ABSTRACT
Metal-based phthalocyanines currently are utilized as a colorant for industrial applications but their unique properties also make them prospective photosensitizers. Photosensitizers are non-toxic drugs, which are commonly used in photodynamic therapy (PDT), for the treatment of various cancers. PDT is based on the principle that, exposure to light shortly after photosensitizer administration predominately leads to the production of reactive oxygen species for the eradication of cancerous cells and tissue. This in vitro study investigated the photodynamic effect of gallium (GaPcCl), indium (InPcCl) and iron (FePcCl) phthalocyanine chlorides on human lung adenocarcinoma cells (A549). Experimentally, 2 × 10(4)cells/ml were seeded in 24-well tissue culture plates and allowed to attach overnight, after which cells were treated with different concentrations of GaPcCl, InPcCl and FePcCl ranging from 2 µg/ml to 100 µg/ml. After 2h, cells were irradiated with constant light doses of 2.5 J/cm(2), 4.5 J/cm(2) and 8.5 J/cm(2) delivered from a diode laser (λ = 661 nm). Post-irradiated cells were incubated for 24h before cell viability was measured using the MTT Assay. At 24h after PDT, irradiation with a light dose of 2.5 J/cm(2) for each photosensitizing concentration of GaPcCl, InPcCl and FePcCl produced a significant decrease in cell viability, but when the treatment light dose was further increased to 4.5 J/cm(2) and 8.5 J/cm(2) the cell survival was less than 40%. Results also showed that photoactivated FePcCl decreased cell survival of A549 cells to 0% with photosensitizing concentrations of 40 µg/ml and treatment light dose of 2.5 J/cm(2). A 20 µg/ml photosensitizing concentration of FePcCl in combination with an increased treatment light dose of either 4.5 J/cm(2) or 8.5 J/cm(2) also resulted in 0% cell survival. This PDT study concludes that low concentrations on GaPcCl, InPcCl and FePcCl activated with low level light doses can be used for the effective in vitro killing of lung cancer cells.
Subject(s)
Chlorides/pharmacology , Coordination Complexes/pharmacology , Gallium/chemistry , Indium/chemistry , Indoles/chemistry , Iron/chemistry , Photosensitizing Agents/pharmacology , Adenocarcinoma/drug therapy , Adenocarcinoma of Lung , Cell Line, Tumor , Cell Survival/drug effects , Chlorides/chemistry , Coordination Complexes/chemistry , Coordination Complexes/therapeutic use , Humans , Isoindoles , Lung Neoplasms/drug therapy , Photochemotherapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/therapeutic useABSTRACT
In the title compound, C21H13Cl2NO2S, the benzo-thia-zole ring makes dihedral angles of 0.94â (1) and 70.65â (5)° with the 4-chloro-phenyl-methanone unit and the 5-chloro-phenyl ring, respectively. The dihedral angle between the 4-chloro-phenyl-methanone unit and the 5-chloro-phenyl ring is 66.20â (5)°. The crystal structure consists of dimeric units generated by C-Hâ¯N hydrogen bonds, further linked by C-Hâ¯O and C-Hâ¯π inter-actions, leading to a three-dimensional network.
ABSTRACT
In the title compound, C21H14BrNO2S, the dihedral angle between the planes of the benzo-thia-zole and phenyl-methanone groups is 63.4â (2)°. In the crystal, pairs of C-Hâ¯N hydrogen bonds link the mol-ecules to form inversion dimers, which are further linked by C-Hâ¯O inter-actions into chains along the c axis. C-Hâ¯π and π-π inter-actions [centroid-centroid distance = 3.863â (1)â Å] further stabilize the mol-ecular assembly.
ABSTRACT
Coumarin (2H-1-benzopyran-2-one) is a plant-derived natural product known for its pharmacological properties such as anti-inflammatory, anticoagulant, antibacterial, antifungal, antiviral, anticancer, antihypertensive, antitubercular, anticonvulsant, antiadipogenic, antihyperglycemic, antioxidant, and neuroprotective properties. Dietary exposure to benzopyrones is significant as these compounds are found in vegetables, fruits, seeds, nuts, coffee, tea, and wine. In view of the established low toxicity, relative cheapness, presence in the diet, and occurrence in various herbal remedies of coumarins, it appears prudent to evaluate their properties and applications further.
