Grayson Lipford - Academia.edu (original) (raw)

Papers by Grayson Lipford

Research paper thumbnail of Aspergillosis TLRs Govern Neutrophil Activity in

Research paper thumbnail of Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine production, and an immunogenic phenotype in chronic lymphocytic leukemia B cells

Blood, 2000

Bacterial DNA and synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted atten... more Bacterial DNA and synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the immune system in a sequence-dependent manner. Here we investigated the potential of CpG-ODNs to cause proliferation, cytokine production, and regulation of surface molecules in human B-chronic lymphocytic leukemia (CLL) cells. CpG-ODN induced proliferation in both B-CLL cells and normal B cells; however, only B-CLL cells increased proliferative responses when CpG-ODN was added to co-cultures of CD40-ligand transfected mouse fibroblasts (CD40LF) and B cells. Production of interleukin-6 and tumor necrosis factor alpha was detectable at borderline levels, using CpG-ODN as the only stimulus. In contrast, when CpG-ODN was added to co-cultures of B cells and CD40LF, a strong increase in cytokine production occurred in B-CLL cells as well as in normal B cells. The surface molecules CD40, CD58, CD80, CD86, CD54, and MHC class I molecules were up-regulated ...

Research paper thumbnail of Differential expression regulation of the alpha and beta subunits of the PA28 proteasome activator in mature dendritic cells

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2005

Activation of dendritic cells (DC) by Th-dependent (CD40) or -independent (LPS, CpG, or immune co... more Activation of dendritic cells (DC) by Th-dependent (CD40) or -independent (LPS, CpG, or immune complexes) agonistic stimuli strongly enhances the expression of the proteasome activator PA28alphabeta complex. Upon activation of DC, increased MHC class I presentation occurred of the melanocyte-associated epitope tyrosinase-related protein 2(180-188) in a PA28alphabeta-dependent manner. In contrast to other cell types, regulation of PA28alphabeta expression in DC after maturation was found to be IFN-gamma independent. In the present study, we show that expression of PA28alpha and beta subunits was differentially regulated. Firstly, PA28alpha expression is high in both immature and mature DC. In contrast, PA28beta expression is low in immature DC and strongly increased in mature DC. Secondly, we show the presence of a functional NF-kappaB site in the PA28beta promoter, which is absent in the PA28alpha promoter, indicating regulation of PA28beta expression by transcription factors of the...

Research paper thumbnail of Dendritic Cells PA28 Proteasome Activator in Mature the {alpha} and {beta} Subunits of the Differential Expression Regulation of

Research paper thumbnail of Therapeutic vaccination with papillomavirus E6 and E7 long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model

Vaccine, 2005

This study was performed to test the therapeutic efficacy of overlapping long E6 and E7 peptides,... more This study was performed to test the therapeutic efficacy of overlapping long E6 and E7 peptides, containing both CD4+ T-helper and CD8+ CTL epitopes, on CRPV-induced lesions, which is an appropriate pre-clinical model for HPV diseases, including recurrent respiratory papillomatosis (RRP). Therapeutic peptide vaccination was able to significantly control wart growth (p < 0.01) and abrogate latent CRPV infection (p = 0.0006) compared to controls. Vaccination was associated with a T(H)1 T cell response, as suggested by a strong DTH skin test, antigen-specific proliferation of PBMC and a minimal IgG antibody response. Thus, this study shows promise for treatment of RRP by vaccination with long peptides.

Research paper thumbnail of Immunostimulatory CpG oligodeoxynucleotides and antibody therapy of cancer

Seminars in Oncology, 2003

Synthetic oligodeoxynucleotides containing CG motifs (CpG ODN) have potent immunostimulatory prop... more Synthetic oligodeoxynucleotides containing CG motifs (CpG ODN) have potent immunostimulatory properties, and have potential as immunotherapeutic agents in cancer. Animal models suggest CpG ODN can activate a variety of immune effector cells such as natural killer (NK) cells, and also enhance the efficacy of tumor immunization when used as immune adjuvants or to directly activate antigen-presenting cells. CpG ODN are also capable of altering the expression of a number of antigens by malignant B-cells, including those targeted by monoclonal antibodies (moAbs) and those involved in communication with T cells. The ability of CpG ODN to activate the immune effector cells that participate in antibody-dependent cellular cytotoxicity (ADCC), upregulate target antigen, and perhaps induce development of an active immune response, suggest these agents may be capable of enhancing the efficacy of antitumor moAb therapy. Such enhanced efficacy has been demonstrated in animal models and is now undergoing evaluation in clinical trials. Semin Oncol 30:476-482.

