Psychological and cognitive effects of laser printer emissions: A controlled exposure study.

The possible impact of ultrafine particles from laser printers on human health is controversially discussed although there are persons reporting substantial symptoms in relation to these emissions. A randomized, single-blinded, cross-over experimental design with two exposure conditions (high-level and low-level exposure) was conducted with 23 healthy subjects, 14 subjects with mild asthma, and 15 persons reporting symptoms associated with laser printer emissions. To separate physiological and psychological effects, a secondary physiologically based categorization of susceptibility to particle effects was used. In line with results from physiological and biochemical assessments, we found no coherent, differential, or clinically relevant effects of different exposure conditions on subjective complaints and cognitive performance in terms of attention, short-term memory, and psychomotor performance. However, results regarding the psychological characteristics of participants and their situational perception confirm differences between the participants groups: Subjects reporting symptoms associated with laser printer emissions showed a higher psychological susceptibility for adverse reactions in line with previous results on persons with multiple chemical sensitivity or idiopathic environmental intolerance. In conclusion, acute psychological and cognitive effects of laser printer emissions were small and could be attributed only to different participant groups but not to differences in exposure conditions in terms of particle number concentrations.

Health effects of laser printer emissions: a controlled exposure study.

Ultrafine particles emitted from laser printers are suspected to elicit adverse health effects. We performed 75-minute exposures to emissions of laser printing devices (LPDs) in a standardized, randomized, cross-over manner in 23 healthy subjects, 14 mild, stable asthmatics, and 15 persons reporting symptoms associated with LPD emissions. Low-level exposures (LLE) ranged at the particle background (3000 cm-3 ) and high-level exposures (HLE) at 100 000 cm-3 . Examinations before and after exposures included spirometry, body plethysmography, transfer factors for CO and NO (TLCO, TLNO), bronchial and alveolar NO, cytokines in serum and nasal secretions (IL-1ß, IL-5, IL-6, IL-8, GM-CSF, IFNγ, TNFα), serum ECP, and IgE. Across all participants, no statistically significant changes occurred for lung mechanics and NO. There was a decrease in volume-related TLNO that was more pronounced in HLE, but the difference to LLE was not significant. ECP and IgE increased in the same way after exposures. Nasal IL-6 showed a higher increase after LLE. There was no coherent pattern regarding the responses in the participant subgroups or single sets of variables. In conclusion, the experimental acute responses to short but very high-level LPD exposures were small and did not indicate clinically relevant effects compared to low particle number concentrations.

Development of a simplified urban water balance model (WABILA).

During the last decade, water sensitive urban design (WSUD) has become more and more accepted. However, there is not any simple tool or option available to evaluate the influence of these measures on the local water balance. To counteract the impact of new settlements, planners focus on mitigating increases in runoff through installation of infiltration systems. This leads to an increasing non-natural groundwater recharge and decreased evapotranspiration. Simple software tools which evaluate or simulate the effect of WSUD on the local water balance are still needed. The authors developed a tool named WABILA (Wasserbilanz) that could support planners for optimal WSUD. WABILA is an easy-to-use planning tool that is based on simplified regression functions for established measures and land covers. Results show that WSUD has to be site-specific, based on climate conditions and the natural water balance.

UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation.

Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction. Uridine diphosphate-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway. UDP-GlcNAc 2-epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules.

Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis.

Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the "fireman's grip" interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the "fireman's grip" is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the "fireman's grip" is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme.

The age dependence of intracellular proteolysis: changes of the substrate proteins.

Liver cytosol proteins of young (4--6 months) and old (18--27 months) rats were degraded in vitro by papain, pronase, trypsin, pepsin, cathepsin D from rat liver and a soluble lysosomal enzyme mixture from rat liver. We could demonstrate the capability of the latter enzyme mixture to degrade proteolytically the cytosol proteins of young animals about 20% faster than those of the older animal group. Digesting radioactive labelled "young" cytosol in the presence of unlabelled "old" cytosol the possibility could be excluded, that this effect was due to an inhibitor of macromolecular size present in the "old" cytosol.

Interleukin-17 stimulates the expression of IkappaB alpha mRNA and the secretion of IL-6 and IL-8 in glioblastoma cell lines.

Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ activated memory T cells. In an effort to elucidate the biological effects of IL-17 in glial cells, we investigated the ability of this cytokine in order to activate nuclear factor (NF)-kappaB, which is being discussed as one of the most important transcription factors in the regulation of neuronal and glial cell function. Activation of NF-kappaB involves the degradation of its cytoplasmatic inhibitor IkappaB-alpha, which allows the nuclear translocation of NF-kappaB, and ensures transcriptional activation of genes including IkappaB-alpha itself. Using a competitive RT-PCR, we examined the IL-17-induced IkappaB-alpha mRNA expression in glioblastoma cells, and we examined IL-17 up-regulated IkappaB-alpha mRNA expression in a dose- and time-dependent fashion with a maximum time between 1 and 3 h. This induction could be inhibited by Calphostin C (protein kinase C inhibitor) and genistein (tyrosine kinase inhibitor). After 60 min of IL-17 stimulation, a degradation of the IkappaB-alpha protein was detectable. Furthermore, IL-17 stimulated the secretion of IL-6 and IL-8 in glial cells, and IL-17 and IL-1beta in combination showed a superadditive effect. We suggest IL-17 to play a role as an immune factor, possibly involved in complex pathophysiological interactions of neurodegenerative diseases.

Expression of aminopeptidase N/CD13 in tumour-infiltrating lymphocytes from human renal cell carcinoma.

We have previously demonstrated the expression of aminopeptidase N (APN, CD13) on synovial T cells from patients with different forms of arthritis. T cells of peripheral blood and serous body fluids are CD13-negative but can be stimulated to express CD13 after activation, e.g., with Con A. In the present report, double-labelling and flow cytometry analyses were performed to characterize the phenotype of tumour-infiltrating lymphocytes (TIL). A large panel of antibodies specific for different activation-associated molecules on T cells was used. In contrast to TIL of lung cancer, TIL of renal cell carcinoma (RCC) consisted of significantly higher percentages of T cells expressing CD13, dipeptidylpeptidase N (DPIV, CD26) and HLA-DR, whereas T cells of lung cancer expressed more CD25, CD69 and CD54/ICAM1. No differences could be found in the expression of CD45RO, CD49a/VLA-1 and CD62L/L-selectin. Our results demonstrate that T cells in RCC and lung cancer differ in their phenotype, especially with respect to surface aminopeptidases. Investigations into the function of APN on T cells could be of help in gaining deeper insight into tumour defence as well as into general mechanisms of T cell functions.

Constitutive expression of HLA class II mRNA in synovial fibroblast-like cells from patients with rheumatoid arthritis .

We describe the quantification of the absolute amounts of HLA class II mRNA and class II transactivator (CIITA) mRNA by competitive reverse transcription polymerase chain reaction in cultured synovial fibroblast-like cells (SFC) of patients with rheumatoid arthritis. High basal levels of transcription of class II mRNA (10(7)-10(9) molecules/microgram total RNA) and CIITA mRNA were detected in cultured SFC, with DPB < DRB = DQB, although SFC only express small amounts of MHC class II proteins. In contrast to SFC, we did not detect class II mRNA nor CIITA mRNA in skin fibroblasts. After treatment with IFN-gamma, we observed a 3- to 28-fold increase in class II mRNA in SFC and an increase of DRB and DPB in skin fibroblasts from undetectable levels to 10(8)-10(9) molecules/microgram total RNA.

Characterization of the immunophenotype and functional properties of fibroblast-like synoviocytes in comparison to skin fibroblasts and umbilical vein endothelial cells.

