Defective production of monocyte-activating cytokines in lepromatous leprosy

We have examined the capacity of monocytes from patients with leprosy to undergo activation and the capacity of mononuclear cells from these patients to incorporate [3H]thymidine and produce monocyte-activating cytokines. Monocytes from patients with either lepromatous or tuberculoid leprosy were activated by concanavalin A (Con A)-induced mononuclear cell supernatants generated from the leukocytes of a normal person. Monocytes activated by these supernatants strongly inhibited L. pneumophila multiplication, and the degree of inhibition was comparable in both groups of patients. Mononuclear cells from patients with either form of leprosy responded comparably to Con A with vigorous [3H]thymidine incorporation. Mononuclear cells from patients with tuberculoid leprosy also vigorously incorporated [3H]thymidine in response to M. leprae antigens. In contrast, mononuclear cells from patients with lepromatous leprosy did not exhibit significant [3H]thymidine incorporation in response to M. leprae antigens. The capacity of mononuclear cells to generate monocyte-activating cytokines generally paralleled their capacity to incorporate [3H]thymidine in response to Con A and M. leprae. Mononuclear cells from patients with either form of leprosy responded to Con A with the production of cytokines (supernatants) able to activate normal monocytes, expressed by inhibition of L. pneumophila multiplication. However Con A-induced supernatants from patients with lepromatous leprosy were less potent than Con A-induced supernatants from patients with tuberculoid leprosy. Mononuclear cells from patients with tuberculoid leprosy responded to M. leprae antigens with the production of potent monocyte-activating supernatants. In contrast, mononuclear cells from patients with lepromatous leprosy did not produce monocyte-activating cytokines in response to M. leprae antigens. These studies support the hypothesis that the immunological defect in lepromatous leprosy results from a failure to activate mononuclear phagocytes rather than from an intrinsic inability of these cells to be activated. We suggest that the failure to activate mononuclear phagocytes stems from defective production of monocyte-activating cytokines in response to M. leprae antigens.     

Inhibition of lymphocyte proliferation stimulated by lectins and allogeneic cells by normal plasma lipoproteins.

Lipoproteins, isolated by sequential flotation at densities 1.006, 1.019, 1.063, and 1.21, were examined for their ability to inhibit human lymphocytes stimulated by allogeneic cells and by lectins (phytohemagglutinin-P and concanavalin A). All the classes of normal plasma lipoproteins inhibited lymphoproliferation when peripheral blood lymphocytes were cultured in autologous, heterologous, or lipoprotein-deficient plasma (d greater than 1.21). The rank order of inhibitory potency was intermediate density lipoprotein (IDL) greater than very low density lipoproteins (VLDL) greater than low density lipoproteins (LDL) greater than high density lipoproteins (HDL), regardless of the mode of stimulation. The concentrations of IDL, VLDL, and LDL required for complete inhibition of stimulated lymphoproliferation were considerably below the levels of each of these lipoproteins normally found in human plasma. In addition, the concentration of HDL required for 50-90% inhibition was in the range of HDL levels normally found in human plasma. Moreover, at relatively higher concentrations, lipoproteins suppressed the incorporation of [3H]thymidine into DNA below the levels seen with reseting, unstimulated lymphocytes. The results suggest that circulating lymphocytes may normally be highly suppressed by the combined effects of all the endogenous lipoproteins and that the lipoproteins may play important roles in vivo in modulating lymphocyte functions and responses.     

Effects of basic fibroblast growth factor on bone formation in vitro.

Basic fibroblast growth factor (bFGF) was studied for its effects on bone formation in cultured rat calvariae. bFGF at 0.1-100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to 4.4-fold. bFGF also increased the number of colcemid-induced metaphase arrested cells and the DNA content. Transient (24 h) treatment with bFGF enhanced [3H]-proline incorporation into collagen 24-48 h after the factor was removed; this effect was DNA synthesis dependent and blocked by hydroxyurea. The collagen stimulated by bFGF was type I, and this effect was observed primarily in the periosteum-free bone. In contrast, continuous treatment with bFGF for 24-96 h inhibited [3H]proline incorporation into type I collagen. bFGF did not alter collagen degradation. In conclusion, bFGF stimulates calvarial DNA synthesis, which causes an increased number of collagen-synthesizing cells, but bFGF has a direct inhibitory effect on collagen synthesis.     

