Hepatocytes form a crucially important cell layer that separates sinusoidal blood from the canalicular bile. They have a uniquely organized polarity with a basal membrane facing liver sinusoidal endothelial cells, while one or more apical poles can contribute to several bile canaliculi jointly with the directly opposing hepatocytes. Establishment and maintenance of hepatocyte polarity is essential for many functions of hepatocytes and requires carefully orchestrated cooperation between cell adhesion molecules, cell junctions, cytoskeleton, extracellular matrix and intracellular trafficking machinery. The process of hepatocyte polarization requires energy and, if abnormal, may result in severe liver disease.A number of inherited disorders affecting tight junction and intracellular trafficking proteins have been described and demonstrate clinical and pathophysiological features overlapping those of the genetic cholestatic liver diseases caused by defects in canalicular ABC transporters. Thus both structural and functional components contribute to the final hepatocyte polarity phenotype. Many acquired liver diseases target factors that determine hepatocyte polarity, such as junctional proteins. Hepatocyte depolarization frequently occurs but is rarely recognized because hematoxylin-eosin staining does not identify the bile canaliculus. However, the molecular mechanisms underlying these defects are not well understood. Here we aim to provide an update on the key factors determining hepatocyte polarity and how it is affected in inherited and acquired diseases. A defining feature of metazoans is the existence of polarized layers of epithelium which give rise to the three dimensional shapes of body parts and types. The formation and maintenance of a polarized epithelium is complex and requires specific cell adhesion molecules, cytoskeletal factors and intracellular trafficking components Hepatocyte polarity mechanisms may be divided into structural and functional components. Structural polarity includes morphologic integrity of TJs and apical plasma membranes with their microvilli, and BC network formation. In cultured hepatocytes and cell lines, reversion to planar polarity phenotype and loss of BC has been demonstrated as a result of deletion or inhibition of individual components of the complex polarization machinery in vivo  and TNF-alpha, and cholangiocytes, which are signaled by NOTCH and TGF-beta Drosophila and which specify future polarization domains in differentiating epithelial cells Acquisition of polarity requires an evolutionarily conserved interconnected network of determinants that have been discovered in Hepatocyte polarization and canalicular network formation require coordinated expression of several key evolutionarily conserved elements each of which consists of many components. These elements are extracellular matrix (ECM), adherens and tight junctions, intracellular protein trafficking machinery including recycling endosomes, cytoskeleton and energy production.in vitro that the trapping of hepatocyte secreted ECM proteins, such as collagen IV, laminin and fibronectin, and possibly growth factors on both non-opposing surfaces is required for the development of hepatocyte polarity phenotype The ECM is a complex macromolecular structural network which forms a scaffold for adhesion and provides a signaling platform by sequestering or releasing cytokines, anchoring processing enzymes and activating hepatocyte surface integrins which trigger intracellular signaling Experiments in MDCK and WIF-B cells demonstrated that Par1b, a serine/threonine kinase activated by LKB1 (see below), is a crucial determinant of hepatocyte-like polarity phenotype Hepatocytes connect through intercellular tight, anchoring (adherens and desmosomes) and gap junctions in vivo knockdown of Rab5 homologues. In mice with Rab5 knockdown DPPIV remained at the basolateral membrane, whilst ABCB11 relocated to REs Apical protein trafficking in hepatocytes involves at least two distinct pathways . HepatocLargely based on studies in cell lines and schematized in MYO5B encoding Myosin 5b, which acts as a molecular motor not only for Rab11a, but also Rab11b, Rab25, and Rab8, cause MicroVillus Inclusion Disease (MVID) in which malabsorption results from the absence of the intestinal brush border MYO5B mutations also manifest cholestasis and progressive liver disease The RE component of the secretory pathway plays a critical role in apical as well as basolateral localization of various proteins Proper endosomal trafficking and recycling of proteins to all plasma membrane domains requires an intact actin and microtubular cytoskeletal system Drosophila, polarized cell lines, neurons and hepatocytes in vitro studies reveal that, after stress, hepatocytes preferentially restore polarity even at low ATP levels, suggesting that polarity is a prime requirement for cellular activity Hepatocyte polarization is energy-dependent but the mechanism is unclear. AMPK, a serine threonine kinase containing a catalytic alpha subunit and a regulatory beta and gamma subunits, controls energy metabolism within cells by sensing the cellular AMP to ATP ratio Typically, diseased liver loses its polarized structure. Polarity defects are seen in single-gene rare inherited disorders  as well CLDN1 and TJP2 encode integral TJ proteins; mutations in CLDN1 were found in neonatal ichthyosis-sclerosing cholangitis syndrome (NISCH) and TJP2 mutations cause familial hypercholanemia (FHC) and a newly described subtype of Progressive Familial Intrahepatic Cholestasis syndrome (PFIC-4) Defects in several apical junction proteins have been associated with inherited human liver diseases . CLDN1 aTJP2 with a heterozygous BAAT (Bile acid-CoA:amino acid N-acyltransferase) M76V mutation .FHC patients have elevated serum bile acid concentrations, pruritus and fat malabsorption. FHC can be caused by defects in several proteins including TJP2 and also enzymes involved in bile acid biogenesis. FHC patients have normal liver enzymes apart from increased serum alkaline phosphatase activity, and variable findings on liver biopsy that include canalicular cholestasis and minimally active chronic hepatitis. Symptoms usually respond to treatment with ursodeoxycholic acid. Some patients have a combination of homozygous missense V48A mutations in TJP2 mutations identified in all patients with PFIC-4 so far are predicted to result in the absence of the protein product TJP2 mutations were reported to have developed hepatocellular carcinoma at 24 and 26 months of age Frame shift deletions and duplications, and splice site Defects in four intracellular trafficking machinery proteins  cause related inherited disorders with polarity defects VPS33B and VIPAS39 encoding VPS33B (vacuolar protein sorting 33 homologue B) and VIPAR . Characteristic presentation of ARC includes neonatal cholestatic jaundice, renal tubular acidosis, arthrogryposis and severe failure to thrive. Patients have normal gamma-GT but significantly increased alkaline phosphatase activity, consistent with the canalicular damage ARC is an autosomal recessive multisystem disorder caused by mutations in Defects in hepatocyte transporter proteins are the most common collective cause of inherited forms of cholestasis. A scope of severity has been demonstrated with more severe protein defects manifesting in infants, with milder abnormalities conferring susceptibility to drug induced and pregnancy induced cholestasis.ATP8B1 (PFIC-1) and ABCB11 (PFIC-2) although patients with a similar phenotype and mutations in TJP2 (PFIC-4) were described recently (see above) ABCB11 mutations are at increased risk of hepatobiliary malignancy Progressive Familial Intrahepatic Cholestasis types I and II (PFIC-1 and PFIC-2) are characterized by persistent cholestasis with normal gamma-GT and progressive liver damage that often requires liver transplantation in childhood. Reduced concentrations of primary BA are found in bile ABCB11 or ATP8B1 that may confer partial instability to the protein display a benign recurrent intrahepatic cholestasis (BRIC) phenotype, in which cholestasis can completely resolve between relapses Patients with milder missense mutations in ABCB4 encoding ABCB4 are associated with PFIC-3 Mutations in ABCC2 cause Dubin-Johnson syndrome. Patients with this condition have recurrent episodes of jaundice without plasma bile acid accumulation. The liver biopsy demonstrates intrahepatocyte deposits of dark pigment often without any obvious hepatobiliary injury Mutations in ABCC2 encodes ABCC2 or MRP2, which is a member of the multidrug resistance protein subfamily that exports anionic glutathione and glucuronate conjugates (including bilirubin) from hepatocytes into canaliculi SLCO1B1 and SLCO1B3 genes and phenotypically is similar to Dubin-Johnson syndrome SLCO1B1 and SLCO1B3 encode organic anion transporting polypeptides OATP1B1 and OATP1B3, which localize to the sinusoidal membrane of hepatocytes and mediate sodium-independent cellular uptake of multiple compounds, including bilirubin glucuronide, bile acids, steroid and thyroid hormones, as well as numerous drugs Rotor syndrome is caused by simultaneous recessive mutations in MicroRNAs (miRNAs), are small 18–24 nucleotide noncoding RNAs that regulate gene expression by binding to mRNAs and interfering with translation CTNNB1, which encodes Beta-catenin are associated with increased Wnt signaling were identified in ∼ 20% of patients with hepatocellular carcinoma (HCC), including a patient with primary PFIC-2 diagnosis Cancer cells become depolarized and there is evidence for the role of disturbed polarity pathways in oncogenesis. Beta-catenin is a constituent protein of adherens junctions and is critical for establishment and maintenance of epithelial polarity. It can dissociate from the junctions and is translocated to the nucleus where it may transmit the contact inhibition signal. Mutations that lead to accumulation of intracytoplasmic and nuclear Beta-catenin were identified in more than half of patients with sporadic hepatoblastoma (HB), a malignant childhood liver tumor in vivo mouse model of HCC with Beta-catenin mutations was recently demonstrated after treatment with a “locked nucleic acid” antisense approach to inactivation of Beta-catenin driven Wnt signaling in vivo nanoparticle mediated siRNA inhibition of integrin subunits, which slowed down progression of HCC and was proposed as a possible novel treatment for this tumor PTEN and more recently PIK3CA and AKT1 were found in patients with “Cowden” or “multiple hamartoma” syndrome, an inherited disorder that leads to multiple hamartomas including biliary hamartomas, and predisposes patients to several types of cancer. PIK3CA encodes the catalytic subunit of PI3K, which adds a phosphate to phosphatidylinositol-4,5-biphosphate (PIP2) to form phosphatidylinositol-3,4,5-triphosphate (PIP3) at the cellular membrane. PTEN dephosphorylates PIP3, which is required for recruitment of AKT1 to the cell membrane where it is phosphorylated. The PIP3 pathway is important for apical membrane formation. PI3KCA somatic mutations are also occasionally seen in HCCs and other cancers Inactivating mutations in APC cause familial adenomatous polyposis and approximately 10% of patients with HB have germline APC mutations Hepatitis B and C both promote hepatocarcinogenesis, which is associated with E-cadherin downregulation and Beta-catenin activation. Hepatitis C virus (HCV) enters hepatocytes using the TJ proteins claudin1 and occludin as co-receptors and a tetraspanin CD81 The work in hepatoma cell lines demonstrated that HCV infection upregulates VEGF which disrupts tight junction integrity promoting viral transmission HCV core protein expression was associated with disrupted apical polarity in MDCK cells, which may be a result of deactivation of PI phosphatase SHIP2 which converts PtdInsP3 to PtdInsP2 HCV entry into via hepatocyte TJs was targeted by the novel approach to therapy. Inhibition of HCV entry using anti-CLDN1 antibody in human liver-chimeric mice successfully treated chronic HCV infection and provides a novel therapeutic approach to this devastating disease Genome wide association studies are helping to unravel pathways involved in the pathogenesis of common human diseases. Primary biliary cirrhosis is the most common autoimmune liver disease primarily affecting women over the age of 40. Recent studies found significant