Inhibition Of 3H-thymidine Incorporation Research Articles

Dipyridamole and Platelet Release of Platelet-derived Growth Factor

Platelet-derived growth factor (PDGF) and β-thromboglobulin (β-TG) are released from platelet alpha-granules during platelet activation. PDGF is a potent chemoattractant and mitogen for human vascular smooth muscle cells, and may be important in the development of late restenosis following angioplasty and in atherogenesis. In recent studies, where PDGF release into serum was evaluated indirectly by measuring (3)H-thymidine incorporation into fibroblasts, it was reported that the antiplatelet drug dipyridamole (DPM) decreased serum levels of PDGF. Such selective inhibition of the PDGF-release would have potential important implications for patients with atherosclerosis and for patients undergoing angioplasty. We therefore measured platelet content of PDGF and β-TG as well as platelet release of PDGF using a newly developed radioimmunoassay in healthy volunteers before and immediately after ingestion of DPM 100 mg t.i.d. for 3 days. We found no significant differences in platelet content of PDGF or β-TG before and after DPM. PDGF release from platelets isolated from plasma by gel filtration and stimulated with thrombin as well as platelet release of PDGF into serum was also unaffected by DPM. In conclusion, treatment with DPM does not affect platelet content of PDGF or β-TG. The treatment did not inhibit the platelet-release of PDGF as previously reported, neither via direct effects on platelets nor on inhibitory plasma components. DPM may, however, inhibit (3)H-thymidine incorporation into fibroblasts.

Captopril Inhibits Proliferation of Human Lung Fibroblasts in Culture: A Potential Antifibrotic Mechanism

The angiotensin converting enzyme (ACE) inhibitor captopril, a free-thiol compound used widely as an antihypertensive agent, also inhibits radiation-induced pulmonary fibrosis in rats (Ward et al., Int J Radiat Oncol Biol Phys 19:1405, 1990). In an attempt to clarify the antifibrotic mechanism of captopril in vivo, the present study examined the effect of the drug on proliferation of human lung fibroblasts in culture. Captopril produced a drug dose-dependent reduction in fibroblast proliferation and 3H-thymidine incorporation during a 24-72-hr incubation. This cytostatic action of captopril was not the result of cytotoxicity as assessed by trypan blue exclusion, or by 51Cr or lactate dehydrogenase (LDH) release. Fibroblasts stimulated to proliferate by basic FGF were more sensitive to the antimitotic effect of captopril than were unstimulated cells. The ability of captopril to inhibit 3H-thymidine incorporation was not reversed by exogenous angiotensin 2, and was not mimicked by the nonthiol ACE inhibitor lisinopril. These data indicate that the cytostatic effect of captopril was not attributable to ACE inhibition. Penicillamine, a thiol compound with virtually no ACE inhibitory activity, also reduced fibroblast 3H-thymidine incorporation, indicating that the antimitotic action of captopril may represent a nonspecific sulfhydryl effect. This study suggests that the antifibrotic activity of captopril in irradiated lung may result in part from a direct inhibition of fibroblast proliferation, particularly in fibroblasts responding to mitogenic stimuli.

Biological Receptors Mediate Anti-proliferative Action of Atrial Natriuretic Peptide in Cultured Mesangial Cells

The mechanism of anti-proliferative action of atrial natriuretic peptide (ANP) was examined in cultured mesangial cells. HS-142-1, a selective antagonist for ANP biological receptors, inhibited ANP-induced cellular cGMP accumulation and prevented ANP-induced inhibition of both serum-stimulated incorporation of 3H-thymidine and cell proliferation. Des-[Gln 18, Ser 19, Gly 20, Leu 21, Gly 22]-ANP 4-23-NH 2 (C-ANP) failed to inhibit 3H-thymidine incorporation. These results indicate that anti-proliferative action of ANP is mediated by the biological receptors in cultured mesangial cells.

Effects of finasteride on the rat ventral prostate.