Biochemical mechanisms in the Killmann experiment: critique of the deoxyuridine suppression test.

The degree of inhibition of [3H]thymidine incorporation into DNA by exogenous deoxyuridine is assayed in a procedure known as the deoxyuridine suppression test. We report studies of the biochemical basis of this phenomenon in phytohemagglutinin-stimulated lymphocytes, which suggest that its mechanism has not been fully understood. Results show that inhibition by deoxyuridine is caused only in part by expansion of the intracellular pools of nonradioactive dTMP and dTTP, which dilutes the specific radioactivity of the [3H]dTMP and [3H]dTTP derived from [3H]thymidine. Increased dTTP levels also inhibit thymidine kinase. In addition, thymidine kinase is competitively inhibited by intracellular deoxyuridine. Inhibition of thymidine kinase activity by both mebolites further decreases the specific radioactivity of [3H]dTMP and [3H]dTTP. Deoxyuridine also inhibits the incorporation of [3H]deoxyadenosine and [3H]deoxyguanosine into DNA in these cells. Exogenous deoxyuridine still inhibits [3H]thymidine incorporation in cells whose de novo thymidylate synthesis has been strongly inhibited by 5-fluorodeoxyuridine or methotrexate. In such drug-treated cells, exposure to high concentrations of exogenous deoxyuridine can partially overcome the inhibition of thymidylate synthetase with resulting increase in the severely depleted dTTP pools. This increase is associated with enhanced DNA synthesis, as measured by incorporation into DNA of labeled deoxyribonucleosides other than [3H]thymidine. We conclude that exogenous deoxyuridine has multiple effects on [3H]thymidine incorporation, which must be considered in interpretations of deoxyurindine suppression test results.     

A new type of vasodilator, HA1077, an isoquinoline derivative, inhibits proliferation of bovine vascular smooth muscle cells in culture

The effects of a newly developed vasodilator agent, HA1077 [1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride], were investigated on the proliferation of cultured bovine aortic vascular smooth muscle cells (VSMC). HA1077 (10-100 microM) inhibited both fetal calf serum-induced proliferation and [3H]thymidine incorporation into DNA of the growth-arrested VSMC in a dose-dependent manner. When quiescent cells were stimulated with platelet-derived growth factor followed by insulin, HA1077 (1-30 microM), administered together with either stimulation, showed dose-dependent inhibition of [3H]thymidine incorporation. Further reduction of [3H]thymidine incorporation was observed when HA1077 was present at both stimulations, suggesting that HA1077 suppresses DNA synthesis acting in both competence and progression stages. After stimulation with fetal calf serum, quiescent VSMC started and ceased DNA synthesis in 15 to 18 hr and 24 hr, respectively. HA1077 inhibited [3H]thymidine incorporation when it was added either from 12 hr to 15 hr or from 21 hr to 24 hr after serum stimulation. In addition, when percent inhibition of [3H]thymidine incorporation by continuous exposure to HA1077 was examined as a function of the time it was added, reductions of the value were observed at 0 to 3 hr, 12 to 18 hr and 21 to 24 hr. Thus, we concluded that HA1077 suppresses DNA synthesis of bovine VSMC acting at the G0/G1 and the G1/S phase transitions and also in the S phase of the cell cycle. It is suggested that this agent may act as a potent inhibitor of VSMC proliferation as well as a vasodilator.     

4-Quinolone drugs affect cell cycle progression and function of human lymphocytes in vitro.

