TONE AND REACTIVITY OF VASCULAR SMOOTH MUSCLE IN GERMFREE RAT MESENTERY

The immune responses to sheep erythrocytes of mouse spleen cell suspensions from immune and nonimmune donors were compared in vitro. In vivo immunity was only transiently reflected in vitro, and 8 wk after in vivo immunization the responses of cultures from immunized and nonimmunized mice were virtually identical. There appeared to be two mechanisms for an antibody response to sheep erythrocytes. The first was responsible for the early primary response and is unmodified in the immune animal though contributing little to subsequent in vivo responses due to its suppressibility by specific antibody. The second was expressed in the in vivo secondary response but not on in vitro challenge of spleen cells from mice immunized many weeks previously; spleen cell cultures from such immune mice, freed from the antibody of the in vivo environment, once again demonstrate a pure primary-type response.     

Mechanism of allopurinol-mediated increase in enzyme activity in man

Allopurinol therapy in man interferes with pyrimidine biosynthesis de novo by inhibition of one or both of the two enzymes, orotate phosphoribosyltransferase (OPRT) and orotidylic decarboxylase (ODC), responsible for the conversion of orotic acid to uridine-5'-monophosphate. Inhibition of this pathway in vivo is followed in 1-3 wk by an increase in the activity of both of these enzymes in erythrocytes and of ODC in circulating leukocytes. This drug-mediated increase in enzyme activity in erythrocytes could not be attributed to enzyme stabilization or induction in vivo but appeared to be due to enzyme "activation." "Activation" of the OPRT enzyme was directly demonstrated in erythrocytes studied in vitro after incubation with oxipurinol, and to a lesser extent, with allopurinol. No evidence for "activation" of the ODC enzyme was demonstrated in vitro. This response to allopurinol therapy provides an excellent model for examining the mechanism of increased enzyme activity in response to drug administration.     

In vivo requirement for a radiation-resistant cells in the immune response to sheep erythrocytes

Experiments have been done to establish whether the radiation-resistant or A cell has a specific function in the initiation of an immune response in mice to sheep erythrocytes (SRBC). All previous demonstrations using accessory (A) cells have involved in vitro assays and are possibly explainable as tissue culture artifacts. If A cells are essential, it should be possible to demonstrate their requirement in vivo. Therefore we first established such conditions. Two methods were found for creating an A-cell deficiency in vivo: (a) A cells disappear gradually from the spleens of irradiated mice, presumably by migration since A-cell function was shown not to be decreased by irradiation. If 3 days elapse between irradiation and transplantation of mixtures of bone marrow and thymus cells (which provide B and T but few A cells), the usual synergistic response does not occur. Addition of large numbers of freshly irradiated spleen cells to the mixture of bone marrow and thymus completely restores the immune response. (b) Injection of 10¹⁰ horse erythrocytes into mice suppresses A-cell activity in these mice 24 hr later; a much reduced response to SRBC is obtained when they are given at this time. The response can be partially restored if irradiated spleen cells are given with the SRBC. This observation formed the basis for a quantitative in vivo assay for A cells in which the magnitude of restoration by various suspensions of irradiated cells was used to estimate the A-cell activity of that suspension. A quantitative in vitro assay for A cells was also developed. It was essential for this assay that the total cell number, B-cell number, and T-cell number be kept constant and that only the number of A cells be allowed to vary. Only under these conditions was the response a linear function of the number of A cells added. If the in vivo and in vitro assays are detecting the same class of radiation-resistant cells, the physical properties of the cells active in each assay should be identical. Spleen cells were separated on the basis of both density and sedimentation velocity. Fractions from both separation methods were tested for their content of A cells using both the in vivo and in vitro assays. The density and sedimentation profiles of A cells were similar in both assays. The demonstration that a radiation-resistant cell is required in vivo and that this cell has properties identical to the radiation-resistant cell required in vitro indicates that this cell (the A cell) is directly involved in the initiation of an immune response to erythrocyte antigens.     

