A molecular approach for the rapid, selective and sensitive detection of Exophiala jeanselmei in environmental samples: development and performance assessment of a real-time PCR assay.

Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR®green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR®green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days.

Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air.

Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical analytical monitoring methods, based on cultivation and microscopic identification, depend on the growth of the fungi. Consequently, they are biased by difficulties to grow some species on certain culture media and under certain conditions or by noncultivable or dead fungi that can consequently not be identified. However, they could have an impact on human health as they might be allergenic. Since molecular methods do not require a culture step, they seem an excellent alternative for the monitoring of indoor fungal contaminations. As a case study, we developed a SYBR® green real-time PCR-based assay for the specific detection and identification of Aspergillus versicolor, which is frequently observed in indoor environment and known to be allergenic. The developed primers amplify a short region of the internal transcribed spacer 1 from the 18S ribosomal DNA complex. Subsequently, the performance of this quantitative polymerase chain reaction (qPCR) method was assessed using specific criteria, including an evaluation of the selectivity, PCR efficiency, dynamic range, and repeatability. The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor. In order to demonstrate that this SYBR® green qPCR assay is a valuable alternative for monitoring indoor fungal contamination with A. versicolor, environmental samples collected in contaminated houses were analyzed and the results were compared to the ones obtained with the traditional methods.

Real-time PCR for quantification in soil of glycoside hydrolase family 6 cellulase genes.

UNLABELLED: Cellulose is the main structural component of the cell walls of higher plants, representing c. 35-50% of a plant's dry weight; after decomposition and transformation, and constituting a large part of soil organic matter. Telluric micro-organisms able to use cellulose as carbon and energy sources for growth are widely distributed in the environment, but the factors controlling the rate of cellulose degradation are not well understood. In this study, we have developed a quantitative real-time PCR (qPCR) primer set to quantify the glycoside hydrolase family 6 (GH6 family) cellulase genes in soil samples. The qPCR assays were linear over 8 orders of magnitude and sensitive down to 10 copies per assay. qPCR analysis of contrasted soil samples showed densities between 2·47 × 10(7) and 1·48 × 10(10) copies per gram of soil. Cloning and sequencing of the PCR products from environmental DNA confirmed both specific amplification (more than 96%) and the wide diversity targeted by the primer set, throughout nearly all the GH6 family, including sequences of bacteria and fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Telluric micro-organisms able to use cellulose as carbon and energy sources for growth are widely distributed in the environment, but the factors controlling the rate of cellulose degradation are not well understood. The objective of our study was to develop a qPCR for rapid quantification of GH6 cellulase genes in soil. This qPCR could be applied to study the potential for cellulose degradation in different soils in order to better understand the factors controlling the stability of the soil organic matter.

A hybrid stochastic model and its Bayesian identification for infectious disease screening in a university campus with application to massive COVID-19 screening at the University of Liège.

Amid the COVID-19 pandemic, universities are implementing various prevention and mitigation measures. Identifying and isolating infectious individuals by using screening testing is one such a measure that can contribute to reducing spread. Here, we propose a hybrid stochastic model for infectious disease transmission in a university campus with screening testing and its surrounding community. Based on a compartmental modeling strategy, this hybrid stochastic model represents the evolution of the infectious disease and its transmission using continuous-time stochastic dynamics, and it represents the screening testing as discrete stochastic events. We also develop, in a Bayesian framework, the identification of parameters of this hybrid stochastic model, including transmission rates. These parameters were identified from the screening test data for the university population and observed incidence counts for the surrounding community. We implement the exploration of the Bayesian posterior using a machine-learning simulation-based inference approach. The proposed methodology was applied in a retrospective modeling study of a massive COVID-19 screening conducted at the University of Liège in Fall 2020. The emphasis of the paper is on the development of the hybrid stochastic model to assess the impact of screening testing as a measure to reduce spread. The hybrid stochastic model allows various factors to be represented and examined, such as interplay with the surrounding community, variability of the transmission dynamics, the rate of participation in the screening testing, the test sensitivity, the test frequency, the diagnosis delay, and compliance with isolation. The application in the retrospective modeling study suggests that a high rate of participation and a high test frequency are important factors to reduce spread.

Interactions between ethylenediaminetetraacetic acid (EDTA) and iron absorption pathways, in the Caco-2 model.

