of Materials Science and Nanotechnology, Bilkent University, Ankara 06800 (Turkey); Scrace, T.; Zhang, P.; Norden, T.; Petrou, A., E-mail: petrou@buffalo.edu [Department of Physics, State University of New York, University at Buffalo, Buffalo, New York 14260 (United States); Thomay, T.; Cartwright, A. N. [Department of Electrical Engineering, State University of New York, University at Buffalo, Buffalo, New York 14260 (United States)

2016-06-13

We used photoluminescence spectroscopy to resolve two emission features in CdSe/CdMnS/CdS and CdSe/CdS core/multi-shell nanoplatelet heterostructures. The photoluminescence from the magnetic sample has a positive circular polarization with a maximum centered at the position of the lower energy feature. The higher energy feature has a corresponding signature in the absorption spectrum; this is not the case for the low-energy feature. We have also studied the temporal evolution of these features using a pulsed-excitation/time-resolved photoluminescence technique to investigate their corresponding recombination channels. A model was used to analyze the temporal dynamics of the photoluminescence which yielded two distinct timescales associated with these recombination channels. The above results indicate that the low-energy feature is associated with recombination of electrons with holes localized at the core/shell interfaces; the high-energy feature, on the other hand, is excitonic in nature with the holes confined within the CdSe cores.

Roles of the kinase TAK1 in TRAF6-dependent signaling by CD40 and its oncogenic viral mimic, LMP1.

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Kelly M Arcipowski

Full Text Available The Epstein-Barr virus (EBV-encoded protein latent membrane protein 1 (LMP1 is essential for EBV-mediated B cell transformation and plays a critical role in the development of post-transplant B cell lymphomas. LMP1 also contributes to the exacerbation of autoimmune diseases such as systemic lupus erythematosus (SLE. LMP1 is a functional mimic of the tumor necrosis factor receptor (TNFR superfamily member CD40, and relies on TNFR-associated factor (TRAF adaptor proteins to mediate signaling. However, LMP1 activation signals to the B cell are amplified and sustained compared to CD40 signals. We previously demonstrated that LMP1 and CD40 use TRAF molecules differently. Although associating with CD40 and LMP1 via separate mechanisms, TRAF6 plays a significant role in signal transduction by both. It is unknown whether TRAF6 mediates CD40 versus LMP1 functions via distinct or shared pathways. In this study, we tested the hypothesis that TRAF6 uses the kinase TAK1 to trigger important signaling pathways following both CD40 and LMP1 stimulation. We determined that TAK1 was required for JNK activation and interleukin-6 (IL-6 production mediated by CD40 and LMP1, in both mouse and human B cells. Additionally, TRAF3 negatively regulated TRAF6-dependent, CD40-mediated TAK1 activation by limiting TRAF6 recruitment. This mode of regulation was not observed for LMP1 and may contribute to the dysregulation of LMP1 compared to CD40 signals.

[Evaluation of percentage of lymphocytes B with expression of co-receptors CD 40, CD22 and CD72 in hypertrophied adenoid at children with otitis media with effusion].

Science.gov (United States)

Wysocka, Jolanta; Zelazowska-Rutkowska, Beata; Ratomski, Karol; Skotnicka, Bozena; Hassmann-Poznańska, Elzbieta

2009-01-01

In hypertrophied adenoid lymphocytes B make up about 60% all lymphocytes. When the lymphocytes B come in interaction with antigens this membranes signal be passed through their receptor (BCR) to interior of cell. This signal affect modulation on gene expression, activation from which depends activation, anergy or apoptosis of lymphocyte B. Accompany BCR co-receptors regulate his functions influence stimulate or inhibitive. To the most important co-receptors stepping out on lymphocyte B belong: CD40, CD22, CD72. The aim of study was evaluation of lymphocytes B (CD19) with co-expression with CD72 and CD40 receptors in hypertrophied adenoid with at children with otitis media with effusion. An investigation was executed in hypertrophied adenoids with or without otitis media with effusion. By flow cytometry percentage of lymphocytes B with co-receptors CD 40, CD22 and CD72 in was analyzed. The percentages of CD19+CD72+ lymphocytes in the group of children with adenoid hypertrophy and exudative otitis media were lower as compared to the reference group. However, the percentages of CD19+CD22+, CD19+CD40+ in the study group was approximate to the reference group. The lower percentage of lymphocytes B CD72 + near approximate percentages of lymphocytes B CD40+ and BCD22+ at children with otitis media with effusion can be the cause of incorrect humoral response in hypertrophied adenoid at children. Maybe it is cause reduced spontaneous production IgA and IgG through lymphocyte at children with otitis media with effusion.

Evaluation of CD40 and CD80 receptors in the colonic mucosal membrane of children with inflammatory bowel disease

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Barbara Kamińska

2015-12-01

Full Text Available [b][/b][b]Introduction. [/b]The most prevalent inflammatory bowel diseases (IBD include ulcerative colitis (UC and Crohn’s disease (CD. Immune processes play a vital role in the etiopathogenesis of these conditions, involving both cellular and humoral response mechanisms. The aim of this study was to quantify CD40- and CD80-positive cells in the biopsy specimens of large intestinal mucosa from children with IBD. [b]Materials and method. [/b]The study comprised 38 children aged between 3–17 years (mean 11.5±3.7 years – 20 boys (52.6 % and 18 girls (47.4%. Eighteen patients were diagnosed with UC on the basis of clinical manifestation, endoscopic and histopathological findings. Mean age of this subgroup was 11.55±4.07 years. A group of 10 children (mean age 12.30±2.83 diagnosed with CD was also included. The control group comprised 10 IBD-free children (mean age 10.28±4.07 years. The surface expressions of CD40 and CD80 were analyzed in large intestine mucosa biopsy specimens, fixed in formaldehyde, embedded in paraffin, and cut with a microtome into 4 µm slices. [b]Results. [/b]The number of CD40- and CD80-positive cells in the large intestinal mucosa of children with Crohn’s disease and ulcerative colitis was significantly higher than in the controls. The highest number of CD40+ and CD80+ cells was observed in the caecal mucosal membrane of Crohn’s disease patients and in the rectal mucosa of individuals with ulcerative colitis. [b]Conclusion.[/b] IBD is characterized by elevated, segment-specific, expression of CD40 and CD80.

Tetranectin, a trimeric plasminogen-binding C-type lectin

DEFF Research Database (Denmark)

Holtet, T L; Graversen, Jonas Heilskov; Clemmensen, I

1997-01-01

-linking analysis and SDS-PAGE to be a homo-trimer in solution as are other known members of the collectin family of C-type lectins. Biochemical evidence is presented showing that an N-terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization....

Recombination in the human Pseudoautosomal region PAR1.

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Anjali G Hinch

2014-07-01

Full Text Available The pseudoautosomal region (PAR is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome.

Comparative investigation of toxicity and bioaccumulation of Cd-based quantum dots and Cd salt in freshwater plant Lemna minor L.

Science.gov (United States)

Modlitbová, Pavlína; Novotný, Karel; Pořízka, Pavel; Klus, Jakub; Lubal, Přemysl; Zlámalová-Gargošová, Helena; Kaiser, Jozef

2018-01-01

The purpose of this study was to determine the toxicity of two different sources of cadmium, i.e. CdCl 2 and Cd-based Quantum Dots (QDs), for freshwater model plant Lemna minor L. Cadmium telluride QDs were capped with two coating ligands: glutathione (GSH) or 3-mercaptopropionic acid (MPA). Growth rate inhibition and final biomass inhibition of L. minor after 168-h exposure were monitored as toxicity endpoints. Dose-response curves for Cd toxicity and EC50 168h values were statistically evaluated for all sources of Cd to uncover possible differences among the toxicities of tested compounds. Total Cd content and its bioaccumulation factors (BAFs) in L. minor after the exposure period were also determined to distinguish Cd bioaccumulation patterns with respect to different test compounds. Laser-Induced Breakdown Spectroscopy (LIBS) with lateral resolution of 200µm was employed in order to obtain two-dimensional maps of Cd spatial distribution in L. minor fronds. Our results show that GSH- and MPA-capped Cd-based QDs have similar toxicity for L. minor, but are significantly less toxic than CdCl 2 . However, both sources of Cd lead to similar patterns of Cd bioaccumulation and distribution in L. minor fronds. Our results are in line with previous reports that the main mediators of Cd toxicity and bioaccumulation in aquatic plants are Cd 2+ ions dissolved from Cd-based QDs. Copyright © 2017 Elsevier Inc. All rights reserved.

CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.

