Effect of anticancer drugs on the release of interferon γ in vitro

Summary Some conventional and experimental anticancer drugs were tested for their effect on concanavalin-A-induced interferon release from rat splenocytes in vitro. When 2.5 × 10⁶ rat splenocytes/ml, stimulated with 1 µg/ml concanavalin A, were incubated with various non-cytotoxic doses of the vinca alkaloid vincristine, there was an inhibition of the release of interferon in culture supernatants. The antitumour antibiotics bleomycin and Adriamycin, alkylating agents 4-hydroperoxycyclophosphamide and mafosfamide, and the immunoactive peptides FK 156 and FK565 did not affect the release of interferon under similar conditions. However, cyclosporin A, in similar experiments, markedly inhibited the release of interferon .     

Effect of anticancer drugs on the release of interleukin-6 in vitro

Summary This study investigates the effects of anticancer drugs and immunomodulating agents on the release of interleukin-6 (IL-6) from lipopolysaccharide-stimulated human peripheral blood mononuclear leucocytes in vitro. The addition of non-cytotoxic concentrations of Adriamycin (doxorubicin), vincristine and 4-OOH-cyclophosphamide (the in vitro active analogue of cyclophosphamide) resulted in suppression of IL-6 release. The drugs bleomycin, FK156 [d-lactoyl-l-alanyl--d-glutamyl-(l)-meso-diaminopimelyl-(l)-glycine], FK565 [heptanoyl--d-glutamyl-(l)-meso-diaminopimelyl-(d)-alanine] and the immunosuppressive agent cyclosporin A did not alter the release of IL-6 in the same experimental system.     

Enhancement of interleukin-2 release in rats by treatment with bleomycin and Adriamycin in vivo

Summary Spleen cells from rats previously injected with bleomycin (10 mg/kg) or Adriamycin (1 mg/kg) were able to release higher levels of interleukin-2 (IL-2) than cells from untreated animals. The difference in IL-2 release was detected after the cells were exposed to a suboptimal dose of concanavalin A (0.5 g/ml) for 24 h. By cytofluorimetry, these drugs did not change the proportion of W3/25⁺ (helper) or OX-8⁺ (suppressor) T-cell subsets. In contrast, the immunosuppressive drug cyclophosphamide inhibited the IL-2 release from spleen cells under the same conditions. It is suggested that some anti-cancer antibiotics may be able to enhance the release of IL-2 while other cytotoxic drugs with more immunosuppressive potential could inhibit the release of this mediator.     

Effect of Adriamycin on liposomal muramyl tripeptide's ability to up-regulate monocyte cytokine expression

Liposomal muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) is a biological agent in phase I and II trials for osteosarcoma and melanoma. Its mechanism of action has been linked to its ability to activate monocyte tumoricidal function and to stimulate monocyte production of tumor necrosis factor (TNF) and interleukins(IL)-1, –6, and –8. Our ultimate goal is to combine L-MTP-PE with chemotherapy. The purpose of this study was to determine whether doxorubicin (Adriamycin) interfered with the ability of L-MTP-PE to activate monocyte cytokine production. Human monocytes were cultured with or without 5–500 ng/ml of Adriamycin for 3 h and washed before being exposed to 2 g/ml L-MTP-PE for 16 h. Cultured supernatants were collected and assayed for TNF, IL-1, IL-6, and IL-8. The messenger RNA expression of IL-1, IL-1, TNF, IL-6, and IL-8 was quantified with northern blot analysis. Adriamycin did not suppress the upregulation of any of these cytokines. We concluded that combination therapy with L-MTP-PE and Adriamycin is feasible and that this combination warrents further investigation in a clinical setting.     

Effect of ibuprofen on monocyte activation by liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (CGP 19835A): Can ibuprofen reduce fever and chills without compromising immune stimulation?

