Cryptosporidium cell culture infectivity assay design.

Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

Flow cytometric assessment of distinct physiological stages within Cryptosporidium parvum sporozoites post-excystation.

Cryptosporidium parvum are protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring within Cryptosporidium sporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37 degrees C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.

Optical homodyne PSK demonstration of 1.5 photons per bit at 156 Mbps with rate-(1/2) turbo coding.

We report the first demonstration of a near quantum-limited optical homodyne PSK receiver combined with powerful forward-error-correction coding, achieving 1.5 photons/bit sensitivity, within 4.5 dB of the Shannon limit. Phase-locking was achieved at 1.55 microm using an analog dither-based optical phase-locked loop with an external phase modulator. Analysis for this configuration with arbitrary loop damping is given showing a performance advantage for the overdamped case.

Antimalarial properties of n-3 and n-6 polyunsaturated fatty acids: in vitro effects on Plasmodium falciparum and in vivo effects on P. berghei.

The polyunsaturated fatty acids docosahexaenoic acid (C22:6,n-3), eicosapentaenoic acid, arachidonic acid, and linoleic acid caused marked in vitro growth inhibition of Plasmodium falciparum, assessed by a radiometric assay. In contrast, negligible parasite killing was seen with oleic acid or docosanoic acid. Parasite killing was significantly increased when oxidized forms of polyunsaturated fatty acids were used. Antioxidants greatly reduced the fatty acid-induced killing. Mice infected with P. berghei and treated for 4 d with C22:6,n-3 showed marked reduction in parasitemia. The anemia associated with the infection was also alleviated by treatment with C22:6,n-3. The data provide new information that could be explored in order to develop new strategies in malaria treatment.

Neutrophil migration inhibitory properties of polyunsaturated fatty acids. The role of fatty acid structure, metabolism, and possible second messenger systems.

The n-3 polyunsaturated fatty acids (PUFA) appear to have antiinflammatory properties that can be partly explained by their biological activity on leukocytes. Since leukocyte emigration is an essential component of the inflammatory response, we have examined the effects of the n-3 PUFA (eicosapentaenoic and docosahexaenoic acids) on neutrophil random and chemotactic movement. Preexposure of neutrophils for 15-30 min to 1-10 micrograms/ml PUFA reduced the random and chemotactic migration to both FMLP- and fungi-activated complement. The inhibitory effect diminished with increasing saturation and carbon chain length, and methylation abolished this activity. Arachidonic and docosahexaenoic acids were the most active fatty acids. The PUFA concentration required to inhibit migration was dependent on cell number, suggesting that the fatty acid effects on leukocyte migration in vivo may be governed by the stage of the inflammatory response. It was concluded that the PUFA rather than their metabolites were responsible for the inhibition since: (a) antioxidants did not prevent the PUFA-induced migration inhibition and the hydroxylated intermediates were less active, and (b) inhibitors of the cyclooxygenase and lipoxygenase pathways were without effect. Inhibitors of protein kinases and calmodulin-dependent enzyme system did not prevent the PUFA-induced migration inhibition, which was also independent of phospholipase D-catalyzed hydrolysis of phospholipids. It is also shown that PUFA decrease the FMLP-induced Ca2+ mobilization.

Altered responses of human macrophages to lipopolysaccharide by hydroperoxy eicosatetraenoic acid, hydroxy eicosatetraenoic acid, and arachidonic acid. Inhibition of tumor necrosis factor production.

The regulation of allergic and autoimmune inflammatory reactions by polyunsaturated fatty acids and their metabolic products (eicosanoids) continues to be of major interest. Our data demonstrate that arachidonic acid 5,8,11,14-eicosatetraenoic acid (20:4n-6) and its hydroxylated derivatives 15(s)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) and 15(s)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) regulate agonist-induced tumor necrosis factor alpha (TNF) production, a cytokine that plays a role in inflammatory diseases. Although 20:4n-6 and 15-HETE caused a reduction in production of TNF in mononuclear leukocytes stimulated with phytohaemagglutinin, pokeweed mitogen, concanavalin A, and Staphylococcus aureus, 15-HPETE was far more active. 15-HPETE was also found to dramatically depress the ability of bacterial lipopolysaccharide to induce TNF production in monocytes and the monocytic cell line Mono Mac 6. These fatty acids depressed the expression of TNF mRNA in Mono Mac 6 cells stimulated with LPS; 15-HPETE was fivefold more active than 20:4n-6 and 15-HETE. While 15-HPETE treatment neither affected LPS binding to Mono Mac 6 cells nor caused a decrease in CD14 expression, the fatty acid significantly reduced the LPS-induced translocation of PKC (translocation of alpha, betaI, betaII, and epsilon isozymes), suggesting that 15-HPETE acts by abrogating the early signal transduction events. The findings identify another molecule that could form the basis for development of antiinflammatory pharmaceuticals.

