Stephan Sudowe | Johannes Gutenberg-Universität Mainz (original) (raw)

Papers by Stephan Sudowe

Research paper thumbnail of A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid

Blood, 2007

maturation in the presence of glucocorticoid differentiated into a mature state, but exerts toler... more maturation in the presence of glucocorticoid differentiated into a mature state, but exerts tolerogenic function upon A newly established murine immature dendritic cell line can be http://bloodjournal.hematologylibrary.org/content/109/9/3820.full.html Updated information and services can be found at: (5019 articles) Immunobiology Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.

Research paper thumbnail of Allergen-induced IgE-dependent gut inflammation in a human PBMC–engrafted murine model of allergy

Journal of Allergy and Clinical Immunology, 2012

Background: Humanized murine models comprise a new tool to analyze novel therapeutic strategies f... more Background: Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine. Objective: In this study we developed a human PBMCengrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms. Methods: Nonobese diabetic (NOD)-scid-gc 2/2 mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically. Results: Using the aeroallergens birch or grass pollen as model allergens and, for some donors, also hazelnut allergen, we show that allergen-specific human IgE in murine sera and allergenspecific proliferation and cytokine production of human CD4 1 T cells recovered from spleens after 3 weeks could only be measured in mice treated with PBMCs plus allergen. Importantly, these mice had the highest endoscopic scores evaluating translucent structure, granularity, fibrin, vascularity, and stool after oral or rectal allergen challenge and a strong histologic inflammation of the colon. Analyzing the underlying mechanisms, we demonstrate that allergen-associated colitis was dependent on IgE, human IgE receptor-expressing effector cells, and the mediators histamine and platelet-activating factor. Conclusion: These results demonstrate that allergic gut inflammation can be induced in human PBMC-engrafted mice, allowing the investigation of pathophysiologic mechanisms of allergic diseases of the intestine and evaluation of therapeutic interventions. (J Allergy Clin Immunol 2012;129:1126-35.)

Research paper thumbnail of Efficacy of recombinant adenovirus as vector for allergen gene therapy in a mouse model of type I allergy

Gene Therapy, 2002

DNA-based immunization represents an attractive alternative approach to the current treatment of ... more DNA-based immunization represents an attractive alternative approach to the current treatment of allergic diseases by specific immunotherapy with allergen extracts. In this study, we used a replication-deficient adenovirus vector (AdCMV), to examine the in vivo efficacy of preventive and therapeutic genetic immunization in a mouse model of type I allergy. Primary immunization with a recombinant adenovirus expressing the model antigen

Research paper thumbnail of Induction of Regulatory T Cells by Leflunomide in a Murine Model of Contact Allergen Sensitivity

Journal of Investigative Dermatology, 2006

Allergic contact dermatitis and contact hypersensitivity (CHS) are characterized by allergen-spec... more Allergic contact dermatitis and contact hypersensitivity (CHS) are characterized by allergen-specific activation of CD8 þ and CD4 þ T cells and the production of cytokines resulting in an inflammatory response and tissue damage. We show here that the immunosuppressive compound leflunomide (N-[4-trifluoro-methylphenyl]-5methylisoxazol-4 carboxamide, HWA 486) (LF) inhibited the contact allergic response induced in mice by epicutaneous application of the haptens dinitrofluorobenzene (DNFB) and oxazolone. The extent of ear swelling remained significantly reduced following repeated challenge with DNFB for up to 18 weeks. LF and DNFB had to be applied simultaneously for inhibition to occur. The loss of CHS responses was shown to be antigen-specific. Adoptive transfer of leukocytes from LF-treated mice into naïve mice resulted in a loss of CHS responsiveness. Transfer of both CD4 þ and CD8 þ cells was required for maximal loss of CHS responses, with CD8 þ cells playing a major role. Significantly enhanced levels of IL-10 mRNA were detected in CD8 þ T cells, but not in CD4 þ T cells, following LF treatment of mice. LF also suppressed CHS responses in mice previously sensitized and challenged with hapten, when administered together with the hapten. Our data suggest that LF induces a long-lived tolerance in mice by inducing CD8 þ and CD4 þ regulatory T cells.

