Assessment of routine malaria diagnosis in the Venezuelan Amazon.

The quality of routine malaria diagnosis is a crucial topic of malaria control. The aim of this assessment was to monitor and evaluate the quality of routine malaria diagnosis in Amazonas (Venezuela) and to improve the quality control system. The traditional non-blinded quality control system was found to be overburdened with diagnostic samples. A modified sampling system with fewer samples to be tested was proposed. Expert microscopists blindly double-checked 1000 slides and 550 rapid diagnostic tests (RDT) (OptiMAL-IT) from health posts (HP). For Plasmodium vivax, HP microscopy and OptiMAL-IT showed sensitivies of 86% and 63%, respectively. For P. falciparum, HP microscopy and OptiMAL-IT showed sensitivities of 68% and 89%, respectively. Both methods lost accuracy when fewer parasites occurred in the sample. HP microscopists from different municipalities displayed significant differences in diagnostic quality. Overall, quality of routine malaria diagnosis in the Venezuelan Amazon is good but not optimal. The change from the traditional non-blinded quality control system to blinded cross-checking of a minimal selection of samples is - comparatively - a low cost intervention with possibly high impact on the quality of routine malaria diagnosis. The introduction of RDTs should be discussed carefully in order not to displace an existing network of HP microscopists.

Simplified culture techniques for growth and differentiation of murine and human pre-adipocytes for translational applications.

BACKGROUND: Adipose tissue has become a promising source of adult stem cells. Looking for optimal culture conditions, we evaluated the ability of L15, a free-gas exchange culture medium, to support cell proliferation and adipogenesis of murine 3T3-F442A and human normal (HNPA) and lipoma-derived (HLPA) pre-adipocytes. METHODS: 3T3-F442A, HNPA and HLPA cell proliferation were compared in short-term cultures and along multiple passages in Dulbecco's modified Eagle medium (DMEM) or DMEM-F12 under a 5% CO(2) atmosphere or L15 medium under a free-gas exchange atmosphere. Adipogenesis in these cells was evaluated by quantifying lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity, and by assaying the expression of adipogenic markers by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: 3T3 pre-adipocytes grew at similar rates in serum-supplemented L15 or DMEM, but L15 induced higher colony-forming efficiency in these cells. HNPA and HLPA grew more actively in L15 than in DMEM-F12 for more than 10 successive passages and reached higher colony-forming efficiency in L15 medium. On the other hand, while high-glucose DMEM and L15 supplemented with glucose 1 g/L induced similar levels of 3T3 adipogenesis, L15 with no added glucose increased HNPA and HLPA adipogenesis with respect to DMEM-F12, as measured by lipid accumulation, GPDH activity and expression of adipogenic markers C/EBPalpha, GLUT-4, LPL and aP2. DISCUSSION: The free-gas exchange medium L15 supports cell proliferation and adipogenesis of murine 3T3 and normal and lipoma-derived human subcutaneous pre-adipocytes to a greater extent than DMEM or DMEM-F12. The routine use of L15 will optimize translational applications of adipose cells.

Screening of biological activities present in honeybee (Apis mellifera) royal jelly.

Bioactivities present in three honeybee royal jelly (RJ) protein fractions were screened for diverse in vitro model systems. RJCP, a RJ crude protein extract, stimulated cell growth of Tn-5B1-4 insect cells inducing 6.5 population doublings per mg of protein added to culture medium, meanwhile fetal bovine serum, the usual growth supplement, gave rise only to 2.55 population doublings. RJCP, as well as the fractions RJP30 and RJP60 obtained by precipitation with ammonium sulfate, also affected Tn-5B1-4 cell shape and stimulated adhesion of these cells to the substrate. RJP30 also increased the percent of mature adipocytes in cultures of 3T3-F442A murine preadipocytes twofold with respect to insulin treatment, without inducing additional cell growth of confluent preadipocytes. Fractions RJCP and RJP60 showed similar capacity as that of insulin to stimulate the formation of mature 3T3 adipocytes. Fraction RJP30 also was cytotoxic for HeLa human cervicouterine carcinoma cells, diminishing 2.5-fold the initial cell density after seven days of treatment. Our results show the presence of diverse bioactivities in RJ affecting cell growth, cell differentiation and cell survival of insect, murine, and human cancer cells.

