In vitro heat exposure induces a redistribution of nuclear matrix proteins in human K562 erythroleukemia cells.

By using both conventional and confocal laser scanning microscopy with three monoclonal antibodies recognizing nuclear matrix proteins we have investigated by means of indirect fluorescence whether an incubation of isolated nuclei at the physiological temperature of 37 degrees C induces a redistribution of nuclear components in human K562 erythroleukemia cells. Upon incubation of isolated nuclei for 45 min at 37 degrees C, we have found that two of the antibodies, directed against proteins of the inner matrix network (M(r) 125 and 160 kDa), gave a fluorescent pattern different from that observed in permeabilized cells. By contrast, the fluorescent pattern did not change if nuclei were kept at 0 degrees C. The difference was more marked in case of the 160-kDa polypeptide. The fluorescent pattern detected by the third antibody, which recognizes the 180-kDa nucleolar isoform of DNA topoisomerase II, was unaffected by heat exposure of isolated nuclei. When isolated nuclear matrices prepared from heat-stabilized nuclei were stained by means of the same three antibodies, it was possible to see that the distribution of the 160-kDa matrix protein no longer corresponded to that observable in permeabilized cells, whereas the fluorescent pattern given by the antibody to the 125-kDa polypeptide resembled that detectable in permeabilized cells. The 180-kDa isoform of topoisomerase II was still present in the matrix nucleolar remnants. We conclude that a 37 degrees C incubation of isolated nuclei induces a redistribution of some nuclear matrix antigens and cannot prevent the rearrangement in the spatial organization of one of these antigens that takes place during matrix isolation in human erythroleukemia cells. The practical relevance of these findings is discussed.

Repeated exposure to Lutzomyia intermedia sand fly saliva induces local expression of interferon-inducible genes both at the site of injection in mice and in human blood.

During a blood meal, Lutzomyia intermedia sand flies transmit Leishmania braziliensis, a parasite causing tegumentary leishmaniasis. In experimental leishmaniasis, pre-exposure to saliva of most blood-feeding sand flies results in parasite establishment in absence of any skin damages in mice challenged with dermotropic Leishmania species together with saliva. In contrast, pre-immunization with Lu. intermedia salivary gland sonicate (SGS) results in enhanced skin inflammatory exacerbation upon co-inoculation of Lu. intermedia SGS and L. braziliensis. These data highlight potential unique features of both L. braziliensis and Lu. intermedia. In this study, we investigated the genes modulated by Lu. intermedia SGS immunization to understand their potential impact on the subsequent cutaneous immune response following inoculation of both SGS and L. braziliensis. The cellular recruitment and global gene expression profile was analyzed in mice repeatedly inoculated or not with Lu. intermedia. Microarray gene analysis revealed the upregulation of a distinct set of IFN-inducible genes, an immune signature not seen to the same extent in control animals. Of note this INF-inducible gene set was not induced in SGS pre-immunized mice subsequently co-inoculated with SGS and L. braziliensis. These data suggest the parasite prevented the upregulation of this Lu. intermedia saliva-related immune signature. The presence of these IFN-inducible genes was further analyzed in peripheral blood mononuclear cells (PBMCs) sampled from uninfected human individuals living in a L. braziliensis-endemic region of Brazil thus regularly exposed to Lu. intermedia bites. PBMCs were cultured in presence or absence of Lu. intermedia SGS. Using qRT-PCR we established that the IFN-inducible genes induced in the skin of SGS pre-immunized mice, were also upregulated by SGS in PBMCs from human individuals regularly exposed to Lu. intermedia bites, but not in PBMCs of control subjects. These data demonstrate that repeated exposure to Lu. intermedia SGS induces the expression of potentially host-protective IFN-inducible genes.

Urinary hexane diamine to assess respiratory exposure to hexamethylene diisocyanate aerosol: a human inhalation study

The use of urinary hexane diamine (HDA) as a biomarker to assess human respiratory exposure to hexamethylene diisocyanate (HDI) aerosol was evaluated. Twenty-three auto body shop workers were exposed to HDI biuret aerosol for two hours using a closed exposure apparatus. HDI exposures were quantified using both a direct-reading instrument and a treated-filter method. Urine samples collected at baseline, immediately post exposure, and every four to five hours for up to 20 hours were analyzed for HDA using gas chromatography and mass spectrometry. Mean urinary HDA (microg/g creatinine) sharply increased from the baseline value of 0.7 to 18.1 immediately post exposure and decreased rapidly to 4.7, 1.9 and 1.1, respectively, at 4, 9, and 18 hours post exposure. Considerable individual variability was found. Urinary HDA can assess acute respiratory exposure to HDI aerosol, but may have limited use as a biomarker of exposure in the workplace. [Authors]