Subject(s)
Coumarins/chemistry , Coumarins/classification , Coumarins/therapeutic use , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/therapeutic use , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/therapeutic use , Anticoagulants/chemistry , Anticoagulants/therapeutic use , Antifungal Agents/chemistry , Antifungal Agents/therapeutic use , Antihypertensive Agents/chemistry , Antihypertensive Agents/therapeutic use , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Coumarins/adverse effects , Coumarins/pharmacokinetics , HumansABSTRACT
Extracts of selected flowering plants, which are considered eco-friendly, are used for the treatment of numerous ailments and vector control worldwide. This has resulted in approximately 25 per cent of currently used drugs being derived from herbal sources. The aqueous and methanolic extracts of twelve plant species, Psidium guajava (pink fruit), Psidium guajava (white fruit), Psidium cattleianum var. cattleianum, Psidium guineense and Psidium X durbanensis, Achyranthes aspera, Alternanthera sessilis, Guilleminea densa, Capparis tomentosa, Leonotis leonurus, Dichrostachys cinerea and Carpobrotus dimidiatus, were tested for insecticidal activity, including larvicidal, adulticidal and repellent activities against the adult female mosquito, Anopheles arabiensis. The extracts of P. guajava (white fruit), C. tomentosa, L. leonurus,D. cinerea, and C. dimidiatus exerted a pronounced inhibitory effect on adult insects, while those of P. guajava (pink fruit), P. X durbanensis, P. cattleianum var. cattleianum, P. guineense, A. aspera, A. sessilis, and G. densa were ineffective and failed to satisfy the criteria set by the World Health Organization. In the tests for repellency against An. arabiensis, all the tested aqueous and methanolic plant extracts except those of A. sessilis repelled 80-100% of mosquitoes. The most effective mosquito repellents were the methanol and aqueous extracts of P. guajava (pink fruit), P. X durbanensis, P. cattleianum var. cattleianum, P. guineense, G. densa,L. leonurus and D. cinerea, which are potential sources of cost effective mosquito repellents to be utilized in malarial endemic areas.
Subject(s)
Anopheles/drug effects , Insecticides/pharmacology , Plant Extracts/pharmacology , Animals , Biological Assay , Female , Humans , Insect Repellents/isolation & purification , Insect Repellents/pharmacology , Insecticides/isolation & purification , Plant Extracts/isolation & purification , South Africa , Survival AnalysisABSTRACT
Photodynamic therapy is a medical treatment that uses an inactive dye/drug and lasers as a light source to activate the dye/drug to produce a toxic form of oxygen that destroys the cancer cells. This study aimed at investigating the cytotoxic effects of different concentrations of aluminum tetrasulfophthalocyanines in its inactive and active state (laser induced) on melanoma skin cancer cells, healthy normal skin fibroblast and keratinocyte cells. Experimentally, 3 × 104 cells/ml were seeded in 24-well plates before treatment with different concentrations of aluminum tetrasulfophthalocyanines. After 2h, cells were irradiated with a light dose of 4.5 J/cm². Post-irradiated cells were incubated for 24h before cell viability was measured using the CellTiter-Blue Viability Assay. Results showed that aluminum tetrasulfophthalocyanines at high concentrations were cytotoxic to melanoma cells in the absence of laser activation. In the presence of laser activation of aluminum tetrasulfophthalocyanines at a concentration of 40 µg/ml decreased cell viability of melanoma cells to 45%, fibroblasts to 78% and keratinocytes to 73%. At this photosensitizing concentration of aluminum tetrasulfophthalocyanines the efficacy of the treatment light dose 4.5 J/cm² and the cell death mechanism induced by photoactivated aluminum tetrasulfophthalocyanines was evaluated. A light dose of 4.5 J/cm² was more efficient in killing a higher number of melanoma cells and a lower number of fibroblast and keratinocyte cells than the other light doses of 2.5 J/cm², 7.5 J/cm² and 10.5 J/cm². Apoptosis features such as blebbing, nucleus condensation, nucleus fragmentation and the formation of apoptotic bodies were seen in the photodynamic therapy treated melanoma skin cancer cells. This in vitro photodynamic therapy study concludes that using aluminum tetrasulfophthalocyanines at a photosensitizing concentration of 40 µg/ml in combination with a laser dose of 4.5 J/cm² was potentially lethal for melanoma skin cancer cells and less harmful for the normal healthy skin cells.
