Occurrence of Giardia cysts and Cryptosporidium oocysts in activated sludge samples in Campinas, SP, Brazil

Giardia and Cryptosporidium have caused several outbreaks of gastroenteritis in humans associated with drinking water. Contaminated sewage effluents are recognized as a potential source of waterborne protozoa. Due to the lack of studies about the occurrence of these parasites in sewage samples in Brazil, we compared the efficiency of two procedures for concentrating cysts and oocysts in activated sludge samples of one sewage treatment plant. For this, the samples were submitted to i) concentration by the ether clarification procedure (ECP) and to ii) purification by sucrose flotation method (SFM) and aliquots of the pellets were examined by immunofluorescence. Giardia cysts were present in all samples (100.0%; n = 8) when using ECP and kit 1 reagents, while kit 2 resulted in six positive samples (85.7%; n = 7). As for SFM, cysts were detected in 75.0% and 100.0% of these samples (for kit 1 and 2, respectively). Regarding Cryptosporidium, two samples (25.0%; kit 1 and 28.5% for kit 2) were detected positive by using ECP, while for SFM, only one sample (examined by kit 1) was positive (12.5%). The results of the control trial revealed Giardia and Cryptosporidium recovery efficiency rates for ECP of 54.5% and 9.6%, while SFM was 10.5% and 3.2%, respectively. Considering the high concentration detected, a previous evaluation of the activated sludge before its application in agriculture is recommended and with some improvement, ECP would be an appropriate simple technique for protozoa detection in sewage samples.

Fecal specimens preparation methods for PCR diagnosis of human taeniosis

Sample preparation and DNA extraction protocols for DNA amplification by PCR, which can be applied in human fecal samples for taeniasis diagnosis, are described. DNA extracted from fecal specimens with phenol/chloroform/isoamilic alcohol and DNAzol® reagent had to be first purified to generate fragments of 170 pb and 600 pb by HDP2-PCR. This purification step was not necessary with the use of QIAmp DNA stool mini kit®. Best DNA extraction results were achieved after eggs disruption with glass beads, either with phenol/chloroform/isoamilic alcohol, DNAzol® reagent or QIAmp DNA stool mini kit®.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

SUMMARYThe use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

CORAL SNAKE ANTIVENOM PRODUCED IN CHICKENS (Gallus domesticus)

The production of anti-snake venom from large mammal's blood has been found to be low-yielding and arduous, consequently, antivenom immunoglobulins for treatment are achieved regularly as polyvalent serum. We have standardized an undemanding technique for making purified immunoglobulin IgY antivenom consisting of polyclonal antibodies against coral snake venom in the egg yolk of immunized hens. We have adapted a reported process of antibody purification from egg yolks, and achieved 90% antibody purity. The customized technique consisted of the removal of lipids from distilled water-diluted egg yolks by a freeze–thaw sequence. The specific immunoglobulins were present in the egg yolk for up to 180 days postimmunization. Therefore, by means of small venom quantities, a significant amount of immunoglobulins were found in an adequately purified state (The obtained material contained about 90% pure IgY). The antigen binding of the immunoglobulins was detected by a double immunodiffusion test. Titers of antibodies in the yolk were estimated with a serum protection assay (Median effective dose = ED50) (ED50= 477 mg/kg). Given that breeding hens is economically feasible, egg gathering is noninvasive and the purification of IgY antibodies is quick and easy, chicken immunization is an excellent alternative for the production of polyclonal antibodies. To the best of our knowledge, this is the first coral snake antivenom prepared in birds.

Microsatellite instability in solitary and sporadic gastric cancer

Recently, the presence of microsatellite instability (MSI) has been reported in gastric cancer and associated with older age of presentation, distal tumor location, early disease staging, and better overall prognosis. Different characteristics in presentation and in tumor behavior may be explained by different genetic alterations during carcinogenesis of gastric cancer. Identification of specific genetic pathways in gastric cancer may have direct impact on prognosis and selection of treatment strategies. PATIENTS AND METHODS: All 24 patients were treated by radical surgery. Fragments of normal and tumor tissues were extracted from the specimen and stored at -80ºC before DNA purification and extraction. PCR amplification utilizing microsatellite markers was performed. Tumors presenting PCR products of abnormal sizes were considered positive for microsatellite instability (MSI+). RESULTS: Five patients (21%) had tumors that were MSI+ in at least 1 marker. In the group of patients with Lauren's intestinal-type gastric carcinoma, 3 had tumors that were MSI+ (23%), while in the group of diffuse-type gastric cancer, 2 patients had tumors that were MSI+ (19%). The mean age of presentation and the male:female ratio was similar in both groups. Tumors that were MSI+ were more frequently located in proximal portion of the stomach compared to microsatellite-stable (MSS) tumors (40% vs. 16%). Although there was a trend of patients with MSI+ tumors towards a proximal gastric tumor location, early staging, and negative lymph node metastasis, there was no statistical significance compared to those with MSS tumors (P >.1). Comparison of overall and disease-free survival between gastric tumors that were MSI+ and those that were MSS found no statistically significant differences (P >.1). CONCLUSIONS: Microsatellite instability is a frequent event in gastric carcinogenesis and shows a trend towards distinct clinical and pathological characteristics of gastric cancer.