Sex differences in urinary levels of several biological indicators of exposure: a human volunteer study

The aim of the study was to quantify the variability on biological indicators of exposure between men and women for three well known solvents: methyl ethyl ketone, 1-methoxy-2-propanol and 1,1,1-trichloroethane. Another purpose was to explore the effect of selected CYP2E1 polymorphisms on the toxicokinetic profile. Controlled human exposures were carried out in a 12m(3) exposure chamber for each solvent separately, during 6h and at half of the threshold limit value. The human volunteers groups were composed of ten young men and fifteen young women, including ten women using hormonal contraceptive. An analysis of variance mainly showed an effect on the urinary levels of several biomarkers of exposure among women due to the use of hormonal contraceptive, with an increase of more than 50% in metabolites concentrations and a decrease of up to 50% in unchanged substances concentrations, suggesting an increase in their metabolism rate. The results also showed a difference due to the genotype CYP2E1*6, when exposed to methyl ethyl ketone, with a tendency to increase CYP2E1 activity when volunteers were carriers of the mutant allele. Our study suggests that not only physiological differences between men and women but also differences due to sex hormones levels can have an impact on urinary concentrations of several biomarkers of exposure. The observed variability due to sex among biological exposure indices can lead to misinterpretation of biomonitoring results. This aspect should have its place in the approaches for setting limits of occupational exposure. [Authors]

Human leukocyte antigen class II variants and adult-onset asthma: does occupational allergen exposure play a role?

Recently, a locus centred on rs9273349 in the HLA-DQ region emerged from genome-wide association studies of adult-onset asthma. We aimed to further investigate the role of human leukocyte antigen (HLA) class II in adult-onset asthma and a possible interaction with occupational exposures. We imputed classical HLA-II alleles from 7579 single-nucleotide polymorphisms in 6025 subjects (1202 with adult-onset asthma) from European cohorts: ECRHS, SAPALDIA, EGEA and B58C, and from surveys of bakers and agricultural workers. Based on an asthma-specific job-exposure matrix, 2629 subjects had ever been exposed to high molecular weight (HMW) allergens. We explored associations between 23 common HLA-II alleles and adult-onset asthma, and tested for gene-environment interaction with occupational exposure to HMW allergens. Interaction was also tested for rs9273349. Marginal associations of classical HLA-II alleles and adult-onset asthma were not statistically significant. Interaction was detected between the DPB1*03:01 allele and exposure to HMW allergens (p = 0.009), in particular to latex (p = 0.01). In the unexposed group, the DPB1*03:01 allele was associated with adult-onset asthma (OR 0.67, 95%CI 0.53-0.86). HMW allergen exposures did not modify the association of rs9273349 with adult-onset asthma. Common classical HLA-II alleles were not marginally associated with adult-onset asthma. The association of latex exposure and adult-onset asthma may be modified by DPB1*03:01.

Gas-chromatography mass-spectrometry determination of phthalic acid in human urine as a biomarker of folpet exposure.

Agricultural workers are exposed to folpet, but biomonitoring data are limited. Phthalimide (PI), phthalamic acid (PAA), and phthalic acid (PA) are the ring metabolites of this fungicide according to animal studies, but they have not yet been measured in human urine as metabolites of folpet, only PA as a metabolite of phthalates. The objective of this study was thus to develop a reliable gas chromatography-tandem mass spectrometry (GC-MS) method to quantify the sum of PI, PAA, and PA ring-metabolites of folpet in human urine. Briefly, the method consisted of adding p-methylhippuric acid as an internal standard, performing an acid hydrolysis at 100 °C to convert ring-metabolites into PA, purifying samples by ethyl acetate extraction, and derivatizing with N,O-bis(trimethylsilyl)trifluoro acetamide prior to GC-MS analysis. The method had a detection limit of 60.2 nmol/L (10 ng/mL); it was found to be accurate (mean recovery, 97%), precise (inter- and intra-day percentage relative standard deviations <13%), and with a good linearity (R (2) > 0.98). Validation was conducted using unexposed peoples urine spiked at concentrations ranging from 4.0 to 16.1 μmol/L, along with urine samples of volunteers dosed with folpet, and of exposed workers. The method proved to be (1) suitable and accurate to determine the kinetic profile of PA equivalents in the urine of volunteers orally and dermally administered folpet and (2) relevant for the biomonitoring of exposure in workers.

