Toxoplasma gondii is a ubiquitous parasite and a prevalent food-borne parasitic pathogen. Infection of the host occurs principally through oral consumption of contaminated food and water with the gastrointestinal tract being the primary route for entry into the host. To promote infection, T. gondii has evolved highly specialized strategies for rapid traversal of the single cell thick intestinal epithelial barrier. Parasite transmigration via the paracellular pathway between adjacent cells enables parasite dissemination to secondary sites of infection where chronic infection of muscle and brain tissue is established. It has recently been proposed that parasite interactions with the integral tight junction (TJ) protein occludin influences parasite transmigration of the intestinal epithelium. We review here the emerging mechanisms of T. gondii transmigration of the small intestinal epithelium alongside the developing role played in modulating the wider TJ-associated proteome to rewire host cell regulatory systems for the benefit of the parasite. Toxoplasma gondii, first isolated in 1908 from the African rodent Ctenodactylus gundi, is a protozoan obligate intracellular parasite of the phylum Apicomplexa that infects virtually all warm-blooded animals.T. gondii infection is widespread throughout the world and in the United States and the United Kingdom it is estimated that 16–40% of the population have been infected by T. gondii, whereas in Central and South America and continental Europe, estimates of infection range from 50–80%.T. gondii infection is high, with the exception of the immunocompromised and pregnant women, individuals usually present no signs of clinical infection other than mild flu-like symptoms with 80–90% of infections going unrecognised.T. gondii also has a considerable economic veterinary impact being a major cause of abortion and stillbirth in livestock.T. gondii encounters the semi-permeable barrier of the single layer of intestinal epithelial cells,T. gondii transmigrate the intestinal epithelium though it is essential for understanding disease pathogenesis. This review aims to build an understanding of how the success of T. gondii in global pathogenesis is linked with its ability to rapidly cross the epithelial barrier of the small intestine (SI), and to provide insights into how technological advances have enabled in-depth examination of how T. gondii interacts with the host cell, potentiating the design of novel intervention and protection strategies.Following ingestion, The continuous ring of the epithelial junctional complex comprises 3 distinct morphological structures: the apical sealing TJ, adhesive adherens junction (AJ) and desmosomes that maintain cell-cell contacts.Now, 50 y since its discovery, the TJ is known to be more dynamic than the original perception as a static, rigid structure that simply sealed the paracellular space with considerable progress being made in the understanding of TJ structure, function and regulation. Dynamic interactions between the integral transmembrane proteins and the cytoplasmic plaque of peripheral adaptor, scaffold and signaling proteins also link the junctional membrane to the actomyosin cytoskeleton.T. gondii has a complex life cycle involving a sexual life cycle that occurs only in the feline host with parasites ultimately being released as oocytes into the environment and an asexual lytic cycle of intracellular growth and multiplication within epithelial cells that occurs in all infected, intermediate hosts. Once ingested from the contaminated environment by intermediate hosts such as livestock, mice or humans the oocysts resist degradation in the stomach and eventually rupture and release bradyzoites into the intestinal lumen.T. gondii establishment of infection and subsequent parasite survival and proliferation is parasite attachment to, and transmigration of the intestinal epithelial barrier. The disease outcome of T. gondii infection is therefore highly dependent on parasite virulence, although surprisingly T. gondii population biology has identified a limited number of dominant strains. Serology samples from infected humans and domestic or farm animals from North America and Europe were used to group T. gondii serovars into clonal lineages I, II and III,3031 and the recently discovered haplotype 12.The crucial step for In silico approaches are being increasingly used to identify new genes of interest or genetic pathways. A recent study using whole genome sequencing of 62 strains of T. gondii found both large regions of conserved genes and specific regions showing enhanced variation. These regions were associated with secretory pathogenicity determinants (SPDs); genes encoding secretory proteins from micronemes (MICs), dense granules (GRA), rhoptries (ROPs) and the SAG-1-related sequence (SRS) superfamily that are associated with host transmission and infection. Highly diverse regions were particularly linked to GRA, ROP and SAG genes such as ROP17, ROP5, GRA3 and SAG3 and SAG2A, which have been previously implicated in murine virulence differences between strains. In contrast, parasite