Effects of neuromuscular blocking drugs on viability of human umbilical vein endothelial cells (HUVECs).

OBJECTIVE: This study aimed to investigate the effects of neuromuscular blocking drugs on the viability of human umbilical vein endothelial cells (HUVECs) and to investigate whether they cause vascular complications due to cell proliferation. METHODS: HUVECs were cultivated with 5% CO2 at 37°C in a predefined supplemented medium over 7 days until confluence of cell monolayers. Assays were conducted during the exponential growth phase. Suxamethonium chloride, vecuronium bromide, atracurium besylate, and rocuronium bromide were used at concentrations of 10-5, 10-6, and 10-7 M in proliferation assays in which cells were incubated with these drugs for 24, 48, and 72 hours. All experiments were performed in four replicates. RESULTS: The neuromuscular blocking drugs used had comparable effects on the survivability of HUVECs. Overall, no significant difference was observed in the survivability of HUVECs in a dose-dependent manner after exposure to the study drugs. However, some significant differences in the viability of HUVECs were found among the different measurement times. CONCLUSIONS: The findings of the current study support the safety of the studied neuromuscular blocking drugs in clinically relevant concentrations regarding their effects on endothelial cell proliferation.

Evaluation of the in vitro activity of ceragenins against Trichomonas vaginalis.

Trichomonosis, caused by the protozoan parasite Trichomonas vaginalis, is a curable sexually transmitted disease that is most commonly encountered worldwide. Increasing importance of trichomoniasis and emerging of resistance against metronidazole lead to search for alternative drugs with different mode of activity. The purpose of this study was to determine in vitro activity of ceragenins (CSA-13, CSA-44, CSA-13, and CSA-138) against the metronidazole-susceptible (ATCC 30001) and metronidazole-resistant (ATCC 50138) strains of T. vaginalis. The effective concentrations were evaluated using two strains of T. vaginalis with different metronidazole susceptibilities (ATCC 30001 and ATCC 50138) in the presence of dilution series of ceragenins in 24-well microtitre assays. Overall, all the ceragenins killed the metronidazole-susceptible (ATCC 30001) and metronidazole-resistant (ATCC 50138) strains of T. vaginalis (p>0.05). With regard to the their effects against the studied strains of T. vaginalis, in order of effectiveness, overall, the ceragenins ordered as CSA-13 (the most effective), CSA-131 and CSA-138 (effective similarly), and CSA-44 (the least effective) (p<0.05). All of the ceragenins reduced the trophozoite numbers of both of studied strains of T. vaginalis with a time- and dose- dependent manner (p<0.05). Although all of the study ceragenins, CSA-13, CSA-44, CSA-13, and CSA-138, killed the studied strains of T. vaginalis. CSA-13 is the leading ceragenin as the most effective anti-trichomonas compound, followed by CSA-131 and CSA-138. They have a potential to have a place in the armemantarium of gynecologic and urologic practice for the management of sexually transmitted diseases.

In vitro comparison of the cytotoxicity and water sorption of two different denture base systems.

PURPOSE: Denture base resins have the potential to cause cytotoxicity in vivo, and the mechanical properties of resins are affected by water sorption. There is a correlation between residual monomer and water sorption. Thus, the purpose of this study was to evaluate water sorption and cytotoxicity of light-activated urethane dimethacrylate (UDMA) denture base resin compared to a conventional heat-activated polymethyl methacrylate (PMMA) resin. MATERIALS AND METHODS: Two denture base resins, heat-activated PMMA (Meliodent) and light-activated UDMA (Eclipse), were used in this study. Cytotoxicity (5 × 1 mm(2) ) and water sorption (1 × 1 mm(2) ) specimens were made following the manufacturers' instructions (n = 10). Cytotoxicity tests of denture base resins were performed according to ISO10993-5:1999, and water sorption was evaluated according to ISO 1567:1997. ANOVA tests were employed for evaluating data (α = 0.05). RESULTS: There was no cytotoxic effect in either the PMMA or UDMA group. In addition, contrary to short-term water storage, a significantly lower water sorption value was shown for UDMA resins compared to PMMA resins in both 3- and 6-month storage periods (p = 0.043 and p = 0.002, respectively). CONCLUSION: The tested denture base materials adhered to the ISO standards for both cytotoxicity and water sorption. The cytotoxicity of the light-activated UDMA resin tested was statistically similar to that of the heat-activated PMMA resin; however, the UDMA resin exhibited decreased water sorption in long-term water storage.