A New Approach for the Carbon Monoxide (CO) Exposure Diagnosis: Measurement of Total CO in Human Blood Versus Carboxyhemoglobin (HbCO).

The aim of the study is to present the application of a headspace-gas chromatography-mass spectrometry (HS-GC-MS) method for the determination of the carbon monoxide (CO) blood concentration and to compare it with carboxyhemoglobin (HbCO) saturation. In postmortem cases, the HbCO measured by spectrophotometry frequently leads to inaccurate results due to inadequate samples or analyses. The true role of CO intoxication in the death of a person could be misclassified. The estimation of HbCO from HS-GC-MS CO measurements provides helpful information by determining the total CO levels (CO linked to hemoglobin (HbCO) and CO dissociated from hemoglobin). The CO concentrations were converted in HbCO saturation levels to define cutoff blood CO values. CO limits were defined as less than 1 μmol/mL for living persons, less than 1.5 μmol/mL for dead persons without CO exposure, and greater than 3 μmol/mL for dead persons with clear CO poisoning.

A toxicokinetic model to assess exposure to folpet fungicide from urinary biomarkers

Folpet is one of the most widely employed fungicides in agriculture. It is typically used in the culture of vegetables, fruits and ornamental plants. Once absorbed in the human body, it has been found to be very reactive, especially in acid conditions. According to various in vitro and in vivo experiments in animals, Folpet is first fractioned at the N-S link when in contact with aqueous solutions and thiol groups. From this non-enzymatic process a phthalimide (PI) molecule is formed, which may be used as a biomarker of exposure, along with the short-lived thiophosgene. We have built a human toxicokinetic model to account for the biotransformation of Folpet into PI and its subsequent excretion while accounting for other non-monitored metabolites. The mathematical parameters of the model were determined accordingly from best-fits to the time courses of PI in blood and urine of five volunteers administered orally 1 mg/kg and dermally 10 mg/kg of Folpet. In both cases, the mean elimination half-life of PI from the body (either through faeces, urine or metabolism) was found to be 31.6 h. The average final fractions of administered dose recovered in urine as PI were 0.025% and 0.002%, for oral and dermal administration, respectively after 96 h. According to the model, when orally administered, PI rapidly hydrolyzes to phthalamic and phthalic acids such that only 0.04% of the PI found in the gastrointestinal tract is absorbed into the blood stream. Likewise, after dermal application, model predicts that only 7.4% of the applied Folpet dose crosses the epidermis. In the model, the PI initial metabolite of Folpet is formed in the dermis and further metabolized prior to reaching systemic circulation, such that only 0.125% of PI formed at the site-of-entry reaches systemic blood. Our mathematical model is in accordance with both measures of blood (R2=0.57 for dermal and R2=0.66 for oral) and urine (R2 =0.98 for dermal and R2=0.99 for oral).

Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases.

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.