Science.gov (United States)

Kasinrerk, W; Baumruker, T; Majdic, O; Knapp, W; Stockinger, H

1993-01-15

In this paper we demonstrate that granulocyte-macrophage CSF (GM-CSF) specifically induces the expression of CD1 molecules, CD1a, CD1b and CD1c, upon human monocytes. CD1 molecules appeared upon monocytes on day 1 of stimulation with rGM-CSF, and expression was up-regulated until day 3. Monocytes cultured in the presence of LPS, FMLP, PMA, recombinant granulocyte-CSF, rIFN-gamma, rTNF-alpha, rIL-1 alpha, rIL-1 beta, and rIL-6 remained negative. The induction of CD1 molecules by rGM-CSF was restricted to monocytes, since no such effect was observed upon peripheral blood granulocytes, PBL, and the myeloid cell lines Monomac1, Monomac6, MV4/11, HL60, U937, THP1, KG1, and KG1A. CD1a mRNA was detectable in rGM-CSF-induced monocytes but not in those freshly isolated. SDS-PAGE and immunoblotting analyses of CD1a mAb VIT6 immunoprecipitate from lysate of rGM-CSF-activated monocytes revealed an appropriate CD1a polypeptide band of 49 kDa associated with beta 2-microglobulin. Expression of CD1 molecules on monocytes complements the distribution of these structures on accessory cells, and their specific induction by GM-CSF strengthens the suggestion that CD1 is a family of crucial structures required for interaction between accessory cells and T cells.

Long term influence of regular intake of high dose n-3 fatty acids on CD40-ligand, pregnancy-associated plasma protein A and matrix metalloproteinase-9 following acute myocardial infarction.

Science.gov (United States)

Aarsetøy, Hildegunn; Brügger-Andersen, Trygve; Hetland, Øyvind; Grundt, Heidi; Nilsen, Dennis W T

2006-02-01

Pregnancy-associated plasma protein A (PAPP-A) and matrix metalloproteinase 9 (MMP-9), both zinc-binding endopeptidases, are abundantly expressed in ruptured and eroded plaques in patients with acute coronary syndromes (ACS). The adhesion molecule CD-40 ligand (CD40L), expressed on activated platelets and T-lymphocytes, can activate metalloproteinases and thereby promote plaque-rupture. N-3 fatty acids, through their anti-inflammatory and anti-thrombotic properties, might reduce the levels of these proatherosclerotic markers and thereby the development of ACS. 300 patients were randomized on day 4 to 6 following an acute myocardial infarction (MI) to receive either 4 g of n-3 fatty acids or a similar daily dose of corn oil for at least one year. We compared levels of PAPP-A, MMP-9 and sCD-40 L at baseline and 12 months in each group, and also looked for inter-group changes. In the omega-3 group, the median level of PAPP-A rose from 0.47 mU/l to 0.56 mU/l (p < 0.001). In the same group, sCD-40 L decreased from a mean baseline value of 5.19 ng/ml to 2.45 ng/ml (p < 0.001) and MMP-9 decreased nonsignificantly from 360.50 ng/ml to 308.00 ng/ml. Corresponding values for the corn oil group were 0.54 mU/l to 0.59 mU/l for PAPP-A (p = 0.007), 5.27 ng/ml to 2.84 ng/ml for sCD-40 L (p < 0.001) and 430.00 ng/ml to 324.00 ng/ml for MMP-9 (p = ns), respectively. In conclusion; both interventions resulted in a significant rise in PAPP-A, a significant decrease in sCD40L and a non-significant decrease in MMP-9 after 12 months of treatment in MI survivors. No inter-group differences were noted.

Human CD141+ Dendritic Cell and CD1c+ Dendritic Cell Undergo Concordant Early Genetic Programming after Activation in Humanized Mice In Vivo

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Yoshihito Minoda

2017-10-01

Full Text Available Human immune cell subsets develop in immunodeficient mice following reconstitution with human CD34+ hematopoietic stem cells. These “humanized” mice are useful models to study human immunology and human-tropic infections, autoimmunity, and cancer. However, some human immune cell subsets are unable to fully develop or acquire full functional capacity due to a lack of cross-reactivity of many growth factors and cytokines between species. Conventional dendritic cells (cDCs in mice are categorized into cDC1, which mediate T helper (Th1 and CD8+ T cell responses, and cDC2, which mediate Th2 and Th17 responses. The likely human equivalents are CD141+ DC and CD1c+ DC subsets for mouse cDC1 and cDC2, respectively, but the extent of any interspecies differences is poorly characterized. Here, we exploit the fact that human CD141+ DC and CD1c+ DC develop in humanized mice, to further explore their equivalency in vivo. Global transcriptome analysis of CD141+ DC and CD1c+ DC isolated from humanized mice demonstrated that they closely resemble those in human blood. Activation of DC subsets in vivo, with the TLR3 ligand poly I:C, and the TLR7/8 ligand R848 revealed that a core panel of genes consistent with DC maturation status were upregulated by both subsets. R848 specifically upregulated genes associated with Th17 responses by CD1c+ DC, while poly I:C upregulated IFN-λ genes specifically by CD141+ DC. MYCL expression, known to be essential for CD8+ T cell priming by mouse DC, was specifically induced in CD141+ DC after activation. Concomitantly, CD141+ DC were superior to CD1c+ DC in their ability to prime naïve antigen-specific CD8+ T cells. Thus, CD141+ DC and CD1c+ DC share a similar activation profiles in vivo but also have induce unique signatures that support specialized roles in CD8+ T cell priming and Th17 responses, respectively. In combination, these data demonstrate that humanized mice provide an attractive and tractable model to study

The diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (ELISA) using recombinant cathepsin L protease.

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Gonzales Santana, Bibiana; Dalton, John P; Vasquez Camargo, Fabio; Parkinson, Michael; Ndao, Momar

2013-01-01

Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected.

Genetic recombination between human and animal parasites creates novel strains of human pathogen.

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Gibson, Wendy; Peacock, Lori; Ferris, Vanessa; Fischer, Katrin; Livingstone, Jennifer; Thomas, James; Bailey, Mick

2015-03-01

Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.

Genetic recombination between human and animal parasites creates novel strains of human pathogen.

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Wendy Gibson

2015-03-01

Full Text Available Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr responsible for sleeping sickness (Human African Trypanosomiasis, HAT in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.

Human SolCD39 Inhibits Injury-induced Development of Neointimal Hyperplasia

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Drosopoulos, Joan H. F.; Kraemer, Rosemary; Shen, Hao; Upmacis, Rita K.; Marcus, Aaron J.; Musi, Elgilda

2010-01-01

SUMMARY Blood platelets provide the initial response to vascular endothelial injury, becoming activated as they adhere to the injured site. Activated platelets recruit leukocytes, and initiate proliferation and migration of vascular smooth muscle cells (SMC) within the injured vessel wall, leading to development of neointimal hyperplasia. Endothelial CD39/NTPDase1 and recombinant solCD39 rapidly metabolize nucleotides, including stimulatory ADP released from activated platelets, thereby suppressing additional platelet reactivity. Using a murine model of vascular endothelial injury, we investigated whether circulating human solCD39 could reduce platelet activation and accumulation, thus abating leukocyte infiltration and neointimal formation following vascular damage. Intraperitoneally-administered solCD39 ADPase activity in plasma peaked 1 hr post-injection, with an elimination half-life of 43 hr. Accordingly, mice were administered solCD39 or saline 1 hr prior to vessel injury, then either sacrificed 24 hr post-injury or treated with solCD39 or saline (3X weekly) for an additional 18 days. 24 hr post-injury, solCD39-treated mice displayed a reduction in platelet activation and recruitment, P-selectin expression, and leukocyte accumulation in the arterial lumen. Furthermore, repeated administration of solCD39 modulated the late stage of vascular injury by suppressing leukocyte deposition, macrophage infiltration and SMC proliferation/migration, resulting in abrogation of neointimal thickening. In contrast, injured femoral arteries of saline-injected mice exhibited massive platelet thrombus formation, marked P-selectin expression, and leukocyte infiltration. Pronounced neointimal growth with macrophage and SMC accretion was also observed (intimal-to-medial area ratio 1.56±0.34 at 19 days). Thus, systemic administration of solCD39 profoundly affects injury-induced cellular responses, minimizing platelet deposition and leukocyte recruitment, and suppressing

Enhancement of bone formation in rabbits by recombinant human growth hormone

International Nuclear Information System (INIS)

Ehrnberg, A.; Brosjoe, O.; Laaftman, P.; Nilsson, O.; Stroemberg, L.

1993-01-01

We studied the effect of human recombinant growth hormone on diaphyseal bone in 40 adult rabbits. The diaphyseal periosteum of one femur in each animal was mechanically stimulated by a nylon cerclage band. The bands induced an increase in bone formation, bone mineral content, and maximum torque capacity of the diaphyseal bone at 1 and 2 months. Growth hormone enhanced the anabolic effect of the cerclage bands on bone metabolism, evidenced by a further increase in torsional strength of the femurs. (au) (32 refs.)