Surface-specific effects of a PPARgamma agonist, darglitazone, on bone in mice.

It has been hypothesized that activation of peroxisome-proliferator-activated receptor-gamma (PPARgamma) by thiazolidinedione drugs can increase adipogenesis at the expense of osteogenesis, leading to bone loss. However, the reported skeletal effects of these compounds are varied and their effects on cortical bone are unknown. In this study, we examined the changes in both cancellous and cortical bone of 6-month-old male mice treated with darglitazone, a potent and selective PPARgamma agonist, at 10 mg/kg/day by dosing the compound in a food mixture for 2 or 8 weeks. At 2 weeks, we observed significantly increased marrow adipose tissue area, decreased trabecular bone density of distal femur, and decreased surface referent bone formation rate of lumbar vertebrae in the mice treated with darglitazone compared with controls. At 8 weeks, lower cancellous bone mass was seen at both distal femurs and lumbar vertebrae of the mice treated with darglitazone. In addition, mineralizing surface was significantly lower, whereas osteoclast surface and number were significantly higher in the lumbar vertebrae of darglitazone-treated mice. At the femoral diaphysis, darglitazone treatment caused bone loss on the endocortical surface. Interestingly, periosteal mineral apposition rate and surface referent bone formation rate were significantly increased in darglitazone-treated mice. In bone marrow cell cultures, darglitazone suppressed alkaline phosphatase activity, osteoblastic gene expression, and mineralized nodule formation while increasing adipogenic gene expression and lipid accumulation. In summary, darglitazone enhanced adipogenesis and caused cancellous bone loss by increasing bone resorption and decreasing bone formation in mice. In addition, darglitazone induced cortical bone loss on the endocortical surface but increased bone formation on the periosteal surface. These data suggest that PPARgamma plays a role in regulating bone resorption and formation and reveal surface-specific effects of a PPARgamma agonist on bone.

Differences in the metabolism of eicosatetraenoic (20:4(n - 6)), tetracosatetraenoic (24:4(n - 6)) and triacontatetraenoic (30:4(n - 6)) acids in human neutrophils.

The metabolism of [1-14C]eicosatetraenoic (arachidonic, 20:4(n - 6)), [1-14C]tetracosatetraenoic (24:4(n - 6)) and [1-14C]triacontatetraenoic (30:4(n - 6)) acids was studied in intact human neutrophils. [1-14C]20:4(n - 6) and [1-14C]24:4(n - 6) were efficiently taken up by the neutrophils, esterified into neutral lipids and phospholipids, and elongated by up to four carbon units. In contrast, [1-14C]30:4(n - 6) was poorly incorporated into the cells and remained predominantly in the original unesterified form. The [1-14C]tetraenoic fatty acids were mainly esterified into triacylglycerol, suggesting that this lipid class is important in the intracellular trafficking of polyunsaturated fatty acids. The leukocytes demonstrated a low capacity to beta-oxidize and desaturate the fatty acid substrates. In the presence of calcium ionophore A23187 the neutrophils converted [1-14C]20:4(n - 6) to a variety of radiolabelled oxygenated fatty acid derivatives including prostaglandins, thromboxanes, mono- and dihydroxylated fatty acids and leukotrienes. The major eicosanoid products were 5-monohydroxy-20:4(n - 6) and leukotriene B4. In contrast, [1-14C]24:4(n - 6) was metabolized to radiolabelled monohydroxylated fatty acids (predominantly the 9-hydroxy positional isomer) but not to other lipoxygenase or cyclooxygenase products by the calcium ionophore-stimulated cells. Negligible oxygenated fatty acid compounds were formed from [1-14C]30:4(n - 6), indicating that it is a poor substrate for the neutrophil cyclooxygenase and lipoxygenase enzymes.