The objectives of this study were to compare changes in the ventral prostate (VP) of young adult Sprague Dawley rats after 28 days of treatment with finasteride (F), a potent 5 alpha-reductase inhibitor, with those caused by castration (Cx). VP concentrations of DHT were reduced to 20.2% and 6.6% of controls (1,947 +/- 207 pg/VP, mean +/- SE) by F (5 or 20 mg/kg/day) and to 2.6% of controls by Cx. VP weights were reduced 49% and 54% by F and 88% by Cx. DNA/VP fell 25% and 15% with F treatment and 72% after Cx, whereas RNA and protein/VP were reduced 37-51% by F and 91-93% by Cx. The RNA/DNA and the protein/DNA ratios fell to 30-36% of controls after F treatment and to 70% of controls after Cx. The mRNA concentrations of the C3 subunit of prostatein 28S ribosomal RNA fell after treatment with F (5 mg/kg/day) and after Cx, whereas the mRNA for TRPM-2, an androgen-suppressed protein associated with apoptosis, was increased only after castration. To examine further the effects of F on the rate of DNA synthesis, 7-day regressed adult rats were treated for 3 days with testosterone propionate +/- F, and incorporation of 3H-thymidine in minced ventral prostates was determined. F inhibited 3H-thymidine incorporation. We conclude that Cx causes a greater reduction in cell number/VP and a greater reduction in RNA and protein cell than F and that the differences between F treatment and castration probably result from differences in prostatic concentrations of T.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual regulation by cAMP of beta-hexosaminidase-induced mitogenesis in bovine tracheal myocytes.

beta-Hexosaminidases, potent mitogens in bovine tracheal myocytes (BTM), stimulate a rapid and transient increase in intracellular cyclic adenosine monophosphate (cAMP) accumulation. The objective of this study was to elucidate the contribution of cAMP in hexosaminidase-induced airway muscle proliferation. Rate of DNA synthesis was measured by 3H-thymidine incorporation in quiescent cells prepared by a low-serum treatment (0.4%) for 48 h after reaching confluency in microtiter wells. cAMP accumulation was measured in acetylated cell extracts in the presence of isobutyl methylxanthine (100 microM) by radioimmunoassay using 125I-cAMP as tracer. Exposure of quiescent cells to purified human placental hexosaminidase B (5 micrograms/ml, 50 nM) caused a significant transient increase in cAMP accumulation (49 to 107 fmol/micrograms protein, or a 20- to 70-fold increase from basal level). Maximum increase occurred at 15 min followed by a rapid decline in cAMP accumulation within 30 min after exposure to hexosaminidase. Similar results were obtained in cells treated with neoglycoprotein mannose bovine serum albumin (100 to 500 nM). The increase in cAMP accumulation was inhibited by mannan (mannose receptor blocker, 0.1 mg/ml), as well as phenylisopropyladenosine (PIA; A1 receptor agonist that inhibits adenylyl cyclase, 0.1 to 1.0 microM). The increase in 3H-thymidine incorporation induced by hexosaminidase B was also inhibited by mannan and PIA. Exposure to 8-(4-chlorophenylthio)-cAMP (cpt-cAMP; a cell-permeable analog of cAMP, 100 microM) or forskolin (a direct activator of catalytic subunit of adenylyl cyclase, 24 microM) up to 6 h enhanced 3H-thymidine incorporation. In contrast, a prolonged exposure (18 to 30 h) to these agents inhibited 3H-thymidine incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between human monocytes and tumour cells. Monocytes can either enhance or inhibit the growth and survival of K562 cells.