MICs which play a central role in cellular attachment by binding to host receptors, were found to be highly conserved, suggesting T. gondii strains may utilize a conserved repertoire of these host MIC receptors.T. gondii strains covering the 3 major clonal lineages and all isolates could be grouped into their respective genotypes based on their MIC16 sequence.T. gondii tachyzoites, bradyzoites and sporozoites using various molecular and imaging-based techniques,T. gondii lacks cilia or flagella, the early stages of host cell entry involves an unusual form of gliding motility that was first observed over 100 y ago [For a review seeT. gondii types I, II and II has been directly linked to parasite virulence.T. gondii expression of surface antigens that demonstrate crossover functions or redundancy may account for the multitude of parasite-host interactions and broad parasite host range.Parasite dissemination across distinct biologic barriers requires control of the diverse cohort of surface and secreted proteins. The transcellular or active penetration invasion mechanism has been well described for 45 and the host cell membrane. The parasite then actively enters the cell through the MJ into the non-fusogenic parasitophorous vacuole (PV) using its actomyosin motor complex before separating from the host plasma membrane and initiation of replication.T. gondii does not actively invade all cells it attaches to. It has therefore been proposed that neighboring cells are prepared for a later wave of transmigration or invasion through organelle secretion of ROP and MIC proteins.Following host cell surface attachment, the parasite undergoes a series of stages of reorientation and penetration depicted in T. gondii are currently available and only one vaccine, Toxovax is available for use in sheep and goats,T. gondii attachment and invasion may also provide a starting point for the discovery of novel therapeutics; preliminary screening of a library of 1,120 compounds identified several encouraging targets. Pimozide, an inhibitor of dopamine signaling, for instance reduced parasite invasion by ∼50% but caution should be taken to rule out any off-target effects.Despite its clinical importance, relatively few drugs against in vitro.in vivoParacellular transmigration of the SI epithelial barrier presents a fast-track route of parasite dissemination to underlying tissues, avoiding replication and cytolysis that can lead to tissue injury and initiation of an acute immune response. This initial transmigration process is known to be rapid, taking only 20–40 seconds T. gondii tachyzoites located in close contact with the apical cellular junction of cultured intestinal epithelial cells in the clear non-stained region between host cells. The parasite subsequently re-orientates and rapidly transmigrates through the paracellular space between host cells within 52 seconds, comparable with previous studies.Exploitation of the paracellular pathway as a mechanism of infection and the role of TJ proteins, particularly the transmembrane proteins, in infection is not a new concept. In 2002, Barragan and Sibley first suggested this early wave of rapidly migrating parasites may be particularly important to ensure dissemination before activation of the immune system.T. gondii is known to exploit the motility of infiltrating SI immune cells in a Trojan Horse-like mechanism to spread throughout the body to secondary sites of infection, such as the muscle tissues or brain.Following transmigration of the SI epithelium, T. gondii infection.in vivo change in occludin localization to the apical TJ was identified in mice lacking a specific γδT-cellpopulation of iIELs whereas in γδiIEL knockout mice no such redistribution occurred implying that γδiIEL interaction with SI epithelial cells played a central role in maintaining TJ integrity and barrier function in response to T. gondii infection.T. gondii take advantage of a breach in the epithelial barrier provided in the absence of functional IELs was enhanced by a recent report using both γδT-cell deficient mice and those expressing migration-deficient, occludin-deficient, γδT-cells. Compared to WT mice, both KO models infected orally with T. gondii demonstrated increased translocation of parasites into the lamina propria.We identified a role for intestinal epithelial lymphocytes (iIELs), which intimately associate with SI epithelial cells, in T. gondii to cross the barriers of the SI epithelium, placenta, blood-brain and blood-retina crucially enable parasites to breach host immune defenses and reach deeper muscle tissues. It is therefore somewhat surprising that little is known about the interactions between T. gondii and these host cellular junctions. In 2011, parasite infection of murine skeletal muscle cells (SkMC) demonstrated loss of the AJ protein cadherin and disruption of cell-cell contacts.T. gondii on cellular junctions of various tissues, the role of the host TJ-associated network of proteins is still not well established.During natural infection the capacity for T. gondii paracellular route of infection using m-ICc12, an in