Biomedical subjects
J Thalhamer
Publications and source records attributed to J Thalhamer.
Nitrocellulose-based immunoaffinity chromatography.
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Use of genetically modified glial cells overexpressing laminin alpha1-chain peptides in neurite outgrowth studies.
1. C6 glioma cells were transfected with two constructs carrying C-terminal laminin alpha1-chain sequences of 117 and 114 bp length, respectively. These sequences are specifically known to code for peptides which have neurite-promoting activity. 2. The stable expression and secretion of the two peptides was detected by Northern and Western blot analysis. 3. Primary neuronal cultures derived from embryonic mouse forebrain were cocultured with these transfected cells and exhibited a substantial increase in neurite outgrowth and in survival time. Conditioned media from the transfected cells generated similar effects. 4. Organotypic cultures from embryonic mouse brain were used as a second system as being closer to the in vivo situation. Again, coculture of brain slices with transfected cells or treatment with laminin peptide-containing media increased neuronal outgrowth.
Genetic vaccination against malaria infection by intradermal and epidermal injections of a plasmid containing the gene encoding the Plasmodium berghei circumsporozoite protein.
The circumsporozoite protein (CSP) from the surface of sporozoite stage Plasmodium sp. malaria parasites is among the most important of the malaria vaccine candidates. Gene gun injection of genetic vaccines encoding Plasmodium berghei CSP induces a significant protective effect against sporozoite challenge; however, intramuscular injection does not. In the present study we compared the immune responses and protective effects induced by P. berghei CSP genetic vaccines delivered intradermally with a needle or epidermally with a gene gun. Mice were immunized three times at 4-week intervals and challenged by a single infectious mosquito bite. Although 50 times more DNA was administered by needle than by gene gun, the latter method induced significantly greater protection against infection. Intradermal injection of the CSP genetic vaccine induced a strong Th1-type immune response characterized by a dominant CSP-specific immunoglobulin G2a (IgG2a) humoral response and high levels of gamma interferon produced by splenic T cells. Gene gun injection induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio and significant IgE production. Neither method generated measurable cytotoxic T lymphocyte activity. The results indicate that a gene gun-mediated CS-specific Th2-type response may be best for protecting against malarial sporozoite infection when the route of parasite entry is via mosquito bite.
Retinoic acid receptor status in mouse spleen during a primary immune response against beta-galactosidase.
OBJECTIVE: Evaluation of the dynamics of all-trans retinoic acid receptor binding properties in mouse spleen nuclear extracts during a primary immune response against beta-galactosidase. METHODS: Female BALB/c mice, aged between 5 and 6 weeks were immunized intradermally into the shaved back (4 spots each) with 100 microg beta-galactosidase in 100 microl sterile phosphate buffered saline (pH 7.2) and blood was taken by tail bleeding on days 0 (preimmune serum), 4 and 6. Production of antibody in serum and the detection of cytokines (IL-4, IFN-gamma) from proliferation supernatants were determined by ELISA. Antigen-specific proliferation assay of isolated spleen cells was based on [3H]-thymidine incorporation measured in a liquid scintillation counter. Both, the maximal binding capacity (Bmax) and the affinity (Ka) of all-trans retinoic acid nuclear receptors (RAR) were evaluated according to Brtko (1994). RESULTS AND CONCLUSIONS: Injection of beta-galactosidase induced the first detectable antibody responses on day 4 (IgM) and on day 6 (IgG). These points of time, reflecting the early and the mature immune response served to measure the antigen-specific proliferation and production of IL-4 and IFN-gamma in the supernatants of the proliferation cultures as well as all-trans retinoic acid receptor (RAR) binding characteristics in spleen nuclear proteins. The RAR Bmax was significantly (P<0.05) decreased only at the time of the first specific IgG antibody production. CONCLUSIONS: The data obtained indicate the involvement of RAR in the late phase of an in vivo immune response.
Improvement of the immune response against plasmid DNA encoding OspC of Borrelia by an ER-targeting leader sequence.
