The pathogenesis of schistosomiasis is controlled by cooperating IL-10-producing innate effector and regulatory T cells

IL-10 reduces immunopathology in many persistent infections, yet the contribution of IL-10 from distinct cellular sources remains poorly defined. We generated IL-10/recombination-activating gene (RAG)2-deficient mice and dissected the role of T cell- and non-T cell-derived IL-10 in schistosomiasis by performing adoptive transfers. In this study, we show that IL-10 is generated by both the innate and adaptive immune response following infection, with both sources regulating the development of type-2 immunity, immune-mediated pathology, and survival of the infected host. Importantly, most of the CD4(+) T cell-produced IL-10 was confined to a subset of T cells expressing CD25. These cells were isolated from egg-induced granulomas and exhibited potent suppressive activity in vitro. Nevertheless, when naive, naturally occurring CD4(+)CD25(+) cells were depleted in adoptive transfers, recipient IL-10/RAG2-deficient animals were more susceptible than RAG2-deficient mice, confirming an additional host-protective role for non-T cell-derived IL-10. Thus, innate effectors and regulatory T cells producing IL-10 cooperate to reduce morbidity and prolong survival in schistosomiasis.     

Liver-specific physiology of immortal,functionally differentiated hepatocytes and of deficient hepatocyte-like variants

Summary Five different immortalized transgenic hepatocyte cell lines derived from mice were investigated with respect to their potential to maintain the physiological properties of primary hepatocytes using chemically defined medium. This research completes a previous study by Klocke and coworkers in 2002, using gene expression analysis of the same cell lines by the respective physiological analysis for investigating the hepatocyte-like function. Three transgenic cell lines harboring a fusion gene derivative (construct 202), consisting of the complete SV40 early region, including the coding sequences for the transforming large and small tumor antigens, placed under the control of the murine metallothioneine 1-promotor/ enhancer element, showed a hepatocyte-like function and physiology. They grew as a monolayer with a polygonal cell shape, consumed lactate, and secreted albumin at a cell-specific rate of 1.5 pg/h, which is in the range of primary hepatocytes. In addition, the potential of detoxifying ammonium could be maintained. Ammonium was metabolized and urea was produced and released into the medium. A complete urea cycle could be determined. A cell line established from neonatal transgenic mice and expressing a secretory variant of the human epidermal growth factor (IgEGF) under the control of the albumin promoter was characterized by an incomplete urea cycle. Another cell line isolated from the liver of homozygote neonatal p53-knockout mice showed no hepatocyte-specific functions but only properties of continuous cell lines. Specific nucleoside triphosphate (NTP) and uridine (U) ratios were used to characterize the differentiation status of the particular cell lines. A low NTP-U value was found for the thre cell lines containing construct 202, which was identical to that observed for primary hepatocytes. In contrast, the cell line harvested from the liver of homozygote neonatal p53-knockout mice presented a NTP-U ratio characteristic for continuous cell lines. This study demonstrates that the four transgenic and the p53-knockout, hepatocyte-derived cell lines can be used as models for investigating the conservation of tissue-specific functions in immortalized cells.     

Visualization of the uptake of individual HDL particles in living cells via the scavenger receptor class B type I

The scavenger receptor class B type I (SR-BI) plays an important role in mediating selective uptake of high-density lipoprotein (HDL)-derived cholesterol and cholesteryl ester in liver and steroidogenic tissues. The molecular mechanism by which this receptor mediates selective cholesteryl ester uptake remains still enigmatic. We applied ultrasensitive fluorescence microscopy to visualize the intracellular transport routes of HDL particles taken up via SR-BI in a Chinese hamster ovarian cell line. Although diffusion of the receptor bound particles on the cell surface is slow, internalization is accompanied by a dramatic increase in the mobility of the particles. HDL particles are endocytosed as clusters and actively transported to the perinuclear region of the cell. Costaining with organelle markers confirmed the involvement of an acidic compartment and the Golgi apparatus in the uptake process; finally, resecretion of the HDL particles was observed.     

N,N',N"-triferuloylspermidine, a new UV absorbing polyamine derivative from pollen of Hippeastrum x hortorum

A new hydroxycinnamoyl polyamine derivative, N,N',N"-triferuloylspermidine (= (E)-N-(4-aminobutyl) -3,3',3"-tris(4-hydroxy-3-methoxyphenyl)-N,N',N"- (butane-1,4-diyl)tris [prop-2-enamide]) (1) was detected in the H2O/MeOH extract of pollen from Hippeastrum x hortorum. The compound was identified by on-line-coupled high-performance liquid chromatography and atmospheric-pressure chemical-ionization mass spectrometry (HPLC-UV(DAD)/APCI-MS and MS/MS). The structure was proven by comparing the HPLC/MS data after UV-induced (E) <==> (Z) photoisomerization and catalytic hydrogenation of the natural compound and the synthetic reference compound. This is the first report of a triferuloylspermidine in nature.     

