Tetrahydrophthalazine derivative "sodium nucleinate" exert its anti-inflammatory effects through inhibition of oxidative burst in human monocytes

We described the use of a new chemical substance Sodium nucleinate (SN) as an immunomodulatory substance exhibiting antiinflammatory properties. Sodium nucleinate (SN) registrated in Russian Federation as Tamerit, is 2-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate, derivative of well known chemical substance luminol. To comprehend the mechanisms of SN immunomodulatory activity, we examined the SN modulation of the oxidative burst responses of whole blood human monocytes and polimorphonuclear cells (PMC) stimulated with phorbol 12-myristate 13-acetate (PMA) or E. coli suspension in vitro. SN did not inhibit the proportion of neutrophils and monocytes phagocytosing E. coli. Oxidative burst responses of monocytes stimulated with PMA were strongly inhibited at SN concentration ranging from 10-500 mg/ml, less efficient inhibitor was SN in E. coli stimulated monocytes (inhibition range was from 50-500 mg/ml SN). SN inhibited PMC oxidative burst only in range 100-500 mg/ml SN. In conclusion, we found SN as an efficient inhibitor of oxidative burst in monocytes. Since ROS generation in monocytes/macrophages has been found to be important for LPS-driven production of several proinflammatory cytokines, SN may exsert its antiinflammatory effects through monocyte/macrophage oxidative burst inhibition.     

Assay methodology for the quantitation of unbound ertapenem,a new carbapenem antibiotic,in human plasma

Ertapenem is a new once-a-day antibiotic with excellent coverage of common community gram negative and gram positive aerobes and anaerobes. It demonstrates nonlinear protein binding in human plasma (about 94% bound). An assay for unbound drug was developed to study the pharmacokinetics of unbound ertapenem in plasma. Unbound drug is separated from plasma samples (1.0 ml) by ultrafiltration using a Centrifree((R)) centrifugal filter device. Ertapenem (vulnerable to hydrolysis of the beta-lactam moiety) is stabilized in the filtrate by adding an equal volume of 0.1 M MES buffer, pH 6.5 and then is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection (300 nm). Non-specific binding to the Centrifree((R)) device is <3%. A suitable internal standard is not available. The assay is specific and linear over the concentration range of 0.25 to 100 microgram/ml in plasma filtrate. The lower limit of quantitation (LLOQ) is 0.25 microgram/ml. Intra-day precision is C.V.<10% and accuracy ranges from 97 to 101% of nominal concentration. Inter-day precision and accuracy were determined using quality control samples (QCs) prepared in plasma ultrafiltrate at 0.5, 12 and 80 microgram/ml and stored at -70 degrees C with stabilizer. Inter-day assay accuracy and precision ranged from 100 to 111% of nominal concentration and 1.8 to 5.3% C.V. (n=40), respectively. The assay has been used to analyze plasma samples from subjects receiving 500 and 2000 mg i.v. doses of ertapenem (30 min infusion).     

The effect of betel nut extract on cell growth and prostaglandin endoperoxide synthase in human epidermoid carcinoma cells

The objective of this study was to find out whether prostaglandin endoperoxide synthase (PHS) involves the action of betel nut extract (BNE) on the growth of oral cancers. Therefore, growth and PHS activity were examined in two human oral carcinoma cell lines (OEC-M1 and KB) and one normal fibroblast cell line (NF) in the presence of increasing BNE concentration. BNE at concentrations above 50 microg/ml significantly inhibited the cell growth of OEC-M1 after 72 h in culture, of KB and NF after 48 h in culture. The IC50 of BNE in OEC-M1, KB and NF at 24 h in culture was about 406, 37.5 and 140 microg/ml respectively. PHS activity in OEC-M1 was significantly increased by low BNE concentrations (50 microg/ml, 114%; 100 microg/ml, 33%; 150 microg/ml, 30%) but significantly reduced at higher BNE concentrations (300 microg/ml, 33%; 500 microg/ml, 61%). The PHS activity in KB was significantly inhibited by BNE and this effect was intensified as concentrations increased (50 microg/ml, 31%; 100 microg/ml, 24%; 150 microg/ml, 43%; 300 microg/ml, 60%; 500 microg/ml, 92%). Similar to that in OEC-M1, the PHS activity in NF was significantly increased at low BNE concentrations (50 microg/ml, 139%; 100 microg/ml, 87%;150 microg/ml, 77%) but reduced at higher concentrations (300 microg/ml, 55%; 500 microg/ml, 72%). The PHS activity in all cell lines was almost completely blocked by indomethacin (5 x 10(-6) M). We conclude that these findings suggest that PHS may be an important biochemical mediator of the effect of BNE on the growth of two human oral carcinoma cell lines.     

