Fibrinogen Gamma Chain Research Articles

Fibrinogen Gamma Chain Mutations Provoke Fibrinogen and Apolipoprotein B Plasma Deficiency and Liver Storage.

p.R375W (Fibrinogen Aguadilla) is one out of seven identified mutations (Brescia, Aguadilla, Angers, Al du Pont, Pisa, Beograd, and Ankara) causing hepatic storage of the mutant fibrinogen γ. The Aguadilla mutation has been reported in children from the Caribbean, Europe, Japan, Saudi Arabia, Turkey, and China. All reported children presented with a variable degree of histologically proven chronic liver disease and low plasma fibrinogen levels. In addition, one Japanese and one Turkish child had concomitant hypo-APOB-lipoproteinemia of unknown origin. We report here on an additional child from Turkey with hypofibrinogenemia due to the Aguadilla mutation, massive hepatic storage of the mutant protein, and severe hypo-APOB-lipoproteinemia. The liver biopsy of the patient was studied by light microscopy, electron microscopy (EM), and immunohistochemistry. The investigation included the DNA sequencing of the three fibrinogen and APOB–lipoprotein regulatory genes and the analysis of the encoded protein structures. Six additional Fibrinogen Storage Disease (FSD) patients with either the Aguadilla, Ankara, or Brescia mutations were investigated with the same methodology. A molecular analysis revealed the fibrinogen gamma p.R375W mutation (Aguadilla) but no changes in the APOB and MTTP genes. APOB and MTTP genes showed no abnormalities in the other study cases. Light microscopy and EM studies of liver tissue samples from the child led to the demonstration of the simultaneous accumulation of both fibrinogen and APOB in the same inclusions. Interestingly enough, APOB-containing lipid droplets were entrapped within the fibrinogen inclusions in the hepatocytic Endoplasmic Reticulum (ER). Similar histological, immunohistochemical, EM, and molecular genetics findings were found in the other six FSD cases associated with the Aguadilla, as well as with the Ankara and Brescia mutations. The simultaneous retention of fibrinogen and APOB-lipoproteins in FSD can be detected in routinely stained histological sections. The analysis of protein structures unraveled the pathomorphogenesis of this unexpected phenomenon. Fibrinogen gamma chain mutations provoke conformational changes in the region of the globular domain involved in the “end-to-end” interaction, thus impairing the D-dimer formation. Each monomeric fibrinogen gamma chain is left with an abnormal exposure of hydrophobic patches that become available for interactions with APOB and lipids, causing their intracellular retention and impairment of export as a secondary unavoidable phenomenon.

ITRAQ-Based Quantitative Proteomics Reveals the New Evidence Base for Traumatic Brain Injury Treated with Targeted Temperature Management.

This study aimed to investigate the effects of targeted temperature management (TTM) modulation on traumatic brain injury (TBI) and the involved mechanisms using quantitative proteomics technology. SH-SY5Y and HT-22 cells were subjected to moderate stretch injury using the cell injury controller (CIC), followed by incubation at TTM (mild hypothermia, 32°C), or normothermia (37°C). Thereal-time morphological changes, cellcycle phase distribution, death, and cell viability were evaluated. Moderate TBI was produced by the controlled cortical impactor (CCI), and the effects of TTM on the neurological damage, neurodegeneration, cerebrovascular histopathology, and behavioral outcome were determined in vivo. Results showed that TTM treatment prevented TBI-induced neuronal necrosis in the brain, achieved a substantial reduction in neuronal death both in vitro and in vivo, reduced cortical lesion volume and neuronal loss, attenuated cerebrovascular histopathological damage, brain edema, and improved behavioral outcome. Using an iTRAQ proteomics approach, proteins that were significantly associated with TTM in experimental TBI were identified. Importantly, changes in four candidate molecules (plasminogen[PLG], antithrombin III[AT III], fibrinogen gammachain [FGG], transthyretin[TTR]) were verified using TBI rat brain tissues and TBI human cerebrospinal fluid (CSF)samples. This study is one of the first to investigate the neuroprotective effects of TTM on the proteome of human and experimental models of TBI, providing an overall landscape of the TBI brain proteome and a scientific foundation for further assessment of candidate molecules associated with TTM for the promotion of reparative strategies post-TBI.

