Nitric oxide inhibits LPS-induced tumor necrosis factor synthesis in vitro and in vivo

The effect of nitric oxide (NO) on LPS-stimulated TNF-alpha synthesis has been studied in vitro and in vivo. The synthesis of TNF-alpha in J774 macrophages stimulated with LPS (0.1 microgram/ml) was increased in concentration-related fashion by NO synthase inhibitor L-NMMA (3-30-300 microM) and reduced by either L-arginine (3-30-300 microM) or the NO donor SIN-1 (1-10-100 microM). The level of TNF-alpha in the serum of LPS-challenged rats (6mg/kg/i.p.) was increased in animals pre-treated s.c. with L-NMMA (10 and 50mg/kg) and reduced in those given L-arginine (100 and 300mg/kg). These results show a negative feedback mechanism exhibited by NO on TNF-alpha synthesis suggesting an important regulatory link between NO and TNF-alpha in pathological processes.     

Invited perspective: in vitro testing in developmental toxicity risk assessment

A surgical intervention is needed in patients with periodontal pouches deeper than 4-5 mm. The results of 239 operations in 182 patients are analyzed. Optimal variants of the operations are described, depending on the patient's status, analgesia, types of secondary adentia, surgical access, and intervals between the operations.     

Evaluation of the developmental toxicity of methacrylonitrile in Sprague-Dawley rats and New Zealand white rabbits

Timed-pregnant Sprague-Dawley (CD) outbred rats and New Zealand White rabbits were dosed by gavage with methacrylonitrile (MACR) in distilled water during major organogenesis. Rats were dosed on Gestational Days (GD) 6 through 15 (0, 5, 25, or 50 mg MACR/kg/day) and rabbits on GD 6 through 19 (0, 1, 3, or 5 mg MACR/kg/day). Maternal clinical status was monitored daily during treatment. At termination (GD 20, rats; GD 30, rabbits), confirmed-pregnant females (25-26 per group, rats; 17-22 per group, rabbits) were evaluated for clinical status and gestational outcome; each live fetus was examined for external, visceral, and skeletal malformations. In rats, no treatment-related maternal clinical signs or mortality were observed, nor was there any adverse effect on maternal body weight or food or water consumption. At necropsy, absolute, relative, and adjusted maternal liver weight was increased at the mid- and high-dose groups, an effect that may be indicative of induction of hepatic enzymes rather than toxicity. In the absence of any indication of maternal toxicity, the no-observed-adverse-effect level (NOAEL) for maternal toxicity in this study was >/=50 mg MACR/kg/day. The NOAEL for developmental toxicity in rats was also >/=50 mg MACR/kg/day. There was no effect of treatment on postimplantation loss, mean fetal body weight per litter, or morphological development. In rabbits, maternal mortality and clinical signs were not dose related. Maternal food consumption, body weight, and liver weight were not adversely affected by treatment. Thus, the maternal NOAEL was >/=5 mg MACR/kg/day. Maternal toxicity, including death, was observed >/=7.5 mg/kg/day in preliminary studies. The developmental NOAEL was also >/=5 mg MACR/kg/day. There was no adverse effect of treatment on postimplantation loss or fetal body weight. A significant decrease in the percentage male fetuses per litter was observed, although there was no effect on total live litter size, suggesting that the reduction in the ratio of live male fetuses in the high-dose group was not biologically significant. MACR had no adverse effect on morphological development. In summary, oral administration of MACR to rats and rabbits during organogenesis, at doses that did not cause persistent maternal toxicity (50 mg MACR/kg/day, rats; 5 mg MACR/kg/day, rabbits), also did not cause any adverse developmental effects.     

In vivo and in vitro antiinflammatory activity of saikosaponins

Buddlejasaponin I and saikosaponin 1 and 2, biologically active compounds from Scrophularia scorodonia and Bupleurum rigidum respectively, exert potent in vivo antiinflammatory effects on mouse ear edema induced by phorbol myristate acetate (PMA). The effects of these compounds on swelling and other inflammatory parameters are described. In screening for in vitro effects of saikosaponins on cellular systems generating cyclooxygenase (COX) and lipoxygenase (LOX) metabolites, we observed that most saikosaponins showed a significant effect. The action is more marked on LOX metabolite LTC4. Our data support the inhibition of arachidonic acid metabolism as one of the biochemical mechanisms that might be the rationale for the putative antiphlogistic activity of these saikosaponins.     

In vitro and in vivo antineoplastic effects of orthovanadate

A liquid chromatographic method for the determination of the macrolide antibiotics, roxithromycin and clarithromycin, in plasma is described. The method is fully automated, employing on-line solid-phase extraction for sample clean-up, using the Prospekt unit. Plasma samples, mixed with internal standard, were injected onto exchangeable CN cartridges. After washing, the compounds were eluted and transferred to a C18 analytical column for separation and electrochemical detection. Clarithromycin was used as internal standard when assaying roxithromycin and vice versa. The recovery of the solid-phase extraction method was 90% and higher, and the relative standard deviation was about 3%. The limit of quantitation was 0.5 mumol/l when 25 microliters of plasma was injected. Comparison with a liquid-liquid extraction method for sample clean-up showed good agreement.     