A Trimeric Lipoprotein Assists in Trimeric Autotransporter Biogenesis in Enterobacteria*

Science.gov (United States)

Grin, Iwan; Hartmann, Marcus D.; Sauer, Guido; Hernandez Alvarez, Birte; Schütz, Monika; Wagner, Samuel; Madlung, Johannes; Macek, Boris; Felipe-Lopez, Alfonso; Hensel, Michael; Lupas, Andrei; Linke, Dirk

2014-01-01

Trimeric autotransporter adhesins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. TAAs form fibrous, adhesive structures on the bacterial cell surface. Their N-terminal extracellular domains are exported through a C-terminal membrane pore; the insertion of the pore domain into the bacterial outer membrane follows the rules of β-barrel transmembrane protein biogenesis and is dependent on the essential Bam complex. We have recently described the full fiber structure of SadA, a TAA of unknown function in Salmonella and other enterobacteria. In this work, we describe the structure and function of SadB, a small inner membrane lipoprotein. The sadB gene is located in an operon with sadA; orthologous operons are only found in enterobacteria, whereas other TAAs are not typically associated with lipoproteins. Strikingly, SadB is also a trimer, and its co-expression with SadA has a direct influence on SadA structural integrity. This is the first report of a specific export factor of a TAA, suggesting that at least in some cases TAA autotransport is assisted by additional periplasmic proteins. PMID:24369174

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

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Pietsch, E C; Dong, J; Cardoso, R; Zhang, X; Chin, D; Hawkins, R; Dinh, T; Zhou, M; Strake, B; Feng, P-H; Rocca, M; Santos, C Dos; Shan, X; Danet-Desnoyers, G; Shi, F; Kaiser, E; Millar, H J; Fenton, S; Swanson, R; Nemeth, J A; Attar, R M

2017-01-01

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices. PMID:28234345

Human natural killer cell maturation defect supports in vivo CD56(bright to CD56(dim lineage development.

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Carolina Inés Domaica

Full Text Available Two populations of human natural killer (NK cells can be identified in peripheral blood. The majority are CD3(-CD56(dim cells while the minority exhibits a CD3(-CD56(bright phenotype. In vitro evidence indicates that CD56(bright cells are precursors of CD56(dim cells, but in vivo evidence is lacking. Here, we studied NK cells from a patient that suffered from a melanoma and opportunistic fungal infection during childhood. The patient exhibited a stable phenotype characterized by a reduction in the frequency of peripheral blood CD3(-CD56(dim NK cells, accompanied by an overt increase in the frequency and absolute number of CD3(-CD56(bright cells. These NK cells exhibited similar expression of perforin, CD57 and CD158, the major activating receptors CD16, NKp46, NKG2D, DNAM-1, and 2B4, as well as the inhibitory receptor CD94/NKG2A, on both CD56(bright and CD56(dim NK cells as healthy controls. Also, both NK cell subpopulations produced IFN-γ upon stimulation with cytokines, and CD3(-CD56(dim NK cells degranulated in response to cytokines or K562 cells. However, upon stimulation with cytokines, a substantial fraction of CD56(dim cells failed to up-regulate CD57 and CD158, showed a reduction in the percentage of CD16(+ cells, and CD56(bright cells did not down-regulate CD62L, suggesting that CD56(dim cells could not acquire a terminally differentiated phenotype and that CD56(bright cells exhibit a maturation defect that might result in a potential altered migration pattern. These observations, support the notion that NK cells of this patient display a maturation/activation defect that precludes the generation of mature NK cells at a normal rate accompanied by CD56(dim NK cells that cannot completely acquire a terminally differentiated phenotype. Thus, our results provide evidence that support the concept that in vivo CD56(bright NK cells differentiate into CD56(dim NK cells, and contribute to further understand human NK cell ontogeny.

The role of the interleukin-10 subfamily members in immunoglobulin production by human B cells

DEFF Research Database (Denmark)

Hummelshoj, L; Ryder, L P; Poulsen, Lars K.

2006-01-01

Interleukin (IL)-10 has been shown to have various effects on B cells, including positively affecting the production of immunoglobulin A (IgA) and IgG. Several human IL-10-related molecules have been identified. These include IL-19, IL-20, IL-22, IL-24, IL-26, IL-28 and IL-29. To determine...... the effects of the IL-10 analogues on the class switch recombination in B cells, we analysed Ig production from naïve B cells stimulated with these cytokines in the presence of anti-CD40. None of the cytokines were found to induce Ig production by themselves in the presence of anti-CD40 Ab. However, all...... cytokines inhibited the production of IgA and IgG induced by anti-CD40 Ab alone. In combination with anti-CD40 Ab and IL-4, IgG4 were inhibited in cultures stimulated with IL-20, IL-22, IL-26, IL-28 and IL-29 compared with IL-4 and anti-CD40 Ab alone, whereas all IL-10 analogues increased the production...

Bcl-xL acts downstream of caspase-8 activation by the CD95 death-inducing signaling complex

NARCIS (Netherlands)

Medema, J. P.; Scaffidi, C.; Krammer, P. H.; Peter, M. E.

1998-01-01

The Bcl-2 family member Bcl-xL has often been correlated with apoptosis resistance. We have shown recently that in peripheral human T cells resistance to CD95-mediated apoptosis is characterized by a lack of caspase-8 recruitment to the CD95 death-inducing signaling complex (DISC) and by increased

The Haemophilus ducreyi trimeric autotransporter adhesin DsrA protects against an experimental infection in the swine model of chancroid.

Science.gov (United States)

Fusco, William G; Choudhary, Neelima R; Routh, Patty A; Ventevogel, Melissa S; Smith, Valerie A; Koch, Gary G; Almond, Glen W; Orndorff, Paul E; Sempowski, Gregory D; Leduc, Isabelle

2014-06-24

Adherence of pathogens to cellular targets is required to initiate most infections. Defining strategies that interfere with adhesion is therefore important for the development of preventative measures against infectious diseases. As an adhesin to host extracellular matrix proteins and human keratinocytes, the trimeric autotransporter adhesin DsrA, a proven virulence factor of the Gram-negative bacterium Haemophilus ducreyi, is a potential target for vaccine development. A recombinant form of the N-terminal passenger domain of DsrA from H. ducreyi class I strain 35000HP, termed rNT-DsrAI, was tested as a vaccine immunogen in the experimental swine model of H. ducreyi infection. Viable homologous H. ducreyi was not recovered from any animal receiving four doses of rNT-DsrAI administered with Freund's adjuvant at two-week intervals. Control pigs receiving adjuvant only were all infected. All animals receiving the rNT-DsrAI vaccine developed antibody endpoint titers between 3.5 and 5 logs. All rNT-DsrAI antisera bound the surface of the two H. ducreyi strains used to challenge immunized pigs. Purified anti-rNT-DsrAI IgG partially blocked binding of fibrinogen at the surface of viable H. ducreyi. Overall, immunization with the passenger domain of the trimeric autotransporter adhesin DsrA accelerated clearance of H. ducreyi in experimental lesions, possibly by interfering with fibrinogen binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rate constant of free electrons and holes recombination in thin films CdSe

International Nuclear Information System (INIS)

Radychev, N.A.; Novikov, G.F.

2006-01-01

Destruction kinetics of electrons generated in thin films CdSe by laser impulse (wave length is 337 nm, period of impulse - 8 nc) is studied by the method of microwave photoconductivity (36 GHz) at 295 K. Model of the process was suggested using the analysis of kinetics of photo-responses decay, and it allowed determination of rate constant of recombination of free electrons and holes in cadmium selenide - (4-6)x10 -11 cm 3 s -1 [ru

Human papilloma virus load and PD-1/PD-L1, CD8+ and FOXP3 in anal cancer patients treated with chemoradiotherapy: Rationale for immunotherapy

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Balermpas, Panagiotis; Martin, Daniel; Wieland, Ulrike; Rave-Fränk, Margret; Strebhardt, Klaus; Rödel, Claus; Fokas, Emmanouil; Rödel, Franz

2017-01-01

ABSTRACT We examined the prognostic role of immune markers programmed cell death protein-1 (PD-1) and its ligand (PD-L1), CD8+ tumor-infiltrating lymphocytes (TILs), FOXP3+ Tregs and phosphorylated Caspase-8 (T273) in patients with anal squamous cell cancer (ASCC) treated with standard chemoradiotherapy (CRT). The baseline immunohistochemical expression of immune markers was correlated with clinicopathologic characteristics, and cumulative incidence of local failure, disease-free survival (DFS) and overall survival (OS) in 150 patients, also in the context of human papilloma virus 16 (HPV16) DNA load and p16INK4a expression. After a median follow-up of 40 mo (1–205 mo), the 5-y cumulative incidence of local failure and DFS was 19.4% and 67.2%, respectively. Strong immune marker expression was significantly more common in tumors with high HPV16 viral load. In multivariant analysis, high CD8+ and PD-1+ TILs expression predicted for improved local control (p = 0.023 and p = 0.007, respectively) and DFS (p = 0.020 and p = 0.014, respectively). Also, high p16INK4a (p = 0.011) and PD-L1 (p = 0.033) expression predicted for better local control, whereas high FOXP3+ Tregs (p = 0.050) and phosphorylated Caspase-8 (p = 0.031) expression correlated with superior DFS. Female sex and high HPV16 viral load correlated with favorable outcome for all three clinical endpoints. The present data provide, for the first time, robust explanation for the favorable clinical outcome of HPV16-positive ASCC patients harboring strong immune cell infiltration. Our findings are relevant for treatment stratification with immune PD-1/PD-L1 checkpoint inhibitors to complement CRT and should be explored in a clinical trial. PMID:28405521

Human dendritic cells sequentially matured with CD4(+) T cells as a secondary signal favor CTL and long-term T memory cell responses.