Isolation, purification, and antibiotic activity of o-methoxycinnamaldehyde from cinnamon.

o-Methoxycinnamaldehyde has been isolated and purified from powdered cinnamon. The compound inhibits the growth and toxin production of mycotoxin-producing fungi. The substance completely inhibited the growth of Aspergillus parasiticus and A. flavus at 100 microgram/ml and A. ochraceus and A. versicolor at 200 microgram/ml. It inhibited the production of aflatoxin B1 by over 90% at 6.25 microgram/ml, ochratoxin A at 25 microgram/ml, and sterigmatocystin at 50 microgram/ml. The substance also displayed a strong inhibitory effect on the growth of five dermatophytoses species, e.g., Microsporum canis (minimum inhibitory concentration, 3.12 to 6.25 microgram/ml). However, no antibacterial effect was observed at concentrations as high as 50 microgram/ml.     

Impaired oxidative burst does not affect human monocyte tumoricidal activity

Human peripheral blood monocytes have been shown to lyse neoplastic cells selectively. It has been suggested that the oxidative burst may mediate monocyte tumoricidal activity. Two patients with chronic granulomatous disease were studied. The first patient had no detectable secretion of hydrogen peroxide and superoxide anion after stimulation with 100 ng/ml PMA. Nevertheless, his tumoricidal activity measured against K562 targets in 4-hr 51Cr-release assays by using highly purified monocytes was reproducibly above the 95th percentile of the normal population. His monocytes lysed WEHI-164 targets pretreated with 1 microgram/ml Actinomycin D with similar efficiency. Another patient whose oxidative burst was 15% of normal nevertheless killed Daudi targets efficiently. In addition, oxygen-deprived monocytes of normal donors manifested normal tumoricidal activity, despite failure to produce any detectable oxidative burst after PMA stimulation. We conclude that reactive oxygen metabolites are not the primary mediators of tumor cytolysis by human peripheral blood monocytes.     

The effects of PGD2 and 9-deoxy-delta 9-PGD2 on colony formation of murine osteosarcoma cells

Effects of antineoplastic prostaglandins (PG), PGD2 and 9-deoxy-delta 9-PGD2, on colony formation of cloned Dunn osteosarcoma (TA 102), normal Swiss 3T3 and V-79 cell lines were evaluated. PGD2 significantly inhibited the colony formation of TA 102 cells in a dose-dependent manner at concentrations between 0.5 and 5 micrograms/ml. The IC50 value was calculated to be 0.72 microgram/ml. A dose-dependent inhibition of TA 102 colony formation was also observed with 9-deoxy-delta 9-PGD2 between 0.01 to 1 microgram/ml, the IC50 value being 0.22 microgram/ml. These prostaglandins did not exert cytocidal effects in vitro on Swiss 3T3 cells at concentrations between 0.01 to 1 microgram/ml. The two agents had no significant cytocidal effects on V-79 cells except for 9-deoxy-delta 9-PGD2 at a concentration of 5 ug/ml. These results suggest that PGD2 and 9-deoxy-delta 9-PGD2 are considered to have cytocidal activity on Dunn osteosarcoma cells in dosages which do not affect non-malignant cells.     

Mutagenicity testing of benomyl, methyl-2-benzimidazole carbamate, streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium in vitro and in rodent host-mediated assays.

