Fibrin-Targeting Immunotherapy for Dementia.

Blood-brain barrier (BBB) disruption is an early event in the development of Alzheimer's disease. It precedes extracellular deposition of amyloid-ß in senile plaques and blood vessel walls, the intracellular accumulation of neurofibrillary tangles containing phosphorylated tau protein, microglial activation, and neuronal cell death. BBB disruption allows the coagulation protein fibrinogen to leak from the blood into the brain, where it is converted by thrombin cleavage into fibrin and deposits in the parenchyma and CNS vessels. Fibrinogen cleavage by thrombin exposes a cryptic epitope termed P2 which can bind CD11b and CD11c on microglia, macrophages and dendritic cells and trigger an inflammatory response toxic to neurons. Indeed, genetic and pharmacological evidence demonstrates a causal role for fibrin in innate immune cell activation and the development of neurodegenerative diseases. The P2 inflammatory epitope is spatially and compositionally distinct from the coagulation epitope on fibrin. Mouse monoclonal antibody 5B8, which targets the P2 epitope without interfering with the clotting process, has been shown to reduce neurodegeneration and neuroinflammation in animal models of Alzheimer's disease and multiple sclerosis. The selectivity and efficacy of this anti-human fibrin-P2 antibody in animal models supports the development of a monoclonal antibody drug targeting fibrin P2 for the treatment of neurodegenerative diseases. THN391 is a humanized, affinity-matured antibody which has a 100-fold greater affinity for fibrin P2 and improved development properties compared to the parental 5B8 antibody. It is currently in a Phase 1 clinical trial.

Identification and initial characterization of matrix metalloproteinases in the yellow fever mosquito, Aedes aegypti.

Aedes aegypti is a major vector for arboviruses such as dengue, chikungunya and Zika viruses. During acquisition of a viremic bloodmeal, an arbovirus infects mosquito midgut cells before disseminating to secondary tissues, including the salivary glands. Once virus is released into the salivary ducts it can be transmitted to another vertebrate host. The midgut is surrounded by a basal lamina (BL) in the extracellular matrix, consisting of a proteinaceous mesh composed of collagen IV and laminin. BL pore size exclusion limit prevents virions from passing through. Thus, the BL probably requires remodelling via enzymatic activity to enable efficient virus dissemination. Matrix metalloproteinases (MMPs) are extracellular endopeptidases that are involved in remodelling of the extracellular matrix. Here, we describe and characterize the nine Ae. aegypti encoded MMPs, AeMMPs 1-9, which share common features with other invertebrate and vertebrate MMPs. Expression profiling in Ae. aegypti revealed that Aemmp4 and Aemmp6 were upregulated during metamorphosis, whereas expression of Aemmp1 and Aemmp2 increased during bloodmeal digestion. Aemmp1 expression was also upregulated in the presence of a bloodmeal containing chikungunya virus. Using polyclonal antibodies, AeMMP1 and AeMMP2 were specifically detected in tissues associated with the mosquito midgut.

Stainless steel surface functionalization for immobilization of antibody fragments for cardiovascular applications.

Stainless steel 316 L material is commonly used for the production of coronary and peripheral vessel stents. Effective biofunctionalization is a key to improving the performance and safety of the stents after implantation. This paper reports the method for the immobilization of recombinant antibody fragments (scFv) on stainless steel 316 L to facilitate human endothelial progenitor cell (EPC) growth and thus improve cell viability of the implanted stents for cardiovascular applications. The modification of stent surface was conducted in three steps. First the stent surface was coated with titania based coating to increase the density of hydroxyl groups for successful silanization. Then silanization with 3 aminopropyltriethoxysilane (APTS) was performed to provide the surface with amine groups which presence was verified using FTIR, XPS, and fluorescence microscopy. The maximum density of amine groups (4.8*10(-5) mol/cm(2)) on the surface was reached after reaction taking place in ethanol for 1 h at 60 °C and 0.04M APTS. On such prepared surface the glycosylated scFv were subsequently successfully immobilized. The influence of oxidation of scFv glycan moieties and the temperature on scFv coating were investigated. The fluorescence and confocal microscopy study indicated that the densest and most uniformly coated surface with scFv was obtained at 37 °C after oxidation of glycan chain. The results demonstrate that the scFv cannot be efficiently immobilized without prior aminosilanization of the surface. The effect of the chemical modification on the cell viability of EPC line 55.1 (HucPEC-55.1) was performed indicating that the modifications to the 316 L stainless steel are non-toxic to EPCs.