Subject(s)
Fluorescent Dyes/pharmacology , Indoles/pharmacology , Melanoma/drug therapy , Organometallic Compounds/pharmacology , Photochemotherapy/methods , Photosensitizing Agents/pharmacology , Skin Neoplasms/drug therapy , Cell Death/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Fibroblasts/drug effects , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/therapeutic use , Humans , Indoles/administration & dosage , Indoles/therapeutic use , Keratinocytes/drug effects , Organometallic Compounds/administration & dosage , Organometallic Compounds/therapeutic use , Photosensitizing Agents/therapeutic useABSTRACT
A series of water-soluble tetrasulfonated metallophthalocyanines (MPcs) dyes have been studied to be used as a drug or photosensitizer (PS) in photodynamic therapy (PDT) for the treatment of cancers. During PDT the PS is administrated intravenously or topically to the patient before laser light at an appropriate wavelength is applied to the cancerous area to activate the PS. The activated PS will react with oxygen typically present in the cancerous tissue to generate reactive oxygen species for the destruction of the cancerous tissue. This in vitro study aimed at investigating the cytotoxic effects of different concentrations of zinc tetrasulfophthalocyanines (ZnTSPc) activated with a diode laser (λ = 672 nm) on melanoma, keratinocyte and fibroblast cells. To perform this study 3 × 104 cells/ml were seeded in 24-well plates and allowed to attach overnight, after which cells were treated with different concentrations of ZnTSPc. After 2h, cells were irradiated with a constant light dose of 4.5J/cm². Post-irradiated cells were incubated for 24 h before cell viability was measured using the CellTiter-Blue Viability Assay. Data indicated high concentrations of ZnTSPc (60-100 µg/ml) in its inactive state are cytotoxic to the melanoma cancer cells. Also, results showed that photoactivated ZnTSPc (50 µg/ml) was able to reduce the cell viability of melanoma, fibroblast and keratinocyte cells to 61%, 81% and 83% respectively. At this photosensitizing concentration the efficacy the treatment light dose of 4.5J/cm² against other light doses of 2.5J/cm², 7.5J/cm² and 10J/cm² on the different cell lines were analyzed. ZnTSPc at a concentration of 50 µg/ml activated with a light dose of 4.5J/cm² was the most efficient for the killing of melanoma cancer cells with reduced killing effects on healthy normal skin cells in comparison to the other treatment light doses. Melanoma cancer cells after PDT with a photosensitizing concentration of 50µg/ml and a treatment light dose of 4.5J/cm² showed certain apoptosis characteristics such as chromatin condensation and fragmentation of the nucleus. This concludes that low concentrations of ZnTSPc activated with the appropriate light dose can be used to induce cell death in melanoma cells with the occurrence of minimal damage to surrounding healthy tissue.
Subject(s)
Fibroblasts/drug effects , Indoles/toxicity , Keratinocytes/drug effects , Melanoma/drug therapy , Organometallic Compounds/toxicity , Photochemotherapy/methods , Photosensitizing Agents/therapeutic use , Cell Line, Tumor , Humans , LasersABSTRACT
In this study, Fusarium species isolated from 29 patients with mycotic keratitis were identified and tested for their ability to produce mycotoxins. Members of the F. solani species complex (Fs complex) were the predominant species isolated, followed by F. verticillioides, F. dimerum, members of the F. oxysporum species complex Fo complex), F. incarnatum, F. chlamydosporum and F. lateritium. Of these, 76% of the Fusarium isolates produced fusaric acid, moniliformin or fumonisin B1. Many of the fusaria isolated are common aetiological agents of mycotic keratitis infections. However, F. incarnatum, F. chlamydosporum and F. lateritium have previously not been found in this infection. These findings indicate that a greater variety of fusarial species are becoming associated with mycotic keratitis infections. This paper further demonstrates the mycotoxin-producing ability of these clinical isolates and assesses cellular cytotoxicity.