The present study outlines the characterization of a DNA-based immune response against the OspC antigen, one of the most promising candidates for a Borrelia vaccine. Balb/c mice were injected intradermally with plasmid DNA encoding the OspC gene (lacking the natural leader sequence) under transcriptional control of the cytomegalovirus (CMV) promotor. Immunization with this construct elicited only a marginal response, which was drastically improved by a fusion construct containing the human tissue plasminogen activator (hTPA) signal sequence. The results indicate that for DNA-based immunization against OspC an ER-targeting signal may be necessary for both antibody production as well as cellular immune responses.
DNA immunization is associated with increased activity of type I iodothyronine 5'-deiodinase in mouse liver.
Inflammatory cytokines in vitro are believed to be involved in the regulation of type I iodothyronine 5'-deiodinase (5'-DI) activity. The present study was undertaken to investigate in vivo effects of DNA immunization of mice on the 5'-DI activity in the liver. A mammalian expression vector encoding the beta-galactosidase (pCMV-betagal) was used for intradermal immunization. Furthermore, immunostimulatory CpG motifs, which induce the expression of IL-6, IL-12, IL-18, TNF-alpha/beta and IFN-gamma were coinjected as oligodeoxynucleotides. From our data we conclude that the activity of 5'-DI in mouse liver when compared to non-immunized animals (100%) was found to be significantly enhanced by DNA immunization 2 weeks (175.7%) or 3 weeks (192.6%) after the plasmid injection. In addition, the activity of the 5'-DI in mouse liver was markedly enhanced 2 weeks (252.4%) or 3 weeks (243.3%) after the injection when CpG motifs were applied together with the plasmid DNA.
Immune responses after immunization with plasmid DNA encoding Bet v 1, the major allergen of birch pollen.
BACKGROUND: Immunization with plasmid DNA encoding various antigens is a promising method in vaccine research. Recent studies also indicate that DNA-based immunization might represent a potential approach in allergen-specific immunotherapy. OBJECTIVE: In this study we have characterized the immune responses induced by recombinant Bet v 1a and plasmid DNA encoding for Bet v 1a, the major allergen of birch pollen in a mouse system. METHODS: Balb/c mice were injected intraperitoneally with recombinant Bet v 1a and intradermally with plasmid DNA encoding for the gene of Bet v 1a (pCMV-Bet). In addition, the effect of immunostimulatory DNA sequences was investigated by appending CpG motifs to the gene of Bet v 1a, coinjecting CpG-oligodeoxynucleotides together with the pCMV-Bet construct, or both. IgE and IgG antibody responses, as well as IgG subclasses, were measured by ELISA in sera after each immunization. IFN-gamma and IL-4 levels were also measured by ELISA in sera and supernatants of allergen-stimulated spleen cells. RESULTS: The primary humoral response to a single treatment with pCMV-Bet was very weak, but the reaction could be boosted to higher levels by 2 additional injections. On the other hand, proliferation assays of spleen cells and measurements of cytokine levels already indicated a cellular response after the first injection of plasmid DNA. After 2 immunizations with pCMV-Bet, the ratio of IgG1 to IgG2a pointed to a TH1 subclass profile. IgE was not detectable in any group at any time during the immune reaction. Accordingly, IL-4 levels were markedly reduced in the serum, as well as in the supernatants, of stimulated spleen cells. Animals immunized with pCMV-Bet containing appended CpG motifs at the 3' end of the Bet v 1a gene and/or with the CpG-ODN GCTAGACGTTAGCGT plus pCMV-Bet displayed reduced humoral responses against Bet v 1a when compared with animals injected with pCMV-Bet alone. The levels of IFN-gamma measured after allergen stimulation of isolated spleen cells were significantly higher in animals immunized with pCMV-Bet plus CpG motifs than with pCMV-Bet alone. Immunization with recombinant Bet v 1a protein elicited a strong TH2 -type response, including IgE production, a high titer of IgG1, and IL-4 production in both serum and supernatants of proliferation cultures. CONCLUSION: In contrast to immunization with protein, DNA immunization induces a strong TH1 -type response against a relevant inhalant allergen. Our data support the concept of developing a novel type of allergen immunotherapy based on plasmid DNA immunization.