In vitro and in vivo calcification of vascular bioprostheses

Efficacy of different chemical treatments on calcification of vascular graft in vitro and in vivo was studied. Culture medium-filled rat aortas were separately treated in 0.2% glutaraldehyde and epoxy compound, and photooxidized in 0.01% methylene blue for a shorter period (group 1). Another group of rat aortas were separately treated in the same chemicals for a longer period (group 2). All fresh and treated aortas of both groups were cultured for 21 days in an organ culture medium and implanted (except for group 1) in weanling rats for five months. Histology and immunohistochemistry revealed that differently treated aortas of group 1 grow and calcify, and the smooth muscle cells between elastin fibers are the primary site of calcium deposition. In contrast, differently treated aortas of group 2 neither grew, nor did calcify in the medium except the epoxy compound cross-linked aorta of group 2 which did not grow but did calcify. Untreated aorta did not calcify. All fresh and differently treated aortic homografts calcified severely in rats. Our whole arterial segment-calcification system would be useful for analyzing the molecular and cellular mechanisms of both bioprosthetic and atherosclerotic calcification of vascular graft. New anticalcification technique is the only hope for better outcome of future vascular bioprostheses.     

In vivo and in vitro macrophage activation by systemic adjuvants

Six systemic adjuvants of immunity were tested for their ability to induce macrophage activation. Four of them: living BCG, hydrosoluble extracts from BCG (HIU II) and from M.smegmatis (IPM), and lipopolysaccharide from E.coli (LPS), when administered to normal mice render macrophages non-specifically cytotoxic for tumor cells in vitro. The intensity of this phenomenon varied according to the route and time of adjuvant administration. In contrast, lentinan extracted from Lentinus edodes, and levamisole which is a synthetic chemical compound, depressed macrophage cytotoxic potential. BCG, IPM and LPS were shown to have a direct action on macrophages. After in vitro exposure to these agents, the cytotoxic potential of normal macrophages was greatly increased. Levamisole was unable to stimulate this macrophage function directly in vitro. On the other hand, such a macrophage activation has been induced in vitro when normal macrophages were cultivated in the presence of MIF coming from the supernatant of human lymphoblastoid cell lines.     

In vivo and in vitro assessment of barium sulphate suspensions

In vitro methods of testing the efficiency of barium sulphate suspensions in delineating mucosal detail using canine cadaveric stomachs have been described in the literature. In this study a comparison is made between in vitro and in vivo tests in the stomach and small intestine of dogs, using several brands of barium sulphate. The results indicate that there is considerable variation in the behavior of these suspensions between the in vitro and in vivo tests particularly in the stomach. It is our view that in vitro tests of this sort are of little value for assessing the relative advantages and disadvantages of these suspensions in demonstrating mucosal detail.     

In vitro and in vivo hematopoietic activities of Betafectin PGG-glucan

Betafectin PGG-glucan is a novel beta-(1,3)glucan that has broad-spectrum anti-infective activities without cytokine induction. Here we report that PGG-glucan also has both in vitro and in vivo hematopoietic activities. In vitro studies with bone marrow target cells from the C3H/HeN mouse revealed that although PGG-glucan alone had no direct effect on hematopoietic colony-forming cell (CFC) growth, when combined with granulocyte colony-stimulating factor (CSF) or granulocyte-macrophage CSF, it increased CFC numbers 1.5- to 2.0-fold over those obtained with CSFs alone. Bone marrow cells cultured for high-proliferative-potential CFCs in the presence of interleukin (IL)-1, IL-3, macrophage CSF, and stem cell factor (SCF), or cultured for erythroid burst-forming units in the presence of IL-3, SCF, and erythropoietin, also exhibited enhanced growth in the presence of PGG-glucan. The synergistic effect of PGG-glucan was specific and could be abrogated by anti-PGG-glucan antibody. The ability of PGG-glucan to modulate hematopoiesis in vivo was evaluated in myelosuppressed rodents and primates. C3H/HeN female mice were intravenously administered saline solution or PGG-glucan (0.5 mg/kg) 24 hours before the intraperitoneal administration of cyclophosphamide (200 mg/kg), and the recovery of bone marrow cellularity and granulocyte-macrophage progenitor cells was evaluated on days 4 and 8 after cyclophosphamide treatment. At both time points, enhanced hematopoietic recovery was observed in PGG-glucan-treated mice compared with saline-treated control mice. In a final series of in vivo experiments, we evaluated the ability of therapeutically administered PGG-glucan to enhance hematopoietic recovery in cyclophosphamide-treated cynomolgus monkeys. Monkeys received intravenous infusions of cyclophosphamide (55 mg/kg) on days 1 and 2, followed on days 3 and 10 by intravenous infusion of PGG-glucan (0.5, 1.0, or 2.0 mg/kg). Compared with those in saline-treated monkeys, accelerated white blood cell recovery and a reduction in the median duration of neutropenia were observed in PGG-glucan-treated monkeys. These studies illustrate that PGG-glucan has both in vitro and in vivo hematopoietic activities and that this agent may be useful in the prevention and/or treatment of chemotherapy-associated myelosuppression.     