Role of human decay-accelerating factor in the evasion of Schistosoma mansoni from the complement-mediated killing in vitro [published erratum appears in J Exp Med 1992 Feb 1;175(2):619]

Decay-accelerating factor (DAF) is a 70-kD membrane glycoprotein that prevents complement (C)-mediated hemolysis by blocking the assembly or accelerating the decay of C3 convertase. Purified DAF is known to incorporate into the membrane of DAF-deficient cells, inhibiting lysis. Since Schistosoma mansoni is a blood-dwelling parasite, we investigated whether DAF can be transferred from human erythrocytes to the worm and protect it against C-mediated killing in vitro. We have found that schistosomula (schla) incubated with normal human erythrocytes (N-HuE), but not with DAF-deficient erythrocytes, become resistant to C damage in vitro. Protected parasites acquire a 70-kD surface protein which can be immunoprecipitated by anti-DAF antibodies. The acquired resistance is abrogated by treatment of N-HuE-incubated parasites with anti-DAF antibody. These results indicate that, in vitro, N-HuE DAF can be transferred to schla, and suggest its participation in preventing their C-mediated killing. This could represent an important strategy of parasites to evade the host's immune response in vivo.     

Functional characteristics of Peyer's patch cells. III. Carrier priming of T cells by antigen feeding

Peyer's patch T cells may serve an important role in the interaction of the host with intraluminal gut antigens. Studies presented in this paper demonstrate that T cells in murine Peyer's patches can be carrier primed for helper function in the induction of an antihapten response by feeding antigen. Carrier priming was assessed by measuring the ability of Peyer's patch cells from mice fed heterologous erythrocytes to enhance an antitrinitrophenyl (TNP) response in vitro when added to normal Peyer's patch cells cultured with TNP coupled to the erythrocyte used for feeding. Priming of T helper cells in Peyer's patches was antigen specific and occurred when erythrocytes were administered orally but not when erythrocytes were injected intravenously or intraperitoneally. Murine Peyer's patches are naturally deficient in a cooperating accessory adherent cell type(s) required for B-cell induction to humoral antibody synthesis in vitro and antigen feeding does not result in significant induction of Peyer's patch B cells to humoral antibody synthesis in vivo. Since Peyer's patch T cells can be carrier-antigen primed for helper function in the absence of B-cell induction to humoral antibody synthesis, these studies may indicate that T-cell priming is less dependent than B-cell induction on cooperating accessory adherent cells.     

THE ROLE OF NONLYMPHOID ACCESSORY CELLS IN THE IMMUNE RESPONSE TO DIFFERENT ANTIGENS

Tissue culture techniques were combined with cell separation procedures to investigate the cellular requirements for a response to antigen, leading to the production of antibody-forming cells. Mouse spleen was dissociated, and the cells were separated into various groups on the basis of density, size, and active adherence. The ability of fractions to initiate a response in vivo, on transfer to an irradiated recipient, was compared to the response in vitro; and this ability was correlated with the presence or absence of phagocytic cells. Two different antigens were studied, sheep erythrocytes (SRC) and polymerized bacterial flagellin (POL). Density distribution analysis of spleen showed a wide density range of cells responding to both antigens in vivo. The same fractions responded to POL in vitro as in vivo. By contrast, only the light density regions responded in vitro to SRC. Response occurred in regions of overlap between lymphocytes and phagocytic macrophages. Separation by active adherence on columns of large glass beads gave a preparation containing large, medium, and small lymphocytes but no detectable phagocytic macrophages and very low levels of phagocytic polymorphs. This lymphocyte preparation responded to both antigens in vivo. In vitro it gave a full response to POL, but no response to SRC. Addition of a small quantity of the adherent fraction, enriched for phagocytic cells, restored response to SRC. The use of strain-specific antisera in a mixed culture containing a C57 phagocytic fraction and CBA lymphocytes showed that the lymphocyte fraction contributed the precursors of the final antibody-forming cells. The accessory cells from C57 spleen banded in the light regions of the density gradient where phagocytic macrophages were found. Irradiated spleen cells also activated the lymphocyte preparation, suggesting that the irradiated host provided the accessory cells for the in vivo response to SRC. Small lymphocytes were purified from spleen by the small glass bead size filtration technique. This sample of small lymphocytes responded less well to POL than the total lymphocyte population, but it responded as well in vitro as in vivo. The small lymphocyte preparation responded in vivo to SRC but not in vitro. Addition of a small quantity of the phagocyte-rich fraction from adherence columns restored the in vitro response to SRC. The results indicated that phagocytic cells are not required in the initiation of an immune response to POL. By contrast some accessory cell, possibly a phagocytic macrophage, is required for a response to SRC. The basis for this marked difference is discussed.     