Isoforms of the major allergen of birch pollen induce different immune responses after genetic immunization.
BACKGROUND: Recent publications indicate that immunization with plasmid DNA encoding allergens might represent a potential approach in allergen-specific immunotherapy. OBJECTIVE: In the present study we have compared the immune responses induced by plasmid DNA encoding for two isoforms of Bet v 1, the major allergen of birch pollen. METHODS: BALB/c mice were injected intradermally with plasmid DNA encoding for the genes of Bet v 1a (pCMV-Beta) and Bet v 1d (pCMV-Betd). In addition, the effect of immunostimulatory DNA sequences was investigated by appending and/or coinjecting CpG motifs. Antibody responses and IFN-gamma and IL-4 levels were measured by ELISA. Allergen-specific proliferation was determined by incorporation of [(3)H]-thymidine. RESULTS: The two isoforms induced a similar humoral response. The lack of any IgE production and the ratio of IgG1 to IgG2a clearly indicated a Th-1-type response. The antisera against both isoforms were highly cross-reactive, which was supported by the energy plot indicating similar folding of the two protein isoforms. However, determination of IFN-gamma and IL-4 in the serum elicited a strikingly different cytokine profile during the course of the immune response. In contrast to pCMV-Beta, pCMV-Betd caused no significant allergen-specific proliferation and induced only marginal levels of the key cytokines. CONCLUSIONS: Based on the assumption that the induction of a strong Th-1 type response is a prerequisite for successful treatment of allergy, our results favor the use of isoform Bet v 1a in combination with CpG motifs for a novel type of allergen immunotherapy based on plasmid DNA immunization. Additionally, the data also confirm the assumption that the antigen itself can have a marked influence on the immune response after genetic immunization.
Cytokeratin 17 as an immunohistochemical marker for intramural cytotrophoblast in human first trimester uteroplacental arteries.
Trophoblast cells, as blastocyst-wall derivatives, are of epithelial origin and differentiate initially into syncytiotrophoblast and cytotrophoblast subpopulations. Cyto- and syncytiotrophoblasts are the two cell types present in the surface cell layers of placental villi. Cytotrophoblastic cells lie in contact with the basal lamina and are the proliferating stem cells that guarantee cytotrophoblast and syncytiotrophoblast persistence. Implantation and placenta formation are mainly based on these two cell types. Villous cytotrophoblasts are the stem cells for all extravillous trophoblast subpopulations, which exhibit strictly regulated invasiveness. One aspect of extravillous trophoblasts is that they invade maternal endometrial spiral arteries and dilate them in order to achieve sufficient fetal blood supply. During this process, trophoblast cells, which are located in the remodelled uteroplacental artery walls, are thus defined as intramural cytotrophoblasts. Trophoblast differentiation is accompanied and defined by alterations in, for example, the translation pattern for cytokeratin genes. In an immunohistochemical study, we have demonstrated that only intramural cytotrophoblasts, from all the trophoblast populations of the junctional zone, express cytokeratin 17. Furthermore, cell shape and vascular architecture indicate that, in human placenta, intra-arterial trophoblast cells reach their destination by migration through the endometrial interstitium with consecutive intravasation. Cytokeratin 17, in particular, can therefore be used as a specific immunohistochemical marker for the intramural trophoblast subpopulation.
The effect of antigen stimulation on splenic transplants.