association between defects in several molecular pathways and primary biliary cirrhosis, including known polarity pathways such as mTOR, PI3K, MAPK, Hh, and Wnt signaling, and adherens junctions Improved understanding of hepatocyte polarization benefited from the work in cell lines, model organisms and discoveries of inherited mutations in patients with cholestatic diseases. The unique role of the canalicular membrane in bile acid secretion and the deleterious intracellular effects of their retention prompt the hypothesis that links polarity, bile acid retention, mitochondrial damage, energy metabolism and cholestasis. Hepatocyte polarization and canalicular membrane formation are likely to be dependent upon stimulation by bile acids and LKB1 phosphorylation of AMPK and Par1b, as well as activation of STAT3 and TNFα transcription factors by cytokine OSM. The unique importance of ECM composition and localization in hepatocyte differentiation has been further defined by research efforts in the liver regeneration field. Hepatocytes have specific trafficking pathways for GPI anchored and single transmembrane domain canalicular membrane proteins that utilise the transcytotic route while polytopic ABC transporters traffic from TGN, either via RE or directly to the canalicular membrane. Cholestasis of various etiologies eventually results in inhibition of the direct and transcytotic protein trafficking pathways resulting in bile acid retention within hepatocytes and damage to mitochondria, Golgi and other organelles in vivo model with Beta-catenin mutations. This treatment can potentially benefit more than 20% of patients with this tumor that is driven by Wnt signaling activation. Furthermore blocking HCV entry via claudin1 with a monoclonal antibody approach eliminated chronic HCV infection, which is one of the most common causes of liver cancer. Better understanding of the miRNA role in liver polarity and disease will provide new RNA based approaches to treatment. For these reasons a better understanding of hepatocyte polarity and the increasing arsenal of potential therapeutic approaches give optimism for future treatment development in liver diseases.Identification of the molecular defects responsible for pathogenesis of rare and common diseases affecting liver polarity provides desirable targets for drug design, and although there are no known therapeutic agents that can restore hepatocyte polarity, the research into drug development has already benefited from the understanding of the involvement of polarity factors in disease. Researchers have successfully tested an antisense oligonucleotide approach to treating HCC in an ERC Starter Grant 337057 CLOC.P.G. is a Wellcome Trust Senior Research Fellow in Clinical Sciences (WT095662MA); P.G. is supported by the The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.Both PG and IMA contributed to design, writing and revising the manuscript.


Phleum pratense), ragweed (Ambrosia artemisifolia), mugwort (Artemisia vulgaris), birch  and pine (Pinus sylvestris) pollens. All pollen species caused a decrease in ionic permeability as monitored trans-epithelial electrical resistance (TER) and induced polarized release of mediators analyzed by ELISA, with grass pollen showing the highest activity. Ultrafiltration showed that the responses were due to components <3kDa. However, lipid mediators, including phytoprostane E1, had no effect on TER, and caused only modest induction of mediator release. Reverse-phase chromatography separated 2 active fractions: the most hydrophilic maximally affected cytokine release whereas the other only affected TER. Inhibitor studies revealed that JNK played a more dominant role in regulation of barrier permeability in response to grass pollen exposure, whereas ERK and p38 controlled cytokine release. Adenosine and the flavonoid isorhamnetin present in grass pollen contributed to the overall effect on airway epithelial barrier responses. In conclusion, bronchial epithelial barrier functions are differentially affected by several low molecular weight components released by pollen. Furthermore, ionic permeability and innate cytokine production are differentially regulated.The bronchial epithelium plays a key role in providing a protective barrier against many environmental substances of anthropogenic or natural origin which enter the lungs during breathing. Appropriate responses to these agents are critical for regulation of tissue homeostasis, while inappropriate responses may contribute to disease pathogenesis. Here, we compared epithelial barrier responses to different pollen species, characterized the active pollen components and the signaling pathways leading to epithelial activation. Polarized bronchial cells were exposed to extracts of timothy grass ( The airway epithelium is the first site of contact for inhaled environmental materials  and its barrier properties are crucial for regulation of innate barrier immunity and control of tissue homeostasis. Epithelial barrier functions can be divided into 3 types: physical, chemical and immunological.The chemical barrier of the pseudostratified airway epithelium is elaborated by the goblet cells and submucosal glands that secrete mucus, a fluid containing hydrated gel-forming mucins and a range of host defense and cytoprotective molecules, including anti-microbial molecules, anti-proteases and antioxidants. This mucus traps and inactivates inhaled particles and facilitates their clearance via the mucociliary escalator. In addition to these physical and chemical barriers, airway epithelial cells play a key role in immune surveillance, contributing to innate immunity by the secretion of immuno-stimulatory and modulatory mediators including cytokines, chemokines, growth factors and lipid mediators.It has been hypothesized that abnormalities in airway epithelial barrier properties are central to many lung diseases, including bronchial asthma.1), have immuno-modulatory properties on dendritic cells favoring TH2-mediated T cell responses.4 increases the physical barrier properties of airway epithelial cells.After inhalation, pollen grains become hydrated on the aqueous surface of the airway epithelium and, within a short period release various substances including allergens and many low molecular weight substances.Phleum pratense), ragweed (Ambrosia artemisifolia), pine (Pinus sylvestris), birch  or mugwort (Artemisia vulgaris) resulted in a significant increase in TER (Ambrosia showing the highest protease activity (Phleum (timothy grass) and Pinus pollen extracts reaching statistical significance. IL-8 release was also observed, however this only reached significance for Phleum pollen extracts which stimulated IL-8 release into both apical and basolateral compartments  and the immunological barrier by analyzing polarized release of mediators by ELISA. Stimulation of polarized 16HBE cells with extracts of pollen from timothy grass  in the pollen extract that were capable of affecting the epithelial barriers, grass pollen extract was separated into fractions lower and higher than 3kDa by ultrafiltration. As shown in 1) was identified as active compound.1 on bronchial epithelial barrier functions. Since barrier improving effects of lipoxin A4 have been reported previously, we also included this lipid mediator into our studies. Similarly, as peroxisome proliferator activated receptor gamma (PPARγ) plays a critical role in the control of epithelial barrier functions1 or lipoxin A4 did not alter the ionic permeability . The effects of ciglitazone on GM-CSF or IL-8 release  were not significant.We also assessed the effects of the lipid mediators on cytokine production. PPESince our data suggested that lipid mediators were not the main class of substances released from pollen that altered epithelial barrier functions, we additionally analyzed the effect of the flavonoid isorhamnetin on bronchial epithelial barrier function. As shown in As a series of known lipid mediators did not mimic the effects of grass pollen, we further characterized the active components of grass pollen, by separating the <3kDa fraction of pollen by reverse phase chromatography to isolate substances according to their hydrophobicity. As shown in In summary, separation of the <3kDa fraction of grass pollen extract by reverse phase chromatography indicated that the substances responsible for inducing mediator release and altering the ionic barrier properties are mainly hydrophilic molecules, since little binding to the reverse phase cartridge was observed. Furthermore, the data suggest that the ionic and immunological barrier properties are differentially regulated, since the TER was mainly increased by the wash fraction whereas GM-CSF release was mainly induced by the flow through fraction. Further analysis by mass spectrometry failed to define the molecular species responsible for these effects.As our data implied differential regulation of the barrier responses to pollen extracts, we hypothesized that different intracellular signaling mechanisms would be involved in these responses. Since MAPK signaling pathways are among the main signaling pathways involved in regulation of epithelial barrier properties, we analyzed the effect of specific pharmacological inhibitors of these pathways on pollen extract mediated responses.2A and A2B adenosine receptors were involved in pollen-mediated changes of bronchial epithelial barrier function. As shown in 2B receptor antagonist caused a small reduction of the pollen-mediated increase in TER while the A2A receptor antagonist had no effect. Consistent with this, addition of adenosine to epithelial cultures caused a small increase in TER which was also blocked by the A2B receptor antagonist. However, the pollen-induced release of IL-8 and TNF-α was not affected by adenosine receptor antagonists . These data indicate that adenosine present in grass pollen may contribute to the pollen-mediated improved physical barrier in airway epithelial cells. However, other yet unidentified substances are also involved and regulate the physical and immunological barrier properties of airway epithelial cells.We previously identified the water-soluble nucleoside adenosine in grass pollen extract and showed that the function of dendritic cells is modulated by pollen-derived adenosine.In this study we found that exposure of bronchial epithelial cells to soluble components derived from pollens of grass, birch, ragweed, mugwort or pine resulted in a decrease in ionic permeability (i.e. increase in TER) of the epithelial barrier, even though protease activity was detectable in the extracts. Additionally, all pollen species induced polarized release of inflammatory mediators by the epithelium. Of the pollens tested, grass pollen derived substances showed the highest potential to affect bronchial epithelial barrier functions. Using ultrafiltration we demonstrated that low molecular weight substances below <3kDa were responsible for altered barrier functions in bronchial epithelial cells. Using ultra-performance liquid chromatography and mass spectrometry, we have previously shown that fraction comprises thousands of substances.Involvement of the JNK pathway in the regulation of epithelial cellular junctions has been shown previously, although effects are cell type, stimulus and duration-specific.Using fully differentiated human PBECs, we previously showed that the TER of differentiated PBECs was increased in the presence of pollen derived substances.1.1 is able to modulate dendritic cell function favoring a TH2 mediated immune response.1 did not significantly affect either cytokine release or the physical barrier of bronchial epithelial cells. Furthermore, although the endogenous lipid mediator lipoxin A4 has been shown to modulate airway epithelial barrier functions by increasing the physical barrier properties,After inhalation, pollen grains are hydrated on the airway mucosa and they release a multitude of substances in a short period of time; these substances range from proteins like allergens and proteases, to carbohydrates and lipid mediators.1 via a PPARγ-dependent mechanism to support TH2 polarization.Epithelial barrier functions can be regulated by PPARγ, a member of the nuclear hormone receptor family and a receptor for many lipid mediators like prostaglandins.Phleum pratense pollen,In addition to lipid mediators, pollen extract contains many flavonoids, polyphenolic molecules which are