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Biomedical subjects

Ulrich Kalinke

Publications and source records attributed to Ulrich Kalinke.

At least 19 recordsLinked to original sources
PubMed2006

Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide.

Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) gamma release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNalpha production in professional APC. Neutralization of IFNalpha as well as the absence of functional IFNalphabeta receptor on APC and T cells interfered with Hsp60-mediated IFNgamma secretion in antigen-dependent T cell activation, strongly suggesting that IFNalpha represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNgamma release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling.

Animals↗
PubMed2006

Protective role of beta interferon in host defense against influenza A virus.

Type I interferon (IFN), which includes the IFN-alpha and -beta subtypes, plays an essential role in host defense against influenza A virus. However, the relative contribution of IFN-beta remains unresolved. In mice, type I IFN is effective against influenza viruses only if the IFN-induced resistance factor Mx1 is present, though most inbred mouse strains, including the recently developed IFN-beta-deficient mice, bear only defective Mx1 alleles. We therefore generated IFN-beta-deficient mice carrying functional Mx1 alleles (designated Mx-BKO) and compared them to either wild-type mice bearing functional copies of both IFN-beta and Mx1 (designated Mx-wt) or mice carrying functional Mx1 alleles but lacking functional type I IFN receptors (designated Mx-IFNAR). Influenza A virus strain SC35M (H7N7) grew to high titers and readily formed plaques in monolayers of Mx-BKO and Mx-IFNAR embryo fibroblasts which showed no spontaneous expression of Mx1. In contrast, Mx-wt embryo fibroblasts were found to constitutively express Mx1, most likely explaining why SC35M did not grow to high titers and formed no visible plaques in such cells. In vivo challenge experiments in which SC35M was applied via the intranasal route showed that the 50% lethal dose was about 20-fold lower in Mx-BKO mice than in Mx-wt mice and that virus titers in the lungs were increased in Mx-BKO mice. The resistance of Mx-BKO mice to influenza A virus strain PR/8/34 (H1N1) was also substantially reduced, demonstrating that IFN-beta plays an important role in the defense against influenza A virus that cannot be compensated for by IFN-alpha.

Animals↗
PubMed2006

TLR-ligand stimulated interleukin-23 subunit expression and assembly is regulated differentially in murine plasmacytoid and myeloid dendritic cells.

Interleukin-23 (IL-23) is a heterodimeric cytokine composed of the p40 and p19 subunits, the first of which is also part of the IL-12 heterodimer. IL-23 induces a unique T helper cell subset to produce IL-17, which plays a critical and IL-12/IFN-gamma-independent role in autoimmunity. Plasmacytoid dendritic cells (pDC), as opposed to myeloid DC (mDC) and the closely related epidermal Langerhans cells (LC), exhibit a specific and broad range of pro-inflammatory cytokine secretion, with type I interferons representing a typical difference to classical mDC and LC. In this study we show that upon treatment with a selection of ligands for Toll-like receptor (TLR) 3, 4, 7, and 9, only mDC and LC but not pDC secreted IL-23. While pDC produced both mRNA and protein of the p40 subunit, the lack of bioactive heterodimeric IL-23 protein release was related to the fact that in these cells only the p19 mRNA was expressed which was not translated into protein. In addition to these differential findings in both DC subsets a novel p19 splice variant was identified. This analysis of transcriptional and/or post-transcriptional regulation of the IL-23 subunits p40 and p19 may help to understand the complex regulation of heterodimeric cytokines and the overlapping but distinct functions of IL-12 and IL-23. It supports the hypothesis of a coordinated adaptive immune response based on a finely tuned contribution of these cytokines by different mouse DC subsets.

Alternative Splicing↗
PubMed2006

Type I interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia.