Regulation of the akt signalling in human skeletal muscle atrophy

Abstract : The term "muscle disuse" is often used to refer collectively to reductions in neuromuscular activity as observed with sedentary lifestyles, reduced weight bearing, cancer, chronic obstructive pulmonary disease, chronic heart failure, spinal cord injury, sarcopenia or exposure to microgravity (spaceflight). Muscle disuse atrophy, caused by accelerated proteolysis, is predominantly due to the activation of the ATP-dependent ubiquitin (Ub) proteasome pathway. The current advances in understanding the molecular factors contributing to the Ub-dependent proteolysis process have been made mostly in rodent models of human disease and denervation with few investigations performed directly in humans. Recently, in mice, the genes Atrogin-1 and MuRF1 have been designated as primary candidates in the control of muscle atrophy. Additionally, the decreased activity of the Akt/GSK-3ß and Akt/mTOR pathways has been associated with a reduction in protein synthesis and contributing to skeletal muscle atrophy. Therefore, it is now commonly accepted that skeletal muscle atrophy is the result of a decreased protein synthesis concomitant with an increase in protein degradation (Glass 2003). Atrogin-1 and MuRF1 are genes expressed exclusively in muscle. In mice, their expression has been shown to be directly correlated with the severity of atrophy. KO-mice experiments showed a major protection against atrophy when either of these genes were deleted. Skeletal muscle hypertrophy is an important function in normal postnatal development and in the adaptive response to exercise. It has been shown, in vitro, that the activation of phosphatidylinositol 3-kinase (PI-3K), by insulin growth factor 1 (IGF-1), stimulates myotubes hypertrophy by activating the downstream pathways, Akt/GSK-3ß and Akt/mTOR. It has also been demonstrated in mice, in vivo, that activation of these signalling pathways causes muscle hypertrophy. Moreover, the latter were recently proposed to also reduce muscle atrophy by inhibiting the FKHR mediated transcription of several muscle atrophy genes; Atrogin-1 and MuRF1. Therefore, these targets present new avenues for developing further the understanding of the molecular mechanisms involved in both skeletal muscle atrophy and hypertrophy. The present study proposed to investigate the regulation of the Akt/GSK-3ß and Akt/mTOR signalling pathways, as well as the expression levels of the "atrogenes", Atrogin-1 and MuRF1, in four human models of skeletal muscle atrophy. In the first study, we measured the regulation of the Akt signalling pathway after 8 weeks of both hypertrophy stimulating resistance training and atrophy stimulation de-training. As expected following resistance training, muscle hypertrophy and an increase in the phosphorylation status of the different members of the Akt pathway was observed. This was paralleled by a concomitant decrease in FOXO1 nuclear protein content. Surprisingly, exercise training also induced an increase in the, expression of the atrophy genes and proteins involved in the ATP-dependant ubiquitin-proteasome system. On the opposite, following the de-training period a muscle atrophy, relative to the post-training muscle size, was measured. At the same time, the phosphorylation levels of Akt and GSK-3ß were reduced while the amount of FOXO1 in the nucleus increased. After the atrophy phase, there was also a reduction in Atrogin-1 and MuRF1 contents. In this study, we demonstrate for the first time in healthy human skeletal muscle, that the regulation of Akt and its downstream targets GSK-3ß, mTOR and FOXO1 are associated with both thé skeletal muscle hypertrophy and atrophy processes. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the loss of both upper and lower motor neurons, which leads to severe muscle weakness and atrophy. All measurements were performed in biopsies from 22 ALS patients and 16 healthy controls. ALS patients displayed an increase in Atrogin-1 mRNA and protein content which was associated with a decrease in Akt activity. However there was no difference in the mRNA and phospho-protein content of FOXO1, FOXO3a, p70S6K and GSK-3ß. The transcriptional regulation of human Atrogin-1 may be controlled by an Akt-mediated transcription factor other than FKHR or via an other signalling pathway. Chronic complete spinal cord injury (SCI) is associated with severe muscle atrophy which is linked to co-morbidity factors such as diabetes, obesity, lipid disorders and cardiovascular diseases. Molecular mechanisms associated with chronic complete SCI-related muscle atrophy are not well understood. The aim of the present study was to determine if there was an increase in catabolic signalling targets such as Atrogin-1, MuRF1, FOXO and myostatin, and decreases in anabolic signalling targets such as IGF, Akt, GSK-3ß, mTOR, 4E-BP1 and p-70S6K in chronic complete SCI patients. All measurements were performed in biopsies taken from 8 complete chronic SCI patients and 7 age matched healthy controls. In SCI patients when compared with controls, there was a significant reduction in mRNA levels of Atrogin1, MuRF1 and Myostatin. Protein levels for Atrogin-1, FOX01 and FOX03a were also reduced. IGF-1 and both phosphorylated GSK-3ß and 4E-BP1 were decreased; the latter two in an Akt and mTOR independent manner, respectively. Reductions in Atrogin-1, MuRF1, FOXO and myostatin suggest the existence of an internal mechanism aimed at reducing further loss of muscle proteins during chronic SCI. The downregulation of signalling proteins regulating anabolism such as IGF, GSK3ß and 4E-BP1 would reduce the ability to increase protein synthesis rates in this chronic state of muscle wasting. The molecular mechanisms controlling age-related skeletal muscle loss in humans are poorly understood. The present study aimed to investigate the regulation of several genes and proteins involved in the activation of key signalling pathways promoting muscle hypertrophy such as GH/STAT5/IGF, IGF/Akt/GSK-3ß/4E-BP1 and muscle atrophy such as TNFα/SOCS3 and Akt/FOXO/Atrogin-1 or MuRF1 in muscle biopsies from 13 young and 16 elderly men. In the older, as compared with the young subjects, TNFα and SOCS-3 were increased while growth hormone receptor protein (GHR) and IGF-1 mRNA were both decreased. Akt protein levels were increased however no change in phosphorylated Akt content was observed. GSK-3ß phosphorylation levels were increased while 4E-BP1 was not changed. Nuclear FKHR and FKHRL1 protein levels were decreased, with no changes in their atrophy target genes, Atrogin-1 and MuRF1. Myostatin mRNA and protein levels were significantly elevated. Human sarcopenia may be linked to a reduction in the activity or sensitivity of anabolic signalling proteins such as GHR, IGF and Akt. TNFα, SOCS-3 and myostatin are potential candidates influencing this anabolic perturbation. In conclusion our results support those obtained in rodent or ín vitro models, and demonstrate Akt plays a pivotal role in the control of muscle mass in humans. However, the Akt phosphorylation status was dependant upon the model of muscle atrophy as Akt phosphorylation was reduced in all atrophy models except for SCI. Additionally, the activity pattern of the downstream targets of Akt appears to be different upon the various human models. It seems that under particular conditions such as spinal cord injury or sarcopenia, .the regulation of GSK-3ß, 4eBP1 and p70S6K might be independent of Akt suggesting alternative signalling pathways in the control of these the anabolic response in human skeletal muscle. The regulation of Atrogin-1 and MuRF1 in some of our studies has been shown to be also independent of the well-described Akt/FOXO signalling pathway suggesting that other transcription factors may regulate human Atrogin-1 and MuRF1. These four different models of skeletal muscle atrophy and hypertrophy have brought a better understanding concerning the molecular mechanisms controlling skeletal muscle mass in humans.