The present paper deals with the regeneration of splenic tissue after autologous transplantation. Control and transplanted rats (60 days after operation (10(6) cells per injection). The effect of a primary response was studied by a single injection, long-lasting bacteraemia was imitated by 5 injections in weekly intervals. Spleens and transplants were investigated by flow-cytometry and immunohistochemistry. Additionally, the proliferation activity and the specific antibody production against Escherichia coli proteins were tested. Flow-cytometric analysis showed altered behaviour of T-helper cells and B-cells in transplants following a primary response, whereas in the multiple injection group a difference between the splenic and transplant response was restricted to macrophages and MHC II+ cells. The results of the morphometric analysis revealed that the cellular composition of unstimulated transplants was very similar to that of the spleen with some subtle alterations. Only the marginal zone showed more striking differences concerning the homing of several cell classes. Under stimulatory conditions, these subtle alterations became more drastic so that CD5+ cells, B-cells and macrophages responded in an abnormal manner in both groups. The analysis of thymidine kinase disclosed decreased activity in the spleen after weekly antigen stimulation. The stimulation index of all transplant groups was significantly lower than that of the spleen. The specific antibody (IgG) production after a single immunization was highest in the transplant group. All groups responded after the multiple challenge. In conclusion, the results demonstrate that splenic transplants differs in several, but subtle aspects from normal splenic tissue. The main reason for most of these alterations may be a slightly misguided recirculation and/or homing of cells.
A non-expressed transgene as a cell marker for the investigation of cellular traffic after splenic autotransplantation.
The investigation of cellular interactions during the regeneration of splenic transplants depends upon distinguishing host cells from graft derived cells. As a cell marker which, in contrast to many other marker systems, does not affect histocompatibility, the non-expressed transgene mMT-HGHRH-1 was introduced by mating into NMRI inbred mice to generate the transgenic mouse line BSM. By transplanting non-transgenic NMRI splenic tissue into transgenic BSM hosts, host derived cells could be identified in the transplants by in situ hybridization after regeneration. In a reciprocal approach, implant derived cells were detected in a transgenic transplant after 3 weeks of regeneration in a non-transgenic host. Relative amounts of transgenic cells in transplant cross-sections were estimated from the signal intensity of autoradiographs by densitometry and computer analysis. This approach could be broadly applied in studies of transplantation systems.
The lipocalin Xlcpl1 expressed in the neural plate of Xenopus laevis embryos is a secreted retinaldehyde binding protein.
The cellular and structural properties and binding capabilities of a lipocalin expressed in the early neural plate of Xenopus laevis embryos and the adult choroid plexus have been investigated. It was found that this lipocalin, termed Xlcpl1, binds retinal at a nanomolar concentration, retinoic acid in the micromolar range, but does not show binding to retinol. Furthermore, this protein also binds D/L thyroxine. The Xlcpl1 cDNA was expressed in cell culture using the vaccinia virus expression system. In AtT20 cells, Xlcpl1 was secreted via the constitutive secretory pathway. We therefore assume that cpl1 binds retinaldehyde during the transport through the compartments of the secretory pathway that are considered to be the storage compartments of retinoids. Therefore, cpl1-expressing cells will secrete the precursors of active retinoids such as retinoic acid isomers. These retinoids may enter the cytosol by diffusion or receptor-controlled mechanisms, as has been shown for exogenously applied retinoids. Based on these data, it is suggested that cpl1 is an integral member of the retinoid signaling pathway and, therefore, it plays a key role in pattern formation in early embryonic development.
Removal of template after random priming yields DNA probes of maximal specific activity.
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Human first-trimester placenta intra-arterial trophoblast cells express the neural cell adhesion molecule.
The supposed influence of endometrial natural killer (NK) cells on the trophoblast invasion activities especially on intravasation of uteroplacental arteries in the non-pathogenic human first-trimester placenta was studied by means of immunohistochemistry. To identify extravillous trophoblast cells, smooth muscle cells, endothelia, endometrial glands, decidual stroma cells and endometrial NK cells, antibodies against cytokeratins, vimentin, smooth muscle cells, epithelium specific antigen and endothelial cells were employed. Furthermore, the immunohistochemical distribution patterns of CD56, CD57 and CD94 were studied and compared with the localization of invading trophoblast cells. Remodelling and dilatation of uteroplacental arteries starts before trophoblast cells can be found in the vicinity of the vessels. Nevertheless, subsequent trophoblast invasion of the arterial wall will lead to media destruction and intravasation only on focally restricted areas. This process is accompanied by the disappearance of endothelial cells and the immediate expression of the neural cell adhesion molecule (N-CAM, CD56) by intra-arterial trophoblast cells, which are eventually beginning to form intraluminal plugs. These findings led us to the conclusion that in the human pregnancy-induced physiological changes of the uteroplacental blood flow and the peripheral blood NK cell activity is not only, but also, due to the effect of CD56 expression by intra-arterial trophoblast cells.