Research paper thumbnail of Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition

Proceedings of the National Academy of Sciences, 2001

The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins... more The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins, which function as mediators of innate immunity for recognition of pathogen-derived ligands and subsequent cell activation via the Toll/IL-1R signal pathway. Here, we show that human TLR9 (hTLR9) expression in human immune cells correlates with responsiveness to bacterial deoxycytidylate-phosphate-deoxyguanylate (CpG)-DNA. Notably "gain of function" to immunostimulatory CpG-DNA is achieved by expressing TLR9 in human nonresponder cells. Transfection of either human or murine TLR9 conferred responsiveness in a CD14- and MD2-independent manner, yet required species-specific CpG-DNA motifs for initiation of the Toll/IL-1R signal pathway via MyD88. The optimal CpG motif for hTLR9 was GTCGTT, whereas the optimal murine sequence was GACGTT. Overall, these data suggest that hTLR9 conveys CpG-DNA responsiveness to human cells by directly engaging immunostimulating CpG-DNA.

Research paper thumbnail of CpG-DNA-Mediated Transient Lymphadenopathy Is Associated with a State of Th1 Predisposition to Antigen-Driven Responses

The Journal of Immunology, 2000

Research paper thumbnail of Role of Interleukin-18 (IL-18) during Lethal Shock: Decreased Lipopolysaccharide Sensitivity but Normal Superantigen Reaction in IL-18-Deficient Mice

Infection and Immunity, 2000

Lethal shock can be associated with excessive secretion of cytokines such as tumor necrosis facto... more Lethal shock can be associated with excessive secretion of cytokines such as tumor necrosis factor (TNF) and gamma interferon (IFN-␥). IFN-␥ mediates macrophage activation and appears to be controlled by interleukin (IL)-12 and IL-18. To investigate the role of IL-18 in vivo, we generated IL-18-deficient mice by gene targeting. IL-18 ؊/؊ mice showed decreased sensitivity towards lipopolysaccharide (LPS)-induced shock. LPS-induced IFN-␥ production was abrogated, yet induction of IL-12 and TNF was not affected. Both wild-type and IL-18deficient mice succumbed to LPS-induced lethal shock after sensitization with D-galactosamine. However, in marked contrast to LPS, the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) induced comparable serum levels of IFN-␥ in IL-18 ؉/؉ and IL-18 ؊/؊ mice, accompanied by an upregulation of cell surface markers CD14, CD122 (IL-2R␤), and CD132 (IL-2R␥) on peritoneal macrophages. Moreover, SEB injection rendered IL-18-deficient mice sensitive for subsequent challenge with LPS. The degree of sensitization was comparable to that in wild-type controls with respect to lethality. However, LPS-induced TNF levels in serum were significantly reduced in SEB-sensitized IL-18-deficient mice. These results imply that IL-18 plays an important role in induction of IFN-␥ and lethality in response to LPS.

Research paper thumbnail of CpG-oligodeoxynucleotides enhance T-cell receptor-triggered interferon-gamma production and up-regulation of CD69 via induction of antigen-presenting cell-derived interferon type I and interleukin-12

Immunology, 2000

Bacterial cytidine±phosphate±guanosine (CpG-DNA) activates antigen-presenting cells (APC) and dri... more Bacterial cytidine±phosphate±guanosine (CpG-DNA) activates antigen-presenting cells (APC) and drives T helper 1 (Th1)-polarized immune responses in the mouse. Claims have been made that CpG-DNA costimulates murine T cells. We examined the direct and indirect effects of CpGoligodeoxynucleotides (CpG-ODN) on human T-cell activation. CpG-ODN failed to costimulate puri®ed human T cells activated with a-CD3 or a-T-cell receptor (TCR)ab antibodies. In contrast, CpG-ODN sequence-speci®cally caused increased expression of CD69 on CD4 and CD8 T cells when peripheral blood mononuclear cells (PBMC) were stimulated via a-CD3. CpG-ODN and a-CD3 stimulation synergized to induce interferon-c (IFN-c) in T cells and natural killer (NK) cells, as shown by intracellular¯uorescence-activated cell sorter (FACS) staining. These effects of CpG-ODN on human T cells were caused by the release of IFN type I (IFN-I) and interleukin-12 (IL-12) from PBMC. Enhancement of CD69 expression on a-CD3-triggered T cells could be reproduced in a coculture transwell system of puri®ed T cells and PBMC, was inhibited by neutralizing antibodies to IFN-I and could be mimicked by adding exogenous IFN-I. Furthermore, neutralization of either IFN-I or IL-12 diminished, and in combination abolished, IFN-c production. These ®ndings show that CpG-ODN potentiate TCR-triggered activation of human T cells in an APC-dependent manner.

Research paper thumbnail of Bacterial DNA as an evolutionary conserved ligand signalling danger of infection to immune cells