Early viral infection is often associated with lymphopenia, a transient reduction of blood lymphocyte counts long before the onset of clinical symptoms. We have investigated lymphopenia in mice infected with vesicular stomatitis virus (VSV) or treated with the Toll-like receptor (TLR) agonists poly(I:C) and R-848. In all cases analyzed, lymphopenia was critically dependent on type I interferon receptor (IFNAR) signaling. With the use of bone marrow-chimeric mice, radioresistant cells, such as stroma and endothelium, could be excluded as type I interferon (IFN-alpha/beta) targets for the induction of lymphopenia. Instead, adoptive transfer experiments and studies in conditionally gene-targeted mice with a B- or T-cell-specific IFNAR deletion demonstrated that IFN-alpha/beta exerted a direct effect on lymphocytes that was necessary and largely sufficient to induce lymphopenia. Furthermore, after treatment with R-848, we found that other cytokines such as TNF-alpha also played a role in T-cell lymphopenia. Investigation of the molecular mechanism revealed that lymphopenia was mainly independent of G protein-coupled receptors (GPCRs) and chemokines. In an adhesion assay, B cells of poly(I:C)-treated mice showed moderately increased adhesion to ICAM-1 but not to VCAM-1. In conclusion, our data identify a new effect of direct IFN-alpha/beta stimulation of lymphocytes that profoundly affects lymphocyte redistribution.

Animals↗
PubMed2006

Selective gene transfer to T lymphocytes using coreceptor-specific [MLV(HIV)] pseudotype vectors in a transgenic mouse model.

The coreceptor usage of HIV-1 envelope proteins (Env) is mainly dependent on a defined variable region within the V3-loop of Env. Thus, retroviral vectors derived from murine leukemia virus (MLV), which have been pseudotyped with HIV-1 envelope proteins holding different V3-loops, enable selective gene delivery into either CXCR4 or CCR5 positive cultured cells. Here, we tested the distribution of CD4/CCR5-tropic [MLV(HIV)]-pseudotype vectors in transgenic mice expressing CD4 and either CXCR4 or CCR5 of human origin. The specificity of gene transfer was analyzed by ex vivo transduction of spleen cells as well as after i.v. or i.p. injection of transgenic mice. Expression of the transferred marker gene EGFP and vector sequences could be detected exclusively in lymphocytes expressing (hu)CD4 and (hu)CCR5, whereas MLV vectors pseudotyped with the VSV-G envelope glycoprotein mediated gene transfer in mice of all genotypes investigated. These data demonstrated that cell-specific gene delivery via [MLV(HIV)]-pseudotyped vectors, as previously shown for cultured cells, is also achievable in vivo.

Animals↗
PubMed2006

CD8 T cells specific for lymphocytic choriomeningitis virus require type I IFN receptor for clonal expansion.

The role of type I IFN signaling in CD8 T cells was analyzed in an adoptive transfer model using P14 TCR transgenic CD8 T cells specific for lymphocytic choriomeningitis virus (LCMV) but deficient in type I IFNR. In the present study, we demonstrate severe impairment in the capacity of P14 T cells lacking type I IFNR to expand in normal type I IFNR wild-type C57BL/6 hosts after LCMV infection. In contrast, following infection of recipient mice with recombinant vaccinia virus expressing LCMV glycoprotein, P14 T cell expansion was considerably less dependent on type I IFNR expression. Lack of type I IFNR expression by P14 T cells did not affect cell division after LCMV infection but interfered with clonal expansion. Thus, direct type I IFN signaling is essential for CD8 T cell survival in certain viral infections.

Adoptive Transfer↗
PubMed2006

Direct stimulation of T cells by type I IFN enhances the CD8+ T cell response during cross-priming.

Type I IFN (IFN-alphabeta), which is produced rapidly in response to infection, plays a key role in innate immunity and also acts as a stimulus for the adaptive immune response. We have investigated how IFN-alphabeta induces cross-priming, comparing CD8+ T cell responses generated against soluble protein Ags in the presence or absence of IFN-alphabeta. Injection of IFN-alpha was found to prolong the proliferation and expansion of Ag-specific CD8+ T cells, which was associated with marked up-regulation of IL-2 and IL-15 receptors on Ag-specific cells and expression of IL-15 in the draining lymph node. Surprisingly, neither IL-2 nor IL-15 was required for IFN-alpha-induced cross-priming. Conversely, expression of the IFN-alphabetaR by T cells was shown to be necessary for effective stimulation of the response by IFN-alpha. The finding that T cells represent direct targets of IFN-alphabeta-mediated stimulation reveals an additional mechanism by which the innate response to infection promotes adaptive immunity.