Dithiothreitol-based protein equalization technology to unravel biomarkers for bladder cancer

This study aimed to assess the benefits of dithiothreitol (DTT)-based sample treatment for protein equalization to assess potential biomarkers for bladder cancer. The proteome of plasma samples of patients with bladder carcinoma, patients with lower urinary tract symptoms (LUTS) and healthy volunteers, was equalized with dithiothreitol (DTT) and compared. The equalized proteomes were interrogated using two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry. Six proteins, namely serum albumin, gelsolin, fibrinogen gamma chain, Ig alpha-1 chain C region, Ig alpha-2 chain C region and haptoglobin, were found dysregulated in at least 70% of bladder cancer patients when compared with a pool of healthy individuals. One protein, serum albumin, was found overexpressed in 70% of the patients when the equalized proteome of the healthy pool was compared with the equalized proteome of the LUTS patients. The pathways modified by the proteins differentially expressed were analyzed using Cytoscape. The method here presented is fast, cheap, of easy application and it matches the analytical minimalism rules as outlined by Halls. Orthogonal validation was done using western-blot. Overall, DTT-based protein equalization is a promising methodology in bladder cancer research.

Inter-alpha Inhibitor H4 as a Potential Biomarker Predicting the Treatment Outcomes in Patients with Hepatocellular Carcinoma.

PurposeEarly prediction of treatment outcomes represents an essential step towards increased treatment efficacy and survival in patients with hepatocellular carcinoma (HCC). In this study, we performed two-dimensional electrophoresis (2-DE) followed by protein profiling to identify biomarkers predictive of therapeutic outcomes in patients with HCC who received liver-directed therapy (LDTx) involving local radiotherapy (RT), and studied the underlying mechanisms of the identified proteins.Materials and Methods2-DE analysis was conducted by pooling sera from patients with a good or poor prognosis; serum proteomic profiles of the two groups were compared and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Identified proteins were confirmed via enzyme-linked immunosorbent assay. An invasion assay was performed after overexpression and knockdown of target protein in Huh7 cells.ResultsLevels of inter-alpha inhibitor H4 (ITIH4), fibrinogen gamma chain, keratin 9/1 complex, carbonic anhydrase I, and carbonmonoxyhemoglobin S were changed by more than 4-fold in response to LDTx. In particular, pre-LDTx ITIH4 expression was more than 5-fold higher in patients with a good prognosis, compared to patients with a poor prognosis. The migration ability of Huh7 cells was significantly suppressed and enhanced by ITIH4 overexpression and knockdown, respectively. The tumors of patients with HCC and a good prognosis expressed high levels of ITIH4, compared to those of patients with a poor prognosis.ConclusionTaken together, ITIH4 may be a potential therapeutic target that could inhibit cancer metastasis, as well as a prognostic marker for patients with HCC who are receiving LDTx.

Abstract 2825: Gene expression profile in genetically engineered mouse model of lung cancer correlates with that in human lung adenocarcinoma

