Prior exposure of mice to Fusobacterium nucleatum modulates host response to Porphyromonas gingivalis.

Multiple periodontal pathogens sequentially colonize the subgingival niche during the conversion from gingivitis to destructive periodontal disease. An animal model of sequential immunization with key periodontal pathogens has been developed to determine whether T- and B-lymphocyte effector functions are skewed and fail to protect the host from pathogenic challenge. The present study was performed to evaluate the immunomodulatory effect of exposure to Fusobacterium nucleatum prior to Porphyromonas gingivalis. Group 1 (control) mice were immunized with phosphate-buffered saline, group 2 were immunized with F. nucleatum prior to P. gingivalis and group 3 were immunized with P. gingivalis alone. All the T-cell clones derived from group 2 demonstrated type 2 helper T-cell clone (Th2 subsets), whereas those from group 3 mice demonstrated Th1 subsets. Exposure of mice to F. nucleatum prior to P. gingivalis interfered with the opsonophagocytosis function of sera against P. gingivalis. In adoptive T-cell transfer experiments, in vivo protective capacity of type 2 helper T-cell clones (Th2) from group 2 was significantly lower than type 1 helper T-cell clones (Th1) from group 3 against the lethal dose infection of P. gingivalis. Western blot analysis indicated a different pattern of recognition of P. gingivalis fimbrial proteins between sera from group 2 and group 3. In conclusion, these studies suggest that exposure of a host to F. nucleatum prior to the periodontal pathogen P. gingivalis modulates the host immune responses to P. gingivalis at the humoral, cellular and molecular levels.

Lethality-based selection of recombinant genes in mammalian cells: application to identifying tumor antigens.

Many biological processes result in either cell death or cessation of cell growth. However, plasmid- and retrovirus-based mammalian expression vectors in which it has been possible to construct representative cDNA libraries cannot be readily recovered from cells that are not actively dividing. This has limited the efficiency of selection of recombinant genes that mediate either lytic events or growth arrest. Examples include genes that encode the target antigens of cytotoxic T cells, genes that promote stem-cell differentiation and pro-apoptotic genes. We have successfully constructed representative cDNA libraries in a poxvirus-based vector that can be recovered from cells that have undergone lethality-based selection. This strategy has been applied to selection of a gene that encodes a cytotoxic T-cell target antigen common to several independently derived tumors.

The relationship of CD5+ B lymphocytes to macrophages: insights from normal biphenotypic B/macrophage cells.

For decades, numerous investigators have reported derivation of macrophage-like cells from CD5(+) pre-B cell lymphomas. Recently, it has become clear that biphenotypic CD5(+) B/macrophage cells are not a spurious result of malignancy. Indeed, the existence of normal biphenotypic cells with CD5(+) B lymphocyte and macrophage characteristics has been demonstrated in the mouse. This review considers normal B/macrophage cell function in an evolutionary context where a primitive, flexible cell type could perform dual roles in adaptive and innate immunity.

Polarization of Porphyromonas gingivalis-specific helper T-cell subsets by prior immunization with Fusobacterium nucleatum.

Antigen-specific T-cell clones were obtained from mice immunized with Fusobacterium nucleatum ATCC 10953 and/or Porphyromonas gingivalis 381. 10 BALB/c mice per group were immunized with F. nucleatum followed by P. gingivalis, or with P. gingivalis alone by intraperitoneal injection of viable microorganisms. Spleen T cells were isolated and stimulated in vitro with viable P. gingivalis cells to establish P. gingivalis-specific T-cell clones. T-cell phenotypes and cytokine profiles were determined along with T-cell responsiveness to F. nucleatum or P. gingivalis. Serum immunoglobulin G antibody titers to F. nucleatum or P. gingivalis were also determined by enzyme-linked immunosorbent assay. All the T-cell clones derived from mice immunized with F. nucleatum followed by P. gingivalis demonstrated Th2 subsets, while those from mice immunized with P. gingivalis alone demonstrated Th1 subsets based on the flow cytometric analysis and cytokine profiles. All T-cell clones from both groups were cross-reactive to both P. gingivalis and F. nucleatum antigens. Phenotypes of T-cell clones were all positive for CD4. Mean post-immune serum IgG antibody levels to F. nucleatum or P. gingivalis were significantly higher than the pre-immune levels (P < 0.05, P < 0.01, respectively). There were no significant differences in the antibody titers between the two groups. It was concluded that P. gingivalis-specific T cells initially primed by cross-reactive F. nucleatum antigens were polarized to Th2 subset, while T cells stimulated with P. gingivalis alone maintained the profile of Th1 subset.

