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Biomedical subjects

E Claassen

Publications and source records attributed to E Claassen.

At least 127 records · Page 7Linked to original sources
PubMed1987

In vivo effects of lipopolysaccharide on lymphoid and non-lymphoid cells in the mouse spleen. Suppressive and adjuvant effects of LPS on the development of specific antibody forming cells in situ.

Mice were immunized i.v. with 2,4,6-trinitrophenyl (TNP)-carrier conjugates and received lipopolysaccharide (LPS) before, simultaneously with or after these antigens, in order to study the effects of LPS on the in situ development of anti-TNP antibody forming cells in the spleen. LPS given before, simultaneously with or shortly after the thymus dependent (TD) antigen TNP-keyhole limpet haemocyanin (KLH) suppressed the development of anti-TNP antibody forming cells in the spleen, but serum IgM-anti-TNP titres were not reduced, suggesting a selective inhibiting effect of LPS on the local immune response in the spleen. Splenic responses to thymus independent (TI) antigens TNP-Ficoll and TNP-LPS were not reduced when these antigens were administered one day after LPS. LPS given 4 days after TNP-KLH and 1 day before killing of the animals had induced severe alterations in splenic histology, but we found no marked influence on numbers and distribution pattern of anti-TNP forming cells. Results indicate that the LPS induced suppression of the immune response to TD antigens in the spleen is not caused by a direct effect of LPS on antigen reactive B-lymphocytes or on the anti-TNP antibody forming cells themselves. Possible explanations for the LPS-induced immunosuppression are discussed.

Adjuvants, Immunologic↗
PubMed1987

Immunomodulation with liposomes: the immune response elicited by liposomes with entrapped dichloromethylene-diphosphonate and surface-associated antigen or hapten.

Macrophages in the murine spleen were eliminated by the intravenous administration of dichloromethylene-diphosphonate (DMDP) encapsulated in liposomes. The immune response against an antigen (bovine serum albumin, BSA) or hapten (dinitrophenyl, DNP) associated with the surface of the DMDP liposomes was studied. Significant effects of macrophage elimination on the primary (IgM), but not on the secondary (IgG), anti-BSA response were found. Dramatic effects on the response against liposome-associated DNP were observed in macrophage-depleted mice. The number of anti-DNP antibody-forming cells in the spleen decreased from 300 to 28 per section, and anti-DNP serum titres dropped to 12% of their normal values. Since a similar phenomenon was observed for TNP-Ficoll, a thymus-independent type-2 antigen (and not for thymus-dependent or thymus-independent type-1 antigens), we suggest that this response should be classified as thymus-independent type-2 on grounds of its in vivo behaviour. We conclude that adjuvant activity (and memory formation) of liposomes with antigen exposed on their surface occurs irrespective of the presence or absence of splenic macrophages, and that DMDP liposomes could be useful in drug targeting with antigenic liposomes.

Animals↗
PubMed1987

Functional and histological studies of adoptive immunity in neonatally transplanted rabbits.

The adoptive transfer of immunity by means of spleen and lymph node cells has been analysed in rabbits of defined major histocompatibility (RLA) types and immunoglobulin (Ig) allotypes. Previous results had shown that chimaeras formed by transplanting adult cells into RLA-matched newborn hosts become stable B-lymphocyte chimaeras, as determined by their continuous production of donor type Ig. However, specific antibodies made by adult chimaeras were demonstrably only of recipient origin, unless the donor had been primed. The present report describes early events after the transfer of cells from donors primed with trinitrophenylated keyhole limpet haemocyanin (TNP-KLH). Double-staining with enzyme-labelled antigen or antibody conjugates was applied to identify lymphocytes of donor or recipient origin in spleen sections of transplanted rabbits and also to identify cells producing anti-TNP. Lymphocytes with membrane-bound Ig (mIg+ cells) of donor allotype were readily identified scattered singly or in small clusters throughout the recipients' B-lymphocyte areas within 5 days after transfer. Groups of donor-derived cells producing anti-TNP were demonstrable only in animals that had been specifically stimulated. Serological analysis and lymphocyte marker and function studies correlated well with results obtained by the histocytochemical approach. These results show that a systematic study of the role and importance of mature B lymphocytes in transplantation is now feasible.

Animals↗
PubMed1986

Influence of carriers on the development and localization of anti-trinitrophenyl antibody-forming cells in the murine spleen.