Gram-scale isolation of isobrucein B and neosergeolide from Picrolemma sprucei Hook. f.

Quassinoids neosergeolide and isobrucein B, obtained from Picrolemma sprucei, have proven in vitro antitumor, antimalarial, anthelminthic, cytotoxic, insecticide and leishmanicidal activities. There is interest in the in vivo pharmacological study of these natural compounds and their semi-synthetic derivatives, however, the quantities obtained in previous extraction processes have been shown to be a limiting factor for continuation of these studies. Herein, we describe a method for obtaining grams of these quassinoids whose purification relies only on recrystallization.

The use of monoclonal antibodies for the characterization and production of Mycobacterium leprae antigens

Similar immunizations of mice and hybridoma technology were used by several investigators to raise monoclonal antibodies which identified a limited range of epitopes and antigenic molecules. Further studies would have the scope for revealing yet more novel structures. The existing MABs are agreed standard reagents, avaiable to investigators and valuable for several applications. At least six epitopes specific for M. leprae were defined in molecular terms. Monoclonal antibody based immunoassays proved to be invaluable for the screening of recombinant DNA clones and for the topographic study of individual epitopes. Purification of antigens using affinity chromatography requires further development of techniques whilst serology of leprosy is open for clinical and epidemiological evaluation.

Pharmacokinetic aspects of Tert-Butylaminoethyl disulfide, an experimental drug against schistosomiasis in mice

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethyl disulfide was performed after administration of two different single doses (35 and 300 mg/kg) of either the cold or labelled drug. Plasma or blood samples were treated with dithiothreitol, perchloric acid, and, after filtration, submitted to further purification with anionic resein. In the final step, the drug was retained on a cationic resin column, eluted with NaCl 1M and detected according to the method of Ellman (1958). Alternatively, radioactive drug was detected by liquid scintillation counting. The results corresponding to the smaller dose of total drug suggested a pharmacokinetic behavior related to a one open compartment model with the following parameters: area under the intravenous curve (AUC i.v.):671 ± 14; AUC oral: 150 ± 40 µg.min. ml [raised to the power of -1]; elimination rate constant: 0.071 min [raised to the power of -1]; biological half life: 9.8 min; distribution volume: 0.74 ml/g. For the higher dose, the results seemed to obey a more complex undertermined model. Combining the results, the occurence of a dose-dependent pharmacokinetic behavior is suggested, the drug being rapidly absorbed and rapidly eliminated; the elimination process being related mainly to metabolization. The drug seems to be more toxic when administered I.V. because by this route it escapes first pass metabolism, while being quickly distributed to tissues. The maximum tolerated blood level seems to be around 16 µg/ml.

Pharmacokinetic profile of tert-butylaminoethanethiol

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethanethiol (TBAESH) was performed after administration of a single dose (35 mg/kg) either orally or intravenously. Plasma or blood samples were treated with dithiothreitol, perchloric acid and, after filtration, submitted to further purification with anionic resin. In the final step the drug was retained on a cationic resin column, eluted with NaCl lM and detected according to the method of Ellman (1958). The results suggested a pharmacokinetic behavior related to a one open compartment model with the following values for the total drug: area under the intravenous curve (AUC i.v.): 443(+ ou -) 24.0; AUC oral: 85.5(+ ou -) 14.5 ug min.ml(elevado a -1); elimination rate constant: 0.069(+ ou -) 0.0055 min(elevado a -1), biological half-life: 10.0(+ ou -) 0.80 min; distribution volume 1.15(+ ou -) 0.15 ml/g; biodisponibility: 0.19(+ ou -) 0.02. From a pharmacokinetic standpoint, TBAESH seems to have no advantage over the analogous disulfide compound.

Toxoplasma gondii: a rapid method for the isolation of pure tachyzoites: preliminary characterization of its genome

A rapid and simple technique for the purification of Toxoplasma gondii tachyzoites was developed. Highly purified parasites were obtained from the peritoneal exudates of infected mice by means of two consecutive discontinous sucrose gradients run at low speed (10,000xg, 30 min). Parasites obtained by this method conserved its biological activity. Hybridizations tudies with DNA from healthy mice and from purified tachyzoites preparations demonstrated that Toxoplasma gondii tachyzoites DNA could be obtained with better than 90 per cents purity. Preliminary studies with DNA endonucleases showed the presence in the tachyzoites genome of highly repetitives sequences.

Analgesic activity of a triterpene isolated from Scoparia dulcis L. (vassourinha)

Analgesic and anti-inflammatory activities of water (WE) and ethanolic (EE) extracts of Scoparia dulcis L. were investigated in rats and mice, and compared to the effects induced by Glutinol, a triterpene isolated by purification of EE. Oral adminsitration (p.o.) of either WE or EE (up to 2 g/Kg) did not alter the normal spontaneous activity of mice and rats. The sleeping time induced by sodium pentobarbital (50 mg/Kg, i.p.) was prolonged by 2 fold in mice pretreated with 0.5 g/Kg EE, p.o. Neither extract altered the tail flick response of mice in immersion test, but previous administration of EE (0.5 g/Kg, p.o.) reduced writhings induced by 0.8% acetic acid (0.1 ml/10 g, i.p.) in mice by 47% EE (0.5 and 1 g/Kg, p.o.) inhibited the paw edema induced by carrageenan in rats by respectively 46% and 58% after 2 h, being ineffective on the paw edema induced by dextran. No significant analgesic or anti-edema effects were detected in animals pretreated with WE (1 g/Kg, p.o.). Administration of Glutinol (30 mg/Kg, p.o.) reduced writhing induced by acetic acid in mice by 40% and the carrageenan induced paw edema in rats by 73%. The results indicate that the analgesic activity of S dulcis L. may be explained by explained by an anti-inflammatory activity probably related to the triterpene Glutinol.

Histopathological alterations induced by non-viable cells and biochemical fractions from Paracoccidioides brasiliensis in mice

Non-viable cells and biochemical fractions from Paracoccidioides brasiliens were obtained for experimental inoculation in mice and posterior histopatological analysis. Dead total fungus, total fungus disrupted by sonorous waves, lipids of the fungus, supernatant of the lipid purification, integral and disrupted fungus free of lipids were obtained. The six preparations arised from masses of lyophilized yeasts of a recent isolate of P. brasiliensis (strain JT-1) and from a "Pool" equitably constituted by four strains maintained in laboratory for a long time (SN, 2, 18 and 192). Different doses of the 12 preparations were intraperitonially inoculated and histopathological analysis were done 30 days later. This analysis showed that all the inoculated preparations gave origin to inflamatory foci, except the one designated "supernatant of lipid purification". The alterations were detected exclusively in the liver of the animals and occurred from the smallest dose tested (1 mg), with exception of the lipids of the fungus, where the foci appeared only from a 3 mg dose onwards. No difference in the capacity of inducing histopathological alterations was found between the preparations obtained from the recent isolate (JT-1) and from the older ones ("Pool"). On the other hand, an increase of the number of inflammatory foci in function of the inoculated dose was observed.

Partial isolation and some properties of enterotoxin produced by Bacillus cereus strains

Extracellular proteins produced by Bacillus cereus AL-42 and AL-15 were fractioned by chromatography on QAE-Sephadex and Sephadex G75. This last chromatographic process resulted in three peaks. The major peak showed vascular permeability activity to rabbits, lethality to mice, and cytotoxicity to Vero and Hela cells. The analysis by SDS-PAGE after ultrafiltration confirm recent findings that the enterotoxin is a compound with molecular mass > 30.000.

The imidazoline receptors and the central regulation of the arterial blood pressure: a minireview

Recently, we proposed the hypothesis according to wich the central hypotensive effect of clonidine and related substances could be related to an action upon specific receptors, requiring the imidazoline or imidazoline-like structures, rather than alpha2-adrenoceptors. Since then, direct evidences have been accumulated to confirm the existence of a population of imidazoline specific binding sites in the brainstem of animals and man, more precisely in the Nucleus Reticularis Lateralis (NRL) region of the ventrolateral medulla (VLM), site of the antihypertensive action of clonidine. The purification of the putative endogenous ligand of the imidazoline receptors - named endazoline - is currently being attempted from human brain extracts. This new concept might at last lead to the expected dissociation of the pharmacological mechanisms involved, on the one hand, in the therapeutic antihypertensive effect, and on the other, in their major side-effect, which is sedation. In fact, it has been recently confirmed that hypotension is mediated by the activation of imidazoline preferring receptors (IPR) within the NRL region, while sedation is attributed to the inhibition of alpha2-adrenergic mechanisms in the locus coeruleus, which is involved in the control of the sleep-waking cycle. The IPRmay constitute on interesting target for new drugs in the treatment of arterial hypertension. Finally, dysfunctions of this modulatory system which could be involved in the pathophysiologyof some forms of the hypertensive disease are under investigation.