Biochemical characterization of detergent-resistant membranes: a systematic approach

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be 'investigator-specific'. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the 'brick-like' raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data.

Host cell responses of Salmonella typhimurium infected human dendritic cells

Live attenuated Salmonella are attractive vaccine candidates for mucosal application because they induce both mucosal immune responses and systematic immune responses. After breaking the epithelium barrier, Salmonella typhimurium is found within dendritic cells (DC) in the Peyer's patches. Although there are abundant data on the interaction of S. typhimurium with murine epithelial cells, macrophages and DC, little is known about its interaction with human DC. Live attenuated S. typhimurium have recently been shown to efficiently infect human DC in vitro and induce production of cytokines. In this study, we have analysed the morphological consequences of infection of human DC by the attenuated S. typhimurium mutant strains designated PhoPc, AroA and SipB and the wild-type strains of the American Type Culture Collection (Manassas, VA, USA), ATCC 14028 and ATCC C53, by electron microscopy at 30 min, 3 h and 24 h after exposure. Our results show that genetic background of the strains profoundly influence DC morphology following infection. The changes included (i) membrane ruffling; (ii) formation of tight or spacious phagosomes; (iii) apoptosis; and (iv) spherical, pedunculated membrane-bound microvesicles that project from the plasma membrane. Despite the fact that membrane ruffling was much more pronounced with the two virulent strains, all mutants were taken up by the DC. The microvesicles were induced by all the attenuated strains, including SipB, which did not induce apoptosis in the host cell. These results suggest that Salmonella is internalized by human DC, inducing morphological changes in the DC that could explain immunogenicity of the attenuated strains.

Dephosphorylation of p-ERK1/2 in relation to tumor remission after HER-2 and Raf1 blocking therapy in a conditional mouse tumor model

Several studies have shown that HER-2/neu (erbB-2) blocking therapy strategies can cause tumor remission. However, the responsible molecular mechanisms are not yet known. Both ERK1/2 and Akt/PKB are critical for HER-2-mediated signal transduction. Therefore, we used a mouse tumor model that allows downregulation of HER-2 in tumor tissue by administration of anhydrotetracycline (ATc). Switching-off HER-2 caused a rapid tumor remission by more than 95% within 7 d of ATc administration compared to the volume before switching-off HER-2. Interestingly, HER-2 downregulation caused a dephosphorylation of p-ERK1/2 by more than 80% already before tumor remission occurred. Levels of total ERK protein were not influenced. In contrast, dephosphorylation of p-Akt occurred later, when the tumor was already in remission. These data suggest that in our HER-2 tumor model dephosphorylation of p-ERK1/2 may be more critical for tumor remission than dephosphorylation of p-Akt. To test this hypothesis we used a second mouse tumor model that allows ATc controlled expression of BXB-Raf1 because the latter constitutively signals to ERK1/2, but cannot activate Akt/PKB. As expected, downregulation of BXB-Raf1 in tumor tissue caused a strong dephosphorylation of p-ERK1/2, but did not decrease levels of p-Akt. Interestingly, tumor remission after switching-off BXB-Raf1 was similarly efficient as the effect of HER-2 downregulation, despite the lack of p-Akt dephosphorylation. In conclusion, two lines of evidence strongly suggest that dephosphorylation of p-ERK1/2 and not that of p-Akt is critical for the rapid tumor remission after downregulation of HER-2 or BXB-Raf1 in our tumor model: (i) dephosphorylation of p-ERK1/2 but not that of p-Akt precedes tumor remission after switching-off HER-2 and (ii) downregulation of BXB-Raf1 leads to a similarly efficient tumor remission as downregulation of HER-2, although no p-Akt dephosphorylation was observed after switching-off BXB-Raf1.