Effect of fatty acid structure on neutrophil adhesion, degranulation and damage to endothelial cells.

Neutrophils have been implicated in ischaemic heart disease, unstable angina pectoris and acute myocardial infarction. Alterations in dietary levels of specific 18- and 20-carbon polyunsaturated fatty acids have significant clinical benefits in cardiovascular disease. However, to date there has been no concerted effort to identify the structural basis for polyunsaturated fatty acid-induced alterations in key neutrophil functions. We have investigated the influence of fatty acid structure and involvement of lipoxygenase/cyclooxygenase pathways on fatty acid-induced neutrophil functions. When neutrophils were incubated with 18-carbon fatty acids containing one to four double bonds (10-33 mumol/l), a significant increase in adherence and release of specific granule constituents occurred compared with control cells. In general, as the number of double bonds in the 18-carbon fatty acid increased, so did its ability to stimulate these functions. There was less stimulation of adherence and specific granule release by 18:3(n-3) than its isomer 18:3(n-6). Smaller effects were seen on azurophilic granule release. A further increase in adherence and degranulation was observed with increasing carbon chain length (20:3(n-6) and 20:4(n-6)). Differences were found in the ability of isomers of 20:3 to stimulate neutrophil function. Of the fatty acids tested only 20:4(n-6) was able to induce significant neutrophil-mediated endothelial detachment. Introduction of either internal hydroperoxy or hydroxyl groups into 20:4(n-6) abolished its adherence stimulating activity and considerably reduced its ability to stimulate release of both specific and azurophilic granules. Preincubation of neutrophils with either lipoxygenase (caffeic acid) or cyclooxygenase (indomethacin) inhibitors had no effect on 20:4(n-6) stimulated function. These studies show that the number and position of double bonds, carbon chain length and oxidation state can be critical to the neutrophil stimulatory properties of these fatty acids.

Inhibitory effects of arachidonic acid (20:4,n-6) and its monohydroperoxy- and hydroxy-metabolites on procoagulant activity in endothelial cells.

The procoagulant response of endothelium to pathophysiological agents such as tumour necrosis factor alpha (TNF alpha) and phorbol myristate acetate (PMA) alters the expression of proteins such as tissue factor. The modulation of such procoagulant activity (PCA) by the polyunsaturated fatty acid arachidonic acid (20:4,n-6) and its 15-hydroperoxy (15-HPETE) and 15-hydroxy (15-HETE) metabolites was examined since this may have important implications in cardiovascular disease and atherosclerosis. Treatment of human umbilical vein endothelial cells (HUVEC) for 30 min with 20:4, 15-HPETE or 15-HETE before induction of PCA with TNF alpha (100 U) or PMA (10(-7) M) caused a significant inhibition of PCA. This inhibition was seen at 2-5 microM fatty acids. Dose response curves with TNF alpha indicated that the inhibition was greatest at higher concentrations of TNF alpha (> or = 250U TNF alpha/ml). The mode of administration of the fatty acid was not critical as fatty acids presented as DPC-fatty acid micelles or solubilised in ethanol gave similar inhibitions of PCA. 20:4, 15-HPETE or 15-HETE did not alter the binding of I125-labelled TNF alpha to its surface receptors on HUVEC, suggesting that the effect of these fatty acids was not mediated by events at the cell surface receptor level. In support of this, these fatty acids were found to inhibit PCA induced by PMA which bypasses cell surface receptors to activate protein kinase C directly.(ABSTRACT TRUNCATED AT 250 WORDS)

The amoeba-to-flagellate transformation test is not reliable for the diagnosis of the genus Naegleria. Description of three new Naegleria spp.