Abstract Introduction: Despite recent advances in the understanding of the biology of lung cancer, there are no agents that can target Kras, the most common mutation seen in lung adenocarcinoma. Development of genetically engineered mouse (GEM) models with krasG12Dand p53R172H+ will help us to understand the biology of kras mutated lung cancer and develop specific targeted therapeutic agents. Material and Methods: We have developed two GEM models of lung adenocarcinoma harboring the KrasG12D mutation [KrasG12D;AdCre (KA) and KrasG12D; p53R172H;AdCre (KPA)] by conditional activation in the lung. We performed RNA-seq analysis from KA and KPA tumor tissues and compared them to normal lungs from littermate controls. The results were compared with publicly available human data (TCGA, cBioportal). In addition, we have developed two novel syngeneic cell lines from KA and KPA tumors, which will be used to test the efficacy of candidate drugs. Results: Hyaluronan binding protein (Habp2) was top most upregulated gene [LogFC 12.7 (KA) 10.4 (KPA)] in both KA and KPA tumor tissues compared to normal. The TCGA and cBioportal data suggest Habp2 is overexpressed in lung adenocarcinoma as opposed to other histologic subtypes. We then analyzed Habp2 associated gene signature and observed that fibrinogen alpha chain (FGA), fibrinogen gamma chain (FGG), hyaluronan synthase 1 (HES1), serpin peptidase inhibitor, clade E1(Serpine1) were also upregulated in both models. On comparison of KA and KPA tumors, we observed that St6galnac1(ST6 N Acetylgalactosaminide Alpha-2,6 Sialyltransferase 1) was upregulated only in the KPA model, suggesting that p53R175H mutation leads to St6galnac1 overexpression. Conclusion: Hyaluronan binding protein (Habp2) was top most upregulated gene in the KA and KPA models, while St6galnac1expression appeared to be associated with p53R175H mutation. Gene expression data from our GEM models of lung cancer agree with the TCGA and cBioportal analyses. Thus these GEM models seem to replicate human lung cancer and can be used to test drugs for in vivo therapeutic efficacy, prior to use in humans. Citation Format: Imayavaramban Lakshmanan, Satyanarayana Rachagani, Moorthy Ponnusamy, Seema Chugh, Samikshan Dutta, Sakthivel Muniyan, Surinder Batra, Apar Kishore. Gene expression profile in genetically engineered mouse model of lung cancer correlates with that in human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2825. doi:10.1158/1538-7445.AM2017-2825

EX-VIVO VALIDATION OF PLASMA PROTEIN FIBRINOGEN GAMMA

The clinical hallmarks of AD become apparent when the disease is advanced, and the challenge is to spot the disease in its infancy, before any symptoms. One avenue being explored is a blood-based biomarker for AD. Recently we demonstrated that a simple test consisting of a reduction in Fibrinogen Gamma Chain (FGG) along with age has potential for predicting neocortical amyloid burden (NAB; Ashton et al., 2015). Recent publications have also shown that a blood test to predict NAB is possible (Burnham et al., 2012; Kiddle et al., 2013). Several publications have reported that Fibrinogen and it's subtypes are normally rejected from the brain by the blood-brain barrier (BBB), yet still have been detected in AD mice models and AD post-mortem human brain tissue (Cortes-Canteli et al., 2010; Ryu., 2009). Furthermore, Fibrinogen has been shown to accumulate over time as AD pathology progresses, co-deposit with Aβ in brain tissue, and enhance aggregation and increase Aβ fibrillisation (Klohs et al., 2012; Ahn et al., 2010). It is encouraging that our blood protein of interest has been independently shown to move across a damaged BBB and directly interact and co-localise with amyloid. We now aim to replicate FGG and other promising plasma biomarker candidates of amyloid pathology in larger cohorts and corroborate deposition in post mortem brain tissue. The study includes multiple time point plasma samples (n=115), with additionall sub-set of post mortem brain tissue and sections (n=30; Braak I-VI). Immunoassays for candidate proteins will be performed on the plasma. Immunohistochemistry will be performed on the brain sections while Western blot will be conducted to quantify levels of the targets in brain homogenates. Statistical analysis will be performed to elucidate the association between plasma and brain FGG. By measuring FGG over multiple time points we have confirmed the protein trajectory and have determined its ability to predict the cases positive for amyloid burden. Post mortem brain tissue and sections have been used to confirm the prediction accuracy of plasma FGG. This study will be the first of its kind to demonstrate the development of a candidate protein from plasma to brain.

Using Coexpression Protein Interaction Network Analysis to Identify Mechanisms of Danshensu Affecting Patients with Coronary Heart Disease.

