Pharmacokinetics and pharmacodynamics of tepoxalin after single oral dose administration to healthy volunteers.

This study was conducted to examine the pharmacokinetics and pharmacodynamics of tepoxalin in healthy volunteers, an antiinflammatory compound that inhibits cyclooxygenase and lipoxygenase. Tepoxalin was absorbed after oral administration of single doses from 35 to 300 mg, after which it was rapidly converted to an acidic metabolite, RWJ 20142, which inhibits cyclooxygenase but not lipoxygenase. The areas under the concentration-time curve (AUC) of tepoxalin and RWJ 20142 in plasma increased in a dose-dependent fashion. Administration of the lowest dose of tepoxalin completely inhibited whole blood cyclooxygenase for the entire period of observation. This inhibition correlated closely with that of secretion and aggregation induced by collagen of platelets obtained from these subjects. Similarly, administration of tepoxalin was associated with significant inhibition of lipoxygenase in whole blood. Lipoxygenase was inhibited a maximum of 60% in a time-dependent fashion, and the duration of inhibition was dose-dependent. These studies demonstrate that tepoxalin inhibits whole blood cyclooxygenase, lipoxygenase, and platelet function after oral administration in humans.

Activities of human acidic fibroblast growth factor in an in vitro dermal equivalent model.

Acidic fibroblast growth factor is a potent mitogen for human dermal fibroblasts in an in vitro three-dimensional collagen matrix, the "dermal equivalent." Both cell numbers and DNA synthesis are optimally stimulated by daily doses of 1 ng/ml of the pure human mitogen in the presence of heparin, which binds to, and stabilizes, the protein. Under daily treatment by 1 ng/ml aFGF, the fibroblast mitogenic response is marked but transient, and decreases steadily when fibroblasts mature in the collagen matrix. aFGF mitogenic stimulation also results in a decrease in cellular volume and inhibition of fibroblast-mediated contraction of the collagen gel. Various dosing regimes indicate that, although the greatest mitotic response was generated by daily dosing, nearly optimal responses can also be achieved with either a short duration of early daily dosing or longer-term intermittent treatment.

The interaction of interleukin 2 with its receptor in the generation of suppressor T cells in antigen-specific and antigen-nonspecific systems in vitro.

The role of interleukin-2 (IL-2) in the activation of suppressor T cells was investigated using the monoclonal antibody anti-Tac, which blocks the binding of IL-2 to the 55-kDa IL-2-binding peptide. The addition of anti-Tac during a preculture period inhibited the generation of Epstein-Barr virus (EBV)-induced suppressor T cells and of suppressor T cells induced in an antigen-specific system by a high antigen (sheep red blood cell) concentration. The cells activated by a short 2- or 7-day preculture period with EBV to become suppressor effectors were of the T8, Tac-positive phenotype. However, the T8-positive T cells obtained from peripheral blood mononuclear cells precultured with EBV for 14 days continued to manifest suppressor function, even though they were no longer Tac positive. In summary, our studies indicate that anti-Tac, by producing a functional blockade of human IL-2 receptors, inhibits the generation of suppressor T cells in antigen-specific as well as antigen-nonspecific systems and that cells that no longer express the Tac antigen may also function as suppressors.

Blockade of the interleukin-2 receptor by anti-Tac antibody inhibits the generation of antigen-nonspecific suppressor T cells in vitro.

The role of interleukin 2 (IL-2) in the activation of suppressor T cells was investigated by using the monoclonal antibody anti-Tac, which blocks the binding of IL-2 to the 55-kDa peptide of the high-affinity IL-2 receptor. Anti-Tac was added to an antigen-nonspecific suppressor system in which Con A-induced suppressor T cells were generated during a preculture period, and their effects on immunoglobulin production were assessed in second, indicator cultures containing pokeweed mitogen and peripheral blood mononuclear cells. Anti-Tac added during the preculture period inhibited Con A-induced suppressor T-cell generation. Cells activated by a short (2-day) preculture period to become effectors of suppression were primarily of the Tac-positive, T8 (CD8)-positive phenotype. Tac-positive, T8-negative T cells might also contribute to the suppressor activity. Our studies indicate that anti-Tac, by producing a functional blockade of human high-affinity IL-2 receptors, inhibits the generation of antigen-nonspecific suppressor T cells.

Humoral immune function in severe, active rheumatoid arthritis.