Mice were i.v. immunized with various 2,4,6-trinitrophenyl (TNP)-carrier conjugates, and the development and localization of specific (i.e. anti-TNP) antibody-forming cells (AFC) in the spleen was studied. The nature of the carrier used, thymus dependent (TD) or thymus independent type 1 or type 2 (TI-1, TI-2), had marked effects on the number and time of appearance of AFC. However, no influence of the different carriers on the localization of specific AFC in the spleen was observed. By using a double immunocytochemical technique we could simultaneously determine specificity (anti-TNP) and isotype (class and subclass) of the AFC. The TNP-carrier conjugates evoked (intracellular) immunoglobulins with carrier-characteristic isotype distribution. No carrier-dependent localization pattern, in the lymphoid compartments of the spleen, of these AFC was found. These results show for the first time the actual in situ effects of various TI and TD carriers on the development of specific AFC. Based on the present findings, no direct evidence for a special role of the marginal zone in the immune response against TI-2 antigens could be demonstrated. We suggest that this new method for in vivo investigation, in combination with existing standard enzyme- and immunohistochemical techniques, can provide more insight into the precise role of lymphoid and nonlymphoid cells in TD and TI immune responses.

Animals↗
PubMed1986

Influence of carriers on the development and localization of anti-2,4,6-trinitrophenyl (TNP) antibody-forming cells in the murine spleen. II. Suppressed antibody response to TNP-Ficoll after elimination of marginal zone cells.

The macrophages in the marginal zones in mice spleens were eliminated by i.v. injection of dichloromethylene diphosphonate encapsulated in liposomes. After immunization with a thymus-dependent (trinitrophenylated keyhole limpet hemocyanin; TNP-KLH) or thymus-independent type 1 (TNP-lipopolysaccharide) antigen, no differences in antibody responses in treated and untreated mice were observed. However, immunization with a thymus-independent type 2 (TI-2) antigen (TNP-Ficoll) resulted in a strong decrease of the antibody response in macrophage-depleted animals. Anti-TNP serum levels dropped 10-fold and the number of antibody-forming cells in the spleen 30-60-fold. These results confirm the special role of the marginal zone macrophages in the processing of TI-2 antigens, as earlier suggested in splenectomy studies.

Animals↗
PubMed1986

Recent advances in the detection and characterization of specific antibody-forming cells in tissue sections.

A new general approach has been developed for the detection of one or more different specific antibody producing cells and the simultaneous determination of their Ig isotype in tissue sections, after immunization of animals. Specificity of intracellular antibodies is demonstrated after incubation of the sections with an antigen-enzyme conjugate and the isotype of the antibodies is determined using an anti-immunoglobulin (Fc chain-specific)-enzyme conjugate followed by histochemical revelation of the two different enzymes. The principles of the method, the required antigen- and antibody-enzyme conjugates and their application in single, double or triple staining studies are reviewed. The method allows the detection of specific antibody-forming cells against protein antigens as well as against haptens. By means of haptens such as trinitrophenyl (TNP), immune responses against thymus dependent, thymus independent, and particulate antigens can be studied. In a limited number of cases the method can also be used to study the localization of antigen-antibody complexes.

Animals↗
PubMed1986

Effects of mitomycin C on the rate of DNA synthesis in normal and Fanconi anaemia cells.

The effect of low doses mitomycin C (MMC) on DNA synthesis of fibroblast cell lines derived from normal individuals or patients with Fanconi anaemia (FA) was studied. Using low doses of MMC (12 ng/ml), little or no effect was observed on DNA synthesis of normal cells, whereas DNA synthesis of FA cells was greatly inhibited 24 and 48 h after treatment. This effect was due to a decrease in the number of DNA-synthesizing cells, while the amount of radioactivity incorporated per cell (as measured with grain counting in autoradiograms) remained the same. These findings indicate that the inhibition of semiconservative DNA synthesis induced by MMC in FA cells is not due to an inhibitory effect of unrepaired lesions on the rate of DNA synthesis but rather to a block in cell cycle progression.

Anemia, Aplastic↗
PubMed1986

Detection of specific anti-hapten antibody-producing hybridoma cells in tissue sections of spleen and liver by hapten-enzyme conjugates using an immunoenzyme approach.

A method is presented for the detection of hybridoma cell growth in vivo by making use of the specific antigen (hapten) recognition properties of the produced monoclonal antibodies. Horseradish peroxidase (HRP)-labeled antigen was used for demonstration of intracellular specific antibodies in hybridoma cells. It appeared that intravenously injected anti-penicilloyl-producing hybridoma cells developed mainly in the red pulp of the spleen; to a lesser extent they were found in the liver.

Animals↗
PubMed1986

Double-enzyme conjugates, producing an intermediate color, for simultaneous and direct detection of three different intracellular immunoglobulin determinants with only two enzymes.