Salvia miltiorrhiza, known as Danshen, has attracted worldwide interest for its substantial effects on coronary heart disease (CHD). Danshensu (DSS) is one of the main active ingredients of Danshen on CHD. Although it has been proven to have a good clinical effect on CHD, the action mechanisms remain elusive. In the current study, a coexpression network-based approach was used to illustrate the beneficial properties of DSS in the context of CHD. By integrating the gene expression profile data and protein-protein interactions (PPIs) data, two coexpression protein interaction networks (CePIN) in a CHD state (CHD CePIN) and a non-CHD state (non-CHD CePIN) were generated. Then, shared nodes and unique nodes in CHD CePIN were attained by conducting a comparison between CHD CePIN and non-CHD CePIN. By calculating the topological parameters of each shared node and unique node in the networks, and comparing the differentially expressed genes, target proteins involved in disease regulation were attained. Then, Gene Ontology (GO) enrichment was utilized to identify biological processes associated to target proteins. Consequently, it turned out that the treatment of CHD with DSS may be partly attributed to the regulation of immunization and blood circulation. Also, it indicated that sodium/hydrogen exchanger 3 (SLC9A3), Prostaglandin G/H synthase 2 (PTGS2), Oxidized low-density lipoprotein receptor 1 (OLR1), and fibrinogen gamma chain (FGG) may be potential therapeutic targets for CHD. In summary, this study provided a novel coexpression protein interaction network approach to provide an explanation of the mechanisms of DSS on CHD and identify key proteins which maybe the potential therapeutic targets for CHD.

Protein Complex of Fibrinogen Gamma Chain and Complement Factor H in Ovarian Cancer Patient Plasma.

In the plasma of an advanced cancer patient, fibrinogen is sometimes increased with possible effects on red blood cells (RBCs). The plasma fraction deteriorating osmotic resistance of RBCs was separated from a patient's plasma with advanced ovarian cancer by phenyl-sepharose column chromatography and analyzed with gel filtration chromatography. In the plasma fraction, we found a protein reactive against whole fibrinogen with a molecular weight higher than that of intact fibrinogen from a healthy volunteer. The-high molecular weight protein was immunoractive to an antibody against fibrinogen gamma chain but not to an antibody against alpha or beta chain. Complement factor H, identified by N-terminal sequencing of a 150-kDa protein separated from the protein, was also eluted from anti-fibrinogen gamma immunoaffinity column. Fibrinogen gamma chain and complement factor H were found to be bound as a protein complex in the plasma of a patient with advanced ovarian cancer.

Comparative analysis of the serum proteome for biomarker discovery to reveal hepatotoxicity induced by iron ion radiation in mice

AimsProteomic analysis of serum biomarkers to determine liver toxicity after exposure to cosmic radiation has not been performed previously. This study was to identify serum biomarkers associated with hepatotoxicity following exposure to iron ion radiation. Main methodsMale mice were whole-body irradiated with a 2grayunit (Gy) iron ion beam, and after 3months, serum and liver samples were collected. Two-dimensional electrophoresis (2-DE) was used to separate the identified serum proteins, and matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-TOF-TOF) was performed to identify differentially expressed proteins. Enzyme-linked immunosorbent assays and immunoblotting were applied to evaluate protein expression, and immunohistochemistry and immunofluorescence were used to investigate protein localization. Real-time polymerase chain reaction (PCR) was performed to confirm altered gene expression. Key findingsA total of 11 spots that showed differential expression were screened and identified as seven proteins. Of these, six proteins were in the same bioinformatics network and included complement component 3, serum amyloid P-component, apolipoprotein E, alpha-2-macroglobulin, fibrinogen alpha chain, and fibrinogen gamma chain. All of these proteins are synthesized by the liver, and may play an important role in liver toxicity. We also confirmed the mRNA transcription, and found that mRNA expression of the six identified proteins increased in the liver in irradiated mice. SignificanceThese results suggest that these proteins may be potential biomarkers of hepatotoxicity in astronauts enduring long space missions.

Sex-specific vitamin D effects on blood coagulation among overweight adults.