Subject(s)
Cornea/microbiology , Corneal Ulcer/microbiology , Eye Infections, Fungal/microbiology , Fusarium/metabolism , Mycotoxins/biosynthesis , Mycotoxins/isolation & purification , Animals , Cell Line , Corneal Ulcer/pathology , Cyclobutanes/analysis , Cyclobutanes/isolation & purification , Cyclobutanes/metabolism , Cyclobutanes/toxicity , Cytotoxins/analysis , Cytotoxins/biosynthesis , Cytotoxins/isolation & purification , Cytotoxins/toxicity , Epithelial Cells/pathology , Eye Infections, Fungal/pathology , Fumonisins/analysis , Fumonisins/isolation & purification , Fumonisins/metabolism , Fumonisins/toxicity , Fusaric Acid/analysis , Fusaric Acid/biosynthesis , Fusaric Acid/toxicity , Fusarium/classification , Fusarium/isolation & purification , Humans , Macaca mulatta , Mycotoxins/analysis , Mycotoxins/toxicityABSTRACT
The control of cancer, the second leading cause of death worldwide, may benefit from the potential that resides in alternative therapies. The primary carcinogens stem from a variety of agricultural, industrial, and dietary factors. Conventional therapies cause serious side effects and, at best, merely extend the patient's lifespan by a few years. There is thus the need to utilise alternative concepts or approaches to the prevention of cancer. This review focuses on the many natural products that have been implicated in cancer prevention and that promote human health without recognisable side effects. These molecules originate from vegetables, fruits, plant extracts, and herbs.
Subject(s)
Anticarcinogenic Agents/therapeutic use , Neoplasms/prevention & control , Plant Extracts/therapeutic use , Clinical Trials as Topic , Humans , Neoplasms/epidemiology , Neoplasms/etiologyABSTRACT
Phenolic compounds from plants offer a means for both the prevention and detoxification of mycotoxins that affect human health. This research investigates the control of fungal growth and toxin production by Fusarium verticillioides with plant phenolic compounds, namely chlorophorin, iroko and maakianin, benzoic acid, caffeic acid, ferulic acid, and vanillic acid. Inhibition by these compounds of fungal growth was determined by the agar overlay method and their effect on fumonisin B(1) (FB(1)) production was determined by high-performance liquid chromatography. Chlorophorin was the most effective compound in inhibiting fungal growth, followed by iroko, maakianin, vanillic acid and caffeic acid. Chlorophorin also was the most effective compound in reducing toxin production (94% reduction), followed by caffeic acid, ferulic acid, vanillic acid and iroko, which reduced FB(1) levels by 90-91%. The widespread occurrence of fumonisins world-wide and the lack of adequate prevention of fumonisins require 'biologically safe' alternatives to prevent the transfer of fungi and their health hazardous toxins into our daily foods and environment.
Subject(s)
Fumonisins/metabolism , Fusarium/drug effects , Phenols/pharmacology , Food Contamination/prevention & control , Fusarium/growth & development , Fusarium/metabolism , Humans , Microbial Sensitivity Tests , Plant Extracts/pharmacologyABSTRACT
Industrial effluents and leachates from hazardous landfill sites were tested for toxicity using the anaerobic toxicity assay. This test was done on several industrial effluents (brewery spent grain effluent, a chemical industry effluent, size effluent), and several hazardous landfill leachates giving vastly different toxicity results. The brewery effluent, spent grain effluent and size effluent were found to be less toxic than the chemical effluent and hazardous landfill leachate samples. The chemical industry effluent was found to be most toxic. Leachate samples from the H:h classified hazardous landfill site were found to be less toxic at high concentrations (40% (v/v)) while the H:H hazardous landfill leachate samples were found to be more toxic even at low concentrations of 4% (v/v). The 30 d biochemical methane potential tests revealed that the brewery effluent, organic spent grain effluent and size effluent were 89%, 63%, and 68% biodegradable, respectively. The leachate from Holfontein hazardous landfill site was least biodegradable (19%) while the chemical effluent and Aloes leachate were 29% and 32% biodegradable under anaerobic conditions.
