Flow cytometry in the study of cell death

In this report we present a concise review concerning the use of flow cytometric methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. The applications of these techniques to clinical and basic research are also considered. The following cell features are useful to characterize the mode of cell death: (1) activation of an endonuclease in apoptotic cells results in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, leads to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content make it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of apoptotic process; (2) plasma membrane integrity, which is lost in necrotic but not in apoptotic cells; (3) the decrease in forward light scatter, paralleled either by no change or an increase in side scatter, represent early changes during apoptosis. The data presented indicate that flow cytometry can be applied to basic research of the molecular and biochemical mechanisms of apoptosis, as well as in the clinical situations, where the ability to monitor early signs of apoptosis in some systems may be predictive for the outcome of some treatment protocols.

The beginning and expansion of flow cytometry in Brazil

The author describes and make comments on the favorable conditions that made possible the creation and organization of the first laboratory of flow cytometry in Brazil and South America installed at the Oswaldo Cruz Institute-Fiocruz, Rio de Janeiro, in November 1988.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuber-culosis infection.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.

Evaluation of Giardia duodenalis viability after metronidazole treatment by flow cytometry

Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability.

Intervening factors in attention flow of professionals injured by biological material

Objective: To describe the barriers and facilitator factors to follow the attention flow of professionals injured by biological material in the worker perspective. Method: Qualitative descriptive study with data collected through individual interviews with 18 injured workers, assisted in reference public units in the city of Goiânia. The content analysis was carried out with assistance of the ATLAS.ti 6.2 software, under the work organization and subjective perspectives. Results: From the interviews regarding the barriers and facilitator factors emerged the categories: organizational structure, Support from close people, and Knowledge influence. Conclusion: The organized services have enabled more qualified consultations and the workers follow-up, which caused a satisfaction feeling in relation to the working environment.


Trauma in the elderly caused by traffic accident: integrative review

OBJECTIVE To describe the scientific knowledge produced about trauma in the elderly caused by traffic accidents in healthcare area studies. METHODS Integrative review of studies from 2003 to 2013 searched in LILACS, SciELO, PubMed and CINHAL databases. We used combination of the descriptors injuries, wounds and accidents, in English, Portuguese and Spanish languages. RESULTS 32 studies were selected. In the thematic analysis, three categories emerged: epidemiological data from traffic accidents involving elderly; traffic accidents with elderly pedestrians; and trauma care in the elderly. We observed increased incidence of trauma in most countries and pedestrians represented a large part of the victims. Among these, the elderly are the most vulnerable group. CONCLUSION Studies showed that trauma care in the elderly need protocols and professionals with training in gerontology specialized in trauma care services.

Soil compaction and eucalyptus growth in response to forwarder traffic intensity and load

During timber exploitation in forest stands harvesting machines pass repeatedly along the same track and can cause soil compaction, which leads to soil erosion and restricted tree root growth. The level of soil compaction depends on the number of passes and weight of the wood load. This paper aimed to evaluate soil compaction and eucalyptus growth as affected by the number of passes and wood load of a forwarder. The study was carried out in Santa Maria de Itabira county, Minas Gerais State - Brazil, on a seven-year-old eucalyptus stand planted on an Oxisol. The trees were felled by chainsaw and manually removed. Plots of 144 m² (four rows 12 m long in a 3 x 2 m spacing) were then marked off for the conduction of two trials. The first tested the traffic intensity of a forwarder which weighed 11,900 kg and carried 12 m³ wood (density of 480 kg m-3) and passed 2, 4, and 8 times along the same track. In the second trial, the forwarder carried loads of 4, 8, and 12 m³ of wood, and the machine was driven four times along the same track. In each plot, the passes affected four rows. Eucalyptus was planted in 30 x 30 x 30 cm holes on the compacted tracks. The soil in the area is clayey (470 clay and 440 g kg-1 sand content) and at depths of 0-5 cm and 5-10 cm, respectively, soil organic carbon was 406 and 272 g kg-1 and the moisture content during the trial 248 and 249 g kg-1. These layers were assessed for soil bulk density and water-stable aggregates. The infiltration rate was measured by a cylinder infiltrometer. After 441 days the measurements were repeated, with additional analyses of: soil organic carbon, total nitrogen, N-NH4+, N-NO3-, porosity, and penetration resistance. Tree height, stem diameter, and stem dry matter were measured. Forwarder traffic increased soil compaction, resistance to penetration and microporosity while it reduced the geometric mean diameter, total porosity, macroporosity and infiltration rate. Stem dry matter yield and tree height were not affected by soil compaction. Two passes of the forwarder were enough to cause the disturbances at the highest levels. The compaction effects were still persistent 441 days after forwarder traffic.

Nutrient supply by mass flow and diffusion to maize plants in response to soil aggregate size and water potential

Nutrients are basically transported to the roots by mass flow and diffusion. The aim of this study was to quantify the contribution of these two mechanisms to the acquisition of macronutrients (N, P, K, Ca, Mg, and S) and cationic micronutrients (Fe, Mn, Zn, and Cu) by maize plants as well as xylem exudate volume and composition in response to soil aggregate size and water availability. The experiment was conducted in a greenhouse with samples of an Oxisol, from under two management systems: a region of natural savanna-like vegetation (Cerradão, CER) and continuous maize under conventional management for over 30 years (CCM). The treatments were arranged in a factorial [2 x (1 + 2) x 2] design, with two management systems (CER and CCM), (1 + 2) soil sifted through a 4 mm sieve and two aggregate classes (< 0.5 mm and 0.5 - 4.0 mm) and two soil matric potentials (-40 and -10 kPa). These were evaluated in a randomized block design with four replications. The experiment was conducted for 70 days after sowing. The influence of soil aggregate size and water potential on the nutrient transport mechanisms was highest in soil samples with higher nutrient concentrations in solution, in the CER system; diffusion became more relevant when water availability was higher and in aggregates < 0.5 mm. The volume of xylem exudate collected from maize plants increased with the decrease in aggregate size and the increased availability of soil water in the CER system. The highest Ca and Mg concentrations in the xylem exudate of plants grown on samples from the CER system were related to the high concentrations of these nutrients in the soil solution of this management system.