Research paper thumbnail of A novel plasmid DNA electroporation method allows transfection of murine DC

Journal of Immunological Methods, 2009

Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent... more Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent immunogenic function due to strong upregulation of costimulatory molecules and proinflammatory cytokines. In numerous studies the potential of modified DC to induce tolerance or immune reactions towards a distinct antigen has been demonstrated. However, DC are refractory to transfection with plasmid DNA by non-viral methods. In this study we have tested the suitability of a newly developed electroporation device to transfect immature murine bone-marrow derived DC (BM-DC). Transfected BM-DC expressed reporter molecules at considerable extent which renders this method suitable to perform all kinds of promoter studies. While electroporation did not alter the low allostimulatory capacity of immature BM-DC, it impaired the stimulation-associated increase in allostimulatory potency of transfectants. However, stimulated transfected BM-DC pulsed with myelin oligodendrocyte protein (MOG)derived peptide induced proliferation of MOG-reactive CD4 + T cells as potently as did nontransfected MOG peptide-pulsed BM-DC. BM-DC transfected with an expression construct encoding MOG efficiently stimulated MOG peptide-specific T cell proliferation. Transfection of BM-DC with an IL-10 encoding expression construct resulted in high IL-10 expression and strongly diminished allogeneic T cell proliferation. Therefore, this method also allows to study functional properties of genetically altered DC.

Research paper thumbnail of Prophylactic and therapeutic intervention in IgE responses by biolistic DNA vaccination primarily targeting dendritic cells

Journal of Allergy and Clinical Immunology, 2006

Background: Allergen gene transfer represents an alternative approach to specific immunotherapy w... more Background: Allergen gene transfer represents an alternative approach to specific immunotherapy with allergen extracts. Gene gun-mediated DNA immunization with plasmid vectors expressing a transgene under control of the promoter of the fascin gene (pFascin) allows for antigen production predominantly by dendritic cells and resulted in the generation of CD8 1 cytotoxic T lymphocytes as well as in the development of a type 1 immune response. Objective: We compared the in vivo efficiency of biolistic transfection with pFascin and plasmids containing the cytomegalovirus promoter (pCMV) in a mouse model of type I allergy. Methods: BALB/c mice were sensitized with the model allergen bgalactosidase to induce a distinctive type 2 immune response. The effect of prophylactic as well as therapeutic biolistic transfection with b-galactosidase-encoding plasmids on the development of antibody titers was followed, and anaphylactic potential of sera was determined. Spleen cells were stimulated in vitro to analyze cytokine production and induction of CD8 1 effector T cells. Results: Protective allergen gene transfer with pFascin efficiently prevented specific IgE production accompanied by immune deviation toward a T H 1-polarized immune response as well as by the induction of IFN-g-producing CD8 1 effector T cells, being comparable to vaccination with pCMV. In a therapeutical setting, biolistic DNA vaccination with pFascin or pCMV transiently protected allergen-sensitized mice against the strong increase in specific IgE production caused by subsequent allergen challenge. Conclusion: We demonstrate for the first time that restricting transgene expression primarily to dendritic cells after DNA vaccination suffices to cause inhibition of IgE production prophylactically and therapeutically. (J Allergy Clin Immunol 2006;117:196-203.)

Research paper thumbnail of Prevention of long-term IgE antibody production by gene gun–mediated DNA vaccination

Journal of Allergy and Clinical Immunology, 2004

Vaccination with allergen-encoding DNA represents a promising approach for the treatment of aller... more Vaccination with allergen-encoding DNA represents a promising approach for the treatment of allergic diseases. In a mouse model of type I allergy, we analyzed the ability of biolistic transfection to inhibit antigen-specific IgE production and to modulate TH2 responses. BALB/c mice were vaccinated by means of gene gun-mediated DNA immunization with plasmid vector pCMV-betaGal, encoding beta-galactosidase as a model allergen. Subsequently, mice were immunized by means of repeated intraperitoneal injection of beta-galactosidase adsorbed to the adjuvant aluminum hydroxide. Development of IgE, IgG1, and IgG2a antibody titers during the course of immunization was followed, and anaphylactic potential of sera was determined by using RBL-2H3 degranulation assay. Spleen cells of vaccinated mice and unvaccinated control animals were stimulated in vitro to analyze cytokine production and induction of CD8 + effector T cells. Gene gun-mediated DNA immunization with pCMV-betaGal very efficiently prevented IgE antibody production on a long-term basis. Concomitantly, IgG1 antibody levels in vaccinated mice were strongly reduced, whereas IgG2a antibody production was increased. Analysis of cytokine profiles indicated immune deviation from a TH2-biased response in control mice toward a mixed TH1/TH2 response in vaccinated mice. In addition, substantial numbers of IFN-gamma-producing CD8 + effector T cells were found in vaccinated mice. Gene gun-mediated DNA vaccination prevents the induction of long-lasting IgE antibody production.

