The impact of prehistoric mining activities on the environment: a multidisciplinary study at the fen Schwarzenbergmoos (Brixlegg, Tyrol, Austria)

The exploitation of copper ore deposits of the northern Greywacke Zone was initiated by the implementation of metallurgic technologies in the Eastern Alps thousands of years ago. This multi-proxy study aimed to detect prehistoric mining phases in the vicinity of a prominent copper ore deposit in the Lower Inn Valley. Therefore we studied a peat core from a fen using pollen, micro charcoal and geochemical analyses. In the same fen, an archaeological investigation revealed an ore beneficiation site, well dated by dendrochronological analysis to the Late Bronze Age (9th century b.c.). First hints of mining activities reflected by the occurrence of anthropogenic indicators in the pollen diagram, associated with elevated metal values, at the beginning of the Bronze Age might result from early mineral prospecting and metallurgical experiments around the use of fahlore. The local ore deposit was then abandoned until during the Bronze Age mining activities started to increase. This is reflected by an expansion of the pioneer species Pinus and Larix on mine spoil heaps in the proximity. Concomitantly metal ratios and micro charcoal increase. From about 1000 to 850 b.c. a strong impact of mining activities is displayed in the multi-proxy data. The local forest was partly cleared on and in the vicinity of the fen. According to dendrochronological data the ore beneficiation plant was in use from about 900 to 870 b.c. Until about 700 b.c. another period with moderate impact by mining activities in the further vicinity of the fen shows up.     

Schistosoma mansoni: Assessment of effects of oleic acid,cercarial age and water temperature on parasite-host attraction

Although the lifecycle of Schistosoma spp. and pathophysiology of schistosomiasis have been established, the mechanism by which cercariae find their host is not well understood. Speculatively, host infection by random and accidental host contact is not as biologically plausible as a biochemical mechanism of mammalian attraction. A few studies have indicated that biochemical cues and temperature gradients may play a role in host identification, attraction and attachment triggers. This study aimed to elucidate these mechanisms more specifically through evaluation of biochemical, age and temperature influences leading to Schistosoma mansoni cercariae attraction and attachment behaviors. Oleic acid, a common unsaturated free fatty acid in the outer layer of human skin, was tested for cercariae attraction across biologically relevant concentrations. Influence of media type (beeswax, nail varnish and agar), age-dependent behavior variability and environmentally appropriate temperatures (22 and 30 °C) were also evaluated. Results indicated that oleic acid at concentrations of 0.3, 0.9 and 1.8 g/mL in beeswax significantly increased median attachment to media (median attachment of 7.50%, 4.20% and 3.71%, respectively, P < 0.001), compared with plain beeswax, with maximal attachment of 30.30% at 0.3 g/mL of oleic acid. In media containing 0.3 g/mL of oleic acid, cercarial attachment was highest for freshly emerged cercariae to 5 h post-emergence, with a significant decrease in attachment behavior at 10 h post-emergence (P < 0.01). Aquatic temperature at which cercariae were exposed to media did not yield significant results (P value >0.05). Biochemical, age and environmental factors influencing cercarial host attraction and attachment behavior have been elucidated by this study. This information will inform further development of devices for environmental surveillance and potentially improve cercarial exposure prevention strategies.     

The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a “buffering” effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0–7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.     

Health Care Utilization and Symptom Severity in Ghanaian Children – a Cross-Sectional Study

The aim of this study was to identify factors influencing health care utilization behavior for children with mild or severe disease symptoms in rural Ghana. Between March and September 2008 a cross-sectional health care utilization survey was conducted and 8,715 caregivers were interviewed regarding their intended behavior in case their children had mild or severe fever or diarrhea. To show associations between hospital attendance and further independent factors (e.g. travel distance or socio-economic status) prevalence ratios were calculated for the four disease symptoms. A Poisson regression model was used to control for potential confounding. Frequency of hospital attendance decreased constantly with increasing distance to the health facility. Being enrolled in the national health insurance scheme increased the intention to attend a hospital. The effect of the other factors diminished in the Poisson regression if modeled together with travel distance. The observed associations weakened with increasing severity of symptoms, which indicates that barriers to visit a hospital are less important if children experience a more serious illness. As shown in other studies, travel distance to a health care provider had the strongest effect on health care utilization. Studies to identify local barriers to access health care services are important to inform health policy making as they identify deprived populations with low access to health services and to early treatment.     