Biphenotypic B/macrophage cells express COX-1 and up-regulate COX-2 expression and prostaglandin E(2) production in response to pro-inflammatory signals.

B/macrophage cells are biphenotypic leukocytes of unknown function that simultaneously express B lymphocyte (IgM, IgD, B220, CD5) and macrophage (phagocytosis, F4/80, Mac-1) characteristics. B/macrophage cells can be generated from purified mouse B lymphocytes incubated in fibroblast-conditioned medium. A potential role for B/macrophage cells in inflammation was shown by their ability to express prostaglandin H synthase-1 (COX-1) and prostaglandin H synthase-2 (COX-2) and by their production of prostaglandin (PG) E(2). COX-1 and COX-2 mRNA expression is not observed in the precursor B lymphocytes and is not known to be a property of B lineage cells. In contrast, COX-2 and the prostanoids PGE(2), PGF(2alpha) and PGD(2) are highly inducible in B/ macrophage cells upon stimulation with lipopolysaccharide, CD40 ligand, or via engagement of surface IgM, supporting a role for these cells in inflammation. PGD(2) and its metabolites are of interest because they activate the nuclear receptor PPARgamma that regulates lipid metabolism. The B/macrophage represents the first instance of a normal B-lineage cell capable of expressing COX-2. Importantly, B/macrophage cells were identified in vivo, providing evidence that they may play a significant role in immune responses. Since PGE(2) blunts IL-12 production, its synthesis by B/macrophage cells may shift the balance of an immune response towards Th2 and humoral immunity.

Fibroblast-secreted macrophage colony-stimulating factor is responsible for generation of biphenotypic B/macrophage cells from a subset of mouse B lymphocytes.

Normal and malignant CD5+ B lymphocytes can develop macrophage-like characteristics. One stimulus of this phenotypic shift is culture of normal mouse splenic B lymphocytes with splenic fibroblasts or their conditioned media. These biphenotypic B/macrophage (B/M phi) cells simultaneously display macrophage characteristics, such as phagocytosis and F4/80 expression, while retaining B cell features, including expression of surface Ig, CD5, B220, and rearranged Ig genes. The present study investigated the fibroblast-secreted factor that promotes this phenotypic change from B cell to B/M phi cell. RT-PCR analysis demonstrated that mRNA for M-CSF is produced by splenic fibroblasts. Recombinant M-CSF (CSF-1) could replace fibroblast-conditioned medium to elicit the development and survival of B/M phi cells from splenic B lymphocytes. In addition, neutralization of fibroblast-secreted M-CSF with specific mAbs abrogated the ability of conditioned supernatants to promote outgrowth of B/M phi cells. The transition from B lymphocyte to B/M phi cell was marked by the kinetic appearance of mRNA for the M-CSF receptor, c-fms, at day 3 following culture initiation. These results demonstrate that M-CSF is important in the development and physiology of mouse B/M phi cells and potentially in the growth of human biphenotypic hematological malignancies. Interestingly, the presence of IFN-gamma in splenic B lymphocyte cultures abrogated the effect of fibroblast-conditioned medium or M-CSF on outgrowth of B/M phi cells. Furthermore, these findings suggest that a Th1 microenvironment favored by typical macrophages is detrimental to the outgrowth of B/M phi cells.

Expression of mouse CD43 in the B cell lineage of transgenic mice causes impaired immune responses to T-independent antigens.