Phenotypic and functional analysis of human fetal liver hematopoietic stem cells in culture

Steady-state hematopoiesis and hematopoietic transplantation rely on the unique potential of stem cells to undergo both self-renewal and multilineage differentiation. Fetal liver (FL) represents a promising alternative source of hematopoietic stem cells (HSCs), but limited by the total cell number obtained in a typical harvest. We reported that human FL nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) could be expanded under simple stroma-free culture conditions. Here, we sought to further characterize FL HSC/SRCs phenotypically and functionally before and following culture. Unexpanded or cultured FL cell suspensions were separated into various subpopulations. These were tested for long-term culture potential and for in vivo repopulating function following transplantation into NOD/SCID mice. We found that upon culture of human FL cells, a tight association between classical stem cell phenotypes, such as CD34(+) /CD38(-) and/or side population, and NOD/SCID repopulating function was lost, as observed with other sources. Although SRC activity before and following culture consistently correlated with the presence of a CD34(+) cell population, we provide evidence that, contrary to umbilical cord blood and adult sources, stem cells present in both CD34(+) and CD34(-) FL populations can sustain long-term hematopoietic cultures. Furthermore, upon additional culture, CD34-depleted cell suspensions, devoid of SRCs, regenerated a population of CD34(+) cells possessing SRC function. Our studies suggest that compared to neonatal and adult sources, the phenotypical characteristics of putative human FL HSCs may be less strictly defined, and reinforce the accumulated evidence that human FL represents a unique, valuable alternative and highly proliferative source of HSCs for clinical applications.

Differential expression of CD45 on canine microglial cells

CD45, also called leucocyte common antigen is a transmembrane protein tyrosine phosphatase on the surface of nearly all white blood cells and has a functional role in signal transduction. In the brain, the expression of CD45 can be used to distinguish microglial cells with a characteristic phenotype of CD11b/c+ and CD45(low) from other central nervous system (CNS) macrophages which show an expression of CD11b/c+ and CD45(high). In the course of pathological changes in the CNS, microglia in rodents is known to readily upregulate expression of various surface molecules, such as CD45. Understanding the mechanisms that regulate expression of surface molecules is essential to study the pathogenesis of CNS diseases. In the present study, the expression of CD45 on microglia of 42 dogs was examined ex vivo by means of flow cytometry. The dogs were classified in two groups according to the histopathological diagnosis in the CNS. All dogs without changes in the CNS (group I; n = 22) only showed low percentages of CD45+ microglial cells. In group II consisting of 20 dogs with different intracranial diseases varying results were obtained. Thirteen dogs showed a low percentage of CD45+ microglial cells whereas seven dogs exhibited high percentages of microglial cells expressing CD45. Evaluation of expression intensity in these seven dogs revealed two subpopulations of CD45+ microglial cells: a large subpopulation with CD45(low) and a small subpopulation with CD45(high). The expression intensity of CD45(high) was comparable with that of canine monocytes. It was attempted to correlate these findings to age of the animals, underlying disease, duration of clinical signs, medical treatment, occurrence of seizure activity and the expression of other surface molecules. It appeared that dogs with high percentages of CD45+ suffered from long-lasting CNS disease with seizures. In future studies, the reason and consequences for upregulated CD45 in long-lasting CNS diseases has to be further evaluated.

Biological safety concepts of genetically modified live bacterial vaccines

Live vaccines possess the advantage of having access to induce cell-mediated and antibody-mediated immunity; thus in certain cases they are able to prevent infection, and not only disease. Furthermore, live vaccines, particularly bacterial live vaccines, are relatively cheap to produce and easy to apply. Hence they are suitable to immunize large communities or herds. The induction of both cell-mediated immunity as well as antibody-mediated immunity, which is particularly beneficial in inducing mucosal immune responses, is obtained by the vaccine-strain's ability to colonize and multiply in the host without causing disease. For this reason, live vaccines require attenuation of virulence of the bacterium to which immunity must be induced. Traditionally attenuation was achieved simply by multiple passages of the microorganism on growth medium, in animals, eggs or cell cultures or by chemical or physical mutagenesis, which resulted in random mutations that lead to attenuation. In contrast, novel molecular methods enable the development of genetically modified organisms (GMOs) targeted to specific genes that are particularly suited to induce attenuation or to reduce undesirable effects in the tissue in which the vaccine strains can multiply and survive. Since live vaccine strains (attenuated by natural selection or genetic engineering) are potentially released into the environment by the vaccinees, safety issues concerning the medical as well as environmental aspects must be considered. These involve (i) changes in cell, tissue and host tropism, (ii) virulence of the carrier through the incorporation of foreign genes, (iii) reversion to virulence by acquisition of complementation genes, (iv) exchange of genetic information with other vaccine or wild-type strains of the carrier organism and (v) spread of undesired genes such as antibiotic resistance genes. Before live vaccines are applied, the safety issues must be thoroughly evaluated case-by-case. Safety assessment includes knowledge of the precise function and genetic location of the genes to be mutated, their genetic stability, potential reversion mechanisms, possible recombination events with dormant genes, gene transfer to other organisms as well as gene acquisition from other organisms by phage transduction, transposition or plasmid transfer and cis- or trans-complementation. For this, GMOs that are constructed with modern techniques of genetic engineering display a significant advantage over random mutagenesis derived live organisms. The selection of suitable GMO candidate strains can be made under in vitro conditions using basic knowledge on molecular mechanisms of pathogenicity of the corresponding bacterial species rather than by in vivo testing of large numbers of random mutants. This leads to a more targeted safety testing on volunteers and to a reduction in the use of animal experimentation.

