In Vitro Desensitization of Human Skin Mast Cells

Desensitization is a clinical procedure whereby incremental doses of a drug are administered over several hours to a sensitive patient until a therapeutic dose and clinical tolerance are achieved. Clinical tolerance may occur in part by attenuating the mast cell response. In the present study, primary human skin mast cells were used to establish and characterize an in vitro model of desensitization. Mast cells in culture were armed with allergen-specific (4-hydroxy-3-nitrophenylacety and Der p2) and non-specific IgE antibodies, and then desensitized by incremental exposures to 4-hydroxy-3-nitrophenylacety-BSA. This desensitization procedure abrogated the subsequent degranulation response to the desensitizing allergen, to an unrelated allergen, and to IgG anti-FcεRI, but not to C5a, substance P, compound 48/80, and calcium ionophore. Desensitized cells regained their FcεRI-dependent degranulation capability by 24–48 h after free allergen had been removed. Therefore, sensitized human skin mast cells are reversibly desensitized in vitro by exposure to incremental doses of that allergen, which also cross-desensitizes them to an unrelated allergen.     

Conditions for Inducing T Helper Cells In Vitro

Lymphoid cells of CBA mice were triggered to act as specific helper cells by incubation with protein antigen (usually keyhole limpet haemocyanin (KLH)) in Marbrook cultures in vitro. After optimum priming, these helper cells, at optimal numbers, stimulated B cells (from unprimed spleens) to respond to trinitrophenyl-KLH in vitro. The in vitro-induced helper cells were carrier-specific. B cell depletion before helper cell induction increased the efficiency of helper cell induction and thus provided further proof of the T-cell nature of in vitro helper cells. Heterogeneity within T helper cell precursors is suggested on the basis of differences in antigen dose requirements of precursor cells in cortisone-resistant thymocytes, spleen, or lymph nodes.     

Assessing the In Vitro Suppressive Capacity of Regulatory T Cells

Regulatory T cells (Treg) play a vital role in controlling peripheral immune responses in order to prevent autoimmunity and control inflammation. Altered Treg activities have been associated with the pathogenesis of multiple disorders including autoimmunity, allergy, cancer, and infection with persistent pathogens. As such, a great deal of interest has recently been directed towards developing additional tools and methods to better understand the mechanisms of suppression employed by Treg. The in vitro suppression assay has emerged as a valuable means by which to assess the functional capacity and activity of Treg. In this review, we summarize the merits and limitations of the various in vitro assays that have been utilized to assess Treg activity and present a novel two color proliferation assay that allows simultaneous monitoring of both regulatory and effector T cell activity. As further immunomodulatory therapies are explored, the need for additional methodologies to understand the cellular and molecular mechanisms of immune regulation conferred by Treg will play an increasingly important role.     

Assessing the In Vitro Suppressive Capacity of Regulatory T Cells

Regulatory T cells (Treg) play a vital role in controlling peripheral immune responses in order to prevent autoimmunity and control inflammation. Altered Treg activities have been associated with the pathogenesis of multiple disorders including autoimmunity, allergy, cancer, and infection with persistent pathogens. As such, a great deal of interest has recently been directed towards developing additional tools and methods to better understand the mechanisms of suppression employed by Treg. The in vitro suppression assay has emerged as a valuable means by which to assess the functional capacity and activity of Treg. In this review, we summarize the merits and limitations of the various in vitro assays that have been utilized to assess Treg activity and present a novel two color proliferation assay that allows simultaneous monitoring of both regulatory and effector T cell activity. As further immunomodulatory therapies are explored, the need for additional methodologies to understand the cellular and molecular mechanisms of immune regulation conferred by Treg will play an increasingly important role.     