ubiquitously present in plants. Biological effects have been described for many flavonoids, ranging from anti-inflammatory, anti-oxidant to anti-microbial.2B receptor antagonist partially reduced the pollen induced increase in physical barrier functions indicating that adenosine may be one of many substances in pollen extract that influence the physical barrier properties of airway epithelial cells. However, other yet unidentified substances might be more effective or interact with each other to affect the epithelial barrier.Separation of the low molecular weight fraction of grass pollen extract by reverse phase chromatography showed that substances with a hydrophilic nature are responsible for the observed effects on epithelial barrier functions. In addition to flavonoids, potential candidate substances with a low molecular weight are purine and pyrimidine nucleosides. Adenosine, a purine nucleoside identified in grass pollen, has been shown to have immune-modulatory function on dendritic cells and thus influence T cell polarization.In summary we have shown that a variety of hydrophilic pollen substances, many yet to be identified, are able to differentially modulate the physical or immunological barrier properties of bronchial epithelial cells. Potential candidates that contribute to the overall effect of pollen extract on the airway barrier functions include the flavonoid isorhamnetin which modulates the immunological barrier and adenosine which affects the physical barrier. By using high performance liquid chromatography to further separate the active compounds it may be possible to identify specific components with distinct effects on epithelial cells rather than the mixed effect observed with the total pollen extract. Identification of specific substances in pollen altering the physical or immunological barrier of bronchial epithelial cells should increase our understanding of the regulatory mechanisms of epithelial barrier functions. Furthermore, identification of natural products that improve the physical barrier function might potentially be of benefit in the therapy of airway diseases where the physical barrier is dysfunctional.The human bronchial epithelial cell line, 16HBE14o- , was maintained in minimum essential medium (MEM) with Glutamax and supplemented with 10% foetal bovine serum and penicillin/streptomycin (Life technologies) (growth medium); cell culture flasks were coated with PureCol collagen I (Advanced BioMatrix). For experiment, 16HBE cells were cultured in collagen coated Transwell® permeable supports . Cells were seeded at a density of 1.5 × 10Phleum pratense), Ragweed (Ambrosia artemisifolia), Mugwort (Artemisia vulgaris), Birch  and Pine (Pinus sylvestris) were obtained from Allergon. Extracts of pollen were prepared by resuspending pollen grains in MEM without supplements at 30 mg/ml. After incubation at 37°C with agitation for 30 min, extracts were centrifuged, sterile filtered and stored in aliquots at −80°C until use.Pollen from Timothy grass , lipoxin A4 (Enzo Life Sciences), isorhamnetin (Sigma) and adenosine (Sigma) were diluted in MEM and applied apically. T0070907 was used as a PPARγ antagonist and ciglitazone as PPARγ agonist . SCH-442416 and MRS-1754 (Sigma) were used as an adenosine A2A and A2B receptor antagonist, respectively.Polarized 16HBE cells were stimulated apically with pollen extract. Concentrations cited in the results refer to the weight of pollen grains per ml before the extraction process. Directly after stimulation, TER was measured and monitored over time. TER measurements were normalized against the TER directly after stimulation (t = 0h). After 24 h, apical and basolateral supernatants were harvested and clarified by centrifugation; mediator release was analyzed by ELISA . Involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the regulation of 16HBE barrier functions was investigated by using specific pharmacological inhibitors. U0126 was used as an inhibitor of the ERK1/2 pathway, SB203580 for blocking the p38 pathway and SP600125 for inhibiting the JNK pathway . Cells were treated apically with 25 µM of each inhibitor 30min before and during APE stimulation. DMSO was used as a vehicle control. Where tested, PPEAfter 24 h of stimulation cells were fixed with 4% paraformaldehyde, permeabilised with 0.1% Triton X-100, blocked with 1% BSA in PBS and triple stained with an AlexaFluor647 labeled mouse monoclonal anti-ZO-1 antibody (clone ZO1-1A12), phalloidin-AlexaFluor488 and SytoxOrange . Stained membranes were mounted on slides using ProLong Gold antifade reagent (Life technologies) and analyzed with a LSM6000 microscope (Leica Microsystems). On z-stacks, a deconvolution was performed using Leica Application Suite software and orthogonal views were performed using ImageJ software.4OH, applied to the conditioned cartridge and the flow through collected. After washing the cartridge with 2% NH4OH, bound substances were eluted with 100% methanol and isopropanol. Each fraction was collected and lyophilized for mass spectrometry (see below) and assessment of bioactivity. In the latter case, the fractions were redissolved in half the volume of the original extract in MEM to account for the loss during the separation process and used for cell stimulation.Timothy grass pollen extract was fractionated by ultrafiltration using centrifugal filter units with a molecular weight cut-off of 3kDa (Millipore). Low molecular weight substances of the <3kDa fraction were further separated by reverse phase chromatography using BondElut Plexa cartridges (Agilent Technologies). Briefly, the <3kDa fraction was made alkaline with 2% NH


CLDN14 gene  after multiple testing correction. The three associated variants were followed-up in the Chinese cohort; one SNP rs170183 demonstrated consistent evidence of association in diverse ethnic populations (Pcombined = 2.43 × 10−5). Notably, expression quantitative trait loci (eQTL) data revealed that rs170183 was correlated with a decline in CLDN14 expression in both lymphoblastoid cell lines and T cells . In conclusion, our study identified a novel PBC susceptibility variant that has been shown to be strongly associated with BMD, highlighting the potential of pleiotropy to improve gene discovery.Primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, has been associated with increased incidence of osteoporosis. Intriguingly, two PBC susceptibility loci identified through genome-wide association studies are also involved in bone mineral density (BMD). These observations led us to investigate the genetic variants shared between PBC and BMD. We evaluated 72 genome-wide significant BMD SNPs for association with PBC using two European GWAS data sets (n = 8392), with replication of significant findings in a Chinese cohort . Our analysis identified a novel variant in the intron of the  Primary biliary cirrhosis (PBC) is the most common autoimmune liver disease that approximately affects 1 in 1000 women over the age of 40156891011MAPT, another gene associated with BMD also confers risk of PBC12A recent Immunochip analysis in European populations identified a novel PBC locus located upstream of the TNFSF11 gene, which encodes RANKL15161718Epidemiological and clinical studies have shown that people with PBC have an increased risk of developing osteoporosis and fracture2324CLDN14 that is likely to have pleiotropic effect on PBC and BMD.In the current study, we examine whether the genetic variants previously shown to be associated with BMD at genome-wide significance level also predispose to PBC. Using two European PBC GWAS data sets and a Chinese Han PBC cohort , we identified a novel variant in −8) using the WTCCC PBC GWAS and Italian PBC GWAS data (12Pfdr = 1.58 × 10-5) in an intron of MAPT gene at 17q21.1, and rs9533090  located between AKAP11 and TNFSF11 at 13q14. The third SNP, rs170183  located in an intron of CLDN14 is a novel PBC association.We investigated 72SNPs significantly associated with BMD  identified in the current study were independent signals at these two loci. Rs17564829 and rs1864325 were in complete linkage disequilibrium (r2 = 1). However, the SNP rs9533090 remained significant after conditional analyses adjusting for rs3862738, suggesting rs9533090 was an independent association signal. To search for potential independent signals at CLDN14 locus, we performed conditional logistic regression analysis, including rs170183 as a covariate, using the two PBC GWAS datasets. However, this analysis did not uncover any additional independent signals . It was noteworthy that rs9533090 and rs1864325 showed substantial differences in allele frequencies between Europeans and Chinese .The three significantly associated SNPs from stage 1 were taken forward for replication in an independent Chinese Han PBC cohort. The novel SNP, rs170183 was the only variant that was successfully replicated in this additional cohort .2 > 0.9) using Haploreg v2.0. There were no variants in LD with rs170183 in the coding sequence of CLDN14.To explore potential causal variants with deleterious functions in coding regions, we checked SNPs in strong LD with rs170183 To examine how rs170183 contributes to disease susceptibility, we explored the potential biological function of rs170183. Notably, rs170183 was located in the binding motif for HNF4, a nuclear receptor protein widely expressed in liver. Furthermore, expression quantitative trait locus (eQTL) analysis using Genevar databaseTNFSF11 and MAPT, using European GWAS data sets. Our analysis highlighted a novel variant within the CLDN14 gene that showed consistent evidence of association with PBC in the individuals of Chinese Han as well as European ancestry.A recent study revealed that ~5% of genome-wide significant SNPs and ~17% of genes included in NHGRI had cross-phenotype effects, suggesting the abundant pleiotropic effects on human complex traits3132Although we observed some shared genetic loci between PBC and BMD, we found no association between the BMD-derived GRS and PBC status, implying that BMD and PBC have distinct genetic architectures. Therefore, the observed epidemiological correlation between reduced BMD and PBC risk is likely to depend on secondary effects or shared environmental factors.CLDN14, a member of the claudin family, encodes an integral membrane protein and a component of tight junction strands. Claudins act as crucial factors for epithelial barrier and transport, and therefore are considered the most important components of tight junction35TJP2 gene, encoding tight junction protein2, disrupted tight junctions and led to severe cholestatic liver diseasehttp://www.biogps.org/). In addition, the variants in CLDN14 were suggested to be associated with kidney stone and reduced BMD15I2 = 0). However, we did observe dramatic discrepancies in the allele frequencies of rs9533090 and rs1864325 between Europeans and Chinese. Accordingly, these two variants were not replicated in the Chinese cohort. This is not surprising in that recent GWAS studies of PBC from European and Japanese populations have already indicated that PBC is characterized by genetic heterogeneity. Many PBC risk loci reported in European populations were not validated in Japanese GWAS, including IL12A, SPIB and TNFRSF1A. On the other hand, POU2F1 and TNFSF15, identified in Japanese GWAS, were not associated with PBC in European populationsThe association at the newly identified SNP rs171803 did not show heterogeneity among UK, Italian and Chinese samples  in disparate population groups. Finally, genetic variations in the sex chromosomes were not considered in the current study. In some recent findings, enhanced rates of X chromosome monosomy and Y chromosome loss have been observed in PBC patients4344CLDN14 at 21q22.13, associated with PBC in ethnically diverse populations. Further evaluation of the risk variant and the gene may help elucidate the mechanism of co-manifestation of PBC and osteoporosis. Our findings indicate searching for pleiotropic genes may present an important opportunity for identifying more of the missing heritability of the complex traits.In conclusion, we identified a new locus, rs170183 in http://www.wtccc.org.uk/). The Italian data set was obtained from the database of Genotypes and Phenotypes  through accession number phs000444.v1.p1. Details about sample characteristics, genotyping and quality control can be found in the original papers. All experiments were approved and