We characterized the immunophenotype as well as functional properties--phagocytosis, the uptake of acetylated LDL, and the expression of HLA class II antigens, adhesion molecules, and cytokine mRNA--of fibroblast-like synoviocytes from rheumatoid arthritis synovium. Skin fibroblasts (FB) and umbilical vein endothelial cells (HUVEC) were studied in parallel. Cytofluorometric immunophenotyping by use of 84 mAb and 2 lectins and immunofluorescence microscopy indicated a high degree of homology between the three cell types. Only staining with mAb to von Willebrand factor (vWF) and CD31 and the lectin UEA-I appeared specific to HUVEC, whereas the mAb 5B5 to prolyl 4-hydroxylase that has been reported to be specific to FB stained HUVEC as well as synoviocytes and FB. All of the cells phagocytosed fluorescent latex beads of 1.7 and 2.6 microns in size. The uptake of acetylated LDL could be shown by HUVEC and, surprisingly, by synoviocytes, but not by FB. The induction of HLA-DR, -DP, and -DQ by IFN-gamma on the three cell types showed a similar dose-dependence. The upregulation of ICAM-1 by IL-1 alpha, TNF-alpha, and IFN-gamma appeared similar, whereas the induction of VCAM-1 by IL-1 alpha, IL-4, TNF-alpha, and IFN-gamma showed differences between the three cell types. ELAM-1 was expressed only on HUVEC after treatment with IL-1 alpha and TNF-alpha. The capacity of the cells to produce cytokines was studied at the level of mRNA by reverse transcription and PCR. All three cell types expressed the mRNA of IL-1 alpha, IL-6, IL-8, GM-CSF, and TGF-beta 1 spontaneously or after LPS stimulation, but never TNF-alpha mRNA. Our results indicate a high degree of relationship between the three cell types. In contrast to HUVEC, none of the markers and functional properties investigated appear specific to FB. Therefore, the issue of the origin of fibroblast-like synoviocytes and the role of vascular endothelial cells in the inflamed synovium is discussed.

Demonstration of CD13/aminopeptidase N on synovial fluid T cells from patients with different forms of joint effusions.

Cytofluorometric analysis was performed to characterize the immunophenotype of lymphocytes of the synovial fluid (SF) and the peripheral blood (PB) from patients suffering from juvenile chronic arthritis (JCA) or rheumatoid arthritis (RA). The most obvious difference could be found in expression of the surface protease aminopeptidase N (AP N/CD13). Whereas monoclonal antibodies specific to CD13 failed to reveal surface expression on lymphocytes of the PB; 63 +/- 15% of SF T cells gave positive staining for CD13 using Leu-M7. No correlation between CD13 expression and joint disease could be found in patients who had different types of inflammatory joint effusions. CD13 expression of T cells was also found in synovial tissue and inflammatory serous cavity effusions. Fixation of T cells revealed the presence of intracellular CD13 antigen already located in the PB T cells of healthy individuals. Induction of CD13 expression on PB T cells could be demonstrated after incubation with Con A/IL-2 or SF from patients with RA. Our findings suggest a role for AP N as a new activation-associated molecule of T lymphocytes.

Cell-cell contact of human T cells with fibroblasts changes lymphocytic mRNA expression: increased mRNA expression of interleukin-17 and interleukin-17 receptor.

Using random arbitrarily primed-reverse transcribed-PCR and sequence analysis, we investigated changes in lymphocytic molecules after cell-cell contact with fibroblasts. An mRNA species which was upregulated in Jurkat T cells by cell-cell contact with MRHF cells (a human foreskin fibroblast line) was identified as coding for the human interleukin-17 receptor. This finding was confirmed by quantitative RT-PCR for the HUT78 and Jurkat T cell lines, for peripheral blood lymphocytes, and for tonsillar T cells. Furthermore, the interleukin-17 mRNA, coding for a proinflammatory cytokine, was also upregulated in peripheral blood lymphocytes and tonsillar T cells after cell-cell contact with fibroblasts. Supernatants obtained from cell-cell contact-stimulated peripheral blood lymphocytes enhance the production of interleukin-6 and interleukin-8 by fibroblast-like synoviocytes and this effect could be blocked by interleukin-17 antibodies. Changes in the mRNA levels of Jurkat T cells induced by cell-cell contact with adherent cells were also found for M-type pyruvate kinase, for tropomyosin TM30 and for the p54nrb gene product.

Increased expression of interleukin-8 and aminopeptidase N by cell-cell contact: interleukin-8 is resistant to degradation by aminopeptidase N/CD13.

In rheumatic joints, high concentrations of interleukin-8 (IL-8) have been measured in synovial fluid and in pannus tissue. In both locations aminopeptidase N (APN)/CD13, an exopeptidase with reported activity towards IL-8 is also present. The surprising stability of IL-8 in the presence of an alleged IL-8-degrading peptidase prompted us to undertake the present study. Cocultivation of fibroblast-like synoviocytes (SFC) with T cells or with T lymphocytic cell membranes, or of T cells with SFC cell membranes, all resulted in increased IL-8 mRNA expression and IL-8 secretion into the medium, and an increase of APN expression on lymphocytes. IL-8 degradation was monitored by Western blots and HPLC. IL-8(72), as a partially processed form, was used throughout this study since it is abundant in tissues and has increased biological activity in comparison to IL-8(77). Thus its degradation/inactivation is considered of high biological significance. Whereas trypsin as a positive control rapidly degraded IL-8, we did not see any IL-8 degradation, either by a variety of soluble APNs, by leucine aminopeptidase or by APN expressed on the surface of SFC, or on ECV304 cells transfected with an APN expression vector. The much more sensitive HPLC technique resulted in negative results as well.