Peripheral blood mononuclear cells (PBMC) from 30 patients with definite or classic active rheumatoid arthritis who were on no remittive drugs were studied for spontaneous and pokeweed mitogen (PWM)-stimulated immunoglobulin plaque-forming cell frequency (IgPFC), spontaneous IgM-rheumatoid factor (IgM-RF) secretion, and in vitro proliferative responses to soluble recall antigens. Rheumatoid spontaneous total (IgG + IgM + IgA) IgPFCs were higher than those of normal controls when assayed after 7 days in culture. Spontaneous and PWM-stimulated IgM-PFCs, in contrast, were significantly less than normal regardless of when assayed. Spontaneous synthesis of IgM-RF was observed in 56% of the RA patients, but absolute amounts produced were widely heterogeneous. Spontaneous IgM-RF production by RA PBMC was associated with low or absent spontaneous IgM-PFC production. Moreover, a strong association was found between the median amount of IgM-RF secreted and depressed proliferative responses to soluble recall antigens. Our results define several abnormalities of immunoglobulin production in a clinically homogeneous and highly active rheumatoid population and delineate methodologic variations that can complicate the interpretation of similar data in the literature. In addition, our findings suggest that subgroups of rheumatoid patients that show distinct cellular and humoral immune abnormalities can be identified.

In vitro antigen-specific antibody response to the species-specific surface protein antigens of typhus group rickettsiae by human peripheral blood mononuclear cells: generation of an antigen-dependent suppressor T cell.

We defined conditions suitable for in vitro synthesis of rickettsia-specific antibody by human PBMC cultured with the SPA of Rickettsia typhi or R. prowazekii and without addition of mitogens or polyclonal stimulators. Antibody synthesis, as measured by an enzyme-linked immunosorbent assay, was cycloheximide-inhibitable and antigen-specific. PBMC from individuals with prior rickettsial infection made antibody, whereas PBMC from those receiving vaccine or with undetectable levels of serum anti-SPA antibody did not. Antibody production was T helper cell-dependent because isolated B cells did not generate antigen-specific antibody in the absence of autologous T cells. Furthermore, prior exposure of T cells to high concentrations of SPA led to the generation of an antigen-dependent population of cells capable of suppressing the anti-SPA response when co-cultured with autologous PBMC and optimal SPA concentrations. This system should serve as an effective tool for analyzing the cellular interactions involved in the in vitro regulation of antigen-specific antibody synthesis by human PBMC.

In vitro interaction with antigen suppresses the differentiation of chronic lymphocytic leukemia cells secreting monoclonal anti-sheep red blood cell antibody.

Leukemic B cells from a patient with chronic lymphocytic leukemia had membrane-bound IgM that reacted against a sheep red blood cell (SRBC) antigen. A small proportion of these cells was able to differentiate into anti-SRBC hemolytic plaque-forming cells when cultured in vitro in the absence of polyclonal stimulators. The addition of pokeweed mitogen or of x-irradiated allogeneic T cells further enhanced their differentiation. The addition of antigen (SRBC) to unstimulated cultures of the patient's mononuclear cells suppressed the anti-SRBC antibody production. Marked suppression was induced by concentrations of SRBC that were found to stimulate anti-SRBC antibody production optimally by normal peripheral blood mononuclear cells. Two lines of evidence suggest this inhibition was not due to the activation of suppressor cells. First, depletion of T cells with the phenotype reported to be associated with suppressor cells, namely OKT8+ cells, did not prevent suppression. Secondly, the patient's mononuclear cells pulsed with SRBC did not suppress anti-SRBC antibody production by untreated autologous cells in co-culture. These findings suggest that leukemic cells were inhibited by the direct interaction with antigen. This suppression might be the result of intrinsic abnormalities of leukemic B cells. Alternatively, it could be the reflection of a tolerogenic response to antigen stimulation displayed by the patient's leukemic cells, which have a surface phenotype, IgM+D-, that is comparable to that of immature normal B cells.

In vitro generation of antigen-specific hemolytic plaque-forming cells from human peripheral blood mononuclear cells.

We have described a culture and assay system for the sensitization of human peripheral blood mononuclear cells with a T cell-dependent antigen, sheep erythrocytes, in the absence of nonspecific stimulatory agents and with the subsequent generation of macroscopic hemolytic plaques. We have shown that the antibody produced by the plaque-forming cells generated in this culture system is specific for the sensitizing antigen, and that the plaques created are not false plaques because their formation is inhibited by cycloheximide. The success of this system can be attributed to several critical factors including large numbers of peripheral blood mononuclear cells (5 x 10(6) culture), a prolonged period of incubation (10-11 d), continuous rocking during the entire period of incubation, culturing in large (35-mm) flat-bottomed culture dishes in the presence of human plasma, and the appropriate antigen concentration (5 x 10(6) sheep erythrocytes/culture). Furthermore, the generation of macroscopic hemolytic plaques requires plaquing sensitized peripheral blood mononuclear cells in target cell monolayers fixed in an agarose matrix with an incubation period of 2-3 h. We have further shown that the antigen-specific response measured by this system is dependent on adherent cells and T lymphocytes. At least one population of the helper T cells is sensitive to 2,000 rad irradiation. This system is simple, sensitive, and should serve as an effective tool for the analysis of cellular interactions involved in the generation of human antigen-specific plaque-forming cells, the genetic control the human immune response, and the pathophysiology of altered immunoregulation in disease.