European Journal of Clinical Microbiology & Infectious Diseases, 1998

During infection, the innate limb of the immune system senses danger (pathogens) via constitutive... more During infection, the innate limb of the immune system senses danger (pathogens) via constitutively expressed pattern-recognition receptors, and responds with activation and secretion of pro-inflammatory cytokines. Cell-wall components of gram-positive and gram-negative bacteria, such as peptidoglycan, endotoxin or lipoteichoic acid, activate via CD14, a prototypic pattern-recognition receptor for carbohydrates. This review article focuses on an alternative recognition system of the innate immune system for the recognition of bacterial DNA. Bacterial DNA differs from eukaryotic DNA in its frequency of the dinucleotides CG and its lack of methylation. These structural differences appear to be sensed by cells of the innate immune system such as antigen-presenting cells. As a consequence bacterial DNA serves as an alternate ligand to signal danger of infection. Bacterial DNA and (synthetic) oligonucleotides (ODN) derived thereof are as efficient as endotoxin in activating macrophages and dendritic cells and in triggering release of pro-inflammatory cytokines. In mice sensitized with D-galactosamine (D-GalN), high doses of bacterial DNA from either gram-positive or gram-negative pathogens induce a lethal cytokine syndrome (lethal shock). Therefore, bacterial DNA may represent a hitherto unrecognized pathophysiological entity in host-parasite interactions. Moreover, recent evidence suggests that bacterial DNA or immunostimulating ODN triggers the immunostimulation of antigen-presenting cells, and can be utilized as adjuvant to enhance immune responses of the adaptive immune system towards poorly immunogenic antigens. In fact, foreign DNA might be useful as immunotherapeutically active adjuvant to direct adaptive immune responses towards Thl-dominated immune reactions. If these findings are operative in humans, immunostimulating ODN might be used to influence Th2-dominated diseases such as allergy.

Research paper thumbnail of The Toll‐like receptor 7 (TLR7)‐specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily

European journal of …, 2003

Loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine) acts as synthetic adjuvant in anti-tumor respons... more Loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine) acts as synthetic adjuvant in anti-tumor responses. Here we first demonstrate that loxoribine activates cells of the innate immune system selectively via the Toll-like receptor (TLR) 7/MyD88-dependent signaling pathway. TLR7-and MyD88-deficient immune cells fail to proliferate or produce cytokines in response to loxoribine, and genetic complementation of TLR7-deficient cells with murine or human TLR7 confers responsiveness. Subsequently we show that cellular activation by loxoribine and resiquimod (R-848), a stimulus for TLR7 and TLR8, depends on acidification and maturation of endosomes and targets MyD88 to vesicular structures with lysosomal characteristics. This mode of TLR7 and TLR8 action resembles CpG-DNA-driven TLR9 activation. We thus conclude that TLR7, 8 and 9 form a functional subgroup within the TLR family that recognizes pathogen-associated molecular patterns in endosomal/lysosomal compartments.

Research paper thumbnail of Monoclonal antibody AG7 inhibits fertilization post sperm-zona binding

Molecular Reproduction and Development, 1992

Monoclonal antibodies (mAbs) against sperm cells are currently being used in an effort to define ... more Monoclonal antibodies (mAbs) against sperm cells are currently being used in an effort to define spermatozoal antigens involved in the fertilization process. We have produced a number of anti-human sperm mAbs by immunization of female mice with the 100,000 x g supernatant of octylglycoside-solubilized washed human sperm. From a panel of mAbs, 1 antibody, AG7, was selected and characterized due to its fertilization-inhibiting characteristics. MAb AG7 defines a sperm acrosome antigen-1 (SAA-1) located in the acrosomal region of human sperm as evaluated by indirect immunofluorescence. Staining of life sperm cells indicated that the antigen is present on the sperm surface. SAA-1 was also found on sperm of several other mammalian species, implying evolutionary conservation of the antigen. SAA-1 was first observed on testicular sperm and can be followed through epididymal transit, ejaculation, and capacitation. When applied in a mouse in vitro fertilization assay, mAb AG7 inhibits fertilization by greater than 95%, and inhibition is dose dependent, with half-maximal inhibition at 0.8 micrograms/ml. The block to fertilization could not be attributed to sperm agglutination, inhibition of motility, interference with adhesion to the zona pellucida, or inhibition of fusion with the oocyte membrane. MAb AG7 was demonstrated to inhibit calcium influx in spermatozoa in vitro (measured using the fluorescent indicator fura 2), a prerequisite for the acrosome reaction. Initial biochemical characterization of the antigen suggests it is proteinlike in nature, with a molecular weight of approximately 220 kD. The results suggest that SAA-1, identified by mAb AG7, is a sperm antigen crucially involved in the fertilization process, possibly an atypical steroid receptor or ion channel located within the sperm plasma membrane.

Research paper thumbnail of Calcium oxalate and calcium phosphate capacities of cardiac sarcoplasmic reticulum