Overweight adults are at increased risk for cardiovascular disease and vitamin D deficiency, whereas an important feature to vitamin D physiology is its sex dependence. The aim of this study was to examine whether vitamin D status improvement exerts a sexually dimorphic effect on serum proteins associated with cardiovascular risk among overweight adults. Unprocessed serum from age- and BMI-matched men (n = 26) and premenopausal women (n = 24) with vitamin D deficiency and after they achieved sufficiency through a 12-month nutritional intervention was analysed using our previously published depletion-free quantitative proteomics method. Key findings were verified with ELISA. Differentially expressed proteins were subjected to in silico bioinformatics assessment using principal component analysis, hierarchical clustering and Metacore™ pathway analysis. All mass spectrometry proteomic data are available via ProteomeXchange (identifier: PXD003663). A total of 282 proteins were differentially expressed after the intervention between men and women (P-value ≤ 0·05), in which the blood coagulation pathway was significantly enriched. In agreement with the proteomics findings, ELISA measurements showed vitamin K-dependent protein C, von Willebrand factor, fibrinogen gamma chain and multimerin-1 proteins, of relevance to blood coagulation, to be differentially affected (P-value ≤ 0·05) between sexes after vitamin D status correction. This study identified novel protein-level molecular indicators on the sexually dimorphic effect of vitamin D status correction associated with blood coagulation among overweight adults. These sex-mediated vitamin D effects should be factored in the design and interpretation of vitamin D observational and interventional studies testing cardiometabolic outcomes.

Molecular dynamics-based analyses of the structural instability and secondary structure of the fibrinogen gamma chain protein with the D356V mutation

Mutations in the fibrinogen gamma chain (FGG) gene have been associated with various disorders, such as dysfibrinogenemia, thrombophilia, and hypofibrinogenemia. A literature survey showed that a residue exchange in fibrinogen Milano I from γ Asp to Val at position 330 impairs fibrin polymerization. The D356V (D330V) mutation located in the C-terminus was predicted to be highly deleterious and to affect the function of the protein. The pathogenicity of the altered gene and changes in protein functions were predicted using in silico methods, such as SIFT, PolyPhen 2, I-Mutant 3.0, Align GV–GD, PhD–SNP, and SNPs&GO. The secondary structure of the mutant protein was unwound by the end of the 50-ns simulation period, and a structural change in the helix-turn transition of the alpha-helical (352–356) region residues was observed. Moreover, a change in the length of the helical region was visualized in the mutant trajectory file, indicating the local transient unfolding of the protein. The obtained computational results suggest that the substitution of the neutral amino acid valine for the acidic amino acid aspartic acid at position 356 results in an unwound conformation within 50 ns, which might contribute to defective polymerization. Our analysis also provides insights into the effect of the conformational change in the D356V (D330V) mutant on protein structure and function.

Patients with calcific aortic stenosis exhibit systemic molecular evidence of ischemia, enhanced coagulation, oxidative stress and impaired cholesterol transport

BackgroundThe most common valve diseases are calcific aortic stenosis (AS) and aortic regurgitation (AR). The former is characterized by thickening of valve leaflets followed by progressive calcification, which produces progressive aortic valve (AV) narrowing, increased pressure afterload on the left ventricle (LV) and subsequent LV hypertrophy. On the other hand, AR is due to malcoaptation of the valve leaflets with resultant diastolic reflux of blood from aorta back to the LV producing volume and pressure overload and progressive LV dilatation. In order to isolate the molecular mechanisms taking place during AS, we have used an integrated “–omic” approach to compare plasma samples from AS and from AR patients used as controls. The final purpose of this work is to find molecular changes in response to the calcification of the AV, diminishing the effects of the AV dysfunction. Methods and resultsUsing two-dimensional difference gel electrophoresis (2D-DIGE) and gas chromatography coupled to mass spectrometry (GC-MS) in a cohort of 6 subjects, we have found differences in 24 protein spots and 19 metabolites, respectively. Among them, 7 proteins and 3 metabolites have been verificated by orthogonal techniques (SRM or turbidimetry): fibrinogen beta and gamma chain, vitronectin, apolipoprotein C-II, antithrombin III, haptoglobin, succinic acid, pyroglutamic acid and alanine. Classification according to their main function showed alterations related to coagulation, inflammation, oxidative stress, response to ischemia and lipid metabolism, defining 4 different molecular panels that characterize AS with high specificity and sensitivity. ConclusionThese results may facilitate management of these patients by making faster diagnostics of the disease and better understand these pathways for regulating its progression.