Occurrence of acetaldehyde protein adducts formed in various organs of chronically ethanol fed rats: an immunohistochemical study.

We investigated the pathophysiological role of acetaldehyde protein adducts formed in vivo in organs of chronically alcohol fed male Wistar rats. Thirty male Wistar rats were fed on rodent pellets and 15% alcohol (V/V) for 5, 8 and 12 months, respectively before they were sacrificed. Further 30 male rats were chosen as the control group. We tested several organs by histological and immunohistochemical methods. Using immunohistological analysis, in the 12 months groups the basal membranes of glomerula, the membranes of liver, skeleton muscle and heart cells, and the gut were stained positively for acetaldehyde adducts. Using Western blotting of liver cell fractions (mitochondria/ lysosomes; microsomes; cytosol) adducts in charateristic molecular weight regions were detected. Approximately 30% of the sera of experimental rats contained antibodies against the acetaldehyde adducts formed in vivo. Immunologically detectable acetaldehyde adducts could be found in all rat organs tested. The stage of alcohol disease attained in this experiment after 12 months of ethanol feeding is described as the initial phase of manifestations of disturbances that are seen also in the carbohydrate metabolism.

Comparative determination of the antiproteolytic potential of therapeutically used blood protein preparations.

Clinical situations with release of proteinases from blood cells or tissues into the circulation may result in a marked decrease of blood proteinase inhibitor content which in turn may result in a capillary leak syndrome, shock and even in exitus letalis. Replenishment of blood proteinase inhibitors is of benefit in such situations. In this study the inhibitory potential of fresh plasma, fresh frozen plasma and the liquid plasma protein preparation Biseko has been tested with the following enzymes: human leukocyte elastase, human plasmin, human matrix metalloproteinase-9, bovine trypsin, bovine chymotrypsin and rat liver lysosomal cathepsins. The concentration of the blood protein preparations resulting in 50% inhibition of constant amounts of each of the enzymes has been determined by plotting residual activity vs. log of concentration of blood protein preparation in enzyme assays. From these IC50 values inactivation ratios for 1 mg and 1 ml of the blood protein preparations was calculated. These inactivation ratios show an equal suitability of fresh plasma, fresh frozen plasma or full plasma proteins for replenishment of plasma proteinase inhibitory potential in vitro. As additional finding, commercial preparations of human serum albumin exert a surprisingly high inhibitory potential to lysosomal cathepsins.

Effects of twelve months ethanol feeding on rat liver metabolic activity.

Thirty male Wistar rats were fed with 15% ethanol (V/V) for one year. Thirty other male animals were the control group. To determine the possible metabolic disturbances caused by chronic ethanol feeding in blood we measured in blood metabolic parameters, and in a liver perfusion assay the hepatic insulin clearance and hepatic urea production in these animals. Between the ethanol-fed and the control animals there were significant differences in the following parameters: blood insulin concentration (47 vs. 2 microU/ml) and activities of amino acid transferases in liver homogenates at the end of the perfusion experiments (ASAT, 5950 vs. 70; ALAT, 3632 vs. 93 U/l). The other parameters were still normal in the ethanol-fed animals. Thus in these experiments after 12 months of 15% (V/V) ethanol feeding the rats still showed only a state of beginning metabolic disturbances in the liver. The results are discussed under consideration of the formation of acetaldehyde protein adducts in all rat organs investigated, namely, liver, kidney, heart and skeleton muscle, gut and spleen.

Endopeptidase 24.11/CD10 is down-regulated in renal cell cancer.