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1985

Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reti... more Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reticulum initially increase linearly with time but fall to a steady-state level within 20 min. The departure from linearity could be due to a decrease in influx or to an increase in efflux of calcium. Because Ca2+-ATPase activity is linear, a decrease in the influx of calcium is an unlikely cause of the non-linear calcium uptake curves. A possible cause of an increase in calcium efflux is rupture of the vesicles. This hypothesis was tested by investigating the amount of calcium which could be released upon addition of 5 mM EGTA. The amount of rapidly releasable calcium was zero until a threshold calcium uptake of about 4-6 pmol calcium oxalate or calcium phosphate per mg was reached. After that point the rapidly releasable calcium continued to increase with calcium oxalate to reach more than 23 #mol/mg, but stayed constant at about 0.7 p mol/mg for calcium phosphate. The rapidly releasable calcium was attributed to calcium oxalate or calcium phosphate crystals externalized by vesicle rupture. The differences in the amounts of rapidly releasable calcium were attributed to different kinetics of calcium phosphate and calcium oxalate dissolution. Addition of ryanodine caused a marked increase in the threshold for rapidly releasable calcium oxalate. Transmission electron micrographs showed that vesicles can become filled with calcium oxalate crystals, but the vesicles were heterogeneous with respect to their size and their sensitivity to ryanodine. These observations support the hypothesis that calcium oxalate and calcium phosphate capacities are limited by vesicle rupture and that ryanodine increases the capacity by closing a calcium channel in a subpopulation of vesicles that otherwise would not accumulate calcium.

Research paper thumbnail of Mechanism of action of ryanodine on cardiac sarcoplasmic reticulum

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1985

Ryanodine was found to initially inhibit calcium uptake by cardiac sarcoplasmic reticulum. This i... more Ryanodine was found to initially inhibit calcium uptake by cardiac sarcoplasmic reticulum. This initial depression was followed by a later marked stimulation of calcium uptake. These effects were noted when calcium uptake was measured in the presence or absence of oxalate. The requirement for preincubation with ryanodine was highly dependent on ryanodine concentration and temperature. The mechanism of action of ryanodine clearly was not an effect on oxalate entry or calcium oxalate precipitation because the effects were also observed in the absence of oxalate. Ryanodine also had no effect on passive calcium efflux from actively loaded vesicles. Because ryanodine had no effect on Ca2+-ATPase activity under defined conditions of an ATP-regenerating system and no calcium gradient, we suggest ryanodine does not change the stoichiometry of the pump. Our results are consistent with the hypothesis that ryanodine closes a calcium channel in a subpopolation of the vesicles.

Research paper thumbnail of Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8

Science (New York, N.Y.), Jan 5, 2004

Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infectio... more Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.

Research paper thumbnail of Vaccination with immunodominant peptides encapsulated in Quil A-containing liposomes induces peptide-specific primary CD8+ cytotoxic T cells

Vaccine, 1994

Immunostimulating complexes (ISCOMs), containing lipids, the saponin Quil A, and proteinaceous an... more Immunostimulating complexes (ISCOMs), containing lipids, the saponin Quil A, and proteinaceous antigens, have been proven to vaccinate effectively CD8 + cytolytic T cells m vivo. However, conventional 1SCOM technology is restricted to hydrophobic proteins or fatty acid-derivatized proteins or peptides. We therefore analysed whether Quil A-containing liposomes are an effective vehicle to shuttle hydrophilic proteins or peptides into the MHC class I pathway of antigen presentation resulting in the in vivo induction of antigen-specific cytolytic T cells ( CTL ). Liposomes were formed by a lipid dry-down method followed by resuspension with an aqueous solution containing protein/peptide and Quil A and then an extrusion step. Quil A-containing liposomes are an effective means to elicit a CD8 + CTL response to peptide antigen in vivo. CTL could be raised in C57B1/6 mice against ovalbumin (OVA) peptide 257-264 and vesicular stomatitis virus nucleoprotein 52-59, as well as in Balb/c mice against listeriolysin peptide 91-99 and cytomegalovirus pp89 168-176, demonstrating the versatility of this approach. The elicited response was peptide-specific, peptide dose-dependent and Quil A was necessary.

Research paper thumbnail of Postexposure prophylaxis against prion disease with a stimulator of innate immunity

Lancet, Jan 20, 2002

The absence of an immune response to prions--the infectious agents of scrapie, bovine spongiform ... more The absence of an immune response to prions--the infectious agents of scrapie, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease--might be related to the fact that these agents do not contain nucleic acids. We aimed to use CpG oligodeoxynucleotides, which have been shown to stimulate innate immunity, as a form of postexposure prophylaxis in mice. We inoculated healthy mice with brain homogenates from mice infected with the RML scrapie prion, and then injected CpG oligodeoxynucleotides. This postexposure prophylaxis with CpG oligodeoxynucleotides resulted in 38% longer survival times than treatment with saline (p<0.0001), or even longer after repeated application. The explanation for this finding remains to be elucidated, but the most likely is stimulation of TLR9-expressing cells of the innate immune system such as macrophages, monocytes, and dendritic cells. CpG oligodeoxynucleotides have not been shown to have adverse effects to human health and could theref...

Research paper thumbnail of The role of immunostimulatory CpG-DNA in septic shock

Springer Seminars in Immunopathology, 2000

... The role of immunostimulatory CpG-DNA in septic shock Hermann Wagner, Grayson B. Lipford, Han... more ... The role of immunostimulatory CpG-DNA in septic shock Hermann Wagner, Grayson B. Lipford, Hans H~icker ... Two groups independently demonstrated that macrophages ingest and are activated by bacterial DNA. Stacey et al. ...

