In vitro and in vivo antioxidant properties of gliclazide

Diabetes is a state of increased oxidant stress and there is evidence that oxidation may play a role in the genesis of complications. Gliclazide, a sulfonylurea hypoglycemic drug, has been shown to possess free radical scavenging properties. This study examined the effects of in vitro supplementation with gliclazide and other sulfonylureas as on low-density lipoprotein (LDL) oxidation and the total plasma antioxidant capacity (TPAC). In a separate study, the effects of 10 months of oral gliclazide therapy on oxidative parameters were assessed in 44 type 2 diabetic patients. Gliclazide, but not glibenclamide, glimepiride, glipizide or tolbutamide, inhibited LDL oxidation and enhanced TPAC. With the addition of 1 microM gliclazide, oxidation lag time increased from 53.6+/-2.6 to 113.6+/-5.1 min (p<0.001), and TPAC increased from 1. 09+/-0.11 to 1.23+/-0.11 mM (p<0.01). Administration of either modified release or standard gliclazide to type 2 diabetic patients resulted in a fall in 8-isoprostanes, a marker of lipid oxidation, and an increase in the antioxidant parameters TPAC, SOD and thiols. These studies show that gliclazide possesses antioxidant properties that produce measurable clinical effects at therapeutic doses.     

Evaluation of in vivo and in vitro anti-inflammatory activity of Ajuga bracteosa Wall ex Benth

Ethnopharmacological relevance: Ajuga bracteosa Wall Ex Benth. (Labiateae) is described in Ayurveda for the treatment of rheumatism, gout, palsy and amenorrhea.ObjectivePresent study was aimed to investigate the in vivo and in vitro anti-inflammatory activity of Neelkanthi (whole plant) and to support its traditional use.MethodsMethanolic extract of plant Ajuga bracteosa (ABE) was investigated for its anti-inflammatory activity in carrageenan induced rat paw oedema, egg albumin induced inflammation in rats and the study was further supported with in vitro anti-inflammatory study by using Human red blood cell membrane stabilization (HRBC) method. Three doses of the extract (ABE-250, 500 and 750 mg/kg, i.p.) were used in the study and diclofenac sodium (5mg/kg, i.p.) was used as standard.ResultsABE (500 and 750 mg/kg, i.p.) significantly (P < 0.05) reduced increased in paw volume induced by carrageenan and egg albumin. ABE also showed significant stabilization toward HRBC membrane.ConclusionsABE at the dose of 500 and 750 mg/kg showed potent action on comparison with the standard drug diclofenac sodium.     

Immunopharmacological activity of cefodizime in young and elderly subjects: In vitro and ex vivo studies

The biological response modifying activities of cefodizime (CDZ), a new third-generation cephalosporin, were investigatedin vitro andex vivo. In vitro investigations using cells isolated from the blood of young healthy donors showed no stimulating activity of CDZ on peripheral blood lymphocytes, natural killer cell activity, IL-1 production by adherent mononuclear cells, PMN chemiluminescence or PMN chemotaxis. A slight but statistically insignificant increase in PMN phagocytosis and phagocytic index was observed in the same population. IL-1 production was increased in three subjects with low resting state values. In a controlledex vivo study, 20 health elderly subjects selected on the basis of depressed phagocytic function were treated with CDZ 1 g i.m. b.i.d. or placebo for eight days. PMN function was determined at baseline and on the day after the last dose. In the CDZ group a significant increase in both phagocytosis and phagocytic index was found, while there were no changes in the placebo group. In conclusion, CDZ restored depressed PMN phagocytic function in a population of elderly subjects. Patients with impaired PMN function who require antibiotic treatment may benefit from this activity of CDZ.Die immunologischen Wirkungen von Cefodizim (CDZ), einem neuen Cephalosporin, wurden untersucht. In einerin vitro Studie mit Zellen, die aus dem Blut junger, gesunder Spender isoliert wurden, zeigte CDZ keine stimulierenden Wirkungen auf Lymphozyten, NK-Zell-Aktivität, IL-1 Produktion durch haftende mononukleäre Zellen, die Chemilumineszenz und Chemotaxis von polymorphkernigen neutrophilen Granulozyten (PMN). Ein geringer, statistisch nicht signifikanter Anstieg der PMN Phagozytose und des Phagozytose-Index wurde beobachtet. Bei drei Personen mit niedriger IL-1 Produktion in Ruhe konnte ein Anstieg derselben induziert werden. Im Rahmen einer kontrolliertenex vivo Studie erhielten 20 gesunde ältere Versuchspersonen, die aufgrund einer verminderten PMN Phagozytose ausgewählt worden waren, CDZ 2 × 1 g i.m. oder Plazebo über einen Zeitraum von acht Tagen. Die Funktion der PMN wurde vor Behandlungsbeginn und am Tag nach der letzten Dosis bestimmt. In der CDZ-Gruppe kam es zu einem signifikanten Anstieg der Phagozytose und des Phagozytose-Index, während in der Plazebo-Gruppe keine Veränderungen beobachtet wurden. Schlußfolgerung: CDZ konnte bei älteren gesunden Versuchspersonen eine verminderte Phagozytosetätigkeit der PMN wiederherstellen. Patienten mit gestörter PMN-Funktion, die einer antibiotischen Behandlung bedürfen, könnte diese Eigenschaft von CDZ zugute kommen.     