The fungicide benomyl and its commercial preparations Fundazol 50WP and Benlate 50WP and the benomyl metabolite methyl-2-benzimidazole carbamate and its commercial preparation MBC 50WP were tested for mutagenicity in in vitro spot tests, in microsomal plate assay, in liquid-culture treatments, or in rodent host-mediated assay. The base-pair substitution Salmonella typhimurium mutant hisG46 and the hisG46-bearing uvrB excision-repair-deficient mutants TA100, TA1530, TA1535 or TA1950 were used as test organisms. Complete genotypic information of these mutants is given in Ames et al. [2]. Captain 50WP, streptozotocin (SZN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-aminopurine and N-acetylaminofluorene were used as positive control compounds. In nonoverlay spot tests Benlate 50WP was not mutagenic over a dose range of 50-5000 microgram/spot in hisG46 and TA1535. In overlay spot tests 50 or 100 microgram/spot Benomyl, MBC, Fundazol 50WP, Benlate 50WP and MBC 50WP were tested in hisG46, TA1530 or TA1950. Only a non-commercial MBC sample at 100 microgram/spot showed weak mutagenic activity in hisG46. In microsomal activation plate assay MBC, benomyl, Fundazol 50WP and Benlate 50WP were tested in TA100 over a dose range of 50-2000 microgram/plate. None of the compounds showed mutagenicity. In a 20-h liquid-culture treatment 10, 100, 1000 and 10 000 microgram/ml Fundazol 50WP were not mutagenic in TA 30. In 1-h liquid-culture treatments benomyl, Benlate 50WP or Fundazol 50WP failed to induce mutations in hisG46, TA100 or TA1950 over a dose range of 0.25-1000 microgram/ml. Appropriate positive controls were mutagenic in each experiment. The consistently negative results in this study with commercial MBC and benomyl preparations are contrary to positive results reported earlier with similar methods and similar commercial preparations. Possible reasons to explain the different results are presented. The alkylating agents SZN and MNNG induced fewer mutations in TA1530 and TA1950 uvrB excision-repair-deficient strains than in the hisG46 excision-proficient strain, indicating that with these mutagens excision-repair is also a mutation-prone process. In rodent host-mediated assays with Fundazol 50WP in mice 3 consecutive subcutaneous hourly doses of 500 mg/kg in hisG46 and TA1950 and in rats or mice an oral dose of 4000 mg/kg in TA1950 were not mutagenic. The positive control SZN was mutagenic.     

The effect of GD3 ganglioside obtained from bovine lymphosarcoma on bovine normal mononuclear cell

The effect of immunological function of GD3 on normal bovine lymphocyte was examined with various in vitro assay systems. The GD3 level in sera from enzootic bovine leukemia (EBL) cattle was significantly increased compared with that of normal cattle (EBL: 0.62 +/- 0.24 microgram/ml; normal cattle: 0.33 +/- 0.09 microgram/ml, P less than 0.05). Lymphocyte blastogenesis elicited by concanavalin A was inhibited by addition of a 50 micrograms/ml concentration, or more, of GD3. Inhibitory effect of GD3 in IL-2-dependent T cell line and EBL tumor cell line was hardly observed compared with normal peripheral blood mononuclear cells. GD3 also inhibited mixed lymphocyte reaction and allo cytotoxic T lymphocyte reaction.     