The role of landuse and natural determinants for grassland vegetation composition in the Swiss Alps

The Alps provide a high habitat diversity for plant species, structured by broad- and fine-scale abiotic site conditions. In man-made grasslands, vegetation composition is additionally affected by the type of landuse. We recorded vegetation composition in 216 parcels of grassland in 12 municipalities representing an area of 170 x 70 km in the south-eastern part of the Swiss Alps. Each parcel was characterized by a combination of altitudinal level (valley, intermediate, alp). traditional landuse (mown. grazed), current management (mown, grazed, abandoned). and Fertilization (unfertilized, fertilized). For each parcel we also assessed the abiotic factors aspect, slope, pH value, and geographic coordinates, and for each municipality annual precipitation and its cultural tradition. We analysed vegetation composition using (i) variation partitioning in RDA. (ii) cover of graminoids. non-legume forbs, and legumes, and (iii) dominance and frequency of species. Species composition was determined by, in decreasing order of variation explained. landuse, broad-scale abiotic factors, fine-scale abiotic factors. and cultural tradition. Current socio-economically motivated landuse changes, such as grazing of unfertilized former meadows or their abandonment, strongly affect vegetation composition. In our study, the frequency of characteristic meadow species was significantly smaller in grazed and even smaller in abandoned parcels than in still mown ones, suggesting less severe consequences of grazing for vegetation composition than of abandonment. Therefore. low-intensity grazing and mowing every few years should be considered valuable conservation alternatives to abandonment. Furthermore. because each landuse type was characterized by different species. a high variety of landuse types should be promoted to preserve plant species diversity in Alpine grasslands. (C) 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

Early serum procalcitonin, interleukin-6, and 24-hour lactate clearance: useful indicators of septic infections in severely traumatized patients

BACKGROUND: Elevated lactate and interleukin-6 (IL-6) levels were shown to correlate with mortality and multiple organ dysfunction in severely traumatized patients. The purpose of this study was to test whether an association exists between 24-hour lactate clearance, IL-6 and procalcitonin (PCT) levels, and the development of infectious complications in trauma patients. METHODS: A total of 1757 consecutive trauma patients with an Injury Severity Score (ISS) > 16 admitted over a 10-year period were retrospectively analyzed over a 21-day period. Exclusion criteria included death within 72 h of admission (24.5%), late admission > 12 h after injury (16%), and age < 16 years (0.5%). Data are stated as the median (range). RESULTS: Altogether, 1032 trauma patients (76.2% male) with an average age of 38 years, a median ISS of 29 (16-75), and an Acute Physiology, Age, and Chronic Health Evaluation (APACHE) II score of 14 (0-40) were evaluated. The in-hospital mortality (>3 days) was 10%. Patients with insufficient 24-hour lactate clearance had a high rate of overall mortality and infections. Elevated early serum procalcitonin on days 1 to 5 after trauma was strongly associated with the subsequent development of sepsis (p < 0.01) but not with nonseptic infections. The kinetics of IL-6 were similar to those of PCT but did differentiate between infected and noninfected patients after day 5. CONCLUSIONS: This study demonstrates that elevated early procalcitonin and IL-6 levels and inadequate 24-hour lactate clearance help identify trauma patients who develop septic and nonseptic infectious complications. Definition of specific cutoff values and early monitoring of these parameters may help direct early surgical and antibiotic therapy and reduce infectious mortality.