CD43 (leukosialin), a sialylated glycoprotein expressed on the surface of most hematopoietic cells, has been implicated in cell adhesion and signaling. However, its precise physiological function remains unclear. We used mouse CD43 (mCD43)-immunoglobulin enhancer-transgenic (TG) mice to study the role of mCD43 in vivo. Previous work revealed that mCD43 expression on mature B cells in these mice resulted in immunodeficiency to T-dependent (TD) antigens (Ag), possibly by impairing B-T cell interactions. In the present study we have immunized the TG mice with the T-independent (TI) Ag fluorescein-(Fl) lipopolysaccharide (LPS) (TI type 1 Ag) and Fl-Ficoll (TI type 2 Ag). Surprisingly, the mCD43-Ig enhancer expressing mice were impaired in their ability to mount humoral responses to both Fl-LPS and Fl-Ficoll, and had decreased numbers of cells responding to Ag in vivo. Flow cytometric analysis was performed on peritoneal B-1 cells, a population which often plays a major role in humoral responses to TI Ag such as bacterial Ag. This analysis revealed similar B220, IgM and CD5 expression patterns for the TG and nontransgenic (NTG) B-1 cells. In addition, purified peritoneal B-1 cells from TG and NTG mice were able to respond to LPS. Stimulation of splenic B cells in vitro with Fl-LPS and Fl-Ficoll revealed that, in contrast to NTG B cell responses, TG B cell responses could not be enhanced by co-culture with T cells. However, soluble T cell factor enhancement of the TG B cell responses was normal. These data suggest that the mCD43 expression on B cells may inhibit cell interactions that are important for enhanced TI Ag responses. The anti-adhesive forces of mucins in general may thus be critical in regulating both TD and TI humoral responses.

Fibroblast heterogeneity in the periodontium and other tissues.

Fibroblasts are the major resident cells which inhabit the periodontal tissues. As such, they are crucial for maintaining the connective tissues which support and anchor the tooth. Little is known of their origins, synthesis of regulatory cytokines and growth factors in health and disease, and importance in soft tissue regeneration. An emerging concept is that fibroblasts are not homogeneous, but instead consist of subsets of cells which can regulate bone marrow-derived cells such as T lymphocytes. Fibroblasts can be separated into subsets on the basis of morphology, size and expression of intermediate filaments as well as collagen subtypes. Differential surface marker expression has also been a key feature to distinguish fibroblast subsets from many tissues. Antigens such as Thy-1, class II MHC, and C1q are among those surface proteins which have been employed successfully to separate fibroblasts. Importantly, these fibroblast subsets are not only antigenically diverse, but also possess distinct functions. Thy 1+ pulmonary fibroblasts can display class II MHC antigens, synthesize IL-1 and can activate T lymphocytes, whereas the Thy 1+ subset is devoid of these functions. Recently, fibroblasts from the human orbit have also been shown to be separable on the basis of Thy 1 surface marker expression. Fibroblasts derived from human gingiva and periodontal ligament also appear to be composed of subsets with a heritable pattern of surface markers which will permit their separation into functional subpopulations. This paper will review findings of fibroblast heterogeneity in periodontal and other tissues. Evidence will be presented for the use of surface markers to delineate functional subsets. The ability to discriminate subsets of fibroblasts will aid in studies of periodontal disease pathogenesis and wound healing.

Differential Thy-1 expression by splenic fibroblasts defines functionally distinct subsets.