B-Cell Activation In Vitro by Helper T Cells Specific to a Protein Region that is Recognized Both by T Cells and by Antibodies

This laboratory had previously mapped the regions of T and B cell recognition on sperm whale myoglobin (Mb). Mb has five regions (E1-E5) that are recognized by both T cells and B cells (i.e. antibodies, Abs) and an additional region (E6) that is recognized exclusively by T cells (i.e., T_E6) and to which no Abs are detectable. The responses to the site are each under separate genetic control. Recently, we showed in an H-2^d haplotype that T_E6 cells preferentially activated Mb-primed B cells (B_Mb) that made Abs against sites within E3 and E4 on the same protein. In the present work, we established, from Mb-primed SJL mice, an E4-specific T cell line (T_E4) by passage in vitro with synthetic peptide E4. At relatively low numbers, these T cells activated syngeneic B_Mb cells in vitro to produce anti-Mb Abs that recognized each of the antigenic sites within regions E1, E2, E3, E4 and E5. We confirmed the ability of T_E4 to activate B cells that produce Abs against each of these regions by allowing T_E4 to activate in vitro syngeneic B cells that had been primed with E1, E2, E3, E4 or E5. The helper activity of T_E4 cells was dependent on the in vitro concentration of the challenge Ag (intact Mb or peptide E4). Thus, T cells against an epitope may provide help restricted to B cells that make Abs against selected antigenic sites or they may activate B cells that make Abs against all the antigenic sites of a protein. This might depend on the site-specificity of the T cell and/or on the host.     

B-Cell Activation In Vitro by Helper T Cells Specific to a Protein Region that is Recognized Both by T Cells and by Antibodies

This laboratory had previously mapped the regions of T and B cell recognition on sperm whale myoglobin (Mb). Mb has five regions (E1-E5) that are recognized by both T cells and B cells (i.e. antibodies, Abs) and an additional region (E6) that is recognized exclusively by T cells (i.e., T_E6) and to which no Abs are detectable. The responses to the site are each under separate genetic control. Recently, we showed in an H-2^d haplotype that T_E6 cells preferentially activated Mb-primed B cells (B_Mb) that made Abs against sites within E3 and E4 on the same protein. In the present work, we established, from Mb-primed SJL mice, an E4-specific T cell line (T_E4) by passage in vitro with synthetic peptide E4. At relatively low numbers, these T cells activated syngeneic B_Mb cells in vitro to produce anti-Mb Abs that recognized each of the antigenic sites within regions E1, E2, E3, E4 and E5. We confirmed the ability of T_E4 to activate B cells that produce Abs against each of these regions by allowing T_E4 to activate in vitro syngeneic B cells that had been primed with E1, E2, E3, E4 or E5. The helper activity of T_E4 cells was dependent on the in vitro concentration of the challenge Ag (intact Mb or peptide E4). Thus, T cells against an epitope may provide help restricted to B cells that make Abs against selected antigenic sites or they may activate B cells that make Abs against all the antigenic sites of a protein. This might depend on the site-specificity of the T cell and/or on the host.     

In Vitro Sensitization of Human T Lymphocytes to Hapten (TNP)-Conjugated and Non-treated Autologous Cells is Restricted by Self-HLA-D

By co-culturing human T lymphocytes with TNP-treated autologous B lymphocytes and macrophages for 10 days in vitro, sensitization to TNP-treated autologous cells could be detected in a proliferative assay. By restimulation with different types of allogeneic cells and with cells from donors compatible or incompatible for the HLA-ABC or -D determinants, results were obtained suggesting that the TNP-specific response was restricted by the HLA-D but not the -ABC antigens of the autologous priming cells. Furthermore, our experiments demonstrate that T lymphocytes can also be primed against non-treated autologous cells in vitro and suggest that the HLA-D determinants may be involved also in this auto-sensitization.     

