Comparison of five methods of malaria detection in the outpatient setting.
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In eastern Africa where 90% of the malaria is due to Plasmodium falciparum, the accuracy of malaria diagnosis at the outpatient level is becoming increasingly important due to problems of drug resistance and use of alternative, costly antimalarial drugs. The quantitative buffy coat (QBC) technique, acridine orange staining with an interference filter system, and the ParaSight-F test have been introduced as alternative methods to conventional microscopy for the diagnosis of malaria. Two hundred thirteen outpatients were tested using these alternative methods and conventional microscopy by five experienced technologists; two were randomly allocated to read the results of each test. Paired results showed the highest level of agreement with the ParaSight-F test (99%), followed by Field stain (92%). The results of the QBC technique showed the least agreement (73%). Using conventional microscopy as the reference standard, the ParaSight-F test had a sensitivity range of 90-92% and a specificity of 99%, staining with acridine orange had a sensitivity range of 77-96% and a specificity range of 81-98% and the QBC technique had a sensitivity range of 88-98% and a specificity range of 58-90%. All microscopic tests showed lower sensitivities (as low as 20% using staining with acridine orange) in detecting low parasitemias (< or = 320/microl) than the ParaSight-F test (70%). Due to the high cost of the ParaSight-F test, Field-stained blood films remain the most appropriate method for diagnosis of P. falciparum in eastern Africa. The ParaSight-F test may be used in situations where no trained microscopists are available, or where malaria is strongly suspected and the results of microscopy are negative. (+info)
Destruction of protamine in human sperm inhibits sperm binding and penetration in the zona-free hamster penetration test but increases sperm head decondensation and male pronuclear formation in the hamster-ICSI assay.
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PURPOSE: Our purpose was to investigate the fertilizing ability of human protamine-damaged sperm in a heterologous system using hamster oocytes. METHODS: The protamine of the sperm were damaged by exposure to dithiothreitol, a disulfide-reducing agent. Their ability to penetrate and form male pronuclei were investigated using the zona-free hamster penetration test and the hamster-intracytoplasmic sperm injection assay, respectively. RESULTS: The zona-free hamster penetration test revealed that protamine-damaged sperm are unable to bind and penetrate the hamster oocyte. On the other hand, hamster-intracytoplasmic sperm injection assay results showed that 56.9% and 39.2% of the injected oocytes developed male pronuclei in protamine-damaged and live-intact sperm groups, respectively, with a significant difference in these rates (P < 0.01). CONCLUSIONS: This study shows that protamine-damaged sperm are able to undergo sperm head decondensation and male pronuclear formation only when injected into the ooplasm, although they cannot bind and penetrate through the zona and enter the ooplasm. (+info)
Reactive oxygen species-induced apoptosis and necrosis in bovine corneal endothelial cells.
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PURPOSE: The loss of corneal endothelial cells associated with aging and possibly other causes has been speculated to be related to exposure to reactive oxygen species (ROS). The current study was conducted to investigate, by use of photosensitizers, the underlying mechanisms involved in the death of bovine corneal endothelial cells (BCENs) caused by ROS. METHODS: BCEN cells in primary culture were treated with a photosensitizer (riboflavin or rose bengal) with light exposure. The patterns of cell damage and death were assessed using an acridine orange-ethidium bromide differential staining method, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and transmission electron microscopy. The cytotoxicity was assayed by mitochondrial function using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) testing. Antioxidants, including catalase, L-histidine, salicylic acid, and superoxide dismutase, were used to determine the types of ROS involved. Activation of nuclear factor (NF)-kappaB was examined by fluorescent immunocytochemistry with anti-p65 antibody. RESULTS: Light-irradiated riboflavin or rose bengal resulted in a significant decrease in viability of BCEN cells. Chromosomal condensation and fragmentation were observed in apoptotic cells, and membrane lysis and damage of cell ultrastructures were observed in necrotic cells. Riboflavin induced apoptosis at 30 minutes and thereafter and induced necrosis after 2 hours. Rose bengal was shown to cause similar effects within half the time required for the effects of riboflavin. Catalase and salicylic acid were found to provide protection for BCENs from cytotoxic effects of riboflavin, and L-histidine was found to protect BCENs from cytotoxicity induced by rose bengal. Kinetic studies using immunocytochemistry showed that NF-kappaB was translocated into the nucleus within 15 minutes and 30 minutes after treatment with rose bengal and riboflavin, respectively. CONCLUSIONS: The cytotoxic effects of photo-irradiated riboflavin and rose bengal are shown to be mediated by two distinct but parallel pathways, one leading to apoptosis and the other to necrosis. Possible involvement of NF-kappaB in cell death is suggested. These findings provide potential leads for future investigation into the molecular mechanisms of loss of corneal endothelial cells related to aging, oxidative stress, and possibly other similar causes. (+info)
Differential chemosensitivity of breast cancer cells to ganciclovir treatment following adenovirus-mediated herpes simplex virus thymidine kinase gene transfer.