IL-13 induces expression of CD36 in human monocytes through PPARgamma activation.

The class B scavenger receptor CD36 is a component of the pattern recognition receptors on monocytes that recognizes a variety of molecules. CD36 expression in monocytes depends on exposure to soluble mediators. We demonstrate here that CD36 expression is induced in human monocytes following exposure to IL-13, a Th2 cytokine, via the peroxisome proliferator-activated receptor (PPAR)gamma pathway. Induction of CD36 protein was paralleled by an increase in CD36 mRNA. The PPARgamma pathway was demonstrated using transfection of a PPARgamma expression plasmid into the murine macrophage cell line RAW264.7, expressing very low levels of PPARgamma, and in peritoneal macrophages from PPARgamma-conditional null mice. We also show that CD36 induction by IL-13 via PPARgamma is dependent on phospholipase A2 activation and that IL-13 induces the production of endogenous 15-deoxy-Delta12,14-prostaglandin J2, an endogenous PPARgamma ligand, and its nuclear localization in human monocytes. Finally, we demonstrate that CD36 and PPARgamma are involved in IL-13-mediated phagocytosis of Plasmodium falciparum-parasitized erythrocytes. These results reveal a novel role for PPARgamma in the alternative activation of monocytes by IL-13, suggesting that endogenous PPARgamma ligands, produced by phospholipase A2 activation, could contribute to the biochemical and cellular functions of CD36.

Effect of intermittent hypoxic training on HIF gene expression in human skeletal muscle and leukocytes

Intermittent hypoxic exposure with exercise training is based on the assumption that brief exposure to hypoxia is sufficient to induce beneficial muscular adaptations mediated via hypoxia-inducible transcription factors (HIF). We previously demonstrated (Mounier et al. Med Sci Sports Exerc 38:1410-1417, 2006) that leukocytes respond to hypoxia with a marked inter-individual variability in HIF-1alpha mRNA. This study compared the effects of 3 weeks of intermittent hypoxic training on hif gene expression in both skeletal muscle and leukocytes. Male endurance athletes (n = 19) were divided into an Intermittent Hypoxic Exposure group (IHE) and a Normoxic Training group (NT) with each group following a similar 3-week exercise training program. After training, the amount of HIF-1alpha mRNA in muscle decreased only in IHE group (-24.7%, P < 0.05) whereas it remained unchanged in leukocytes in both groups. The levels of vEGF(121) and vEGF(165) mRNA in skeletal muscle increased significantly after training only in the NT group (+82.5%, P < 0.05 for vEGF(121); +41.2%, P < 0.05 for vEGF(165)). In leukocytes, only the IHE group showed a significant change in vEGF(165) (-28.2%, P < 0.05). The significant decrease in HIF-1alpha mRNA in skeletal muscle after hypoxic training suggests that transcriptional and post-transcriptional regulations of the hif-1alpha gene are different in muscle and leukocytes.

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells.

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 degrees C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents.