Clastogenic activity of integerrimine determined in mouse micronucleus assays

The pyrrolizidine alkaloid integerrimine, obtained from Senecio brasiliensis, was tested by acute dosing at two concentrations (18.75 and 37.5 mg/kg), and at different times, to establish its ability to induce micronuclei in mouse erythrocytes. This alkaloid was able to increase the frequency of micronucleated polychromatic erythrocytes in both, bone marrow and peripheral blood erythrocytes

A priori estimation of accuracy and of the number of wells to be employed in limiting dilution assays

The use of limiting dilution assay (LDA) for assessing the frequency of responders in a cell population is a method extensively used by immunologists. A series of studies addressing the statistical method of choice in an LDA have been published. However, none of these studies has addressed the point of how many wells should be employed in a given assay. The objective of this study was to demonstrate how a researcher can predict the number of wells that should be employed in order to obtain results with a given accuracy, and, therefore, to help in choosing a better experimental design to fulfill one's expectations. We present the rationale underlying the expected relative error computation based on simple binomial distributions. A series of simulated in machina experiments were performed to test the validity of the a priori computation of expected errors, thus confirming the predictions. The step-by-step procedure of the relative error estimation is given. We also discuss the constraints under which an LDA must be performed.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Lignification of the plant and seed quality of RR soybeans sprayed with herbicide glyphosate

Differences in levels of lignin in the plant between conventional and transgenic cultivars RR has been reported by several authors, however, there are few studies evaluating the influence of spraying of glyphosate on the lignin in the plant and RR soybean seeds. The aim of this study was to evaluate the physiological quality of RR transgenic soybean seeds and the lignin contents of plants sprayed with the herbicide glyphosate. The assays were conducted both in greenhouse and field in the municipality of Lavras, MG, in the agricultural year 2007/08. The experiment was arranged in a splitplot design with four replicates, considering the treatments hand weeding and herbicide glyphosate as plots, and five RR soybean cultivars (BRS 245 RR, BRS 247 RR, Valiosa RR, Silvânia RR and Baliza RR) as splitplots. In the greenhouse, the cultivars tested were BRS 245 RR and Valiosa RR in a randomized block design with four replicates. The sprayings were carried out at stages V3, V7 and early R5 (3L/ha). The 1000 seed weight, mechanical injury, germination and germination velocity index, emergence velocity index, accelerated aging, electrical conductivity and water soaking seed test, lignin content in the seed coat, in the stem and legumes were determined. The spraying of glyphosate herbicide, in greenhouse and field, did not alter the physiological quality of seeds and the lignin contents in the plant.

Hydraulic conductivity in response to exchangeable sodium percentage and solution salt concentration

Hydraulic conductivity is determined in laboratory assays to estimate the flow of water in saturated soils. However, the results of this analysis, when using distilled or deionized water, may not correspond to field conditions in soils with high concentrations of soluble salts. This study therefore set out to determine the hydraulic conductivity in laboratory conditions using solutions of different electrical conductivities in six soils representative of the State of Pernambuco, with the exchangeable sodium percentage adjusted in the range of 5-30%. The results showed an increase in hydraulic conductivity with both decreasing exchangeable sodium percentage and increasing electrical conductivity in the solution. The response to the treatments was more pronounced in soils with higher proportion of more active clays. Determination of hydraulic conductivity in laboratory is routinely performed with deionized or distilled water. However, in salt affected soils, these determinations should be carried out using solutions of electrical conductivity different from 0 dS m-1, with values close to those determined in the saturation extracts.

Specific circulating immune complexes in acute chagas' disease

The presence of circulating immune complexes formed by IgM and IgG (CIC-IgM and CIC-IgG) was investigated, using antigen-specific enzyme-immunoassays (ELISA), in 30 patients with acute Chagas' disease who showed parasitemia and inoculation chagoma. Control population consisted of patients with chronic T. cruzi infection (30), acute toxoplasmosis 10), leishmaniasis (8), rheumatoid arthritis (3) and healthy individuals with negative serology for Chagas* disease (30). Acute chagasic patients were 100% CIC-IgG and 96.66% CIC-IgM positive whereas immunofluorescence tests yielded 90% and 86.66% of positivity for specific IgG and IgM antibodies, respectively. Chronic patients were 68% CIC-IgG and 0% CIC-IgM positive. The 30 negative and the 21 cross-reaction controls proved negative for ELISA (CIC-IgM and CIC-IgG). The high sensitivity of ELISA assays would allow early immunologic diagnosis, as well as prompt treatment, of acute T. cruzi infection, thus eliminating the problem of the false-positive and false-negative results which affects traditional methods for detection of circulating antibodies.