Cardiac Donor Risk Factors Predictive of Short-Term Heart Transplant Recipient Mortality: An Analysis of the United Network for Organ Sharing Database.

INTRODUCTION: To address the shortage of donor hearts for transplantation, there is significant interest in liberalizing donor acceptance criteria. Therefore, the aim of this study was to evaluate cardiac donor characteristics from the United Network for Organ Sharing (UNOS) database to determine their impact on posttransplantation recipient outcomes. METHODS: Adult (≥18 years) patients undergoing heart transplantation from July 1, 2004, to December 31, 2012, in the UNOS Standard Transplant Analysis and Research (STAR) database were reviewed. Patients were stratified by 1-year posttransplantation status; survivors (group S, n = 13,643) and patients who died or underwent cardiac retransplantation at 1-year follow-up (group NS/R = 1785). Thirty-three specific donor variables were collected for each recipient, and independent donor predictors of recipient death or retransplantation at 1 year were determined using multivariable logistic regression analysis. RESULTS: Overall 1-year survival for the entire cohort was 88.4%. Mean donor age was 31.5 ± 11.9 years, and 72% were male. On multivariable logistic regression analysis, donor age >40 years (odds ratio [OR] 1.44, 95% confidence interval [CI] 1.27 to 1.64), graft ischemic time >3 hours (OR 1.32, 1.16 to 1.51), and the use of cardioplegia (OR 1.17, 1.01 to 1.35) or Celsior (OR 1.21, 1.06 to 1.38) preservative solution were significant predictors of recipient death or retransplantation at 1 year posttransplantation. Male donor sex (OR 0.83, 0.74 to 0.93) and the use of antihypertensive agents (OR 0.88, 0.77 to 1.00) or insulin (OR 0.84, 0.76 to 0.94) were protective from adverse outcomes at 1 year. CONCLUSIONS: These data suggest that donors who are older, female, or have a long projected ischemic time pose greater risk to heart transplant recipients in the short term. Additionally, certain components of donor management protocols, including antihypertensive and insulin administration, may be protective to recipients.

Effect of iron oxidation state on the electrical conductivity of the Earth's lower mantle.

Iron can adopt different spin states in the lower mantle. Previous studies indicate that the dominant lower-mantle phase, magnesium silicate perovskite (which contains at least half of its iron as Fe(3+)), undergoes a Fe(3+) high-spin to low-spin transition that has been suggested to cause seismic velocity anomalies and a drop in laboratory-measured electrical conductivity. Here we apply a new synchrotron-based method of Mössbauer spectroscopy and show that Fe(3+) remains in the high-spin state in lower-mantle perovskite at conditions throughout the lower mantle. Electrical conductivity measurements show no conductivity drop in samples with high Fe(3+), suggesting that the conductivity drop observed previously on samples with high Fe(2+) is due to a transition of Fe(2+) to the intermediate-spin state. Correlation of transport and elastic properties of lower-mantle perovskite with electromagnetic and seismic data may provide a new probe of heterogeneity in the lower mantle.

Portable double-sided laser-heating system for Mössbauer spectroscopy and X-ray diffraction experiments at synchrotron facilities with diamond anvil cells.

The diamond anvil cell (DAC) technique coupled with laser heating is a major method for studying materials statically at multimegabar pressures and at high temperatures. Recent progress in experimental techniques, especially in high-pressure single crystal X-ray diffraction, requires portable laser heating systems which are able to heat and move the DAC during data collection. We have developed a double-sided laser heating system for DACs which can be mounted within a rather small (~0.1 m(2)) area and has a weight of ~12 kg. The system is easily transferable between different in-house or synchrotron facilities and can be assembled and set up within a few hours. The system was successfully tested at the High Pressure Station of White Beam (ID09a) and Nuclear Resonance (ID18) beamlines of the European Synchrotron Radiation Facility. We demonstrate examples of application of the system to a single crystal X-ray diffraction investigation of (Mg(0.87),Fe(3+) (0.09),Fe(2+) (0.04))(Si(0.89),Al(0.11))O(3) perovskite (ID09a) and a Synchrotron Mössbauer Source (SMS) study of (Mg(0.8)Fe(0.2))O ferropericlase (ID18).

BX90: a new diamond anvil cell design for X-ray diffraction and optical measurements.

