Differential effect of culture epimastigotes and blood-form trypomastigotes on normal mouse splenocyte responsiveness to mitogens

Blood form trypomastigotes of the Y strain of T. cruzi, produced a strong inhibition of the blastogenic response to T and B cell mitogens, of the C3H/He, C57BL/6 and BALB/cJ strains of mice, while culture epimastigotes of the Y strain kept in a medium that allows parasite growth at 26 degrees, 30 degrees, 34 degrees and 37 degrees C produced a strong stimulatory effect that was even higher than the effect of the mitogens alone. Both the inhibitory or the stimulatory effects were dose-dependent. The stimulatory effect of epimastigotes was also temperature-dependent producing increased stimulation indexes as the temperature of parasite cultures was raised. Metabolically active, living parasites seemed to be necessary for an improved lymphocyte stimulation suggesting a potential role of secreted metabolites as polyclonal activators of mouse lymphocytes.     

Macrophages and lymphoid cells exposed to antigenic stimulation

After antigenic stimulation (intraperitoneal administration of 6% emulsion of sheep erythrocytes) interaction of macrophages and lymphoid cells has been studied in non-inbred mice spleen, lymph nodes, lungs and skin. Histological, morphometrical and radiographic techniques demonstrate that dermal macrophages possess the least reactivity. In 15 days of the experiment activation of the macrophagial link of the spleen and lymph node coinsides with the most intensive transformation of lymphoid elements into the antibody-producing plasma cells. In the lung the phenomena described are observed on the 9th day of the experiment.     

Thymopoietin-induced acquisition of responsiveness to T cell mitogens

A population of murine spleen cells, enriched by flotation in discontinuous bovine serum albumin gradients, was induced to differentiate in vitro by incubation with the purified thymic polypeptide hormone thymopoietin. These cells, normally unresponsive to both T and B cell mitogens, acquired the capacity to respond to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) but remained unresponsive to the B cell mitogen lipopolysaccharide from Escherichia coli. The acquisition of responsiveness to mitogens was not impaired by treatment with anti-Thy-1 serum + complement before induction but was prevented by this treatment after induction; thus the cells acquiring the functional capacity to respond to T cell mitogens had also been induced to express the T cell alloantigen Thy-1. Like the expression of T cell alloantigens, the capacity to respond to Con A developed rapidly and reached its maximum within 6 hr. Responses to Con A always greatly exceeded those to PHA. Our data suggest that committed precursor cells, which we believe to be prothymocytes, are induced by thymopoietin to differentiate to cells with an antigenic phenotype and mitogen responsiveness similar to cortical thymocytes.     

Differential effect of trypsin and trypsin inhibitors on lipopolysaccharide stimulation of hamster lymphoid cells

Activation of hamster lymphoid cells by the mitogen lipopolysaccharide was shown to be greatly potentiated by addition of the protease trypsin. However, activation of cells by the polyanion, dextran sulfate, was not altered by this enzyme. Addition of the soybean trypsin inhibitor inhibited lipopolysaccharide stimulation of lymph node cells from animals 1–4 months of age but only partially inhibited stimulation of splenocytes from young animals (5–6 weeks) and did not inhibit stimulation of splenocytes from older animals. Dextran sulfate stimulation was not inhibited by soybean trypsin inhibitor.     

The effect of dibromodulcitol on resting and dividing lymphoid cells

The effect of dibromodulcitol (DBD) on the incorporation of labelled precursors into DNA and RNA fractions of PHA-stimulated human lymphocytes and of P388F lymphoma cells at various stages of their growth was studied. Both cell systems showed sensitivity to the drug within the concentration range of 1–10 μg/ml.When DBD was added before phytohaemagglutinin (PHA), human lymphocytes showed a DNA labelling that was more affected than RNA. In contrast, by adding DBD after PHA, RNA labelling was much more inhibited than DNA. In the latter case, the decrease in DNA labelling occurred only 24 h after drug treatment whereas RNA labelling was decreased 1 h after treatment. Levels of DBD which normally produced 30% inhibition in plating efficiency of P388F lymphoma cells affected uridine-5-T incorporation to a different extent at different stages of growth of the culture. Enhanced RNA labelling occurred in early exponential stage while at later stages of growth, RNA synthesis was depressed.     

Immunological selection of variant mouse lymphoid cells with altered glucocorticoid responsiveness.