Toxoplasmosis serology: an efficient hemagglutination procedure to detect IgG and IgM antibodies

In search of an efficient but simple, low cost procedure for the serodiagnosis of Toxoplasmosis, especially suited for routine laboratories facing technical and budget limitations as in less developed countries, the diagnostic capability of Hematoxo® , an hemagglutination test for toxoplasmosis, was evaluated in relation to a battery of tests including IgG- and IgM-immunofluorescence tests, hemagglutination and an IgM-capture enzymatic assay. Detecting a little as 5 I.U. of IgG antitoxoplasma antibodies, Hematoxo® showed a straight agreement as to reactivity and non-reactivity for the 443 non-reactive and the 387 reactive serum samples, included in this study. In 23 cases presenting a serological pattern of acute toxoplasmosis and showing IgM antibodies, Hematoxo® could detect IgM antibodies in 18, indicated by negativation or a significant decrease in titers as a result of treating samples with 2-mercapto-ethanol. However, a neat increase in sensitivity for IgM specific antibodies could be achieved by previously removing IgG from the sample, as demonstrated in a series of acute toxoplasmosis sera. A simple procedure was developed for this purpose, by reconstituting a lyophilized suspension of Protein A - rich Staphylococcus with the lowest serum dilution to be tested. Of low cost and easy to perform, Hematoxo® affords not only a practical qualitative procedure for screening reactors and non-reactors, as in prenatal services, but also quantitative assays that permit to titrate antibodies as well as to identify IgM antibodies.

Screening the mutagenic activities of coomonly used antiparasite drugs by the Simultest, a simplified Salmonella/microsome plate incorporation assay

The mutagenic activities of 16 anti-parasite drugs were screened by the Simultest in both qualitative (spot test) and quantitative (plate incorporation) assays with a Salmonella typhimurium pool composed by the indicator strains TA97, TA 98, TA100 and TA102. Four anti Chagas' disease drugs (nifurtimox, benznidazole, CL 64,855, and MK 436) and two anti-amebae drugs (metronidazole and tinidazole) gave positive results in qualitative tests and incorporation of rat liver microsomes did not alter the results. Comparative dose response curves of the mutagenic activities of CL 64,855, metronidazole and benznidazole obtained by the simultest and by individual Salmonella indicator strains demonstrated that both approachs have similar sensitivities. The results corroborate the validity of the Simultest, as a simplified, fast and economic version of the Ames test in preliminary screening of potential mutagenic drugs.

Evaluation of temporal, seasonal and geographic stability of the molluscicidal property of Euphorbia splendens latex

Laboratory tests with aqueous solutions of Euphorbia splendens var. hislopii latex have demonstrated seasonal stability of the molluscicidal principle, with LD90 values of 1.14 ppm (spring), 1.02 ppm (fall), 1.09 ppm (winter), and 1.07 ppm (summer) that have been determined against Biomphalaria tenagophila in the field. Assays on latex collected in Belo Horizonte and Recife yielded LD90 values similar to those obtained with the reference substance collected in Rio de Janeiro (Ilha do Governador), demonstrating geographic stability of the molluscicidal effect. The molluscicidal action of aqueous dilutions of the latex in natura, centrifuged (precipitate) and lyophilized, was stable for up to 124 days at room temperature (in natura) and for up to 736 days in a common refrigerator at 10 to 12ºC (lyophilized product). A 5.0 ppm solution is 100% lethal for snails up to 13 days after preparation, the effect being gradually lost to almost total inactivity by the 30th day. This observation indicated that the active principle is instable. These properties together with the wide distribution of the plant, its resistance and adaptation to the tropical climate, its easy cultivation and the easy obtention of latex and preparation of the molluscicidal solution, make this a promising material for large-scale use in the control of schistosomiasis

Simplified fluorescent inhibition microtest for the titration of rabies neutralizing antibodies

A simplified fluorescence inhibition microtest (SFIMT) was standardized for the evaluation of antirabies serum neutralizing antibodies based on the rapid fluorescent focus inhibition test (RFFIT) and the fluorescence inhibition microtest (FIMT). The simplified test showed reproductibility similar to that of the FIMT with advantages as easier executation and quicker reading. A simple pre-treatment of Brazilian microplates produced for immune enzymatic assays (PROSIL) gave equivalent results and substantial coast reduction, in relation to imported plates (DIFCO). The simplified test can be easily implemented in less sophisticated laboratories, as alternative to the mouse serum neutralization test, still the most largely employed in Brazil, or even to others as RFFIT and FIMT.