We present a new design of a universal diamond anvil cell, suitable for different kinds of experimental studies under high pressures. Main features of the cell are an ultimate 90-degrees symmetrical axial opening and high stability, making the presented cell design suitable for a whole range of techniques from optical absorption to single-crystal X-ray diffraction studies, also in combination with external resistive or double-side laser heating. Three examples of the cell applications are provided: a Brillouin scattering of neon, single-crystal X-ray diffraction of α-Cr(2)O(3), and resistivity measurements on the (Mg(0.60)Fe(0.40))(Si(0.63)Al(0.37))O(3) silicate perovskite.

Superhard semiconducting optically transparent high pressure phase of boron.

An orthorhombic (space group Pnnm) boron phase was synthesized at pressures above 9 GPa and high temperature, and it was demonstrated to be stable at least up to 30 GPa. The structure, determined by single-crystal x-ray diffraction, consists of B12 icosahedra and B2 dumbbells. The charge density distribution obtained from experimental data and ab initio calculations suggests covalent chemical bonding in this phase. Strong covalent interatomic interactions explain the low compressibility value (bulk modulus is K300=227 GPa) and high hardness of high-pressure boron (Vickers hardness HV=58 GPa), after diamond the second hardest elemental material.

A proteomic study of serum from children with autism showing differential expression of apolipoproteins and complement proteins.

Modern methods that use systematic, quantitative and unbiased approaches are making it possible to discover proteins altered by a disease. To identify proteins that might be differentially expressed in autism, serum proteins from blood were subjected to trypsin digestion followed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) on time-of-flight (TOF) instruments to identify differentially expressed peptides. Children with autism 4-6 years of age (n=69) were compared to typically developing children (n=35) with similar age and gender distributions. A total of 6348 peptide components were quantified. Of these, five peptide components corresponding to four known proteins had an effect size >0.99 with a P<0.05 and a Mascot identification score of 30 or greater for autism compared to controls. The four proteins were: Apolipoprotein (apo) B-100, Complement Factor H Related Protein (FHR1), Complement C1q and Fibronectin 1 (FN1). In addition, apo B-100 and apo A-IV were higher in children with high compared to low functioning autism. Apos are involved in the transport of lipids, cholesterol and vitamin E. The complement system is involved in the lysis and removal of infectious organisms in blood, and may be involved in cellular apoptosis in brain. Despite limitations of the study, including the low fold changes and variable detection rates for the peptide components, the data support possible differences of circulating proteins in autism, and should help stimulate the continued search for causes and treatments of autism by examining peripheral blood.

Peripheral blood gene expression profiling in rheumatoid arthritis.

We carried out gene expression profiling of peripheral blood mononuclear cells (PBMCs) in 29 patients with active rheumatoid arthritis (RA) and 21 control subjects using Affymetrix U95Av2 arrays. Using cluster analysis, we observed a significant alteration in the expression pattern of 81 genes (P<0.001) in the PBMCs of RA patients compared with controls. Many of these genes correlated with differences in monocyte counts between the two study populations, and we show that a large fraction of these genes are specifically expressed at high levels in monocytes. In addition, a logistic regression analysis was performed to identify genes that performed best in the categorization of RA and control samples. Glutaminyl cyclase, IL1RA, S100A12 (also known as calgranulin or EN-RAGE) and Grb2-associated binding protein (GAB2) were among the top discriminators. Along with previous data, the overexpression of S100A12 in RA patients emphasizes the likely importance of RAGE pathways in disease pathogenesis. The altered expression of GAB2, an intracellular adaptor molecule involved in regulating phosphatase function, is of particular interest given the recent identification of the intracellular phosphatase PTPN22 as a risk gene for RA. These data suggest that a detailed study of gene expression patterns in peripheral blood can provide insight into disease pathogenesis. However, it is also clear that substantially larger sample sizes will be required in order to evaluate fully gene expression profiling as a means of identifying disease subsets, or defining biomarkers of outcome and response to therapy in RA.

Whole blood microvolume laser scanning cytometry for monitoring resting and activated platelets.

Enumerating and phenotyping of platelets, resting and activated, from whole blood is important for both the identification and verification of many disease states. Microvolume laser scanning cytometry (MLSC) has been shown to be a simple method for enumerating and phenotyping peripheral blood cells. Here, the utility of MLSC, in conjunction with an anticoagulant containing platelet activation inhibitors, for simultaneously measuring platelet count, phenotype and responsiveness directly from non-fixed whole blood was examined. CTAD or EDTA anticoagulated blood was collected from five to 20 healthy volunteers, stained with fluorescence-labeled antibodies specific for platelet antigens, and run on an in-house modified MSLC device. MLSC was able to measure antigens CD9, CD29, CD36, CD41, CD42a, CD42b, and CD61 on platelets and determine an average of 2.3 x 10(5) +/- 7 x 10(4) platelets per microliter. Counts correlated well with those obtained from the Cell-Dyn 3500 (r(2)=0.84). Agreeing with previous data, less than 2% of platelets from peripheral blood of normal individuals expressed the activation markers CD62P or CD63. After in vitro thrombin activation, >93% of the platelets expressed activation markers. Data presented here shows the benefits of using MLSC in combination with platelet inhibitors to quantitate and phenotype platelets while maintaining a viable responsive state.