Adoptive Transfer↗
PubMed2006

Cutting edge: enhancement of antibody responses through direct stimulation of B and T cells by type I IFN.

Type I IFN (IFN-alphabeta) is induced rapidly by infection and plays a key role in innate antiviral defense. IFN-alphabeta also exerts stimulatory effects on the adaptive immune system and has been shown to enhance Ab and T cell responses. We have investigated the importance of B and T cells as direct targets of IFN-alphabeta during IFN-alpha-mediated augmentation of the Ab response against a soluble protein Ag. Strikingly, the ability of IFN-alpha to stimulate the Ab response and induce isotype switching was markedly reduced in mice in which B cells were selectively deficient for the IFN-alphabetaR. Moreover, IFN-alpha-mediated enhancement of the Ab response was also greatly impaired in mice in which T cells were selectively IFN-alphabetaR-deficient. These results indicate that IFN-alphabetaR signaling in both B and T cells plays an important role in the stimulation of Ab responses by IFN-alphabeta.

Animals↗
PubMed2006

Double-stranded RNA-binding protein E3 controls translation of viral intermediate RNA, marking an essential step in the life cycle of modified vaccinia virus Ankara.

Infection of human cells with modified vaccinia virus Ankara (MVA) activates the typical cascade-like pattern of viral early-, intermediate- and late-gene expression. In contrast, infection of human HeLa cells with MVA deleted of the E3L gene (MVA-DeltaE3L) results in high-level synthesis of intermediate RNA, but lacks viral late transcription. The viral E3 protein is thought to bind double-stranded RNA (dsRNA) and to act as an inhibitor of dsRNA-activated 2'-5'-oligoadenylate synthetase (2'-5'OA synthetase)/RNase L and protein kinase (PKR). Here, it is demonstrated that viral intermediate RNA can form RNase A/T1-resistant dsRNA, suggestive of activating both the 2'-5'OA synthetase/RNase L pathway and PKR in various human cell lines. Western blot analysis revealed that failure of late transcription in the absence of E3L function resulted from the deficiency to produce essential viral intermediate proteins, as demonstrated for vaccinia late transcription factor 2 (VLTF 2). Substantial host cell-specific differences were found in the level of activation of either RNase L or PKR. However, both rRNA degradation and phosphorylation of eukaryotic translation initiation factor-2alpha (eIF2alpha) inhibited the synthesis of VLTF 2 in human cells. Moreover, intermediate VLTF 2 and late-protein production were restored in MVA-DeltaE3L-infected mouse embryonic fibroblasts from Pkr(0/0) mice. Thus, both host-response pathways may be involved, but activity of PKR is sufficient to block the MVA molecular life cycle. These data imply that an essential function of vaccinia virus E3L is to secure translation of intermediate RNA and, thereby, expression of other viral genes.

2',5'-Oligoadenylate Synthetase↗
PubMed2006

Short-term, but not post-exposure, protection against lethal orthopoxvirus challenge after immunization with modified vaccinia virus Ankara.

Safety-tested vaccinia virus (VACV) MVA serves as a candidate third-generation vaccine against smallpox. Here, MVA immunization of mice shortly before or after lethal respiratory challenge with VACV Western Reserve was investigated. Whilst post-exposure treatment failed to protect animals, immunizations on day 2 prior to challenge were fully protective. On the day of challenge, MVA inoculation may prevent death, but not onset of severe respiratory disease. After intranasal MVA application, massive influx of leukocytes (such as neutrophils, macrophages, natural killer cells and T cells) was found in the lungs of the animals, indicating the contribution of innate responses to protection. Correspondingly, in RAG-1-/- mice, MVA inoculation delayed onset of disease significantly, but did not prevent fatal infection. Thus, short-term protection required a tight interplay of both innate and adaptive antiviral immunity. These data suggest that, in addition to conventional vaccination, MVA may serve for potent emergency prophylaxis against orthopoxvirus infection.

Animals↗
PubMed2006

Effects of type I interferons on Friend retrovirus infection.