Research paper thumbnail of Immunostimulatory DNA sequences help to eradicate intracellular pathogens

Springer Seminars in Immunopathology, 2000

Research paper thumbnail of Aspergillosis TLRs Govern Neutrophil Activity in

Research paper thumbnail of Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine production, and an immunogenic phenotype in chronic lymphocytic leukemia B cells

Blood, 2000

Bacterial DNA and synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted atten... more Bacterial DNA and synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the immune system in a sequence-dependent manner. Here we investigated the potential of CpG-ODNs to cause proliferation, cytokine production, and regulation of surface molecules in human B-chronic lymphocytic leukemia (CLL) cells. CpG-ODN induced proliferation in both B-CLL cells and normal B cells; however, only B-CLL cells increased proliferative responses when CpG-ODN was added to co-cultures of CD40-ligand transfected mouse fibroblasts (CD40LF) and B cells. Production of interleukin-6 and tumor necrosis factor alpha was detectable at borderline levels, using CpG-ODN as the only stimulus. In contrast, when CpG-ODN was added to co-cultures of B cells and CD40LF, a strong increase in cytokine production occurred in B-CLL cells as well as in normal B cells. The surface molecules CD40, CD58, CD80, CD86, CD54, and MHC class I molecules were up-regulated ...

Research paper thumbnail of Differential expression regulation of the alpha and beta subunits of the PA28 proteasome activator in mature dendritic cells

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2005

Activation of dendritic cells (DC) by Th-dependent (CD40) or -independent (LPS, CpG, or immune co... more Activation of dendritic cells (DC) by Th-dependent (CD40) or -independent (LPS, CpG, or immune complexes) agonistic stimuli strongly enhances the expression of the proteasome activator PA28alphabeta complex. Upon activation of DC, increased MHC class I presentation occurred of the melanocyte-associated epitope tyrosinase-related protein 2(180-188) in a PA28alphabeta-dependent manner. In contrast to other cell types, regulation of PA28alphabeta expression in DC after maturation was found to be IFN-gamma independent. In the present study, we show that expression of PA28alpha and beta subunits was differentially regulated. Firstly, PA28alpha expression is high in both immature and mature DC. In contrast, PA28beta expression is low in immature DC and strongly increased in mature DC. Secondly, we show the presence of a functional NF-kappaB site in the PA28beta promoter, which is absent in the PA28alpha promoter, indicating regulation of PA28beta expression by transcription factors of the...

Research paper thumbnail of Dendritic Cells PA28 Proteasome Activator in Mature the {alpha} and {beta} Subunits of the Differential Expression Regulation of

Research paper thumbnail of Therapeutic vaccination with papillomavirus E6 and E7 long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model

Vaccine, 2005

This study was performed to test the therapeutic efficacy of overlapping long E6 and E7 peptides,... more This study was performed to test the therapeutic efficacy of overlapping long E6 and E7 peptides, containing both CD4+ T-helper and CD8+ CTL epitopes, on CRPV-induced lesions, which is an appropriate pre-clinical model for HPV diseases, including recurrent respiratory papillomatosis (RRP). Therapeutic peptide vaccination was able to significantly control wart growth (p &amp;amp;lt; 0.01) and abrogate latent CRPV infection (p = 0.0006) compared to controls. Vaccination was associated with a T(H)1 T cell response, as suggested by a strong DTH skin test, antigen-specific proliferation of PBMC and a minimal IgG antibody response. Thus, this study shows promise for treatment of RRP by vaccination with long peptides.

Research paper thumbnail of Immunostimulatory CpG oligodeoxynucleotides and antibody therapy of cancer

Seminars in Oncology, 2003

Synthetic oligodeoxynucleotides containing CG motifs (CpG ODN) have potent immunostimulatory prop... more Synthetic oligodeoxynucleotides containing CG motifs (CpG ODN) have potent immunostimulatory properties, and have potential as immunotherapeutic agents in cancer. Animal models suggest CpG ODN can activate a variety of immune effector cells such as natural killer (NK) cells, and also enhance the efficacy of tumor immunization when used as immune adjuvants or to directly activate antigen-presenting cells. CpG ODN are also capable of altering the expression of a number of antigens by malignant B-cells, including those targeted by monoclonal antibodies (moAbs) and those involved in communication with T cells. The ability of CpG ODN to activate the immune effector cells that participate in antibody-dependent cellular cytotoxicity (ADCC), upregulate target antigen, and perhaps induce development of an active immune response, suggest these agents may be capable of enhancing the efficacy of antitumor moAb therapy. Such enhanced efficacy has been demonstrated in animal models and is now undergoing evaluation in clinical trials. Semin Oncol 30:476-482.