The purpose of this study was to determine the effects of ibuprofen on the ability of liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) to activate human blood monocytes in vitro. We undertook these experiments because the major toxic side-effects following L-MTP-PE infusion, fever and chills, could be prevented when ibuprofen was given orally immediately before L-MTP-PE infusion. It was therefore important to determine whether ibuprofen interfered with the macrophage-activation properties of L-MTP-PE. Peripheral blood monocytes were isolated from normal donors, then incubated with L-MTP-PE in the presence or absence of ibuprofen. The cytotoxic properties of the monocytes were assessed by a radioisotope-release assay against A375 cells. Ibuprofen at dose levels of 40 µg/ml suppressed the generation of the cytotoxic phenotype but did not interfere with the killing process once the cells were activated. Interleukin-1 (IL-1) and tumor necrosis factor (TNF) production, as well as the mRNA expression of these cytokines, was suppressed by 40 µg/ml ibuprofen. Since IL-1 and TNF play a crucial role in the cytotoxic function of monocytes, these findings may explain the mechanism by which ibuprofen inhibited the generation of the cytotoxic phenotype by L-MTP-PE. By contrast, ibuprofen dose levels up to 10 µg/ml had no effect on the generation of monocyte-mediated cytotoxicity by L-MTP-PE and no effect on the production, secretion, or mRNA expression of TNF and IL-1. Therefore, we concluded that if ibuprofen is to be used to control the side-effects of L-MTP-PE, blood levels of up to 10 µg/ml are desirable. In two of three patients, we determined that an oral dose of 200 mg given immediately before L-MTP-PE infusion could achieve these desired blood levels.     

Tacrolimus (FK506) Suppresses TREM-1 Expression at an Early but Not at a Late Stage in a Murine Model of Fungal Keratitis

Purpose

To investigate the efficacy and mechanism of tacrolimus(FK506), which is a novel macrolide immunosuppressant, in inhibiting triggering receptor expressed on myeloid cells-1 (TREM-1) expression in a murine keratitis model induced by Aspergillus fumigatus.

Method

TREM-1 was detected in 11 fungus-infected human corneas by quantitative real-time PCR (qRT-PCR). RAW264.7 macrophages were divided into four groups, which received treatment with zymosan (100 µg/ml), zymosan (100 µg/ml) + mTREM-1/Fc protein (1 µg/ml), or zymosan (100 µg/ml) + FK506 (20 µM) or negative-control treatment. After this treatment, the expression of TREM-1, interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) was assayed using qRT-PCR and ELISA. The mouse model of fungal keratitis was created by intrastromal injection with Aspergillus fumigatus, and the mice were divided into 2 groups: group A received vehicle eye drops 4 times each day, and group B received 4 doses of FK506 eye drops each day. Corneal damage was evaluated by clinical scoring and histologic examination,and myeloperoxidase (MPO) protein levels were also detected by ELISA. The expression of TREM-1, IL-1β and TNFα was then determined at different time points using qRT-PCR and ELISA.

Results

TREM-1 expression dramatically increased in the human corneas with fungal keratitis. In contrast, FK506 reduced the expression of TREM-1, IL-1β and TNFα in RAW264.7 macrophages stimulated with zymosan. In the mouse model, at day 1 post-infection, the corneal score of the FK506-treated group was lower than that of the control, and polymorphonuclear neutrophil (PMN) infiltration was diminished. TREM-1, IL-1β and TNFα expression was significantly reduced at the same time point. However, the statistically significant differences in cytokine expression, clinical scores and infiltration disappeared at 5 days post-infection.

Conclusions

FK506 may inhibit the inflammation induced by fungi and alleviate the severity of corneal damage at an early stage of fungal keratitis by downregulating TREM-1 expression.     

Dehalogenation of chlorinated aromatic compounds using a hybrid bioinorganic catalyst on cells of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis>

A novel bioinorganic catalyst was obtained via reduction of Pd(II) to Pd⁰ on to the surface of cells of Desulfovibrio desulfuricans at the expense of H₂. Palladised biomass, supplied with formate or H₂ as an electron donor, catalysed the dehalogenation of 2-chlorophenol and polychlorinated biphenyls. In the example of 2,3,4,5-tetrachlorobiphenyl, the bioinorganic catalyst promoted a rate of chloride release of 9.33 ± 0.17 nmol min^–1 mg ^–1and only ~5% of this value was obtained using chemically reduced or commercially available Pd ⁰. In the case of 2,2,4,4,6,6-hexachlorobiphenyl the rate was more than four orders of magnitude faster than the degradation reported using a sulfidogenic culture. Negligible chloride release occurred from any of the chloroaromatic compounds using biomass alone, or from palladised biomass challenged with hexane carrier solvent only. Analysis of the spent solution showed that in addition to catalysis of reductive dehalogenation the new material was able to remove very effectively the organic residua, with neither any PCB nor any breakdown products identifiable by GC/MS.Revisions requested 8 September 2004; Revisions received 21 October 2004;     