Effects of tyrosine kinase inhibitor E7080 and eNOS inhibitor L-NIO on colorectal cancer alone and in combination.

OBJECTIVE: To investigate the effects of E7080 and N (5)-(1-iminoethyl)-L-ornithine dihydrochloride (L-NIO) on colorectal cancer alone and in combination. METHODS: HT29 colorectal cancer cell line from Sap Institute was used. Real-time cell analysis (xCELLigence system) was performed to determine the effects of E7080 and L-NIO on colorectal cell proliferation. While apoptosis was determined with Annexin V staining, and the effect of agents on angiogenesis was determined with chorioallantoic membrane (CAM) model. RESULTS: We found that E7080 has a strong antiproliferative effect with an half maximum inhibition of concentration (IC50) value of 5.60×10(-8) mol/L. Also it has been observed that E7080 showed antiangiogenic and apoptotic effects on HT29 colorectal cancer cells. Antiangiogenic scores of E7080 were 1.2, 1.0 and 0.6 for 100, 10 and 1 nmol/L E7080 concentrations, respectively. Furthermore, apoptosis has been detected in 71% of HT29 colorectal cancer cells after administration of 100 nmol/L E7080 which may indicate strong apoptotic effect. Meanwhile administration of L-NIO alone did not show any effect, but the combination of E7080 with L-NIO increased the antiproliferative, antiangiogenic and apoptotic effects of E7080. CONCLUSIONS: Results of this study indicate that E7080 may be a good choice in treatment of colorectal tumors. Furthermore the increased effects of E7080 when combined with L-NIO raise the possibility to use a lower dose of E7080 and therefore avoid/minimize the side effects observed with E7080.

Effects of low molecular weight heparins and unfractionated heparin on viability of human umbilical vein endothelial cells.

AIM: The aim of this study was to investigate the effects of bemiparin, nadroparin, enoxaparin, and heparin on viability of human umbilical vein endothelial cells (HUVEC). METHODS: Cultivation of HUVEC was performed in an incubator having 5 % CO(2) at 37 °C and with predefined supplemented medium, until cell monolayers attained confluence which occurred after 7 days. The assays were performed in the exponential growth phase of the cells. The cell viability was assessed using the cleavage of tetrazolium salts added to the culture medium. Heparin sodium, enoxaparin sodium, bemiparin sodium, and nadroparin calcium with concentrations of 100, 10, and 1 IU/100 µL were used for the proliferation assay in which cells were incubated for 24, 48, and 72 h with these drugs. The experiments were conducted in four replicates. RESULTS: Among the study drugs with maximal concentration used in the experiments (100 IU/100 µL), heparin was found to be associated with the lowest viability score in 24 and 48 h, while bemiparin showed the lowest at 72 h. Bemiparin 100 IU/100 µL was significantly associated with lower viability score than that of bemiparin 10 IU/100 µL and bemiparin 1 IU/100 µL at every time interval. Among gradual concentrations of enoxaparin, however, concentration of 1 IU/100 µL was associated with the lowest viability scores at every time point. Heparin 1 IU/100 µL, nadroparin 100 IU/100 µL, and enoxaparin 100 IU/100 µL groups had the highest viability score after 72 h of incubation. CONCLUSION(S): Among low molecular weight heparins (LMWHs), 100 IU/100 µL concentration of bemiparin was associated with a more pronounced effect on reducing viability of HUVEC after 72 h of incubation, while nadroparin 100 IU/100 µL and enoxaparin 100 IU/100 µL showed the least effects. LMWHs differ both from each other and heparin with respect to their effects on cellular viability of HUVEC in dose- and time-dependent manner.

Comparison of the antiangiogenic effects of heparin sodium, enoxaparin sodium, and tinzaparin sodium by using chorioallantoic membrane assay.