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Simon, Thomas; Tanguy-Royer, Séverine; Royer, Pierre-Joseph; Boisgerault, Nicolas; Frikeche, Jihane; Fonteneau, Jean-François; Grégoire, Marc

2012-01-01

Dendritic cells (DCs) are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL) responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As first signals, we used TNF-α/polyI:C mimicking inflammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4+ T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.

Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs.

Science.gov (United States)

Farahmand, Mahin; Nahrevanian, Hossein

2016-07-01

Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs.

Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

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This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

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This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

Science.gov (United States)

This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant.

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Cameron Martin

Full Text Available Lack of safe and effective adjuvants is a major hindrance to the development of efficacious vaccines. Signaling via CD40 pathway leads to enhanced antigen processing and presentation, nitric oxide expression, pro-inflammatory cytokine expression by antigen presenting cells, and stimulation of B-cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation. Agonistic anti-CD40 antibodies have shown promising adjuvant qualities in human and mouse vaccine studies. An anti-CD40 monoclonal antibody (mAb, designated 2E4E4, was identified and shown to have strong agonistic effects on primary cells from multiple livestock species. The mAb recognize swine, bovine, caprine, and ovine CD40, and evoked 25-fold or greater proliferation of peripheral blood mononuclear cells (PBMCs from these species relative to cells incubated with an isotype control (p<0.001. In addition, the mAb induced significant nitric oxide (p<0.0001 release by bovine macrophages. Furthermore, the mAb upregulated the expression of MHC-II by PBMCs, and stimulated significant (p<0.0001 IL-1α, IL6, IL-8, and TNF-α expression by PBMCs. These results suggest that the mAb 2E4E4 can target and stimulate cells from multiple livestock species and thus, it is a potential candidate for adjuvant development. This is the first study to report an anti-swine CD40 agonistic mAb that is also broadly reactive against multiple species.

The influence of recombination on human genetic diversity.

Directory of Open Access Journals (Sweden)

Chris C A Spencer

2006-09-01

Full Text Available In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution.

Viral aggregating and opsonizing activity in collectin trimers

DEFF Research Database (Denmark)

Hartshorn, Kevan L; White, Mitchell R; Tecle, Tesfaldet

2010-01-01

of bovine serum collectin CL-46 induces aggregation of influenza A virus (IAV) and potently increases IAV uptake by neutrophils. CL-46-NCRD showed calcium-dependent and sugar-sensitive binding to both neutrophils and IAV. Replacement of specific residues of the CRD of human SP-D with those found in bovine...... were further increased by cross-linking of hSP-D-NCRD/R343V trimers with mAbs directed against areas of the hSP-D-NCRD not involved in viral binding. Unlike the wild-type human SP-D hSP-D-NCRD, hSP-D-NCRD/R343V also induced viral aggregation. These results indicate that collectins can act as opsonins...

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Directory of Open Access Journals (Sweden)

Xiaodong Weng

2011-03-01

Full Text Available Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12 in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA. Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4% and CD86 (80.13 ± 2.81%] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL and IL-12 (249.57 ± 12.51 pg/mL production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05 than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018, indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Formation of highly stable chimeric trimers by fusion of an adenovirus fiber shaft fragment with the foldon domain of bacteriophage t4 fibritin.

Science.gov (United States)

Papanikolopoulou, Katerina; Forge, Vincent; Goeltz, Pierrette; Mitraki, Anna

2004-03-05

The folding of beta-structured, fibrous proteins is a largely unexplored area. A class of such proteins is used by viruses as adhesins, and recent studies revealed novel beta-structured motifs for them. We have been studying the folding and assembly of adenovirus fibers that consist of a globular C-terminal domain, a central fibrous shaft, and an N-terminal part that attaches to the viral capsid. The globular C-terminal, or "head" domain, has been postulated to be necessary for the trimerization of the fiber and might act as a registration signal that directs its correct folding and assembly. In this work, we replaced the head of the fiber by the trimerization domain of the bacteriophage T4 fibritin, termed "foldon." Two chimeric proteins, comprising the foldon domain connected at the C-terminal end of four fiber shaft repeats with or without the use of a natural linker sequence, fold into highly stable, SDS-resistant trimers. The structural signatures of the chimeric proteins as seen by CD and infrared spectroscopy are reported. The results suggest that the foldon domain can successfully replace the fiber head domain in ensuring correct trimerization of the shaft sequences. Biological implications and implications for engineering highly stable, beta-structured nanorods are discussed.

p62 regulates CD40-mediated NFκB activation in macrophages through interaction with TRAF6

Energy Technology Data Exchange (ETDEWEB)

Seibold, Kristina; Ehrenschwender, Martin, E-mail: martin.ehrenschwender@ukr.de

2015-08-14

CD40 is a member of the tumor necrosis factor (TNF) receptor family. Activation-induced recruitment of adapter proteins, so-called TNF-receptor-associated factors (TRAFs) to the cytoplasmic tail of CD40 triggers signaling cascades important in the immune system, but has also been associated with excessive inflammation in diseases such as atherosclerosis and rheumatoid arthritis. Especially, pro-inflammatory nuclear factor κB (NFκB) signaling emanating from CD40-associated TRAF6 appears to be a key pathogenic driving force. Consequently, targeting the CD40-TRAF6 interaction is emerging as a promising therapeutic strategy, but the underlying molecular machinery of this signaling axis is to date poorly understood. Here, we identified the multifunctional adaptor protein p62 as a critical regulator in CD40-mediated NFκB signaling via TRAF6. CD40 activation triggered formation of a TRAF6-p62 complex. Disturbing this interaction tremendously reduced CD40-mediated NFκB signaling in macrophages, while TRAF6-independent signaling pathways remained unaffected. This highlights p62 as a potential target in hyper-inflammatory, CD40-associated pathologies. - Highlights: • CD40 activation triggers interaction of the adapter protein TRAF6 with p62. • TRAF6-p62 interaction regulates CD40-mediated NFκB signaling in macrophages. • Defective TRAF6-p62 interaction reduces CD40-mediated NFκB activation in macrophages.

Ex Vivo Expanded Human Non-Cytotoxic CD8+CD45RClow/− Tregs Efficiently Delay Skin Graft Rejection and GVHD in Humanized Mice

Directory of Open Access Journals (Sweden)

Séverine Bézie

2018-01-01

Full Text Available Both CD4+ and CD8+ Tregs play a critical role in the control of immune responses and immune tolerance; however, our understanding of CD8+ Tregs is limited while they are particularly promising for therapeutic application. We report here existence of highly suppressive human CD8+CD45RClow/− Tregs expressing Foxp3 and producing IFNγ, IL-10, IL-34, and TGFβ to mediate their suppressive activity. We demonstrate that total CD8+CD45RClow/− Tregs can be efficiently expanded in the presence of anti-CD3/28 mAbs, high-dose IL-2 and IL-15 and that such expanded Tregs efficiently delay GVHD and human skin transplantation rejection in immune humanized mice. Robustly expanded CD8+ Tregs displayed a specific gene signature, upregulated cytokines and expansion in the presence of rapamycin greatly improved proliferation and suppression. We show that CD8+CD45RClow/− Tregs are equivalent to canonical CD4+CD25highCD127low/− Tregs for suppression of allogeneic immune responses in vitro. Altogether, our results open new perspectives to tolerogenic strategies in human solid organ transplantation and GVHD.

Honokiol trimers and dimers via biotransformation catalyzed by Momordica charantia peroxidase: novel and potent α-glucosidase inhibitors.

Science.gov (United States)

He, Ye; Wang, Xiao-Bing; Fan, Bo-Yi; Kong, Ling-Yi

2014-01-15

Ten honokiol oligomers (1-10), including four novel trimers (1-4) and four novel dimers (5-8), were obtained by means of biotransformation of honokiol catalyzed by Momordica charantia peroxidase (MCP) for the first time. Their structures were established on the basis of spectroscopic methods. The biological results demonstrated that most of the oligomers were capable of inhibiting α-glucosidase with significant abilities, which were one to two orders of magnitude more potent than the substrate, honokiol. In particular, compound 2, the honokiol trimer, displayed the greatest inhibitory activity against α-glucosidase with an IC50 value of 1.38μM. Kinetic and CD studies indicated that 2 inhibited α-glucosidase in a reversible, mixed-type manner and caused conformational changes in the secondary structure of the enzyme protein. These findings suggested that 2 might be exploited as a promising drug candidate for the treatment of diabetes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ovarian response to recombinant human follicle-stimulating hormone

DEFF Research Database (Denmark)

Arce, Joan-Carles; Andersen, Anders Nyboe; Fernández-Sánchez, Manuel

2014-01-01

OBJECTIVE: To evaluate the dose-response relationship of a novel recombinant human FSH (rhFSH; FE 999049) with respect to ovarian response in patients undergoing IVF/intracytoplasmic sperm injection treatment; and prospectively study the influence of initial antimüllerian hormone (AMH) concentrat......OBJECTIVE: To evaluate the dose-response relationship of a novel recombinant human FSH (rhFSH; FE 999049) with respect to ovarian response in patients undergoing IVF/intracytoplasmic sperm injection treatment; and prospectively study the influence of initial antimüllerian hormone (AMH...