Human Monocytic U937 Cells Kill Salmonella In Vitro by NO-Independent Mechanisms

Nitric oxide (NO) has a central role in host defense against intracellular microbes. HLA-B27 has been shown to directly modulate host-microbe interaction in vitro, leading to the impaired elimination of Salmonella in human monocytic U937 cells. Here, we studied whether impaired elimination of Salmonella would result from differences in NO production between HLA-B27- and HLA-A2-transfected U937 cells. Both human monocytic transfectants produced NO equally well and killed Salmonella via NO-independent mechanisms.     

Differentiation of Human Embryonic Stem Cells to Cardiomyocytes for In Vitro and In Vivo Applications

The ability of human embryonic stem cells to differentiate into spontaneously contracting cardiomyocyte-like cells has attracted substantial interest from the scientific community over the last decade. From having been difficult to control, human cardiomyogenesis in vitro is now becoming a process which, to a certain extent, can be effectively manipulated and directed. Although much research remains, new and improved protocols for guiding pluripotent stem cells to the cardiomyocyte lineage are accumulating in the scientific literature. However, the stem cell derived cardiomyocytes described to date, generally resemble immature embryonic/fetal cardiomyocytes, and they are in some functional and structural aspects different from adult cardiomyocytes. Thus, a future challenge will be to design strategies that eventually may allow the cells to reach a higher degree of maturation in vitro. Nevertheless, the cells which can be prepared using current protocols still have wide spread utility, and they have begun to find their way into the drug discovery platforms used in the pharmaceutical industry. In addition, stem cell derived cardiomyocytes and cardiac progenitors are anticipated to have a tremendous impact on how heart disease will be treated in the future. Here, we will discuss recent strategies for the generation of cardiomyocytes from human embryonic stem cells and recapitulate their features, as well as highlight some in vitro applications for the cells. Finally, opportunities in the area of cardiac regenerative medicine will be illustrated.     

Adhesion of Subsets of Human Blood Mononuclear Cells to Endothelial Cells In Vitro, as Quantified by Flow Cytometry

Binding of leucocytes to endothelial cells (EC) is essential as an initial step in inflammatory responses. We present a rapid, non-radioactive method to measure adhesion of human lymphoid cells to EC using flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMC) were allowed to adhere to EC grown in 24-well plates. Non-adhering cells were removed, after which adhering cells and EC were dissociated using trypsin/EDTA. These samples were subsequently analysed by flow cytometry, using scatter properties to distinguish between adhering cells and EC. The ratio of the number of adhering leucocytes and EC was calculated to quantify adhesion. Results of the flow cytometric adhesion assay were comparable to those obtained with a conventional adhesion assay using chromium-labelled cells. We additionally show that by using the flow cytometric adhesion assay, adhesion of lymphocytes and monocytes present within the adhering PBMC can be quantified simultaneously. As a model, the contribution of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in adhesion of PBMC to EC was studied. It was found that adhesion of lymphocytes and monocytes is regulated differently by phorbol ester and that the relative contribution of LFA-1 and ICAM-1 differs for both cell types.     

Cytotoxic Cells Directed Against Placental Cells Detected In Human Habitual Abortions by an In Vitro Terminal Labeling Assay

PROBLEM: From the clinical point of view, it has been proposed that an immunological imbalance between the mother and the fetus might exist in one of the mechanisms for human habitual abortion. However, in the definition of habitual abortion, we have no distinct immunological criteria for this clinical entity at the moment. METHOD: We employed aborted placental cells as the target cells in an in vitro terminal labeling (IVTL) assay system, in which the cytotoxic activity of maternal peripheral blood mononuclear cells (PBMCs) against the placental cell was examined. RESULTS: Our results showed that the cytotoxic activity of maternal PBMCs was significantly higher in the habitual aborters (the mean target cell destruction: %TCD = 34.9%, N = 14) than that in the women with a normal first trimester (the mean %TCD = 8.9%, N= 14, P<.01). The results from the IVTL assay did not correlate with other assays using paternal lymphocytes as the target cells. The surface marker analyses revealed that CD16+ cells, CD14 + cells, and CD5+ cells were involved in the cytotoxic response against the placental cells in various degrees among the cases. CONCLUSIONS: The above evidence suggests that a variety of cytotoxic cells participate in the phenomenon of human habitual abortion.     