Research paper thumbnail of Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition

Proceedings of the National Academy of Sciences, 2001

The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins... more The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins, which function as mediators of innate immunity for recognition of pathogen-derived ligands and subsequent cell activation via the Toll/IL-1R signal pathway. Here, we show that human TLR9 (hTLR9) expression in human immune cells correlates with responsiveness to bacterial deoxycytidylate-phosphate-deoxyguanylate (CpG)-DNA. Notably &quot;gain of function&quot; to immunostimulatory CpG-DNA is achieved by expressing TLR9 in human nonresponder cells. Transfection of either human or murine TLR9 conferred responsiveness in a CD14- and MD2-independent manner, yet required species-specific CpG-DNA motifs for initiation of the Toll/IL-1R signal pathway via MyD88. The optimal CpG motif for hTLR9 was GTCGTT, whereas the optimal murine sequence was GACGTT. Overall, these data suggest that hTLR9 conveys CpG-DNA responsiveness to human cells by directly engaging immunostimulating CpG-DNA.

Research paper thumbnail of CpG-DNA-Mediated Transient Lymphadenopathy Is Associated with a State of Th1 Predisposition to Antigen-Driven Responses

The Journal of Immunology, 2000

Research paper thumbnail of Role of Interleukin-18 (IL-18) during Lethal Shock: Decreased Lipopolysaccharide Sensitivity but Normal Superantigen Reaction in IL-18-Deficient Mice

Infection and Immunity, 2000

Lethal shock can be associated with excessive secretion of cytokines such as tumor necrosis facto... more Lethal shock can be associated with excessive secretion of cytokines such as tumor necrosis factor (TNF) and gamma interferon (IFN-␥). IFN-␥ mediates macrophage activation and appears to be controlled by interleukin (IL)-12 and IL-18. To investigate the role of IL-18 in vivo, we generated IL-18-deficient mice by gene targeting. IL-18 ؊/؊ mice showed decreased sensitivity towards lipopolysaccharide (LPS)-induced shock. LPS-induced IFN-␥ production was abrogated, yet induction of IL-12 and TNF was not affected. Both wild-type and IL-18deficient mice succumbed to LPS-induced lethal shock after sensitization with D-galactosamine. However, in marked contrast to LPS, the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) induced comparable serum levels of IFN-␥ in IL-18 ؉/؉ and IL-18 ؊/؊ mice, accompanied by an upregulation of cell surface markers CD14, CD122 (IL-2R␤), and CD132 (IL-2R␥) on peritoneal macrophages. Moreover, SEB injection rendered IL-18-deficient mice sensitive for subsequent challenge with LPS. The degree of sensitization was comparable to that in wild-type controls with respect to lethality. However, LPS-induced TNF levels in serum were significantly reduced in SEB-sensitized IL-18-deficient mice. These results imply that IL-18 plays an important role in induction of IFN-␥ and lethality in response to LPS.

Research paper thumbnail of CpG-oligodeoxynucleotides enhance T-cell receptor-triggered interferon-gamma production and up-regulation of CD69 via induction of antigen-presenting cell-derived interferon type I and interleukin-12

Immunology, 2000

Bacterial cytidine±phosphate±guanosine (CpG-DNA) activates antigen-presenting cells (APC) and dri... more Bacterial cytidine±phosphate±guanosine (CpG-DNA) activates antigen-presenting cells (APC) and drives T helper 1 (Th1)-polarized immune responses in the mouse. Claims have been made that CpG-DNA costimulates murine T cells. We examined the direct and indirect effects of CpGoligodeoxynucleotides (CpG-ODN) on human T-cell activation. CpG-ODN failed to costimulate puri®ed human T cells activated with a-CD3 or a-T-cell receptor (TCR)ab antibodies. In contrast, CpG-ODN sequence-speci®cally caused increased expression of CD69 on CD4 and CD8 T cells when peripheral blood mononuclear cells (PBMC) were stimulated via a-CD3. CpG-ODN and a-CD3 stimulation synergized to induce interferon-c (IFN-c) in T cells and natural killer (NK) cells, as shown by intracellular¯uorescence-activated cell sorter (FACS) staining. These effects of CpG-ODN on human T cells were caused by the release of IFN type I (IFN-I) and interleukin-12 (IL-12) from PBMC. Enhancement of CD69 expression on a-CD3-triggered T cells could be reproduced in a coculture transwell system of puri®ed T cells and PBMC, was inhibited by neutralizing antibodies to IFN-I and could be mimicked by adding exogenous IFN-I. Furthermore, neutralization of either IFN-I or IL-12 diminished, and in combination abolished, IFN-c production. These ®ndings show that CpG-ODN potentiate TCR-triggered activation of human T cells in an APC-dependent manner.

Research paper thumbnail of Bacterial DNA as an evolutionary conserved ligand signalling danger of infection to immune cells