In vitro and ex vivo retina angiogenesis assays

Pathological angiogenesis of the retina is a key component of irreversible causes of blindness, as observed in proliferative diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Seminal studies in the early 1980 s about the angiogenic activity exerted by mammalian retinal tissue extracts on the chick embryo chorioallantoic membrane and the later discovery of vascular endothelial growth factor (VEGF) accumulation in eyes of patients with diabetic retinopathy paved the way for the development of anti-angiogenic VEGF blockers for the treatment of retinal neovascularization. Since then, numerous preclinical and clinical studies about diabetic retinopathy and other retinal disorders have opened new lines of angiogenesis inquiry, indicating that limitations to anti-VEGF therapies may exist. Moreover, the production of growth factors other than VEGF may affect the response to anti-VEGF approaches. Thus, experimental models of retinal angiogenesis remain crucial for investigating novel anti-angiogenic therapies and bringing them to patients. To this aim, in vitro and ex vivo angiogenesis assays may be suitable for a rapid screening of potential anti-angiogenic molecules before in vivo validation of the putative lead compounds. This review focuses on the different in vitro and ex vivo angiogenesis assays that have been developed over the years based on the isolation of endothelial cells from the retina of various animal species and ex vivo cultures of neonatal and adult retina explants. Also, recent observations have shown that eye neovascularization in zebrafish (Danio rerio) embryos, an in vivo animal platform experimentally analogous to in vitro/ex vivo models, may represent a novel target for the identification of angiogenesis inhibitors. When compared to in vivo assays, in vitro and ex vivo models of retina neovascularization, including zebrafish embryo, may represent cost-effective and rapid tools for the screening of novel anti-angiogenic therapeutics.     

Visualization of live endothelial cells ex vivo and in vitro

The present study describes quick and effective methods that allow visualization of the vascular endothelium in living networks within dissected pieces of human tissue or in primary cultures containing heterogeneous cell populations. Fresh human uterine and subcutaneous gluteal fat tissues were directly labelled using fluorescently conjugated Ulex Europaeus Agglutinin I (UEA-1) to visualize the three-dimensional nature of the vascular network. Using conventional epi-illuminescence microscopy, the convoluted architecture demonstrating branch points within capillaries, between capillaries and larger vessels, were clearly observed in uterine and subcutaneous gluteal fat samples. In adult endometrial tissue where angiogenesis occurs on a monthly basis, complex anastamosis of vessels and tenuous structures were clearly seen. Three-dimensional rendered surface models formed by examination of confocal z-stacks demonstrated the existence of the lumen within microvessels. Tissue prelabelled with UEA-1 was used to assist and verify the presence of endothelial cells in culture, during and after the isolation procedure. Additionally, UEA-1 was added to a uterine fibroblast-microvascular-endothelial cell coculture model to allow daily vital observations of changes in the phenotype of the endothelium. The simple techniques described here demonstrate the ease with which fluorescently labelled UEA-1 can be used as a vital marker of endothelial cells either in tissue or in a tube-forming human uterine microvascular culture model.     

Electrical and mechanical activity in the ex vivo perfused total canine colon.