Malaria serology: performance of six Plasmodium falciparum antigen extracts and of three ways of determining serum titers in IgG and IgM-ELISA

The study evaluated six Plasmodium falciparum antigen extracts to be used in the IgG and IgM enzyme-linked immunosorbent assays (ELISA), for malaria diagnosis and epidemiological studies. Results obtained with eighteen positive and nine negative control sera indicated that there were statistically significant differences among these antigen extracts (Multifactor ANOVA, p< 0.0001). Urea, sodium deoxycholate and Zwittergent antigen extracts performed better than did the three others, their features being very similar for the detection of IgG antibodies. Urea, alkaline and sodium deoxycholate antigen extracts proved to be better than the others for the detection of IgM antibodies. A straight line relationship was found between the optical densities (or their respective log 10) and the log 10 of antibody dilutions, with a very constant slope. Thus serum titers could be determined by direct titration and by two different equations, needing only one serum dilution. For IgM antibody detections, log 10 expression gave results that better correlated with direct titration (95% Bonferroni). For IgG antibody detections, the titer differences were not significant. The reproducibility of antibody titers and antigen batches was also evaluated, giving satisfactory results.

Measles serodiagnosis: standardization and evaluation of a dot-ELISA

A Dot-ELISA using a measles virus (MV) antigen obtained by sodium deoxycholate treatment was standardized and evaluated for IgM and IgG antibody detection in measles patients and measles-vaccinated subjects. A total of 192 serum samples were studied, comprising 47 from patients with acute and convalescent measles, 55 from 9-month old children prior to measles vaccination and 41 from children of the same age after vaccination, and 49 from patients with unrelated diseases. The diagnostic performances of the IgG Dot-ELISA and IgG immuno fluorescence test (IFT) were found to be close, varying from 0.97 to 1.00 in sensitivity and the specificities were maximum (1.00). Nevertheless, the sensitivity of the IgM Dot-ELISA (0.85) was higher than that (0.63) of the IgM IFT, although both assays had comparably high (1.00) specificities. The IgM Dot-ELISA in particular proved to be more sensitive in relation to other assays studied by revealing antibodies in 80.0% (12/15) of vaccinated children on the 15th day after immunization. In contrast the IgM IFT, failed to detect antibodies in the same group of vaccinated children. The stability of the MV antigen was longer than that of the IFT antigen, and the reproducibility of the Dot-Elisa was satisfactory.

Legionnaires' disease in the renal transplant unit of "Hospital das Clinicas, FMUSP": during a five year period (1988-1993)

Several reports have related Legionella pneumophila with pneumonia in renal transplant patients, however this association has not been systematically documented in Brazil. Therefore this paper reports the incidence, by serologycal assays, of Legionella pneumophila serogroup 1 in these patients during a five year period. For this purpose sera from blood samples of 70 hospitalized patients with pneumonia from the Renal Transplant Unit of Hospital das Clinicas, FMUSP collected at the acute and convalescent phase of infection were submitted to indirect immunofluorescence assay (IFA) to demonstrate anti-Legionella pneumophila serogroup 1 antibodies. Of these 70 patients studied during the period of 1988 to 1993,18 (25.71 %) had significant rises in specific antibody titers for Legionella pneumophila serogroup 1. Incidence was interrupted following Hospital water decontamination procedures, with recurrence of infections after treatment interruption. In this study, the high susceptibility (25.71%) of immunodepressed renal transplant patients to Legionella pneumophila serogroup 1 nosocomial infections is documented. The importance of the implementation and maintenance of water decontamination measures for prophylaxis of the infection is also clearly evident.

Difficulties in diagnosing atypical primary HIV-1 infection

Several cases of primary HIV-1 infection are not identified, either because the diagnosis is not suspected or because they test negative for HIV-1 antibody. This work presents an uncommon case of primary HIV-1 infection in an young parenteral drug abuser man, who presented symptoms of acute hepatitis. During the initial acute phase the serum sample of the patient tested negative for the presence of antibodies against several viruses, including HIV-1. Nevertheless, the diagnosis of primary HIV-1 infection was suspected by using an alternative method for"in vitro" induced antibody production (IVIAP), and confirmed by p24 antigen serum positivity and seroconversion in serial plasma samples of the patient. The authors suggest the use of the IVIAP and others complementary assays to help the diagnosis of acute HIV-1 infection in persons at high risk conditions.