Most antibacterial agents do not affect human lymphocyte function, but a few are inhibitory. In contrast, a pronounced increase in the incorporation of [3H]thymidine in the presence of 4-quinolones was observed in these studies. The uptake of [3H]thymidine into DNA (trichloroacetic acid precipitable) was significantly increased in phytohemagglutinin-stimulated human lymphocytes when they were exposed to eight new 4-quinolone derivatives, ciprofloxacin, norfloxacin, ofloxacin, A-56619, A-56620, amifloxacin, enoxacin, and pefloxacin, at 1.6 to 6.25 micrograms/ml for 5 days. Four less antibacterially active 4-quinolones (nalidixic acid, cinoxacin, flumequine, and pipemidic acid) stimulated [3H]thymidine incorporation only at higher concentrations or not at all. Kinetic studies showed that incorporation of [3H]thymidine was not affected or slightly inhibited by ciprofloxacin 2 days after phytohemagglutinin stimulation but was increased on days 3 to 6. The total incorporation of [3H]thymidine from day 1 to day 6 after phytohemagglutinin stimulation was increased by 42 to 45% at 5 to 20 micrograms of ciprofloxacin per ml. Increased [3H]thymidine incorporation was also seen when human lymphocytes were stimulated with mitogens other than phytohemagglutinin. Ciprofloxacin added at the start of the culture had a more pronounced effect on [3H]thymidine incorporation than when added later. In spite of the apparent increase in DNA synthesis, lymphocyte growth was inhibited by 20 micrograms of ciprofloxacin per ml, and cell cycle analysis showed that ciprofloxacin inhibited progression through the cell cycle. In addition, immunoglobulin secretion by human lymphocytes stimulated by pokeweed mitogen for Epstein-Barr virus was inhibited by approximately 50% at 5 micrograms of ciprofloxacin per ml. These results suggest that the 4-quinolone drugs may also affect eucaryotic cell function in vitro, but additional studies are needed to establish an in vivo relevance.     

Modulation of human lymphocyte function by C3a and C3a(70-77)

Human C3a and the synthetic octapeptide C3a (70-77), which retains the activities of an anaphylatoxin, inhibit in a concentration-dependent manner the generation of leukocyte inhibitory factor (LIF) activity by human mononuclear leukocytes and T lymphocytes cultured with the mitogens phytohemagglutinin (PHA) or concanavalin A (Con A) or the antigen streptokinase-streptodornase (SK-SD). The generation of LIF activity was inhibited by 50% by 10(-8) M C3a or C3a(70-77) with PHA or Con A as the stimulus, whereas a more than 10-fold higher concentration of C3a(70-77) than C3a was required to achieve the same level of suppression with SK-SD as the stimulus. Similar concentrations of C3a(70-77) inhibited to the same extent the migration of T lymphocytes stimulated by alpha-thioglycerol of Con A. Neither C3a nor C3a(70-77) altered significantly the uptake of [3H]thymidine by human mononuclear cells exposed to PHA, Con A, or SK-SD. The capacity of C3a(70-77)- Sepharose,m but not Sepharose alone, to adsorb or inactivate mononuclear leukocytes required for the generation of LIF activity established a direct interaction. Analysis of the lymphocytes in the effluent from C3a(70-77)-Sepharose columns, using monoclonal antibodies to surface antigens, showed a selective depletion of the helper/inducer population of lymphocytes. C3a might represent an important mediator of the functionally selective regulation of human T lymphocyte activities by the complement system.     

Hodgkin''s disease. An immunodepleting and immunosuppressive disorder.

Irradiated leukocytes or mononuclear leukocytes, from 16 out of 30 patients with Hodgkin's disease and from one patient with the Sézary syndrome, stimulated in culture subnormal (3H)thymidine incorporation by allogeneic lymphocytes from normal individuals. This abnormality was not demonstrated in any of 30 other patients with non-Hodgkin's lymphomas. Subnormal mixed leukocyte culture reaction activation was caused by suppression of the mixed leukocyte reaction by patients' cells. Inhibition of the reaction by patient mononuclear leukocytes was corrected when adherent cells were removed or when protein synthesis was inhibited with cycloheximide. The inhibitory cells were probably lymphocytes since selective removal of phagocytic cells did not remove the inhibition by other patient mononuclear leukocytes. The presence in culture of as few as 2,500 granulocytes per mm3 also reduced responses when target cells were from patients with Hodgkin's disease. Patient cells no longer suppressed the mixed leukocyte reaction after patients entered clinical remission which suggests that suppression is a reversible, disease-related abnormality. Thus, the immune deficiency with advance Hodgkin's disease caused by ly lymphocyte depletion may be compounded by a relative excess of suppressor lymphocytes. The overall immunodeficiency may be further compounded by suppression of immune response by granulocytes at even physiologic concentrations.     