Electrical and mechanical activity were studied in the ex vivo total canine colon, supported in a perfusion chamber by a supporting dog. Regular electrical activity could be recognized visually in 50--70% of the records. The frequency of this activity was in the range of 7--10/min. Fourier analysis also revealed dominant frequencies in this range. Two types of mechanical activity were observed. The first consisted of small amplitude repetitive contractions, which appeared both during quiescent periods and during vigorous contractions. The frequency of this rippling, when periodic, usually approximated that of the electrical activity. The second type consisted of large amplitude prolonged (30--135 sec) contractions. Without stimulation, the large amplitude contractions were asynchronous and not propagated. Following neostigmine, however, the large amplitude contractions were always propagated aborally and were usually associated with evacuation of the colon. The rippling contractions had a frequency similar to the regular electrical activity and are probably under their control. The large amplitude, prolonged duration contractions are unlikely, however, to be solely under myogenic control.     

Antibacterial activity of human neutrophil peptide-1 against Mycobacterium tuberculosis H37Rv: in vitro and ex vivo study.

The aim of the study was to investigate the activity of human neutrophil peptide (HNP)-1 to kill Mycobacterium tuberculosis H37Rv in vitro and ex vivo in the murine macrophage cell line J744A.1 on the basis of colony forming units. Macromolecular biosynthesis was studied by monitoring the incorporation of radioactive precursors into different macromolecules. The binding and localization studies were carried out with radioiodinated HNP-1 whereas the cytotoxicity of HNP-1 to macrophages was determined by trypan blue exclusion assay. A concentration dependent inhibition in the growth of M. tuberculosis H37Rv was observed in the presence of HNP-1. The minimum inhibitory concentration and median inhibitory concentration of HNP-1 were found to be 2.5 microg x mL(-1) and 0.8 microg x mL(-1). Treatment of both in vitro grown and phagocytosed mycobacterial cells with HNP-1 resulted in generalized inhibition in the macromolecular biosynthesis with maximum inhibition in deoxyribonucleic acid and lipid biosynthesis. HNP-1 exhibited equilibrium binding with respect to time and two-thirds of bound radioactivity was shown to be present inside the macrophages. Approximately 50% and 98% killing of intracellular mycobacteria was observed after 3 days of treatment with 5 microg x mL(-1) and 40 microg x mL(-1) of HNP-1, respectively. HNP-1 exhibited low cytotoxicity towards the macrophage cell line at the bactericidal concentration to mycobacteria. From the results of this study, it is concluded that human neutrophil peptide-1 possesses potent bactericidal activity against virulent mycobacteria in vitro as well as mycobacteria replicating within macrophages.     

The microfilaricidal activity of ivermectin in vitro and in vivo

Ivermectin has been tested against the microfilariae of Onchocerca lienalis, Brugia pahangi and Dirofilaria immitis in vitro and in vivo. All in vitro tests were performed on larvae incubated for 48 hours at 37 degrees C in Hepes buffered medium 199 containing 20% serum, benzylpenicillin and streptomycin. In vivo tests were performed on larvae in female BALB/C mice dosed with ivermectin, 5 mg/kg, orally. The microfilariae of B. pahangi in vitro were insensitive to ivermectin at concentrations to 30 ng/ml. In vivo, an 87% reduction in the level of microfilaraemia was obtained by 24 hours after drugging but no reduction was observed in the numbers of peritoneal microfilariae. O. lienalis microfilariae in vitro were killed by ivermectin at 3 ng/ml and the larvae of this species within the subcutaneous and cutaneous tissues of the mouse were also eliminated by ivermectin at 5 mg/kg. D. immitis larvae within the bloodstream of the mouse were also sensitive to ivermectin at the dosage employed but were unaffected by ivermectin in vitro at concentrations up to 30 ng/ml.     

Phytochemical composition and in vitro antioxidant activity of aqueous extract of Aerva lanata (L.) Juss. ex Schult. Stem (Amaranthaceae)