The type I interferon (IFN) response plays an important role in the control of many viral infections. However, since there is no rodent animal model for human immunodeficiency virus, the antiviral effect of IFN-alpha and IFN-beta in retroviral infections is not well characterized. In the current study we have used the Friend virus (FV) model to determine the activity of type I interferons against a murine retrovirus. After FV infection of mice, IFN-alpha and IFN-beta could be measured between 12 and 48 h in the serum. The important role of type I IFN in the early immune defense against FV became evident when mice deficient in IFN type I receptor (IFNAR(-/-)) or IFN-beta (IFN-beta(-/-)) were infected. The levels of FV infection in plasma and in spleen were higher in both strains of knockout mice than in C57BL/6 wild-type mice. This difference was induced by an antiviral effect of IFN-alpha and IFN-beta and was most likely mediated by antiviral enzymes as well as by an effect of these IFNs on T-cell responses. Interestingly, the lack of IFNAR and IFN-beta enhanced viral loads during acute and chronic FV infection. Exogenous IFN-alpha could be used therapeutically to reduce FV replication during acute but not chronic infection. These findings indicate that type I IFN plays an important role in the immediate antiviral defense against Friend retrovirus infection.

Animals↗
PubMed2006

Prion protein-specific antibodies for therapeutic intervention of transmissible spongiform encephalopathies.

Prion diseases, also called transmissible spongiform encephalopathies, are a group of fatal neurodegenerative conditions that affect humans and a wide variety of animals. There is no therapeutic or prophylactic approach against prion diseases available at present. The causative infectious agent is the prion, also termed PrPSc, which is a pathological conformer of the cellular prion protein PrPC. Passive immunisation studies with PrPC-specific antibodies indicated that immunotherapeutic strategies directed against PrPC can prevent prion disease. In this review, putative mechanisms of antibody-mediated prion inactivation, as well as active immunisation strategies, are discussed. Special attention is given to the problem of immunological self-tolerance against PrP.

Animals↗
PubMed2005

Novel functions of tyrosine kinase 2 in the antiviral defense against murine cytomegalovirus.

We have recently reported that tyrosine kinase 2 (Tyk2)-deficient mice have a selective defect in the in vivo defense against certain viruses. In our current study we show that Tyk2 is essential for the defense against murine CMV (MCMV). In vivo challenges with MCMV revealed impaired clearance of virus from organs and decreased survival of mice in the absence of Tyk2. Our in vitro studies demonstrate that MCMV replicates to dramatically higher titers in Tyk2-deficient macrophages compared with wild-type cells. We show an essential role of type I IFN (IFN-alphabeta) in the control of MCMV replication, with a prominent role of IFN-beta. MCMV infection leads to the activation of STAT1 and STAT2 in an IFN-alphabeta receptor 1-dependent manner. Consistent with the role of Tyk2 in IFN-alphabeta signaling, activation of STAT1 and STAT2 is reduced in Tyk2-deficient cells. However, lack of Tyk2 results in impaired MCMV-mediated gene induction of only a subset of MCMV-induced IFN-alphabeta-responsive genes. Taken together, our data demonstrate a requirement for Tyk2 in the in vitro and in vivo antiviral defense against MCMV infection. In addition to the established role of Tyk2 as an amplifier of Jak/Stat signaling upon IFN-alphabeta stimulation, we provide evidence for a novel role of Tyk2 as a modifier of host responses.

Animals↗
PubMed2005

Phenotypic and biochemical analyses of BACE1- and BACE2-deficient mice.

Beta-secretase (BACE1) is the rate-limiting protease for the generation of the amyloid beta-peptide (Abeta) in Alzheimer disease. Mice in which the bace1 gene is inactivated are reported to be healthy. However, the presence of a homologous gene encoding BACE2 raises the possibility of compensatory mechanisms. Therefore, we have generated bace1, bace2, and double knockout mice. We report here that BACE1 mice display a complex phenotype. A variable but significant number of BACE1 offspring died in the first weeks after birth. The surviving mice remained smaller than their littermate controls and presented a hyperactive behavior. Electrophysiologically, subtle alterations in the steady-state inactivation of voltage-gated sodium channels in BACE1-deficient neurons were observed. In contrast, bace2 knockout mice displayed an overall healthy phenotype. However, a combined deficiency of BACE2 and BACE1 enhanced the bace1-/- lethality phenotype. At the biochemical level, we have confirmed that BACE1 deficiency results in an almost complete block of Abeta generation in neurons, but not in glia. As glia are 10 times more abundant in brain compared with neurons, our data indicate that BACE2 could indeed contribute to Abeta generation in the brains of Alzheimer disease and, in particular, Down syndrome patients. In conclusion, our data challenge the general idea of BACE1 as a safe drug target and call for some caution when claiming that no major side effects should be expected from blocking BACE1 activity.

