Biomedical subjects

J Thalhamer

Publications and source records attributed to J Thalhamer.

50 records · Page 3Linked to original sources

PubMed1992

A method for the detection of serologically crossreacting antigens both within and between protein mixtures: the western cross blot.

The method described in the present paper permits the detection of antigenically related proteins within or between different antigen mixtures. For this purpose two separate SDS-polyacrylamide gels are run (the antigen mixture is applied to the entire width of the gel) and each gel is blotted on to a separate nitrocellulose paper. One sheet ('donor sheet') is incubated with antiserum and placed on to the other blot ('receptor sheet') so that the antigen bands are perpendicular to each other. Subsequently the antibodies from the donor sheet are blotted on to the receptor sheet in alkaline borate buffer containing 1 M NaSCN, which dissociates antigen-antibody complexes. After the removal of the donor sheet the receptor sheet containing the transblotted antibodies is equilibrated in phosphate-buffered saline in order to reconstitute the binding conditions. Antibodies which have bound to receptor antigens during this equilibration period are detected by the use of a peroxidase labelled 2nd antibody. Because each band on the donor sheet crosses each band on the receptor sheet during the transfer, the antibodies from each band of the donor sheet are able to react with each antigen band of the receptor sheet. The validity of the method was established by demonstrating the crossreactivity of a polyclonal antiserum against the intact bovine gamma globulin molecule with the heavy and light chain subunits (and partial reduction products of the molecule) and the absence of crossreactivity between heavy and light chains. In a second experiment crossreacting antigens of different molecular weight were detected within several strains of Escherichia coli. In addition, comparisons of different strains of Escherichia coli revealed crossreacting antigens of identical as well as of different molecular weight.

Blotting, Western↗

PubMed1992

Regeneration of autotransplanted splenic tissue at different implantation sites.

Inbred animals (Lewis rats) were used to investigate the regeneration of autologously implanted splenic tissue at intra-omental and subcutaneous sites. Quantitative immunohistology with monoclonal antibodies against lymphocytes and macrophages was performed to analyse the cell density of red pulp (RP), periarteriolar lymphoid sheath (PALS), marginal zone (MZ) and follicle, 7-180 days after transplantation. Antigenic, allogeneic and mitogenic stimulation and Northern blotting were also performed. Transplant groups differed from spleen only in the reduced size of PALS; however, quantitative analysis demonstrated subtle differences between spleen and transplants. The cell density of B-cells and ED-1+ macrophages was reduced in the RP, Tsupp/cyt-cells were decreased and B-cells increased in PALS, and B-cells and Thelper-cells reduced in the MZ. No differences could be detected between the transplant groups. Flow-cytometric analysis of cell suspensions from spleen and transplants revealed a reduction of T-cells (OX-19+), MHC-I and transferrin-receptor-bearing cells in both transplant groups, and a decrease in the number of Thelper-cells and ED-3+ macrophages in subcutaneous transplants. Both transplant groups were defective regarding the allogeneic and pokeweed mitogen response. Aberration of the lipopolysaccharide response was restricted to subcutaneous transplants, which additionally showed abnormal expression of interferon-gamma, interleukin-5 and interleukin-6 mRNA. Thus, subtle alterations of the newly developed microenvironment and/or lymphocyte-homing may influence the regeneration of splenic tissue; the implantation site may represent an important parameter in functional reorganisation.

Animals↗

PubMed1992

Immunoarchitecture and specific functions of splenic autotransplants at different implantation sites.

The present study deals with the morphological and functional development of intraomentally and subcutaneously implanted splenic tissue. Spleens and splenic transplants from 138 Lewis rats were investigated with immunohistological, immunological and molecular biological methods at different times after operation (up to 200 days postoperatively). The analysis of the development revealed a nonsignificant reduction concerning the weight of subcutaneous replants and a nonsignificant decrease of the weight of female transplants of both groups at different phases after operation. The cell composition of cell suspensions from spleen and both transplant types showed a deficiency of T, B, MHC-I+ cells and a certain macrophage subset (ED-3+ cells) in transplants. In a quantitative immunohistological analysis of compartments (red pulp, periarteriolar lymphoid sheaths, marginal zone and follicles) the T cell reduction was related to the Tsupp/cyt cells and T cell receptor bearing cells in the periarteriolar lymphoid sheaths, whereas the density of T helper cells was normal. In addition, a different homing of kappa-light chain positive and leukocyte common antigen (B cell type)-positive B cells in follicles and marginal zone was detected. The amount of two macrophage subsets (ED-1+ and ED-2+ cells) was increased in the red pulp. Only minor differences in the immunoarchitecture of transplants at different implantation sites were measured. A functional analysis of spleen compared to both transplant groups elicited a B cell defect after LPS stimulation in subcutaneous transplants and a reduced allogeneic response of both transplant types but a normal proliferation of T cells after ConA stimulation and a correct IgM antibody response against sheep red blood cells. The in vivo mRNA expression and the expression kinetics of interferon-gamma and granulocyte-macrophage colony-stimulating factor after antigen stimulation differed in both transplant groups with a remarkable permanent expression of both mediators in subcutaneous transplants. It can be summarized that the results clearly indicate a development of spleen-like immunoarchitecture of intraomental replants with subtle cellular, functional and molecular alterations. In contrast, despite a comparable development, some severe functional defects occurred in subcutaneous implants pointing out the important role of interactions between the regenerating splenic tissue and the target tissue on a functional and molecular level.