Subject(s)
Industrial Waste , Refuse Disposal , Soil Pollutants/metabolism , Soil Pollutants/toxicity , Bacteria, Anaerobic/physiology , Beer , Biodegradation, Environmental , Bioreactors , Chemical IndustryABSTRACT
This study was undertaken to determine the effectiveness of modified atmospheres and packaging materials on the growth of Penicillium expansum and patulin production in apples. Granny Smith apples were surface sterilized with 76% ethanol and inoculated with 0.1 ml of a 1.1 x 10(7) spore/ml P. expansum spore suspension. The apples were packaged either in polyethylene (PE) or polypropylene (PP) and treated with three different gas combinations, viz., 58% CO2/42% N2, 48% CO2/52% N2, and 88% CO2/12% N2, and were then incubated for 14 days at 25 degrees C. Fungal growth was monitored every 2 to 4 days by measuring radial growth from the point of inoculation. After the 14th day, apples were pulped, and patulin was extracted, purified, and quantified by high-performance liquid chromatography. PP did not inhibit fungal growth in any of the atmospheres tested, and it only inhibited patulin production in atmospheric gas and 58% CO2/42% N2. PE was very effective and inhibited fungal growth by four- or fivefold, depending on the modified atmosphere. Patulin production in PE-packaged apples was almost completely inhibited by all three gas combinations. Gas chromatographic analysis of the PE-packaged samples before and after the incubation period showed that CO2 levels dropped and N2 levels increased for all of the atmospheres tested. Our studies showed conclusively that PE is an excellent packaging material for the storage of apples since it inhibited the growth of P. expansum, thereby allowing <3.2 microg/ml of patulin to be produced, regardless of gaseous environment.
Subject(s)
Food Packaging/methods , Malus/microbiology , Patulin/biosynthesis , Penicillium/growth & development , Carbon Dioxide , Chromatography, Gas , Food Handling , Mutagens , Nitrogen , Oxygen , Penicillium/metabolism , Time FactorsABSTRACT
The effect of nine different oils was evaluated on the growth of Aspergillus parasiticus and Fusarium moniliforme. The experimental design to examine the inhibition of mycotoxins involved the incorporation of each of seven oils into broth and patty cultures. The fungal mycotoxin was identified by high-pressure liquid chromatography. Clove oil (eugenol) was the most inhibitory to the growth of A. parasiticus and F. moniliforme, followed by cinnamon (cinnamic aldehyde), oregano (thymol and carvacol) and mace oils (myristin). Neem and eucalyptus oil (cineole) did not affect fungal growth. The feasibility of implementing the results of this study to control mycotoxin toxicity was examined by costoring whole and ground cloves with mycotoxin-infected grain. Addition of both whole and ground cloves markedly reduced the aflatoxin contamination of the grain. These results clearly suggest that commonly occurring mycotoxigenic fungi can be controlled with clove oil (eugenol), thus spice oil successfully inhibited the growth of A. parasiticus and F. moniliforme, regulated the production of fumonisins. and prevented the formation of aflatoxins. The social implication of this finding is that rural communities can prevent the formation of fungal toxins in contaminated grain by simple measures.