In vitro and in vivo immunopharmacological profile of Sch 40120

Sch 40120 (10-(3-chlorophenyl) - 6,8,9,10- tetrahydrobenzo [b] [1,8] naphthyridin-5 (7H)-one) is a leukotriene inhibitor that is also a potent inhibitor of acute inflammatory responses in rodent systems. In the present study, we have evaluated the effects of this drug on immune function as well as its activity in models of immune mediated chronic inflammatory disease. Sch 40120 was particularly effective in suppressing T cell proliferative responses in vitro. Antigen-specific and poly-clonally-induced in vitro antibody responses were also inhibited by the drug. However, the in vivo potency of Sch 40120 in suppressing immune responses and in inhibiting the pathological changes seen in rodent models of autoimmune disease (EAE and adjuvant arthritis) was somewhat less than that previously observed in models of acute inflammation. Nevertheless, the spectrum of activities exhibited by Sch 40120 suggests that it will be particularly useful in the treatment of psoriasis where T lymphocytes have been implicated in the development of disease and leukotrienes appear to have a role in the persistence of psoriatic plaques.     

The theoretical and empirical case for in vitro developmental toxicity screens, and potential applications

In vitro assays for the screening of developmental toxicity potential have been under development for approximately 15 years. During that period, we have learned that assays consisting of primary cultures of embryonic tissues or cells, intact embryos in culture, or free-living embryos are capable of distinguishing between mammalian developmental toxicants and nondevelopmental toxicants with an accuracy of > or = 80%. Despite this level of performance, there is still considerable reluctance among the scientific community to employ these assays for preliminary screening. In this paper, I review the theoretical basis for the predictiveness of these assays, outline the empirical data indicating their utility in screening toxicants, discuss the major limitations of in vitro assays and how they can be managed, and suggest applications for in vitro pre-screens. The embryo-derived assays should work because they continue to develop in vitro, and the underlying cellular and molecular processes driving this development are the same as those in the mammalian embryo in situ, and therefore, susceptible to the same insults. The assays do work, as specific mechanisms of developmental toxicity have been demonstrated in vitro, and because extensive validation studies have shown them to be highly concordant with traditional in vivo screens. The assays are inherently limited by the fact that they do not include all the levels of complexity of the maternal-embryonic unit; however, these limitations can be minimized by thoughtful assay selection, study design, and interpretation. Potential applications are suggested that complement but do not replace in vivo testing. Pre-screens will make product development more efficient and add to our knowledge about the developmental toxicity of previously untested compounds. In vivo screening would still be conducted on all classes of substances that are currently tested for developmental toxicity; however, fewer chemicals with high likelihood of being developmentally toxic, and therefore not appropriate for further commercial consideration, would be evaluated in these costly screens.     

In vitro and in vivo antipseudomonal activity, acute toxicity, and mode of action of a newly synthesized fluoroquinolonyl ampicillin derivative

Compounds N-(6,7-difluoroquinolonyl)-ampicillin (AU-1) and N-(6-fluoroquinolonyl)-ampicillin (FQ-1), synthesized by coupling of the carboxyl group of 6,7-difluoroquinolone (FP-3) and 6-fluoroquinolone (FP4), respectively, with the alpha-amino-group of ampicillin side chain, exhibit antipseudomonal activity similar to and lower acute toxicity than that of norfloxacin, whereas neither ampicillin nor the fluoroquinolone moieties, compound FP-3 or FP4, alone have such activity. Also, AU-1 and FQ-1 are active against tested clinical isolates of Pseudomonas aeruginosa that are highly resistant to norfloxacin, gentamicin, or both. The therapeutic efficacies of FQ-1 and norfloxacin were assessed and compared in neutropenic mice infected with a 90% lethal dose of P aeruginosa. Mice intraperitoneally administered FQ-1 (10 mg/kg) 4, 8, 24, and 48 hours after infection had survival rates as high as 80%, comparable to those of mice treated with norfloxacin at the same dosage and dosing schedule. The study of protoplast formation revealed that FQ-1 did not inhibit cell-wall biosynthesis but did induce cell filamentation of Bacillus subtilis at a level close to its minimal inhibition concentration. Both AU-1 and FQ-1 were able to intercalate into the double-stranded DNA. However, that FQ-1 lost such activity after it was treated with penicillinase suggests that the lactam-ring structure in ampicillin moiety of FQ-1 was hydrolyzed by penicillinase and that the hydrolyzed structure of FQ-1 does not own DNA-intercalation activity.     