ObjectiveTo analyze the phytochemical composition and in vitro antioxidant properties of aqueous extract of Aerva lanata (A. lanata) stem.MethodsDuring the preliminary phytochemical analysis, the aqueous extract of A. lanata was screened for the presence of carbohydrates, proteins, phenolic compounds, oil and fats, saponins, flavonoids, alkaloids, tannins and phytosterols. Antioxidant activity of the extract was determined by 2, 2-diphenyl-1-picrylhydrazyl radical scavenging activity, metal chelating activity, reducing power activity and DNA damage inhibition activity. Analysis of phenolic compounds was performed by Folin-Ciocalteau reagent method and gradient high performance liquid chromatography technique.ResultsPreliminary phytochemical analysis exhibited the presence of phenolic compounds, saponins, flavonoids, tannins and phytosterols as major phytochemical groups. The extract exhibited high 2, 2-diphenyl-1-picrylhydrazyl radical scavenging activity (IC₅₀= 110.74 μg/mL), metal chelating activity (IC₅₀= 758.17 μg/mL), reducing power activity and DNA damage inhibition efficiency. The extract was reported to possess a high amount of total phenolic content and some of them were identified as gallic acid (3,4,5-OH), apigenin-7-O-glucoside (apigetrin), quercetin-3-O-rutinoside (rutin) and myricetin (3,5,7,3,4,5-OH) by high performance liquid chromatography analysis. The extract was found non toxic towards human erythrocytes in the hemolytic assay (IC₅₀ = 24.89 mg/mL).ConclusionsThese results conclud that A. lanata stem possesses high antioxidant activity and can be used for the development of natural and safe antioxidant compounds.     

In vivo and in vitro antioxidant activity of ghrelin: Attenuation of gastric ischemic injury in the rat

BACKGROUND AND AIM: Gherlin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is produced by stomach cells. It regulates food intake, gastric secretion and motility. However, its role as a protective agent in gastric ischemia/reperfusion (I/R) injury has not yet been investigated. Therefore, the objectives of the present study were to: (i) test the in vivo effect of peripherally administered ghrelin on gastric I/R-induced lesions in rats; and (ii) investigate in vitro the effect of ghrelin on reactive oxygen species (ROS) production by human polymorphoneuclear (PMN) cells. METHODS: The present study was carried out on three groups of rats (six per group): control (sham-operated), I/R (clamping of celiac artery for 30 min and reperfusion for 1 h), and I/R + ghrelin (200 ng/kg i.v., 15 min before ischemia and before reperfusion, respectively). Histological assessment of hematoxylin and eosin stained sections was performed and immunostaining with inducible nitric oxide (iNOS) antibody were performed on a gastric paraffin embedded section. Oxidative stress markers thiobarbituric acid reactive substance (TBARS) and glutathione (GSH) were measured in gastric tissue homogenates. Serum lactic acid dehydrogenase (LDH) was determined. Tumor necrosis factor-alpha (TNF-alpha) was assayed in gastric tissue homogenate. Gastric permeability was assessed calorimetrically using Evans blue dye. In vitro studies were carried out on isolated human PMN cells incubated with ghrelin and tested for ROS generation as measured by chemiluminecence (CL). RESULTS: Peripheral administration of ghrelin attenuated gastric injury by reducing ulceration, tissue congestion, cellular infiltration and vascular permeability. Serum level of LDH and tissue content of TNF-alpha were markedly reduced. A decrement in TBARS and an increment in GSH were observed. Ghrelin treatment attenuated iNOS protein expression which was upregulated by gastric ischemic injury. In vitro studies showed for the first time that ghrelin inhibited ROS generation by human PMN in a dose-dependent manner. CONCLUSIONS: These results provide evidence that peripherally administered ghrelin protects against gastric I/R injury. We also demonstrated that this protection is possibly accomplished through the antioxidant activity of ghrelin observed in vivo and in vitro.     

Highly potent,synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo

Highly active antiretroviral therapy (HAART) decreases plasma viremia below the limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable to eliminate latently infected, resting CD4⁺ T cells. Such infected cells are potentially capable of reinitiating virus replication upon cessation of HAART, thus leading to viral rebound. Agents that would eliminate these reservoirs, when used in combination with HAART, could thus provide a strategy for the eradication of HIV. Prostratin is a preclinical candidate that induces HIV expression from latently infected CD4⁺ T cells, potentially leading to their elimination through a virus-induced cytopathic effect or host anti-HIV immunity. Here, we report the synthesis of a series of designed prostratin analogs and report in vitro and ex vivo studies of their activity relevant to induction of HIV expression. Members of this series are up to 100-fold more potent than the preclinical lead (prostratin) in binding to cell-free PKC, and in inducing HIV expression in a latently infected cell line and prostratin-like modulation of cell surface receptor expression in primary cells from HIV-negative donors. Significantly, selected members were also tested for HIV induction in resting CD4⁺ T cells isolated from infected individuals receiving HAART and were found to exhibit potent induction activity. These more potent agents and by extension related tunable analogs now accessible through the studies described herein should facilitate research and preclinical advancement of this strategy for HIV/AIDS eradication.     