Subject(s)
Aspergillus/growth & development , Fusarium/growth & development , Plant Oils/pharmacology , Aspergillus/drug effects , Eugenol/pharmacology , Food Handling/methods , Food Microbiology , Fusarium/drug effects , Mycotoxins/biosynthesis , SpicesABSTRACT
Traditionally brewed alcoholic beverages are regularly consumed by most ethnic black South Africans. Maize and barley, both of which are used for producing locally brewed alcoholic beer, are frequently contaminated by mycotoxin-producing moulds. The study was undertaken to investigate whether these toxins are present in raw grains and the traditional beers imbibed by the local black African population. It was established that the raw ingredients (sorghum, sorghum malt grains, maize grits), commercially produced traditional beers (Utshwala and Utshwala special) and home-brewed beers (Umqombotha, Isiqatha, Imfulamfula) were contaminated by bacteria and fungi (both yeasts and moulds). The contaminating moulds were isolated and identified. The contaminated samples were analysed for aflatoxins B1, B2, G1 and G2, zearalenone, citrinin, deoxynivalenol, and ochratoxin A using a multi-mycotoxin thin-layer chromatography screening method and the toxins were quantified by high-performance liquid chromatography. Grain samples were infected by. Aspergillus flavus, A. alliaceus, A. clavatus, Penicillium spp., Rhizopus spp. and Mucor spp. Sorghum malt grain samples contained the toxin zear alenone. No mycotoxin-producing fungi were present in the fermented beers but two of six commercial beer samples contained aflatoxins (200 and 400 microg l(-1) and 45% (13 of 29) of the home-brewed beers had zear alenone (range 2.6-426 microg l(-1) and/or ochratoxin A (3-2340 microg l(-1).
Subject(s)
Beer/analysis , Food Contamination/analysis , Mycotoxins/analysis , Bacteria/isolation & purification , Chromatography, High Pressure Liquid/methods , Chromatography, Thin Layer/methods , Edible Grain/chemistry , Edible Grain/microbiology , Food Microbiology , Fungi/isolation & purification , Humans , South AfricaABSTRACT
The anti-amoebic activity of four plant extracts: maackiain and formononetin from Virgilia oroboides and chlorophorin and Iroko from Chlorophora excelsa, were evaluated. Anti-protozoal tests conducted on trophozoites of Entamoeba histolytica established that all four compounds had an affect on the trophozoites to some degree. Chlorophorin showed the highest anti-protozoal activity with an MIC of 0.25 microg/ml followed by maackiain and Iroko with MICs of 1 microg/ml. Chlorophorin and Iroko induced the release of acid phosphatase. Chlorophorin reduced alpha amylase levels by 89%. Formononetin and maackiain had a minimal effect on the enzyme levels. Ultrastructural changes occurred in trophozoites treated with plant compounds. The degree of destruction of the trophozoites increased with an increase in compound concentration. Trophozoite destruction was initiated by the disintegration of the nucleus and culminated with the rupture of the cytoplasmic membrane. Maackiain was the only compound that showed some level of mutagenicity. Formononetin and Iroko were very slightly mutagenic, while chlorophorin was non-mutagenic. In addition, none of the compounds tested showed cytopathic effects on any of the cell lines tested. Chlorophorin and Iroko exhibit the potential to be exploited as natural multi-functional safe control agents in the treatment of bacterial, fungal and protozoal infections.
Subject(s)
Amebicides/toxicity , Entamoeba histolytica/drug effects , Plants, Medicinal/chemistry , Animals , Cell Survival/drug effects , Entamoeba histolytica/enzymology , Entamoeba histolytica/ultrastructure , Epithelial Cells/drug effects , Humans , Lysosomes/drug effects , Lysosomes/enzymology , Microscopy, Electron , Mutagenicity Tests , Mutagens/toxicity , Plant Extracts/toxicityABSTRACT
Patulin is a mycotoxin produced by a number of molds involved in fruit spoilage. This compound is carcinogenic and teratogenic. Various methods are currently used to reduce the levels of patulin in apple juice, namely, charcoal treatment, chemical preservation (sulfur dioxide), gamma irradiation, fermentation, and trimming of fungus-infected apples. Many of these processes are expensive and time-consuming. Therefore, there is a need to find a convenient and economical process to control patulin levels. This study was undertaken to evaluate the effectiveness of several clarification processes for the reduction of patulin. Clarification was carried out on a laboratory scale. Apple pulp was spiked with patulin, pressed, and clarified using four different processes, namely, fining with bentonite, enzyme (pectinase) treatment, paper filtration, and centrifugation. Patulin was recovered from the clarified juice by liquid-liquid extraction, and solid-phase chromatography was used for sample clean-up prior to analysis by high-performance liquid chromatography (HPLC). The minimum detectable limit using HPLC was 20 microg/liter. Pressing followed by centrifugation resulted in an average toxin reduction of 89%. Total toxin reduction using filtration, enzyme treatment, and fining were 70, 73, and 77%, respectively. Patulin reduction was due to the binding of the toxin to solid substrates that was verified by analyzing the clarified juice as well as the filter cake, pellet, and sediment. The combined concentrations correlated to the spiked concentration. These results reveal that clarification was successful in the reduction of patulin levels in apple juice. However, clarification resulted in high levels of patulin in the pressed pulp after filtration and centrifugation, and this could be harmful if they are used as animal feeds.
