Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 µ L and photomicrographied. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10² promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

The effect of isolation on reproduction and growth of Pseudosuccinea columella (Pulmonata: Lymnaeidae): a snail-conditioned water experiment

A snail-conditioned water experiment was conducted in Pseudosuccinea columella to test the possible role of a chemical interaction between snails on the diminished growth and fecundity rates found for snails raised in pairs compared to those raised in complete isolation. The results permit to discard the hypothesis of an inhibition of growth and reproduction between snails due to factors released into the water.

Comparison between hydrographically conditioned digital elevation models in the morphometric charaterization of watersheds

The aim of this study was to compare the hydrographically conditioned digital elevation models (HCDEMs) generated from data of VNIR (Visible Near Infrared) sensor of ASTER (Advanced Spaceborne Thermal Emission and Reflection Radiometer), of SRTM (Shuttle Radar Topography Mission) and topographical maps from IBGE in a scale of 1:50,000, processed in the Geographical Information System (GIS), aiming the morphometric characterization of watersheds. It was taken as basis the Sub-basin of São Bartolomeu River, obtaining morphometric characteristics from HCDEMs. Root Mean Square Error (RMSE) and cross validation were the statistics indexes used to evaluate the quality of HCDEMs. The percentage differences in the morphometric parameters obtained from these three different data sets were less than 10%, except for the mean slope (21%). In general, it was observed a good agreement between HCDEMs generated from remote sensing data and IBGE maps. The result of HCDEM ASTER was slightly higher than that from HCDEM SRTM. The HCDEM ASTER was more accurate than the HCDEM SRTM in basins with high altitudes and rugged terrain, by presenting frequency altimetry nearest to HCDEM IBGE, considered standard in this study.

Development of conditioned taste aversion to Mascagnia rigida in goats

The aim of this study was to determine whether goats could be averted from consuming Mascagnia rigida, a toxic plant found in the semiarid region of northeastern Brazil. Fourteen male goats not previously familiarized to M. rigida were randomly allocated to two treatment groups: control (treated with 5.5mL water orally by a drenching gun) and lithium group (treated with 100mg LiCl/kg body weight orally by a drenching gun). For conditioning, goats were allowed to feed on M. rigida leaves for 15 min, followed by LiCl or water administration. The time spent on eating M. rigida leaves was measured. The conditioning was repeated daily until the LiCl-treated goats stopped eating M. rigida. On the 10th, 17th, and 24th day after conditioning, extinction trials of the M. rigida aversion were performed in goats by using single-choice tests. There was no difference between the two treatment groups with respect to the consumption of M. rigida on the first day of aversion conditioning, however, controls ingested increasing amounts of the plant on consecutive conditioning days. On the second day, five out of the seven goats in the lithium group did not eat the leaves, but on the third day, all the goats in the lithium group did not ingest M. rigida. This aversion persisted throughout all evaluated days. This indicates that goats can be easily conditioned by using lithium chloride to avoid eating M. rigida temporarily.

Conditioned food aversion to Ipomoea carnea var. fistulosa induced by Baccharis coridifolia in goats

Baccharis coridifolia is a plant that induces strong conditioned food aversion in ruminants. This research aimed to induce a conditioned food aversion to Ipomoea carnea var. fistulosa in goats, using B. coridifolia as an aversive agent, and to compare the aversion induced by this plant with the aversion induced by lithium chloride (LiCl). Thirteen goats were allotted into two groups: Group 1 with six goats was averted with 175mg/kg of body weight of LiCl and Group 2 with seven goats was averted with 0,25g/kg of bw of dried B. coridifolia. All goats were averted on day 1 after the ingestion of I. carnea. The aversion procedure with LiCl or B. coridifolia in goats from Groups 1 and 2, respectively, was repeated in those goats that again consumed the plant during tests on days 2, 3, and 7. The goats of both groups were challenged in pens on 23 and 38 days after the last day of aversion and challenged in the pasture on days 11, 15, 18, 20, 22, 25, 27 and 29 after the last day of aversion. After this period goats were challenged every 15 days on pasture until the 330º day after the last day of aversion (7th day). Two goats from Group 1 ingested I. carnea on the first day of the pasture challenge, 4 days after the last day of aversive conditioning in the pen. In addition, another goat in Group 1 started to consume the plant on day 18, and other two goats ate it on day 20. One goat in Group 1 that had never eaten I. carnea died on day 155. One goat from Group 2 started to ingest I. carnea on the first day of the pasture challenge, and a second goat started to consume this plant on day 182. At the end of the experiment, on day 330, the other five goats averted with B. coridifolia remained averted. These results suggest that B. coridifolia or an active compound from the plant could be used to induce aversion to toxic plants. Using B. coridifolia would be cheaper and, particularly in flocks with large number of animals, possibly easier than using LiCl, which requires the use of oral gavage and qualified personnel for its implementation.