Genetic factors predisposing to chronic lymphocytic leukemia and to autoimmune disease.

Among 320 relatives of 28 patients with chronic lymphocytic leukemia (CLL), 4 had CLL, 1 had lymphosarcoma, and 2 other adults had leukemia of unknown type. Autoimmune disease including hyperthyroidism, pernicious anemia, rheumatoid arthritis and systemic lupus erythematosus affected 18 relatives. HLA typing of members of two families demonstrated that within each family the patient with CLL shared a common haplotype with relatives with autoimmune disease, but the haplotype was different in the two kindreds. In contrast, CLL was encountered in only 1 and autoimmune disease in 4 of 396 relatives of a group of 28 control patients. These data together with information from earlier reports support the hypothesis that genetic factors distrubing the regulation of the immune system may predispose both to lymphoid neoplasms and to autoimmune disease. The concept is supported by an array of experimental studies in animals.

Regulation of hematopoiesis: helper and suppressor influences of the thymus.

Thymocytes from normal mice strains as well as from genetically determined stem cell defective W/Wv anemic mice were cocultured with syngeneic (or congeneic) bone marrow cells. We assayed these cocultures for the proliferation of erythroid progenitor cell types (BFU-E and CFU-E) using the plasma clot technique. Results indicate that when concentrations of thymocytes were lower than bone marrow cells, significant suppression of erythroid growth was observed. However, when the concentration of thymocytes exceeded that of the bone marrow cells in culture (greater than 1:1), significant enhancement of erythroid growth was demonstrated. The W/Wv anemic bone marrow appears to respond to this interaction by enhancement at all concentrations of added normal thymocytes. The regulatory functions observed can be diminished by treatment of the thymocytes in vitro with anti-theta serum plus complement. Thus, we establish regulatory functions for anti-theta-sensitive regulatory cells (TSRC) with both positive (enhancement) and negative (suppression) components.

Erythropoiesis in vitro. Role of calcium.

The in vitro plasma clot technique was employed to examine the role of calcium during the interaction of erythropoietin and mouse erythroid progenitor cells. Erythropoietin-induced erythroid colony formation was increased 24% by the carboxylic ionophore A23187 (10 nM), whereas a 35% increase was produced by the carboxylic ionophore Ro 2-2985/1 (1 nM). EGTA (3 mM) inhibited erythropoietin-induced erythroid colony formation. Inhibition of erythroid colony formation by EGTA could be reversed by Ca2+, but not by Mn2+, Mg2+, Zn2+, or Fe2+. At least 30 min exposure of marrow cells to erythropoietin in vitro was required for production of erythroid colonies. EGTA substantially inhibited erythropoietin-induced erythroid colony formation even when the marrow cells were exposed to the hormone for up to 2 h before addition of the chelator. Marrow cells incubated first in calcium-free medium with erythropoietin and then cultured in the presence of calcium but not erythropoietin, failed to form erythroid colonies although colony formation occurred when erythropoietin was provided. Taken together, the data indicate that calcium is required for both extracellular and intracellular events during the interaction of erythropoietin with its target cells.

Separation of erythroid progenitor cells in mouse bone marrow by isokinetic-gradient sedimentation.

Isokinetic-gradient sedimentation employing a shallow linear gradient of Ficoll in tissue culture medium was used to isolate erythroid progenitor cells (CFU-e) from mouse bone marrow. Following gradient sedimentation, 34% of the total nucleated cells and 48% of the CFU-e applied to the gradient were recovered, and three distinct modal populations of CFU-e could be distinguished. The slowest-migrating population did not require exposure to exogenous erythropoietin in order to form erythroid colonies in vitro. The other two modal populations of CFU-e required exposure to exogenous erythropoietin for differentiation. One of these, constituting 64% of the hormone-dependent CFU-e recovered, migrated with the bulk of the marrow cells, whereas the other migrated ahead of the bulk of the marrow cells. This latter population, which contained 34% of the CFU-e, was recovered with 11% of the marrow cells, representing a twofold to threefold enrichment. BFU-e migrated more slowly than the erythropoietin-dependent CFU-e. Resedimentation studies suggested that the two erythropoietin-dependent CFU-e populations were distinct modal populations. When cells from the fastest-migrating population of erythropoietin-dependent CFU-e were cocultured with unseparated marrow cells, a further twofold to threefold enhancement of erythroid colony formation was obtained. Comparison of isokinetic-gradient sedimentation with discontinuous and continuous albumin density-gradient sedimentation revealed that isokinetic-gradient sedimentation was a more efficient method than the former and a more rapid method than the latter for isolating CFU-e from mouse bone marrow.