Use of isotopic and molecular techniques to link toluene degradation in denitrifying aquifer microcosms to specific microbial populations

Microcosms were inoculated with sediments from both a petroleum-hydrocarbon (PHC)-contaminated aquifer and from a nearby pristine aquifer and incubated under anoxic denitrifying conditions with [methyl-13C]toluene. These microcosms served as a laboratory model system to evaluate the combination of isotope (13C-labeling of polar-lipid-derived fatty acids) and molecular techniques (16S rRNA-targeting gene probes) to identify the toluene-metabolizing population. After total depletion of toluene, the following bacterial phospholipid fatty acids (PLFA) were 13C-enriched: 16:1omega7c, 16:1omega7t, 16:0, cy17:0, and 18:1omega7c. Pure culture experiments demonstrated that these compounds were also found in PLFA profiles of PHC-degrading Azoarcus spp. (beta-Proteobacteria) and related species. The origin of the CO2 evolved in the microcosms was determined by measurements of stable carbon isotope ratios. Toluene represented 11% of the total pool of mineralized substrates in the contaminated sediment and 54% in the pristine sediment. The microbial community in the microcosm incubations was characterized by using DAPI staining and whole-cell hybridization with specific fluorescently labeled 16S rRNA-targeted oligonucleotide probes. Results revealed that 6% of the DAPI-stained cells in the contaminated sediment and 32% in the pristine sediment were PHC-degrading Azoarcus spp. In biotic control microcosms (incubated under denitrifying conditions, no toluene added), Azoarcus spp. cells remained at less than 1% of the DAPI-stained cells. The results show that isotope analysis in combination with whole-cell hybridization is a promising approach to identify and to quantify denitrifying toluene degraders within microbial communities.     

Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures

During recent years, biopharmaceutical products manufactured by processes that use mammalian cell cultures have gained increasing importance. At the same time, a strong awareness of the importance of the safety and quality of such products has also emerged. This has led to improvements in cultivation and production technology, validation procedures and process organization.     

Measurement of Apolipoprotein E (apoE) in Cerebrospinal Fluid

Apolipoprotein E (apoE) is a protein involved in transport of lipids and has been implicated to play an important role in regeneration after nerve injury. Determination of apoE in cerebrospinal fluid (CSF) thus have a potential interest when studying different forms of brain damage and as a marker of ongoing regenerative processes in the brain. However, previous studies on CSF-ApoE in Alzheimer's disease (AD) have given inconclusive results. Such inconsistant results might be related to confounding factors interfering with sample handling and/or analyses, which have not been fully elucidated. We therefore examined different potential confounding factors for analyses of apoE in CSF and also developed a new enzyme linked immunosorbent assay (ELISA). The hydrophobic character of ApoE resulted in adsorbtion to different types of test tubes commonly used for collection of CSF at lumbar puncture, resulting in falsly low levels. This makes CSF handling critical, especially if samples are taken in different types of tubes, or is transferred to new tubes. Taking this confounding factors in consideration and analysing patient and control CSF handled in the same way and using the new ELISA, we could confirm our previous finding of reduced levels of ApoE in AD, (3.4 ± 1.3mg/l) compared with controls (4.5 ± 2.7mg/l) (p = 0.045). Both in the AD and in the control group, higher levels of CSF-ApoE was found in individuals possessing the ApoE4 alleles. Our results support that CSF-ApoE is reduced in AD, and that handling of CSF is a critical factor, which may explain the discrepant results from previous studies. Differences in the amount of patients and controls possessing the ApoE4 allele included might also increase the variance between different studies.     

Regulation and distribution of body fluid during a 6-day head-down tilt study in a randomized cross-over design

Head down tilt (-6 degrees HDT) examinations are commonly used simulation models for various microgravity induced changes in body functions. Body fluid distribution (by means of dye dilution and two independent multifrequency impedance techniques), water- and sodium-handling, and the plasma/serum concentrations of fluid balance related hormones have been determined in a randomized, controlled, cross-over study in 8 healthy test subjects. The comparison of responses to HDT and an upright control position with respective experiences from space shows some similarities but also various discrepancies between the terrestrial simulation and real microgravity.     

Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione

The coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1. 30), was introduced into the genome of potato plants under the control of the cauliflower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5'-signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.