Gelatin of Limulus amoebocyte lysate by simple polysaccharides

The Limulus lysate gelatin activity of several simple polysaccharides including yeast mannans and bacterial dextrans was investigated. The mannans from Saccharomyces cerevisiae wild and mutant strains possessing dense branches showed positive gelatin activity at concentrations of 1 microgram/ml or more regardless of differences in their chemical structure. However, two synthetic mannans possessing linear structures with alpha 1 leads to 2 and alpha 1 leads to 6 linkages also gave positive reactions at concentrations of 10 microgram/ml or more and 500 microgram/ml or more, respectively. The dextran from Leuconostoc mesenteroides IAM 1046 consisting of a dense branching moiety displayed reactivity at concentrations of 100 microgram/ml or more, while the dextrans devoid of such branches were negative in this reaction. The optimal concentration for Limulus lysate gelatin could not be determined for any of the polysaccharides and lipopolysaccharides (LPS) tested in this study. The gelation activity of the polysaccharides was stable to treatment with 100 mM NaOH at 30 C for 72 hr. The minimum concentration for the gelation activity of LPS treated with 100 mM NaOH under the same conditions was reduced from 10(-6) approximately(-9) microgram/ml to 1-10 microgram/ml. The above findings demonstrate that the major part of Limulus lysate gelation activity of LPS depends on the alkali-degradable lipid A moiety, and that such simple polysaccharides are also able to participate in this activity even though the extent of participation is very low.     

Unexpected suppression of free phenytoin concentration by salicylate in uremic sera due to the presence of inhibitors: MALDI mass spectrometric determination of molecular weight range of inhibitors

Salicylate displaces phenytoin from protein binding leading to an increase in free phenytoin concentration. We observed unexpected decreases in free phenytoin concentration in the presence of salicylate. Serum pools containing no phenytoin or salicylate were supplemented with the same concentrations of phenytoin. Then to the aliquots of the individual pool, no salicylate (control), 150, 300 and 500 microg/ml of salicylate (therapeutic range: 15-300 microg/ml) were added. Specimens were incubated at 37 degrees C for 2 h and after re-equilibration at room temperature for 20 min, total and free phenytoin (in the protein free ultrafiltrates) concentrations were measured using fluorescence polarization immunoassay on the TDx/FLX analyzer. We observed an increase in free phenytoin concentration from 1.91 microg/ml (in the absence of salicylate) to 2.39 microg/ml in the presence of 500 microg/ml salicylate (total phenytoin: 13.3 microg/ml) in the normal pool. In sharp contrast, the free phenytoin concentrations decreased from an initial concentration of 3.82 microg/ml to 2.52 microg/ml in the presence of 500 microg/ml of salicylate (total phenytoin: 13.2 microg/ml) in the uremic pool. We also treated the uremic pool with activated charcoal. In the original uremic pool, the initial free phenytoin concentration was 3.05 microg/ml and the free concentrations then decreased to 2.28 microg/ml in the presence of 300 microg/ml of salicylate. In contrast, in the charcoal treated pool, the initial free phenytoin concentration increased from 1.61 microg/ml to 3.23 microg/ml in the presence of 300 microg/ml of salicylate. More interestingly when uremic toxins were extracted back from charcoal with methanol and the dry residue was added to an aliquot of normal serum, the normal serum behaved like a uremic serum and free phenytoin concentration was significantly decreased in the presence of salicylate. When an aliquot of methanol extract was studied by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (scan up to 10,000), we observed no peak at molecular weight over 551, indicating that these inhibitors are small molecules. We also identified hippuric acid as one of the inhibitors.     

Effect of thyrotropin releasing hormone thyroxine on the activity of cytochrome oxidase in rat adenohypophysis]

Thyrotropin releasing-hormone (TRH) increased the activity of cytrochrome C oxidase in a concentration of 0.01 and 1.0 microgram/ml in the adenohypophysis of rats fed methylthiouracil for 6 weeks. This effect of TRH on the activity of the enzyme was blocked with T4 added to the incubation medium in a concentration of 20 microgram/ml. Actinomycin D (20 MICrogram/ml) prevented the block of the enzyme with thyroxin. In a concentration of 0.01 microgram/ml TRH, and in a concentration of 2.0 microgram/ml T4 failed to change the activity of cytochrome oxidase in the adenohypophysis of normal and partially thyroidectomized rats.     