Research paper thumbnail of Allergen-induced IgE-dependent gut inflammation in a human PBMC–engrafted murine model of allergy

Journal of Allergy and Clinical Immunology, 2012

Background: Humanized murine models comprise a new tool to analyze novel therapeutic strategies f... more Background: Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine. Objective: In this study we developed a human PBMCengrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms. Methods: Nonobese diabetic (NOD)-scid-gc 2/2 mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically. Results: Using the aeroallergens birch or grass pollen as model allergens and, for some donors, also hazelnut allergen, we show that allergen-specific human IgE in murine sera and allergenspecific proliferation and cytokine production of human CD4 1 T cells recovered from spleens after 3 weeks could only be measured in mice treated with PBMCs plus allergen. Importantly, these mice had the highest endoscopic scores evaluating translucent structure, granularity, fibrin, vascularity, and stool after oral or rectal allergen challenge and a strong histologic inflammation of the colon. Analyzing the underlying mechanisms, we demonstrate that allergen-associated colitis was dependent on IgE, human IgE receptor-expressing effector cells, and the mediators histamine and platelet-activating factor. Conclusion: These results demonstrate that allergic gut inflammation can be induced in human PBMC-engrafted mice, allowing the investigation of pathophysiologic mechanisms of allergic diseases of the intestine and evaluation of therapeutic interventions. (J Allergy Clin Immunol 2012;129:1126-35.)

Research paper thumbnail of Interleukin-10-Treated Dendritic Cells Do Not Inhibit Th2 Immune Responses in Ovalbumin/Alum-Sensitized Mice

International Archives of Allergy and Immunology, 2006

It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory fu... more It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory function of human dendritic cells (DC) in vitro. Recently, we have shown that these IL-10 DC inhibit the production of T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines by T cells from atopic individuals in vitro. The current study was set out to analyze whether IL-10 DC also exert inhibitory effects in vivo in a murine model of allergy to ovalbumin adsorbed to the adjuvant aluminium hydroxide (OVA/alum). OVA-pulsed or unpulsed bone marrow-derived DC, treated with IL-10 or left untreated during generation, were injected intravenously into BALB/c mice prior to and during OVA/alum sensitization, and sera and immune responses of mesenterial lymph node cells were analyzed. Additionally, bronchoalveolar lavage was performed after intranasal challenge with OVA. Treatment of BALB/c mice with OVA-pulsed DC led to a significantly enhanced proliferation as well as Th2 (IL-4, IL-5), Th1 (interferon-gamma) and IL-10 cytokine production after restimulation of lymph node cells with OVA in vitro compared with OVA immunization alone. In contrast, using OVA-pulsed IL-10 DC for transfer, proliferation and cytokine production by lymph node cells were not enhanced. OVA-specific immunoglobulin G1 (IgG1) and IgG2a production were significantly increased after transfer of OVA-pulsed DC and OVA-pulsed IL-10 DC, respectively, whereas anti-OVA IgE production and airway eosinophilia remained unchanged. Our data indicate that IL-10 treatment of DC decreases the Th1 and Th2 stimulatory capacity of DC but does not actually inhibit systemic (IgE) and local (airway inflammation) allergen-specific immune responses in a murine model of allergy.

Research paper thumbnail of Formalin-Fixed <i>Staphylococcus aureus</i> Particles Prevent Allergic Sensitization in a Murine Model of Type I Allergy