The role of intercostal nerve preservation in pain control after thoracotomy

OBJECTIVES Pain control after thoracotomy is an important issue that affects the outcome in thoracic surgery. Intercostal nerve preservation (ICNP) has increased interest in the outcomes of conventional thoracotomy. The current study critically evaluates the role of preservation of the intercostal nerve in early and late pain control and its benefit in patients undergoing thoracotomy. METHODS Data obtained prospectively between January 2006 and December 2010 by a study colleague at our division of General Thoracic Surgery were retrospectively analysed. There were 491 patients who underwent thoracotomy. Eighty-one patients were excluded from the study due to incompatible data. Patients were divided into two groups according to the intercostal nerve state: Group I consisted of patients with ICNP and Group II consisted of patients with intercostal nerve sacrifice. RESULTS Group I consisted of 288 patients [206 male (71%), P < 0.001, mean age 66 years]. Group II consisted of 122 patients [79 male (64%), P = 0.001, mean age 66 years]. There was less use of opiate in Group I (P = 0.019). Early mobilization of the patients was significantly higher in Group I (P = 0.031). The rate of pneumonia and re-admission to the intensive care unit was higher in Group II (P = 0.017 and 0.023, respectively). The rate of pain-free patients at discharge was significantly higher in Group I (P = 0.028). A 2-week follow-up after hospital discharge showed parasternal hypoesthesia to be more in Group II (P = 0.034). Significant patient contentment in Group I was noticed (P = 0.014). Chronic post-thoracotomy pain (CPTP) was higher in Group II (P = 0.016). CONCLUSIONS ICNP without harvesting an intercostal muscle flap achieves excellent outcomes in controlling acute post-thoracotomy pain and CPTP. ICNP is an effective, simple method to perform, and it should be considered as standard in performing thoracotomy.

Surveillance of bovine tuberculosis and risk estimation of a future reservoir formation in wildlife in Switzerland and Liechtenstein.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis or M. caprae has recently (re-) emerged in livestock and wildlife in all countries bordering Switzerland (CH) and the Principality of Liechtenstein (FL). Comprehensive data for Swiss and Liechtenstein wildlife are not available so far, although two native species, wild boar (Sus scrofa) and red deer (Cervus elaphus elaphus), act as bTB reservoirs elsewhere in continental Europe. Our aims were (1) to assess the occurrence of bTB in these wild ungulates in CH/FL and to reinforce scanning surveillance in all wild mammals; (2) to evaluate the risk of a future bTB reservoir formation in wild boar and red deer in CH/FL. Tissue samples collected from 2009 to 2011 from 434 hunted red deer and wild boar and from eight diseased ungulates with tuberculosis-like lesions were tested by direct real-time PCR and culture to detect mycobacteria of the Mycobacterium tuberculosis complex (MTBC). Identification of suspicious colonies was attempted by real-time PCR, genotyping and spoligotyping. Information on risk factors for bTB maintenance within wildlife populations was retrieved from the literature and the situation regarding identified factors was assessed for our study areas. Mycobacteria of the MTBC were detected in six out of 165 wild boar (3.6%; 95% CI: 1.4-7.8) but none of the 269 red deer (0%; 0-1.4). M. microti was identified in two MTBC-positive wild boar, while species identification remained unsuccessful in four cases. Main risk factors for bTB maintenance worldwide, including different causes of aggregation often resulting from intensive wildlife management, are largely absent in CH and FL. In conclusion, M. bovis and M. caprae were not detected but we report for the first time MTBC mycobacteria in Swiss wild boar. Present conditions seem unfavorable for a reservoir emergence, nevertheless increasing population numbers of wild ungulates and offal consumption may represent a risk.

Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs).

Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.

2-methiopropamine, a thiopene analogue of metamphetamine: studies on its metabolism abt detectability in the rat and human using