A mechanism for the stimulation by inorganic mercury of [3H] thymidine incorporation into DNA in cultured Molt-4F cells

Mercuric chloride at a narrow range of concentration (2 to 2.5 X 10(-5)M) facilitated [3H]thymidine incorporation into acid-insoluble material (DNA fraction) of cultured human T lymphoid cells, Molt-4F, after 72-hr culture with the metal. This effect by mercury was observed in spite of the decrease in growth rate and DNA contents of the cells. Thymidine kinase activity in Molt-4F cells treated with 2 X 10(-5)M mercury decreased to 50 to 60% of the control activity. The stimulation of [3H]thymidine incorporation into the cells by mercury, therefore, might be independent of the increase in thymidine kinase activity. 3H-Thymidine incorporation by the control cells decreased as culture time passed. In contrast to the control, [3H]thymidine incorporation by mercury-treated cells increased until 72-hr culture. [3H]Thymidine uptake by the control cells after 24, 48, or 72-hr culture increased until 20 min of incubation period, but thereafter no increase in the uptake was observed until 60 min. On the other hand, [3H]thymidine uptake by the cells treated with mercury for 24 to 72 hr increased linearly until 60 min of incubation period. These results seemed to indicate that the mercury stimulation of [3H]thymidine incorporation might be attributable not to the actual increase of DNA synthesis but to the suppression of the culture time-dependent decrease in the incorporation by the control cells.     

Liberation of a fibrogenic factor from human blood monocytes, ascites cells, cultured histiocytes and transformed mouse macrophages by treatment with SiO2

Human monocytes and ascites macrophages from cirrhotic patients were isolated in Percoll-gradient and cultured with and without silica. Similar experiments were carried out also with cultured malignant human histiocytes and transformed mouse macrophages. The fibrogenic activity of the culture media was tested by measuring the incorporation of [3H]proline and [3H]thymidine into cultured rat granuloma and human synovial cells. Media from silica-treated monocytes, ascites macrophages and certain histiocyte and mouse macrophage lines caused an increase in the incorporation of both [3H]proline and [3H]thymidine into collagen and DNA, respectively, in both cell systems. Alkaline RNase activities were decreased markedly in the media from silica-treated ascites macrophages but not in the media of the monocytes or histiocytes.     

Inhibition of immune functions by antiviral drugs.

Immune functions were evaluated in vitro for PBMC isolated from healthy donors and cultured with the antiviral agents, 3'-azido-3'-deoxythymidine (AZT), ribavirin, ganciclovir, 2'3'-dideoxyinosine (ddI), or acyclovir. To identify methods for assessing the effects of antiviral drugs on immune cells, the PBMC response to mitogens, Con A, or phytohemagglutinin was evaluated from measurements of [3H]thymidine and [14C]-leucine incorporation, cell growth, cellular RNA, DNA, and protein levels, and the PBMC proliferative cycle (i.e., progression from G0----G1----S----G2 + M). At clinically relevant concentrations, AZT, ribavirin, or ganciclovir diminished PBMC responsiveness to mitogen. The numbers of proliferating cells in G1, S, and G2 + M phases of the cell cycle, DNA content, and [3H]thymidine uptake were decreased in cultures treated with AZT, ribavirin, or ganciclovir. AZT or ribavirin but not ganciclovir reduced RNA and protein in the cultures and inhibited cell growth. Whereas AZT, ribavirin, or ganciclovir were antiproliferative, ddI or acyclovir had little, if any, effect on PBMC mitogenesis. The inhibitory effects of antivirals on immune cells may contribute to the immune deterioration observed in patients following prolonged use of the drugs.     