We have devised an immunological procedure to separate cells on the basis of expression of mouse mammary tumor virus (MMTV) gene products. Plastic petri dishes coated with specific antibodies against MMTV proteins bind cells with an efficiency that correlates with the level of MMTV gene expression. Glucocorticoid-sensitive mouse thymoma cell line W7 was infected with MMTV. Clones from the infected population retain the relatively slow cytolytic glucocorticoid response and, in addition, exhibit a rapid induction of MMTV-specific RNA and proteins. By combining our immunological selection with the selection for resistance to hormone-mediated cytolysis, we have isolated variant cells which are resistant to the cytotoxic effect of glucocorticoids but which retain the induction of viral gene products and must therefore have a functional glucocorticoid receptor protein.     

Dendritic cells and T lymphocyte interactions in patients with lymphoid malignancies

Dendritic cell (DC) vaccination is an attractive approach to the treatment of patients with lymphoid tumors. To evaluate its feasibility, we have tested the functional properties of DC and T-lymphocytes in patients with treated and untreated chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). Healthy volunteers were used both as controls and as a source of cells for allogeneic mixed leukocyte reaction (MLR). In these reactions, dendritic cells from both untreated and treated patients were comparable to dendritic cells from healthy volunteers. In all the untreated patients studied, autologous dendritic cells promoted the survival and proliferation of both CD4 and CD8 lymphocytes (though the proliferation response was much better in the CD4 subset), whereas only 3 out of 5 treated patients were able to mount this response with CD4 lymphocytes and 4 out of 5 with CD8 lymphocytes. In 3 out of 5 untreated patients, pulsing of DCs with tetanus toxoid promoted a better CD4 response than was achieved with unpulsed DCs, while none of 5 treated patients had an additional response after pulsing with tetanus toxoid. None of patients studied, either treated or untreated, had a better CD8 response to pulsed DCs than to unpulsed ones. During CD4 lymphocyte proliferation, more CD4(+)CD25(hi) lymphocytes were generated in both treated and untreated patients than in healthy controls. Poor proliferation of cytotoxic cells and preferential proliferation of CD4(+)CD25(hi) T-regulatory cells in response to self and/or foreign antigens might be one of the mechanisms responsible for immunosuppression and impaired tumor surveillance in patients with lymphoid malignancies.     

Mechanical interactions between dendritic cells and T cells correlate with T cell responsiveness

Ag recognition is achieved through the communication across intercellular contacts between T cells and APCs such as dendritic cells (DC). Despite remarkable progress in delineating detailed molecular components at the intercellular contacts, little is known about the functional roles of physical cross-junctional adhesion between T and DC in shaping T cell responses. In addition, the mechanisms underlying sensitivity and specificity of Ag discrimination by T cells at intercellular contacts remain to be elucidated. In this study, we use single-cell force spectroscopy to probe the mechanical interactions between DC and T cells in response to stimulation with a panel of altered peptide ligands. The results show that intercellular interactions of DC-T cell conjugates exhibited different ranges of interaction forces in peptide-dependent manners that match the ability of the peptides to activate T cells. Elevated calcium mobilization and IL-2 secretion by T cells were only promoted in response to antigenic peptides that induce strong interaction forces, suggesting that mechanically stable DC-T cell contacts are crucial for driving T cell activation. Strong interactions were not solely dependent on cell-surface molecules such as TCRs and the adhesion molecule LFA-1, but were also controlled by cytoskeletal dynamics and the integrity of membrane lipid rafts. These data provide novel mechanical insights into the effect of Ag affinity on intercellular contacts that align with T cell responsiveness.     

The effect of thymosin on glucocorticoid receptors in lymphoid cells

The present study investigates the effect of a partially purified thymus factor, thymosin Fraction 5, and an homogeneous polypeptide component of Fraction 5, thymosin α₁, on glucocorticoid resistance and glucocorticoid receptors in mouse thymocytes. Treatment of thymocytes in vitro with thymosin Fraction 5 or α₁, results in an increase in the percentage of glucocorticoid-resistant cells. Studies on the specific whole-cell binding of [³H]dexamethasone and steroid competition experiments demonstrate the existence of high-affinity (K_d = 1.0 × 10^?8M) specific glucocorticoid receptors in mouse thymocytes. Preincubation with thymosin Fraction 5 or α₁ appears to cause a reduction in the specific [³H]dexamethasone binding to intact thymocytes.     

Cell-cell interactions modulate the responsiveness of PC12 cells to nerve growth factor

The growth of PC12 cells on a collagen substratum or on monolayers of several non-neuronal cell types was studied by measuring nerve growth factor (NGF)-dependent increases in the expression of a 150 X 10(3) (Mr) neurofilament protein subunit and the membrane glycoprotein Thy-1. Both responses were found to be greatly suppressed in cultures of fibroblasts as compared to the C2 and G8-1 muscle cell lines and the C6 glioma cell line. This suppression was associated with an inhibition of NGF-dependent neuritic outgrowth from PC12 cells grown on fibroblast monolayers. There was no evidence that fibroblasts secrete soluble molecules that directly inhibit these responses or neutralize NGF. In addition, there was no difference in the neurofilament protein response from PC12 cells that had been treated with NGF prior to coculture, and the now primed PC12 cells readily extended axons over fibroblast monolayers. These data demonstrate that cell-cell and/or cell-matrix interactions can modulate biochemical responses to NGF and suggest that responsiveness of neuronal cells to environmental cues is not immutable. Control of the latter may be at the level of expression of receptor molecules for cell-surface- or matrix-associated macromolecules and a similar mechanism operating during development could play a role in growth cone guidance.     

