Photodegradation of the UV filter ethylhexyl methoxycinnamate under ultraviolet light: Identification and in silico assessment of photo-transformation products in the context of grey water reuse.

To prevent water shortages in the future and to reduce domestic water consumption, decentralized grey water (GW) reuse has become increasingly important. This water has, however, to be free of pollutants. Conventional treatment of GW does not fully eliminate micropollutants such as the UV filter substance ethylhexyl methoxycinnamate (EHMC). EHMC, which is commonly used in sunscreens and personal care products, is an endocrine disruptor and shows potential to bioaccumulation, which is also reflected in its low water solubility. Photolysis has been proposed as an alternative treatment method for other micropollutants, but it is not clear yet whether it can also be used to eliminate EHMC. One goal of this study was to better understand the basic pathways involved in this process. It aimed to identify photo-transformation products (photo-TPs) by using, in the test conditions, an initial concentration of EHMC higher than those expected in the environment. Acetonitrile (ACN) was added in low concentrations to the aqueous solution to overcome the low aquatic solubility of EHMC. The influence of this co-solvent on the degradation kinetics was studied. The photolysis experiments were carried out using a medium pressure mercury lamp, which emits UV light in the range of 200-400nm. The quantum yield of the photolysis of EHMC was 0.0042 and 0.0023mol·Einstein-1 (for 0.2 and 0.5% ACN (v/v), respectively), and the relative and absolute UV photon fluxes were determined. HPLC was used to monitor the elimination kinetics of EHMC, which followed first-order kinetics. The results of LC-MSn analyses revealed that beside others, several oxidized and hydroxylized EHMC isomers were formed as photo-TPs in aqueous solution. Using a set of in silico quantitative structure-activity relationship (QSAR) models, this study also offered new insights concerning the environmental fate and toxicity of the TPs of EHMC.

Monitoring of methotrexate chlorination in water.

Anti-cancer drugs are an important class of pharmaceutical products. Methotrexate (MTX) is a folic acid antagonist used in high doses as antimetabolite in anti-cancer treatment as well as in low doses for the treatment of rheumatoid arthritis and adults' psoriasis. In the past, several anti-cancer drugs, including methotrexate, have been found in the environment. Their presence in water, especially if used for the production of drinking water, is even in low concentrations of particular interest, due to the risk to retrieve them in the consumed water and their high activity and grave effects. But prior to usage as drinking water, raw waters are treated and chlorination is a common practice in several countries. As such a treatment can lead to the formation of organochlorine in water, the study of the fate of MTX during chlorination in a batch trial was carried out. The reaction was monitored by dissolved organic carbon (DOC) and by fluorescence and UV spectroscopy. Investigation of by-products formed was done with liquid chromatography/mass spectrometry (LC/MS). Under the given experimental conditions, Methotrexate was eliminated rapidly (t1/2 around 21 min). However, DOC elimination was incomplete. Monitoring with LC-MS showed the formation of a monochlorinated transformation product of MTX. In silico analysis of the proposed transformation products for different carcinogenic, mutagenic and genotoxic endpoints with different software platforms provided no clear evidence that the possible transformation products after chlorination might be more toxic than the parent compound. However, since a number of alerts is altered after chlorination, it cannot be excluded that the toxicity of these transformation products might be modulated compared with the parent compound.

The recombinant bifunctional protein αCD133-GPVI promotes repair of the infarcted myocardium in mice.

BACKGROUND: Bone-marrow-derived progenitor cells are important in myocardial repair mechanisms following prolonged ischemia. Cell-based therapy of diseased myocardium is limited by a low level of tissue engraftment. OBJECTIVES: The aim of this study was the development of the bifunctional protein αCD133-glycoprotein (GP)VI as an effective treatment for supporting vascular and myocardial repair mechanisms. RESULTS: We have generated and characterized a bifunctional molecule (αCD133-GPVI) that binds both to the subendothelium of the injured microvasculature and to CD133(+) progenitor cells with high affinity. αCD133-GPVI enhances progenitor cell adhesion to extracellular matrix proteins and differentiation into mature endothelial cells. In vivo studies showed that αCD133-GPVI favors adhesion of circulating progenitor cells to the injured vessel wall (intravital microscopy). Also, treatment of mice undergoing experimental myocardial infarction with αCD133-GPVI-labeled progenitor cells reduces infarction size and preserves myocardial function. CONCLUSIONS: The bifunctional trapping protein αCD133-GPVI represents a novel and promising therapeutic option for limiting heart failure of the ischemic myocardium.

Clonal expansions of pathogenic CD8+ effector cells in the CNS of myelin mutant mice.

Tissue damage in the CNS is critically influenced by the adaptive immune system. Primary oligodendrocyte damage (by overexpression of PLP) leads to low-grade inflammation of high pathological impact, which is mediated by CD8+ T cells. To yield further insight into pathogenesis and nature of immune responses in myelin mutated mice, we here apply a detailed immunological characterization of CD8+ T cells in PLP-transgenic and aged wild type mice. We provide evidence that T effector cells accumulate in the CNS of PLP-transgenic and wild-type mice and show a higher level of activation in mutant mice, indicated by surface markers and clonal expansions, as demonstrated by T cell receptor CDR3-spectratype analysis. Vbeta-Jbeta similarities suggest specificity against a common antigen, albeit we could not find specific responses against myelin-antigen-derived peptides. The association of primary oligodendrocyte damage with secondary expansions of pathogenic cells underlines the role of adaptive immune reactions in neurodegenerative and neuroinflammatory diseases.

Expression of CD28-related costimulatory molecule and its ligand in inflammatory neuropathies.