In vitro developmental toxicity of concanavalin A in rat embryos: analysis of neural crest cell migration using monoclonal antibody HNK-1

The developmental toxicity of concanavalin A (Con A) was evaluated in vitro using a rat whole embryo culture system, and the distributions of the neural crest cells were immunohistochemically investigated in embryos with monoclonal antibody HNK-1. In addition, binding sites of Con A in the embryos were observed according to the avidin-biotin peroxidase complex method with biotin-labeled anti-Con A. The rat embryos on embryonic day 8 were exposed to a final concentration of 12.5, 25, 50, or 100 micrograms/ml of Con A for a 72 hr culture period. Exposure to Con A concentration-dependently resulted in lower viability, decreases in yolk sac diameter, crown-rump length, and number of somites, and increases in the incidence of morphological abnormalities characterized by neural tube defects in the embryos. In the Con A-treated embryos, the distributions of the neural crest cells were restricted in the dorsal and cranial regions, and the migration into the interventricular chamber was delayed in the cardiac region. The Con A-treated embryos were confirmed to have Con A binding on the wall of the outflow tract in the cardiac region and in the mesenchyme of the cranial region, which are thought to be migration pathways of neural crest cells. These findings suggested that Con A inhibited the early migration of neural crest cells by binding directly to some substrates distributed along the pathways in the embryos, so that the neural crest cells could not punctually reach the locations where they would proliferate and differentiate into the destined cell types.     

In vitro and in vivo protection against kainate-induced excitotoxicity by melatonin

In this study, the protective effect of melatonin against kainate (KA)-induced neurotoxicity was evaluated in vitro and in vivo. In rat brain synaptosomes, KA-induced oxidative stress was measured as shown by significant increases in both the basal generation of reactive oxygen species (ROS), assessed by a fluorescent method, and lipid peroxidation, evaluated as malondialdehyde (MDA) levels. Melatonin decreased, in a concentration-dependent manner, KA-induced lipid peroxidation. The intrinsic fluorescence of melatonin molecule hindered the evaluation of its protective effect against KA-induced ROS generation. However, melatonin was able to reduce FeSO4/ascorbate-induced ROS generation. The melatonin protective effect was confirmed by in vivo experiments: 73% of rats injected with KA (10 mg/kg i.p.) died within 5 days; melatonin administration i.p. significantly reduced mortality of the animals. The present results suggest that melatonin might be considered a pharmacological agent for the treatment of neurodegenerative pathologies.     

In vivo and in vitro analysis of baculovirus ie-2 mutants

Upon transient expression in cell culture, the ie-2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: trans activation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived from Spodoptera frugiperda but not in a cell line derived from Trichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. The ie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form of ie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus, ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.     

On toxicity question of synthetic polymers and their extracts tested in vitro and in vivo

The increasing use of polymer materials in medicine as much as the undesirable effects observed in patients during their application made us to study not only their functional properties but also their toxicological and biostability parameters, before their clinical application. Some of the aspects of this wide-ranging problem have been studied in our laboratory. Our aim was to try and identify the effect synthetic polymers and their extract have on different biological systems. The comparison of the results obtained through some of the biological methods was aimed at finding out to what extent such methods were suitable for testing of the polymer toxicity on living tissue.     

In vivo and in vitro evaluation of marginal integrity in ceramic inlays

The marginal integrity is an important factor for the long-term success of ceramic inlays. The long term clinical performance of porcelain inlays depends on a number of factors of which the marginal adaptation is of significant interest. The aim of this study was to determine the margin quality of adhesively luted sintered porcelain inlays both in vivo and in vitro. MOD cavities without bevels were prepared on 10 extracted human mandibular molar teeth. Using the Ducera inlay system, inlays were fired on refractory dies and luted with a dual-curing composite resin. After polishing, each, tooth was sectioned in buccal/lingual and mesial/distal directions and marginal adaptation was assessed microscopically. The mean marginal gap of 78.77 +/- 14.85 microns recorded for occlusal margins was significantly smaller than that of 128.85 +/- 34.34 microns seen at the approximal margins. For in vivo evaluation, 25 fired porcelain inlays, including 7 onlays, were placed in Class II cavities. The assessment of the marginal adaptation of inlays was made according to the scaling system used by Aberg et al. (Acta Odontol Scand 1994; 52:140-149). In 19 of the clinical cases, the restoration was contiguous with the existing anatomic form. Both in vivo and in vitro evaluations showed the margin quality of porcelain inlays to be high.