E. Claassen

Preparation and characteristics of dichloromethylene diphosphonate-containing liposomes

AbstractDichloromethylene diphosphonate (DMDP), encapsulated in liposomes and administered intravenously in mice, will eliminate all macrophages in spleen and liver. DMDP can be incorporated in liposomes to a maximum of 15 mM, if less than 12mM is encapsulated not all macrophages will be eliminated (since the animals could not be injected with more liposomes). To determine DMDP content of the liposomes before in vivo administration, an in vitro test system was developed. This method is based on the competition for calcium binding by either DMDP or murexide. Murexide is a metallochromatic indicator which gives a distinct wavelength shift after binding of calcium. The decrease in absorbance at 510 nm of the murexide-calcium complex, due to the addition of DMDP, was used as a reliable (s.d. 5 per cent) value for measurement of DMDP concentrations. The possible use of this system in the study of calcium binding and transport over artificial biomembranes is discussed.

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.

TNP-enzyme conjugates for the detection of anti-TNP antibody producing cells in vivo

After injection of TNP-KLH in mice and TNP-BGG-PEN in rabbits, anti-TNP antibody-forming cells were observed in the spleen. When cryostat sections of the stimulated spleen were incubated with TNP-HRP conjugate, and then treated for HRP cytochemistry, the cytoplasm of anti-TNP-forming cells was stained red. When similar sections were incubated with TNP-AP conjugate, and then treated for AP cytochemistry, the cytoplasm of anti-TNP-forming cells was stained blue. After simultaneous incubation with TNP-AP conjugate and PEN-HSA-HRP conjugate, followed by treatment for HRP cytochemistry and AP cytochemistry, anti-TNP-forming cells with a blue cytoplasm and anti-PEN-forming cells with a red cytoplasm could be distinguished in the same spleen section.

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.

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

SummaryA 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.

INADEQUATE ANTI-POLYSACCHARIDE ANTIBODY RESPONSES IN THE CHICKEN

Chickens are notorious for the fact that they carry bacteria such as Salmonellae and Campylobacter, which can cause zoonoses by contamination of the end product, without hampering growth and development of the chicken itself. This carrier status can only been explained by the inability of the chickens immune system to eliminate the pathogen, this in turn being due to insufficient humoral responses towards the polysaccharides of the bacterial capsule. In a previous study, we demonstrated that in chickens a model thymus-independent type 2 (TI-2) polysaccharide antigen, trinitrophenylated Ficoll (TNP-Ficoll), hardly evokes a humoral immune response. Furthermore this TI-2 antigen was shown to exhibit a very specific initial localization pattern after intravenous injection, i.e. in the periellipsoidal lymphocyte sheaths (PELS) and the surrounding ring of macrophages. The functional equivalent of these macrophages in mammals, the marginal zone macrophages, were shown to suppress the humoral responses against TI-2 antigens. Therefore we investigated whether other standard TI-2 antigen models also induce low antibody responses, whether this low response is dose-dependent, and whether macrophages are responsible for this low response. It was found that other TI-2 antigens, such as hydroxyethyl starch and detoxified lipopolysaccharides, also induced very low IgM and IgG responses, indicating a general phenomenon that could not be overcome by using a higher dose of antigen. In addition, selective depletion of splenic macrophages with liposomes containing dichloromethylene diphosphonate prior to immunization increased the specific humoral response to TD and TI-1 antigens, but failed to do so for TI-2 antigen. This result indicates that the low humoral responses are not (only) due to a macrophage suppressive activity but also to other yet unknown mechanisms, for example the lack of responsive B cells in the splenic PELS.

In vitro and in vivo elimination of macrophage tumor cells using liposome-encapsulated dichloromethylene diphosphonate

SummaryIt is shown in the present study that RAW 264 tumor cells can be killed by liposomeentrapped dichloromethylene diphosphonate (DMDP), both in vitro and in vivo. DMDP is ingested by phagocytic cells when entrapped in liposomes. Once phagocytized the liposomal membranes are digested and the drug is released into the cell and is ready for action. In vitro, even low doses of liposome-entrapped DMDP caused an significant reduction in cell numbers. In vivo, liposome-encapsulated DMDP markedly reduced tumor formation in the liver, when given 1 day after injection of 1 × 106 RAW 264 tumor cells. Liposome-encapsulated DMDP, given 4 or more days after injection of the tumor cells had no significant effect. We concluded that tumor formation by RAW 264 cells is only susceptible to in vivo treatment with liposome-entrapped DMDP during a short period of time after injection of the cells. Obviously, phagocytosis of the tumor cells is reduced after this period making the cells less susceptible to treatment with the liposome-entrapped drug.

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.

Double immunocytochemical staining for in vivo detection of epitope specificity and isotype of antibody-forming cells against synthetic peptides homologous to human immunodeficiency virus-1

Many infections evoke a strong humoral immune response. Some (e.g., HIV-1, EBV, CMV) also lead to disorders of the B-cell system. Data concerning cell dysfunction are largely derived from in vitro studies, which necessarily exclude all microenvironmental influences. The aim of this study was to develop a tool for the investigation of epitope specific humoral immune responses in vivo. Mice were immunized with one of two synthetic peptides, both 21 amino acids long and homologous to regions of the HIV-1 gp160. Cryostat sections of spleen and lymph nodes were incubated with the corresponding peptide coupled to alkaline phosphatase and simultaneously incubated with peroxidase-conjugated rabbit antisera specific for mouse immunoglobulin isotypes. We were able to show simultaneous detection of epitope specificity, isotype, and localization of antibody-forming cells and immune complexes in tissue sections. It should prove useful for in vivo investigation of the development of specific (e.g., anti-HIV-1) humoral immune response, the determination of B-cell specificity in lymph node infiltrates, and the role of immune complexes in lymph node pathology.

Fixation of cryo-sections under HIV-1 inactivating conditions: integrity of antigen binding sites and cell surface antigens.

SummaryCryostat-sections of biopsies from HIV-infected patients or HIV/SIV-infected experimental animals pose a biohazard risk to laboratory workers. The objective of this study was to select a procedure that appropriately fixes cryo-sections and reduces the risk of HIV-1 infectivity. This inactivation procedure should preserve antigen binding capacity of host-produced antibodies and the antigenic structure of epitopes present in these tissues, while retaining sufficient morphologic detail. We tested the effect of seven different established fixation-inactivation procedures for HIV-1 on the detection of specific antibodies and membrane markers, compared to acetone fixation as a reference. Frozen sections of spleens from mice immunized with trinitrophenyl (TNP)-Ficoll were incubated with TNP-alkaline phosphatase to detect specific antibody-forming cells and follicular immune complexes containing TNP-specific antibodies. In addition, sections were stained with monoclonal antibodies directed against IgM (187-1), T-cells (anti Thy-1), and marginal metallophilic macrophages (MOMA-1). Five procedures proved useful as they gave results similar to regular acetone fixation. In contrast, two procedures with a methanol-containing fixative obscured both antigen binding sites and membrane antigens. Subsequently, these five selected procedures were tested on glass slide preparations of HIV-1 infected cell lines, expressing HIV-1 determinants defined by monoclonal antibodies. Finally, the procedures were tested on sections of an HIV-1 infected human lymph node. for detection of HIV-specific B-cells. We show that fixation-inactivation in 0.37% (v/v) formaldehyde in PBS for 10 min at room temperature and 0.5% paraformaldehyde (w/v) in PBS for 10 min at room temperature are the methods of choice, combining preservation of antigen binding sites (Fab), membrane antigens, and HIV-1 determinants with good tissue morphology.