Fibroblasts have an important structural role in the spleen, as they provide a scaffold of extracellular matrix in which cells of the immune system reside. Aside from their vague recognition as "stromal" or "reticular" components of the spleen, these cells have not been characterized. In this study, normal fibroblast lines from mouse [B6D2(F1)] spleen were established. The fibroblast phenotype of these lines was confirmed by their morphology, expression of vimentin, as well as their lack of epithelial and endothelial cell markers, their failure to display the hematopoietic marker CD45, and their inability to phagocytize. Interestingly, 50-65% of the splenic fibroblasts expressed the Thy-1 antigen, while a subpopulation of Thy-1-negative fibroblasts existed. FACS on the basis of Thy-1, as well as limiting dilution cloning, yielded stable lines and clones of Thy-1+ and Thy-1- splenic fibroblasts. Phenotypic characterization revealed that both subsets synthesized collagen and expressed class I MHC, ICAM-1, VCAM-1, and CD44 constitutively. However, intriguing differences existed between the fibroblast subpopulations. Thy-1+ splenic fibroblasts produced significantly greater levels of IL-6 than did their Thy-1- counterparts. After treatment with IFN-gamma (150 U/ml, 72 hr), Thy-1-, but not Thy-1+, splenic fibroblasts expressed class II MHC and presented antigen to an I-A(b)-restricted T cell line. This suggests that the Thy-1- fibroblasts may present antigen to T lymphocytes in vivo under inflammatory conditions. Thus, splenic fibroblasts are a heterogeneous and dynamic cell type poised in an immunologically relevant location to interact with bone marrow-derived cells under normal and fibrotic conditions.

Fibroblasts support outgrowth of splenocytes simultaneously expressing B lymphocyte and macrophage characteristics.

B lymphocytes and macrophages are considered to be derived from separate lineages and to have specialized functions. However, some malignant B lymphocytes can generate descendants with macrophage-like properties and monocytoid B cells are described in certain diseases. In this report, we demonstrate that normal biphenotypic cells can be isolated by incubating mouse splenic fibroblasts or their conditioned media with purified splenic B lymphocytes. Phagocytic vascular cells were isolated that simultaneously displayed typical B cell (B220, surface IgM, surface IgD, and CD5) and macrophage (F4/80 and Mac-1) markers and contained rearranged Ig genes. F(ab')2 anti-mu Ab inhibited the proliferation of these biphenotypic cells in a dose-dependent manner, indicating that functional IgM was expressed. The adherent B/macrophage cells also displayed CD40 and BCL-2 and were sensitive to ionizing radiation, consistent with having a B cell origin. In the presence of splenic fibroblast-conditioned medium, lines of B/macrophage cells can be propagated for months in vitro. The ability to derive these biphenotypic cells from normal sources suggests that certain B lymphocytes and macrophages share a closer lineage relationship than is predicted by current models of hematopoietic differentiation. Alternatively, these biphenotypic cells may represent a primitive B lymphocyte lineage that possesses greater flexibility to adapt to infectious agents than dedicated lymphoid or myeloid cells and may be prone to malignant transformation.

Fibroblast heterogeneity in the healing wound.

Although fibroblasts are traditionally described as static cells providing framework and support for tissues, there is an accumulating body of evidence showing that fibroblasts are a dynamic cell type which exist in functionally and morphologically heterogeneous subpopulations. Fibroblast subsets have been shown to play a critical role in the production and regulation of extracellular matrix components, in wound repair and regeneration, and have been implicated in the pathogenesis of fibrotic conditions. We have reviewed the evidence supporting heterogeneity of fibroblasts from pulmonary, periodontal, and dermal tissues. In addition, we will explore the role fibroblast subpopulations may play in the complex process of wound repair and regeneration.

Simulating gas flow through the exhalation leg of a respirator's patient circuit.

The effectiveness of prescribed respiratory therapy is often dependent upon the choice of a respirator (ventilator) that excels for a particular mode of ventilation. The exhalation value of a ventilator is most often the key to a strong or weak performance. A computer model of the patient's gas flow through the expiratory circuit and exhalation valve is not only beneficial for design, but can also be used to study the optimum performance for a particular mechanical system. For this paper, the system that was used incorporated a linear voice coil actuator suspended by flat spider springs. The details of the modelling are given on a theoretical basis (with the appropriate equations), and the packaged simulation is described. Results are presented for simple computer algorithms with the intention of demonstrating the proper behaviour of the system. There are suggestions for further detailed studies to compare the linear voice coil model with other common exhalation valve mechanical designs, under various modes of ventilation.