UNLABELLED: EDTA is a well known enhancer of iron absorption; however, the precise way of absorption of iron ingested in presence of EDTA is not known; some data suggest it could use a passive, non regulated paracellular way. Iron (sulphate or gluconate) absorption by Caco-2 cells was assessed in presence of EDTA. EDTA did not change the apical uptake of iron; transport in the basal chamber increased by 98% for FeSO4 and 95% for Fe gluconate. By contrast, intracellular storage decreased by 31% for FeSO4 and 64% for Fe gluconate. In addition EDTA induced a significant increase of permeability of the cell monolayer assessed by a decrease of transepithelial electrical resistance: 314+/-34 Omegacm(-2) to 235+/-57 Omegacm(-2) for sulphate, 414+/-33 Omegacm(-2) to 223+/-36 Omegacm(-2) for gluconate; iron free control: 410+/-10 Omegacm(-2). CONCLUSIONS: These results suggest that in presence of EDTA iron absorption occurs mainly by the paracellular instead of the regulated cellular way, that could potentially enhance its toxicity.

Effects of a P-class CpG-ODN administered by intramuscular injection on plasma cytokines and on white blood cells of healthy horses.

Cytosine-phosphate-guanosine (CpG-ODN) has been described as a potent immunostimulatory agent in different species. No study reported the effect of a P-class CpG when administered systemically in healthy horses. The aim of this study was to evaluate the tolerance and the effect of an intramuscularly administered P-class CpG-ODN on hematology and on plasma cytokines (IFN-α, IL-10, TNF-α, IFN-γ) in 8 healthy horses. Intra-muscular CpG-ODN or placebo (PBS) was administered twice at a 7 days-interval. Groups were inversed after 2 months of washout period. A physical examination, complete blood count (CBC) and plasma cytokine measurements were performed from 2 days before injection up to 21 days after injection. P-class CpG-ODN injection was well tolerated with minor side effects. After the first injection a significant transient drop in circulating total leukocytes, lymphocytes and an increase in monocytes were observed. A transient drop in eosinophils was also noted after each CpG injection. P-class CpG-ODN at a dose of 5 mg did not create major side effects in 7 horses, one horse showed transient pyrexia. A redistribution of white blood cells was observed in horses receiving CpG, but no change in plasma cytokines was observed at the indicated dose, route of administration and sampling times.

Helenalin reduces Staphylococcus aureus infection in vitro and in vivo.

Staphylococcus (S.) aureus is a major udder pathogen causing bovine mastitis. Some pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), enhance extracellular and intracellular growth of S. aureus, indicating that the inflammatory process favors S. aureus infection. Helenalin is a sesquiterpene lactone with potent anti-inflammatory properties. This study was designed to evaluate the effects of helenalin on S. aureus infection. First, in vitro experiments were conducted. These studies revealed that proliferation of S. aureus in bovine mammary epithelial MAC-T cells treated in the presence or absence of TNF-alpha was markedly reduced in the presence of helenalin. Secondly, in vivo effects of helenalin were investigated. Lactating mice treated in the presence or absence of helenalin were challenged by the intramammary route with S. aureus and the bacteria in the mammary glands were counted 12 h after infection. Significantly less numbers of bacteria were recovered from the infected glands of helenalin-treated mice compared with untreated mice. Moreover, histological examination of mammary tissue from helenalin-treated mice that were challenged with S. aureus indicated that helenalin is able to significantly reduce leukocyte infiltration in the mammary gland following S. aureus inoculation. Our results show that helenalin reduces S. aureus intracellular growth and experimental S. aureus infection. We conclude that helenalin may be of potential interest in the treatment of S. aureus-induced mastitis in the bovine species.

Prolactin-induced activation of nuclear factor kappaB in bovine mammary epithelial cells: role in chronic mastitis.