International Archives of Allergy and Immunology, 2007

Bacterial infections are supposed to act counterregulatory to the development of allergen-specifi... more Bacterial infections are supposed to act counterregulatory to the development of allergen-specific Th2 immune responses. We analyzed whether administration of extracellular Staphylococcus aureus inhibited experimental sensitization against allergens. BALB/c mice were immunized with alum-adsorbed ovalbumin (OVA) together with formalin-fixed Staphylococcus particles. OVA-specific antibody production and cytokine synthesis by spleen cells was analyzed. Airway reactivity and cellular infiltration into the airways was assessed after intranasal challenge of mice with OVA. In addition, the capacity of Staphylococcus particles to modulate cytokine production by bone marrow-derived dendritic cells was analyzed in vitro. Simultaneous application of OVA and Staphylococcus particles very efficiently inhibited production of specific IgE and IgG1 as well as secretion of IL-4 and IL-5 by splenocytes, while enhancing IgG2a formation and production of IFN-gamma, indicating a shift from a Th2 response towards a Th1-biased response. This effect was not dependent on the expression of protein A by Staphylococcus. An enhanced frequency or activity of regulatory T cells after administration of Staphylococcus particles was not apparent. Treatment of mice with Staphylococcus particles during the sensitization phase prevented lung inflammation (airway hyperreactivity, eosinophilia) after local challenge with OVA. Culture of bone marrow-derived dendritic cells with Staphylococcus particles induced IL-12p35 and p40 mRNA expression as well as secretion of IL-12p70, and increased production of IL-10 mRNA and protein. Administration of formalin-fixed Staphylococcus particles induced Th1-biased immune responses and prevented allergic sensitization.

Research paper thumbnail of Uptake and presentation of exogenous antigen and presentation of endogenously produced antigen by skin dendritic cells represent equivalent pathways for the priming of cellular immune responses following biolistic DNA immunization

Research paper thumbnail of Antigen dose-dependent differences in IgE antibody production are not due to polarization towards Th1 and Th2 cell subsets

European Journal of Immunology, 1998

The quality of the humoral immune response against protein antigens in CBA/J mice is dependent on... more The quality of the humoral immune response against protein antigens in CBA/J mice is dependent on the antigen dose used for immunization: low doses induce high titers of IgE antibodies, whereas high doses promote the production of IgG2a antibodies but inhibit IgE formation. To investigate whether the reciprocal regulation of antibody production is possibly due to a differential activation of Th1 and Th2 cell populations in the two immunization groups, the cytokine pattern of spleen cells from both groups, cultured with antigen in vitro, was analyzed by measurement of intracellular and secreted cytokine levels. The data presented show that in vitro restimulated spleen cells from mice primed with low as well as with high doses of antigen produce predominantly the Th2 cytokines IL-4 and IL-10 but reduced levels of IL-12. The release of IFN-gamma is only slightly enhanced compared to unstimulated control cultures. The results indicate that CD4+ T cells in both groups belong mainly to the Th2 cell subset. This finding is contradictory to the general allegation that the antigen dose is decisive for the polarization of Th1 versus Th2 immune responses and shows that the antigen dose-dependent regulation of IgE antibody production is not due to differential polarization towards Th1 and Th2 cells.

Research paper thumbnail of Antigen dose-dependent suppression of murine IgE responses is mediated by CD4−CD8− double-negative T cells

Clinical & Experimental Allergy, 2010

Background The IgE response against protein antigens is profoundly influenced by the dose used fo... more Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. Objective The aim of the study was to identify immune cells that are involved in antigen dosedependent regulation of IgE formation. Methods Wild-type mice as well as T helper (Th)1-deficient IL-12p40 À/À and IFN-g À/À mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen-dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT-PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. Results Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL-4, IL-5 and IL-13, but no induction of IFN-g production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen-and isotype-specific manner. Neither CD4 1 nor CD8 1 T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4 À CD8 À double-negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. Conclusion Our data demonstrate that CD4 À CD8 À dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.

Research paper thumbnail of Comparison of adjuvant and adjuvant-free murine experimental asthma models

Clinical & Experimental Allergy, 2009

Research paper thumbnail of A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid

Blood, 2007

maturation in the presence of glucocorticoid differentiated into a mature state, but exerts toler... more maturation in the presence of glucocorticoid differentiated into a mature state, but exerts tolerogenic function upon A newly established murine immature dendritic cell line can be http://bloodjournal.hematologylibrary.org/content/109/9/3820.full.html Updated information and services can be found at: (5019 articles) Immunobiology Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.

Research paper thumbnail of Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin

Gene Therapy, 2003

Cutaneous dendritic cells (DC) are pivotal for the elicitation of antigen-specific immune respons... more Cutaneous dendritic cells (DC) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection of the skin. We transcriptionally targeted transgene expression to DC using vectors containing the murine fascin promoter (pFascin) to control antigen production and compared the immune response elicited with conventional DNA immunization using plasmid constructs with the ubiquitously active CMV promoter (pCMV).