in accordance by the Human Subjects and Ethics Committee of the State Key Laboratory for Oncogenes and Related Genes, Division of Gastroenterology and Hepatology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Cancer Institute, Shanghai Institute of Digestive Disease. Informed consent was obtained from all subjects.We used a two-stage design to analyze data from three independent cohorts . Stage 1We conducted replication analysis (stage 2) involving 685 PBC cases and 1152 controls from South China. The Chinese PBC cases were 86% female and mean age of 53.8 years (SD: 11.6). The diagnosis of PBC was based on the following three criteria: (1) abnormal liver biochemistry ; (2) a positive test for antimitochondrial antibodies (titer 1:40 or greater); or (3) a liver biopsy with histological abnormalities consistent with PBC. The patients fulfilled at least two of the three criteria were included in the study. The control data set was obtained from a previous GWAS study, in which the controls were recruited in the same region as was the PBC subjects www.genome.gov/gwastudies and accessed in December 2014). Eighty-nine SNPs met genome-wide significance (P < 5.0 × 10−8) in at least one previously published Bone mineral density GWAS or meta-analysis of GWAS. Of the 89 SNPs, 25 were genotyped directly in two PBC GWAS datasets. The SNPs not genotyped were pre-phased by SHAPEIT and then imputed by IMPUTE2, using 1000 Genomes release 20101123 reference panel472 > 0.9) with each other in each locus, only one SNP that was either directly genotyped or imputed with higher confidence was retained in the study. Thus, 13 SNPs were replaced by their proxies. After quality control, a total of 72 independent SNPs were taken forward for the association analysis and genetic risk score construction using WTCCC and Italian PBC GWAS data.We selected SNPs that showed significant association with BMD according to genome-wide association studies catalog http://www.r-project.org/).Case-control association tests were conducted using logistic regression analysis implemented in PLINK v1.07 ( We used MuTHER pilot data from lymphoblastoid cell lines (LCL) for eQTL analysis using GENEVAR (v3.3.0)http://www.broadinstitute.org/mammals/haploreg/)The regulatory elements overlapped with rs170183 were annotated with HaploReg v3 ( P < 5 × 10−8). We calculated the GRS by simply summing risk allele counts and dividing them by non-missing SNPs for each individual.The genetic risk score was constructed in the two PBC GWAS datasets using the 72 SNPs associated with BMD at genome-wide level of significance .


Significant differences were found for 25(OH)D levels (mean: control 74 nmol/L (29.5 ng/ml); SLE 58 nmol/L (23.1 ng/ml), P = 0.04), 25(OH)D deficiency (P = 0.02). Regression models indicate that, for a twofold rise in 25(OH)D level, the odds ratio (OR) for ANA-positivity drops to 36% of the baseline OR. No link was found between SRF-days and 25(OH)D levels. Our results support links between vitamin D deficiency and expression of serological autoimmunity and clinical autoimmunity (SLE). However, no demonstrable association between 25(OH)D and SRF was confirmed, suggesting independent influences of other flare-inducing factors. Results indicate that SLE patients have high risk of 25(OH)D deficiency and therefore supplementation with regular monitoring should be considered as part of patient management.This study explores links between vitamin D deficiency (25(OH)D = 50 nmol/L) and serological autoimmunity (ANA > 1 : 80) and frequency of self-reported flares (SRF) in participants with clinical autoimmunity (SLE). 25(OH)D levels of 121 females were quantified and compared. The cohort consisted of 80 ACR defined SLE patients and 41 age and sex matched controls. Association analysis of log2 (25(OH)D) levels and ANA 80 positivity was undertaken via two-sample  Systemic lupus erythematosus (SLE) is a systemic autoimmune illness with a complex and multifactorial pathogenesis . PatientAlthough the use of UV protective measures is important as a management strategy, it is undertaken with reservation, as adopting such measures may result in vitamin D deficiency and insufficiency reducing individual patient capacity to maintain vitamin D synthesis within the skin. Mechanisms of UV-related influences in SLE range from induction of anti-double stranded DNA (anti-dsDNA) autoantibody production resulting in skin lesion exacerbation  through Deficiency of vitamin D is also demonstrated in association with a range of immune disturbances, including allergy and autoimmune illnesses such as SLE, ulcerative colitis, rheumatoid arthritis, and multiple sclerosis , 11–15. In our cohort of patients with SLE, we set out to explore the determinants of flare events from a patient's perspective with the primary aim being to determine whether lower vitamin D levels were predictive of an increased risk of SRF in SLE patients. The secondary goal was to assess whether any such link was mediated by the serological expression of autoimmunity, as reflected by ANA positivity with a cutoff of ANA 1 : 80.The overall hypothesis of this study was that, through immunomodulating activity, vitamin D (25(OH)D) levels could influence the risk of an SLE patient experiencing and reporting a flare event. The hypothesis proposes a 2-step pathway: (i) from low vitamin D levels to expression of serological autoimmunity (expressed by ANA-positive ratio ≥1 : 80) and (ii) from ANA positivity to frequent flare events. The overall pathogenic pathway  examinedVitamin D (25(OH)D) levels of a cohort of 121 female participants were quantified and compared with reference to vitamin D deficiency, insufficiency, or normal levels  across tAll participants had blood collected for assessment of general measures of health and immunological markers relevant to autoimmune illness and specifically SLE diagnosis, including ANA, DNA, and ENA testing. ANA positivity was recorded with ratios ≥1 : 80 and used to investigate any link between vitamin D levels and serological autoimmunity.SLE participants also shared their flare experience in an interview designed to examine lupus flare history over the 12-month period prior to interview. Flare data was of a self-reported retrospective nature with SRF specific methods detailed previously  and brieEthical review and approval of study according to Declaration of Helsinki 2008  were recParticipants were recruited from the Hunter and Central Coast areas of New South Wales (NSW), Australia. SLE participants were recruited through the Autoimmune Resource and Research Centre (ARRC) and private immunology clinics. Inclusion criteria were specified as being 18–80 years of age and having a confirmed diagnosis of SLE. Confirmation of participant SLE diagnosis was obtained through auditing of health records according to ACR SLE classification . LimitedAs part of study specific questionnaires of a wider study ), participants self-reported their personal medical history including medication and supplement use, home environment characteristics, and lifestyle practices. Of particular interest were usage patterns of ultraviolet (UV) protective products such as SS and FM, as well as hours spent undertaking activities in the outdoors during daylight hours.Participants attended a scheduled study clinic appointment in the 2006 spring season (September–November) to collect questionnaires and complete study requirements including diagnostic pathology testing and assessment of relevant health measurements. Biological samples were collected by a qualified phlebotomist, processed immediately, and stored at −20°C until specific analysis was required. Height and weight measures were used for calculation of body mass index .SLE participant SRF-days and 25(OH)D level relationship was assessed with negative binomial regression models due to overdispersion of flare data. All variables of interest were included in the initial model with a backward stepwise approach with covariates of age; diagnosis years; ACR criteria, outdoors hours; SES; BMI; FMSS, vitamin D supplementation, use of hormones, and ITM. All significant P values). Groups were found to be well matched with no significant differences found in demographic characteristics. Participant mean age was 50 years for control group and 48 years with mean disease duration of 7.7 years (SD 6.2) for the SLE group. The majority of participants were Caucasian (97.5%) with 2 SLE participants and a single control participant identifying with Asian ethnicity. Both groups within the cohort reported educational levels to advanced or vocational level and above (62%), and an SES of either “above Australian average” or “Australian average” was reported in 110 (91%) of the combined cohort. Participants identifying as a current smoker did differ in percentage (control 2.4% : SLE 7.5%) however not significantly with Fisher's exact analysis. UV exposure through sunlight has impacts on vitamin D levels; therefore participants were asked whether they spent regular periods in the sun and also asked to estimate the average weekday and weekend hours spent in the outdoors . Within our cohort, 68% of control participants as compared to 49% SLE participants reported that they experienced regular periods in the sun. This resulted in a calculated significant difference between the groups (P = 0.05). However this difference was not found when comparing the groups' calculated estimate of annual outdoor hours. The use of SS was reported in our cohort in similar proportions (control 78% : SLE 81%) reflecting general adherence to sun protective measures. Individual SS product sun protection factor (SPF) was not collected due to retrospective nature of study and multiple SS products used across the year; however health promotion advice within Australia recommends SS of a minimum SPF 30+ and the majority of SS products within Australia adhere to a minimum of SPF 30+ standard with broad spectrum UVA and UVB protection [Demographic data for the 80 SLE participants and 41 controls (without autoimmune illness) are shown in otection .P = 0.04), 25(OH)D deficiency (P = 0.02), and levels categorised as abnormal (≤72.5 nmol/L) representing vitamin D levels considered to be both deficient and insufficient (P = 0.01). Deficiency level differences between groups were not observed when vitamin D supplementation use was considered; however, this did not extend to insufficient vitamin D 25(OH)D levels with insignificant differences found in unadjusted values but significant differences (P = 0.01) when participant use of supplementation was factored into analysis.As highlighted in SLE participant individual ACR criteria were confirmed within audit phase of the study and included within association analysis to explore potential links between criteria and levels of 25(OH)D. No significant differences were found for any of the 11 ACR criteria in cross analysis with 25(OH)D levels or any subcategory of 25(OH)D levels. Due to reports of leucopoenia being a potential consequence of low vitamin D levels  a separaP = 0.01) and combined ITM group (P = 0.05) and prednisolone (P = 0.03) were all associated with deficient levels of vitamin D. Association P values between 25(OH)D level, ACR criteria analysis, and medications of interest are outlined in The potential for pharmaceutical regimes to also be associated with altered 25(OH)D levels was tested with reference to quantified 25(OH)D levels and also 25(OH)D categories of deficient, insufficient, and a combined abnormal level. Medications and supplements were analysed as single variables and also as combined groups of variables as in ITM. It is of interest that hormone supplementation  indicates that participants with ANA positivity are more likely to be diagnosed with autoimmune SLE. Likewise, two-sample t-test analysis of ANA positivity and 25(OH)D levels clearly demonstrates that lower 25(OH)D levels are associated with a greater risk of autoimmunity  of our cohort (control (17 : 41%) and SLE (63 : 79%)). Unsurprisingly, a significant difference . Two SLE participants had experienced major flares requiring extended periods in hospital, whilst 9 experienced SRF monthly and 2 on a weekly basis. The flare evaluation phase did not link individual SRF events with individual symptoms; therefore the count could represent an unresolved single symptom SRF and/or multiple events representing exacerbation of multiple or different symptoms. This study was