European Journal of Clinical Microbiology & Infectious Diseases, 1998

During infection, the innate limb of the immune system senses danger (pathogens) via constitutive... more During infection, the innate limb of the immune system senses danger (pathogens) via constitutively expressed pattern-recognition receptors, and responds with activation and secretion of pro-inflammatory cytokines. Cell-wall components of gram-positive and gram-negative bacteria, such as peptidoglycan, endotoxin or lipoteichoic acid, activate via CD14, a prototypic pattern-recognition receptor for carbohydrates. This review article focuses on an alternative recognition system of the innate immune system for the recognition of bacterial DNA. Bacterial DNA differs from eukaryotic DNA in its frequency of the dinucleotides CG and its lack of methylation. These structural differences appear to be sensed by cells of the innate immune system such as antigen-presenting cells. As a consequence bacterial DNA serves as an alternate ligand to signal danger of infection. Bacterial DNA and (synthetic) oligonucleotides (ODN) derived thereof are as efficient as endotoxin in activating macrophages and dendritic cells and in triggering release of pro-inflammatory cytokines. In mice sensitized with D-galactosamine (D-GalN), high doses of bacterial DNA from either gram-positive or gram-negative pathogens induce a lethal cytokine syndrome (lethal shock). Therefore, bacterial DNA may represent a hitherto unrecognized pathophysiological entity in host-parasite interactions. Moreover, recent evidence suggests that bacterial DNA or immunostimulating ODN triggers the immunostimulation of antigen-presenting cells, and can be utilized as adjuvant to enhance immune responses of the adaptive immune system towards poorly immunogenic antigens. In fact, foreign DNA might be useful as immunotherapeutically active adjuvant to direct adaptive immune responses towards Thl-dominated immune reactions. If these findings are operative in humans, immunostimulating ODN might be used to influence Th2-dominated diseases such as allergy.

Research paper thumbnail of The Toll‐like receptor 7 (TLR7)‐specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily

European journal of …, 2003

Loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine) acts as synthetic adjuvant in anti-tumor respons... more Loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine) acts as synthetic adjuvant in anti-tumor responses. Here we first demonstrate that loxoribine activates cells of the innate immune system selectively via the Toll-like receptor (TLR) 7/MyD88-dependent signaling pathway. TLR7-and MyD88-deficient immune cells fail to proliferate or produce cytokines in response to loxoribine, and genetic complementation of TLR7-deficient cells with murine or human TLR7 confers responsiveness. Subsequently we show that cellular activation by loxoribine and resiquimod (R-848), a stimulus for TLR7 and TLR8, depends on acidification and maturation of endosomes and targets MyD88 to vesicular structures with lysosomal characteristics. This mode of TLR7 and TLR8 action resembles CpG-DNA-driven TLR9 activation. We thus conclude that TLR7, 8 and 9 form a functional subgroup within the TLR family that recognizes pathogen-associated molecular patterns in endosomal/lysosomal compartments.

Research paper thumbnail of Monoclonal antibody AG7 inhibits fertilization post sperm-zona binding

Molecular Reproduction and Development, 1992

Monoclonal antibodies (mAbs) against sperm cells are currently being used in an effort to define ... more Monoclonal antibodies (mAbs) against sperm cells are currently being used in an effort to define spermatozoal antigens involved in the fertilization process. We have produced a number of anti-human sperm mAbs by immunization of female mice with the 100,000 x g supernatant of octylglycoside-solubilized washed human sperm. From a panel of mAbs, 1 antibody, AG7, was selected and characterized due to its fertilization-inhibiting characteristics. MAb AG7 defines a sperm acrosome antigen-1 (SAA-1) located in the acrosomal region of human sperm as evaluated by indirect immunofluorescence. Staining of life sperm cells indicated that the antigen is present on the sperm surface. SAA-1 was also found on sperm of several other mammalian species, implying evolutionary conservation of the antigen. SAA-1 was first observed on testicular sperm and can be followed through epididymal transit, ejaculation, and capacitation. When applied in a mouse in vitro fertilization assay, mAb AG7 inhibits fertilization by greater than 95%, and inhibition is dose dependent, with half-maximal inhibition at 0.8 micrograms/ml. The block to fertilization could not be attributed to sperm agglutination, inhibition of motility, interference with adhesion to the zona pellucida, or inhibition of fusion with the oocyte membrane. MAb AG7 was demonstrated to inhibit calcium influx in spermatozoa in vitro (measured using the fluorescent indicator fura 2), a prerequisite for the acrosome reaction. Initial biochemical characterization of the antigen suggests it is proteinlike in nature, with a molecular weight of approximately 220 kD. The results suggest that SAA-1, identified by mAb AG7, is a sperm antigen crucially involved in the fertilization process, possibly an atypical steroid receptor or ion channel located within the sperm plasma membrane.

Research paper thumbnail of Calcium oxalate and calcium phosphate capacities of cardiac sarcoplasmic reticulum

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1985

Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reti... more Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reticulum initially increase linearly with time but fall to a steady-state level within 20 min. The departure from linearity could be due to a decrease in influx or to an increase in efflux of calcium. Because Ca2+-ATPase activity is linear, a decrease in the influx of calcium is an unlikely cause of the non-linear calcium uptake curves. A possible cause of an increase in calcium efflux is rupture of the vesicles. This hypothesis was tested by investigating the amount of calcium which could be released upon addition of 5 mM EGTA. The amount of rapidly releasable calcium was zero until a threshold calcium uptake of about 4-6 pmol calcium oxalate or calcium phosphate per mg was reached. After that point the rapidly releasable calcium continued to increase with calcium oxalate to reach more than 23 #mol/mg, but stayed constant at about 0.7 p mol/mg for calcium phosphate. The rapidly releasable calcium was attributed to calcium oxalate or calcium phosphate crystals externalized by vesicle rupture. The differences in the amounts of rapidly releasable calcium were attributed to different kinetics of calcium phosphate and calcium oxalate dissolution. Addition of ryanodine caused a marked increase in the threshold for rapidly releasable calcium oxalate. Transmission electron micrographs showed that vesicles can become filled with calcium oxalate crystals, but the vesicles were heterogeneous with respect to their size and their sensitivity to ryanodine. These observations support the hypothesis that calcium oxalate and calcium phosphate capacities are limited by vesicle rupture and that ryanodine increases the capacity by closing a calcium channel in a subpopulation of vesicles that otherwise would not accumulate calcium.