BACKGROUND: Activation of effector T lymphocytes, mediated in part by costimulatory molecules, is an important mechanism in the pathogenesis of immune-mediated diseases of the peripheral nervous system (PNS). OBJECTIVE: To analyze the expression and distribution pattern of the inducible costimulator (ICOS), a recently identified costimulatory molecule implicated in T-cell activation, and its unique ligand (ICOS-L), in inflammatory disorders of the PNS. METHODS: We studied RNA and protein expression in sural nerve biopsy specimens from patients with Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and vasculitic neuropathy (VN) vs patients with hereditary neuropathies (HNs) serving as a noninflammatory control using reverse-transcriptase PCR and immunohistochemistry. In addition, in vitro analysis was performed by flow cytometry. RESULTS: ICOS and ICOS-L mRNA was found to be significantly upregulated in samples from patients with GBS, CIDP, and VN compared to HNs. Immunohistochemistry identified T lymphocytes as the cellular source of ICOS, whereas macrophages expressed the corresponding ligand ICOS-L. Further analysis revealed that the distribution of ICOS-expressing T cells did not differ between acute and chronic inflamed PNS diseases. Correspondingly, the expression pattern of ICOS-L was similar in the inflamed tissues but differed significantly when compared to HNs. CONCLUSIONS: Inducible costimulator, expressed by T lymphocytes, and inducible costimulator ligand, expressed by macrophages within the peripheral nerve, might not only be relevant in inducing an acute immune response but might also be critically involved in perpetuating inflammation in chronically immune-mediated disorders of the peripheral nervous system.

Identification of a heparin-binding motif on adeno-associated virus type 2 capsids.

Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary.

Enhancement of capsid gene expression: preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes.

Expression of the structural proteins L1 and L2 of the human papillomaviruses (HPV) is tightly regulated. As a consequence, attempts to express these prime-candidate genes for prophylactic vaccination against papillomavirus-associated diseases in mammalian cells by means of simple DNA transfections result in insufficient production of the viral antigens. Similarly, in vivo DNA vaccination using HPV L1 or L2 expression constructs produces only weak immune responses. In this study we demonstrate that transient expression of the HPV type 16 L1 and L2 proteins can be highly improved by changing the RNA coding sequence, resulting in the accumulation of significant amounts of virus-like particles in the nuclei of transfected cells. Data presented indicate that, in the case of L1, adaptation for codon usage accounts for the vast majority of the improvement in protein expression, whereas translation-independent posttranscriptional events contribute only to a minor degree. Finally, the adapted L1 genes demonstrate strongly increased immunogenicity in vivo compared to that of unmodified L1 genes.

[Histomorphology after transmyocardial laser revascularization]. / Histomorphologie nach transmyokardialer Laserrevaskularisation.

From 11/1994 to 4/1997 we enrolled 140 patients with diffuse CAD refractory to maximum antianginal therapy who are not candidates for PTCA or CABG for transmyocardial laser revascularisation (TMLR). Of these patients aged 63.5 +/- 15 years, 98 had coronary 3-vessel disease, and the average left ventricular ejection fraction was 44%. Eleven out of these 140 patients died from different reasons (pneumonia, myocardial infarction, septicemia). Seven patients who died between the 1st and 20th postoperative day underwent a postmortem examination with histological analysis of the areas treated by TMLR. On the seven investigated ventricles a total of 220 channels were created. The predominant finding in specimens within five days after TMLR was recently closed channels. Furthermore, a zone of necrosis with an average extension of 500 microns on each side of the channel was evident. Many changes were noticeable in specimens from patients who died two or three weeks after TMLR. Freshly clotted material had been replaced by a granular tissue of variable density. High macrophage and monocyte activity was evident. The extent of this cellular activity could be depicted by staining with a special proliferation marker, such as MiB. On the one hand numerous dividing macrophages were observed, on the other, active fibroblasts indicative for the transformation into scar-like tissue. After staining for type-4-collagen, typical for the basal membrane of capillaries, a large number of stained structures was noticeable in the closed channel lumen. Numerous garlandlike structures became visible under higher magnification. By CD 31 incubation, these structures, were found to be lined with endothelium. Further research will be required to indicate whether the laser channels later are partially or completely open, from where the capillaries are supplied, and whether they even connect to the ventricle lumen. But in conclusion, it seems unlikely, that TMLR follows the mechanism of the amphibian heart.

Histological findings after transmyocardial laser revascularization.

In recent time, it has become more and more probable that patients with severe diffuse coronary artery disease, who are not candidates for aortocoronary bypass surgery or percutaneous transluminal coronary angioplasty procedures, can benefit from transmyocardial laser revascularization (TMR). But the underlying principle of TMR still remains unclear. This study reports on a histological analysis of eight patients, in whom a total of 250 channels had been created, who died after TMR. The TMR channels were created by a CO2 laser surrounded by a zone of necrosis with an extent of about 500 microns. In the hearts of patients who died in the early postoperative period (1 to 7 days postoperative), almost all channels were closed by fibrin clots, erythrocytes, and macrophages. There were no obvious connections between the channels and the ventricular cavity. In specimens from patients, who died 2 or more weeks after the procedure, a granular tissue with high macrophage and monocyte activity was observable. Within this tissue, we observed a developing network of capillaries. Otherwise, the tissue filling the channels did not substantially differ from scar tissue. We failed to observe connections between the ventricular cavity and the new capillaries. Whether these vessels within the closed channels have any impact on myocardial perfusion remains unclear, but it seems unlikely that the clinical effects of TMR are based on the principle of the amphibian heart.