A rapid bioassay to determine stabilities of anticancer agents under conditions of the clonogenic assay

Summary We developed a rapid bioassay to determine in vitro drug stabilities in the clonogenic assay. The in vitro half-lives of 11 standard antitumor agents, actinomycin D, Adriamycin, bleomycin, cis-Platinum, dacarbazine, 5-fluorouracil, melphalan, mitomycin C, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), vinblastine, and vincristine, and four investigational drugs, Aziridinylbenzoquinone (AZQ) (NSC 182986), diamminecyclobutane-dicarboxylatoplatinum (CBDCA) (NSC 241240), Dihydroxyanthracenedione dihydrochloride (DHAD) (NSC 301739), and 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea) (PCNU) (NSC 95466), were determined under conditions of the clonogenic assay as well as under storage conditions at −40° and−196°C. BCNU and AZQ at−40°C had t1/2 of 4.7 d and 2.5 d, respectively. All other drugs were stable at −40° and −196°C with t1/2>6 wk. Under assay conditions at 37°C, actinomycin D, bleomycin, dacarbazine, 5-fluorouracil, mitomycin C, vinblastine, vincristine, and DHAD were stable, with t1/2>14 d. CBDCA, AZQ, Adriamycin, cis-Platinum, melphalan, BCNU, and PCNU had t1/2 of 94,72,29,18.5,1.8,1, and 0.5 h, respectively. This work was supported by Veterans Administration Medical Research Service and by Contract CM57710 of the National Cancer Institute.     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor     

No inhibition of interferon γ release in human lymphocytes by Ciamexone

Summary In previous experiments Ciamexone, derivative of 2-cyan-aziridine, was able to influence T-cell-mediated regulatory mechanisms but seemed to have no or only little effect on T cell effector mechanisms. On the basis of these observations Ciamexone seems to be a highly selective immunosuppressive agent. In order to evaluate further possible mechanisms of Ciamexone it was the aim of our investigation to study its influence on interferon (IFN) production in phytohaemogglutinin (PHA)-stimulated T lymphocytes of 15 tumour patients and 12 healthy reference subjects. The IFN concentration of the cell supernatant was measured using an enzyme-linked immunosorbent assay. When the cells were stimulated with PHA at 7.5 µg/ml the IFN concentration rose to significantly different values in the reference group (1.0 ng/ml) as compared to the tumour patients (0.4 ng/ml) (P <0.05). An addition of Ciamexone (at any of the concentrations administered) to PHA stimulation of peripheral blood mononuclear cells (PBMC) showed no influence on the IFN release in either test group. The influence of hydrocortisone on the stimulation of PBMC with PHA resulted in a dose-dependent suppression of IFN production in both test groups, again with significant differerences between them. The IFN concentration was 0.95 ng/ml in the reference group and 0.2 ng/ml in the tumour patients when 0.01 µg/ml hydrocortisone was added (P <0.05). At 10 µg/ml hydrocortisone suppressed IFN production completely in both groups. Our results corroborate those investigations that showed no influence of the compound on T cell effector mechanisms. The attenuation of humoral immunophenomena, however, suggest a very specific point of action within the immune system by Ciamexone.     

Interleukin-2-dependent long-term cultures of low-density lymphocytes allow the proliferation of lymphokine-activated killer cells with natural killer,Tiγ/δ or TNK phenotype

Summary We have developed a culture system for longterm growth of human lymphokine-activated killer (LAK) cells exhibiting an elevated, wide-spectrum antitumor cytotoxicity. The system allows the exponential growth of monocyte-depleted low-density lymphocytes in the presence of human serum and recombinant human interleukin-2 (10³ U/ml), alone or in combination with interleukin-1 or (both at 10 U/ml). Eighteen cultures were established from 18 normal adult donors. The membrane phenotypes of the final LAK cell population, assessed by a panel of monoclonal antibodies (mAb), consist of three main types: (a) NKH-1⁺, Ti/^–, Ti/^–, and CD3^– lymphocytes; (b) NKH-1⁺, Ti/^–, Ti/⁺, and CD3⁺ lymphocytes and (c) NKH-1⁺, Ti/⁺, Ti/^– and CD3⁺ lymphocytes. Northern blot analysis showed that all these cell populations express relatively high levels of perforin RNA, particularly cells exhibiting the first phenotype. This culture system may provide a tool for cellular and molecular studies on the mechanisms of antitumor cytotoxicity, as well as the basis for new adoptive immunotherapy protocols in advanced cancer.     