Human bloodstream monocytes can kill cultured tumour cells (K562), as assessed by specific release of 51Cr from the targets and by inhibition of 3H-thymidine incorporation. Confluent monolayers of monocytes were required for maximal cytotoxicity, and the density of the K562 cells was also an important factor. For example, when K562 cells were seeded at high cell densities, they were killed during incubation with monocytes, but when seeded at low cell densities their growth and survival was enhanced during culture with monocytes. The factor(s) which promoted the survival and division of low density K562 cultures was endogenously secreted from monocytes as it was present in monocyte-conditioned medium, whereas the cytotoxic factor(s) were only expressed during co-culture of monocytes with K562 cells. Conditioned medium from HL 60, U-937, HeLa and K562 could also enhance the growth and survival of low density K562 cultures, and a similar effect was also observed upon the addition of catalase and superoxide dismutase to such cultures. Thus, the monocyte:target ratio is important in determining whether monocytes exhibit cytotoxic or growth-promoting effects and hence tumour-derived or monocyte-derived reactive oxidant species may play a role in tumour cell cycle regulation.

Growth Factor-Dependent Proliferative Stimulation of Hematopoietic Cells Is Associated With the Modulation of Cytoplasmic and Nuclear 68-Kd Calmodulin-Binding Protein

AbstractCalcium and calmodulin (CaM) are known to play critical roles in controlling cell cycle progression in a variety of cells. We observed that the CaM antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride (W-7), inhibited 3H-thymidine incorporation into DNA of factor-depen-dent hematopoietic cells. To delineate the role of CaM in proliferation of hematopoietic cells, we have investigated intracellular distribution of specific CaM-binding proteins (CaM-BPs) in response to hematopoietic growth factors in FDC-P1,32D, NFS-60, and T1165 cells. Each of these cell lines, when deprived of cytokines for 16 to 18 hours, essentially ceased proliferation, even in the presence of fetal calf serum. Concomitant to the cessation of proliferation, there was a dramatic depletion of a specific CaM-BP of about 68 Kd in both their cytoplasmic and nuclear fractions. Within 6 to 12 hours of reexposure to proliferation-specific cytokines, there was a restoration of the nuclear as well as cytoplasmic 68-Kd CaM-BP. Furthermore, such an induction and nuclear localization of the 68-Kd CaM-BP by the cytokines coincided temporally with the progression of synchronized FDC-P1 cells from G1 to S phase. By contrast, colony-stimulating factor-1 (CSF-1)-dependent bone marrow macrophages and BAC-1 cells did not exhibit 68-Kd CaM-BP in the nuclear or cytoplasmic fractions. These studies suggest that while hematopoietic growth factor granulocyte CSF-, granulocyte-macro-phage CSF-, interleukin-3 (IL-3)-, or IL-6-, whose receptors are members of the hematopoietin receptor family, induced cell proliferation is associated with a common mechanism involving nuclear localization of the 68-Kd CaM-BP, the CSF-1-induced proliferation seems to involve 68-Kd CaM-BP-independent pathways.

Growth factor-dependent proliferative stimulation of hematopoietic cells is associated with the modulation of cytoplasmic and nuclear 68- Kd calmodulin-binding protein

Calcium and calmodulin (CaM) are known to play critical roles in controlling cell cycle progression in a variety of cells. We observed that the CaM antagonist, N-(6-aminohexyl)-5-chloro-1- naphthalensulfonamide hydrochloride (W-7), inhibited 3H-thymidine incorporation into DNA of factor-dependent hematopoietic cells. To delineate the role of CaM in proliferation of hematopoietic cells, we have investigated intracellular distribution of specific CaM-binding proteins (CaM-BPs) in response to hematopoietic growth factors in FDC- P1, 32D, NFS-60, and T1165 cells. Each of these cell lines, when deprived of cytokines for 16 to 18 hours, essentially ceased proliferation, even in the presence of fetal calf serum. Concomitant to the cessation of proliferation, there was a dramatic depletion of a specific CaM-BP of about 68 Kd in both their cytoplasmic and nuclear fractions. Within 6 to 12 hours of reexposure to proliferation-specific cytokines, there was a restoration of the nuclear as well as cytoplasmic 68-Kd CaM-BP. Furthermore, such an induction and nuclear localization of the 68-Kd CaM-BP by the cytokines coincided temporally with the progression of synchronized FDC-P1 cells from G1 to S phase. By contrast, colony-stimulating factor-1 (CSF-1)-dependent bone marrow macrophages and BAC-1 cells did not exhibit 68-Kd CaM-BP in the nuclear or cytoplasmic fractions. These studies suggest that while hematopoietic growth factor granulocyte CSF-, granulocyte-macrophage CSF-, interleukin-3 (IL-3)-, or IL-6-, whose receptors are members of the hematopoietin receptor family, induced cell proliferation is associated with a common mechanism involving nuclear localization of the 68-Kd CaM-BP, the CSF-1-induced proliferation seems to involve 68- Kd CaM-BP-independent pathways.

Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro.

Immunohistochemical observations indicate that human myometrial smooth muscle cells express epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-AB and contain EGF and PDGF-beta receptors with no variation in intensity with phases of the menstrual cycle. Furthermore, immunofluorescent microscopic studies revealed that primary myometrial smooth muscle cell cultures also express EGF, PDGF-AB, and contain EGF and PDGF-beta, but not alpha-receptor. Incubation of subconfluent smooth muscle cells in serum-free medium leads to quiescence within 48 h as demonstrated by 3H-thymidine incorporation and labeling index. Exposure of quiescent cells to 10% fetal bovine serum stimulates resumption of DNA synthesis and proliferation in a time-dependent manner with a doubling time of 41.6 h. EGF (1.5-50 ng/ml) and PDGF-AB (1-10 ng/ml) in a dose- and time-dependent manner significantly stimulated 3H-thymidine incorporation by quiescent myometrial smooth muscle cells (P less than 0.05). Combinations of EGF (15 ng/ml) and PDGF-AB (10 ng/ml) significantly increased 3H-thymidine incorporation induced by either growth factor alone (P less than 0.05). PDGF-BB at 10 ng/ml also stimulated 3H-thymidine incorporation and its effect was similar to that induced by PDGF-AB at the same concentration. 17 beta-Estradiol (E2) at 1 microM inhibited 3H-thymidine incorporation by the smooth muscle cells (P less than 0.05). E2 also reduced the stimulatory effect of EGF (15 ng/ml) and PDGF (3 ng/ml). Progesterone at 1 microM either alone or in combination with E2 did not have any effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF. The effect of EGF and PDGF on cell growth and 3H-thymidine incorporation by myometrial smooth muscle cells was independent of phases of the menstrual cycle. In summary, the results of present studies indicate that human myometrial tissue and myometrial smooth muscle cells in primary culture locally produce EGF and PDGF-AB and contain EGF and PDGF-beta, but not alpha-receptors. Moreover, the myometrial smooth muscle cells in culture respond to the mitogenic action of EGF and PDGF.

The inhibition of DNA synthesis in chronic lymphocytic leukaemia cells by chlorambucil in vitro

The inhibition of 3H-thymidine incorporation into the DNA of mitogen-stimulated lymphocytes from patients with chronic lymphocytic leukaemia by chlorambucil was measured in vitro and the results related to clinical drug resistance. The assay proved to be both sensitive and specific showing a clear separation of those patients with responsive disease from those with disease resistant to treatment. There was evidence of primary drug resistance in untreated patients. In almost all patients who received treatment this led to increasing resistance to chlorambucil in vitro. The assay is predictive of clinical responsiveness and provides a potential means whereby new therapeutic agents and treatment modifiers may be investigated.

Effect of rufloxacin on in-vitro proliferation and differentiation of human mononuclear cells.