Interaction between LIS1 and doublecortin, two lissencephaly gene products.

Mutations in either LIS1 or DCX are the most common cause for type I lissencephaly. Here we report that LIS1 and DCX interact physically both in vitro and in vivo. Epitope-tagged DCX transiently expressed in COS cells can be co-immunoprecipitated with endogenous LIS1. Furthermore, endogenous DCX could be co-immunoprecipitated with endogenous LIS1 in embryonic brain extracts, demonstrating an in vivo association. The two protein products also co-localize in transfected cells and in primary neuronal cells. In addition, we demonstrate homodimerization of DCX in vitro. Using fragments of both LIS1 and DCX, the domains of interaction were mapped. LIS1 and DCX interact with tubulin and microtubules. Our results suggest that addition of DCX and LIS1 to tubulin enhances polymerization in an additive fashion. In in vitro competition assays, when LIS1 is added first, DCX competes with LIS1 in its binding to microtubules, but when DCX is added prior to the addition of LIS1 it enhances the binding of LIS1 to microtubules. We conclude that LIS1 and DCX cross-talk is important to microtubule function in the developing cerebral cortex.

Who represents managed care?

Managed care-related trade associations are more than just legislative advocates. Faced with a competitive marketplace and an overlap in membership from the sheer number of associations, these groups now offer a multitude of services, such as on-line job matching, resource guides, discounts on related professional services, and public relations assistance.

Predominant VH genes expressed in innate antibodies are associated with distinctive antigen-binding sites.

Antibodies to phosphatidylcholine (PtC), a common constituent of mammalian and bacterial cell membranes, represent a large proportion of the natural antibody repertoire in mice. Previous studies of several mouse strains (e.g., C57BL/6) have shown that anti-PtC antibodies are mainly encoded by the VH11 and VH12 immunoglobulin heavy chain variable region gene families. We show here, however, that VH11 and VH12 encode only a small proportion of the anti-PtC antibodies in BALB/c mice. Instead, VHQ52-encoded antibodies predominate in this strain. In addition, two-thirds of the cells expressing VHQ52 family genes use a single gene (which, interestingly, has been previously shown to predominate in the anti-oxazolone response). We also show here that in anti-PtC antibodies from all strains, the distinctive antigen-binding sites associated with VHQ52 differ substantially from those associated with VH11 and VH12. That is, VHQ52-containing transcripts preferentially use the joining region JH4 rather than JH1 and exhibit more diverse complementarity-determining region 3 (CDR3) junctions with more N-region nucleotide additions at the gene segment junctions. Thus, the VH gene family that predominates in the anti-PtC repertoire differs among mouse strains, whereas the distinctive VHDJH rearrangements (CDR3, JH) associated with each VH gene family are similar in all strains. We discuss these findings in the context of a recent hypothesis suggesting that CDR3 structure, independent of VH framework, is sufficient to define the specificity of an antibody.

Presence of germline and full-length IgA RNA transcripts among peritoneal B-1 cells.

Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgA in vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline C alpha mRNA and mature C alpha mRNA transcripts. Germline C alpha and mature C alpha transcripts were readily detectable in peritoneal B-1 cells (defined as IgMbright/IgDdull), but not, or very little, in peritoneal B-2 cells (defined as IgMdull/IgDbright). Moreover, by subdividing the B-1-cell population in CD5+ B-1a cells and CD5- B-1b cells, it was shown that in vivo expression of germline C alpha and mature C alpha transcripts was largely restricted to the B-1b-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-1b-cell subset.

[Serotonin re-uptake inhibitors as primary therapy for carotid sinus hypersensitivity].

Carotid sinus syndrome is a well-recognized cause of unexplained syncope in older patients, and may, lead to significant morbidity due to trauma from falls. Dual chamber pacing has been shown to be effective in relieving symptoms due to bradycardia, but not due to vasodepressor response. We report an 84-year-old man with recurrent syncope due to carotid sinus hypersensitivity. He was treated only with a serotonin reuptake inhibitor and was symptom-free after 3 weeks of therapy. He has remained symptom-free for the past year.