vitro murine SI epithelial cell line.T. gondii tachyzoites were shown to both co-localize with TJ-associated occludin and cause re-distribution of occludin from an apical TJ location to the intracellular compartment; an effect more recently observed using the colorectal adenocarcinoma-derived Caco-2 cell line after 24 hours of infection.T. gondii paracellular transmigration of the SI epithelium.As the first point of contact between parasite and host, the SI epithelial barrier has been more widely studied. We recently confirmed a role for TJ-associated occludin in the T. gondii and the extracellular domains of occludin. This together with the recently captured high-resolution 2-photon live-cell imaging of in vitro paracellular transmigration of the SI epithelium, is consistent with T. gondii interacting directly with these domains to initiate rapid paracellular transmigration of SI epithelium.With the knowledge that occludin is crucial for transmigration, we also provided evidence for occludin acting as a receptor during parasite paracellular transmigration. Cell-free binding studies using recombinant TJ occludin proteins identified the potential interaction between T. gondii to attach and invade almost any nucleated cell together with the expression of occludin in a broad range of cell types including endothelial cells of blood vessels and the blood-brain barrier,T. gondii and occludin may be a universal means of parasite paracellular transmigration of host tissue barriers including the SI epithelium. Further analysis of T. gondii binding to, and the effect on the junctional complex is required to reveal if drugs targeting this interaction could be a therapeutic option to block the early transmigration stage of infection and prevent toxoplasmosis.The ability of T. gondii transmigration of the SI epithelium several experimental approaches have been used. For instance immunoprecipitation with a soluble ICAM-1 antibody that identified T. gondii MIC2, a microneme adhesion protein discharged to the parasite surface and involved in parasite transmigration of the host epithelium.T. gondii proteomeT. gondii infection and novel potential host-parasite effector proteins including parasite-derived kinases, phosphatases and proteases.To investigate the involvement of host proteins during T. gondii represents an important step toward understanding the molecular mechanisms of paracellular parasite infection and may aid the development of therapeutic strategies against T. gondii. However, this is complicated by an incomplete understanding of TJ composition and architecture, and by additional constituents yet to be identified.It has to date been difficult to dissect the complex interactions between parasites and host cells as both share similar components such as actin cytoskeleton. However, recent advances in proteomics promise to provide new insights into the potential mechanisms of host cell subversion. Identifying components of the TJ protein complex modulated in response to T. gondii tachyzoite infection (unpublished observations). This methodology involves growing 2 populations of cells in culture media with either ‘light’  or ‘heavy’  essential amino acids that are incorporated into all newly synthesized proteins. Here one cell population was infected with T. gondii and the other served as a non-infected control cell population. Post-infection, the 2 cell populations were mixed and the abundance of cellular proteins quantified by mass spectrometry. Using the known difference in molecular weight between ‘heavy’ and ‘light’ labeled peptides, termed the ‘mass shift’,Accordingly, we recently deployed a quantitative stable isotope labeling with amino acids in cells culture (SILAC) proteomics methodology to examine the small intestinal epithelial (IEC-6) cell response to Subsequent comparison of the SILAC data set of host-derived proteins with a compiled TJ protein network, constructed by examination of current literature and integration of functional annotations, protein-protein interactions and known signaling pathways, identified 8 host TJ-associated proteins that significantly changed in abundance in response to parasite infection. Functional classification identified changes in catalytic, binding and structural proteins as well as statistically significant enrichment of differentially regulated proteins involved in cell metabolism, glycolysis, organelle organization, cellular transport, cell cycle, transcription, cell structure and the cellular stress response; analogous with findings from previous studies. For example within 24 hours of infection, host Bcar1 , Ybx3 , Mras (Ras related protein) and Cstf2  increased in abundance. Whereas Akt , Arhgef11 (Rho guanine nucleotide exchange factor (GEF) 11, also known as PDZ-RhoGEF), Cldn15 (Claudin-15) and Prkcι  all decreased in abundance.T. gondii modulated TJ-associated proteins identified by the SILAC study. Identification of the newly discovered T. gondii MIC protein, claudin-like apicomplexan microneme protein (CLAMP), as showing structural similarity to claudins -15 and -19, is particularly intriguing as although