Research paper thumbnail of A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid

Blood, 2007

maturation in the presence of glucocorticoid differentiated into a mature state, but exerts toler... more maturation in the presence of glucocorticoid differentiated into a mature state, but exerts tolerogenic function upon A newly established murine immature dendritic cell line can be http://bloodjournal.hematologylibrary.org/content/109/9/3820.full.html Updated information and services can be found at: (5019 articles) Immunobiology Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.

Research paper thumbnail of Allergen-induced IgE-dependent gut inflammation in a human PBMC–engrafted murine model of allergy

Journal of Allergy and Clinical Immunology, 2012

Background: Humanized murine models comprise a new tool to analyze novel therapeutic strategies f... more Background: Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine. Objective: In this study we developed a human PBMCengrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms. Methods: Nonobese diabetic (NOD)-scid-gc 2/2 mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically. Results: Using the aeroallergens birch or grass pollen as model allergens and, for some donors, also hazelnut allergen, we show that allergen-specific human IgE in murine sera and allergenspecific proliferation and cytokine production of human CD4 1 T cells recovered from spleens after 3 weeks could only be measured in mice treated with PBMCs plus allergen. Importantly, these mice had the highest endoscopic scores evaluating translucent structure, granularity, fibrin, vascularity, and stool after oral or rectal allergen challenge and a strong histologic inflammation of the colon. Analyzing the underlying mechanisms, we demonstrate that allergen-associated colitis was dependent on IgE, human IgE receptor-expressing effector cells, and the mediators histamine and platelet-activating factor. Conclusion: These results demonstrate that allergic gut inflammation can be induced in human PBMC-engrafted mice, allowing the investigation of pathophysiologic mechanisms of allergic diseases of the intestine and evaluation of therapeutic interventions. (J Allergy Clin Immunol 2012;129:1126-35.)

Research paper thumbnail of Efficacy of recombinant adenovirus as vector for allergen gene therapy in a mouse model of type I allergy

Gene Therapy, 2002

DNA-based immunization represents an attractive alternative approach to the current treatment of ... more DNA-based immunization represents an attractive alternative approach to the current treatment of allergic diseases by specific immunotherapy with allergen extracts. In this study, we used a replication-deficient adenovirus vector (AdCMV), to examine the in vivo efficacy of preventive and therapeutic genetic immunization in a mouse model of type I allergy. Primary immunization with a recombinant adenovirus expressing the model antigen

Research paper thumbnail of Induction of Regulatory T Cells by Leflunomide in a Murine Model of Contact Allergen Sensitivity

Journal of Investigative Dermatology, 2006

Allergic contact dermatitis and contact hypersensitivity (CHS) are characterized by allergen-spec... more Allergic contact dermatitis and contact hypersensitivity (CHS) are characterized by allergen-specific activation of CD8 þ and CD4 þ T cells and the production of cytokines resulting in an inflammatory response and tissue damage. We show here that the immunosuppressive compound leflunomide (N-[4-trifluoro-methylphenyl]-5methylisoxazol-4 carboxamide, HWA 486) (LF) inhibited the contact allergic response induced in mice by epicutaneous application of the haptens dinitrofluorobenzene (DNFB) and oxazolone. The extent of ear swelling remained significantly reduced following repeated challenge with DNFB for up to 18 weeks. LF and DNFB had to be applied simultaneously for inhibition to occur. The loss of CHS responses was shown to be antigen-specific. Adoptive transfer of leukocytes from LF-treated mice into naïve mice resulted in a loss of CHS responsiveness. Transfer of both CD4 þ and CD8 þ cells was required for maximal loss of CHS responses, with CD8 þ cells playing a major role. Significantly enhanced levels of IL-10 mRNA were detected in CD8 þ T cells, but not in CD4 þ T cells, following LF treatment of mice. LF also suppressed CHS responses in mice previously sensitized and challenged with hapten, when administered together with the hapten. Our data suggest that LF induces a long-lived tolerance in mice by inducing CD8 þ and CD4 þ regulatory T cells.