Antigen-Pulsed, Interleukin-4-Treated B Cells Activate Primed T Cells In Vitro but not Naive T Cells In Vivo

The ability of B cells to act as effective antigen-presenting cells is a source of debate which centres on the degree of activation of either B cells or T cells. We have investigated whether B cells treated with interleukin 4 (IL-4) can express the two signals required to activate T cells: MHC Class 2/antigenic peptide complexes(signal 1) and the costimulatory molecules B7-1 and B7-2 (signal 2). We have also determined whether these cells could activate atitigen-experienced T cells in vitro and whether they could prime naive T cells in vivo . We found that B cells expressed abutidant MHC Class 2 molecules and moderate levels of B7-2 after 24 h culture in IL-4 with or without purified protein derivative (PPD) but B7-1 was not detectable. PPD-pulsed, IL-4 treated B cells induced antigeti-experienced T cells to proliferate in vitro but these cells failed to prime naive T cells in vivo when injected itito mice. We conclude that signals, in addition to those mduced with IL-4, are required for B cells to initiate an immutie respotise to atitigen.     

In Vitro Production of Granular T Cells and Mast Cells by Use of Different Conditioned Media

Normal mouse spleen cells were cultured in different conditioned media (CM). Mixed lymphocyte culture supernatant (MLC SN) was shown to promote the proliferation of cytotoxic, Thy-1+, Lyt-1+, Lyt-2+, asialo-GM-1+, weakly adherent cells with numerous vacuoles and lysosome-like cytoplasmic granules. In contrast, the Con A SN induced the proliferation of non-cytotoxic, Thy-1-, Lyt-1-, Lyt-2-, asialo-GM-1-, non-adherent cells with numerous cytoplasmic granules. The ultrastructural morphology of these cells and the cytochemical characteristics of their granules enable us to identify them as mast cells. The different effects of both CM could be related to their T-cell growth factor (TCGF) content. When the amount of TCGF of these two CM was determined (by assaying growth-stimulating activity for T-cell colonies), it appeared that the MLC SN contained larger amounts of TCGF than the Con A SN used in these experiments.     

In Vitro Development of Neural Progenitor Cells from Human Embryos

Behavior of stem/progenitor cells from the brain of human embryos during in vitro culturing was studied. Cultured cells from human embryonic brain developed and formed neurospheres heterogeneous by their cell composition. In a serum-containing medium some cells underwent differentiation by the neuronal pathway, while others remained in the stem state.     

E-Selectin Involvement in In Vitro Adhesion of Blood Dendritic Cells to Human Umbilical Cord Endothelial Cells

Peripheral blood dendritic cells (BDC) are potent antigen-presenting lymphoid cells. In the present study, we have examined the in vitro adhesion of BDC to human umbilical cord venous endothelial cells (HUVEC) and studied the expression of CD molecules and oligosaccharide haptens on BDC and endothelial cells. Immunohistochemistry showed that BDC were strongly positive for antibodies against HLA-DR, CD 11c, CD 18, CD44 and CD54, and moderately positive for anti-CD 11a, CD31,CD43 and CD58. In addition, BDC were moderately positive for anti-Sialyl Lewis a and strongly positive for anti-Sialyl Lewis x and CD77 (Galα l 4Galβl- 4Glc) Non-stimulated HUVEC were positive for anti-CD29, CD31 and CD77. An in vitro adhesion assay showed that only a small percentage of radiolabelled BDC bound to non-stimulated HUVEC (16.9 ± 5.9%, mean±SD). Stimulation of the HUVEC with IL-1 for 4 h produced a significant increase (P<0.002) in the percentage of radiolabelled BDC that bound to HUVEC (42.3 ± 7.1%). Preincubation of HUVEC with antibodies against E-selectin (10μg/ml) significantly inhibited (P<0.02) the binding of radiolabelled BDC to activated HUVEC (32.2±1.3%) whereas preincubution of BDC with antibodies against CD54, CD18, CD11b, CD11c and Sialyl Lewis x did not produce any significant inhibition. Preincubation of BDC with Sialyl Lewis a antibody and with isotype-matched control antibodies did not affect the increased binding of BDC to IL-l-activated HUVEC. Thus, E-selectin seems to be involved in adhesion of BDC to IL-1-stimulated HUVEC.     