the interaction between claudins and TJ transmembrane proteins such as occludin is not fully understood, claudins are known to interact in both cis and trans at the TJ and the occludin-claudin interaction may be important in regulation of the TJ barrier [For a review seeT. gondii that shows intimate interaction with the host cell during infection therefore presents a promising candidate for interaction with host TJs during the initial stages of infection. Furthermore, disruption of the claudin-occludin complex may induce or inhibit various downstream signaling pathways such as programmed cell death, termed apoptosis. Primarily initiated by the host-cell to maintain the epithelial barrier by extruding damaged cells, parasite triggering of host-cell apoptosis may additionally allow parasite entry into the sub-epithelial compartment by creating gaps in the epithelial monolayer during acute infection. In contrast, the well-documented anti-apoptotic effect of intracellular, replicating parasites demonstrates the wide array of strategies evolved by T. gondii to manipulate the host cell and emphasizes the difficulties in unravelling the complex mechanisms of host-cell subversion.By combining our current knowledge of how these proteins regulate the function of the epithelial TJ, either directly via integral transmembrane proteins or indirectly through their downstream network of cytoplasmic plaque, scaffolding, adaptor and signaling proteins enables the model shown in T. gondii.Further work is now needed to provide functional studies corroborating these findings and answer a key question arising from this study; the degree of conservation of the hypothesized host-cell proteome response in the broad range of host tissue barriers targeted by T. gondii infection following consumption of contaminated food and water presents a significant worldwide health problem, potentiated by the parasites highly specialized strategies for rapidly crossing the barrier of intestinal epithelial cells that contributes to its success in infecting a wide range of hosts. Our understanding of T. gondii transmigration of the SI epithelium has been progressed by recent advances in high resolution microscopy and proteomics that have uncovered new questions that need to be answered to achieve a mechanistic model of T. gondii transmigration of the SI epithelium. Further in depth pathway reconstruction maps and network analysis of the proteins involved in signaling pathways downstream of the integral TJ proteins is required to fully understand interactions between the modulated host proteins and the impact of changes in abundance or redistribution. Modeling of the TJ signaling complex is challenging when considering TJ-associated protein-protein interactions may be weak or transient and TJ assembly and disassembly is highly dynamic occurring within seconds or minutes. Elucidating how the modulation of these signaling mechanisms contributes to T. gondii disease development may hold the key to developing successful future therapeutics.


Optic neuritis is highly correlated with multiple sclerosis and is a major cause of acute visual loss and long-term neuronal degeneration. Primary cerebral hypoperfusion has been reported in brain demyelinating diseases. This study investigated whether peripapillary perfusion is changed in patients with acute optic neuritis (AON).This three-year cohort study was conducted from September 1 2012, to August 31, 2015. Two hundred and forty-one patients with non-glaucomatous acute optic neuropathy were screened, and 42 non-highly myopic patients who had suffered their first episode of unilaterally idiopathic AON were studied. All cases received spectral-domain optical coherence tomography (OCT) examination, general survey, and standard corticosteroid therapy. OCT images were analyzed using a customized MATLAB program for measuring peripapillary choroidal thickness (PCT). Multivariate regression models were constructed to identify factors that are significantly related to peripapillary perfusion.Decreased PCT was found in eyes experiencing AON combined with disc swelling . In comparison to the healthy fellow eyes, approximately every 26% increase in the thickness of the retinal nerve fiber layer due to axonal swelling was associated with a 10% decreased thickness of PCT. Thinner PCT is also correlated with poorer trough vision, which may lead to poorer final vision. These findings were obvious in patients with optic papillitis but not in patients with retrobulbar neuritis.Peripapillary vascular hypoperfusion was found in patients experiencing AON combined with disc swelling. These findings are unlike those for other ocular inflammatory diseases but are consistent with cerebral hypoperfusion, which is found in brain demyelinating diseases; thus, these findings may represent a new neurovascular model in this field. Optic neuritis (ON) is usually a primary demyelinating process and is a major cause of acute visual loss in young to middle-aged adults. Isolated acute optic neuritis is among the most common first manifestations of multiple sclerosis (MS) and neuromyelitis optica (NMO), which are leading causes of neurologic disability with important socio-economic impacts.