Evaluation and physiological correlation of plasma proteomic fingerprints for deltamethrin-induced hepatotoxicity in Wistar rats

AimsUprising reports towards deltamethrin (DLM)-induced toxicity in non-target species including mammals have raised a worldwide concern. Moreover, in the absence of any identified marker, the prediction of DLM elicited early toxic manifestations in non-targets remains elusive. Main methodsComprehensive approach of proteome profiling along with conventional toxico-physiological correlation analysis was performed to classify novel protein based markers in the plasma of DLM exposed Wistar rats. Animals were exposed orally to DLM (low dose: 2.56mg/kg b.wt. and high dose: 5.12mg/kg b.wt.) up to seven consecutive days. Key findingsThe UPLC-MS/MS analysis revealed a dose-dependent dissemination of DLM and its primary metabolite (3-Phenoxy benzoic acid) in rat plasma. Through 2-DE-MS/MS plasma profiling and subsequent verification at the transcriptional level, we found that 6 liver emanated acute phase proteins (Apolipoprotein-AIV, Apolipoprotein E, Haptoglobin, Hemopexin, Vitamin D Binding protein, and Fibrinogen gamma chain) were significantly (p<0.05) modulated in DLM treated groups in a dose-dependent manner. Accordingly, DLM exposure resulted in adverse effects on body growth (body weight & relative organ weight), serum profile, liver function and histology, inflammatory changes (enhanced TNF-ɑ, TGF-β and IL6 level), and oxidative stress. Moreover, these toxic manifestations were suppressed upon N-acetyl cysteine (NAC) supplementation in DLM treated animals. Thus, DLM-induced inflammatory response and subsequent oxidative injury to liver grounds the altered expression of identified acute phase proteins. SignificanceIn conclusion, we proposed these six liver emanated plasma proteins as novel candidate markers to assess the early DLM-induced hepatotoxicity in non-target species with a minimal invasive mean.

Analysis of a family with congenital dysfibrinogenemia caused by an Arg275His mutation in the gamma chain of fibrinogen

To explore the clinical phenotype of a family affected with congenital dysfibrinogenemia and potential mutations underlying the disease. Coagulation testing and hepatorenal function testing were conducted on 18 individuals from three generations. Plasma fibrinogen was extracted and analyzed with SDS-PAGE electrophoresis. All of the exons and flanking sequences of fibrinogen FGA, FGB, FGG genes were analyzed by PCR, and the products were subjected to Sanger sequencing. Hepatorenal function, prothrombin time and activated partial thromboplastin time of the proband were all normal. However, his thrombin time was significantly prolonged. Fibrinogen activity was decreased, while the concentration of antigen was in the normal range. The results of his mother, brother, and nephew were similar. DNA sequencing has confirmed that the proband, his mother, brother, and nephew have all carried a g.5877G>A mutation in the exon 8 of the FGG gene, which resulted in replacement of arginine (Arg) by histidine (His) at position 275. The Arg275His mutation of the fibrinogen gamma chain probably underlies the pathogenesis of congenital dysfibrinogenemia in this family.

Identification of differentially expressed plasma proteins in atherosclerotic patients with type 2 diabetes