Fluorescent in situ hybridization (FISH) on paraffin-embedded placental tissues as an adjunct for understanding the etiology of early spontaneous abortion.

OBJECTIVES: An investigation of first-trimester spontaneous abortions (SAs) for those cases in which karyotype is not available was designed to test the efficiency of fluorescence in situ hybridization (FISH) on paraffin-embedded tissues combined with pathological examination for understanding the etiology of SAs. METHODS: Pathological examination of 202 placental tissues from SAs was performed. FISH analysis was then carried out on paraffin-embedded tissue sections from the same abortion products with probes specific for chromosomes 13, 16, 18, 21, X, Y. RESULTS: FISH could be achieved in 196 cases (97%). After pathological analysis alone, the etiology of SAs was evoked in 40 cases. The suspected diagnosis was confirmed by FISH in 26 cases (13.2%). After combined pathological and FISH analysis, the etiology of SAs was identified in 83 from the 196 cases (42.3%) with the probe set used. CONCLUSION: The present study demonstrates the value of FISH on paraffin-embedded tissues as an adjunct for understanding the etiology of SAs for those cases in which karyotype is not available. Combination of pathological and FISH analysis increases the yield of diagnosis by a factor of 3.2. The results also demonstrate that predictions of the karyotype from pathological examination should be avoided.

[Antenatal metastatic neuroblastoma: prognostic criteria. A case report]. / Le neuroblastome métastatique anténatal: critères pronostiques. A propos d'un cas.

Neuroblastoma, the most common malignant tumor in the neonatal period is metastatic in 25 to 50% of cases. While the prognosis of antenatal neuroblastoma is often considered as favorable, included in the most common metastatic stage (Stage IV S), it can lead to fetal or neonatal death. We report a case of a fetus with a stage IV neuroblastoma who died in utero. The most important prognostic factor is tumor stage, making sonographic detection of metastasis essential. Nevertheless, accurate staging remains difficult by sonography. When metastatic neuroblastoma is suspected, sonographic survey has to be reinforced, and if serious criteria such as massive hepatomegaly, placentomegally or hydrops appear, delivery must be considered.

Glycerol-3-Phosphate dehydrogenase (E.C.1.1.1.8) is expressed in cultured chicken embryonic adipofibroblasts and upregulated by embryonic chicken serum.

Chicken embryonic adipofibroblasts (CEA) accumulate intracytoplasmic lipids when cultured in medium containing chicken serum (CS), but not in medium with fetal bovine serum (FBS). To characterize this process of lipid accumulation, we evaluated the expression of the enzyme glycerol-3-phosphate dehydrogenase (E.C.1.1.1.8) (GPDH), first in chicken tissues and then in CEA cultured under diverse conditions. GPDH activity in adipose depots from 4-wk-old broiler chickens was similar or higher than that shown by liver, the main organ for fatty acid synthesis in chickens, while skeletal muscle had the lowest levels of GPDH. In vitro, GPDH activity increased in CEA cultured in the presence of CS but not in medium with FBS, paralleling the lipid accumulation by these cells. Both lipid accumulation and GPDH activity were further increased in CEA cultured in the presence of embryonic CS. Our results show that GPDH is highly expressed in avian tissues related to lipid metabolism and therefore can be a reliable marker for avian adipogenesis, and suggest that ECS is an optimum source for the purification of avian adipogenic factors.

Colorimetric quantitation of in vitro cell density using carmine, a chromosome-specific stain.

Cell number is usually evaluated during in vitro studies to estimate metabolic or pharmacological effects of specific compounds. However, estimation of in vitro cell density by direct cell counting is a laborious and time-consuming task, whereas indirect methods for cell quantitation have serious disadvantages such as environmental costs or inaccuracies derived from non-specific interferences. We developed a new method for in vitro cell density quantitation which employs carmine, a natural dye widely used for chromosome staining in cytological studies. Normal or transformed murine fibroblasts, avian normal fibroblasts, human epithelial HeLa cells, and insect cells, inoculated at a range of cell densities, were fixed with 4% formaldehyde/PBS and stained with 0.4% alcoholic-HCl carmine. The stain retained in cell monolayers was extracted with 0.01 M NaOH and spectrophotometrically measured at 531 nm. Invariably, high correlation coefficients between cell number and absorbance were obtained for each cell type, within a range of 5 x 10(3) to 5 x 10(5) cells. Moreover, identical cell growth curves were obtained when cell number was estimated over several days of culture by both direct cell counting and carmine staining methods. Our results show that the carmine staining method represents an easy, precise and reliable alternative for in vitro cell quantitation, avoiding interferences caused by cell components modulable by culture treatments, and over a wide range of cell types and cell densities.