Tumor necrosis factor α modifies resistance to interferon α in vivo: First clinical data

Summary Patients with Philadelphia-positive chronic-phase chronic myelogenous leukemia (CML) resistant to interferon (IFN) were treated in a phase I/II study with recombinant human tumor necrosis factor to overcome IFN resistance. Doses of 40, 80, 120 or 160 µg/m² TNF were given as 2-h infusions on 5 consecutive days every 3 weeks. IFN (4 × 10⁶ IU/m² s.c., daily) treatment was continued. Six patients were treated, completing 1–24 (median, 12) treatment cycles. Five of the six patients achieved partial hematological remission, while the remaining patient had to stop treatment because of WHO grade 4 thrombocytopenia following the first TNF cycle. No complete hematologic remission or cytogenetic improvement was seen. Side-effects were similar to those described for both substances alone. Maximum tolerable TNF doses usually varied between 80 µg/m² and 160 µg/m². To examine possible pathways of TNF activity in these patients, interferon receptor status and (2–5)-oligoadenylate synthetase levels were examined in peripheral blood mononuclear cells. Both parameters remained unchanged during TNF treatment. These preliminary data point to significant clinical efficacy of additionally applyed TNF in IFN-resistant CML patients.     

Effect of recombinant human tumor necrosis factor α on the induction of antibody-dependent cellular cytotoxicity in the treatment of established B16 melanoma liver nodules

Summary Incubation of C3H/Hen thymocytes in the presence of recombinant human tumor necrosis factor (TNF) and interleukin-2 (IL-2) augmented the generation of antibody-dependent cellular cytotoxicity (ADCC) when compared to cells cultured in TNF or IL-2 alone. This effect was optimal when 100–200 units/ml IL-2 was used together with 10³–10⁴ units/ml TNF. TNF alone at any concentration could not mediate the induction of ADCC. Similar to the results obtained in vitro, TNF, when given alone, had no effect on the generation of ADCC in vivo. The addition, however, of TNF to IL-2, given at 10 000 and 20 000 but not 40 000 units, enhanced the IL-2-induced ADCC on a per-cell basis. Furthermore, TNF enhanced the total ADCC activity in various organs including the liver, spleen and thymus as a result of an increase in the number of mononuclear cells isolated from these organs. The increase in total ADCC activity was optimal when 110 000–220 000 units (5–10 µg) TNF were employed together with IL-2. The combined treatment with TNF and IL-2 also increased the intracellular benzyloxycarbonyl-l-l-lysinethiobenzyl-ester esterase content in cells isolated from the livers of mice treated with these cytokines. On the basis of these results we treated mice bearing a single B 16 melanoma nodule with TNF and TNF + IL-2 given with or without anti-B 16 monoclonal antibody. We found that TNF administration augmented the anti-tumor effect of specific anti-B 16 antibodies, and the addition of IL-2 further increased this anti-tumor effect. Offprint requests to: S. A. Rosenberg, Surgery Branch, National Cancer Institute, Building 10, Room 2B42, Bethesda, MD 20892 USA     

Activation by a new synthetic acyltripeptide and its analogs entrapped in liposomes of rat alveolar macrophages to the tumor cytotoxic state

Summary FK-565 (heptanoyl--d-Glu-(l-meso-a, -A₂pm (l)-d-AlaOH) is a synthetic acyltripeptide closely resembling cell wall peptidoglycan peptides of Streptomyces in structure. Alveolar macrophages (AM) lavaged from lungs of F344 rats were activated by in vitro treatment with FK-565 and its derivatives at concentrations of 1–50 g/ml medium, and the activated AM killed syngeneic mammary adenocarcinoma cells. When FK-565 and related compounds were encapsulated in multilamellar (MLV) liposomes composed of phosphatidyl-choline and phosphatidylserine, dose-response experiments showed that they were about 800 times more effective than the free compounds in activating AM. Liposome-encapsulated FK-565 and its analogs caused significant activation of AM within 4 h. These data indicated that acyltripeptide and its analogs encapsulated in liposomes are more efficient than the free compounds in rendering AM tumoricidal.     