Trophozoites of several isolates from one location in Australia have failed consistently to transform into flagellates, although they display all other characteristics of the genus Naegleria. When changing the standard transformation test, flagellates were produced. In phylogenetic trees derived from partial small subunit ribosomal DNA (SSUrDNA) sequences, one of these strains branches close to a cluster comprising N. clarki, N. australiensis, N. italica and N. jadini. It is proposed that these Australian isolates represent a new species, named N. fultoni (strain NG885). Failing to form flagellates since their isolation, even when different transformation procedures are used, are two Naegleria strains from Chile and Indonesia. In SSUrDNA-based phylogenetic trees the Chilean strain clusters with N. pussardi and the Indonesian strain clusters with N. galeacystis, but the degree of sequence difference from these described species (3.5% and 2.2%, respectively) is sufficient to propose that both of the strains represent new species, named N. chilensis (strain NG946) and N. indonesiensis (strain NG945), respectively. The close relationships between each of the new species and the Naegleria species with which they cluster in SSUrDNA-based trees were confirmed by ribosomal internal transcribed spacer region (ITS) sequence comparisons. In France, several non-flagellating N. fowleri strains were isolated from one location. ITS rDNA sequence comparisons indicated that they correspond to a 'type' of N. fowleri found in both Europe and the USA. A redefinition of the genus Naegleria is proposed as a consequence of these and previous findings.

The epidemiology and control of primary amoebic meningoencephalitis with particular reference to South Australia.

This paper reviews the history, epidemiology and control of primary amoebic meningoencephalitis (PAM), caused by Naegleria fowleri, with particular reference to South Australia. The intention has been to outline misconceptions and uncertainties pervading the earlier literature. Although PAM infections elsewhere have been attributed to cysts in air-borne dust, we believe that contact with water in the domestic environment was not adequately considered as a potential source of these infections. Several reports have cast doubt on the effectiveness of chlorine in controlling N. fowleri, although there is laboratory and South Australian field experience to the contrary. These reports can be traced to a misunderstanding of the circumstances surrounding cases of PAM reported by other workers. Provided that a continuous free chlorine residual of 0.5 mg/l can be maintained in water accessible to N. fowleri, the risk of disease should be negligible. The failure of amphotericin B therapy to save recent victims of the disease, despite relatively prompt intervention, is disappointing. Possible reasons for this, and the reports that rifampin or tetracycline combined with amphotericin might be more successful, are discussed.

Echocardiographic visualization of acute myocardial ischemia--in vitro study.

To evaluate whether myocardial texture changes resulting from acute ischemia can be visualized with satisfactory spatial resolution, short axis compound echo images (CEI) (B-scan) were obtained from 12 excised canine hearts in vitro. Seven had myocardial ischemia produced by open chest ligation of the left anterior descending coronary artery (LAD) for 15-30 min prior to excision. The CEI were constructed by compounding 60 simple linear B-scans. Hearts were sectioned after scanning, and gross morphological changes were recorded. Microscopic comparison between grossly abnormal and normal regions were recorded. The CEI from the ischemic group revealed altered myocardial texture seen as bright coarsely granular echoes in the regions normally perfused by the ligated LAD artery. Corresponding anatomic sections revealed increased redness in these regions. Microscopically these regions revealed interstitial and intercellular edema as compared to the normal regions. Acute myocardial ischemia can be visualized in CEI and these regions have significantly increased backscatter, decreased attenuation, and decreased speed of ultrasound relative to normal regions in the same hearts. Myocardial edema is probably responsible for these changes.

Monoenoic fatty acids in human brain lipids: isomer identification and distribution.

The carbon chain length distribution and the double bond positional isomer composition of the monoenoic fatty acids of the lipids of total human brain tissue have been determined using gas chromatography and gas chromatography/mass spectrometry of the fatty acid methyl and picolinyl esters. The even chain length monoenoic C16 to C28 fatty acids contain predominantly two positional isomer series, the n-7 and n-9 cis homologues, whose relative proportion varies significantly with chain length. The odd chain length long-chain fatty acids consist of n-8 and n-10 isomers, whereas the odd chain length very long-chain (more than 22 carbon) fatty acids are n-7 and n-9 isomers.

A temporary flagellate (mastigote) stage in the vahlkampfiid amoeba Willaertia magna and its possible evolutionary significance.