Cyclic water-trimer encapsulation into D2 (22)-C84 fullerene

Science.gov (United States)

Slanina, Zdeněk; Uhlík, Filip; Nagase, Shigeru; Akasaka, Takeshi; Lu, Xing; Adamowicz, Ludwik

2018-03-01

The cyclic water-trimer encapsulations into D2 (22)-C84 fullerene are evaluated. The encapsulation energy is computed at the M06-2X/6-31++G∗∗ level and it is found that the trimer storage in C84 yields the potential-energy gain of 10.4 kcal/mol. The encapsulated trimer can have two different forms, either the conformation known with the free gas-phase water trimer or the arrangement with the three non-hydrogen bonded H atoms on the same side of the O-O-O plane. The latter endohedral isomer is lower in the potential energy by 0.071 kcal/mol and forms about 57% of their equilibrium mixture at room temperature.

CD40-mediated apoptosis in murine B-lymphoma lines containing mutated p53

DEFF Research Database (Denmark)

Hollmann, Annette C; Gong, Qiaoke; Owens, Trevor

2002-01-01

Crosslinking CD40 induces normal B-cells to proliferate and differentiate but causes many tumor cell lines to undergo apoptosis. As p53 is required for many apoptotic pathways, we analyzed the effects of CD40 ligation and their correlation with p53 function in four murine B-lymphoma lines. A20...... of detectable p21 mRNA in A20 and M12 cells. P21 mRNA was increased to detectable levels in M12 cells upon CD40 ligation; however, blocking this effect with the p53 inhibitor pifithrin had no effect on CD40-mediated apoptosis. Sequencing showed that p53 in A20 and M12 cells contained point mutations leading...... to amino acid substitutions in DNA binding regions, but was unmutated in WEHI231 and WEHI 279. These results suggest that CD40-mediated apoptosis can occur in the absence of functional p53....

New perspectives on recombinant human antibodies

NARCIS (Netherlands)

J. de Kruif (John); A.-R. van der Vuurst de Vries (Anne); L. Cilenti (L.); E. Boel (E.); W. van Ewijk (Willem); T. Logtenberg (Ton)

1996-01-01

textabstractThe limited potential of murine monoclonal antibodies for human immunotherapy has driven recent progress in recombinant antibody technology. Here, de Kruif and colleagues report on advances in the development and use of phage-antibody-display libraries.

Human Endometrial CD98 Is Essential for Blastocyst Adhesion

Science.gov (United States)

Domínguez, Francisco; Simón, Carlos; Quiñonero, Alicia; Ramírez, Miguel Ángel; González-Muñoz, Elena; Burghardt, Hans; Cervero, Ana; Martínez, Sebastián; Pellicer, Antonio; Palacín, Manuel; Sánchez-Madrid, Francisco; Yáñez-Mó, María

2010-01-01

Background Understanding the molecular basis of embryonic implantation is of great clinical and biological relevance. Little is currently known about the adhesion receptors that determine endometrial receptivity for embryonic implantation in humans. Methods and Principal Findings Using two human endometrial cell lines characterized by low and high receptivity, we identified the membrane receptor CD98 as a novel molecule selectively and significantly associated with the receptive phenotype. In human endometrial samples, CD98 was the only molecule studied whose expression was restricted to the implantation window in human endometrial tissue. CD98 expression was restricted to the apical surface and included in tetraspanin-enriched microdomains of primary endometrial epithelial cells, as demonstrated by the biochemical association between CD98 and tetraspanin CD9. CD98 expression was induced in vitro by treatment of primary endometrial epithelial cells with human chorionic gonadotropin, 17-β-estradiol, LIF or EGF. Endometrial overexpression of CD98 or tetraspanin CD9 greatly enhanced mouse blastocyst adhesion, while their siRNA-mediated depletion reduced the blastocyst adhesion rate. Conclusions These results indicate that CD98, a component of tetraspanin-enriched microdomains, appears to be an important determinant of human endometrial receptivity during the implantation window. PMID:20976164

Experimental studies of the dissociative recombination processes for the dimethyl ether ions CD3OCD2+ and (CD3)2OD+

Science.gov (United States)

Hamberg, M.; Österdahl, F.; Thomas, R. D.; Zhaunerchyk, V.; Vigren, E.; Kaminska, M.; Af Ugglas, M.; Källberg, A.; Simonsson, A.; Paál, A.; Larsson, M.; Geppert, W. D.

2010-05-01

Aims: Determination of branching fractions, cross sections and thermal rate coefficients for the dissociative recombination of CD3OCD2+ (0-0.3 eV) and (CD3)2OD+ (0-0.2 eV) at the low relative kinetic energies encountered in the interstellar medium. Methods: The measurements were carried out using merged electron and ion beams at the CRYRING storage ring, Stockholm, Sweden. Results: For (CD3)2OD+ we have experimentally determined the branching fraction for ejection of a single hydrogen atom in the DR process to be maximally 7% whereas 49% of the reactions involve the break up of the COC chain into two heavy fragments and 44% ruptures both C-O bonds. The DR of CD3OCD2+ is dominated by fragmentation of the COC chain into two heavy fragments. The measured thermal rate constants and cross sections are k(T) = 1.7±0.5 × 10-6(T/300)-0.77±0.01 cm3 s-1, σ = 1.2±0.4 × 10-15(Ecm[eV])-1.27±0.01 cm2 and k(T) = 1.7±0.6 × 10-6(T/300)-0.70 ± 0.02 cm3 s-1, σ = 1.7±0.6 × 10-15(Ecm[eV])-1.20±0.02 cm2 for CD3OCD2+ and (CD3)2OD+, respectively.

The role of CD40 expression in dendritic cells in cancer biology; a systematic review.

Science.gov (United States)

Lee, Gui Han; Askari, Alan; Malietzis, George; Bernardo, David; Clark, Susan K; Knight, Stella C; Al-Hassi, Hafid Omar

2014-01-01

CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor superfamily and is essential in activation of dendritic cells. Dendritic cells (DCs) are antigen-presenting cells capable of initiating cytotoxic T-lymphocyte immune response against cancer cells. However, there are few studies on the characterization of DCs in cancer, specifically their expression of CD40, despite its implication in cancer immunotherapy. We reviewed available data on the expression of CD40 on DCs in various cancers, and its implications for cancer immunotherapy. A systematic review on CD40 expression on DCs in cancer was performed with reference to preferred reporting items for systematic reviews and meta-analyses (PRISMA). Studies that satisfied the inclusion and exclusion criteria were 21 out of 927. Variations in type and status of the cancers, source of DCs and methodology for detecting CD40 expression amongst the studies resulted in contrasting results. DCs generally expressed low CD40 in tumor infiltrating DCs (tiDCs), in DCs derived by in vitro culture from blood monocytes using cytokine stimulation (MoDCs) and in DCs exposed in vitro to tumor cells lines; the studies suggested that CD40 expression in DCs is impaired in cancer particularly in metastatic disease. However, DCs identified in fresh peripheral blood mononuclear cells (PBMC) expressed higher numbers of CD40 positive cells in some cancer patients, which could be due to tumor-derived factors leading to partially-stimulated DCs. The results provide evidence that some cancer patients may show partial systemic DC activation and expression of increased CD40 in response to the presence of tumor but that such activity may become abortive in the presence of factors produced by the tumor. This review has thus identified key papers on CD40 expression on DCs in various cancers and discusses the limitations and contrasting results of these studies in relation to variations in methodology. The results highlight the need

Protease activity of Per a 10 potentiates Th2 polarization by increasing IL-23 and OX40L.

Science.gov (United States)

Agrawal, Komal; Kale, Sagar L; Arora, Naveen

2015-12-01

Proteases are implicated in exacerbation of allergic diseases. In this study, the role of proteolytic activity of Per a 10 was evaluated on Th2 polarization. Intranasal administration of Per a 10 in mice led to allergic airway inflammation as seen by higher IgE levels, cellular infiltration, IL-17A, and Th2 cytokines, whereas, inactive (Δ)Per a 10 showed attenuated response. There was an increased OX40L expression on lung and lymph node dendritic cells in Per a 10 immunized group and on Per a 10 stimulated BMDCs. Reduction in CD40 expression without any change at transcript level in lungs of Per a 10 immunized mice suggested CD40 cleavage. BMDCs pulsed with Per a 10 showed reduced CD40 expression with lower IL-12p70 secretion as compared to heat inactivated Per a 10. IL-23, TNF-α, and IL-6 levels were significantly higher in Per a 10 stimulated BMDCs supernatant. In DC-T cell coculture studies, Per a 10 pulsed BMDCs showed higher levels of IL-4 and IL-13 that were reduced on blocking of either IL-23 or OX40L. In conclusion, the data suggests a critical role of protease activity of Per a 10 in promoting Th2 polarization by increasing IL-23 secretion and OX40L expression on dendritic cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid modifications of peripheral T-cell subsets that express CD127 in macaques treated with recombinant IL-7.