We investigated the effects of rufloxacin, a new, long acting fluoroquinolone, on the growth and differentiation of human peripheral blood mononuclear cells (MNC) stimulated with T- and B-cell mitogenic agents. Rufloxacin inhibited 3H-thymidine incorporation into MNC stimulated with phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) in a dose-dependent manner. The concentrations of rufloxacin required to inhibit 1/2 maximal proliferation of T- and B-cells were 62 and 33.5 mg/L respectively. Rufloxacin, at clinically achievable serum levels (less than 10 mg/L), was found not to inhibit PHA-induced T-cell differentiation as assessed by IL-2 production, IL-2 receptor expression and the expression of cell differentiation markers (CD4 and CD8). However, higher concentrations of rufloxacin (10 and 50 mg/L) markedly inhibited B-cell differentiation in-vitro as determined by the measurement of immunoglobulin production by MNC stimulated with PWM. The clinical relevance of our in-vitro findings remains to be elucidated.

SELECTIVE CYTOTOXICITY TO LEUKEMIC CELLS OF A MCAB ZH805 DIRECTED TO MYELOID CELL-DIFFERENTIATION ANTIGEN AND ADRIAMYCIN CONJUGATE

Via a dextran brige, adriamycin was conjugated to McAb Zh805. Under the experimental con-dition used, the rate of adriamycin conjugated to McAb Zh805 was 62% with a little loss of the drug activity. The conjugate retained 73% of the original antibody activity. The in vitro cytotoxic activity of the conjugate was assayed by the inhibition of 3H-thymidine incorporation. The results showed that Zh805DA conjugate inhibited 86% of 3H-thymidine incorporation on HL-60 cells after a 20-hour exposure to the conjugate at a concentration of 20μg/ml. In contrast to HL-60 cells, only 24% of 3H-thymidine incorporation in FO cells was inhibited. The specific cytotoxicities of Zh805DA conjugate to various fresh leukemic cells and leukemic cell lines were measured and compared with thal of equimolar amounts of free adriamycin and of untreated Zh805. The results showed that Zh805DA conjugaies exhibited significantly stronger cytotoxicity against the leukemic cells of AML, especially FAB Ml, M2, M3, and HL-60 ,K562 cells than against the leukemic cells of ALL and FO cells,while free adriamycin exhibited more or less the same cytotoxicity to various fresh leukemic cells and leukemic cell lines. Key words: McAb; adriamycis; leukemia; immune directed therapy

CYCLOSPORINEʼS DUAL MECHANISM OF ACTION IN INHIBITING GENERATION OF THE CYTOPLASMIC ACTIVATION SIGNAL AND THE NUCLEAR RESPONSE THERETO

The mechanism of cyclosporine's action was dissected using protocols admixing cytoplasmic extracts to trigger isolated nuclei prepared after phytohemagglutinin, OKT3, or mixed lymphocyte culture stimulation of normal volunteer and renal transplant recipient lymphocytes. CsA at 100-1000 ng/ml doses prevented cytoplasmic extracts of cells activated in vitro by OKT3 or MLC from triggering incorporation of radiolabeled nucleotides by resting isolated nuclei. In addition, nuclei isolated from cells previously treated with 200-1000 ng/ml of CsA were refractory to the potent cytoplasmic signal extracted from OKT3- or MLC-stimulated cells. CsA less effectively inhibited phytohemagglutinin-stimulated responses. CsA, but not its inactive analog cyclosporin H, inhibited 3H-thymidine incorporation into in situ nuclei. The inhibited cytoplasmic signal generation and nuclear responsiveness observed with normal lymphocytes exposed to CsA in vitro also was demonstrated with cells from renal transplant recipients who had been treated with CsA for less than 6 months. The impaired response of isolated patient lymphocyte nuclei to in vitro triggering with activated cytoplasmic extracts correlated with intranuclear CsA concentrations, but not with serum levels assessed with monoclonal antibodies selective for CsA or nonselective for CsA plus metabolites. Furthermore, patients displaying drug-induced nephrotoxicity had elevated intranuclear CsA, but not CsA plus metabolite, concentrations compared with stable transplant recipients. Thus, CsA addition in vitro or administration in vivo causes stereospecific inhibition, particularly for CD3 stimuli, of the cytoplasmic activation signal, and, to a lesser extent, of the subsequent nuclear response.