Infusion flow rates of whole blood and AS-1-preserved erythrocytes: a comparison

We compared the flow rates of whole blood and erythrocytes resuspended in a new preservative solution (AS-1, consisting of adenine, dextrose, mannitol, and saline) which results in an erythrocyte preparation with a hematocrit lower than that of packed erythrocytes. When 100 ml of AS-1 solution is added to erythrocytes, a hematocrit of 59 +/- 5% is consistently obtained, and the resultant product has an improved flow rate. When we compared the infusion flow rates of whole blood and AS-1-preserved erythrocytes in vitro and in vivo, we found that flow times were shorter for AS-1 erythrocytes than for whole blood in vitro and in vivo, the flow rates of AS-1 erythrocytes and whole blood when expressed per volume were similar in vivo, and the flow rate of AS-1 erythrocytes for erythrocyte mass delivery in vivo was superior to that of whole blood. Thus, we conclude that the flow rates of the two products are comparable.     

In vitro and in vivo transport of lithium by human erythrocytes.

An in vitro system that can be used to measure both uptake and efflux of lithium by erythrocytes (RBCs) is described. Using this system, RBC lithium accumulation in vitro was compared with in vivo RBC lithium concentrations observed in 6 normal volunteers. A significant correlation was demonstrated between in vitro RBC lithium accumulation after 48-hr incubation and in vivo RBC lithium concentration at 24, 48, 72, and 96 hr following the beginning of lithium ingestion. In addition, when efflux of lithium from RBCs in vitro was studied, a significant correlation was observed between residual lithium in RBCs and in vitro RBC lithium accumulation. Finally, it has been demonstrated that storage of blood in ice for 5 hr prior to incubation with lithium results in increased RBC lithium accumulation. A potential role for this in vitro incubation system as a model for in vivo RBC lithium accumulation is suggested.     

Immune response in the mutant diabetic C57BL/Ks-dt+ mouse. Discrepancies between in vitro and in vivo immunological assays.

Cell-mediated and humoral immune responses of mutant diabetic db+/db+ mice were evaluated using in vivo and in vitro immunological assays. When compared to lean, nondiabetic db+/m+ or m+/m+ mice, db+/db+ mice demonstrated markedly altered in vivo immune responses characterized by a significantly diminished ability to reject allogeneic skin grafts, a markedly diminished capacity to generate cytotoxic cells after sensitization with allogeneic EL-4 lymphoma cells and a significantly enhanced plaque-forming cell response to sheep erythrocytes. In contrast, spleen cells from db+/db+ mice demonstrated only minimal alterations in in vitro responses to mitogens and allogeneic cells and no alteration in their capacity to generate an in vitro plaque-forming cell response. The spleens and thymuses of db+/db+ mice weighed significantly less than organs from db+/db+ mice. In addition, thymuses from db+/db+ mice demonstrated a marked deficiency in in vivo [125I]UdR uptake. These data suggest that the altered metabolic status of the diabetic host influences immune function in vivo possibly due to abnormal function of lymphocyte subpopulations.     

Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum beta-lactamase CTX-M-15 and of its structurally related beta-lactamase CTX-M-3

The immunomodulatory properties of antimicrobial agents and their clinical impact have been the focus of worldwide interest in recent years. In this study, the effects of different treatments with 14-, 15- and 16-membered ring macrolides on the mitogen-induced proliferative response of lymphocytes and the splenic response to immunization with sheep erythrocytes have been tested by in vitro and ex vivo assays in a murine experimental model. We observed that the in vivo administration of these antibiotics to mice induces a compensatory mechanism that abrogates the suppression observed by in vitro assays. Thus, physiological parameters may be important when testing the immunopharmacological effects of antibiotics.