retrospective and undertaken outside clinical assessment appointments, so validated clinic-based flare assessment tools were not able to be used.The final pathway examined involved modelling SRF-day counts as the outcome variable and independent variables including ANA 1 : 80 and ACR criteria. Due to overdispersion of SRF-day counts, a negative binomial model was undertaken. The analysis was adjusted for demographic and social factors that could exhibit confounding effects for 25(OH)D levels ). Those factors significant in univariate analysis were included in the final multivariate model. Statistically significant associations for both univariate and multivariate models are listed within P values in both univariate and multivariate models. However, despite a significant association between ANA positivity (P = 0.007), the ACR criterion of arthritis (P = 0.003), and SRF frequency, this association was in a negative direction with OR of 0.48 and 0.51, respectively.No link was found between SRF-days and 25(OH)D levels or any sub categorisation of 25(OH)D levels indicating that whilst lower vitamin D levels may influence autoimmune diagnosis as defined by ANA-positivity, the lower levels do not influence flare risk or frequency. Protective associations were found for the use of UV barrier products of FMSS as indicated by OR and Deficiency in vitamin D levels is often reported in general populations and is increasingly linked to increased incidence of autoimmune illnesses such as SLE and other musculoskeletal illnesses , 29. GenLevels of insufficiency within our cohort were found in both healthy (46.3%) and SLE (48.8%) groups without adjustment for supplementation. This finding was considerably reduced when adjusted for supplement use. Insufficiency is not an unusual finding in patients with SLE even with supplementation , 35 withIt is reported that 13–45% of healthy individuals may have ANA positivity during their lifetime –40. PrevThe log2 logistic model related 25(OH)D levels to probability of ANA positivity. Substitution allowed absolute odds values to be calculated. For a 25(OH)D level of 40 nmol/L, the probability of ANA positivity is 79% (odds 3.67 : 1), whilst a 25(OH)D level of 80 nmol/L is associated with an ANA positivity probability of only 57% (odds 1.32 : 1), equating to 36% of the odds of ANA positivity for the lower vitamin D level of 40 . This confirms that as vitamin D levels increase, probability of autoimmune disease expression decreases. This finding, with particular reference to vitamin D deficiency and expression of an autoimmune disease (ANA 1 : 80), is in line with established literature , 44.The negative association between serological magnitude of autoimmunity and SRF frequency was unexpected but consistently demonstrated throughout data subanalysis of ANA patterns (homogeneous only), titres, and a restricted analysis of ANA-positive participants with concurrent ENA and/or DNA positivity. ANA patterns were of particular interest due to reports of dense fine speckled 70 (DFS70) antigen staining, as a potential marker of protection against autoimmunity , 45, 46.This concords with evidence for SLE and autoimmunity representing a multistep process proceeding from self-tolerance to serological “benign” autoimmunity (step 1), followed by later conversion of serological “silent” autoimmunity, to overt autoimmune disease (SLE) (step 2) . CurrentGiven the virtually universal requirement for ANA positivity to exist before a clinical diagnosis of SLE is made, there is an inherent incorporation bias operating in any study attempting to tease out relative pathogenic contributions of various determinants to either “benign”  or “overt”  autoimmunity: this is because the presence of ANA positivity increases the likelihood of a SLE diagnosis being made. This phenomenon occurs despite the decline in immune function with aging (immunosenescence), which is often associated with a rise in ANA expression and titres, in the absence of any clinical autoimmune disease presence.Given the results of separate studies defining links between environmental chemical exposure and SRF frequency , 49, theThe phenomenon of “protective” autoimmunity is well established, with antinuclear antibodies having been shown to operate to limit immune-related pathology in a range of settings. Examples of protective autoimmunity include (i) expression of ENA along with ANA protecting against severe renal lupus ; (ii) anOur study's findings are consistent with a proposed role of ANA in offering protection against lupus flares, and, indeed, expression of some ANAs may be a marker of adaptive regulatory immune function as an allostatic mechanism.Despite the clear autoimmune predisposition conferred by low 25(OH)D levels, we could find no significant association between 25(OH)D levels and frequency of SRF in SLE participants. A lack of correlation has also been reported in other vitamin D/activity studies , 51. In This study provides retrospective data within the context of a relatively homogenous Caucasian population with a validated SLE diagnosis according to ACR criteria. The retrospective design would limit firm establishment of casual relationships. The study was undertaken outside routine clinical management appointments with data collection from a nonclinician; therefore SLE flare data collected was from a self-reported patient perspective rather than according to standard disease activity measures. To minimise bias, the methods followed a standardised protocol which included a flare definition  and struDespite collection of samples within a single spring season, the dynamic and fluctuating nature of vitamin D levels  coupled The capacity for extrapolation of results from this study is limited by the lack of diversity in ethnicity and gender within our study population. The results should be viewed as of a pilot nature with the need for a comprehensive prospective study protocol documenting vitamin D levels over time along with assessment of disease activity and occurrence of flares potentially adding to evidence of any associations.In summary, our paper reinforces existing literature supporting a link between vitamin D deficiency and the expression of autoimmune phenomena (ANA positivity) and clinical conditions (such as SLE). In contrast, there was no demonstrable association between vitamin D levels and flare frequency, consistent with the proposed independent influence of other flare-inducing factors. The results indicate that SLE patients are at a high risk of vitamin D deficiency and vitamin D supplementation along with regular monitoring should be a consideration as part of individual patient health management plans.


APOBEC3G, APOBEC3F, LAT, CD1D and CCL5 represented the top-five core genes. Characterization of immune cell composition with the platform CIBERSORT showed no differences between genders; however, there were differences when tumor tissues were compared to normal tissues. Our results suggest different immune responses in NSCLC between genders that could be related with the different clinical outcome.There are different biological and clinical patterns of lung cancer between genders indicating intrinsic differences leading to increased sensitivity to cigarette smoke-induced DNA damage, mutational patterns of KRAS and better clinical outcomes in women while differences between genders at gene-expression levels was not previously reported. Here we show an enrichment of immune genes in NSCLC in women compared to men. We found in a GSEA analysis  of six public datasets a repeated observation of immune gene sets enrichment in women. “Immune system process”, “immune response”, “defense response”, “cellular defense response” and “regulation of immune system process” were the gene sets most over-represented while  Lung cancer is the most common malignancy and the leading cause of death worldwide where tobacco smoking is the main risk factor for the development of this cancer , 2.It is well known that lung cancer shows different patterns of clinical characteristics and outcomes according to sex. Women have higher susceptibility to cigarette smoke-induced DNA damage and an increased risk for lung cancer, including higher levels of DNA adducts and higher frequency of the KRAS G12C mutation than men , 4. In aDue to different susceptibility to DNA damage, according to sex, our research group previously evaluated differences between genders in gene expression and mutational status in genes involved in DNA repair without observing differences. In contrast, in a global evaluation, some gene sets were differentially enriched in women, including immune gene sets . DespiteWith the aim of exploring transcriptional differences of Non-small cell lung cancer (NSCLC) between the genders, we performed a comprehensive analysis of differentially enriched immune gene sets annotated by Gene Ontology Biological Process (C5 BP).p-values < 0.00000001; FDR's 0.4%–24.2%) and “Immune System Process”  in 06 subsets; “Defense Response” , “Cellular Defense Response”  and “Regulation of Immune System Process”  in 05 subsets  that exerts innate immune activity against retroviruses and has shown tumor suppressive effects in human hepatocellular carcinoma and enhance cell radio resistance in lymphomas;  receptor 2) that mediates monocyte infiltration. The complete list of core genes involved in immune gene sets is shown in Due to the gene redundancy across immune gene sets, we identified “core genes”  over-represented in different subsets for immune biological processes. The top-ten rank core genes included mphomas; , 9 APOBEgration;  CCL5  omposed by mature dendritic cell (DC)/T-cell clusters adjacent to B-cell follicles . DespiteA different composition of tumor infiltrating immune cells between genders might be a good explanation for our findings in the GSEA analyses and could be more easily linked to the outcomes in women patients with NSCLC; however, the similar composition of immune cells suggests differences in activation of immune pathways and interaction between immune and tumoral cells.There are several methodologies for the analysis of various datasets to minimize the batch effect . In thisAPOBEC3G and APOBEC3F seem to play and important role in the immune response in NSCLC, while over-representation of CCL5 , CD1D, LAT, TRAT1, IL32 and others, suggest different regulatory activities of T lymphocytes in NSCLC in women compared to men. Although “Immune Response” and “Immune System Process” where the gene sets more over-represented, which is logical because include more genes that produce redundancy across gene sets, we found differences in more specific subsets such as “Regulation of Immune System Process” “T-Cell Activation”, “Regulation of Cytokine Biosynthetic Process” , providing the first evidence of gender differences of immune regulation by elements that escape X chromosome inactivation and trigger regulatory networks and activation of genes with immune function in other autosomes in a study done in primary humans T cells . Our res015, provRecently, immune checkpoints inhibitors such as nivolumab and pembrolizumab are included in the National Comprehensive Cancer Network (NCCN) guidelines for the treatment of metastatic NSCLC subsequent to first line of chemotherapy . A phaseBiological differences of lung tumors among genders should be deeply explored in order to improve the immunotherapeutic approaches. Our study provides evidence of biological differences of NSCLC between genders and the basis for the distinct clinical outcome.http://www.ncbi.nlm.nih.gov/geo/). One RNASeq Level 3 dataset of lung adenocarcinoma was downloaded from the TCGA website (https://tcga-data.nci.nih.gov/docs/publications/luad_2014/).With the aim of explore differences in immune gene sets in NSCLC between genders in a functional approach, we retrieved normalized gene expression data of five NSCLC datasets  from the NCBI GEO website . Because of the small sample size of the subsets, the GSEA was conducted with 1000 gene set permutations. The GSEA analyses were performed using the Java GSEA implementation downloaded from www.broad.mit.edu/gsea/msigdb/. Gene sets from samples of men vs women were compared. A gene set was considered enriched when it was included in the top 50 rank in at least two subsets with a p-value < 0.05 and a False Discovery Rate (FDR) < 25% and when was represented in only 1 gender.We evaluated 825 gene sets in biological processes annotated from Gene Ontology (C5 BP) in the Molecular Signature Database  and type of tissue  when these samples were available. Patients with unknown smoking status or former smokers were excluded from this analysis, except for patients from the dataset GSE7670 whose samples lack this information Table .https://cibersort.stanford.edu/) was done. CIBERSORT quantify relative levels of the abundances of distinct cell types in a mixed cell population [To investigate if signal of enrichment of immune gene sets in women is related to a different background of immune cells in the samples, gene expression analysis with the online analytical platform CIBERSORT  with the LM22 gene signature that is able to identify 22 immune cell types . For this analysis, labels of affymetrix probes were replaced with gene names. Due to more than one affymetrix probe could represent a gene, genes were collapsed to the highest value. After this step, the data were quintile normalized. Analyses were done with 100 permutations with default statistical parameters. The results were filtered by a maximum