Research paper thumbnail of A novel plasmid DNA electroporation method allows transfection of murine DC

Journal of Immunological Methods, 2009

Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent... more Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent immunogenic function due to strong upregulation of costimulatory molecules and proinflammatory cytokines. In numerous studies the potential of modified DC to induce tolerance or immune reactions towards a distinct antigen has been demonstrated. However, DC are refractory to transfection with plasmid DNA by non-viral methods. In this study we have tested the suitability of a newly developed electroporation device to transfect immature murine bone-marrow derived DC (BM-DC). Transfected BM-DC expressed reporter molecules at considerable extent which renders this method suitable to perform all kinds of promoter studies. While electroporation did not alter the low allostimulatory capacity of immature BM-DC, it impaired the stimulation-associated increase in allostimulatory potency of transfectants. However, stimulated transfected BM-DC pulsed with myelin oligodendrocyte protein (MOG)derived peptide induced proliferation of MOG-reactive CD4 + T cells as potently as did nontransfected MOG peptide-pulsed BM-DC. BM-DC transfected with an expression construct encoding MOG efficiently stimulated MOG peptide-specific T cell proliferation. Transfection of BM-DC with an IL-10 encoding expression construct resulted in high IL-10 expression and strongly diminished allogeneic T cell proliferation. Therefore, this method also allows to study functional properties of genetically altered DC.

Research paper thumbnail of Prophylactic and therapeutic intervention in IgE responses by biolistic DNA vaccination primarily targeting dendritic cells

Journal of Allergy and Clinical Immunology, 2006

Background: Allergen gene transfer represents an alternative approach to specific immunotherapy w... more Background: Allergen gene transfer represents an alternative approach to specific immunotherapy with allergen extracts. Gene gun-mediated DNA immunization with plasmid vectors expressing a transgene under control of the promoter of the fascin gene (pFascin) allows for antigen production predominantly by dendritic cells and resulted in the generation of CD8 1 cytotoxic T lymphocytes as well as in the development of a type 1 immune response. Objective: We compared the in vivo efficiency of biolistic transfection with pFascin and plasmids containing the cytomegalovirus promoter (pCMV) in a mouse model of type I allergy. Methods: BALB/c mice were sensitized with the model allergen bgalactosidase to induce a distinctive type 2 immune response. The effect of prophylactic as well as therapeutic biolistic transfection with b-galactosidase-encoding plasmids on the development of antibody titers was followed, and anaphylactic potential of sera was determined. Spleen cells were stimulated in vitro to analyze cytokine production and induction of CD8 1 effector T cells. Results: Protective allergen gene transfer with pFascin efficiently prevented specific IgE production accompanied by immune deviation toward a T H 1-polarized immune response as well as by the induction of IFN-g-producing CD8 1 effector T cells, being comparable to vaccination with pCMV. In a therapeutical setting, biolistic DNA vaccination with pFascin or pCMV transiently protected allergen-sensitized mice against the strong increase in specific IgE production caused by subsequent allergen challenge. Conclusion: We demonstrate for the first time that restricting transgene expression primarily to dendritic cells after DNA vaccination suffices to cause inhibition of IgE production prophylactically and therapeutically. (J Allergy Clin Immunol 2006;117:196-203.)

Research paper thumbnail of Prevention of long-term IgE antibody production by gene gun–mediated DNA vaccination

Journal of Allergy and Clinical Immunology, 2004

Vaccination with allergen-encoding DNA represents a promising approach for the treatment of aller... more Vaccination with allergen-encoding DNA represents a promising approach for the treatment of allergic diseases. In a mouse model of type I allergy, we analyzed the ability of biolistic transfection to inhibit antigen-specific IgE production and to modulate TH2 responses. BALB/c mice were vaccinated by means of gene gun-mediated DNA immunization with plasmid vector pCMV-betaGal, encoding beta-galactosidase as a model allergen. Subsequently, mice were immunized by means of repeated intraperitoneal injection of beta-galactosidase adsorbed to the adjuvant aluminum hydroxide. Development of IgE, IgG1, and IgG2a antibody titers during the course of immunization was followed, and anaphylactic potential of sera was determined by using RBL-2H3 degranulation assay. Spleen cells of vaccinated mice and unvaccinated control animals were stimulated in vitro to analyze cytokine production and induction of CD8 + effector T cells. Gene gun-mediated DNA immunization with pCMV-betaGal very efficiently prevented IgE antibody production on a long-term basis. Concomitantly, IgG1 antibody levels in vaccinated mice were strongly reduced, whereas IgG2a antibody production was increased. Analysis of cytokine profiles indicated immune deviation from a TH2-biased response in control mice toward a mixed TH1/TH2 response in vaccinated mice. In addition, substantial numbers of IFN-gamma-producing CD8 + effector T cells were found in vaccinated mice. Gene gun-mediated DNA vaccination prevents the induction of long-lasting IgE antibody production.