The effects of a protease inhibitor fraction from tepary bean (Phaseolus acutifolius) on in vitro cell proliferation and cell adhesion of transformed cells.

Some protease inhibitors (PI), such as the soybean Bowman-Birk protease inhibitor (SBBI), have been described as anticarcinogenic agents. Although PI are ubiquitous compounds in living organisms, the anticarcinogenic potential of PIs other than SBBI remain poorly explored. We evaluated the antiproliferative effect of a protein fraction from tepary bean (Phaseolus acutifolius) seeds with protease inhibitor activity (TPIF), on normal and on malignant cells. TPIF was obtained after precipitation with ammonium sulfate and gel filtration, and its bioactivity was assayed in vitro on HeLa cells, normal 3T3 fibroblasts and 3T3/v-mos transformed fibroblasts. TPIF showed antiproliferative and cytotoxic effects on 3T3/v-mos transformed fibroblasts in a dose-dependent way. On the contrary, TPIF was only cytostatic for normal 3T3 cells at the highest doses assayed, and had no effect on epithelial HeLa cells proliferation. Sublethal TPIF doses also stimulated cell adhesion of poorly adherent 3T3/v-mos cell line.

A preadipose 3T3 cell variant highly sensitive to adipogenic factors and to human growth hormone.

We describe a new Swiss 3T3 preadipose clone, 3T3-F442A/C4, which shows higher sensitivity to serum adipogenic factors and to human growth hormone as compared to other 3T3 preadipose clones. The 3T3-F442A/C4 clone exhibited several characteristics different from the parental 3T3-F442A cells, mainly a high extent of adipose conversion under culture conditions that are non-adipogenic for the parental cells. The 3T3-F442A/C4 cells are not committed to undergo adipose differentiation, since they do not differentiate into adipocytes under serum-free or low-serum culture conditions, unless adipogenic factors or growth hormone are added into the culture medium. The 3T3-F442A/C4 cells showed 1.5- to 3.6-fold higher sensitivity to serum adipogenic factors and 5- to 6-fold higher sensitivity to human growth hormone as compared to the 3T3-F442A cells. The 3T3-F442A/C4 variant clone also differed from the parental clone by having a shorter population doubling time, an increased saturation density, and lower activity levels in some biochemical markers of adipose differentiation. On the other hand, the new variant clone has a similar proportion of cells susceptible to become adipocytes, and a similar response to insulin as compared to the parental cells. Our results show that the 3T3-F442A/C4 cells represent a new 3T3 preadipose clone that could be useful as a bioassay to evaluate growth hormone activity, as well as to purify and characterize hormones, adipogenic factors, and those compounds that affect mammalian adipogenesis.

Inhibition of 3T3 adipogenesis by retinoic acid is not mediated by cytoplasmic retinoic acid-binding protein.

Retinoic acid (RA) inhibits 3T3 adipogenesis in a dose-dependent and reversible manner, but its mechanism of action remains unknown. 3T3-F442A cell variants obtained by mutagenesis with nitrosoguanidine and/or selection with high RA concentrations showed different resistance to RA cytotoxicity and underwent adipose conversion of various extents when they were cultured in adipogenic conditions. Commitment to adipose differentiation was also inhibited by RA in these clones. Gel filtration chromatography showed the presence of a cytosolic RA-binding activity in the parental cells but not in three of the variant clones isolated. We demonstrate that cytoplasmic RA-binding activity is not essential for the inhibitory effects of the retinoid on 3T3 adipogenesis, or for resistance to RA cytotoxicity. Other mechanisms should be involved to explain the inhibition of adipose differentiation by RA.