Peptide inhibition of myointimal proliferation by angiopeptin, a somatostatin analogue

Vascular smooth muscle cell hyperplasia is a major component of atherogenesis in various animal models. Angiopeptin, a cyclic octapeptide analogue of somatostatin, markedly inhibits myointimal proliferation in response to endothelial cell injury in the rat carotid artery, rabbit aorta and iliac arteries and in coronary arteries of transplanted rabbit hearts. Angiopeptin does not affect serum lipid profiles in nonhuman primates. It is unlikely, therefore, that its antiproliferative effect is mediated by alterations in cholesterol metabolism.Angiopeptin and other peptide analogues of somatostatin are potent inhibitors of growth hormone release and insulin-like growth factor-1 production. However, inhibition of smooth muscle cell proliferation in vivo is not a property common to all somatostatin analogues. This suggests that plasma growth hormone and growth hormone-dependent insulin-like growth factor-1 production are not physiologic stimuli for myointimal proliferation in vivo.Angiopeptin inhibits 3H-thymidine incorporation into rat carotid artery explants, suggesting a local effect on automne or paracrine mechanisms regulating cell growth. In view of its potent inhibitory effect on smooth muscle cell replication, angiopeptin may have clinical utility in preventing restenosis after percutaneous transluminal coronary angioplasty and in preventing accelerated coronary atherosclerosis after cardiac transplantation.

Differential Alteration of Cellular Lipids in Drug Sensitive and Resistant P388 Leukemia Cells by Clofibrate: Effects on Mitoxantrone Cytotoxicity

Earlier investigations have indicated a difference in the lipid profiles of drug-sensitive and drug-resistant tumor cells. This study was undertaken to evaluate the effect of alterations in the cellular lipid compositions by clofibrate (CPIB), an antihyperlipidemic agent, on mitoxantrone (Mtn) cytotoxicity in murine P388 leukemia cells sensitive (P388/S) and resistant (P388/Adr) to adriamycin and in human chronic myeloid leukemia (CML) cells. CPIB did not elicit any significant alterations in the lipid levels of P388/S cells, whereas in the P388/Adr cells it brought about a 14% and 49% decrease in the levels of cholesterol and triglyceride respectively. Inhibition of 3H-thymidine incorporation was utilized as a measure of cellular cytotoxicity. CPIB caused a dose dependent inhibition of DNA and RNA biosynthesis in P388/S, P388/Adr and CML cells. The combination of CPIB and Mtn induced a greater cytotoxicity in P388/Adr cells as compared to P388/S cells, as shown by enhanced inhibition of 3H-thymidine incorporation in P388/Adr cells. Similar results were observed when 3H-uridine was used as a measure of cellular cytotoxicity. These observations were further confirmed in fresh CML cell samples, in which the combination of CPIB with Mtn induced an irreversible and synergistic inhibition of DNA biosynthesis. Results warrant extensive studies on CPIB as a clinical modulator to enhance the antiproliferative activity of Mtn.

Specific cytotoxic effect of neocarzinostatin conjugated to monoclonal antibody A7 on human pancreatic carcinoma

The anti-cancer drug neocarzinostatin (NCS) was bound covalently to the monoclonal antibody A7 to form the conjugate A7-NCS. This antibody was produced by fusing the spleen cells of a mouse immunized against human colonic carcinoma with murine myeloma cells and reacts with a high percentage of human pancreatic carcinoma cell lines and with no normal human pancreas tissue by immunoperoxidase staining. The cytotoxic effect of A7-NCS was tested by measuring the inhibition of 3H-thymidine incorporation into human pancreatic carcinoma cells. The A7-NCS was approximately 2.7 times as effective as free NCS against human pancreatic carcinoma cells which reacted with the monoclonal antibody A7. A7-NCS had almost the same cytotoxicity a free NCS on human pancreatic carcinoma cells which did not react with the monoclonal antibody A7. A7-NCS appeared to be potentially useful as a conjugate for immunotargeting chemotherapy against pancreatic carcinoma.