Abdominal Muscles↗

PubMed1992

[Autotransplantation of the spleen in rats: development, function and cytokine expression in intra-omental and subcutaneous regeneration].

The implantation of splenic tissue at different implantation sites (intraomental and subcutaneous) into one animal (Lewis rats) results in the development of splenic nodules at both sites. In a quantitative immunohistological analysis of splenic compartments such as red pulp (RP), periarteriolar lymphoid sheaths (PALS), marginal zone (MZ) and follicles (F) the T-cell reduction was related to the T(helper) cells in the MZ and T(supp/cyt) cells in the PALS. In contrast, the cell density of B cells and ED-1+ macrophages in the PALS and T(supp/cyt) cells in the MZ was increased. Significant differences between the implantation sites were restricted to CD5+ cells (thymocytes and T cells) in the MZ and OX-33+ cells (B cells with LCAB antigen) in the PALS. The reorganisation of the compartments of subcutaneous implants showed a delay of one week as compared with omental ones. Functional assays like haemolytic plaque assay, mitogen stimulation and mixed lymphocyte assay elicited an analogous delay of the functional maturation of IgM-positive B cells, a reduced proliferation of both transplant groups after pokeweed mitogen (PWM) stimulation, a decreased response after lipopolysaccharide (LPS) stimulation in solely subcutaneous replants and no differences concerning the mitogens concanavalin A (ConA) and phytohaemagglutinin (PHA). Both transplant groups showed a significantly reduced allogeneic response. The results of the functional analysis and the abnormal mRNA expression of Il-5, Il-6 (Interleukin 5 and 6), GMCSF (Granulocyte-Macrophage-Colony-Stimulation-Factor) and IFN-gamma (Interferon gamma) in subcutaneous replants indicate subtle molecular alterations (independent of a spleen-like immunoarchitecture) at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Incidence of septic and thromboembolic-related deaths after splenectomy in adults.

In a review of 37,012 autopsies over the last 20 years 202 deceased adults who had had a splenectomy were investigated. The incidence of infections and thromboembolic complications related to death in these patients was compared with that of a matched deceased population (n = 403) who had not undergone splenectomy. Death-related pneumonia was diagnosed frequently in the splenectomy group and to a lesser extent in the control group (57.9 versus 24.1 per cent, P less than 0.001). Lethal sepsis with multiple organ failure occurred in 6.9 per cent of the splenectomy group and in 1.5 per cent of the controls (P less than 0.001). Purulent pyelonephritis was observed in 7.9 per cent of the splenectomy group and was significantly more frequent than in the control group with its rate of 2.2 per cent (P less than 0.001). Finally, pulmonary embolism was the major or a contributory cause of death more often in the splenectomy group than in the control group (35.6 versus 9.7 per cent, P less than 0.001). We conclude that splenectomy generates a considerable life-long risk of severe infection and of thromboembolism.

Adult↗

PubMed1989

Alteration of proliferation and subtle changes of protein synthesis in autologous transplanted spleens.

In the case of massive splenic rupture, heterotopic autologous transplantation of splenic tissue into the omentum majus may be used to restore splenic function. Yet little is known about specific functions of the transplants compared to the normal spleen. The goal of this study was to get more information about immunologic functions and protein expression in splenic transplants. As an animal model we used the pig, whose splenic morphology and immunoarchitecture is similar to that of the human spleen. Histologic examination of transplants revealed structures that were comparable to normal spleens (consisting of red and white pulp and including germinal centers). Immunologic tests such as the hemolytic plaque assay and mitogen stimulation revealed that the number of plaque-forming cells was not changed significantly, but the stimulation index for T cells was drastically increased in the autotransplants. Electrophoresis and immunochemical methods showed differences in the protein patterns between both tissues. Several proteins were found to be produced only in the spleen, or were produced in much higher amounts in the spleen than in the splenic transplants. More information about these differences between spleen and splenic transplants is needed before we can recommend a general clinical application of autologous spleen transplantation.