2-Methiopropamine [1-(thiophen-2-yl)-2-methylaminopropane, 2-MPA], a thiophene analogue of methamphetamine, is available from online vendors selling "Research chemicals." The first samples were seized by the German police in 2011. As it is a recreational stimulant, its inclusion in routine drug screening protocols should be required. The aims of this study were to identify the phase I and II metabolites of 2-MPA in rat and human urine and to identify the human cytochrome-P450 (CYP) isoenzymes involved in its phase I metabolism. In addition, the detectability of 2-MPA in urine samples using the authors' well-established gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-linear ion trap-mass spectrometry (LC-MS(n)) screening protocols was also evaluated. The metabolites were isolated from rat and human urine samples by solid-Phase extraction without or following enzymatic cleavage of conjugates. The phase I metabolites, following acetylation, were separated and identified by GC-MS and/or liquid chromatography-high-resolution linear ion trap mass spectrometry (LC-HR-MS(n)) and the phase II metabolites by LC-HR-MS(n). The following Major metabolic pathways were proposed: N-demethylation, hydroxylation at the side chain and at the thiophene ring, and combination of these transformations followed by glucuronidation and/or sulfation. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were identified as the major phase I metabolizing enzymes. They were also involved in the N-demethylation of the analogue methamphetamine and CYP2C19, CYP2D6, and CYP3A4 in its ring hydroxylation. Following the administration of a typical user's dose, 2-MPA and its metabolites were identified in rat urine using the authors' GC-MS and the LC-MS(n) screening approaches. Ingestion of 2-MPA could also be detected by both protocols in an authentic human urine sample.

Denudation rates of small transient catchments controlled by former glaciation: The Hörnli nunatak in the northeastern Swiss Alpine Foreland

Extensive glaciers repeatedly occupied the northern Alpine Foreland during the Pleistocene and left a strongly glacially overprinted low slope landscape. Only few islands appeared as nunataks standing above the surface of the large piedmont glacier lobes. These nunatak areas kept their original shape, manifested in steep catchments with mean slopes up to 33 . Even though not glaciated, these catchments where significantly affected by base-level changes occurring as a consequence of phases of glacier advances and retreats. Both domains, the glacially eroded and non-eroded, are therefore prone to different mechanisms and time-scales of fluvial and colluvial re-adjustment. In this study we investigate these effects by exploring the spatial distribution and magnitude of denudation in the Hörnli region of the eastern Swiss Alpine Foreland in the present Interglacial. The area represents both domains in a relatively small area with largely uniform tectonic, lithologic and climatic conditions. The differences in Holocene andscape evolution are investigated using topographic analyses and catchment-averaged denudation rates derived from 10Be concentrations in fluvial quartz sand. We find that in formerly non-glaciated, fluvially dominated catchments close hillslope-channel coupling prevails and that these catchments yield high average denudation rates of 350 mm/ka. Glacially overprinted catchments yielded catchment-wide denudation rates an order of magnitude lower. These low denudation rates are hypothesized to be the consequence of both (i) a dominance of slow hillslope processes and (ii) admixture of high concentration, pre-LGM glacial sediment. This suggests that a) a careful field investigation must accompany the denudation rate studies and b) that the concept of area-weighted cosmogenic nuclide denudation rates must be considered in light of the predominant catchment processes.

A reconstruction of atmospheric carbon dioxide and its stable carbon isotopic composition from the penultimate glacial maximum to the last glacial inception

The reconstruction of the stable carbon isotope evolution in atmospheric CO2 (δ13Catm), as archived in Antarctic ice cores, bears the potential to disentangle the contributions of the different carbon cycle fluxes causing past CO2 variations. Here we present a new record of δ13Catm before, during and after the Marine Isotope Stage 5.5 (155 000 to 105 000 yr BP). The dataset is archived on the data repository PANGEA® (www.pangea.de) under 10.1594/PANGAEA.817041. The record was derived with a well established sublimation method using ice from the EPICA Dome C (EDC) and the Talos Dome ice cores in East Antarctica. We find a 0.4‰ shift to heavier values between the mean δ13Catm level in the Penultimate (~ 140 000 yr BP) and Last Glacial Maximum (~ 22 000 yr BP), which can be explained by either (i) changes in the isotopic composition or (ii) intensity of the carbon input fluxes to the combined ocean/atmosphere carbon reservoir or (iii) by long-term peat buildup. Our isotopic data suggest that the carbon cycle evolution along Termination II and the subsequent interglacial was controlled by essentially the same processes as during the last 24 000 yr, but with different phasing and magnitudes. Furthermore, a 5000 yr lag in the CO2 decline relative to EDC temperatures is confirmed during the glacial inception at the end of MIS5.5 (120 000 yr BP). Based on our isotopic data this lag can be explained by terrestrial carbon release and carbonate compensation.