Research paper thumbnail of Allergen-induced IgE-dependent gut inflammation in a human PBMC–engrafted murine model of allergy

Journal of Allergy and Clinical Immunology, 2012

Background: Humanized murine models comprise a new tool to analyze novel therapeutic strategies f... more Background: Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine. Objective: In this study we developed a human PBMCengrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms. Methods: Nonobese diabetic (NOD)-scid-gc 2/2 mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically. Results: Using the aeroallergens birch or grass pollen as model allergens and, for some donors, also hazelnut allergen, we show that allergen-specific human IgE in murine sera and allergenspecific proliferation and cytokine production of human CD4 1 T cells recovered from spleens after 3 weeks could only be measured in mice treated with PBMCs plus allergen. Importantly, these mice had the highest endoscopic scores evaluating translucent structure, granularity, fibrin, vascularity, and stool after oral or rectal allergen challenge and a strong histologic inflammation of the colon. Analyzing the underlying mechanisms, we demonstrate that allergen-associated colitis was dependent on IgE, human IgE receptor-expressing effector cells, and the mediators histamine and platelet-activating factor. Conclusion: These results demonstrate that allergic gut inflammation can be induced in human PBMC-engrafted mice, allowing the investigation of pathophysiologic mechanisms of allergic diseases of the intestine and evaluation of therapeutic interventions. (J Allergy Clin Immunol 2012;129:1126-35.)

Research paper thumbnail of Interleukin-10-Treated Dendritic Cells Do Not Inhibit Th2 Immune Responses in Ovalbumin/Alum-Sensitized Mice

International Archives of Allergy and Immunology, 2006

It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory fu... more It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory function of human dendritic cells (DC) in vitro. Recently, we have shown that these IL-10 DC inhibit the production of T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines by T cells from atopic individuals in vitro. The current study was set out to analyze whether IL-10 DC also exert inhibitory effects in vivo in a murine model of allergy to ovalbumin adsorbed to the adjuvant aluminium hydroxide (OVA/alum). OVA-pulsed or unpulsed bone marrow-derived DC, treated with IL-10 or left untreated during generation, were injected intravenously into BALB/c mice prior to and during OVA/alum sensitization, and sera and immune responses of mesenterial lymph node cells were analyzed. Additionally, bronchoalveolar lavage was performed after intranasal challenge with OVA. Treatment of BALB/c mice with OVA-pulsed DC led to a significantly enhanced proliferation as well as Th2 (IL-4, IL-5), Th1 (interferon-gamma) and IL-10 cytokine production after restimulation of lymph node cells with OVA in vitro compared with OVA immunization alone. In contrast, using OVA-pulsed IL-10 DC for transfer, proliferation and cytokine production by lymph node cells were not enhanced. OVA-specific immunoglobulin G1 (IgG1) and IgG2a production were significantly increased after transfer of OVA-pulsed DC and OVA-pulsed IL-10 DC, respectively, whereas anti-OVA IgE production and airway eosinophilia remained unchanged. Our data indicate that IL-10 treatment of DC decreases the Th1 and Th2 stimulatory capacity of DC but does not actually inhibit systemic (IgE) and local (airway inflammation) allergen-specific immune responses in a murine model of allergy.

Research paper thumbnail of Formalin-Fixed <i>Staphylococcus aureus</i> Particles Prevent Allergic Sensitization in a Murine Model of Type I Allergy