Animals↗

PubMed1988

Antigenic competition in the immune response against protein mixtures: strain-specific non-immunogenicity of Escherichia coli antigens.

Antigenic competition is argued to impair the immune response on the level of accessory cell-dependent antigen presentation to responsive T-cells (regulated by MHC encoded Ir gene products). A possible influence of these mechanisms on in vivo immunization with antigen mixtures was investigated by using cytoplasmic extracts of four different strains of Escherichia coli as antigen sources for immunizing rabbits. The immune response against these antigen mixtures was tested by crossed immune electrophoresis (CIE) and immunoblotting (IB) in a homologous system (a given antigen extract of one strain against the corresponding antisera) and in the heterologous system (antigen extract of one strain against the antisera of different other strains). Several proteins were non-immunogenic in the extract of one strain but elicited good antibody responses in other strains. One of the strain-specific non-immunogenic proteins was purified and revealed a normal immune response upon immunization. The data suggest that different antigenic competition effects are induced by different protein compositions of antigen mixtures. This strain-specific competition seems to determine the immunogenicity of certain molecules (and not only the immunogenic properties of the molecules themselves). Furthermore this method offers a practical approach to increase the antibody production against weak immunogens in antigen mixtures.

Animals↗

PubMed1987

Perfusion of autologous splenic grafts in correlation with specific immunological functions--an experimental study in pigs.

The study deals with the immunological capacity and perfusion rate of splenic grafts in pigs after autologous transplantation into the omentum majus. 12 months after the operation, the weight and relative perfusion of the regenerated tissue was clearly reduced. Independent of this fact, the splenic grafts could balance out an IgM-deficiency, observed due to splenectomy, 4 months after the transplantation. Furthermore, the number of antibody forming cells in the grafts was only slightly decreased compared to the normal spleen. These results show that regenerated splenic tissue is able to carry out specific immunological functions. One of the limiting factors in preventing postsplenectomy sequelae (i.e., sepsis) seems to be the poor regeneration weight and low perfusion of the grafts.

Animals↗

PubMed1986

[Immunologic capacity and blood circulation of splenic autotransplants--experimental results in swine].

The study deals with the immunological capacity and perfusion rate of splenic grafts in pigs after autologous transplantation into the omentum majus. 12 months after operation the weight and relative blood flow of the regenerated splenic tissue were clearly reduced. The number of antibody forming cells in the grafts was only slightly decreased compared to the normal spleen, whereas the autotransplants could balance a serum-IgM-deficiency, observed due to splenectomy, 4 months after grafting. Furthermore the mitogenic stimulation of T- and B-lymphocytes showed different proliferation rates for the autotransplants compared to the spleen.

Animals↗

PubMed1986

The role of the spleen and splenic autotransplants in clearing experimental bacteremia caused by the gram-negative bacterium Escherichia coli.

Several clinical and laboratory studies have demonstrated the risk of sepsis in connection with encapsulated bacteria. The importance of clearing these organisms by the spleen is now well accepted. In contrast, the present work deals with the clearance of non-encapsulated gram-negative bacteria (Escherichia coli). The animals used for the experiments (a heterogeneous population of rabbits) were divided into a control group, a splenectomized group, and an autotransplanted group. The histological examination of the splenic transplants revealed typical splenic tissue including lymphatic follicles with germinal activity 42 days after transplantation. For estimation of the clearance capacity different amounts of bacteria were injected i.v. into the rabbits, and colony-forming bacterial cells in the blood were counted at certain intervals. In the control group no bacteria could be detected in the blood after 7 min. All animals of the splenectomized and autotransplanted groups showed a remarkable decrease in clearance efficiency (no bacteria in the blood after 19 min). No difference in the clearance kinetics could be shown between splenectomized and autotransplanted animals. Measuring the uptake of bacterial cells into different organs elicited low incorporation in the spleen as compared to non-immunocompetent organs, but no difference between normal spleen and splenic replants. However, saturation with E. coli cells reached higher limits in the normal spleen than in autotransplants. The immunologic capacity with respect to IgM-producing lymphocytes was measured by the hemolytic plaque assay. The results showed a severe malfunction of the autotransplants as compared to the normal spleen (only 2% of the activity of the control group). Vaccination against E. coli increased the clearance efficiency in all three groups. The data presented in this paper point out that several functions of the spleen cannot be carried out by autotransplants. The reasons, therefore, may be limited transplant mass and/or decreased specific functions.