–3 FollowDespite the ability of the current standard treatment (high-dose intravenous corticosteroid) to speed the resolution of inflammation, the treatment does not seem to influence visual outcome or atrophy of the optic nerve. ScientisCurrently, ON represents a promising target for trials of neuroprotective therapies of multiple sclerosis.–15, 29 Bst, 2012 and August 31st, 2015 were selected for inclusion in this institutional cohort study. Patients suffering from non-glaucomatous acute optic neuropathy (N = 241) who had been referred to our neuro-ophthalmology service were initially included. After excluding patients who did not match the inclusion criteria and/or had a history matching the exclusion criteria  central nervous system disorders including MS and NMO, (2) malignancy, (3) ophthalmological surgery or diseases, (4) autoimmune disorders, or (5) chronic diseases such as diabetes mellitus (DM), hypertension (HTN), cardiac artery disease (CAD), and blood diseases. These data were doubly confirmed by (1) recording the medical history from patients and their family and (2) examining the ICD-9 diagnosis record at Taiwan’s National Health Insurance system. We excluded patients having any of the diseases listed above to eliminate possible interference with measurements of choroidal thickness.™ Ganzfeld, LKC Technologies, Gaithersburg, US).[All participants were given complete ophthalmological examinations including pupillary light reflex, best-corrected visual acuity (BCVA), Ishihara color vision test, cycloplegic refraction, intraocular pressure (IOP), and indirect fundus examination. Visual field (VF) test was examined using the central 30–2 program . Pattern-reversal visual-evoked potential (VEP) test was carried out according to the standards of the International Society for Clinical Electrophysiology of Vision (ISCEV) . Cases whBlood samples were obtained at the time that participants were referred to our service and before they received pulse corticosteroid therapy. We collected data regarding complete blood cell count, differential count, electrolytes (serum sodium and potassium concentrations), liver enzymes (aspartate transaminase (AST) and alanine transaminase (ALT)) and renal function indicators (blood urea nitrogen (BUN) and serum creatinine (Cr)), autoimmune profiles including antinuclear antibody (ANA), rheumatoid factor (RF), complement components C3 and C4, a venereal disease research laboratory test (VDRL), C-reactive protein (CRP), and the erythrocyte sedimentation rate (ESR) for 1 and 2 hours. These tests were used to determine baseline health status. If there were abnormal data indicating specific diseases, we performed a more in-depth survey and referred the patient to relevant specialists. We excluded cases with renal insufficiency, hepatitis, blood diseases, active infections, and ocular inflammation due to syphilis. Positive findings regarding autoimmune profiles that were found in this examination were not used to exclude patients, but these individuals were simultaneously referred to a rheumatologist for further evaluation and follow-up. Aquaporin-4 antibody was tested if the patient did not achieve a visual acuity of better than 20/40 within 3 months. Brain MRI scans with and without contrast were acquired using a Siemens Sonata 1.5-T scanner. The results were read by an experienced radiologist, and the lesion numbers, locations and sizes were recorded within a formal medical report. Cases with brain pathology that might influence the visual pathway were also excluded.Patients underwent disc and RNFL imaging with spectral-domain OCT . The segmental and average thickness of RNFL were calculated in an automated manner, and the results were presented in an ONH (optic nerve head) analysis report. In addition to RNFL imaging, the high-resolution crossline imaging mode of the RTVue was used to obtain a high-resolution cross-sectional image of the peripapillary retina and choroid along an 8-mm line passing through the central part of the disc. Two sets of images for each disc (X-Y axis (0 and 90 degree) and oblique axis (45 and 135 degree)) were recorded to obtain peripapillary images in 8 directions. The outer border of the choroid can be well visualized on the single-line image for most patients. Images with signal strength ≥7 were included in the study.No commercial software is available for the automated measurement of peripapillary choroidal thickness (PCT). Furthermore, more than half of the acute ON eyes (22 of 42) had a swollen disc, which complicated the evaluation of PCT. To unleash the true diagnostic power of OCT, we developed a custom-written MATLAB script for the semi-automated measurement of PCT at high spatial resolution and to visualize whole-field peripapillary choroid thickness using data interpolated from a limited number of cross-line scans. To obtain an original image that would provide the best image-processing results, we first implemented a file reader to extract raw image data from the OCT files