Science.gov (United States)

Dereuddre-Bosquet, Nathalie; Vaslin, Bruno; Delache, Benoit; Brochard, Patricia; Clayette, Pascal; Aubenque, Céline; Morre, Michel; Assouline, Brigitte; Le Grand, Roger

2007-08-01

Interleukin-7 (IL-7) is a key regulator of thymopoiesis and T-cell homeostasis, which increases blood T-cell number by enhancing thymic output of naive cells and peripheral proliferation. We explored the effects of unglycosylated recombinant simian IL-7 (rsIL-7) administration on peripheral T-cell subpopulations in healthy macaques. RsIL-7 was well tolerated. Mean half-life ranged between 9.3 and 13.9 hours. Blood CD3(+)CD4(+) and CD3(+)CD8(+) lymphocyte counts decreased rapidly after each rsIL-7 administration, the duration of these effects being dependent on the frequency of administration. At treatment completion, the increased of CD3(+) lymphocytes was marked at 100 microg/kg every 2 days. CD3(+) lymphocytes that harbour the alpha chain of IL-7 receptor (CD127) and CD3(+)CD8(+) lymphocytes that expressed the proliferation marker Ki-67 exhibited a similar initial profile. The expression of the anti-apoptotic marker Bcl-2 increased in CD3(+) lymphocytes during the treatment and post-treatment period in a dose/frequency dependent manner. RsIL-7 was well tolerated in macaques and induces rapid modifications of T-cells that express CD127.

Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers.

Science.gov (United States)

Blattner, Claudia; Lee, Jeong Hyun; Sliepen, Kwinten; Derking, Ronald; Falkowska, Emilia; de la Peña, Alba Torrents; Cupo, Albert; Julien, Jean-Philippe; van Gils, Marit; Lee, Peter S; Peng, Wenjie; Paulson, James C; Poignard, Pascal; Burton, Dennis R; Moore, John P; Sanders, Rogier W; Wilson, Ian A; Ward, Andrew B

2014-05-15

All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-PGT158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. Because PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer. Copyright © 2014 Elsevier Inc. All rights reserved.

Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds.

Science.gov (United States)

Galeano, Mariarosaria; Altavilla, Domenica; Bitto, Alessandra; Minutoli, Letteria; Calò, Margherita; Lo Cascio, Patrizia; Polito, Francesca; Giugliano, Giovanni; Squadrito, Giovanni; Mioni, Chiara; Giuliani, Daniela; Venuti, Francesco S; Squadrito, Francesco

2006-04-01

Erythropoietin interacts with vascular endothelial growth factor (VEGF) and stimulates endothelial cell mitosis and motility; thus it may be of importance in the complex phenomenon of wound healing. The purpose of this study was to investigate the effect of recombinant human erythropoietin (rHuEPO) on experimental burn wounds. Randomized experiment. Research laboratory. C57BL/6 male mice weighing 25-30 g. Mice were immersed in 80 degrees C water for 10 secs to achieve a deep-dermal second degree burn. Animals were randomized to receive either rHuEPO (400 units/kg/day for 14 days in 100 microL subcutaneously) or its vehicle alone (100 microl/day distilled water for 14 days subcutaneously). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of neoangiogenesis by immunohistochemistry, and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, measurement of VEGF wound content (enzyme-linked immunosorbent assay), expression (Western blot) of endothelial and inducible nitric oxide synthases, and determination of wound nitric oxide (NO) products. rHuEPO increased burn wound reepithelialization and reduced the time to final wound closure. These effects were completely abated by a passive immunization with specific antibodies against erythropoietin. rHuEPO improved healing of burn wound through increased epithelial proliferation, maturation of the extracellular matrix, and angiogenesis. The hematopoietic factor augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31. Furthermore, rHuEPO enhanced the wound content of VEGF caused a marked expression of endothelial and inducible nitric oxide synthases and increased wound content of nitric oxide products. Our study suggests that rHuEPO may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

DEFF Research Database (Denmark)

Shin, Yun-Ji; Hong, Shin-Young; Kwon, Tae-Ho

2003-01-01

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome...... of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system....

Functional expression of a human GDP-L-fucose transporter in Escherichia coli.

Science.gov (United States)

Förster-Fromme, Karin; Schneider, Sarah; Sprenger, Georg A; Albermann, Christoph

2017-02-01

To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes. The heterologous expression of the recombinant and codon-adapted human GDP-L-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-L-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of 3 H-GDP-L-fucose with a V max of 8 pmol/min mg with a K m of 4 µM. The functional expression of SLC35C1 in GDP-L-fucose overproducing E. coli led to the export of GDP-L-fucose to the culture supernatant. The export of GDP-L-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

Synthesis of disk-rod-disk liquid crystal trimers by using click chemistry

Institute of Scientific and Technical Information of China (English)

无

2010-01-01

A series of disk-rod-disk liquid crystal trimers were synthesized.CuI-NEt3 catalyzed alkyne azide cycloaddition in toluene at room temperature connected two triphenylene discogens to a biphenyl rod-shaped mesogen.The trimers were characterized by using 1H NMR,IR,and high resolution mass spectrometry.The mesomorphic properties were investigated using polarized optical microscopy(POM) ,differential scanning calorimetry(DSC) ,and wide-angle X-ray diffraction.The results showed that the trimers exhibited rectangular columnar mesophase(Colr) .The length of the flexible spacer connecting the three segments has prominent influence on the phase transition temperatures of the trimers.

L-Cysteine Capped CdSe Quantum Dots Synthesized by Photochemical Route.

Science.gov (United States)

Singh, Avinash; Kunwar, Amit; Rath, M C

2018-05-01

L-cysteine capped CdSe quantum dots were synthesized via photochemical route in aqueous solution under UV photo-irradiation. The as grown CdSe quantum dots exhibit broad fluorescence at room temperature. The CdSe quantum dots were found to be formed only through the reactions of the precursors, i.e., Cd(NH3)2+4 and SeSO2-3 with the photochemically generated 1-hydroxy-2-propyl radicals, (CH3)2COH radicals, which are formed through the process of H atom abstraction by the photoexcited acetone from 2-propanol. L-Cysteine was found to act as a suitable capping agent for the CdSe quantum dots and increases their biocompatability. Cytotoxicty effects of these quantum dots were evaluated in Chinese Hamster Ovary (CHO) epithelial cells, indicated a significant lower level for the L-cysteine capped CdSe quantum dots as compare to the bare ones.

The Diagnosis of Human Fascioliasis by Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Cathepsin L Protease

Science.gov (United States)

Gonzales Santana, Bibiana; Vasquez Camargo, Fabio; Parkinson, Michael

2013-01-01

Background Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Methodology/Principal findings Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. Conclusions/Significance A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this

Recombinant human endostatin improves tumor vasculature and alleviates hypoxia in Lewis lung carcinoma

International Nuclear Information System (INIS)

Peng Fang; Wang Jin; Zou Yi; Bao Yong; Huang Wenlin; Chen Guangming; Luo Xianrong; Chen Ming

2011-01-01

Objective: To investigate whether recombinant human endostatin can create a time window of vascular normalization prior to vascular pruning to alleviate hypoxia in Lewis lung carcinoma in mice. Methods: Kinetic changes in morphology of tumor vasculature in response to recombinant human endostatin were detected under a confocal microscope with immunofluorescent staining in Lewis lung carcinomas in mice. The hypoxic cell fraction of different time was assessed with immunohistochemical staining . Effects on tumor growth were monitored as indicated in the growth curve of tumors . Results: Compared with the control group vascularity of the tumors was reduced over time by recombinant human endostatin treatment and significantly regressed for 9 days. During the treatment, pericyte coverage increased at day 3, increased markedly at day 5, and fell again at day 7. The vascular basement membrane was thin and closely associated with endothelial cells after recombinant human endostatin treatment, but appeared thickened, loosely associated with endothelial cells in control tumors. The decrease in hypoxic cell fraction at day 5 after treatment was also found. Tumor growth was not accelerated 5 days after recombinant human endostatin treatment. Conclusions: Recombinant human endostatin can normalize tumor vasculature within day 3 to 7, leading to improved tumor oxygenation. The results provide important experimental basis for combining recombinant human endostatin with radiation therapy in human tumors. (authors)

Effects of selenium on the structure and function of recombinant human S-adenosyl-L-methionine dependent arsenic (+3 oxidation state) methyltransferase in E. coli.