Amyloid Precursor Protein Secretases↗
PubMed2005

Circumventing tolerance to the prion protein (PrP): vaccination with PrP-displaying retrovirus particles induces humoral immune responses against the native form of cellular PrP.

Passive immunization with antibodies directed against the cellular form of the prion protein (PrPC) can protect against prion disease. However, active immunization with recombinant prion protein has so far failed to induce antibodies directed against native PrPC expressed on the cell surface. To develop an antiprion vaccine, a retroviral display system presenting either the full-length mouse PrP (PrP209) or the C-terminal 111 amino acids (PrP111) fused to the transmembrane domain of the platelet-derived growth factor receptor was established. Western blot analysis and immunogold electron microscopy of the retroviral display particles revealed successful incorporation of the fusion proteins into the particle membrane. Interestingly, retroviral particles displaying PrP111 (PrPD111 retroparticles) showed higher incorporation efficiencies than those displaying PrP209. Already 7 days after intravenous injection of PrPD111 retroparticles, PrPC-deficient mice (Prnp(o/o)) showed high immunoglobulin M (IgM) and IgG titers specifically binding the native PrPC molecule as expressed on the surface of T cells isolated from PrPC-overexpressing transgenic mice. More importantly, heterozygous Prnp(+/o) mice and also wild-type mice showed PrPC-specific IgM and IgG antibodies upon vaccination with PrPD111 retroparticles, albeit at considerably lower levels. Bacterially expressed recombinant PrP, in contrast, was unable to evoke IgG antibodies recognizing native PrPC in wild-type mice. Thus, our data show that PrP or parts thereof can be functionally displayed on retroviral particles and that immunization with PrP retroparticles may serve as a novel promising strategy for vaccination against transmissible spongiform encephalitis.

Adjuvants, Immunologic↗
PubMed2004

A CpG-rich bidirectional promoter induces the T-cell death-associated gene 51 and downregulates an inversely oriented transcript during early T-cell activation.

The human T-cell death-associated gene 51 (TDAG51) is upregulated upon lymphocyte stimulation and in the context of ER stress. Moreover, TDAG51 plays a role in programmed cell death and tumorigenesis. We performed an extensive TDAG51 promoter analysis and found a strong CpG-rich bidirectional promoter within the first 582 nucleotides of the TDAG51 reference DNA complementary to RNA (cDNA). Upon stimulation of primary human T cells, this promoter modulated the downregulation of a newly detected head-to-head oriented transcript. Mapping of the transcription start points revealed that the 5' regions of the TDAG51 mRNA and of the newly identified transcript did not overlap in T cells. Thus, the TDAG51 locus shows an operon-like organization of two head-to-head oriented transcripts that are inversely regulated in T lymphocytes by a CpG-rich bidirectional promoter.

Base Sequence↗
PubMed2003

Antiviral immune responses in gene-targeted mice expressing the immunoglobulin heavy chain of virus-neutralizing antibodies.

Two gene-targeted immunoglobulin heavy chain transgenic mouse strains, TgH(KL25) and TgH(VI10), expressing neutralizing specificities for lymphocytic choriomeningitis virus and vesicular stomatitis virus, respectively, have been generated. Three days after lymphocytic choriomeningitis virus infection, TgH(KL25) mice showed a thymus-independent neutralizing IgM response followed by thymus-dependent (TD) IgG. In contrast, WT mice mounted only a TD IgG response around day 80. These observations indicated that not only structural properties of the virus but also immunological parameters such as the frequency of B cells were indicative for the induction of thymus-independent versus TD Ig responses. Naïve vesicular stomatitis virusspecific Ig heavy chain transgenic mice displayed greatly elevated natural antibody titers. However, despite these high naïve titers, de novo activation of naïve CD4+ T and B cells was not blocked. Therefore, B cells giving rise to natural antibodies do not participate in virus-induced antibody responses.

Animals↗