Two- Versus Three-Dimensional In Vitro Differentiation of Human Pulp Cells Into Odontoblastic Cells

The spatial organization of the pulp cells may modify the cytodifferentiation process. The purpose of this study was to compare the two- versus three-dimensional cell culture systems for differentiation of human odontoblastic cells in vitro. Pulpal cores from freshly extracted human third molars were cultured in vitro in a perfusion device on two types of membranes: polyester membrane (two-dimensional [2D] cell culture) and nylon mesh (three-dimensional [3D] cell culture). The cells were incubated with minimum essential medium containing (a) substitute serum, (b) 10% fetal calf serum (FCS), (c) 10% fetal calf serum + 2 mM &#103 -glycerophosphate ( &#103 GP), and (d) 10% fetal calf serum + transforming growth factor (TGF) &#103 1. Immunohistochemistry was used to evaluate the expression of collagen I, osteonectin, and nestin. Small differences were observed between 2D and 3D cell culture systems. This was particularly evident in the 10% FCS group. &#103 -Glycerophosphate in the 3D system seems to stimulate the osteogenic cell phenotype, as a considerable induction of osteonectin is observed.     

Interferon-γ Production by Human T Cells and Natural Killer Cells In Vitro in Response to Antigens from the Two Intracellular Pathogens Mycobacterium tuberculosis and Leishmania major

Acquired resistance to both mycobacteria and Leishmania is primarily mediated by interferon-γ (IFN-γ), which triggers mechanisms leading to the death of the microorganism in macrophages. In this study, cell activation and IFN-γ production was investigated in human peripheral blood mononuclear cells (PBMC) from individuals previously sensitized to tuberculin and without known exposure to Leishmania parasites. Immune staining for intracellular IFN-γ and surface markers allowed flow cytometric identification of the cellular sources of IFN-γ in cell cultures incubated with purified protein derivative of tuberculin (PPD) and Leishmania antigens. It was found that IFN-γ was produced in response to both PPD and Leishmania stimulant by T cells in the cultures. Activation of IFN-γ producing natural killer (NK) cells was demonstrated only in some cultures, and only with concomitant T cell activation.     

In Vitro Studies of Human Prostatic Epithelial Cells: Attempts to Identify Distinguishing Features of Malignant Cells

Abstract

Recent advances in culture techniques have enabled routine establishment and propagation of epithelial cells derived from normal and malignant tissues of the human prostate. Comparative studies of the responses of normal and cancer-derived cell populations to various growth and differentiation factors in vitro were undertaken to examine the possibility that cancer cells might respond differentially. Clonal growth assays in serum-free medium demonstrated that optimal proliferation of normal as well as cancer cell strains was generally dependent on the presence of cholera toxin, epidermal growth factor, pituitary extract, hydrocortisone, insulin, and high levels of calcium in the culture medium, and on the use of collagen-coated dishes. Only one cancer strain responded aberrantly to epidermal growth factor and hydrocortisone. Putative differentiation factors (transforming growth factor-β and vitamin A) inhibited the growth of all normal and cancer strains. The origin of a cancer-derived cell strain that responded similarly to normal strains was verified by positive labeling with a prostate cancer-specific antibody, validating the conclusion from these studies that normal and cancer prostatic epithelial cells are not distinguishable on the basis of responses to the tested factors.