Research paper thumbnail of Mechanism of action of ryanodine on cardiac sarcoplasmic reticulum

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1985

Ryanodine was found to initially inhibit calcium uptake by cardiac sarcoplasmic reticulum. This i... more Ryanodine was found to initially inhibit calcium uptake by cardiac sarcoplasmic reticulum. This initial depression was followed by a later marked stimulation of calcium uptake. These effects were noted when calcium uptake was measured in the presence or absence of oxalate. The requirement for preincubation with ryanodine was highly dependent on ryanodine concentration and temperature. The mechanism of action of ryanodine clearly was not an effect on oxalate entry or calcium oxalate precipitation because the effects were also observed in the absence of oxalate. Ryanodine also had no effect on passive calcium efflux from actively loaded vesicles. Because ryanodine had no effect on Ca2+-ATPase activity under defined conditions of an ATP-regenerating system and no calcium gradient, we suggest ryanodine does not change the stoichiometry of the pump. Our results are consistent with the hypothesis that ryanodine closes a calcium channel in a subpopolation of the vesicles.

Research paper thumbnail of Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8

Science (New York, N.Y.), Jan 5, 2004

Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infectio... more Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.

Research paper thumbnail of Vaccination with immunodominant peptides encapsulated in Quil A-containing liposomes induces peptide-specific primary CD8+ cytotoxic T cells

Vaccine, 1994

Immunostimulating complexes (ISCOMs), containing lipids, the saponin Quil A, and proteinaceous an... more Immunostimulating complexes (ISCOMs), containing lipids, the saponin Quil A, and proteinaceous antigens, have been proven to vaccinate effectively CD8 + cytolytic T cells m vivo. However, conventional 1SCOM technology is restricted to hydrophobic proteins or fatty acid-derivatized proteins or peptides. We therefore analysed whether Quil A-containing liposomes are an effective vehicle to shuttle hydrophilic proteins or peptides into the MHC class I pathway of antigen presentation resulting in the in vivo induction of antigen-specific cytolytic T cells ( CTL ). Liposomes were formed by a lipid dry-down method followed by resuspension with an aqueous solution containing protein/peptide and Quil A and then an extrusion step. Quil A-containing liposomes are an effective means to elicit a CD8 + CTL response to peptide antigen in vivo. CTL could be raised in C57B1/6 mice against ovalbumin (OVA) peptide 257-264 and vesicular stomatitis virus nucleoprotein 52-59, as well as in Balb/c mice against listeriolysin peptide 91-99 and cytomegalovirus pp89 168-176, demonstrating the versatility of this approach. The elicited response was peptide-specific, peptide dose-dependent and Quil A was necessary.

Research paper thumbnail of Postexposure prophylaxis against prion disease with a stimulator of innate immunity

Lancet, Jan 20, 2002

The absence of an immune response to prions--the infectious agents of scrapie, bovine spongiform ... more The absence of an immune response to prions--the infectious agents of scrapie, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease--might be related to the fact that these agents do not contain nucleic acids. We aimed to use CpG oligodeoxynucleotides, which have been shown to stimulate innate immunity, as a form of postexposure prophylaxis in mice. We inoculated healthy mice with brain homogenates from mice infected with the RML scrapie prion, and then injected CpG oligodeoxynucleotides. This postexposure prophylaxis with CpG oligodeoxynucleotides resulted in 38% longer survival times than treatment with saline (p<0.0001), or even longer after repeated application. The explanation for this finding remains to be elucidated, but the most likely is stimulation of TLR9-expressing cells of the innate immune system such as macrophages, monocytes, and dendritic cells. CpG oligodeoxynucleotides have not been shown to have adverse effects to human health and could theref...

Research paper thumbnail of The role of immunostimulatory CpG-DNA in septic shock

Springer Seminars in Immunopathology, 2000

... The role of immunostimulatory CpG-DNA in septic shock Hermann Wagner, Grayson B. Lipford, Han... more ... The role of immunostimulatory CpG-DNA in septic shock Hermann Wagner, Grayson B. Lipford, Hans H~icker ... Two groups independently demonstrated that macrophages ingest and are activated by bacterial DNA. Stacey et al. ...

Research paper thumbnail of Immunostimulatory DNA sequences help to eradicate intracellular pathogens

Springer Seminars in Immunopathology, 2000