A new double-enzyme conjugate was synthesized by coupling alkaline phosphatase (AP) to horseradish peroxidase (HRP). After AP (blue) and subsequent HRP (red) cytochemistry, this new conjugate produced a stable intermediate-colored (violet) product. By coupling this double-enzyme conjugate to an antigen (trinitrophenyl, TNP) or an antibody (anti-mouse immunoglobulin G2a), anti-TNP or -IgG2a-producing cells could be demonstrated as violet cells in spleen sections. This led to the development of a rapid one-step incubation--two-step cytochemical procedure for simultaneous detection of three different determinants in a single tissue section. To demonstrate this novel triple staining method, we coupled three different antigens to, respectively, AP, HRP, and AP-HRP. When spleen sections of immunized animals were incubated with a mixture of these three antigen-enzyme conjugates, we could distinguish antibody-forming cells against each of these three antigens simultaneously as red (HRP), blue (AP), and violet (AP-HRP) cells. The simultaneous detection of three different classes of intracellular antibodies in a single section also proved to be possible with this method. With this study we provide a new direct method for detection of three different intracellular immunoglobulins after a one-step incubation and a two-step standard cytochemical procedure.

Alkaline Phosphatase↗
PubMed1986

Double immunocytochemical staining for the in situ study of allotype distribution during an anti-trinitrophenyl immune response in chimeric rabbits.

After incubation of tissue sections with anti-allotype-enzyme conjugates, the localization of immunoglobulin-allotype-bearing cells in the lymphoid tissues of conventional and chimeric rabbits could be established. The use of anti-allotype sera bearing distinct enzyme labels allowed simultaneous recognition of B cells producing immunoglobulin of one or the other parental types in heterozygous rabbits, or of B cells from the donor and recipient in chimeras. After immunization of chimeric rabbits with trinitrophenyl-keyhole limpet hemocyanin, anti-trinitrophenyl antibody-forming cells could be demonstrated through the use of a trinitrophenyl-alkaline phosphatase conjugate. Simultaneous incubation of sections with this reagent and with horseradish peroxidase coupled to (donor or recipient) anti-allotype sera made possible the determination of the origin (donor or recipient) of the antibody-forming cells. In agreement with the results of plaque assays and analyses of serum antibodies, all the anti-TNP producing cells were of donor origin when the chimeras had been created through injection of spleen or lymph node cells from trinitrophenyl primed donors. With this study we introduce a simple, direct method for the simultaneous identification of cells that produce antibody of a given allotype and a given specificity, applicable to appropriate studies in heterozygous or chimeric rabbits. The procedure has various advantages over previously reported methods.

Alkaline Phosphatase↗
PubMed1986

Marginal zone of the spleen and the development and localization of specific antibody-forming cells against thymus-dependent and thymus-independent type-2 antigens.

In order to study the precise localization pattern of anti-TNP antibody-forming cells (AFCs) during the early primary immune response against TNP conjugated TD (thymus-dependent) and TI-2 (thymus-independent type-2) antigens, rats received an intravenous injection with either TNP-keyhole limpet haemocyanin (KLH) or with TNP-Ficoll. Anti-TNP AFCs developed in the spleen already at 2 days after injection of the antigens as demonstrated with our immunoenzyme technique for the detection of specific AFCs. In order to obtain information on the relationship between the non-lymphoid cells in the marginal zone (MZ) and the localization of AFCs, simultaneous staining for marginal metallophils and MZ macrophages (MZM) was performed using the monoclonal antibody ED3. AFCs were not found in the marginal zone (MZ), but the bulk of the cells in the white pulp were found in the outer part of the periarteriolar lymphocyte sheaths (PALS) close to the border between PALS and MZ. The precise localization of the anti-TNP AFCs in the outer part of the PALS resembled the localization of marginal metallophils but the latter cells were mainly present in the outer part of the follicles. So, the present results did not indicate a close relationship between marginal zone macrophages or marginal metallophils and anti-TNP AFCs, neither in the immune response to TD antigens nor in that to T1-2 antigens.

Animals↗
PubMed1986

Anti-TNP-forming cells in bronchus-associated lymphoid tissue (BALT) and paratracheal lymph node (PTLN) of the rat after intratracheal priming and boosting with TNP-KLH.

The aim of the present study was to elucidate the role of the paratracheal lymph node (PTLN) and bronchus-associated lymphoid tissue (BALT) in the generation of antigen-specific antibody-forming cells after intratracheal administration of trinitrophenyl-conjugated keyhole limpet haemocyanin (TNP-KLH). Priming as well as boosting resulted in the occurrence of specific antibody-forming cells in the PTLN, in BALT and in the lung. No anti-TNP-forming cells were observed in Peyer's patch, mesenteric lymph node and popliteal lymph node. From the data on the kinetics of anti-TNP-forming cells, it is concluded that intratracheal administration of antigen leads to a locally confined plasmacellular reaction. This suggests that the migrating and/or homing capacities of memory cells generated in the lung environment (PTLN, BALT) differ from those generated in the gut.