An improved method for measuring soil microbial activity by gas phase flow injection analysis

The rate of carbon dioxide production is commonly used as a measure of microbial activity in the soil. The traditional method of CO2 determination involves trapping CO2 in an alkali solution and then determining CO2 concentration indirectly by titration of the remaining alkali in the solution. This method is still commonly employed in laboratories throughout the world due to its relative simplicity and the fact that it does not require expensive, specific equipment. However, there are several drawbacks: the method is time-consuming, requires large amounts of chemicals and the consistency of results depends on the operator's skills. With this in mind, an improved method was developed to analyze CO2 captured in alkali traps, which is cheap and relatively simple, with a substantially shorter sample handling time and reproducibility equivalent to the traditional titration method. A comparison of the concentration values determined by gas phase flow injection analysis (GPFIA) and titration showed no significant difference (p > 0.05), but GPFIA has the advantage that only a tenth of the sample volume of the titration method is required. The GPFIA system does not require the purchase of new, costly equipment but the device was constructed from items commonly found in laboratories, with suggestions for alternative configurations for other detection units. Furthermore, GPFIA for CO2 analysis can be equally applied to samples obtained from either the headspace of microcosms or from a sampling chamber that allows CO2 to be released from alkali trapping solutions. The optimised GPFIA method was applied to analyse CO2 released from degrading hydrocarbons from a site contaminated by diesel spillage.

Effects of traffic control on the soil physical quality and the cultivation of sugarcane

The cultivation of sugarcane with intensive use of machinery, especially for harvest, induces soil compaction, affecting the crop development. The control of agricultural traffic is an alternative of management in the sector, with a view to preserve the soil physical quality, resulting in increased sugarcane root growth, productivity and technological quality. The objective of this study was to evaluate the physical quality of an Oxisol with and without control traffic and the resulting effects on sugarcane root development, productivity and technological quality. The following managements were tested: no traffic control (NTC), traffic control consisting of an adjustment of the track width of the tractor and sugarcane trailer (TC1) and traffic control consisting of an adjustment of the track width of the tractor and trailer and use of an autopilot (TC2). Soil samples were collected (layers 0.00-0.10; 0.10-0.20 and 0.20-0.30 m) in the plant rows, inter-row center and seedbed region, 0.30 m away from the plant row. The productivity was measured with a specific weighing scale. The technological variables of sugarcane were measured in each plot. Soil cores were collected to analyze the root system. In TC2, the soil bulk density and compaction degree were lowest and total porosity and macroporosity highest in the plant row. Soil penetration resistance in the plant row, was less than 2 MPa in TC1 and TC2. Soil aggregation and total organic carbon did not differ between the management systems. The root surface and volume were increased in TC1 and TC2, with higher productivity and sugar yield than under NTC. The sugarcane variables did not differ between the managements. The soil physical quality in the plant row was preserved under management TC1 and TC2, with an improved root development and increases of 18.72 and 20.29 % in productivity and sugar yield, respectively.

EFFECTS OF COMPACTION DUE TO MACHINERY TRAFFIC ON SOIL PORE CONFIGURATION

Soil compaction has been recognized as a severe problem in mechanized agriculture and has an influence on many soil properties and processes. Yet, there are few studies on the long-term effects of soil compaction, and the development of soil compaction has been shown through a limited number of soil parameters. The objectives of this study were to evaluate the persistence of soil compaction effects (three traffic treatments: T0, without traffic; T3, three tractor passes; and T5, five tractor passes) on pore system configuration, through static and dynamic determinations; and to determine changes in soil pore orientation due to soil compaction through measurement of hydraulic conductivity of saturated soil in samples taken vertically and horizontally. Traffic led to persistent changes in all the dynamic indicators studied (saturated hydraulic conductivity, K0; effective macro- and mesoporosity, εma and εme), with significantly lower values of K0, εma, and εme in the T5 treatment. The static indicators of bulk density (BD), derived total porosity (TP), and total macroporosity (θma) did not vary significantly among the treatments. This means that machine traffic did not produce persistent changes on these variables after two years. However, the orientation of the soil pore system was modified by traffic. Even in T0, there were greater changes in K0 measured in the samples taken vertically than horizontally, which was more related to the presence of vertical biopores, and to isotropy of K0 in the treatments with machine traffic. Overall, the results showed that dynamic indicators are more sensitive to the effects of compaction and that, in the future, static indicators should not be used as compaction indicators without being complemented by dynamic indicators.

Genetic structure and gene flow of Eugenia dysenterica natural populations

This study was carried out to assess the genetic variability of ten "cagaita" tree (Eugenia dysenterica) populations in Southeastern Goiás. Fifty-four randomly amplified polymorphic DNA (RAPD) loci were used to characterize the population genetic variability, using the analysis of molecular variance (AMOVA). A phiST value of 0.2703 was obtained, showing that 27.03% and 72.97% of the genetic variability is present among and within populations, respectively. The Pearson correlation coefficient (r) among the genetic distances matrix (1 - Jaccard similarity index) and the geographic distances were estimated, and a strong positive correlation was detected. Results suggest that these populations are differentiating through a stochastic process, with restricted and geographic distribution dependent gene flow.