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The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirus-mediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment. Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that express mutant p53 and that may have developed resistance to conventional genotoxic agents. (+info)
Evanescent-wave microscopy: a new tool to gain insight into the control of transmitter release.
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Evanescent-wave excitation was used to visualize individual fluorescently labelled vesicles in an optical slice near the plasma membrane of bovine adrenal chromaffin cells. A standard upright microscope was modified to accommodate the optics used for directing a laser beam under a supracritical angle on to the glass-water interface on top of which the cells are grown. Whereas epi-illumination images appeared blurred and structureless, evanescent-wave excitation highlighted acridine orange-labelled vesicles as individual pinpoints. Three-dimensional (3D) trajectories of individual vesicles were obtained from time-resolved image stacks and used to characterize vesicles in terms of their average fluorescence F and mobility, expressed here as the 3D diffusion coefficient D(3). Based on the single-vesicle analysis, two groups of vesicles were identified. Transitions between these states were studied before and after stimulation of exocytosis by repetitive or maintained membrane depolarizations by elevated extracellular [K+]. Findings were interpreted as sequential transitions between the previously characterized pools of vesicles preceding the fusion step. The observed approach of vesicles to their docking sites was not explained in terms of free diffusion: most vesicles moved unidirectionally as if directed to their binding sites at the plasma membrane. Vesicle mobility at the membrane was low, such that the sites of docking and fusion were in close vicinity. Both the rim region and confined areas in the centre of the footprint region were the site of intense vesicle trafficking. (+info)
Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes.
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In insulin-sensitive fat and muscle cells, the major glucose transporter GLUT4 is constitutively sequestered in endosomal tubulovesicular membranes, and moves to the cell surface in response to insulin. While sequence information within GLUT4 appears to be responsible for its constitutive intracellular sequestration, the regulatory elements and mechanisms that enable this protein to achieve its unique sorting pattern under basal and insulin-stimulated conditions are poorly understood. We show here that arrest of endosome acidification in insulin-sensitive 3T3-L1 adipocytes by bafilomycin A1, a specific inhibitor of the vacuolar proton pump, results in the rapid and dose-dependent translocation of GLUT4 from the cell interior to the membrane surface; the effects of maximally stimulatory concentrations of bafilomycin A1 (400-800 nM) were equivalent to 50-65% of the effects of acute insulin treatment. Like insulin, bafilomycin A1 induced the redistribution of GLUT1 and Rab4, but not that of other proteins whose membrane localization has been shown to be insulin-insensitive. Studies to address the mechanism of this effect demonstrated that neither autophosphorylation nor internalization of the insulin receptor was altered by bafilomycin A1 treatment. Bafilomycin-induced GLUT4 translocation was not blocked by cell pretreatment with wortmannin. Taken together, these data indicate that arrest of endosome acidification mimics insulin action on GLUT4 and GLUT1 translocation by a mechanism distal to insulin receptor and phosphatidylinositol 3-kinase activation, and suggest an important role for endosomal pH in the membrane dynamics of the glucose transporters. (+info)
Radiation-induced leukocyte entrapment in the rat retinal microcirculation.
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PURPOSE: To evaluate the effects of irradiation on leukocyte dynamics in the rat retinal microcirculation. METHODS: Thirty-five Brown-Norway rats received a dose of 10 Gy irradiation in one fraction. Leukocyte dynamics were studied with acridine orange digital fluorography, in which a nuclear fluorescent dye of acridine orange is used and examined by scanning laser ophthalmoscope. This technique allowed visualization of fluorescent leukocytes in vivo. The leukocyte dynamics were evaluated at 0, 4, 7, 14, 30, and 60 days after the irradiation. RESULTS: Mean leukocyte velocity in the retinal capillaries decreased immediately. It partially recovered on day 4 but then gradually decreased up to the 2-month mark. Low-dose irradiation induced entrapment of leukocytes in the retinal microcirculation. The number of entrapped leukocytes gradually increased with time. The major retinal vessels significantly constricted immediately after irradiation. The diameter was reduced by 76% in arteries and 75% in veins, 2 months after irradiation. CONCLUSIONS: Entrapped leukocytes may be activated and exacerbate vascular injury and microinfarction and thus may participate in the pathogenesis of radiation retinopathy. (+info)
Tick-borne relapsing fever imported from West Africa: diagnosis by quantitative buffy coat analysis and in vitro culture of Borrelia crocidurae.
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West African tick-borne relapsing fever (TBRF) is difficult to diagnose due to the low number of spirochetes in the bloodstream of patients. Previously, the causative microorganism, Borrelia crocidurae, had never been cultured in vitro. TBRF was rapidly diagnosed for two patients returning from western Africa with fever of unknown origin by quantitative buffy coat (QBC) analysis. Diagnosis was confirmed by intraperitoneal inoculation of blood specimens from patients into laboratory mice. In vitro experiments showed that QBC analysis may be as much as 100-fold more sensitive than thick smear. Spirochetes were also cultured from blood samples from both patients in modified Kelly's medium and were identified as B. crocidurae by partial sequencing of the PCR-amplified rrs gene. (+info)