Pharmacological removal of serum amyloid P component from intracerebral plaques and cerebrovascular Aβ amyloid deposits in vivo

Human amyloid deposits always contain the normal plasma protein serum amyloid P component (SAP), owing to its avid but reversible binding to all amyloid fibrils, including the amyloid β (Aβ) fibrils in the cerebral parenchyma plaques and cerebrovascular amyloid deposits of Alzheimer''s disease (AD) and cerebral amyloid angiopathy (CAA). SAP promotes amyloid fibril formation in vitro, contributes to persistence of amyloid in vivo and is also itself directly toxic to cerebral neurons. We therefore developed (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), a drug that removes SAP from the blood, and thereby also from the cerebrospinal fluid (CSF), in patients with AD. Here we report that, after introduction of transgenic human SAP expression in the TASTPM double transgenic mouse model of AD, all the amyloid deposits contained human SAP. Depletion of circulating human SAP by CPHPC administration in these mice removed all detectable human SAP from both the intracerebral and cerebrovascular amyloid. The demonstration that removal of SAP from the blood and CSF also removes it from these amyloid deposits crucially validates the strategy of the forthcoming ‘Depletion of serum amyloid P component in Alzheimer''s disease (DESPIAD)’ clinical trial of CPHPC. The results also strongly support clinical testing of CPHPC in patients with CAA.     

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.     

Renal Transplant Recipients Treated with Calcineurin-Inhibitors Lack Circulating Immature Transitional CD19+CD24hiCD38hi Regulatory B-Lymphocytes

BackgroundCD19⁺CD24^hiCD38^hi transitional immature B-lymphocytes have been demonstrated to play an important role in regulating the alloimmune response in transplant recipients. Here, we analyzed the effect of calcineurin inhibition on these peripherally circulating regulatory B-cells (Breg) in renal transplant recipients receiving cyclosporine A (CsA) or tacrolimus.MethodsPBMCs from healthy subjects (HS) (n = 16) and renal transplant recipients (n = 46) were isolated. Flow cytometry was performed for CD19, CD24, CD38 and IL-10 either after isolation or after 72 hours of co-culture in presence of PMA/Ionomycin and TLR9-ligand in presence or absence of increasing concentrations of tacrolimus or CsA.ResultsThe amount of CD19⁺ B-cells among lymphocytes was ∼9.1% in HS, ∼3.6% in CsA (n = 11, p<0.05) and ∼6.4% in TAC (n = 35, p<0.05) treated patients. Among B-cells, a distinct subset of Breg was found to be 4.7% in HS, 1.4% in tacrolimus treated patients and almost blunted in patients receiving CsA. Similarily, ∼4% of B-cells in HS and even fewer in CsA or tacrolimus treated patients produced IL-10 (0.5% and 1.5%, p<0.05) and this was confirmed both in non-transplanted CsA-treated healthy subjects and in in vitro co-culture experiments. Among 29 patients with <1% of Breg, 9 cases (31%) displayed an allograft rejection in contrast to only one case of rejection (6%) among 17 patients with >1%.ConclusionCalcineurin inhibitors reduce number and IL-10 production of Bregs in the peripheral circulation of both renal transplant recipients and non-transplanted healthy subjects. CNI induced Breg reduction is not restricted to a solid organ transplant setting and is not mediated by co-medication with steroids or MPA. A low proportion of Breg cells is associated with an elevated frequency of allograft rejection events.     

Non-Random Variability in Functional Composition of Coral Reef Fish Communities along an Environmental Gradient

Changes in the coral reef complex can affect predator-prey relationships, resource availability and niche utilisation in the associated fish community, which may be reflected in decreased stability of the functional traits present in a community. This is because particular traits may be favoured by a changing environment, or by habitat degradation. Furthermore, other traits can be selected against because degradation can relax the association between fishes and benthic habitat. We characterised six important ecological traits for fish species occurring at seven sites across a disturbed coral reef archipelago in Indonesia, where reefs have been exposed to eutrophication and destructive fishing practices for decades. Functional diversity was assessed using two complementary indices (FRic and RaoQ) and correlated to important environmental factors (live coral cover and rugosity, representing local reef health, and distance from shore, representing a cross-shelf environmental gradient). Indices were examined for both a change in their mean, as well as temporal (short-term; hours) and spatial (cross-shelf) variability, to assess whether fish-habitat association became relaxed along with habitat degradation. Furthermore, variability in individual traits was examined to identify the traits that are most affected by habitat change. Increases in the general reef health indicators, live coral cover and rugosity (correlated with distance from the mainland), were associated with decreases in the variability of functional diversity and with community-level changes in the abundance of several traits (notably home range size, maximum length, microalgae, detritus and small invertebrate feeding and reproductive turnover). A decrease in coral cover increased variability of RaoQ while rugosity and distance both inversely affected variability of FRic; however, averages for these indices did not reveal patterns associated with the environment. These results suggest that increased degradation of coral reefs is associated with increased variability in fish community functional composition resulting from selective impacts on specific traits, thereby affecting the functional response of these communities to increasing perturbations.     