To gain insight into the role of altered gene expression in hereditary colorectal polyposis predisposition, in the present study we analyzed absolute and allele-specific expression (ASE) of APC and MUTYH mRNA expression levels were investigated by quantitative Real-Time PCR (qRT-PCR) analysis using TaqMan assay and by ASE assays using dHPLC-based primer extension.We analyzed DNA and RNA extracted from peripheral blood mononuclear cells (PBMC) of 49 familial polyposis patients and 42 healthy blood donors selected according similar gender and age. Patients were studied for germline alterations in both genes using dHPLC, MLPA and automated sequencing. APC gene and six in MUTYH gene. Twenty-nine patients did not show mutations in both genes. Results from qRT-PCR indicated that gene expression of both APC and MUTYH was reduced in patients analyzed. In particular, a significant reduction in APC expression was observed in patients without APC germline mutation vs control group (P < 0.05) while APC expression in the mutation carrier patients, although lower compared to control individuals, did not show statistical significance. On the other hand a significant reduced MUTYH expression was detected in patients with MUTYH mutations vs control group (P < 0.05). Altered ASE of APC was detected in four out of eight APC mutation carriers. In particular one case showed a complete loss of one allele. Among APC mutation negative cases, 4 out of 13 showed a moderate ASE. ASE of MUTYH did not show any altered expression in the cases analyzed. Spearman’s Rho Test analysis showed a positive and significant correlation between APC and MUTYH genes both in cases and in controls (P = 0.020 and P < 0.001).Twenty out of 49 patients showed germline mutations: 14 in APC and MUTYH showed a reduced germline expression, not always corresponding to gene mutation. Expression of APC is decreased in mutation negative cases and this appears to be a promising indicator of FAP predisposition, while for MUTYH gene, mutation is associated to reduced mRNA expression. This study could improve the predictive genetic diagnosis of at-risk individuals belonging to families with reduced mRNA expression regardless of presence of mutation.The online version of this article (doi:10.1186/s13046-015-0244-4) contains supplementary material, which is available to authorized users. The tumor suppressor APC gene, a component of Wnt pathway, encodes a multifunctional protein that regulates many cellular processes, as differentiation, proliferation, life and death decision; it is involved in ephithelial turn-over, then APC loss is an early event in gut epithelium tumorigenesis [APC germline mutations are detected in the majority of FAP patients, even if in a relevant subset of cases the mutations cannot be identified. A fraction (7–23 %) of APC mutation negative cases with phenotypes overlapping with attenuated FAP (AFAP) or classical FAP, is associated with biallelic germline variants of the MUTYH gene. MUTYH together with OGG1 and MTH1 is a component of DNA Base Excision Repair (BER) pathway that removes damaged bases generated by reactive oxygen species (ROS), capable to induce mutations commonly observed in cancerogenesis [APC expression with similar phenotype to patients with truncating APC mutation [APC transcripts may modulate the disease resulting in classic or attenuated phenotype in cases with and without pathogenic mutations [APC and MUTYH mutations and altered expression, in the present study we investigated the role of dosage imbalance influencing the transcriptome of these two colon cancer-predisposing genes, performing an analysis of absolute and allele-specific expression in patients with different degrees of penetrance of hereditary colorectal disease. We analyzed peripheral blood mononuclear cells (PBMCs) from patients with and without mutations and compared the gene expression with control individuals. Finally, to understand the interaction between APC and BER pathway, we investigated the possible mutual modulation. This exploratory study on correlation among mutational spectrum, gene dosage and phenotype, could improve the genetic diagnosis performing predictive testing of at-risk individuals belonging to families with reduced mRNA expression regardless of presence of mutation. The identification of carriers of mutation or/and reduced mRNA expression may be helpful for the appropriate monitoring and clinical management of patients.Colorectal cancer (CRC) is one of the most frequent causes of cancer death worldwide , 2. InheAP) OMIM#08456. ThP OMIM #15100 and ogenesis . A relevogenesis . The varogenesis –14. In umutation . Germlinmutation , 17–19. utations , 20–22. We analyzed 49 polyposis patients recruited in different collaborating Italian Institutions including the Units of Clinical Physiopathology and Medical Genetics of the University of Florence, Medical Genetics of the University “G. d’Annunzio” of Chieti-Pescara, Gastroenterology and Digestive Endoscopy of the “Regina Elena” National Cancer Institute of Rome. This series consisted of patients affected by classical FAP, AFAP, multiple colorectal polyposis (with more than five polyps)  and famiAPC and MUTYH genes for patients analyzed in previous study was conducted with different PCR-based techniques as previously described [APC also multiplex ligation-dependent probe amplification (MLPA) analysis was performed using the SALSA P043 APC MLPA kit  to detect large gene-rearrangements. Mutations are listed in the International Society for Gastrointestinal Hereditary Tumours  database.Mutation screening of escribed . AdditioAPC and MUTYH messenger RNA (mRNA) expression in PBMCs was investigated by TaqMan quantitative real-time PCR (qRT-PCR) analysis using StepOne™ 2.0 (Applied Biosystems). Data were analyzed using the comparative Ct method and were graphically indicated as 2−∆Ct ± SE. In accordance with the method, the mRNA amounts of the target genes  were normalized to the endogenous housekeeping gene GUSB  [The level of systems) . Target APC gene and rs3219489 (c.1014G > C) (MAF = 0.31) in exon 12 of MUTYH gene were selected. An additional specific assay on exon 7 frequent pathogenic mutation of MUTYH gene  was also designed  . This teThe one-way analysis of variance (ANOVA) in the absolute expression of mRNA levels was performed to test the difference in means between different groups. Bonferroni post-hoc test allowed us to identify which group resulted significantly different based on the comparison of multiple values simultaneously.ASE values were summarized as median, mean and standard deviation (SD) separately for the two groups (cases and controls). Shapiro-Wilk test was used to evaluate the normality in the distribution within each group. To assess differences of ASE values between cases and controls the Kruskall-Wallis test was performed followed by other tests carried out to determine if different proportions of individuals in cases and controls group were at a standard distance from the overall mean  by Chi-square test or Fisher’s exact test. To assess a possible correlation between the two genes Spearman’s rho correlation coefficient was evaluated. All analyses were performed with STATA software (version 10).APC and MUTYH germline mutations, absolute expression and ASE. All analyses were performed and compared to a control population.Cases were analyzed for APC gene 14 mutations introducing frameshift were detected, of which 12 previously published [MUTYH gene was performed for cases negative at APC mutation screening and detected sequence variants in six patients: two carriers of heterozygous variants (GD72 and GD155), three compound heterozygotes , and one carrier of exon 12 homozygous deletion (GD91) , carriers of MUTYH mutations  and patients negative at mutation screening for both genes . 2−∆Ct mean values of cases were compared to those of 42 healthy blood donors selected according similar gender and age in order to have a comparable sample size. RNA amounts from ten patients were used up for qRT-PCR analysis.Patients analyzed by qRT-PCR belonged to three different subgroups: carriers of APC gene expression by qRT-PCR showed a statistically significant reduced expression in the 21 APC-/MUTYH- patients  as compared to the control group  (P < 0.05)  Fig. . On the nce Fig. .Fig. 1GeMUTYH gene expression, qRT-PCR analysis showed a statistically significant reduced expression in the 5 MUTYH+ patients  as compared to the control group  (P < 0.05), while the 13 APC+ and the 20 APC-/MUTYH- patients did not show significant MUTYH reduction  cases heterozygous for the SNP rs2229992 (c.1458C > T), of which 7 carrying APC mutation , whereas CRC cases and controls were not  compared to controls (Table P = 0.022). Furthermore the group of the CRC cases deviating more than 1 SD from the overall mean was significantly larger than controls (Table P = 0.001), as noted before [Germline ASE of 229992 c.458C > T,ividuals . Resultsot Table . Only APst Table . Four oud before . Altoget229992 c.458C > T,MUTYH, all APC negative cases, heterozygous at MUTYH c.1014G > C variant, and cases bearing the c.536A > G (Y179C) pathogenic variant, were analyzed for ASE using 2 primer extension assays developed in this study. The ASE assay for MUTYH c.1014G > C variant was performed in 14 out of 49 cases  pathogenic variant was performed in 5 cases  we calculated the overall mean of all ASE values measured in cases and controls with both assays and that was 0.96 ; the range of variation ± 1 SD was <0.82 and >1.10 . This correlation was more strengthened among controls.We also correlated the APC and MUTYH was reduced in hereditary colorectal polyposis patients compared to healthy donors. In particular a reduced APC expression was observed even in patients without APC germline mutation meanwhile a reduced MUTYH expression has been detected in patients with MUTYH mutations. The statistically significant reduction in APC expression detected by qRT-PCR in 21 cases with APC-/MUTYH- genotype  were detected only in two APC+ patients included in this study. Other patients had only modest degrees of imbalances of APC, which, as previously discussed, increase the risk of colorectal cancer [APC mutation. In this case ASE identified the complete loss of an allele that could not be seen with the simple observation by qRT-PCR even if this case, with monoallelic expression, showed an approximately 50 % reduction in the overall expression of APC , except case GD72, who developed a classical FAP with vertical transmission ; as regarding APC, we found a moderately reduced relative expression (0.83) (Additional file MUTYH alleles showed a low but equal expression with a reduction in absolute mRNA expression. This might explain FAP phenotype in a carrier of monoallelic MUTYH mutation. It is noteworthy that case GD72 manifested the disease at the age of 29 years. Individuals carrying monoallelic mutations tend to have a later presentation of disease of at least 5 years compared to biallelic ones but patients with heterozygous Y179C mutation have usually a more severe phenotype with earlier onset as compared with the other frequent G396D mutation [MUTYH, led to the formation of an inactive protein, as it can remove normal interactions with other proteins [Case GD72, carrier of a monoallelic Y179C mutation and showing by qRT-PCR a reduced