Conditioned medium from activated spleen cells supports the survival of rat retinal cells in vitro

Cytokines are a heterogeneous group of molecules that have been associated with several functions in the nervous system, such as survival and differentiation of neuronal and glial cells. In the present study, we demonstrated that conditioned medium from spleen cells activated with concanavalin A increased neuritogenesis and survival of retinal cells, as measured by biochemical and morphological criteria. Our data showed that conditioned medium induced a five-fold increase in the amount of protein after 120 h in vitro. This effect was not inhibited by the blockade of voltage-dependent L-type calcium channels with 5.0 µM nifedipine. However, the use of an intracellular calcium chelator (15.0 µM BAPTA-AM) inhibited this effect. Our results support the idea that factors secreted by activated lymphocytes, such as cytokines, can modulate the maintenance and the differentiation of rat retinal cells in vitro, indicating a possible role of these molecules in the development of retinal cells, as well as in its protection against pathological conditions

Effects of sciatic-conditioned medium on neonatal rat retinal cells in vitro

Schwann cells produce and release trophic factors that induce the regeneration and survival of neurons following lesions in the peripheral nerves. In the present study we examined the in vitro ability of developing rat retinal cells to respond to factors released from fragments of sciatic nerve. Treatment of neonatal rat retinal cells with sciatic-conditioned medium (SCM) for 48 h induced an increase of 92.5 ± 8.8% (N = 7 for each group) in the amount of total protein. SCM increased cell adhesion, neuronal survival and glial cell proliferation as evaluated by morphological criteria. This effect was completely blocked by 2.5 µM chelerythrine chloride, an inhibitor of protein kinase C (PKC). These data indicate that PKC activation is involved in the effect of SCM on retinal cells and demonstrate that fragments of sciatic nerve release trophic factors having a remarkable effect on neonatal rat retinal cells in culture.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), α-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with α-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100%; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6%, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83%, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7% ethanol (62 vs 62%, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62%, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.

Induction of chagasic-like arrhythmias in the isolated beating hearts of healthy rats perfused with Trypanosoma cruzi-conditioned medium

Chagas' myocardiopathy, caused by the intracellular protozoan Trypanosoma cruzi, is characterized by microvascular alterations, heart failure and arrhythmias. Ischemia and arrythmogenesis have been attributed to proteins shed by the parasite, although this has not been fully demonstrated. The aim of the present investigation was to study the effect of substances shed by T. cruzi on ischemia/reperfusion-induced arrhythmias. We performed a triple ischemia-reperfusion (I/R) protocol whereby the isolated beating rat hearts were perfused with either Vero-control or Vero T. cruzi-infected conditioned medium during the different stages of ischemia and subsequently reperfused with Tyrode's solution. ECG and heart rate were recorded during the entire experiment. We observed that triple I/R-induced bradycardia was associated with the generation of auricular-ventricular blockade during ischemia and non-sustained nodal and ventricular tachycardia during reperfusion. Interestingly, perfusion with Vero-infected medium produced a delay in the reperfusion-induced recovery of heart rate, increased the frequency of tachycardic events and induced ventricular fibrillation. These results suggest that the presence of parasite-shed substances in conditioned media enhances the arrhythmogenic effects that occur during the I/R protocol.

Designing competitive international compensation programs

Over the past 25 years, expatriate managers have voiced increased disenchantment with their compensation packages whíle abroad. This paper takes a prescriptive approach, outlíning severa I elements of a successful human resources strategy and stressing key ingredients of effective international compensation programs. Particular ettention is given to the adherence of cultural values and distrlbutive justice when working across nations and cultures.