International Archives of Allergy and Immunology, 2007

Bacterial infections are supposed to act counterregulatory to the development of allergen-specifi... more Bacterial infections are supposed to act counterregulatory to the development of allergen-specific Th2 immune responses. We analyzed whether administration of extracellular Staphylococcus aureus inhibited experimental sensitization against allergens. BALB/c mice were immunized with alum-adsorbed ovalbumin (OVA) together with formalin-fixed Staphylococcus particles. OVA-specific antibody production and cytokine synthesis by spleen cells was analyzed. Airway reactivity and cellular infiltration into the airways was assessed after intranasal challenge of mice with OVA. In addition, the capacity of Staphylococcus particles to modulate cytokine production by bone marrow-derived dendritic cells was analyzed in vitro. Simultaneous application of OVA and Staphylococcus particles very efficiently inhibited production of specific IgE and IgG1 as well as secretion of IL-4 and IL-5 by splenocytes, while enhancing IgG2a formation and production of IFN-gamma, indicating a shift from a Th2 response towards a Th1-biased response. This effect was not dependent on the expression of protein A by Staphylococcus. An enhanced frequency or activity of regulatory T cells after administration of Staphylococcus particles was not apparent. Treatment of mice with Staphylococcus particles during the sensitization phase prevented lung inflammation (airway hyperreactivity, eosinophilia) after local challenge with OVA. Culture of bone marrow-derived dendritic cells with Staphylococcus particles induced IL-12p35 and p40 mRNA expression as well as secretion of IL-12p70, and increased production of IL-10 mRNA and protein. Administration of formalin-fixed Staphylococcus particles induced Th1-biased immune responses and prevented allergic sensitization.

Research paper thumbnail of Uptake and presentation of exogenous antigen and presentation of endogenously produced antigen by skin dendritic cells represent equivalent pathways for the priming of cellular immune responses following biolistic DNA immunization

Research paper thumbnail of Antigen dose-dependent differences in IgE antibody production are not due to polarization towards Th1 and Th2 cell subsets

European Journal of Immunology, 1998

The quality of the humoral immune response against protein antigens in CBA/J mice is dependent on... more The quality of the humoral immune response against protein antigens in CBA/J mice is dependent on the antigen dose used for immunization: low doses induce high titers of IgE antibodies, whereas high doses promote the production of IgG2a antibodies but inhibit IgE formation. To investigate whether the reciprocal regulation of antibody production is possibly due to a differential activation of Th1 and Th2 cell populations in the two immunization groups, the cytokine pattern of spleen cells from both groups, cultured with antigen in vitro, was analyzed by measurement of intracellular and secreted cytokine levels. The data presented show that in vitro restimulated spleen cells from mice primed with low as well as with high doses of antigen produce predominantly the Th2 cytokines IL-4 and IL-10 but reduced levels of IL-12. The release of IFN-gamma is only slightly enhanced compared to unstimulated control cultures. The results indicate that CD4+ T cells in both groups belong mainly to the Th2 cell subset. This finding is contradictory to the general allegation that the antigen dose is decisive for the polarization of Th1 versus Th2 immune responses and shows that the antigen dose-dependent regulation of IgE antibody production is not due to differential polarization towards Th1 and Th2 cells.

Research paper thumbnail of Antigen dose-dependent suppression of murine IgE responses is mediated by CD4−CD8− double-negative T cells

Clinical & Experimental Allergy, 2010

Background The IgE response against protein antigens is profoundly influenced by the dose used fo... more Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. Objective The aim of the study was to identify immune cells that are involved in antigen dosedependent regulation of IgE formation. Methods Wild-type mice as well as T helper (Th)1-deficient IL-12p40 À/À and IFN-g À/À mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen-dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT-PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. Results Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL-4, IL-5 and IL-13, but no induction of IFN-g production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen-and isotype-specific manner. Neither CD4 1 nor CD8 1 T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4 À CD8 À double-negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. Conclusion Our data demonstrate that CD4 À CD8 À dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.

Research paper thumbnail of Comparison of adjuvant and adjuvant-free murine experimental asthma models

Clinical & Experimental Allergy, 2009

Research paper thumbnail of A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid

Blood, 2007

maturation in the presence of glucocorticoid differentiated into a mature state, but exerts toler... more maturation in the presence of glucocorticoid differentiated into a mature state, but exerts tolerogenic function upon A newly established murine immature dendritic cell line can be http://bloodjournal.hematologylibrary.org/content/109/9/3820.full.html Updated information and services can be found at: (5019 articles) Immunobiology Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.

Research paper thumbnail of Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin

Gene Therapy, 2003

Cutaneous dendritic cells (DC) are pivotal for the elicitation of antigen-specific immune respons... more Cutaneous dendritic cells (DC) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection of the skin. We transcriptionally targeted transgene expression to DC using vectors containing the murine fascin promoter (pFascin) to control antigen production and compared the immune response elicited with conventional DNA immunization using plasmid constructs with the ubiquitously active CMV promoter (pCMV).