A temporary flagellate (mastigote) stage has been observed in several isolates of the vahlkampfiid amoeba Willaertia magna. In an Australian isolate studied in detail, flagellates appeared synchronously, although later than in Naegleria fowleri or N. lovaniensis under similar conditions (half-maximal time, t50 = 168 min at 37 degrees C). The flagellates initially have four flagella and lack a cytostome, but undergo several successive divisions, the first of them synchronous, resulting in progressive reduction in cell volume. New flagella appear during and after division, and the number of flagella in daughter cells of later divisions is rather variable. Comparison of these observations with descriptions of other amoeboflagellates confirms that Willaertia is a valid genus. A likely sequence of morphological changes in the evolution of Willaertia and Naegleria from a hypothetical ancestral vahlkampfiid is proposed.

Very long-chain fatty acids in peroxisomal disease.

Fatty acids with from 24 to 28 carbon atoms (very long-chain fatty acids, VLCFA) are present in small amounts in all mammalian tissues. Even longer chain fatty acids with from 30 to 38 carbon atoms (ultra-long-chain fatty acids, ULCFA) are found in certain specialized tissues including retina, brain, and spermatozoa. In patients with inherited defects in peroxisomal structure and/or function, there is an accumulation of VLCFA in most tissues, while VLCFA and ULCFA levels are increased in brain. The most pronounced changes occur in those patients who have defects in peroxisomal assembly (Zellweger syndrome, infantile Refsum's disease, and neonatal adrenoleukodystrophy). In the brain of these individuals, ULCFA are distributed largely in molecular species of phosphatidylcholine with penta-, hexa-, and heptaenoic acids. In contrast, patients with X-linked adrenoleukodystrophy have increased levels of phosphatidylcholine with monoenoic rather than polyenoic ULCFA. A defect in a peroxisomal VLCFA CoA synthetase or ligase has been reported for these patients, but assembly of their peroxisomes is apparently normal. We speculate that ULCFA are normal products of carbon chain elongation. We have confirmed this by demonstrating the elongation of [1-14C]hexacosatetraenoic acid (26:4n-6) by rat brain in vivo to a series of longer chain tetraenoic acids with carbon chain lengths up to 34. Elongation to ULCFA can occur as well in non-neural tissues as shown by detection of labeled saturated and monoenoic fatty acids with up to 32 carbon atoms after incubation of normal and Zellweger syndrome fibroblasts with [2-14C] acetate. Increased labeling of VLCFA and ULCFA is observed in cells from patients with peroxisomal disorders. Our data suggest that ULCFA with up to at least 32 carbon atoms are formed normally, as a part of the elongation process in most mammalian tissues, and that control of carbon chain elongation is a major function of peroxisomes. Impairment of this function as occurs in peroxisomal disease results in the accumulation of VLCFA and ULCFA. The relative enrichment in normal tissues of ULCFA such as 32:6n-3 in ram and bull spermatozoa and 36:4n-6 in human and rat brain suggests a probable physiological role for this class of fatty acids in these tissues.

The scattering of ultrasound by cylinders: implications for diffraction tomography.

The validity of wave equations employed as system models in acoustical diffraction tomography is investigated using simulations and measurements of the scattering of plane ultrasound waves by cylinders. It is demonstrated by simulation and experiment that it can be appropriate to neglect density fluctuations and shear waves, implying that the commonly used form of the wave equation suitably describes scattering by fluctuations of acoustic speed and absorption. Diffraction tomographic reconstructions of simulated data reveal the importance of absorption, the behavior of the real and imaginary parts of the reconstructed refractive index, and the relative advantages and limitations of the Born and Rytov approximate transformations.

Epitope specificities of human serum antibodies reactive with respiratory syncytial virus fusion protein.

Respiratory syncytial (RS) virus continues to cause serious human respiratory disease and no prophylactic vaccine is yet available. Serum antibodies to RS virus fusion protein (F) that have the appropriate specificities and activities could confer protection against severe RS virus infections. To explore human serum antibody responses to RS virus F we first characterised four epitopes on F and then measured the concentrations of human serum antibodies to these sites for 389 sera. Individuals varied in serum antibody concentration to the epitopes. The distribution patterns of the concentrations of antibodies reactive to each epitope were different. Antigenic variation of F at these epitopes in Southampton RS virus isolates was examined by immunofluorescence. The F proteins from different isolates varied within and between RS virus subtypes which co-circulated in the outbreak of winter 1985-1986. Variations in F detected by immunofluorescence were consistent with differences between the strains' susceptibilities to monoclonal antibody antiviral action.