Besides hyperglycaemia and insulin resistance, several factors are associated with a higher cardiovascular risk in type 2 diabetes mellitus (T2DM), many of them being closely related to each other owing to common origins or pathways. The pathophysiological mechanisms underlying vascular dysfunctions in diabetes include reduced bioavailability of nitric oxide, increased ROS and prothrombotic factors production, as well as activation of receptors for advanced glycation end-products. These alterations contribute to create a pro-inflammatory/thrombotic state that ultimately leads to plaque formation and complication.This study aimed at identifying differentially expressed plasma proteins between T2DM and non-diabetic patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis coupled with LC-MS/MS. Before analysis, plasma samples were enriched in low-expression proteins through combinatorial hexapeptide ligand libraries. Both mono- and two-dimensional western blotting were performed for data validation. Differentially expressed proteins were mapped onto STRING v10 to build a protein–protein interaction network.Sixteen differentially expressed spots were identified with a high score. Among them, there were fibrinogen beta and gamma chains, complement C1r, C3 and C4-B subcomponents, alpha-1-antitrypsin (AAT), vitronectin and CD5 antigen-like. Protein–Protein interaction analysis evidenced a network among differentially expressed proteins in which vitronectin seems to represent a potentially pivotal node among fibrinolysis, complement dependent immune responses and inflammation in accordance with a number of in vitro and in vivo evidences for a contributory role of these proteins to the development of diabetic atherosclerosis.

BRCA2 gene mutations and coagulation-associated biomarkers.

Thromboembolic events are the second cause of death in cancer patients, although the mechanisms underlying this increased thromboembolic risk remain unclear. The aims of this study were to examine whether BRCA2 gene mutations may modify the circulating levels of thrombocoagulation biomarkers and whether breast cancer development may influence changes in such circulating biomarkers. The study was performed in 25 women with mutations in the BRCA2 gene (n=12 breast cancer, n=13 breast cancer-free) and in 13 BRCA2 non-mutant controls. Results revealed that plasma levels of fibrinogen gamma chain isotypes 2 and 3, haptoglobin isotypes 4 and 5, serotransferrin isotypes 3 and 4 and convertase C3/C5 isotypes 4 and 5 were significantly higher in BRCA2 mutation carriers compared to controls. However, plasma levels of vitamin D binding protein isotype 1 and alpha1-antitrypsin isotypes 2, 3 and 4 were significantly decreased in BRCA2 mutation carriers compared to controls. Plasma expression of PF4 and P-selectin was significantly higher in BRCA2 mutations carriers than in controls. BRCA2 truncated mutations conserving a binding region for RAD51 were associated with increased plasma levels of alpha1-antitrypsin isotypes 3 and 4 with respect to women showing BRCA2 mutations that loss the binding RD51 region to BRCA2. Only plasma levels of vitamin D binding protein isotypes 1 and 3 were significantly reduced and alpha 1-antitrypsin isotype 1 was increased in cancer-free BRCA2 mutation carriers compared to BRCA2 mutation carriers with breast cancer. The presence of BRCA2 mutations is associated with increased plasma levels of thrombo-coagulating-related proteins, which are independent to breast cancer development.

Visualization of Neutrophil Extracellular Traps and Fibrin Meshwork in Human Fibrinopurulent Inflammatory Lesions: I. Light Microscopic Study

Neutrophil extracellular traps (NETs) are extracellular fibrillary structures composed of degraded chromatin and granules of neutrophil origin. In fibrinopurulent inflammation such as pneumonia and abscess, deposition of fibrillar eosinophilic material is a common histopathological finding under hematoxylin-eosin staining. Expectedly, not only fibrin fibrils but also NETs consist of the fibrillar material. The aim of the present study is to analyze immunohistochemically how NETs are involved in the inflammatory process. Archival formalin-fixed, paraffin-embedded sections accompanying marked neutrophilic infiltration were the target of analysis. Neutrophil-associated substances (citrullinated histone H3, lactoferrin, myeloperoxidase and neutrophil elastase) were evaluated as NETs markers, while fibrinogen gamma chain was employed as a fibrin marker. Light microscopically, the fibrils were categorized into three types: thin, thick and clustered thick. Lactoferrin represented a good and stable NETs marker. Thin fibrils belonged to NETs. Thick fibrils are composed of either mixed NETs and fibrin or fibrin alone. Clustered thick fibrils were solely composed of fibrin. Neutrophils were entrapped within the fibrilllar meshwork of the thin and thick types. Apoptotic cells immunoreactive to cleaved caspase 3 and cleaved actin were dispersed in the NETs. In conclusion, NETs and fibrin meshwork were consistently recognizable by immunostaining for lactoferrin and fibrinogen gamma chain.