Insulin-Like Growth Factor 1 (IGF-1) in Parkinson's Disease: Potential as Trait-, Progression- and Prediction Marker and Confounding Factors

Introduction

Biomarkers indicating trait, progression and prediction of pathology and symptoms in Parkinson''s disease (PD) often lack specificity or reliability. Investigating biomarker variance between individuals and over time and the effect of confounding factors is essential for the evaluation of biomarkers in PD, such as insulin-like growth factor 1 (IGF-1).

Materials and Methods

IGF-1 serum levels were investigated in up to 8 biannual visits in 37 PD patients and 22 healthy controls (HC) in the longitudinal MODEP study. IGF-1 baseline levels and annual changes in IGF-1 were compared between PD patients and HC while accounting for baseline disease duration (19 early stage: ≤3.5 years; 18 moderate stage: >4 years), age, sex, body mass index (BMI) and common medical factors putatively modulating IGF-1. In addition, associations of baseline IGF-1 with annual changes of motor, cognitive and depressive symptoms and medication dose were investigated.

Results

PD patients in moderate (130±26 ng/mL; p = .004), but not early stages (115±19, p>.1), showed significantly increased baseline IGF-1 levels compared with HC (106±24 ng/mL; p = .017). Age had a significant negative correlation with IGF-1 levels in HC (r = -.47, p = .028) and no correlation in PD patients (r = -.06, p>.1). BMI was negatively correlated in the overall group (r = -.28, p = .034). The annual changes in IGF-1 did not differ significantly between groups and were not correlated with disease duration. Baseline IGF-1 levels were not associated with annual changes of clinical parameters.

Discussion

Elevated IGF-1 in serum might differentiate between patients in moderate PD stages and HC. However, the value of serum IGF-1 as a trait-, progression- and prediction marker in PD is limited as IGF-1 showed large inter- and intraindividual variability and may be modulated by several confounders.     

The MIF Antagonist ISO-1 Attenuates Corticosteroid-Insensitive Inflammation and Airways Hyperresponsiveness in an Ozone-Induced Model of COPD

Introduction

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with acute and chronic inflammatory disorders and corticosteroid insensitivity. Its expression in the airways of patients with chronic obstructive pulmonary disease (COPD), a relatively steroid insensitive inflammatory disease is unclear, however.

Methods

Sputum, bronchoalveolar lavage (BAL) macrophages and serum were obtained from non-smokers, smokers and COPD patients. To mimic oxidative stress-induced COPD, mice were exposed to ozone for six-weeks and treated with ISO-1, a MIF inhibitor, and/or dexamethasone before each exposure. BAL fluid and lung tissue were collected after the final exposure. Airway hyperresponsiveness (AHR) and lung function were measured using whole body plethysmography. HIF-1α binding to the Mif promoter was determined by Chromatin Immunoprecipitation assays.

Results

MIF levels in sputum and BAL macrophages from COPD patients were higher than those from non-smokers, with healthy smokers having intermediate levels. MIF expression correlated with that of HIF-1α in all patients groups and in ozone-exposed mice. BAL cell counts, cytokine mRNA and protein expression in lungs and BAL, including MIF, were elevated in ozone-exposed mice and had increased AHR. Dexamethasone had no effect on these parameters in the mouse but ISO-1 attenuated cell recruitment, cytokine release and AHR.

Conclusion

MIF and HIF-1α levels are elevated in COPD BAL macrophages and inhibition of MIF function blocks corticosteroid-insensitive lung inflammation and AHR. Inhibition of MIF may provide a novel anti-inflammatory approach in COPD.     

Population dynamics of a natural red deer population over 200 years detected via substantial changes of genetic variation

Most large mammals have constantly been exposed to anthropogenic influence over decades or even centuries. Because of their long generation times and lack of sampling material, inferences of past population genetic dynamics, including anthropogenic impacts, have only relied on the analysis of the structure of extant populations. Here, we investigate for the first time the change in the genetic constitution of a natural red deer population over two centuries, using up to 200‐year‐old antlers (30 generations) stored in trophy collections. To the best of our knowledge, this is the oldest DNA source ever used for microsatellite population genetic analyses. We demonstrate that government policy and hunting laws may have strong impacts on populations that can lead to unexpectedly rapid changes in the genetic constitution of a large mammal population. A high ancestral individual polymorphism seen in an outbreeding population (1813–1861) was strongly reduced in descendants (1923–1940) during the mid‐19th and early 20th century by genetic bottlenecks. Today (2011), individual polymorphism and variance among individuals is increasing in a constant‐sized (managed) population. Differentiation was high among periods (F_ST > ***); consequently, assignment tests assigned individuals to their own period with >85% probability. In contrast to the high variance observed at nuclear microsatellite loci, mtDNA (D‐loop) was monomorphic through time, suggesting that male immigration dominates the genetic evolution in this population.