Platelet factor 4 modulates the mitogenic activity of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) has been shown to stimulate cell proliferation after vascular injury. The mitogenic activity of bFGF requires interactions with both a high affinity receptor and a cell-surface heparan sulfate proteoglycan. We tested the ability of platelet factor 4 (PF 4) and other platelet heparin-binding proteins to modulate bFGF-stimulated [3H]thymidine incorporation into fibroblasts. The supernatant of thrombin-stimulated platelets contained an inhibitor of bFGF-induced mitogenesis; this activity coeluted with PF 4 upon gel filtration, heparin-agarose, and ion-exchange chromatography. Purified thrombospondin and beta-thromboglobulin did not inhibit the mitogenic activity of bFGF. PF 4 inhibited the activity of 5 pM bFGF with 50% inhibitory concentration of 75 nM. Purified PF 4 also inhibited the basal incorporation of [3H]thymidine into 3T3 fibroblasts and the increased [3H]thymidine incorporation occurring after wounding of a cell monolayer. PF 4 did not affect the mitogenic activity of serum. Inhibition of bFGF activity by PF 4 could be overcome by exogenous heparin or chondroitin-4-sulfate, suggesting that inhibition of mitogenesis is caused by binding of PF 4 to cell-surface glycosaminoglycans. These results indicate that an important role of PF 4 released at sites of vascular injury and platelet activation is to control cellular proliferation caused by the release of bFGF from ruptured cells.     

Application of a serum-free medium in the growth and differentiation of human peripheral blood lymphocytes

To eliminate variability due to nonspecific stimulation or inhibition by different lots of fetal bovine serum, the activation of human peripheral blood mononuclear cells in a modification of Iscove's medium (medium C-IMDM) was compared with the routinely used Roswell Park Memorial Institute (RPMI)-1640 medium containing 15% fetal bovine serum. Stimulation of peripheral blood mononuclear cells was enhanced and occurred at lower mitogen concentrations in C-IMDM compared with cells grown in RPMI-1640 supplemented with fetal bovine serum. Maximum incorporation of [3H]thymidine following stimulation by concanavalin A, phytohemagglutinin, and pokeweed mitogen was more than twice the peak values obtained in RPMI-1640 supplemented with fetal bovine serum. Concentrations of concanavalin A and phytohemagglutinin required for maximum stimulation were 0.5 and 15 micrograms/ml, and 0.3 and 1.0 micrograms/ml . 5 X 10(5) cells in C-IMDM and RPMI-1640, respectively. Cells grown in C-IMDM responded to lower concentrations of pokeweed mitogen and optimal growth in the serum-free medium required 0.4 micrograms/ml . 5 X 10(5) cells. The stimulation of immunoglobulin-producing cells in C-IMDM was enhanced and occurred at lower concentrations of pokeweed mitogen. Less variability of growth (i.e., incorporation of [3H]thymidine) and immunoglobulin synthesis occurred in peripheral blood mononuclear cells cultured in different preparations of C-IMDM than that reported for cells cultured in RPMI-1640 supplemented with different lots or batches of fetal bovine serum. These data suggest that C-IMDM may be an alternative to media supplemented with fetal bovine serum.     

Comparison of flow cytometric analysis and [3H]thymidine incorporation for measurement of the effects of drug toxicity on lymphocyte stimulation.

Human mononuclear cells were exposed to three antiviral agents, stimulated with phytohemagglutinin, and assayed for DNA synthesis with [3H]thymidine uptake and flow cytometric analysis. Cytosine-arabinoside demonstrated inhibition of blastogenic reactivity by both [3H]thymidine uptake and flow cytometric analysis, whereas acyclovir showed no significant suppression. In contrast, (E)-5-(2-bromovinyl)-2'-deoxyuridine, a thymidine analog, demonstrated a lack of correlation between the two methods. The competitive inhibition of some compounds with [3H]thymidine incorporation necessitates the use of other methods to measure DNA synthesis.     

Effect of adenosine deaminase inhibition upon human lymphocyte blastogenesis.