We sought to determine whether prolactin (PRL) could influence the neutrophilic inflammation that characterizes chronic mastitis. Most of the genes encoding inflammatory proteins depend on the nuclear factor kappaB (NF-kappaB) for their expression. We addressed the hypothesis that immunomodulatory activities of PRL might arise from an increase in NF-kappaB activity. MAC-T cells, a bovine mammary epithelial cell line, were stimulated with increasing concentrations of bovine PRL (1, 5, 25, 125, and 1,000 ng/mL). Level of NF-kappaB binding activity was measured and mRNA was evaluated for IL-1beta, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GMCSF), IFN-gamma, and tumor necrosis factor (TNF)-alpha, cytokines known to require NF-kappaB for their maximal transcription. Prolactin activated NF-kappaB; maximal NF-kappaB activation was weaker with PRL than with TNF-alpha at 30 or 180 min poststimulation. In addition, PRL significantly amplified, in a dose-dependent manner, mRNA expression of IL-1beta, IL-6, IL-8, GMCSF, and TNF-alpha. We measured PRL concentrations in blood and milk from healthy and chronic mastitis-infected cows, and studied the relationship between the PRL concentration and the degree of inflammation in the mammary gland as indirectly assessed by somatic cell counts (SCC). Plasma PRL did not differ significantly between healthy and chronic mastitis-affected cows (63.7 and 67.5 ng/mL, respectively). Milk PRL concentration was significantly increased in chronic mastitis-affected quarters with the highest SCC, and had a positive significant correlation between SCC, as well as between the number of neutrophils present in milk samples. The present findings show that PRL promotes an inflammatory response in bovine mammary epithelial cells via NF-kappaB activation, and suggest a role for PRL in the pathogenesis of chronic mastitis.

[Control of gene transcription as a new therapeutic target in asthma]. / Le contrôle de la transcription génique en tant que nouvelle cible thérapeutique dans le traitement de l'asthme.

The recent advances in the knowledge of the molecular mechanisms underlying asthma have lead to a significant improvement of the current treatments of the disease and opened new perspectives for the development of therapeutic alternatives to inhaled corticosteroids. The selective targeting of transcription factors controlling the expression of the genes implicated in the pathogenesis of asthma is one of these privileged strategies. This review aims at describing the most promising new therapeutic targets in the control of asthmatic inflammation at the gene transcription level.

Impaired accumulation of granulocytes in the lung during ozone adaptation.

Respiratory alterations induced by an acute exposure to ozone (O(3)) paradoxically resolve during multiday exposure. This adaptation is characteristically accompanied by a gradual attenuation of lung neutrophilia. As maintenance of neutrophilia at the site of inflammation is due to cytokine-mediated delayed neutrophil apoptosis, which is associated with reduced levels of Bax, a proapoptotic protein, we sought to determine whether defects in these mechanisms could account for O(3) adaptation. Lung granulocytes obtained at different time points from calves exposed to 0.75 ppm O(3) for 12 h/d for 7 consecutive days neither showed enhancement of survival nor Bax deficiency, when compared to blood granulocytes. To further investigate the effects of an exogenous oxidative stress on neutrophil survival, human granulocytes were treated with hydrogen peroxide alone, or in combination with granulocyte/macrophage colony-stimulating factor, an antiapoptotic cytokine. Both treatments led to rapid apoptosis associated with downregulation of Bcl-x(L) and Bcl-2, two antiapoptotic proteins. This study shows that O(3) adaptation is associated with a failure in the mechanisms leading to accumulation of neutrophils at the site of inflammation, and suggests that this defect is due to direct proapoptotic effects of exogenous oxidative stress on granulocytes.

Molybdenum content of term and preterm human milk during the first 2 months of lactation.

To assess the molybdenum supply and requirements of preterm infants, Mo concentration was determined in milk from mothers of 6 term and 11 preterm newborns; no difference was found between fore- and hindmilk and no diurnal variations were found during 24-h collections. Respective values (means +/- SD) of term and preterm milks were 10.2 +/- 3.7 and 4.0 +/- 3.7 micrograms/L (106.2 +/- 38.5 and 41.7 +/- 38.5 nmol/L) at 3-5 d of lactation, 4.8 +/- 3.9 and 3.7 +/- 3.8 micrograms/L (50.0 +/- 4.6 and 38.5 +/- 39.6 nmol/L) at 7-10 d, 1.5 +/- 1.4 and 1.4 +/- 0.9 micrograms/L (15.6 +/- 14.6 and 14.6 +/- 9.6 nmol/L) at 14 d, 2.6 +/- 2.2 and 1.9 +/- 1.4 micrograms/L (27.1 +/- 22.9 and 19.8 +/- 14.6 nmol/L) at 1 mo, and 0.2 and 1.2 +/- 0.5 micrograms/L (2.1 and 12.5 +/- 5.2 nmol/L) at 2 mo. A statistical difference was found between term and preterm milk at 3-5 d of lactation. During lactation significant changes were found between the periods 3-5 d and 7-10 d, 14 d, 1 mo (p less than 0.01) and 2 mo (p less than 0.05) of lactation and between 7-10 d and 14 d (p less than 0.05). According to the requirements of the preterm infant, a supplementation of 2-3 micrograms.kg-1.d-1 by enteral route is suggested.