Inhibition of plant-pathogenic fungi by a corn trypsin inhibitor overexpressed in Escherichia coli

The cDNA of a 14-kDa trypsin inhibitor (TI) from corn was subcloned into an Escherichia coli overexpression vector. The overexpressed TI was purified based on its insolubility in urea and then refolded into the active form in vitro. This recombinant TI inhibited both conidium germination and hyphal growth of all nine plant pathogenic fungi studied, including Aspergillus flavus, Aspergillus parasiticus, and Fusarium moniliforme. The calculated 50% inhibitory concentration of TI for conidium germination ranged from 70 to more than 300 microgram/ml, and that for fungal growth ranged from 33 to 124 microgram/ml depending on the fungal species. It also inhibited A. flavus and F. moniliforme simultaneously when they were tested together. The results suggest that the corn 14-kDa TI may function in host resistance against a variety of fungal pathogens of crops.     

Serum thyrotropin response to thyrotropin-releasing hormone and free thyroid hormone indices in patients with familial thyroxine-binding globulin deficiency.

The response in serum thyrotropin (TSH) to synthetic thyrotropin-releasing hormone (TRH) as well as serum free thyroxine index (FT4I) and free triiodothyronine index (FT3I) was investigated in six patients with familial thyroxine-binding-globulin (TBG) deficiency. The total serum thyroxine (T4) and triiodothyronine (T3) concentrations were significantly decreased, compared with those of normal subjects (3.4 +/- 0.9 microgram/dl, mean +/- SD. vs. 9.0 +/- 1.5 microgram/dl, p less than 0.01 and 87 +/- 27 ng/dl vs. 153 +/- 37 ng/dl, p less than 0.01, respectively). FT4I was lower than the normal range in all but one (5.3 +/- 1.5 vs. 8.9 +/- 1.6, p less than 0.01), whereas FT3I was all in the normal range and of no significant difference from the normal control (132 +/- 22 vs. 148 +/- 25). Serum TSH concentrations in TBG deficiency were all in the normal range (1.0-4.2 muU/ml) and the maximum TSH increments following TRH 500 microgram iv were 8.9 +/- 2.0 muU/ml and of no significant difference from the normal control (10.2 +/- 4.5 muU/ml). These results indicate that the euthyroid state in familial TBG deficiency is more clearly defined by TRH-test and the normal response to TRH in familial TBG deficiency is presumably under the control of the serum free T3 level rather than the serum free T4 level.     

Influence of interferon on human monocyte to macrophage development

The effect of interferon-α (Wellferon) on human monocyte to macrophage maturation in vitro has been investigated. Cell volume and three markers, acid phosphatase, leucine aminopeptidase, and phagocytosis, which increase with maturation, have been studied employing recently developed flow cytofluorometric techniques. The increase in cell volume and in the expression of all three markers was inhibited in a dose-dependent manner in monocyte cultures given 50–300 U/ml of interferon within 2 hr of culture initiation. An initial dose of 300 U/ml of interferon, removed from the cultures after 24 hr, was as effective in inhibiting the development of each of the markers as three 100 U pulses on three consecutive days, and as effective as 300 U interferon left in throughout the culture period. Histogram analysis of marker expression indicated that all monocytes, and not a subpopulation, were affected by the interferon. Cytotoxic activity of freshly isolated monocytes rapidly decayed when the cells were cultured under standard maturation conditions. The addition of interferon to the cultures prevented the loss of this activity while also preventing the development of more mature cells. It appears that maintenance of the cytotoxic state is one influence of interferons; however, it may be that these cells have also been directed toward alternate pathways of macrophage differentiation.     

Interleukin 1 induces interleukin 1. II. Recombinant human interleukin 1 induces interleukin 1 production by adult human vascular endothelial cells