Modulatory effect of zinc on the proliferative response of murine spleen cells to polyclonal T cell mitogens

To study the effect of zinc on the proliferative response to polyclonal T cell mitogens, spleen cells from C57BL/6 mice were cultured with or without ZnCl2 and stimulated with graded doses of concanavalin A or phytohemagglutinin. Addition of 10(-4) M ZnCl2 inhibited proliferation whereas 10(-5) to 10(-6) M ZnCl2 did not modify the response to suboptimal doses of mitogen but increased DNA synthesis in cultures stimulated with high doses of mitogen (10 or 20 micrograms/ml of concanavalin A and 10 or 25 microliters/ml of phytohemagglutinin) which are supraoptimal for C57BL/6 mice, and inhibited proliferation in cultures of spleen cells from animals of this strain, low responder to T cell mitogens. In contrast, supplementation with ZnCl2 did not enhance the response to mitogen of spleen cells from high responder BALB/c mice. The enhancing effects of ZnCl2 on the proliferative response of C57BL/6 cells were not observed following depletion of adherent cells or in cultures supplemented with 5 X 10(-5) M 2-mercaptoethanol, both conditions capable of abrogating the inhibitory effect of high mitogen doses on the response of C57BL/6 cells.     

Ability of monovalent cations to replace potassium during stimulation of lymphoid cells

Stimulation of hamster thymocytes, splenocytes, or lymph node cells occurred to a minimal extent in the absence of K⁺. This observation was found for stimulation by T-cell mitogens (phytohemagglutinin and concanavalin A), A B-cell mitogen (lipopolysaccharide), or antigen (KLH). Marginal restoration of the responses to these stimulants occurred in the presence of 0.1 mM K⁺ and responsiveness returned to near maximal levels on addition of 1 mM K⁺ to the cultures. Attempts to restore the responsiveness with other monovalent cations revealed an order of effectiveness of K⁺ ≥ Rb⁺ ? NH₄⁺ ≥ Li⁺. At the 1 mM level K⁺ and Rb⁺ were equally effective in supporting stimulation by phytohemagglutinin while all concentrations of Li⁺ tested (0.1–10 mM) would not support stimulation. However, addition of Li⁺ to cultures reconstituted with 1 mM K⁺ or Rb⁺ revealed that this ion could enhance the phytohemagglutinin response by approximately 100% in the presence of K⁺ and only 30% in the presence of Rb⁺. These data support the hypotheses that the Na,K ATPase must be active for lymphocyte stimulation to occur and that some of the biological effects of Li⁺ on lymphocyte stimulation are mediated at the level of the Na,K ATPase.     

The effect of ionizing radiation on lipid metabolism in lymphoid cells

Lipid metabolism was studied in lymphoid tissues of rats after whole body irradiation with doses producing damage of different degrees to lymphoid cells (4-10 Gy). The content of free cholesterol, cholesterol esters, and total phospholipids was determined in peripheral blood lymphocytes and thymocytes 1-2 h after exposure. Simultaneously, the rate of in vitro incorporation of 2 14C-acetate into total lipids, phospholipids, and cholesterol of lymphoid cells was estimated. It was shown that exposure of rats to ionizing radiation caused activation of lipogenesis. Cholesterol synthesis was activated after a dose of 4 Gy and decreased with increasing dose.     

The effect of various fixation procedures on the digestability of sialomucins with neuraminidase

Synopsis The histochemical digestability with neuraminidase of sialomucin in mouse sublingual gland was studied in unfixed and formaldehyde vapour-fixed cryostat sections, and in sections prepared from paraffin-embedded material fixed in several alcohol- or formaldehyde-containing fixatives recommended for mucosubstances.The removal of sialic acid residues from sections treated with neuraminidase was followed histochemically with the following staining methods: Azure A pH 3.5, Alcian Blue pH 2.5, Low Iron Diamine and Alcian Blue pH 2.5 followed by periodic acid-Schiff. When Goland's methanolic cyanuric chloride was used as fixative, only a partial loss of tissue basophilia was evident after enzyme incubation, but in tissues fixed in other ways a complete loss of histochemically detectable sialic acid residues was observed.