Role of oxidized human plasma low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation

The effects of oxidized human plasma low density lipoproteins (Ox-LDL) on the proliferation of cultured aortic smooth muscle cells was studied, employing viable cell counting, [³H] thymidine incorporation into DNA, and the release of lactate dehydrogenase (LDH) into the medium. Oxidized LDL (prepared by incubation of LDL with copper sulfate) exerted a concentration-dependent stimulation (2 fold, compared to control) of aortic smooth muscle cell proliferation at low concentrations (0.1 µg – 10 µg/ml medium). On the other hand, at high concentrations (25–200 µg/ml), Ox-LDL produced a pronounced decrease in viable cells, a decrease in the incorporation of [³H] thymidine into DNA, and an increase in the release of LDH in the medium. In this report, the previously postulated biological roles of oxidized-LDL in atherosclerosis are discussed in view of these findings.Abbreviations Ox-LDL Oxidized human plasma Low Density Lipoproteins - SMC Smooth Muscle Cells - LDH Lactate Dehydrogenase - LPC Lysophosphatidycholine - PC Phosphatidylcholine - TNF Tumor Necrosis Factor     

In vitro susceptibilities of clinical yeast isolates to three antifungal agents determined by the microdilution method

A comparative evaluation of the in vitro susceptibilities of 597 clinical yeast isolates to amphotericin B, fluconazole, and 5-fluorocytosine (5FC) was conducted. The broth macrodilution reference method of the National Committee for Clinical Laboratory Standards (NCCLS, M27-P) was adapted to the microdilution method. Microdilution endpoints for amphotericin B were scored as the lowest concentration in which a score of 0 (complete absence of growth) was observed and for 5FC and fluconazole as the lowest concentration in which a score of 2 (prominent decrease in turbidity; MIC-2) was observed compared to the growth control. The MIC values were read after 24 and 48 h incubation. A broad range of MIC values was observed with each antifungal agent. Amphotericin B was very active (MIC₉₀1.0 µg/ml) against all of the yeast isolates with the exception ofC. lusitaniae (MIC₉₀2.0 µg/ml). Fluconazole was most active againstC. parapsilosis (MIC₉₀ of 1.0 µg/ml) and least active againstC. krusei (MIC₉₀ of 32 µg/ml). 5FC was most active againstC. albicans, C. parapsilosis, C. tropicalis, andT. glabrata (MIC₉₀1.0 µg/ml) and was least active againstC. krusei andC. lusitaniae (MIC₉₀16 µg/ml). These data indicate that the microdilution method, performed in accordance with M27-P, provides a means of testing larger numbers of yeast isolates against an array of antifungal agents and allows this to be accomplished in a reproducible and standardized manner. Given these results, it appears that the microdilution method may be a useful alternative to the macrodilution reference method for susceptibility testing of yeasts.     

Chronic effects of cadmium on two species of mysid shrimp:Mysidopsis bahia andMysidopsis bigelowi

Two species of mysid shrimp, the sub-tropicalMysidopsis bahia and the northern temperateMysidopsis bigelowi, were exposed simultaneously to cadmium (as CdCl₂) in a continuous-flow bioassay system to determine the effect on survival and reproductive success. Temperature and salinity were maintained at 21 ± 1°C and 30,respectively. The 96-h LC50 was 110 µg ^–1 for both species. The 23-day life cycle LC50 forM. bahia was 19.5 µg ^–1 and forM. bigelowi the 27-day LC-50 was 14.8 µg ^–1. At 10 µg ^–1 a series of morphological aberrations were observed in both species at the onset of sexual maturity. Carapace malformations apparently prevented molting after the release of the initial brood and resulted in death of brooding females. As a result, although the initial reproductive rate at this concentration was successful, successive broods could not be produced. For both species in this study the no observed effect concentration was 5.1 µg ^–1; the effect concentration was 10.0 µg ^–1. Mechanisms were postulated in this study to explain the effect of cadmium on the molting process and on calcification and enzymatic reactions of osmosis.Contribution No. 257 of the U.S. Environmental Protection Agency.Contribution No. 257 of the U.S. Environmental Protection Agency.     