Visualization of Neutrophil Extracellular Traps and Fibrin Meshwork in Human Fibrinopurulent Inflammatory Lesions: II. Ultrastructural Study.

Neutrophil extracellular traps (NETs) represent an extracellular, spider’s web-like structure resulting from cell death of neutrophils. NETs play an important role in innate immunity against microbial infection, but their roles in human pathological processes remain largely unknown. NETs and fibrin meshwork both showing fibrillar structures are observed at the site of fibrinopurulent inflammation, as described in our sister paper [Acta Histochem. Cytochem. 49; 109–116, 2016]. In the present study, immunoelectron microscopic study was performed for visualizing NETs and fibrin fibrils (thick fibrils in our tongue) in formalin-fixed, paraffin-embedded sections of autopsied lung tissue of legionnaire’s pneumonia. Lactoferrin and fibrinogen gamma chain were utilized as markers of NETs and fibrin, respectively. Analysis of immuno-scanning electron microscopy indicated that NETs constructed thin fibrils and granular materials were attached onto the NETs fibrils. The smooth-surfaced fibrin fibrils were much thicker than the NETs fibrils. Pre-embedding immunoelectron microscopy demonstrated that lactoferrin immunoreactivities were visible as dots on the fibrils, whereas fibrinogen gamma chain immunoreactivities were homogeneously observed throughout the fibrils. Usefulness of immunoelectron microscopic analysis of NETs and fibrin fibrils should be emphasized.

Visualization of Neutrophil Extracellular Traps and Fibrin Meshwork in Human Fibrinopurulent Inflammatory Lesions: III. Correlative Light and Electron Microscopic Study.

Neutrophil extracellular traps (NETs) released from dead neutrophils at the site of inflammation represent webs of neutrophilic DNA stretches dotted with granule-derived antimicrobial proteins, including lactoferrin, and play important roles in innate immunity against microbial infection. We have shown the coexistence of NETs and fibrin meshwork in varied fibrinopurulent inflammatory lesions at both light and electron microscopic levels. In the present study, correlative light and electron microscopy (CLEM) employing confocal laser scanning microscopy and scanning electron microscopy was performed to bridge light and electron microscopic images of NETs and fibrin fibrils in formalin-fixed, paraffin-embedded, autopsied lung sections of legionnaire’s pneumonia. Lactoferrin immunoreactivity and 4'-6-diamidino-2-phenylindole (DAPI) reactivity were used as markers of NETs, and fibrin was probed by fibrinogen gamma chain. Of note is that NETs light microscopically represented as lactoferrin and DAPI-colocalized dots, 2.5 μm in diameter. CLEM gave super-resolution images of NETs and fibrin fibrils: “Dotted” NETs were ultrastructurally composed of fine filaments and masses of 58 nm-sized globular materials. A fibrin fibril consisted of clusters of smooth-surfaced filaments. NETs filaments (26 nm in diameter) were significantly thinner than fibrin filaments (295 nm in diameter). Of note is that CLEM was applicable to formalin-fixed, paraffin-embedded sections of autopsy material.

Blood metabolite markers of neocortical amyloid-β burden: discovery and enrichment using candidate proteins.

We believe this is the first study to investigate associations between blood metabolites and neocortical amyloid burden (NAB) in the search for a blood-based biomarker for Alzheimer's disease (AD). Further, we present the first multi-modal analysis of blood markers in this field. We used blood plasma samples from 91 subjects enrolled in the University of California, San Francisco Alzheimer's Disease Research Centre. Non-targeted metabolomic analysis was used to look for associations with NAB using both single and multiple metabolic feature models. Five metabolic features identified subjects with high NAB, with 72% accuracy. We were able to putatively identify four metabolites from this panel and improve the model further by adding fibrinogen gamma chain protein measures (accuracy=79%). One of the five metabolic features was studied in the Alzheimer's Disease Neuroimaging Initiative cohort, but results were inconclusive. If replicated in larger, independent studies, these metabolic features and proteins could form the basis of a blood test with potential for enrichment of amyloid pathology in anti-amyloid trials.