Potentiation of cisplatin and carboplatin cytotoxicity by amphotericin B in different human ovarian carcinoma and malignant peritoneal mesothelioma cells.

An in vitro study of the combined cytotoxicity of either cisplatin (CDDP) or carboplatin and amphotericin B (AmB) was undertaken on a set of different ovarian carcinoma (IGROVI, IGROVI-C10, OAW42) and peritoneal malignant mesothelioma (CFB-CARP1) cell lines and ascitic cells freshly obtained from ovarian cancer patients so as to investigate the possibility of overcoming their resistance to platinum compounds. Growth-inhibition curves obtained 6 days after a 2-h period of exposure to the drugs showed that AmB at 5-10 mg/l allowed a 5- to 10-fold decrease in the 50% growth-inhibitory concentrations (IC50) of CDDP and carboplatin on either sensitive or resistant cells. Intracellular platinum assays with IGROVI cells showed that AmB acted by increasing dramatically the platinum uptake at a proportion that accounted for the increase in cytotoxicity. In the subline IGROVI-C10, a 10-fold resistant subline of IGROVI, AmB at 10 mg/l allowed recovery to the level of sensitivity seen in the parental cell line in the absence of AmB but not to the level observed in the presence of AmB. Acquisition of resistance mechanisms that are independent of the regulation of platinum uptake might be involved in this cell line. Thus, AmB might act by increasing the intracellular concentration of platinum without modifying the resistance mechanism involved downstream. However, in our models an increase in the intracellular level of platinum was always sufficient for the recovery of chemosensitivity in vitro. We also show that the phosphodiesterase inhibiting methylxanthines act synergistically with AmB. The latter drugs are weakly toxic and could also attenuate the nephrotoxicity of AmB.

Comparison of serum copper determination by colorimetric and atomic absorption spectrometric methods in seven different laboratories. The S.F.B.C. (Société Française de Biologie Clinique) Trace Element Group.

An interlaboratory collaborative trial was conducted on the determination of serum copper using two different methods, based on colorimetry (test combination Copper, Boehringer Mannheim, Mannheim, Germany) and flame atomic absorption spectrometry (FAAS). The general performance of the colorimetric method was below that of FAAS, except for sensitivity and linear range, as assessed by detection limit (0.44 versus 1.32 mumol/L) and upper limit of linearity (150 versus 50 mumol/L). The range of the between-run CVs and the recovery of standard additions were, respectively, 2.3-11.9% and 92-127% for the colorimetric method and 1.1-6.0% and 93-101% for the FAAS method. Interferences were minimal with both methods. The two techniques correlated satisfactorily (the correlation coefficients ranged from 0.945-0.970 among laboratories) but the colorimetric assay exhibited slightly higher results than the FAAS method. Each method was transferable among laboratories.

Mechanisms of absorption of caseinophosphopeptide bound iron.

Binding iron (Fe) to the 1-25 caseinophosphopeptide obtained from enzyme hydrolysis of beta casein (beta CPP) improves Fe bioavailability in the rat. To assess the mechanisms involved in its absorption, a perfused, vascularized duodenal rat loop model was used in controls and in Fe-deficient (bleeding of 25% blood volume) rats. Inhibitors of oxidative phosphorylation [2-4 dinitrophenol (DNP)] and/or of endocytosis [phenylarsine oxide (PAO)] were added to the perfusion solution containing 50 microM Fe as beta CPP bound Fe (Fe-beta CPP) or gluconate (Fe Gluc). Fe-beta CPP enhanced Fe uptake, reduced mucosal storage, and improved net absorption both in controls and in deficient animals. DNP reduced uptake, mucosal storage, and net absorption by the same percentage in Fe-beta CPP and Fe Gluc perfused rats in both control and Fe-deficient animals. PAO decreased uptake, mucosal storage, and net absorption of Fe-beta CPP but not of Fe Gluc. At the end of the experiment Fe serum levels were increased only in Fe Gluc animals. These results confirm the improved bioavailability of beta CPP bound Fe. They suggest that at least part of its absorption can occur by a different pathway than usual Fe salts. Fe-beta CPP can be taken up by endocytosis and absorbed bound to amino acids or peptides.