expression (0.06 ± 0.01, Additional file mutation . The misproteins  and causMUTYH mutations has been considered comparable with that of first-degree relatives of patients with sporadic colorectal cancer by some authors [MUTYH could be attributed to a situation of haploinsufficiency [MUTYH is still to be defined. Studies of absolute and relative gene expression applied to a larger group of monoallelic mutation carriers could provide a clue of possible involved pathways.The risk of colorectal cancer in heterozygous carriers of  authors  or consi authors , 33. Othficiency . A complAPC gene mutation [APC germline reduced expression. The observations in relatives GD82#1 and GD82#2 might be the first evidence of this kind of correlations. It will be very interesting to extend the study to other polyposis families with a larger number of relatives. If confirmed in further studies, the correlation between germline reduced expression and disease manifestations could improve the predictive genetic diagnosis of at-risk individuals belonging to families with reduced mRNA expression regardless of presence of mutation.Correlation between germline altered gene expression and disease, sometimes in absence of mutations, have been reported in chronic inflammatory diseases and in forms of intellectual disability, all compared to control individuals –37. In smutation , 38, 39,APC gene, it was recently discovered its involvement in DNA repair mediated by BER, as it is able to modulate the activity of this pathway [APC and MUTYH expression in 39 cases and 42 controls by Spearman’s test. As reported in Fig. Among the various cellular functions of  pathway . TherefoAPC and MUTYH expression levels in hereditary colorectal polyposis patients carrying either APC mutation, MUTYH mutation or none mutations in both genes, compared to 42 controls. Both genes showed a reduced germline expression, not always corresponding to gene mutation. Expression of APC is decreased in mutation negative cases and this appears to be a promising indicator of FAP predisposition, while for MUTYH gene, reduced mRNA expression is associated to mutation. This is the first evidence of ASE analysis of MUTYH but, as the recessive pattern of hereditary transmission of this gene, altered ASE could be masked by contemporary reduced expression of both alleles. This study could improve the predictive genetic diagnosis of at-risk individuals belonging to families with reduced mRNA expression regardless of presence of mutation. It will be very interesting to extend the study to other genes of the Wnt and BER pathways or to other known cancer predisposing genes.In this study the most significant result comes from the analysis of


Pocillopora damicornis is known to release either sexual or asexual derived planula-larvae in various reef locations. Using microsatellite loci as markers, we documented the release of asexually derived chimeric larvae (CL), originating from mosaicked maternal colonies that were also chimeras, at Thai and Philippines reefs. The CL, each presenting different combinations of maternal genotypic constituents, create genetically-complex sets of asexual propagules. This novel mode of inheritance in corals challenges classical postulations of sexual/asexual reproduction traits, as asexual derived CL represent an alliance between genotypes that significantly sways the recruits’ absolute fitness. This type of inherited chimerism, while enhancing intra-entity genetic heterogeneity, is an evolutionary tactic used to increase genetic-heterogeneity, primarily in new areas colonized by a limited number of larvae. Chimerism may also facilitate combat global change impacts by exhibiting adjustable genomic combinations of within-chimera traits that could withstand alterable environmental pressures, helping Pocillopora become a successful cosmopolitan species.The globally distributed coral species  Life-history strategies, among them sexual and asexual reproductive traits, reveal sets of co-adapted demographic and evolutionary tactics that determine the population dynamics and absolute fitness of specific organisms in any given environment. Variations in reproductive strategies among the populations of any species may influence genetic parameters and population genetic features2Ecological situations that guide asexual vs. sexual modes of reproduction in hermatypic corals have been the focus of scientific interest for many decades, as they directly shape the corals’ life-history strategies66Pocillopora damicornis, a globally distributed branching coral species that inhabits a wide range of depths and habitats, is well known for its labile modes of reproduction, shifting from asexual pathways of fragmentation1112131216P. damicornis may also sustain widely distributed populations by asexually producing planula larvae111218121618P. damicornis12Seriatopora hystrixP. damicornis, genetic heterogeneity is also facilitated by the flow of infused genes by way of congener hybridization, by trans-oceanic dispersal via long competent larval periods1324P. damicornis reproduction reflects the entire spectrum of the reproductive modes characteristic of this species. In fact, the archetypal sets of the biological/environmental conditions that direct clonal versus sexual reproduction in P. damicornis are still ambiguous, despite the fact that it is one of the most extensively studied coral species.P. damicornis colonies that have originated from reefs in Thailand and the Philippines, we were surprised to find in both Indo-Pacific sites, a novel mode of asexual reproduction–chimeric larvae, originating from chimeric maternal colonies. Moreover, in a single clutch, released by a single specific colony, each of the chimeric larvae presented different combinations of maternal genotypic constituents, altogether creating a unique genetic variability by means of asexual reproduction, while also revealing the potential ecological advantages of this reproduction mode compared to commonly known sexual/asexual modes of reproduction.After using microsatellite loci as markers of the genetic diversity of We collected 15–22 planulae from each of 12 mother colonies (six colonies from Thailand and six colonies from the Philippines) as well as two tissue samples that were taken from different branch tips in each mother colony. All samples were subjected to genetic analyses that assessed the genotypic diversity and the identity of the maternal colonies and their offspring using six polymorphic microsatellites  and S1. In the Philippines, chimerism was recorded in two loci, PV2 and PV6. In locus PV2, chimerism was found in all six larval clutches and in branch tips sampled from mother colonies (MC) Nos. 4 and 6. In locus PV6, chimerism was evident in the larval clutch released from MC3  and S1. P. damicornis microsatellite alleles from locus Pd3-002 (For six representative DNA samples (two mother colonies and four offspring), we cloned and sequenced  Pd3-002  and S1.  Pd3-002 . Interes Pd3-002 : branch  Pd3-002 .It should be noted that not a single morphological indication  was noticed during sampling of MCs to support the assumption that the chimeric colonies were a result of two colonies having fused together, rather than colonies having inherited the trait from their parents. In view of the above, while parent-colony chimerism is probably the outcome of tissue fusions between young colonies settling in close proximity (see Discussion), offspring chimerism is most likely the outcome of yet unknown budding processes from chimeric adult tissues, which contain a dissimilar genotypic distribution for each chimeric larva  and S1.For dispersion, coral reef organisms  rely on their motile planula stage, larvae that drift in the water column until they encounter a suitable substratum to settle on and metamorphose into primary polyps6Pursuant to the ongoing discussion about the significance of sexual/asexual modes of reproduction in reef-building organisms28Chimerism in sedentary marine organisms is associated with the trait of gregarious settlement303132332430343536373839404142434445464333While organismal uniclonality is assumed to be functional and beneficial in preventing inner-organism conflicts2931323941455152In light of the marked potential advantages of chimerism, the results of the present study challenge classical models and assumptions regarding dwindling genetic heterogeneity wherever asexual reproduction occurs. Our results, while excluding the possibility of parthenogenesisP. damicornis population genetics significantly , which showed a longer planktonic life and a greater dispersal potential, and by the results of Jiang et al.Platygyra daedalea, which further developed into conjoined larvae.The suggested performance exhibited by asexual chimeric larvae in P. damicornis may emerge as an operative tool for structuring and widening the species’ dispersal capabilities by exposing adjustable genotypic combinations of organismal traits to alterable natural selection operations, further shaping the interactions between the functions and the ecological traits, assisting P. damicornis with becoming a successful cosmopolitan species. The full extent and ecological significance of asexually produced planula larvae in corals that are released from chimeric maternal colonies as established chimera entities remains an open question that deserves additional detailed studies.Considering the functional ecological traits related to our findings, larval chimerism in P. damicornis colonies, six from a reef near the Phuket Marine Station in Thailand (June 2006) and six from a reef near the Bolinao Marine Station, in the North Philippines (September 2006). The colonies were collected at each site from depths of 5–10 m and brought to the laboratory, where they were each placed in a separate aquarium supplied with running seawater and aeration and given a serial number . Coral colonies were kept in the aquaria for 1–2 nights, until 15–22 free-swimming planulae were released. Each planula was carefully collected with a syringe, placed in a separate 1.5 ml plastic vial and given its MC’s number followed by a sequential number. In addition, two small branch-tip pieces (0.5 cm) were removed with a small side cutter from different distanced branches of the same maternal colonies , before the colonies were returned to the collection sites. The side cutter and the other sampling tools were thoroughly washed and cleansed with alcohol and distilled water following each sampling.Planulae were collected from 12 large (each 15–20 cm in diameter) gravid 4, shaken, supplemented with 240 μL phenol-chloroform-isoamyl alcohol , and then shipped to the IOLR laboratory in Israel. Samples were then centrifuged for 5 min at 14,000 rpm; the aqueous upper phase was collected and further mixed with 240 μL chloroform-isoamyl alcohol , centrifuged for 5 min at 14,000 rpm, and then the upper phase was collected and the DNA was precipitated with 500 μL of 100% cold ethanol overnight. Vials were centrifuged for 15 min at 4 °C, 14,000 rpm, washed with 500 μL of 70% ethanol and centrifuged for 5 min. Then the ethanol was removed and the precipitated DNA was dissolved in 40 μL DDWGenomic DNA was isolated from larvae and from maternal tissues following GrahamMicrosatellite typing. Six polymorphic microsatellite loci were used; loci PV2 and PV6et al.et al.The amplification products were identified directly from the chromatographs using the Genotyper software version 3.7 (Applied Biosystems). The data were analyzed using the GenAlEx software version 6.2 All the samples which showed unusual allelic patterns  were repeatedly tested 2 to 3 times in order to authenticate the PCR results. Samples indicating chimerism were re-amplified from the same DNA extract. A sample was considered to represent a chimera only if the second screen generated the same multilocus profile as the first. Studies on experimentally produced urochordate chimerasP. damicornis microsatellite Pd3-002 from two different branch samples of mother colony No. 2 (MC#2 Thailand) and from its four offspring  were cloned into a pDRIVE plasmid (Qiagene PCR cloning kit), which was then used to transform a XL1blue bacteria strain of E. coli. In total, 16 positive white bacteria colonies from each cloned plasmid were picked and sequenced using M13 forward and reveres universal primers. The sequences were analyzed and aligned with the GenBank P. damicornis Pd3-002 sequence .