Animals↗

PubMed1986

[Should the injured spleen be preserved?].

Splenectomy is primarily followed by reduced defence against infection due to encapsulated bacteria, such as pneumococci. Organ preservation, therefore, has been emphasised in the treatment of traumatic rupture of the spleen, especially when it comes to patients in childhood. There are several practicable approaches to orthotopic preservation of the spleen, including conservative treatment of patients for smallest ruptures under stringent clinical control and, even more, fibrin sealing. Growth in accurate knowledge of the segmental structure of the spleen have enabled partial removal of the organ along the poorly vascularised intersegmental regions. Autologous transplantation of splenic pulp into the major omentum should be considered an alternative to splenectomy alone in strictly selected cases of massive splenic rupture.

Antibody Formation↗

PubMed1985

Passive immunization: a method of enhancing the immune response against antigen mixtures.

Antigenic competition is known to be a widespread phenomenon when using crude extracts of antigens (e.g., Escherichia coli cytoplasmic proteins) for immunization. This non-specific form of immune suppression can be partially overcome by passive immunization with antibodies against dominant antigens (which are the suppressive molecules) before injection of the antigenic mixture. Blocking these immunodominant antigens or antigenic determinants by a passively administered antibody permits antibody responses against hitherto weakly or non-immunogenic molecules.

Animals↗

PubMed1984

Cascade immunization: a method of obtaining polyspecific antisera against crude fractions of antigens.

Between 20 and 30 precipitation lines are usually obtained by crossed immune electrophoresis of an Escherichia coli cytoplasmic extract against antisera produced against that extract in individual rabbits. With a combination of several such antisera, the number of precipitation lines increases to 30-40. Nevertheless, extracts as used in this work contain many antigens in addition to those thus detected. Intermolecular immune competition may be avoided by removing strong immunogens from the extracts. The remaining antigens which give no immune response in the primary immunization are used for further immunization. New antibody production against 8-14 additional antigens occurs after one such 'cascade immunization' step. Separation of strong from weak immunogens is performed by preparative CIE and the use of immuno-affinity columns. The procedure is called cascade immunization because it involves repeated removal of antigens and production of further antisera directed against antigens in the remainder.

Animals↗

PubMedDate not supplied

The influence of CpG motifs on a protein or DNA-based Th2-type immune response against major pollen allergens Bet v 1a, Phl p 2 and Escherichia coli-derived beta-galactosidase.

BACKGROUND: DNA immunization and protein immunization with CpG motifs as adjuvants represent promising approaches in allergen-specific immunotherapy. OBJECTIVE: We investigated the effect of coinjection or prepriming with CpG-ODN on Th2-type responses induced by gene gun and protein immunization. METHODS: BALB/c mice were immunized with the gene gun using plasmid DNA containing the cDNAs coding for the genes of Bet v 1a, Phl p 2 and beta-galactosidase or with the purified Al(OH)(3)-adsorbed proteins. In addition, CpG-ODN were applied by coinjection or by prepriming treatment. Antibody and cytokine responses were measured by ELISA, proliferative and cytotoxic responses were determined by standard labeling procedures. Furthermore, the allergenic activity of sera was measured by passive cutaneous anaphylaxis. RESULTS: Gene gun immunization and protein immunization induced a clear Th2-type response for all antigens. The Th1-promoting effect of CpG-ODN coinjection together with gene gun immunization was restricted to beta-galactosidase as indicated by the increase of IgG2a and a marked expression of IFN-gamma. CpG motifs also increased the specific cytotoxic response against beta-galactosidase. Prepriming with CpG-ODN and gene gun or protein immunization with Bet v 1a exhibited no significant difference to the non-CpG control group. However, sera from mice preprimed with CpG-ODN induced no anaphylaxis with gene gun immunization, but with protein immunization. CONCLUSIONS: The effect of CpG motifs in vivo depends on a variety of parameters like the nature of the antigen and the immunization modality. Furthermore, our studies indicate that a combination of CpG + DNA immunization may be more effective in antagonizing Th2 responses than the combination of CpG + protein immunization.

Adjuvants, Immunologic↗