Drosophila cell extracts contain a TGF-β-like activity

Extracts of the Drosophila cell line KCo contain a TGF-β-like activity. This bioactivity, like mammalian TGF-β, induces the anchorage-independent growth of rat NRK-49F cells in the presence of EGF, inhibits 3H-thymidine incorporation in the mink epithelial cell line CCL-64 and is acid and heat stable but destroyed by dithiothreitol. On a reverse phase column a single bioactive peak eluted at 37–40% acetonitrile. This TGF-β-like activity in KCo cells inhibits the growth of the homotypic Drosophila cell line 14-11-23, whereas porcine TGF-β 1 had no effect.

Cytotoxic effects of sera from patients with systemic scleroderma: Comparison of three different in vitro methods

Sera from 93 patients with systemic scleroderma including incipient or prodromal acroscleroderma and from 43 healthy individuals were studied for cytotoxic effects on endothelial cells by means of three different in vitro methods. Inhibition of 3H-thymidine incorporation by endothelium was caused by about 33% of sera, almost exclusively from patients with diffuse scleroderma and the transitional form: acroscleroderma--diffuse scleroderma. An antibody-dependent cellular cytotoxicity assay revealed cytotoxicity of about 37% of sera from patients with diffuse scleroderma and the transitional form but also of a proportion of sera from patients with CREST syndrome with pronounced vascular changes. Serum cytotoxic activity, revealed by both methods, was related with more frequent involvement of muscle and kidney in the patients. A direct 51Cr release assay showed the cytotoxicity only in 4 of 68 cases in diffuse scleroderma with pronounced internal organ involvement. Thus, depending on the method used, various types of cytotoxicity could be detected in sera from patients with systemic scleroderma.

Arrest of DNA Replication of Macrophages in BCG Granuloma and Peritoneal Exudates by Bacteria

Previously we found that bacterial surface substances induced the suppression of DNA synthesis of macrophages. We examined in the present study whether DNA synthesis of macrophages could be similarly suppressed by whole bacteria themselves. For this purpose we isolated macrophages from BCG granuloma of guinea pigs and rats. The macrophages from both of the animals gave essentially the same results. No isolated macrophages containing bacilli were found to incorporate 3H-thymidine when tested by autoradiography. Further, DNA replication of peritoneal exudate macrophages was markedly and rapidly suppressed in vitro upon phagocytosis of various kinds of bacteria but not of non-bacterial preparations. A close correlation was found between granuloma formation and inhibition of 3H-thymidine incorporation by MDP, its analogs, and various bacteria. These findings suggest that macrophages suppress their DNA replication when they phagocytose bacteria and that they can discriminate between bacteria and non-bacterial preparations.

3-Deazaadenosine — An inhibitor of interleukin 1 production by human peripheral blood monocytes

3-Deazaadenosine (c 3Ado) has been reported to have properties of an immunosuppressive and anti-inflammatory agent. This study was designed to investigate whether c 3Ado might exert anti-inflammatory activities through inhibiting Interleukin 1 (IL-1). c 3Ado was found to be a potent inhibitor of IL-1 production by LPS stimulated human peripheral blood monocytes and acted at the level of synthesis rather than secretion. c 3Ado also had direct effects on IL-1 biological activity in two separate assays; thymocyte proliferation and induction of prostaglandin release. Further experiments indicated that c 3Ado also inhibited growth factor dependent proliferation driven by both Interleukin-2 and Interleukin-3 as well as the proliferation of a number of non-growth factor dependent cells. However, short term exposure to c 3Ado resulted in no inhibition of 3H-thymidine incorporation by cells but a significant inhibition of 3H-uridine uptake into TCA precipitable material. These results suggest that c 3Ado is a selective inhibitor of RNA synthesis and inhibits IL-1 production and activity by blocking new messenger RNA production induced by LPS or IL-1. General inhibition of RNA synthesis would also account for its anti-proliferative activity.