sepharose and Granuloma

In the majority of individuals infected with Mycobacterium tuberculosis, the bacilli cause a long-term asymptomatic infection called latent tuberculosis, a state during which the bacilli reside within granulomas. Latently infected individuals have around 10% risk of progression to clinical disease at a later stage. Determining the state of the mycobacteria and the host cells during this latent phase, i.e. within the granulomas, would greatly improve our understanding of the physiopathology of tuberculosis, and thus enable the development of new therapeutic means to treat the one-third of the world's population who are latently infected. We have developed an in vitro model of human mycobacterial granulomas, enabling the cellular and molecular analysis of the very first steps in the host granulomatous response to either mycobacterial compounds or live mycobacterial species. In vitro mycobacterial granulomas mimic natural granulomas very well, with the progressive recruitment of macrophages around live bacilli or mycobacterial antigen-coated beads, their differentiation into multinucleated giant cells and epithelioid cells, and the final recruitment of a ring of activated lymphocytes. Besides morphological similarities, in vitro granulomas also functionally resemble natural ones, with the development of intense cellular co-operation and intracellular mycobactericidal activities.

Topics: Animals; Antigens, Bacterial; BCG Vaccine; Cells, Cultured; Disease Progression; Granuloma; Humans; In Vitro Techniques; Leukocytes, Mononuclear; Lymphocyte Activation; Mycobacterium tuberculosis; Sepharose; Tuberculosis

Granulomas consist of organised collections of macrophages showing evidence of activation in response to an agent localised within a tissue. To date, in vitro models of granuloma formation have failed to reproduce the histological appearance of a granuloma, which forms a characteristic three-dimensional structure. The use of 3-D collagen gels allows agarose beads and human mononuclear cells to be suspended together in a milieu which permit the two to interact. The interaction between agarose beads and mononuclear cells produces monocyte-macrophage aggregates within 24 h which resemble an early granuloma. The monocytes show evidence of activation by direct microscopy, electron microscopy and immunohistochemistry. This model should permit the laboratory testing of hypotheses describing granuloma formation.

Topics: Animals; Cell Aggregation; Collagen; Gels; Granuloma; HLA-DR Antigens; Humans; Microspheres; Models, Biological; Monocytes; Neutrophils; Rats; Sepharose; Tuberculoma

Microencapsulation of adrenal cells is proposed for reducing the nonspecific inflammatory reaction observed around polymer implants. This hypothesis was tested by comparing both host cellular reaction and the surrounding graft cell populations which appeared either when agarose embedded cells or when empty agarose beads were implanted. Our results showed that the fibrotic material that surrounded the implanted empty agarose microbeads was not as severe and important when adrenal cells were present. Similarly, T lymphocyte population surrounding the graft was considerably reduced together with the percentage of CD4 and CD8 positive cell subpopulations. The activation macrophage marker IaD disappeared. Our results support the hypothesis that embedded adrenal cells may be a suitable solution for reducing early inflammatory events due to microcapsules implantation.

Topics: Adrenal Cortex; Animals; Capsules; Flow Cytometry; Graft Survival; Granuloma; Inflammation; Lymphocytes; Male; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Microscopy, Electron; Sepharose

To investigate the basic mechanisms of granuloma formation, in vitro granulomas were induced by culturing murine spleen cells in the presence of artificial microparticles. Large granulomas developed around dextran beads. The lesions were inducible by spleen cells from either normal mice or athymic nude mice. Minimal inflammation was produced around latex beads. The histologic features and time kinetics of granulomas in vitro. Culture supernatants of dextran induced granulomas contained high levels of interleukin-1 (IL-1) activity but not interleukin-2 (IL-2) or interleukin-4 (IL-4) activity. IL-1 activity was correlated with granuloma size. Additionally, granulomas were produced by culturing spleen cells in the presence of agarose beads coupled to recombinant IL-1 or recombinant tumor necrosis factor-alpha (TNF-alpha). Granulomatous lesions also were induced by macrophage-enriched populations in the presence of monokine-coupled beads. Adherent macrophages, but not nonadherent cells, were required for induction of granulomas in vitro. In contrast, very small lesions were seen when spleen cells or adherent cells were cultured in the presence of beads coupled to recombinant IL-2 or recombinant interferon-gamma (IFN-gamma). These results suggest that macrophages and monokines such as IL-1 and TNF-alpha play an essential role in granuloma formation in vitro.

Topics: Animals; Biological Factors; Cell Transformation, Neoplastic; Cells, Cultured; Cytokines; Dextrans; Female; Granuloma; Immunosuppressive Agents; Interleukin-1; Macrophages; Mice; Mice, Inbred Strains; Microspheres; Monokines; Polystyrenes; Sepharose; Spleen; Tumor Necrosis Factor-alpha

A subset of Schistosoma mansoni egg glycoproteins that share a common carbohydrate epitope recognized by monoclonal antibody 128C3 was shown to induced formation of hepatic granulomata when conjugated to Sepharose beads and injected into the portal circulation of naive mice. Concanavalin-binding egg glycoproteins exhibited more granuloma-inducing activity than did total egg extract, although deglycosylated egg proteins also induced granulomata; thus, both amino acid and carbohydrate epitopes appeared to be involved. Glycoproteins derived from adult male worms also were active, indicating that immunological processes responsible for granuloma formation may not be absolutely stage specific.

Topics: Animals; Antibodies, Monoclonal; Antigens, Helminth; Glycoconjugates; Glycoproteins; Granuloma; Liver; Models, Biological; Ovum; Receptors, Concanavalin A; Schistosoma mansoni; Schistosomiasis mansoni; Sepharose

Pulmonary granulomas were induced in immunized BALB/c mice by the intratracheal injection of antigen-coated and plain agarose beads. Prominent lesions developed within 24 hr, reached peak intensity within 3 days, and gradually declined in size thereafter. The hypersensitivity granulomas induced in sensitized mice by antigen-coated beads were much larger than the lesions induced by plain beads. Minimal inflammation was produced in unsensitized mice injected with antigen-coated or plain beads. Aqueous extracts prepared from pulmonary granuloma lesions induced in sensitized mice by antigen-coated beads contained high levels of interleukin 1 (IL 1) and migration inhibition factor (MIF) activities. The kinetics of appearance of these mediators were similar. Lower but detectable activity of both mediators was detected in extracts prepared from sensitized mice injected with plain beads. Neither interleukin 2 (IL 2) nor IL 2 neutralizing activities were detected in the extracts. The presence of IL 1 and MIF in extracts prepared from early and peak pulmonary granulomatous lesions suggests that these soluble factors are produced by cells within the lesions, and that they are involved in mediating the expression and/or maintenance of the granulomas.

Topics: Animals; Antigens; Female; Granuloma; Interleukin-1; Leukocyte Migration-Inhibitory Factors; Lung Diseases; Mice; Mice, Inbred BALB C; Sepharose

Topics: Animals; Antigens; Dinitrophenols; Foreign-Body Reaction; Granuloma; Guinea Pigs; Humans; Hypersensitivity, Delayed; Immunity, Cellular; Immunization; Injections, Intradermal; Lymphocytes; Macrophages; Models, Biological; Sepharose; Serum Albumin; Skin Tests; Time Factors