Animals↗
PubMed1985

Binding of different antigen-enzyme and antibody-enzyme conjugates by intracellular antibodies in cytoplasm and Golgi complex of plasma cells. A double immunocytochemical study.

Intracellular immunoglobulins in plasma cells were characterized by antigen-enzyme conjugates and anti-immunoglobulin antibody-enzyme conjugates applied in a double immunocytochemical approach. After their assemblage, immunoglobulins in the cytoplasm of anti-TNP anti-body producing plasma cells can be demonstrated both by TNP-enzyme conjugates and by anti-immunoglobulin (mu or gamma chain specific) antibody-enzyme conjugates. Once arrived in the Golgi complex (GC) detection with TNP-enzyme conjugates remains possible, but anti-immunoglobulin anti-body-enzyme conjugates did not bind to a detectable degree. Similar results were obtained in experiments where immunoglobulin-enzyme conjugates were used both as an antigen-enzyme conjugate and as an antibody-enzyme conjugate.

Alkaline Phosphatase↗
PubMed1985

A trinitrophenyl(TNP)-poly-L-lysine-horseradish peroxidase conjugate for the detection of anti-TNP antibodies in vivo.

A new conjugate for the detection of anti-trinitrophenyl(TNP) antibodies was developed to study the localization pattern of specific antibody containing cells and extracellular antibody in vivo. By means of a bridging molecule, poly-L-lysine, nine TNP groups and six horseradish peroxidase (HRP) groups were joined in one conjugate. Thus a higher specificity (more hapten) was united with a higher staining intensity (more enzyme) in the same conjugate. This conjugate made possible the simultaneous detection of anti-TNP antibody containing cells and establishment of their class (immunoglobulin M (IgM) and IgG). It was also used for the demonstration of anti-TNP antibodies in tissues where a TNP-alkaline phosphate (AP) conjugate could not be used due to high AP (endogenous) background staining. Thus we demonstrated anti-TNP antibody containing cells in gut associated lymphoid tissue and anti-TNP-(TNP-ovalbumin) immune complexes in the glomeruli of the kidney. We suggest that poly-L-lysine is a suitable bridging molecule for the preparation of hapten-HRP conjugates.

Animals↗
PubMed1985

Intra-intestinal priming leads to antigen-specific IgA memory cells in peripheral lymphoid organs.

The aim of this study was to gain more insight into the mechanism of IgA memory formation by testing the effects of intra-intestinal antigen priming on various booster routes. To obtain a primary immune response trinitrophenyl conjugated keyhole limpet haemocyanin (KLH-TNP) was injected into the lumen of the small intestines of mice. For secondary immune responses mice were boosted intra-intestinally, intravenously or subcutaneously. The distribution of antigen specific cells in situ was demonstrated by enzyme histochemistry whereas quantification of TNP-specific cells was performed with a plaque-forming cell assay. After single or repeated intra-intestinal antigen administrations both primary and secondary immune responses in terms of specific antibody containing cells were mainly located in the spleen. The anti-TNP antibody-containing cells produced predominantly IgM during the primary and IgM, IgG and IgA during the secondary response. In mesenteric lymph nodes and villi antigen-specific cells were detected sporadically. When intra-intestinal priming was followed by intravenous or subcutaneous booster injections most anti-TNP antibody-producing cells were demonstrated in the spleen and in the draining popliteal lymph nodes. In contrast to repeated intravenous or subcutaneous immunizations alone, these organs contained, besides specific IgM and IgG cells, many TNP-specific cells producing IgA antibodies. This result demonstrates that the production of IgA antibodies is not restricted to mucosa-associated lymphoid tissues. IgA memory cells are induced in mucosa associated lymphoid tissues, probably in Peyer's patches, will consecutively migrate throughout the whole lymphoid system and can be triggered by renewed antigen contact to become IgA plasma cells.

Animals↗
PubMed1985

Evidence that macrophages in the marginal zone have no role in the migration of lymphocytes into the periarteriolar lymphocyte sheaths (PALS).

The macrophages in the marginal zones in the spleens of mice were eliminated by the intravenous injection of dichloromethylene diphosphonate encapsulated in liposomes. When syngeneic splenic lymphocytes were labelled with [3H]uridine and injected intravenously into the treated mice, their migration from the marginal zones into the periarteriolar lymphoid sheaths did not differ significantly from than seen in untreated animals. We concluded that the migration of lymphocytes from the marginal zones of the spleen is not mediated by the local macrophages.

Acid Phosphatase↗
PubMed1984

The effect of elimination of macrophages on the tissue distribution of liposomes containing [3H]methotrexate.

In the present study the tissue distribution of [3H]methotrexate was studied after intravenous injection of [3H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [3H]methotrexate liposomes in the blood, due to a decreased uptake of [3H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [3H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.

Animals↗