The regulatory mechanisms responsible for malignant transformation, tumor progression and metastasis in renal cell cancer (RCC) are still unclear, but there is some evidence that biologically active peptides might have regulatory effects on the behavior of this malignancy. Tumor cells can change local concentrations of active peptides by modulating their cell-surface enzymes. Using immunohistochemistry and enzyme-histochemistry, the expression of various membrane peptidases was examined in RCC and adjacent noninvaded renal parenchyma (n = 44). We describe the down-regulation of neutral endopeptidase 24.11 (NEP) protein expression in RCC of the clear cell/chromophilic type when compared with renal parenchyma, and show for the first time the lack of enzyme activity of NEP in RCC. The strongest expression could be found for dipeptidyl peptidase IV (DPIV) which is only decreased in RCC of the chromophobe cell type and is even present in oncocytoma. Aminopeptidase N (APN) and aminopeptidase A (APA) show attenuated expression in up to one third of clear cell/ chromophilic RCC. Chromophobe RCC and oncocytomas do not express APN, APA, NEP and gamma-glutamyltranspeptidase.

IL-10 and TGF-beta differ in their regulation of aminopeptidase N/CD13 expression in monocytes.

Human monocytic cells express considerable amounts of aminopeptidase N (APN)/CD13, a transmembrane protein proposed to play a role in the modulation of kinins, neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Previous studies have shown that APN/CD13 participates in antigen processing and presentation, trimming peptides protruding out of MHC class II molecules. In several inflammatory processes, macrophages have been shown to express especially high amounts of MHC class II molecules and of this peptidase. To learn more about the regulation of APN/CD13 on monocytes we investigated its expression under the influence of cytokines. Here, we report a dose- and time-dependent up-regulation of APN/CD13 mRNA and protein expression by transforming growth factor (TGF)-beta on human monocytes. To the contrary, we found IL-10 down-regulating the expression of APN/CD13 mRNA and protein. Both the regulation of the APN/CD13 protein assessed by immunofluorescence and the gene expression assessed by real-time PCR were highly correlated. Using the Dual-Luciferase reporter assay, we demonstrate that TGF-beta treatment of monocytes results in a higher activity of the APN/CD13 myeloid promoter. Our results implicate differences in the expression of the membrane peptidase APN/CD13 and therefore in the peptide-modulating ability of monocytes after exposure to these two immunosuppressive cytokines, TGF-beta and IL-10.

Correlations on radioimmunoassay, fluorescence polarization immunoassay, and enzyme immunoassay of cannabis metabolites with gas chromatography/mass spectrometry analysis of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in urine specimens.

Results obtained from three commercial immunoassay kits, Abuscreen, TDx, and EMIT, commonly used for the initial test of urine cannabinoids (and metabolites) were correlated with the 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (9-THC-COOH) concentration as determined by GC/MS. Correlation coefficients obtained based on 26 (out of 1359 total sample population) highly relevant samples, are 0.601 and 0.438 for Abuscreen and TDx. Correlation coefficients obtained from a parallel study on a different set of 47 (out of 5070 total sample population) highly relevant specimens are 0.658 and 0.575 for Abuscreen and Emit. The immunoassay concentration levels, that correspond to the commonly used 15 ng/ml GC/MS cutoff value for 9-THC-COOH, as calculated from the regression equations are 82 ng/ml and 75 ng/ml for TDx and EMIT and 120 ng/ml and 72 ng/ml for Abuscreen manufactured at two different time periods. The difference of these calculated corresponding concentrations provides quantitative evidence of the reagent specificity differences.

Transforming growth factor-beta increases the expression of aminopeptidase N/CD13 mRNA and protein in monocytes and monocytic cell lines.

Aminopeptidase N (APN)/CD13 is a membrane-bound surface ectopeptidase with a ubiquitous distribution. In hematopoiesis, APN/CD13 is expressed on stem cells and during most developmental stages of myeloid cells. Because APN/CD13 has been implicated in the trimming on the cell surface of peptides that protrude out of MHC class II molecules, we wanted to study the regulation of this membrane peptidase in antigen presenting cells by TGF-beta. TGF-beta is a potent inducer of the maturation of monocyte precursors towards a macrophage phenotype. Using competitive RT-PCR and cytofluorimetric analyses, we quantified the modulation of the APN/CD13 mRNA as well as protein expression by TGF-beta 1 and -2 and found a stimulation of the APN/CD13 expression in a time- and dose-dependent manner in monocytic cells. In U937 cells, the time course showed a maximum for APN/CD13 mRNA at 24 hours incubation with TGF-beta. In experiments with actinomycin D- treated cells was found a stabilization of APN/CD13 mRNA by TGF-beta 1. Contrary to the IL-4-induced expression of APN/CD13 as well as of MHC class II in monocytic cells, we could show that TGF-beta is able to augment the APN/CD13 expression but decreases the MHC class II expression.