Unfractionated heparin (UFH) and low molecular weight heparins have been used as anticoagulation agents in cardiovascular clinics for decades. However, these molecules also have potent antiangiogenic effects. Whereas, angiogenesis may be the most crucial determinant of the prognosis of cardiovascular diseases, and except some special situation, antiangiogenic effect is not desirable in the most of the cardiovascular disease. In this study, we aimed to compare the antiangiogenic potency of UFH, enoxaparin, and tinzaparin. The antiangiogenic efficacies of UFH, enoxaparin, and tinzaparin were examined in vivo by using the chick chorioallantoic membrane (CAM) model. Twenty fertilized eggs were used for each studied drug. Drug solutions were prepared in 10 and 1 IU/10 µl concentrations. Decreases in the density of the capillaries were assessed and scored. All three drugs showed antiangiogenic effects on the chick CAM at the 10 IU/10 µl concentration. However, the antiangiogenic score of the UFH was significantly higher than that of enoxaparin and tinzaparin at 1 and 10 IU/10 µl concentrations. UFH had stronger and antiangiogenic potential than enoxaparin and tinzaparin. However, tinzaparin showed dose-dependent antiangiogenic effects. We think that an anticoagulant molecule with a less and dose-dependent antiangiogenic effect, as in the case of tinzaparin, may be more desirable in case of cardiovascular disease related with insufficient angiogenesis.

The antiangiogenic effects of levosimendan in a CAM assay.

BACKGROUND: There is a physiological balance between the stimulatory and inhibitory signals for blood vessel growth. In many symptomatic patients with peripheral artery disease, coronary artery disease, and ischemic chronic wounds, there is a pathological insufficiency of angiogenesis. Therefore, determining the angiogenic or antiangiogenic effects of molecules currently used in cardiovascular treatment is crucial. Although levosimendan is the most well studied calcium sensitizer in preclinical and clinical practice, to the best of our knowledge, there are no previous studies investigating its angiogenic or antiangiogenic effects. In the present study, we aimed to investigate the effects of levosimendan on angiogenesis. METHODS: The antiangiogenic efficacy of levosimendan was examined in vivo in the chick chorioallantoic membrane (CAM) model by using 20 fertilized eggs and drug solutions of 1 and 10 µmol/L concentrations. Decreases in the density of the capillaries were assessed and scored. RESULTS: Significant antiangiogenic effects were observed at 1 and 10 µmol/L concentrations of levosimendan. The antiangiogenic scores of levosimendan at 1 and 10 µmol/L concentrations were 0.6 and 1.10, respectively. The antiangiogenic score of bevacizumab, used as a positive control, was 0.95 at 1.0 µmol/L concentration. No significant difference was found between the antiangiogenic scores of levosimendan and bevacizumab (p=0.54). CONCLUSIONS: Our results indicate that levosimendan has antiangiogenic effects on the chorioallantoic membrane. However, these findings must be confirmed in future studies on humans.

Efficacy of miltefosine for topical treatment of Acanthamoeba keratitis in Syrian hamsters.

Acanthamoeba keratitis is a painful corneal infection and difficult to treat because no sufficiently efficient drug has yet been available. The aim of the study therefore was to assess the therapeutic potential of miltefosine on Acanthamoeba keratitis-infected hamster eyes. The cornea of hamsters were infected with Acanthamoeba hatchetti, a human corneal isolate. On the fifth day, all the cornea were microscopically examined in order to determine the degree of infections (G, from 0 to 3). Four groups were then prepared: miltefosine (160 µM); 0.1% propamidine isetionate plus 0.02% polyhexnide; infected control (0.05% ethanol in PBS) and a non-infected control (0.05% ethanol in PBS) groups. The treatment was continued for 28 days. After the treatment, the cornea were excised and used for Acanthamoeba culture to investigate the presence of Acanthamoeba growth. Miltefosine treatment yielded much higher cure scores than propamidine isetionate plus polyhexanide. On the last day of treatment, 85% of the miltefosine-treated eyes were graded as G0; no changes were observed in the uninfected control group eyes; G3 eyes showed only a partial improvement. Furthermore, no Acanthamoeba cells could be recovered from the miltefosine-treated eye samples. Miltefosine appeared to hold necessary therapeutic properties for the treatment of Acanthamoeba keratitis.

Effect of combined chlorhexidine gluconate and neosporin on experimental keratitis with two pathogenic strains of Acanthamoeba.

Acanthamoeba keratitis (AK) is a painful, sight-threatening, and difficult-to-treat corneal infection caused by the ubiquitous free-living amoebae Acanthamoeba species. The aim of the present study was to compare the severity of keratitis, caused by Acanthamoeba hatchetii and Acanthamoeba castellanii infections, and to assess the therapeutic effects of combined chlorhexidine (CHX) and Neosporin® treatment in rats. The rats were first divided into two groups, in which the eyes of the animals were infected with A. hatchetii or A. castellanii trophozoites. On day 5, all corneas were examined in order to determine the degree of infection (grade 0 to 3), and animals were divided into two new groups, treatment and infected control groups. The treatment was continued for 28 days, followed by excision and histological evaluation of the corneas. In conclusion, the clinical picture progressed more rapidly and severely in eyes infected by A. castellanii, while it was non-invasive and slower to progress with A. hatchetii. Moreover, eyes infected by A. hatchetii responded quicker and more positively to therapy, consistent with its clinical course, while a longer recovery was seen with A. castellanii. Histological examinations revealed the presence of A. castellanii and A. hatchetii trophozoites in the stroma of eyes of the treatment and control groups. As a result, our findings suggest that a combination of Neosporin with lower doses of CHX may be beneficial to treat patients with early diagnosis of AK.

Antiangiogenic activities of bemiparin sodium, enoxaparin sodium, nadroparin calcium and tinzaparin sodium.

INTRODUCTION: The low-molecular-weight heparins have been demonstrated to have antiangiogenic effects in various assays. We aimed to demonstrate and compare the antiangiogenic effects of four types of commercially available low-molecular weight heparins in the chick embryo chorioallantoic membrane model. MATERIALS AND METHODS: The antiangiogenic efficacies of bemiparin, enoxaparin, nadroparin, and tinzaparin were examined in vivo in the chick chorioallantoic membrane model. Drug solutions are prepared in three different concentrations (100 IU, 10 IU, or 1 IU/10 µl). For each set of experiment twenty fertilized eggs were used. The decrease of vessel formation is examined and scored according to previous literature. RESULTS: Bemiparin, enoxaparin, nadroparin, and tinzaparin sodium all have antiangiogenic effects on chick chorioallantoic membrane at the concentration of 100 IU/10 µl. This effect was also observed in 10 IU/10 µl concentrations of nadroparin and tinzaparin. CONCLUSIONS: The low molecular weight heparins studied have obvious antiangiogenic effects. There may be a difference in the potency of the drugs that could have a significant implication for further clinical research.

In vitro amoebicidal activity of a ceragenin, cationic steroid antibiotic-13, against Acanthamoeba castellanii and its cytotoxic potential.

Acanthamoeba is a free-living amoeba causing a potentially blinding infection of the cornea. Acanthamoeba keratitis is difficult to treat, without total efficacy in some patients because of cysts that are less susceptible than trophozoites to the usual treatments. Contact lens wearers are most at risk and account for some 95% of cases. Cationic steroid antibiotic (CSA)-13 is a small molecule aminosterol that has been shown to mimic the activity of endogenous antimicrobial peptides and has bactericidal activity based on membrane disruption. We investigated here the in vitro effectiveness of CSA-13 with a concentration of 100, 75, 50, and 25 mg/mL on proliferation of Acanthamoeba castellanii trophozoites and cysts and cytotoxic potential. CSA-13 was evaluated for its amoebicidal activity using an inverted light microscope at 1, 2, 4, 8, 12, and 24 h. For the determination of cytotoxicity of the CSA-13 on L929 cells, agar diffusion tests were performed. CSA-13 inhibited trophozoite growth in dose- and time-dependent ways. At 1 h, no viable trophozoites were observed in the presence of CSA-13 solution in a concentration 100 mg/mL in phosphate-buffered saline. Results of cytotoxicity experiments demonstrated that CSA-13 solution had mild toxicity at 100 mg/mL concentration on cells, whereas it had no toxicity at 75 mg/mL concentration. The findings of this experiment as in vitro ameboebicidal activity for Acanthamoeba suggest that CSA-13 has a potential to be used as a new agent in lens solutions to prevent Acanthamoeba growth and infections.

In vitro amoebicidal activity of Salvia staminea and Salvia caespitosa on Acanthamoeba castellanii and their cytotoxic potentials on corneal cells.

BACKGROUND/AIMS: Amoebic keratitis is difficult to treat with total efficacy in some patients because of cysts, which are less susceptible than trophozoites to the usual treatments. We investigated the in vitro effectiveness of methanolic extract of Salvia staminea and Salvia caespitosa against Acanthamoeba castellanii, as well as their cytotoxicity on corneal cells in vitro. METHODS: Extracts were evaluated for their amoebicidal activities using an inverted light microscope. The effect of Savia species, with concentrations ranging between 1.0 and 32.0 mg/mL, on the proliferation of A. castellanii trophozoites and cysts were examined in vitro. For determining the cytotoxicity of Salvia species on corneal cells, agar diffusion tests were performed. RESULTS: According to the results obtained from these tests, S. staminea showed remarkable amoebicidal effect on A. castellanii. In the case of the cytotoxic activity, methanolic extract of S. staminea showed no cytotoxicity on corneal cells with a concentration of 16 mg/mL. CONCLUSIONS: Methanolic extract of S. staminea could be considered a new natural agent against Acanthamoeba. However, further evaluation by in vivo testing is needed to confirm the efficiency of its biological effect.

Influence of storage media containing Salvia officinalis on survival of periodontal ligament cells.

AIM: The purpose of this study was to determine the ability of Salvia officinalis (S. officinalis) extracts to serve as a storage medium for the maintenance of periodontal ligament (PDL) cell viability of avulsed teeth. METHODS AND MATERIALS: PDL cells were obtained from healthy third molars and cultured in Dulbecco's Modi?ed Eagle's Medium (DMEM). Cultures were subjected to 4, 2.5, 1.5, and 0.5% S. officinalis solutions, Hank's balanced salt solution (HBSS), phosphate buffered saline (PBS), and tap water. Tissue culture plates were incubated with experimental media at 37 masculineC for 1, 3, 6, 12 or 24 hours. PDL cell viability was assessed by trypan blue exclusion. Statistical analysis of the data was performed by one-way analysis of variance (ANOVA) complemented by the Tukey's test. The level of significance was 5% (p< 0.05). RESULTS: The results showed 2.5% S. officinalis was a more effective storage medium than the other experimental solutions (p<0.05). Only at 1 hour and 3 hours was there found similar effect between 2.5% S. officinalis and HBSS. At 24 hours, 2.5% S. officinalis was found to be significantly better than the other solutions tested. CONCLUSION: S. officinalis can be recommended as a suitable transport medium for avulsed teeth. CLINICAL SIGNIFICANCE: The findings of this study support the use of S. officinalis as another option for clinicians to use to store and transport avulsed teeth until reimplantation procedures can be done.

Cytotoxicity analysis of strontium ranelate on cultured human periodontal ligament fibroblasts: a preliminary report.

BACKGROUND/PURPOSE: The aim of this study was to analyze the cytotoxicity of strontium ranelate (SR) on human periodontal ligament fibroblasts (PDL cells) in vitro. METHODS: PDL cells were obtained from healthy human third molars and cultured in Dulbeccos Modified Eagles Medium. The experimental groups were: G1, cultures treated with fresh medium (control); and G2, G3, G4 and G5: treated with SR at 20, 10, 5 and 2.5 mg/mL, respectively. The experimental times were 1, 6, 12 and 24 hours (short-term) for viability, and 2, 4, 6 and 8 days (long-term) for cell survival. The cells were counted using a hemocytometer. Data were then analyzed by one-way ANOVA and Tukey's tests (p < 0.05). RESULTS: Cultures treated with the highest SR concentrations (G2 and G3) had significantly lower cell viability and cell numbers (p < 0.05) than those in G1, G4 and G5. SR at 2.5 mg/mL was non-cytotoxic to PDL cells. CONCLUSION: SR was non-toxic at appropriate concentrations. Preclinical tests are needed to further assess its safety and effectiveness for tooth resorption prior to clinical use.

In vitro evaluation of the amoebicidal activity of garlic (Allium sativum) extract on Acanthamoeba castellanii and its cytotoxic potential on corneal cells.

Free-living protozoa of the genus Acanthamoeba can cause one of the most severe, potentially sight-threatening infections of the eye, the so-called A. keratitis. A. keratitis is difficult to treat because, under adverse conditions, the amoeba encyst and medical therapy is often less effective against cysts than against trophozoites. The aim of this study was to investigate evaluate the in vitro effect of the nonpolar subfraction of the methanol extract of garlic (Allium sativum) on the growth of A. castellanii trophozoites and cysts and also its cytotoxicity on corneal cells in vitro. Extract was evaluated for its amoebicidal activity, using an inverted light microscope. The effect of the nonpolar extract with the concentrations, ranging from 0.78 to 62.5 mg/mL on the proliferation of A. castellanii trophozoites and cysts, were examined in vitro. For the determination of cytotoxicity of the extract on corneal cells, agar diffusion tests were performed. The present study demonstrates the in vitro effectiveness of the garlic against the A. castellanii growth curve. Evaluations revealed that garlic inhibits trophozoite growth in dose- and time-dependent ways. In the case of the cyctotoxic acitivities, it showed no cytotoxicity for the cornea cells in the concentration of 3.90 mg/mL. These findings indicate that nonpolar subfraction of the methanol extracts of garlic has amoebicidal, as well as its cysticidal, properties on Acanthamoeba trophozoites and cysts. Garlic alone, and in combination with other amoebicidal agents, may be used in clinical practices after further investigations.

Evaluation of in vitro effect of Morus rubra (red mulberry) on survival of periodontal ligament cells.

OBJECTIVE: The purpose of this study was to determine the ability of the juice of Morus rubra fruit to serve as a temporary storage medium for the maintenance of periodontal ligament (PDL) cell viability of avulsed teeth. STUDY DESIGN: PDL cells were obtained from healthy third molars and cultured in Dulbecco's Modified Eagle's Medium (DMEM). Cultures were subjected to 4.0%, 2.5%, 1.5%, and 0.5% of the juice of M. rubra fruit, Hank's balanced salt solution (HBSS), phosphate-buffered saline (PBS), and tap water. Tissue culture plates were incubated with experimental media at 37 degrees C for 1, 3, 6, 12, or 24 hours. PDL cell viability was assessed by trypan blue exclusion. Statistical analysis of the data was accomplished using 1-way analysis of variance complemented by the Tukey test. The level of significance was 5% (P < .05). RESULTS: The efficacy of 4.0% and 2.5% M. rubra at 3, 6, and 12 hours was found to be significantly better than HBSS (P < .05). At 24 hours, 4% M. rubra was found to be similar to HBSS, but 2.5% M. rubra was found to be significantly worse than HBSS (P < .005). The results showed that juice of the fruit sample of M. rubra studied at a concentration of 4% was a more effective storage medium than other groups. CONCLUSION: Juice of the fruit of M. rubra can be recommended as a suitable transport medium for avulsed teeth.

The effect of propolis in experimental Acanthamoeba keratitis.

PURPOSE: To examine the effect of propolis in a rat model of Acanthamoeba keratitis and to determine its in vitro cytotoxicity in cultured corneal epithelial cells. METHODS: Eighteen Wistar albino rats were used. Cultured corneal epithelial cells obtained from two healthy rats for in vitro cytotoxicity of propolis. Corneal stromal inoculation was performed in 16 rats with amoebic culture containing 1 x 10(6) amoeba/mL. Rats with Acanthamoeba keratitis 5 days later after the inoculation were divided randomly into four groups, and eight eyes of each group were treated with study drugs. The propolis, chlorhexidine (CHX), propolis plus CHX and control eyes were treated with topical propolis, 0.002% CHX, propolis plus 0.002% CHX and lubricant eye drops, respectively. The study drugs were instilled every one hour for 10 days. All eyes were examined and keratitis graded by slit-lamp biomicroscopy on days 2, 5 and 10 during the administration of the study drugs. After the completion of keratitis grading, all the 16 rats were humanely killed and their corneas were excised and used for Acanthamoeba culture to evaluate presence of Acanthamoeba growth after treatment 14 days later. RESULTS: Concentrations of propolis higher than 7.81 mg/mL cause damage to corneal epithelial cells in the experiment of in vitro cytotoxicity of propolis on corneal epithelial cells. The keratitis grade on day 2 in the CHX eyes was significantly lower than that in the control eyes (P < 0.05). The keratitis grades on days 5 and 10 in the propolis, CHX and propolis plus CHX eyes were significantly lower compared with those on days 5 and 10 in the control eyes (P < 0.05). In the propolis eyes, the keratitis grade on day 5 was significantly lower than that on day 2 (P < 0.05), and it was significantly lower on day 10 compared with that on day 5 (P < 0.05). In the CHX and propolis plus CHX eyes, the keratitis grade on day 10 was significantly lower compared with that on days 2 and 5 (P < 0.05). In the control eyes, there was no significant difference in the keratitis grades on days 2, 5 and 10 (P > 0.05). The culture positivity at Acanthamoeba growth after treatment experiment in the propolis, CHX and propolis plus CHX eyes was significantly lower than that in the control eyes (P < 0.05). CONCLUSIONS: We suggest that propolis had amoebicidal properties in this rat model of Acanthamoeba keratitis. Further investigations to evaluate the antimicrobial activity of the individual fractions of the resin could yield more information about its mechanism of action in treating this disease.