The biochemical mechanisms by which a genetically determined deficiency of adenosine deaminase leads to immunodeficiency are still poorly understood and prompted this study. We have examined the effects of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) upon the response of human peripheral blood mononuclear cells to the mitogen concanavalin A (Con A). Cells isolated from normal volunteers were incubated in microtiter plates in the presence of various inhibitors, and the incorporation of tritrated thymidine or leucine into macromolecular material was measured after 64 h. EHNA at a concentration of 0.3 muM, which inhibited 90% of the adenosine deaminase (ADA) activity in a mononuclear preparation, impaired the incorporation of tritrated leucine into protein; 100 muM EHNA was the minimal concentration that inhibited thymidine uptake. The addition of 15 muM adenosine or 10 muM cyclic AMP to Con A-stimulated lymphocytes inhibited leucine uptake, while millimolar concentrations were required to inhibit thymidine uptake. Lower doses of adenosine and cyclic AMP stimulated thymidine incorporation. The inhibition of thymidine uptake observed with millimolar concentrations of adenosine was independent of the type of mitogen (pokeweed or Con A), the concentration of mitogen, or the medium used, but could be increased if the cells were cultured in a serum with reduced levels of adenosine deaminase. Washout experiments failed to demonstrate a critical period early in immune induction during which adenosine exerted its inhibitory effects. Noninhibitory doses of EHNA potentiated the effects of adenosine and cyclic AMP on leucine and thymidine uptake. EHNA at a concentration of 50 muM also potentiated the inhibitory effects on thymidine uptake of dibutyryl cyclic AMP, butyric acid, norepinephrine, and isoproterenol, but not theophylline. When mitogenesis was assayed by leucine incorporations, no synergy between EHNA and these compounds was apparent. Uridine relieved to some extent the inhibition of blastogenesis produced by adenosine and cyclic AMP, but not by dibutyryl cyclic AMP, norepinephreine, isoproterenol, or theophylline. Neither uridine alone nor uridine plus adenosine protected lymphocytes from the inhibitory effects of EHNA.     

Nitric oxide, atrial natriuretic peptide, and cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fibroblasts.

This study tested the hypothesis that nitric oxide (NO) and atrial natriuretic peptide (ANP) can attenuate the effects of adrenergic agonists on the growth of cardiac myocytes and fibroblasts. In ventricular cells cultured from neonatal rat heart, ANP and the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) caused concentration-dependent decreases in the norepinephrine (NE)-stimulated incorporation of [3H]leucine in myocytes and [3H]thymidine in fibroblasts. In myocytes, the NO synthase inhibitor NG-monomethyl-L-arginine potentiated NE-stimulated [3H]leucine incorporation. In both cell types, ANP and SNAP increased intracellular cGMP levels, and their growth-suppressing effects were mimicked by the cGMP analogue 8-bromo-cGMP. Furthermore, in myocytes, 8-bromo-cGMP attenuated the alpha1-adrenergic receptor-stimulated increases in c-fos. Likewise, ANP and 8-bromo-cGMP attenuated the alpha1-adrenergic receptor- stimulated increase in prepro-ANP mRNA and the alpha1-adrenergic receptor-stimulated decrease in sarcoplasmic reticulum calcium ATPase mRNA. The L-type Ca2+ channel blockers verapamil and nifedipine inhibited NE-stimulated incorporation of [3H]leucine in myocytes and [3H]thymidine in fibroblasts, and these effects were not additive with those of ANP, SNAP, or 8-bromo-cGMP. In myocytes, the Ca2+ channel agonist BAY K8644 caused an increase in [3H]leucine incorporation which was inhibited by ANP. These findings indicate that NO and ANP can attenuate the effects of NE on the growth of cardiac myocytes and fibroblasts, most likely by a cGMP-mediated inhibition of NE-stimulated Ca2+ influx.     

Human parathyroid cell proliferation in response to calcium, NPS R-467, calcitriol and phosphate

It remains uncertain how calcium, phosphate and calcitriol regulate parathyroid cell growth. The present study was aimed at examining possible direct effects of these modulators and of the calcimimetic NPS R-467 on parathyroid cell growth in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell cycle antigen Ki 67 expression in a parathyroid cell culture model derived from uraemic patients. The effect of NPS R-467 on parathyroid hormone (PTH) secretion and intracellular [Ca2+]i response was also examined. Increasing the [Ca2+] in the medium from 0.5 to 1.7 mM increased DNA synthesis (P < 0.005) and the number of Ki 67-positive cells (P < 0.005). However, NPS R-467 (0.01-1 microM) inhibited 3[H]thymidine incorporation by 35% in the presence of 0.5 mM [Ca2+]e. Exposure of cells to Ca2+ or NPS R-467 led to a rapid increase of intracellular Ca2+, although the pattern of increase differed. Addition of calcitriol (10-10-10-7 M) to the culture medium suppressed [3H]thymidine incorporation dose-dependently. Finally, high levels of phosphate (3.5 mM) in the medium led to a significant (P < 0.05) increase in [3H]thymidine incorporation. The observed stimulatory effect of Ca2+ in the medium in vitro appears to be at variance with the inhibitory effect of calcimimetic NPS R-467 in vitro. In an attempt to solve these apparent discrepancies, and based on the notion of a reduced calcium-sensing receptor (CaR) expression in parathyroid tissues of uraemic patients, we hypothesize that Ca2+ may regulate parathyroid cell proliferation via two different pathways, with predominant growth inhibition in cases of high CaR expression or activation, but prevailing stimulation of proliferation in cases of low CaR expression.     