Interleukin 1 (IL-1) alters several potentially pathogenic endothelial cell (EC) functions. The authors report here that recombinant human IL-1 (rIL-1) alpha (0.1 to 10 ng/ml) or IL-1-beta (1 to 100 ng/ml) induce concentration- and time-dependent increases in IL-1-beta mRNA levels in EC derived from adult human saphenous vein. rIL-1 induced IL-1-alpha mRNA only in EC treated concomitantly with cycloheximide (2 micrograms/ml). IL-1-beta mRNA production began within 1 hr of exposure to rIL-1, peaked after 24 hr, and declined thereafter. Actinomycin D prevented the appearance of IL-1 mRNA in rIL-1-treated EC. rIL-1 also induced the release of biologically active IL-1 from EC, which was inhibited by cycloheximide (1 microgram/ml). When compared on the basis of their activity in the thymocyte costimulation assay, rIL-1-alpha and rIL-1-beta were equipotent as inducers of IL-1 production by EC. EC stimulated with rIL-1 produced prostaglandin E2, which inhibits IL-1 production by other cell types and also decreases the responsiveness of thymocytes to IL-1. When EC were exposed to rIL-1 in the presence of indomethacin (1 microgram/ml), which blocked prostaglandin E2 production, greater amounts of rIL-1-induced IL-1 release were detected, although the inhibitor did not affect IL-1-beta mRNA levels. IL-1-induced IL-1 production was unlikely to be caused by endotoxin contamination of tissue culture media or IL-1 preparations, because the lipopolysaccharide (LPS) antagonist polymyxin B (10 micrograms/ml) blocked LPS-induced IL-1 production by EC but did not affect IL-1 release in response to rIL-1-beta (100 ng/ml). The IL-1-inducing property of rIL-1-beta was heat-labile, whereas heated LPS stimulated EC IL-1 production. The source of IL-1 in our cultures was not monocyte/macrophages, as treatment of EC with monoclonal antibody to the monocyte antigen Mo2 under conditions that lysed adherent peripheral blood monocytes did not affect production of IL-1 by EC in response to LPS (1 microgram/ml) or rIL-1-beta (100 ng/ml). IL-1 elicits a coordinated program of altered endothelial function that increases adhesiveness for leukocytes and coagulability. IL-1-induced IL-1 gene expression in human adult EC could thus provide a positive feedback mechanism in the pathogenesis of vascular disease including atherosclerosis, vasculitis, and allograft rejection.     

Effects of cordacin on lymphocyte activity in vitro

The effects of cordacin on lymphocyte activity were studied with two parameters in vitro. The cordacin inhibited T lymphocyte E rosette formation by 93% at the concentration of 1,000 microgram/ml and tritiated-thymidine incorporation into PHA-transformed lymphocytes by 66% at the concentration of 10 microgram/ml. The implication of these results on replicating leukemic as well as normal cells is discussed.     

Sperm concentration and the fertilization of human eggs in vitro

The effect of sperm concentration on the fertilization of preovulatory and immature human eggs was studied in the context of an ongoing in vitro fertilization-embryo transfer (IVF-ET) program. Fertilization success was independent of the follicular recruitment protocol used, and with preovulatory eggs, was inversely related to sperm concentration over the range of 2.5 - 50 X 10(4) motile sperm/ml. Maximum fertilization (80.8%) occurred at a concentration of 2.5 X 10(4) motile sperm/ml. The incidence of polyspermic fertilization was directly related to the sperm concentration, decreasing from 5.5% at 10 X 10(4) to 0% at 1-2.5 X 10(4) motile sperm/ml. Immature eggs cultured in vitro, then inseminated, also demonstrated an inverse relationship between fertilization and sperm concentration with a maximum fertilization rate of 66.6% at 5 X 10(4) motile sperm/ml. The percentage of motile sperm in the inseminating population had no influence on fertilization rates unless the value dropped below 40%. Fertilization success using sperm from oligospermic and polyzoospermic males was also examined. In contrast to males with normal semen parameters, oligospermic males demonstrated highest fertilization success at 50 X 10(4) motile sperm/ml. The IVF of preovulatory eggs using sperm from polyzoospermic males was comparable to that for males with normal semen parameters at equivalent sperm concentrations. The implications of these findings to the application of IVF-ET technology to the infertile couple is discussed.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 micrograms/ml); ascorbic acid, 40 micrograms/ml; L-isoleucine, 50 micrograms/ml; epidermal growth factor, 20 ng/ml; insulin, 5 micrograms/ml; cholera toxin, 5 ng/ml; transferrin, 1 microgram/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37 degrees C in humidified gas containing 5% CO2: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.