Clinical and histologic evaluations of experimental Acanthamoeba keratitis.

Amoebic keratitis, a sight-threatening, progressive corneal disease, is commonly caused by ubiquitous, pathogenic, free-living Acanthamoeba spp., which are widely distributed in the environment. We investigated clinical findings and histology of Acanthamoeba keratitis in a rat cornea model. Experimental Acanthamoeba keratitis was induced in Wistar rats by intrastromal inoculation of Acanthamoeba castellanii trophozoites. The clinic features of Acanthamoeba keratitis by day 70 are observed. All rats inoculated with Acanthamoeba developed keratitis. Histologically, the eyes displayed blood vessels, edema, and amoebae in stroma. A mixed cellular response, including neutrophils, mononuclear cells, and spindle-shaped cells, was seen. In conclusion, progressive, suppurative Acanthamoeba keratitis can be induced in the rat cornea model. This rat cornea model assists researchers who study the pathogenesis of Acanthamoeba keratitis and devise treatment for this difficult condition.

In vitro effectiveness of Thymus sipyleus subsp. sipyleus var. sipyleus on Acanthamoeba castellanii and its cytotoxic potential on corneal cells.

Acanthamoeba species are an important cause of microbial keratitis that may cause severe ocular inflammation and visual loss. Acanthamoeba keratitis is difficult to treat, without total efficacy in some patients because of cysts which is less susceptible than trophozoites to the usual treatments. We investigated here the in vitro amoebicidal activity of methanolic extract of Thymus sipyleus subsp. sipyleus var. sipyleus from Turkish flora against Acanthamoeba castellanii and also its cytotoxicity on corneal cells in vitro. Extract was evaluated for its amoebicidal activity using an inverted light microscope. The effect of the polar extract with the concentrations ranging from 1.0 to 32.0 mg/ml on the proliferation of A. castellanii trophozoites and cysts were examined in vitro. For the determination of cytotoxicity of the extract on corneal cells, agar diffusion tests were performed. According to results obtained from the tests, the extract evaluated here showed remarkable amoebicidal effect on A. castellanii. In the case of the cytotoxic activities, it showed no cytotoxicity for corneal cells in the concentration of 32 mg/ml. As a result, polar subfraction of the methanolic extract of Thymus sipyleus subsp. sipyleus var. sipyleus could be concluded as a new natural agent for the treatment of Acanthamoeba infections. On the other hand, it still needs to be further evaluated by in vivo test systems to confirm the efficiency of its biological effect.

Efficacy of contact lens storage solutions against trophozoite and cyst of Acanthamoeba castellanii strain 1BU and their cytotoxic potential on corneal cells.

Acanthamoeba is a free-living amoeba causing a potentially blinding infection of the cornea. Contact lens wearers are most at risk and account for about 95% of cases. We investigated the in vitro effectiveness of 10 contact lens solutions against Acanthamoeba castellanii and their cytotoxicity on corneal cells in vitro. Contact lens solutions were evaluated for their amoebicidal activities using an inverted light microscope. To determine of their cytotoxicity on corneal cells, agar diffusion tests were performed. According to the results obtained from the tests, AVIZOR Aqua Soft Comfort and Elegance(R) showed the best amoebicidal effect on A. castellanii trophozoites. Cysts were still viable after overnight (8 h) exposure. In the case of the cyctotoxic acitivities, All In One Light, Astek, SOLO-Care Aqua, Maxima, and Horien showed no cytotoxicity on the corneal cells. ReNu MultiPlus, AVIZOR Aqua Soft Comfort, Carrera, and Elegance showed mild cytotoxicity on the corneal cells. Plurisol.M presented moderate cytotoxicity on the corneal cells. All commercial solutions examined in this study are the lack of efficacy against A. castellanii. Improvement or development of new contact lens disinfecting systems by the manufacturers is needed to prevent Acanthamoeba keratitis.