Approximately 65° of 1st MPJ dorsiflexion is required for normal effective walking [st MPJ range of motion is often used in current practice [st MPJ stiffness. This study presents a novel technique to measure joint stiffness using a tactile pressure sensing system . The moment arm was measured from the tuberosity of the first metatarsal head to just beneath the tuberosity of the 1st distal phalanx (Figure st MPJ of the subject through its full range of motion before data collection. For each trial, joint movement is paused briefly at 3 interval points between the resting and maximally dorsiflexed position. At each interval point, the corresponding force applied was measured using a tactile pressure sensing system (Figure st MPJ can be quantified using video analysis. A total of 3 trials were taken, resulting in nine sets of data points to plot a torque-angular displacement graph (Figure 2 indicates that 61% of variability can be explained by this model.To illustrate the method, data were collected on one female flat-footed subject with posterior tibial tendon dysfunction (age 25 yr, body mass index 20.6 kg/mx Figure . A qualim Figure . The prost MPJ stiffness is potentially useful for measuring small joint stiffness in clinical practice. Quantified joint stiffness provides greater accuracy to facilitate clinicians in their diagnoses and prescription of treatment.The proposed method of quantifying 1


The axonal cytoskeleton of neurofilament (NF) is a long-lived network of fibrous elements believed to be a stationary structure maintained by a small pool of transported cytoskeletal precursors. Accordingly, it may be predicted that NF content in axons can vary independently from the transport rate of NF. In the present report, we confirm this prediction by showing that human NFH transgenic mice and transgenic mice expressing human NFL Ser55 (Asp) develop nearly identical abnormal patterns of NF accumulation and distribution in association with opposite changes in NF slow transport rates. We also show that the rate of NF transport in wild-type mice remains constant along a length of the optic axon where NF content varies 3-fold. Moreover, knockout mice lacking NFH develop even more extreme (6-fold) proximal to distal variation in NF number, which is associated with a normal wild-type rate of NF transport. The independence of regional NF content and NF transport is consistent with previous evidence suggesting that the rate of incorporation of transported NF precursors into a metabolically stable stationary cytoskeletal network is the major determinant of axonal NF content, enabling the generation of the striking local variations in NF number seen along axons. NFs ain vivo, we demonstrate that similar grossly abnormal axonal distributions of NF seen in two mouse models are associated with abnormal rates of NF transport in opposite directions. Moreover, in normal mice, the absolute number and regional density of NF varies dramatically proximally to distally along optic axons and we show that NF transport rate is constant along a length of optic axons where NF content varies 3-fold. These observations fulfill predictions of a two NF population model in which the rate of incorporation of a moving population of NF precursors into a stationary metabolically stable NF network is the principal determinant of the varying regional NF content along mature axons.Using genetic mouse models of NF subunit perturbation All experimental protocols were approved by the New York University and Nathan Kline Institute IACUC Committees in conformance with Public Health Service (PHS) policy on the humane care and use of laboratory animals and the NIH Guide for the Care and Use of Laboratory Animals. Mice were sacrificed by decapitation with previous anesthesia using isoflurane.Adult male or female mice of the C57BL/6J strain, aged 3–4 months at the time of injection or sacrifice, were used in all experiments. Mice were housed at 23°C on a 12-h light-dark cycle and were maintained on Lab Chow  supplied at libitum. Production of NFH knockout has been previously described (HKO) . SoutherProduction of human NFH transgenic (hNFH tg) mice  and humaProtein concentrations were determined with bicinchoninic acid (BCA) assay . SDS-PAGE was performed according to Laemmli . SeparatAntibodies used were mAbs to NFL (NR4), NFM (NN18) and NFH (N52) . Antibodies also used were mAbs to human NFH (OC95) and human NFL  and to both human and mouse NFH (N52)  and NFL (24.1) .Mice were anesthetized with halothane gas and the tissue was fixed through intracardial perfusion with 4% paraformaldehyde, 5% glutaraldehye in 0.1 M PBS, pH 7.4, at room temperature. The optic nerve was dissected and processed as described before , segmentBetween 120 and 383 retinal ganglion cell axon profiles were selected at each axonal level for analyses from the larger population of profiles solely on the basis that NF appeared unequivocally in cross-section throughout the axoplasm unless vesicular organelles occupied more than 50% of the cross sectional area (<5% of axons were excluded on this basis). All regions of the optic nerve section were used in obtaining sample profiles and all axons meeting the minimal inclusion criterion were analyzed. Axons were sampled in a caliber distribution that was identical to that in the total fiber population in optic nerves. NFs were counted from randomly assorted photomicrographs at 80,000x by a single investigator. Repetitions were accurate to ± 2.5%.-1 body weight of Avertin (0.5 g tribromoethanol and 0.25 g of 2-methyl-2-butanol in 39.5 ml of distilled water) and received 0.30 μl of phosphate-buffered normal saline (pH 7.4) which contained 50 μCi of L-[35S] methionine . Injections were made into the vitreous of each eye with a glass micropipette (70–100 μm) apparatus.Adult mice were anesthetized with 13–20 μl gSince NFs and many of other proteins are non-uniformly distributed along nerves , 24, thet-test or the Mann-Whitney test. The variance is similar between the groups that are statistically compared.Sample sizes were chosen according to the standard practice in the field. Significance was determined using unpaired two-tailed Student’s in vivo axonal transport rates of pulse-radiolabeled NFs in mice overexpressing human NFH (“hNFH”) at a level 2-fold higher than endogenous levels  methionine to pulse-radiolabel retinal ganglion cell proteins. The distribution of newly synthesized NF proteins was then analyzed at 7 and 16 days after injection in consecutive 1.1 mm segments of the optic pathway by autoradiography after SDS-PAGE. Despite similar NF distributions along axons, the transport of NF proteins relative to that in WT mice was substantially slower in hNFH tg mice but was modestly faster in hNFL Ser 55 tg mice  and 37% , respectively, in HKO mice relative to that in WT mice (Mean ± SEM)  provided another opportunity to investigate the relationship of NF content to transport rate. Morphometric analyses of NF number in 9864 axons from 5 WT and 5 HKO mice were performed at optic nerve levels 50, 700 and 2000 or 4000 μm from the retinal excavation. At the 50 and 700 μm levels, NF density was lowered 44%   consisten ± SEM) . In thisn ± SEM) . Despiten ± SEM)  with comin vivo than routinely used [35S] methionine. Even under these discriminating conditions, the NF transport rate in HKO mice was not detectably different from that of WT mice  axons  varies in length and the largest proportion of RGC has very short initial segments close to the retinal excavation. Because the axon initial segment may contain ribosomes [Previously, investigators of axonal transport have used either the actual radiolabeled NF peak to calculate NF transport rate or the position of the “50ibosomes  capable ibosomes –35, we mibosomes , 37, it In faster NF transport rate is less clear at present. Previous studies suggest that phosphorylation of Ser55 does not, on its own, control assembly of NFL into intermediate filament arrays but might induce subtle changes in NF architecture [Our findings demonstrate that, under a range of experimental conditions, the content and regional distribution of NFs along axons can vary strikingly and independently of any commensurate change in NF transport rates. Consistent with these data are additional recent findings that deletion of the carboxyl terminal tails of NFM and NFH causes NF density to fall 6-fold proximally and 3-fold distally in the absence of any alteration in the NF transport rate or quantity of newly synthesized NF entering axons . This siitecture , which cThe striking differences in NF content and non-uniform axonal distribution patterns seen in different NF mouse models, and the lack of relationship of these differences to changes in NF transport rate, are incompatible with a single NF population model in which NFs undergoing slow axonal transport compose the entire NF content of axons, as proposed in a mathematical modeling study of previously published data on mouse optic axons . Key assOur observations in this report and other existing evidence on NF transport  are bestDespite their non-uniform distribution along axons, NFs appear to be transported at similar rates at different levels of optic axons in mice of a particular age. NFs are transported by kinesin and dynein motors along microtubules  and curr