Risk factors for basal cell carcinoma: a case-control study

A controlled trial was performed with the purpose of investigating which factors could be considered of significant risk for the development of basal cell carcinoma. A total of 259 cases of basal cell carcinoma diagnosed from July 1991 to July 1992 were compared with 518 controls matched for age and sex. All subjects in both groups were white. Protocol data were submitted to statistical analysis by the chi-square test and by multiple conditional logistic regression analysis and the following conclusions were reached: 1) light skin color (types I and II of the Fitzpatrick classification), odds ratio of 2.8; outdoor work under constant sunlight, odds ratio of 5.0; the presence of actinic lesions due to exposure to the sun, odds ratio of 4.9, are risk factors perse. 2) Type III skin in the Fitzpatrick classification only represents a risk factor when the patient reports a history of intense sunburns, but not in the absence of such a history. 3) Sunburns per se do not represent a risk factor althorig the point made in item 2 of these conclusions is valid. 4) Other suspected risk factors whose significance was not confirmed by multiple conditioned logistic regression analysis were: residence in rural areas, light eyes and blond hair color, extent of the awareness of the "sun x skin cancer" relationship, familial occurrence of skin cancer, excessive exposure to the sun, and freckles appearing in childhood.

Observations on the miraxonal attraction exercised by sexually immature or adult Biomphalaria glabrata infected or not by Schistosoma mansoni

OBJECTIVE: The influence of age and the presence of secondary sporocysts in the miraxonal attraction exercised by Biomphalaria glabrata on miracidia of Schistosoma mansoni of the BH strain were studied. MATERIAL AND METHOD: A glass apparatus containing two compartments joined by a tube and previously tested in other experiments, was used. Specimens of B. glabrata or its snail conditioned water (SCW) selected before the first oviposition (sexually immature), after the first oviposition (adult), with or without secondary sporocysts, were used to attract the miracidia. RESULTS: It was noted that snails or their SCW containing secondary sporocysts lost the ability to attract miracidia. The sexual maturity of the snail did not influence miraxonal attraction.

Detection of pathogens from periodontal lesions

OBJECTIVE: To comparatively detect A. actinomycetemcomitans and F. nucleatum from periodontal and healthy sites. METHODS: Subgingival clinical samples from 50 periodontitis adult patients and 50 healthy subjects were analyzed. Both organisms were isolated using a trypticase soy agar-bacitracin-vancomycin (TSBV) medium and detected by PCR. Conventional biochemical tests were used for bacteria identification. RESULTS: A. actinomycetemcomitans and F. nucleatum were isolated in 18% and 20% of the patients, respectively, and in 2% and 24% of healthy subjects. Among A. actinomycetemcomitans isolates, biotype II was the most prevalent. Primer pair AA was 100% sensitive in the detection of A. actinomycetemcomitans from both subject groups. Primers ASH and FU were also 100% sensitive to detect this organism in healthy subject samples. Primer pair FN5047 was more sensitive to detect F. nucleatum in patients or in healthy samples than primer 5059S. Primers ASH and 5059S were more specific in the detection of A. actinomycetemcomitans and F. nucleatum, respectively, in patients and in healthy subject samples. CONCLUSIONS: PCR is an effective tool for detecting periodontal pathogens in subgingival samples, providing a faster and safer diagnostic tool of periodontal diseases. The method's sensitivity and specificity is conditioned by the choice of the set of primers used.

Health-related quality of life and working conditions among nursing providers

OBJECTIVE: To evaluate working conditions associated with health-related quality of life (HRQL) among nursing providers. METHODS: Cross-sectional study conducted in a university hospital in the city of São Paulo, Southeastern Brazil, during 2004-2005. The study sample comprised 696 registered nurses, nurse technicians and nurse assistants, predominantly females (87.8%), who worked day and/or night shifts. Data on sociodemographic information, working and living conditions, lifestyles, and health symptoms were collected using self-administered questionnaires. The following questionnaires were also used: Job Stress Scale, Effort-Reward Imbalance (ERI) and Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36). Ordinal logistic regression analysis using proportional odds model was performed to evaluate each dimension of the SF-36. RESULTS: Around 22% of the sample was found to be have high strain and 8% showed an effort-reward imbalance at work. The dimensions with the lowest mean scores in the SF-36 were vitality, bodily pain and mental health. High-strain job, effort-reward imbalance (ERI>1.01), and being a registered nurse were independently associated with low scores on the role emotional dimension. Those dimensions associated to mental health were the ones most affected by psychosocial factors at work. CONCLUSIONS: Effort-reward imbalance was more associated with health than high-strain (high demand and low control). The study results suggest that the joint analysis of psychosocial factors at work such as effort-reward imbalance and demand-control can provide more insight to the discussion of professional roles, working conditions and HRQL of nursing providers.