Generation of Amyloid-β Is Reduced by the Interaction of Calreticulin with Amyloid Precursor Protein,Presenilin and Nicastrin

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer''s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca²⁺- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β₄₂ levels in culture supernatants, while transfection with the P-domain increases amyloid-β₄₀ levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β₄₀ and amyloid-β₄₂ levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer''s disease.     

The Absence of Mrp4 Has No Effect on the Recruitment of Neutrophils and Eosinophils into the Lung after LPS,Cigarette Smoke or Allergen Challenge

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4^−/− and Mrp4^+/+ mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4^−/− and Mrp4^+/+ mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4^+/+ or Mrp4^−/− mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.     

Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.     

The Loss of Gnai2 and Gnai3 in B Cells Eliminates B Lymphocyte Compartments and Leads to a Hyper-IgM Like Syndrome

B lymphocytes are compartmentalized within lymphoid organs. The organization of these compartments depends upon signaling initiated by G-protein linked chemoattractant receptors. To address the importance of the G-proteins Gα_i2 and Gα_i3 in chemoattractant signaling we created mice lacking both proteins in their B lymphocytes. While bone marrow B cell development and egress is grossly intact; mucosal sites, splenic marginal zones, and lymph nodes essentially lack B cells. There is a partial block in splenic follicular B cell development and a 50-60% reduction in splenic B cells, yet normal numbers of splenic T cells. The absence of Gα_i2 and Gα_i3 in B cells profoundly disturbs the architecture of lymphoid organs with loss of B cell compartments in the spleen, thymus, lymph nodes, and gastrointestinal tract. This results in a severe disruption of B cell function and a hyper-IgM like syndrome. Beyond the pro-B cell stage, B cells are refractory to chemokine stimulation, and splenic B cells are poorly responsive to antigen receptor engagement. Gα_i2 and Gα_i3 are therefore critical for B cell chemoattractant receptor signaling and for normal B cell function. These mice provide a worst case scenario of the consequences of losing chemoattractant receptor signaling in B cells.     

The Systemic Immune Network in Recent Onset Type 1 Diabetes: Central Role of Interleukin-1 Receptor Antagonist (DIATOR Trial)

Background

The hypothesis was tested that the systemic immune milieu in recent-onset type 1 diabetes is associated with residual beta cell function and other metabolic patient characteristics.

Methods and Findings

All patients (n = 89, 40% female) of the Diabetes and Atorvastatin (DIATOR) Trial were analyzed at recruitment, i.e. prior to receiving the study medication. Inclusion criteria were insulin dependent diabetes for 2 weeks to 3 months, age range 18–39 years, and islet cell autoantibodies. Blood samples were analyzed for 14 immune mediators by standard methods. Concentrations of all mediators correlated with at least one other mediator (p<0.05, Spearman correlation) giving rise to a network. Interleukin 1 receptor antagonist (IL1-RA) held a central position and was associated with both pro- and anti-inflammatory mediators. Further central elements were the pro-inflammatory mediators CRP and IL-6, the soluble adhesion molecules sICAM-1 and E-selectin, and MCP-4 which held a central position in the chemokine network. The two Th1-associated mediators IFNγ and IP-10 remained outside the network but correlated with each other. All correlations were positive (r = 0.25–0.72), i.e., high levels of pro-inflammatory mediators were accompanied by increased levels of anti-inflammatory mediators. IL-1RA was the only mediator associated with fasting and liquid mixed meal stimulated C-peptide concentrations (r = 0.31 and 0.24, p = 0.003 and 0.025, after adjustment for age, sex, BMI). There were associations between the immune mediator network and BMI (IL-1RA, CRP, IL-6, MCP-4, MIP-1ß) but few or no associations with HbA1c, insulin dose, lipid parameters, age or sex.

Conclusions

In patients with recent onset type 1 diabetes, systemic acute phase proteins, cytokines, chemokines and soluble adhesion molecules form a network. Among the few central elements IL-1RA has a dominant role. IL-1RA is associated with all other groups of mediators and is the only mediator which correlates (positively) with residual beta cell function.

Trial registration

ClinicalTrials.gov registration number: {"type":"clinical-trial","attrs":{"text":"NCT00974740","term_id":"NCT00974740"}}NCT00974740