Release of RANTES from nasal and bronchial epithelial cells

RANTES is a chemokine with eosinophil attractant and activating activities. This study was undertaken to determine whether primary cultures of human nasal and primate bronchial epithelial cells produce RANTES and the effect of various cytokines and dexamethasone on the release of this chemokine. Nasal epithelial cells from 32 patients (HNE) and bronchial epithelial cells from 17 Macaca nemestrina monkeys (PBE) were cultured in vitro for 24 to 72 h with LPS, TNF-, IL-1, IFN- and TNF- combined with IFN- and/or dexamethasone at 10 to 1000 µg/ml. Culture supernatants were assayed for RANTES by ELISA. RANTES synthesis was measured by immunoprecipitation. HNE and PBE released modest constitutive amounts of RANTES (350 to 1000 pg/ml) which did not increase with time in culture. Release of RANTES was stimulated by all activators except LPS in a time-dependent manner, with the greatest synthesis induced by the combined addition of TNF- and IFN-. The combination of these activators also increased RANTES synthesis as determined by immunoprecipitation. Dexamethasone at 100 and 1000 µg/ml produced significant inhibition of stimulated RANTES release. These data indicate that normal nasal and bronchial epithelial cells release RANTES which is upregulated by various cytokines and inhibited by dexamethasone. The enhanced release is due to stimulation of both synthesis and secretion. Production of RANTES by epithelial cells could contribute to the inflammation that characterizes the respiratory tract in asthma and rhinitis and downregulation of RANTES by glucocorticoids may be one mechanism of the therapeutic effect of these agents.     

Stable production of an analog of human tissue plasminogen activator from culturedDrosophila cells

We have studied the expression of an analog of human tissue plasminogen activator, FK2P, inDrosophila Schneider 2 cells. A number of promoters were tested, including theDrosophila metallothionein promoter (MTd), baculovirus immediate early promoter (IE),Drosophila copia promoter, mouse metallothionein promoter, cytomegalovirus immediate early promoter with or without intron, SV40 immediate early promoter, and human elongation factor 1 promoter. Two of these promoters drove significant expression of FK2P. The MTd promoter is tightly regulated and upon induction with copper or cadmium expression of FK2P increases as much as 180-fold, accumulating in the culture medium to about 7 g FK2P/10⁶ cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 g FK2P/10⁶ cells/day. The production of FK2P in these cell lines remains at about the same level after repeated passages, even in the absence of selective pressure. The FK2P accumulated in the culture medium is fully active in an assay using a chromogenic substrate for serine proteases. Western immunoblot analysis shows that the product remains predominately as single-chain molecules in serum-free medium, while in serum-containing medium two-chain material occurs as expected due to the presence of plasmin in serum. Judged from the size in Western immunoblots, the FK2P produced is glycosylated.     

Differential effects of tumor necrosis factor and asbestos fibers on manganese superoxide dismutase induction and oxidant-induced cytotoxicity in human mesothelial cells

We compared induction of manganese superoxide dismutase (MnSOD) by asbestos fibers and tumor necrosis factor (TNF) using cultures human mesothelial cells. Transformed pleural mesothelial cells (MET 5A) were exposed for 48 h to amosite asbestos fibers (2 g/cm²), to TNF (10 Ng/ml), and to the combination of these two. TNF and amosite+TNF caused significant MnSOD mRNA upregulation. Similarly MnSOD specific activity was increased by TNF (290% increase) and the amosite+TNF combination (313% increase) but not by amosite alone. In cell injury experiments, amosite and amosite+TNF exposures caused significant cell membrane injury when assessed by lactate dehydrogenase release, which was 31% and 57% higher than in the unexposed cells. However, only the amosite+TNF combination caused significant depletion of cellular high-energy nucleotide when expressed as percentage of [¹⁴C]denine labeling in cellular high-energy nucleotides. The nucleotide levels were 91.5 ± 2.0% in the unexposed cells, 89.9 ± 3.9% in amosite-exposed cells, 90.1 ± 2.2% in TNF-exposed cells, and 79.8 ± 9.4% in amosite+TNF-exposed Amosite+TNF-exposed cells were also most sensitive to menadione (20 mol/L, 2 h), a compound which generates superoxide radicals intracellularly. In conclusion, our data suggests that in human mesothelial cells inflammatory cytokines but not asbestos fibers alone can cause MnSOD induction. In this study, however amosite asbestos+TNF treatment rendered these cells more vulnerable to oxidant-induced cell damage despite elevated MnSOD activity.Abbreviations MnSOD manganese superoxide dismutase - TNF tumor necrosis factor - LDH lactate dehydrogenase