Regeneration of splenic stromal elements.
Splenic regeneration represents an interesting phenomenon both in relation to its role as a model system (to study the development of the complex three-dimensional architecture of an immunological organ) and because of the clinical application, namely autotransplantation of spleen. The latter is one of the attempts to restore splenic functions after splenectomy, which is known to increase a life-long risk of fatal sepsis. However, splenic functions of autotransplanted splenic tissue are known to be highly dependent on the recovery of the complex microenvironment and immunoarchitecture of the splenic compartments during the regeneration processes, but the elements inducing splenic reorganization are still unknown. Therefore, the present work investigates whether splenic stroma depleted of cells is able to induce regenerative processes after implantation. In addition, we tried to recombine stromal tissue with selected cell populations to study their influence. Cell-free stromal tissue induced angiogenesis and to a lesser extent also attracted the immigration of lymphocytes during the first 60 days of regeneration. However, after this period of regeneration, the transplants began to degenerate and were resorbed. The recombination of stromal tissue with mitogen-stimulated spleen cells only resulted in intensifying the degenerative processes, and all implants were resorbed after 120 days. Except that in the first 30 days there were some accumulations of lymphocytes that resembled primitive follicles, no splenic compartments such as red pulp, periarteriolar lymphoid sheath, or marginal zone could be detected in any of the transplants. From these results it can be concluded that splenic stroma can induce the primary events of splenic regeneration (like angiogenesis), but is not able to provide an appropriate microenvironment and immunoarchitecture for a correct repopulation and differentiation of cells. Furthermore, the recombination experiments point to a minor role of T-cells and possibly an important role for accessory cells in splenic regeneration.
Particulate nitrocellulose as a solid phase for protein immobilization in immuno-affinity chromatography.
The use of nitrocellulose paper as a solid phase matrix for protein immobilization and its application in immunoaffinity chromatography is described. Pieces of nitrocellulose paper were frozen in liquid nitrogen and ground to a powder (NCP) which was then fractionated according to particle size by repeated sedimentation/resuspension cycles in water. The flow properties of different NCP fractions were compared with those of Sephadex G-50. Protein binding capacity and binding dynamics were investigated in a model system with bovine serum albumin (BSA) in phosphate buffered saline. Applications of the material are illustrated by batch chromatography for the removal of anti-carrier protein antibodies from a hapten antiserum and by affinity purification of a hapten-specific antibody fraction using NaSCN elution from haptenated NCP. Furthermore, the applicability of the material in column chromatography is demonstrated by elution of a monospecific fraction of anti-fluorescein antibodies from a hapten/carrier mixed bed column by excess of soluble hapten. The results demonstrate that NCP chromatography appears to be a cheap and useful alternative to many other chromatographic media used for protein immobilization.
Improvement of antisera raised against complex antigen mixtures by the use of heterologous sources of antigen for immunization.
Polyspecific antisera against antigen mixtures are important tools for many experimental purposes. However, following immunization with extracts containing a great number of different proteins, many antigens remain non-immunogenic. Therefore, the improvement of the antibody spectrum of antisera is an essential goal in maximising the power and applicability of many serological analyses. In the present report we demonstrate that the additional use of heterologous (i.e., antigenically related, but not identical) antigen mixtures for immunization increases the variety of antibodies in polyspecific antisera as well as the antibody titer against weak immunogens.