Insulin-like growth factor I mediates selective anabolic effects of parathyroid hormone in bone cultures.

PTH was studied for its effects on bone formation in cultured rat calvariae. 0.01-10 nM PTH stimulated [3H]thymidine incorporation into DNA by up to 4.8-fold. Although continuous treatment with PTH for 24-72 h inhibited [3H]proline incorporation into collagen, transient (24 h) treatment enhanced [3H]proline incorporation into collagen 24-48 h after the hormone was removed. The collagen stimulated by PTH was type I and the effect was observed in the periosteum-free bone and was not blocked by hydroxyurea. Furthermore, treatment with 1-100 nM PTH for 24 h increased insulin-like growth factor (IGF) I concentrations by two to fourfold, and an IGF I antibody prevented the PTH stimulation of collagen synthesis, but not its mitogenic effect. In conclusion, continuous treatment with PTH inhibits calvarial collagen, whereas transient treatment stimulates collagen synthesis, and the stimulatory effect is mediated by local production of IGF I.     

Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors.

Hepatic lipocytes appear to be central to the pathogenesis of hepatic fibrosis, undergoing activation during inflammation to a matrix-producing, proliferative cell type. We have studied the activation process in culture by examining the response of lipocytes to conditioned medium from hepatic macrophages (Kupffer cells). Lipocytes exposed to Kupffer cell medium (KCM) exhibited cellular and nuclear enlargement associated with up to a threefold increase in collagen and total protein synthesis per cell. Cell proliferation was also stimulated as measured by [3H]thymidine incorporation and direct cell counting. The latter effect was serum dependent and inhibited by antibodies to platelet-derived growth factor (PDGF). Proliferation could be stimulated by recombinant PDGF, but only after preincubation of cells with KCM. These findings suggested that KCM was eliciting expression of the PDGF receptor in lipocytes, and this was confirmed by immunoblot analysis with antibodies to the PDGF receptor. DNA synthesis in lipocytes exposed to KCM occurred at 48 h, which reflected the time required for PDGF receptor expression (24 h) plus initiation of [3H]thymidine incorporation (24 h). These results indicate that KCM has multiple stimulatory effects on cultured lipocytes similar to activation of these cells observed in vivo.     

Activity of P536, a UDP-glucose analog, against Trypanosoma cruzi.

P536, a UDP-glucose analog which was previously described as an antiviral agent (M. J. Camaraza, P. Fernández Resa, M. T. García López, F. G. de las Heras, P. P. Mendez-Castrillón, B. Alarcón, and L. Carrasco, J. Med. Chem. 28:40-46, 1985), has a potent and selective activity against the intracellular and extracellular stages of Trypanosoma cruzi in vitro. It had a 50% inhibitory concentration of less than 5 micrograms/ml for T. cruzi extracellular cultured forms (epimastigote) and of 25 micrograms/ml for T. cruzi intracellular forms (amastigote) growing inside J774G8 macrophagelike cells. In contrast, the 50% inhibitory concentration was 100 micrograms/ml or greater for cultured mammalian cells and 180 micrograms/ml for the proliferation of mouse spleen lymphocytes. Furthermore, the addition of P536 (50 micrograms/ml) to T. cruzi-infected J774G8 cells cured the infected macrophages, making them able to grow and function normally. Studies on the mechanism of action of this drug indicated that it inhibited incorporation of [35S]methionine, [3H]thymidine, [3H]mannose, [14C]-N-acetylglucosamine, and [3H]uridine into macromolecules by T. cruzi epimastigotes, the last being the most sensitive.