Science.gov (United States)

Geng, Zhirong; Song, Xiaoli; Xing, Zhi; Geng, Jinlong; Zhang, Sichun; Zhang, Xinrong; Wang, Zhilin

2009-05-01

The effects of Se(IV) on the structure and function of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT) purified from the cytoplasm of Escherichia coli were studied. The coding region of human AS3MT complementary DNA was amplified from total RNA extracted from HepG2 cell by reverse transcription PCR. Soluble and active human AS3MT was expressed in the E. coli with a Trx fusion tag under a lower induction temperature of 25 degrees C. Spectra (UV-vis, circular dichroism, and fluorescence) were first used to probe the interaction of Se(IV) and recombinant human AS3MT and the structure-function relationship of the enzyme. The recombinant human AS3MT had a secondary structure of 29.0% alpha-helix, 23.9% beta-pleated sheet, 17.9% beta-turn, and 29.2% random coil. When Se(IV) was added, the content of the alpha-helix did not change, but that of the beta-pleated sheet increased remarkably in the conformation of recombinant human AS3MT. Se(IV) inhibited the enzymatic methylation of inorganic As(III) in a concentration-dependent manner. The IC(50) value for Se(IV) was 2.38 muM. Double-reciprocal (1/V vs. 1/[inorganic As(III)]) plots showed Se(IV) to be a noncompetitive inhibitor of the methylation of inorganic As(III) by recombinant human AS3MT with a K (i) value of 2.61 muM. We hypothesized that Se(IV) interacts with the sulfhydryl group of cysteine(s) in the structural residues rather than the cysteines of the active site (Cys156 and Cys206). When Se(IV) was combined with cysteine(s) in the structural residues, the conformation of recombinant human AS3MT changed and the enzymatic activity decreased. Considering the quenching of tryptophan fluorescence, Cys72 and/or Cys226 are deduced to be primary targets for Se(IV).

Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

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Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

2006-01-01

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell

Human recombinant RNASET2-induced inflammatory response and connective tissue remodeling in the medicinal leech.

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Baranzini, Nicolò; Pedrini, Edoardo; Girardello, Rossana; Tettamanti, Gianluca; de Eguileor, Magda; Taramelli, Roberto; Acquati, Francesco; Grimaldi, Annalisa

2017-05-01

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68 + and HmAIF-1 + macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.

Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18.

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Lecoanet-Henchoz, S; Plater-Zyberk, C; Graber, P; Gretener, D; Aubry, J P; Conrad, D H; Bonnefoy, J Y

1997-09-01

CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the alpha chain of the beta 2 integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ-CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ-CD23 and CD11b/CD18-expressing cells were significantly inhibited by anti-CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up-regulation of interleukin-6 (IL-6) production following ZZ-CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL-6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activities of murine myeloid cells.

Human IgG repertoire of malaria antigen-immunized human immune system (HIS) mice.

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Nogueira, Raquel Tayar; Sahi, Vincent; Huang, Jing; Tsuji, Moriya

2017-08-01

Humanized mouse models present an important tool for preclinical evaluation of new vaccines and therapeutics. Here we show the human variable repertoire of antibody sequences cloned from a previously described human immune system (HIS) mouse model that possesses functional human CD4+ T cells and B cells, namely HIS-CD4/B mice. We sequenced variable IgG genes from single memory B-cell and plasma-cell sorted from splenocytes or whole blood lymphocytes of HIS-CD4/B mice that were vaccinated with a human plasmodial antigen, a recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP). We demonstrate that rPfCSP immunization triggers a diverse B-cell IgG repertoire composed of various human VH family genes and distinct V(D)J recombinations that constitute diverse CDR3 sequences similar to humans, although low hypermutated sequences were generated. These results demonstrate the substantial genetic diversity of responding human B cells of HIS-CD4/B mice and their capacity to mount human IgG class-switched antibody response upon vaccination. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Human CD56bright NK Cells

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Michel, Tatiana; Poli, Aurélie; Cuapio, Angelica

2016-01-01

Human NK cells can be subdivided into various subsets based on the relative expression of CD16 and CD56. In particular, CD56(bright)CD16(-/dim) NK cells are the focus of interest. They are considered efficient cytokine producers endowed with immunoregulatory properties, but they can also become c...... NK cell subsets is not fully defined, nor is their precise hematopoietic origin. In this article, we summarize recent studies about CD56(bright) NK cells in health and disease and briefly discuss the current controversies surrounding them....

Phytoavailability of Cadmium (Cd) to Pak Choi (Brassica chinensis L.) Grown in Chinese Soils: A Model to Evaluate the Impact of Soil Cd Pollution on Potential Dietary Toxicity

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Yang, Xiaoe; Xiao, Wendan; Stoffella, Peter J.; Saghir, Aamir; Azam, Muhammad; Li, Tingqiang

2014-01-01

Food chain contamination by soil cadmium (Cd) through vegetable consumption poses a threat to human health. Therefore, an understanding is needed on the relationship between the phytoavailability of Cd in soils and its uptake in edible tissues of vegetables. The purpose of this study was to establish soil Cd thresholds of representative Chinese soils based on dietary toxicity to humans and develop a model to evaluate the phytoavailability of Cd to Pak choi (Brassica chinensis L.) based on soil properties. Mehlich-3 extractable Cd thresholds were more suitable for Stagnic Anthrosols, Calcareous, Ustic Cambosols, Typic Haplustalfs, Udic Ferrisols and Periudic Argosols with values of 0.30, 0.25, 0.18, 0.16, 0.15 and 0.03 mg kg−1, respectively, while total Cd is adequate threshold for Mollisols with a value of 0.86 mg kg−1. A stepwise regression model indicated that Cd phytoavailability to Pak choi was significantly influenced by soil pH, organic matter, total Zinc and Cd concentrations in soil. Therefore, since Cd accumulation in Pak choi varied with soil characteristics, they should be considered while assessing the environmental quality of soils to ensure the hygienically safe food production. PMID:25386790

Phytoavailability of cadmium (Cd) to Pak choi (Brassica chinensis L.) grown in Chinese soils: a model to evaluate the impact of soil Cd pollution on potential dietary toxicity.

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Rafiq, Muhammad Tariq; Aziz, Rukhsanda; Yang, Xiaoe; Xiao, Wendan; Stoffella, Peter J; Saghir, Aamir; Azam, Muhammad; Li, Tingqiang

2014-01-01

Food chain contamination by soil cadmium (Cd) through vegetable consumption poses a threat to human health. Therefore, an understanding is needed on the relationship between the phytoavailability of Cd in soils and its uptake in edible tissues of vegetables. The purpose of this study was to establish soil Cd thresholds of representative Chinese soils based on dietary toxicity to humans and develop a model to evaluate the phytoavailability of Cd to Pak choi (Brassica chinensis L.) based on soil properties. Mehlich-3 extractable Cd thresholds were more suitable for Stagnic Anthrosols, Calcareous, Ustic Cambosols, Typic Haplustalfs, Udic Ferrisols and Periudic Argosols with values of 0.30, 0.25, 0.18, 0.16, 0.15 and 0.03 mg kg-1, respectively, while total Cd is adequate threshold for Mollisols with a value of 0.86 mg kg-1. A stepwise regression model indicated that Cd phytoavailability to Pak choi was significantly influenced by soil pH, organic matter, total Zinc and Cd concentrations in soil. Therefore, since Cd accumulation in Pak choi varied with soil characteristics, they should be considered while assessing the environmental quality of soils to ensure the hygienically safe food production.

Phytoavailability of cadmium (Cd to Pak choi (Brassica chinensis L. grown in Chinese soils: a model to evaluate the impact of soil Cd pollution on potential dietary toxicity.

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Muhammad Tariq Rafiq

Full Text Available Food chain contamination by soil cadmium (Cd through vegetable consumption poses a threat to human health. Therefore, an understanding is needed on the relationship between the phytoavailability of Cd in soils and its uptake in edible tissues of vegetables. The purpose of this study was to establish soil Cd thresholds of representative Chinese soils based on dietary toxicity to humans and develop a model to evaluate the phytoavailability of Cd to Pak choi (Brassica chinensis L. based on soil properties. Mehlich-3 extractable Cd thresholds were more suitable for Stagnic Anthrosols, Calcareous, Ustic Cambosols, Typic Haplustalfs, Udic Ferrisols and Periudic Argosols with values of 0.30, 0.25, 0.18, 0.16, 0.15 and 0.03 mg kg-1, respectively, while total Cd is adequate threshold for Mollisols with a value of 0.86 mg kg-1. A stepwise regression model indicated that Cd phytoavailability to Pak choi was significantly influenced by soil pH, organic matter, total Zinc and Cd concentrations in soil. Therefore, since Cd accumulation in Pak choi varied with soil characteristics, they should be considered while assessing the environmental quality of soils to ensure the hygienically safe food production.