Combined diagnostic value of biochemical markers in acute pancreatitis.

Eighty-three patients suffering from upper abdominal pain were studied to evaluate the contribution of commonly used biochemical markers in the diagnosis of acute pancreatitis. On admission to hospital, serum amylase, lipase, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyl transferase activities were measured. By stepwise logistic discrimination, only two determinations appeared to be of clinical value: lipase and alkaline phosphatase activities. A classification rule was established including these two measurements and its diagnostic performance evaluated by a jackknifed method amounted .83%. ROC curves were used to assess sensitivity and specificity. Our study clearly shows that serum lipase measurements should be preferred to amylase measurements, and that our two-test classification rule provides an efficient aid in clinical decision-making.

NF-kappaB activation in response to toxical and therapeutical agents: role in inflammation and cancer treatment.

The NF-kappaB transcription factor is ubiquitously expressed and controls the expression of a large number of genes. Experimental data clearly indicate that NF-kappaB is a major regulator of the inflammatory reaction by controlling the expression of pro-inflammatory molecules in response to cytokines, oxidative stress and infectious agents. We demonstrated that NF-kappaB activation by IL-1beta follows three distinct cell-specific pathways. Moreover, our studies indicated that in one model of inflammatory diseases, horse recurrent airway obstruction (RAO), the extent of NF-kappaB basal activity correlates with pulmonary dysfunction. Another role of NF-kappaB activity protects cancer cells against apoptosis and could participate in the resistance to cancer treatment. However, we did not observe any increased cytotoxicity after treatment with anticancer drugs or TNF-alpha of cells expressing a NF-kappaB inhibitor. Therefore, we can conclude that the inhibition of apoptosis by NF-kappaB is likely to be cell type and stimulus-dependent. Further studies are required to determine whether NF-kappaB could be a target for anticancer treatments.

Zinc and iron status and growth in healthy infants.

OBJECTIVE: To elucidate the relationships between growth and zinc and iron status in normal infants. STUDY DESIGN: Growth of normal infants (less than 3 y old: n = 66) was prospectively assessed with a mean delay of 24+/-6 weeks between measurements; subjects were free from illness and presented with a normal growth. Growth was compared to serum zinc (s-zinc), IGF-1 and iron status. SETTING: Teaching hospital of Caen. RESULTS: No relation was found between linear or weight growth and s-zinc; when taking into account the effect of age, linear growth was significantly associated with ferritin (P<0.001); weight gain was significantly correlated with IGF-1 (P = 0.034) and ferritin (P = 0.008). No relationship was found between s-zinc and iron status. CONCLUSIONS: In normal infants iron status, more than serum zinc, seems to be correlated with growth.

Relationship between bone mineralization and aluminium in the healthy infant.

OBJECTIVE: This prospective study was designed to assess the relationship between variations of serum Aluminium levels and bone mineralization, which is one of its target tissues, in healthy premature (PT) and fullterm (FT) infants. STUDY DESIGN: Lumbar spine bone mineral density (BMD) and content (BMC) studied by dual energy X-ray absorptiometry were compared to serum aluminium (S-Al), Ca (S-Ca), P (S-P), osteocalcin, alkaline phosphatase activity (S-AP), and 25 OH Vitamin D (25 OH D) by simple and multiple regressions in healthy PT (n = 44) following their hospital discharge and FT (n = 82). PT (gestational age at birth (mean +/- 1 s.d.) 32 +/- 2 weeks) and FT were 43 +/- 39 and 36 +/- 32 weeks old respectively. RESULTS: In PT multiple stepwise regression analysis including gestational age at birth, postconceptional age and postnatal age displayed only a significant correlation between BMD or BMC and postnatal age and a negative one with S-Al. In FT correlations were found between BMD or BMC and age and S-Ca. CONCLUSIONS: In PT, variations in blood Al are associated with developmental delays. Care should be taken to lessen Al levels, even in healthy PT babies.