Subject(s)
Food Contamination/prevention & control , Food Handling/methods , Mutagens/analysis , Patulin/analysis , Rosales/chemistry , Beverages , Centrifugation , Chromatography, High Pressure Liquid , FiltrationABSTRACT
Haemophilus ducreyi is the causative bacterium of genital ulcers, which are collectively known as chancroid. Little is known about the cytotoxicity of H. ducreyi. The virulent strains are relatively resistant to phagocytosis and apoptosis by neutrophils. Therefore, experiments were designed to examine whether neutrophil degranulation caused by H. ducrey would provide insights into the virulence mechanisms through which cellular damage is affected by the organism. Clinical isolates of eight strains of H. ducreyi and the culture strain type CIP542 (Collection Institute Pasteur) were incubated with neutrophils harvested from human donor blood. The release by the organism of lysosomal enzymes from intracellular granules of neutrophils was indicative of degranulation. The results showed that H. ducreyi triggered the release of lysosomal enzymes from human neutrophils, and that the magnitude of the release was dependent both on the ratio of bacteria to neutrophils and the duration of incubation. In vitro experiments involving HeLa cells were designed to determine the manner in which H. ducreyi initiated the process of degranulation. The morphological changes associated with degranulation were visualized by confocal and transmission electron microscopy. This is the first report that describes degranulation of neutrophils induced by H. ducreyi which causes chancroid infection.
Subject(s)
Cytoplasmic Granules/pathology , Haemophilus ducreyi/pathogenicity , Neutrophils/microbiology , Cathepsin D/analysis , Cathepsin D/metabolism , Coculture Techniques , Cytoplasmic Granules/enzymology , Cytoplasmic Granules/metabolism , HeLa Cells , Humans , Microscopy, Confocal , Microscopy, Electron , Muramidase/analysis , Muramidase/metabolism , Necrosis , Neutrophils/pathology , Neutrophils/ultrastructure , Peroxidase/analysis , Peroxidase/metabolismABSTRACT
Seven different agar-based media were compared to determine the optimal set of culture media for primary isolation of Haemophilus ducreyi. Also, a new method for sampling genital ulcers -- with a disposable sterile plastic loop -- and processing specimens that provides a standardised inoculum for culture of H. ducreyi on various media is described. A total of 202 patients with genital ulcer disease was enrolled in this study. A sterile swab or plastic loop was used to sample the base of the ulcers and ulcer material was suspended in sterile phosphate-buffered saline. A 100-microl sample of this suspension was mixed with an equal volume of tryptic soy broth containing IsoVitaleX and centrifuged for 1 min. This suspension was used to inoculate the different media. Plates were incubated at 33 degrees C in micro-aerophilic conditions and examined for growth of H. ducreyi after 48 h. Of the 202 specimens, 77 (38.1%) were culture positive for H. ducreyi. None of the agar bases supported the growth of all H. ducreyi strains. Based on this observation, we recommend the universal use of Mueller-Hinton agar base supplemented with chocolate horse blood and IsovitaleX (MH-HBC) and Columbia agar base supplemented with bovine haemoglobin, activated charcoal, fetal calf serum and IsovitaleX (C-HgCh) to enable comparison of prevalence figures for chancroid. In addition, the novel sampling technique described in this study eliminates sampling bias normally associated with genital ulcer specimens.