The activity of nitrofurazone and furazolidone against Leishmania donovani, L. major and L. enriettii in vitro and in vivo

Furazolidone and nitrofurazone showed in vitro activity against amastigotes of Leishmania donovani, L. enriettii and L. major in macrophages, at concentrations which were also toxic to the macrophages. A low grade of activity was observed against L. donovani infections in BALB/c mice by furazolidone but not with nitrofurazone. Nitrofurazone, in two concentrations, was not active when applied to the lesions of cutaneous leishmaniasis due to L. enriettii (guinea-pig infection) or L. major strain P (BALB/c mouse infection). After systemic administration to BALB/c mice infected with L. major strain JISH 252 clone 1, low-grade activity was observed at the highest level tested.     

Beta-blockers: propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol, manifest atherogenicity on in vitro, ex vivo and in vivo models. Elimination of propranolol atherogenic effects by papaverine.

The addition of the beta-blockers propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol to a culture of peritoneal macrophages or smooth muscle cells induced an increase in the intracellular cholesterol content. Blood serum obtained from a rabbit after a peroral administration of beta-blockers also induced cholesterol accumulation. This property of drug or blood serum obtained after peroral administration is conventionally referred to as atherogenic potential or atherogenicity. Regular administration of propranolol during a 21-day period evoked stable atherogenicity of rabbit blood serum. This was accompanied by stimulation of manifestations of atherosclerosis in the aorta deendothelialized with a balloon catheter. Propranolol increased neointimal thickening, lipid accumulation, an increase in cell number and in the collagen content. In vitro, the combination of propranolol with papaverine eliminated the atherogenic effect of propranolol which manifested itself as stimulation of cholesterol accumulation in cultured cells. Simultaneous peroral administration of propranolol and papaverine prevented the appearance of serum atherogenicity. Papaverine eliminated neointimal thickening, an increase in cell number and in the lipid and collagen contents evoked by propranolol. Papaverine itself had no effect on these parameters. Thus, the atherogenicity of propranolol as well as capacity of papaverine to eliminate beta-blocker atherogenicity revealed in cell culture was confirmed in vivo. We hope that these results may be useful in the development of new drugs and optimization of antiatherosclerotic drug therapy.     

The activity in ex vivo expansion of cord blood myeloid progenitor cells before and after cryopreservation

A total of 50 human umbilical cord blood (UCB) samples were studied. The hematopoietic stem/progenitor (CD34+) populations were isolated from UCB mononuclear cells (MNC) by means of immunomagnetic separation. Double immunofluorescent staining of UCB CD34+ cells revealed that there was a high proportion (82.33 +/- 4.47%) of CD34+ cells co-expressing CD13, while the percentage of CD34+ CD33+ cells was much lower (22.17 +/- 3.35%). In contrast, for co-expressing lymphoid differentiation antigens, the proportion of CD34+CD38+ cells (38.34 +/- 6.09%) was relatively higher than that of CD34+CD10+ cells (11.52 +/- 1.24%) or CD34+CD2+ cells (9.84 +/- 2.30%). For stimulating the ex vivo expansion of UCB progenitor cells, no single hematopoietic growth factor (HGF) was efficacious when used alone, while combination of 4 HGFs, such as GM-CSF, G-CSF, IL-3, and SCF could induce a 55-fold increase in the myeloid progenitor cells, day-14 CFU-GM, in a short term of 7 days' liquid culture. Cryopreservation of UCB as MNC preparations at -196 degrees C could satisfactorily retain the number and activity of CD34+ cells. After thawing, a high recovery rate of about 80% CD34+ cells was obtained. When suspended in liquid cultures containing a combination of 4 HGFs, as shown above, the frozen cord blood progenitor cells could be well expanded, reaching a >50-fold increase in day-14 CFU-GM, which was very similar to that of the fresh UCB samples. In addition, a similar result was also seen in CFU-GEMM, indicating that after cryopreservation the recovered UCB progenitor cells retain an intact clonogeneic ability capable of efficiently responding to hematopoietic growth factors for ex vivo expansion.