Human dendritic cells sequentially matured with CD4+ T cells as a secondary signal favor CTL and long-term T memory cell responses

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Thomas Simon

2012-01-01

Full Text Available Dendritic cells (DCs are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As first signals, we used TNF-α/polyI:C mimicking inflammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4+ T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.

CD1 and mycobacterial lipids activate human T cells.

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Van Rhijn, Ildiko; Moody, D Branch

2015-03-01

For decades, proteins were thought to be the sole or at least the dominant source of antigens for T cells. Studies in the 1990s demonstrated that CD1 proteins and mycobacterial lipids form specific targets of human αβ T cells. The molecular basis by which T-cell receptors (TCRs) recognize CD1-lipid complexes is now well understood. Many types of mycobacterial lipids function as antigens in the CD1 system, and new studies done with CD1 tetramers identify T-cell populations in the blood of tuberculosis patients. In human populations, a fundamental difference between the CD1 and major histocompatibility complex systems is that all humans express nearly identical CD1 proteins. Correspondingly, human CD1 responsive T cells show evidence of conserved TCRs. In addition to natural killer T cells and mucosal-associated invariant T (MAIT cells), conserved TCRs define other subsets of human T cells, including germline-encoded mycolyl-reactive (GEM) T cells. The simple immunogenetics of the CD1 system and new investigative tools to measure T-cell responses in humans now creates a situation in which known lipid antigens can be developed as immunodiagnostic and immunotherapeutic reagents for tuberculosis disease. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Radiative recombination process of high density excitons in CdS crystals

Energy Technology Data Exchange (ETDEWEB)

Dneprovskij, V.S.; Klimov, V.I.; Martynenko, E.D.; Stadnik, V.A.

1983-11-01

The behaviour of the P-, E-, L- and Q-lines of luminescence in CdS is compared with calculated results for the processes of exciton-exciton scattering, exciton-electron scattering, annihilation of equilibrium electron-hole fluid (EHF), annihilation of electron-hole plasma (processes of amplification and reabsorption are taken into account). The comparison permitted to determine parameters of high density exciton gas and EHF. Spectral-kinetic properties of generation are investigated, and amplification factor in CdS is estimated.

Synergy of anti-CD40, CpG and MPL in activation of mouse macrophages.

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Shi, Yongyu; Felder, Mildred A R; Sondel, Paul M; Rakhmilevich, Alexander L

2015-08-01

Activation of macrophages is a prerequisite for their antitumor effects. Several reagents, including agonistic anti-CD40 monoclonal antibody (anti-CD40), CpG oligodeoxynucleotides (CpG) and monophosphoryl lipid A (MPL), can stimulate activation of macrophages. Our previous studies showed synergy between anti-CD40 and CpG and between anti-CD40 and MPL in macrophage activation and antitumor efficacy in mice. In the present study, we asked whether there was synergy among these three reagents. The activation of adherent peritoneal exudate cells (PEC) obtained from mice injected with anti-CD40 and then treated with CpG and/or MPL in vitro was determined by their ability to suppress proliferation of tumor cells and to produce various cytokines and chemokines in vitro. Cell sorting and histology followed by functional testing showed that macrophages were the main cell population in PEC activated by CD40 ligation in vivo. A combination of anti-CD40, CpG or MPL activated PEC to suppress proliferation of B16 cells and produce nitric oxide far greater than the single reagents or any of the double combinations of these reagents. In addition, the combination of all three reagents activated PEC to secrete IL-12, IFN-γ and MCP-1 to a greater degree than any single reagent or any two combined reagents. These results demonstrate that macrophages can be synergistically activated by anti-CD40, CpG and MPL, suggesting that this novel combined approach might be further investigated as potential cancer therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Synergy of anti-CD40, CpG and MPL in activation of mouse macrophages

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Shi, Yongyu; Felder, Mildred A.R.; Sondel, Paul M.; Rakhmilevich, Alexander L.

2015-01-01

Activation of macrophages is a prerequisite for their antitumor effects. Several reagents, including agonistic anti-CD40 monoclonal antibody (anti-CD40), CpG oligodeoxynucleotides (CpG) and monophosphoryl lipid A (MPL), can stimulate activation of macrophages. Our previous studies showed synergy between anti-CD40 and CpG and between anti-CD40 and MPL in macrophage activation and antitumor efficacy in mice. In the present study, we asked whether there was synergy among these three reagents. The activation of adherent peritoneal exudate cells (PEC) obtained from mice injected with anti-CD40 and then treated with CpG and/or MPL in vitro was determined by their ability to suppress proliferation of tumor cells and to produce various cytokines and chemokines in vitro. Cell sorting and histology followed by functional testing showed that macrophages were the main cell population in PEC activated by CD40 ligation in vivo. A combination of anti-CD40, CpG or MPL activated PEC to suppress proliferation of B16 cells and produce nitric oxide far greater than the single reagents or any of the double combinations of these reagents. In addition, the combination of all three reagents activated PEC to secrete IL-12, IFN-γ and MCP-1 to a greater degree than any single reagent or any two combined reagents. These results demonstrate that macrophages can be synergistically activated by anti-CD40, CpG and MPL, suggesting that this novel combined approach might be further investigated as potential cancer therapy. PMID:25829245

PP4 is essential for germinal center formation and class switch recombination in mice.

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Ming-Yu Chen

Full Text Available PP4 is a serine/threonine phosphatase required for immunoglobulin (Ig VDJ recombination and pro-B/pre-B cell development in mice. To elucidate the role of PP4 in mature B cells, we ablated the catalytic subunit of murine PP4 in vivo utilizing the CD23 promoter and cre-loxP recombination and generated CD23(crePP4(F/F mice. The development of follicular and marginal zone B cells was unaffected in these mutants, but the proliferation of mature PP4-deficient B cells stimulated by in vitro treatment with either anti-IgM antibody (Ab or LPS was partially impaired. Interestingly, the induction of CD80 and CD86 expression on these stimulated B cells was normal. Basal levels of serum Igs of all isotypes were strongly reduced in CD23(crePP4(F/F mice, and their B cells showed a reduced efficiency of class switch recombination (CSR in vitro upon stimulation by LPS or LPS plus IL-4. When CD23(crePP4(F/F mice were challenged with either the T cell-dependent antigen TNP-KLH or the T cell-independent antigen TNP-Ficoll, or by H1N1 virus infection, the mutant animals failed to form germinal centers (GCs in the spleen and the draining mediastinal lymph nodes, and did not efficiently mount antigen-specific humoral responses. In the resting state, PP4-deficient B cells exhibited pre-existing DNA fragmentation. Upon stimulation by DNA-damaging drug etoposide in vitro, mutant B cells showed increased cleavage of caspase 3. In addition, the mutant B cells displayed impaired CD40-mediated MAPK activation, abnormal IgM-mediated NF-κB activation, and reduced S phase entry upon IgM/CD40-stimulation. Taken together, our results establish a novel role for PP4 in CSR, and reveal crucial functions for PP4 in the maintenance of genomic stability, GC formation, and B cell-mediated immune responses.

Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes.

Science.gov (United States)

Ponnazhagan, S; Weigel, K A; Raikwar, S P; Mukherjee, P; Yoder, M C; Srivastava, A

1998-06-01

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and

Recombinant Human Parvovirus B19 Vectors: Erythroid Cell-Specific Delivery and Expression of Transduced Genes

Science.gov (United States)

Ponnazhagan, Selvarangan; Weigel, Kirsten A.; Raikwar, Sudhanshu P.; Mukherjee, Pinku; Yoder, Mervin C.; Srivastava, Arun

1998-01-01

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562–566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111–1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited

[A study of recombinant human sestrin 1 and sestrin 2 proteins produced in a prokaryotic system].

Science.gov (United States)

Rai, N; Kumar, R; Haque, Md A; Hassan, Md I; Dey, S

2017-01-01

Sestrins are highly conserved stress-inducible proteins capable of suppressing the production of ROS and signalling through mTORC1. Here we report a study of human sestrin1 (sesn1) and sestrin2 (sesn2) proteins produced in a pET28^(+) vector based prokaryotic system. Mass spectrometry analysis, western blot and surface plasmon resonance (SPR) of affinity purified sesn1 and sesn2 proteins confirmed their identity; biophysical characteristics were observed using circular dichroism (CD) showing that sesn1 and sesn2 have a predominant α-helical structure. Here we describe a simple, one step purification process to purify a large amount of sestrin proteins with significant yield. Further study of recombinant human sestrins may further facilitate the understanding of their roles in eukaryotic cells.

Th cells promote CTL survival and memory via acquired pMHC-I and endogenous IL-2 and CD40L signaling and by modulating apoptosis-controlling pathways.

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Channakeshava Sokke Umeshappa

Full Text Available Involvement of CD4(+ helper T (Th cells is crucial for CD8(+ cytotoxic T lymphocyte (CTL-mediated immunity. However, CD4(+ Th's signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4(+ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4(+ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA-pulsed dendritic cell (DCova. CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K(b/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I complex signaling, CD4(+ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4(+ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2 and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7 molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4(+ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.