generated by RTVue . All folhttps://www.protocols.io/private/553f318d0d6746b96df18b8b448ea365.Choroid thickness was calculated from 4 cross-line images covering eight regions of the eye  . In all A database was established to facilitate data management and statistical analysis. Subsequent statistical analyses were carried out using SPSS 12.0  and SAS 9.1.3  software. Two-sided P-values ≤ 0.05 were considered statistically significant. Two-sample t tests were used to compare the mean values of continuous variables between the optic papillitis and retrobulbar neuritis groups. One-way ANOVA test was used for comparisons among more than two groups. Chi-square tests or Fisher’s exact tests were used to examine the associations between categorical variables listed in Tables The study included 42 patients with idiopathic acute optic neuritis with unilateral involvement. The average age at referral was 38.0 ± 11.9 years (range from 20–58 years old), and there were more female than male patients (F:M = 30:12). All recruited patients demonstrated positive findings of relative afferent pupil defect (RAPD) signs, decreased central vision, impaired color vision, and newly onset visual field defect. In their diseased eye , disc swelling was observed in 22 patients (the optic papillitis (OP) group) but not in the other 20 patients (the retrobulbar neuritis (RN) group). The reverse trends in PCT and RNFL between the OP and RN groups raised the question as to whether peripapillary perfusion is negatively correlated with inflammation of the nearby optic nerve head. Linear regression was conducted to further clarify the relationship between the L/F ratios of PCT and RNFL in our cases . The blaUnivariable and multivariable regression were used to further clarify the risk factors and predictive parameters associated with the thinning of PCT in eyes with acute optic neuritis. All categorized or quantified data were incorporated in the equation for analysis. In the univariable regression analysis, the presence of disc swelling and the L/F ratio of average RNFL were both strong predictors, while the refractive status of the lesion was also a significant predictor but yielded very small estimates. Multiple regression demonstrated that the best-fit model, when controlled for age and gender, was that which used all three variables, and the RNFL ratio was still the most important variable. Statistical results relating to selected important factors are listed in Most of our cases experienced good visual recovery after the episode. Thirty-three of the total 42 patients (79%) presented a BCVA of 20/40 or above within three months after steroid treatment. While analyzing the predictors for final vision after this episode, the only parameter that was found significant was “initial trough BCVA” . Therefore, we used the LogMAR initial trough BCVA as a model to analyze the significant predictors. Statistical results of selected important factors are listed in The principal finding of this study is that peripapillary choroidal thickness (PCT) became thinner in eyes with acute optic neuritis and disc swelling (acute papillitis), while no obvious change was seen for retrobulbar neuritis. The phenomenon was positively correlated with disc swelling severity presenting as increased thickness of the retinal nerve fiber layer (RNFL). Simultaneously, lesser PCT is also indicative of poorer trough vision, which may lead to poorer final vision. In both MS and NMO, cerebral hypoperfusion has been mentioned as a pathophysiology. , 47–48 SThe eye provides a window to intracranial diseases. The development of OCT has facilitated the monitoring of neuronal degeneration in several important diseases, such as Alzheimer's disease, cerebral infarction, occipital injury, and multiple sclerosis. –56 New SSeveral factors at baseline can influence an individual's choroidal thickness, including age, refraction, and chronic diseases, such as diabetes. , 60–61 HOur results revealed that PCT is compromised in acute optic neuritis, especially when inflammation is located nearby . Regional hypoperfusion should not be secondary to axonal degeneration since all the recruited cases were free from previous optic nerve and retinal disorders. The result obtained differs from the results expected based on the previous concept that the choroid tends to thicken in ocular inflammatory diseases, such as uveitis and CSCR. , 63–65 OStatistical analysis also showed that choroidal hypoperfusion is positively correlated with the severity of swelling of RNFL, which raises another possibility—that regional hypoperfusion arises due to physical compression from the swelling anterior optic nerve. However, this possibility is less likely since the choroid receives many inputs, including long and short posterior ciliary arteries. Many of these arteries leave the dura mater far from the optic nerve head and are unlikely to be restricted. Second, patients in the RN group, for whom the inflammation was located in a more posterior part of the nerve, showed no real-time change in PCT. Furthermore, a segmental analysis of RNFL, PCT, and the correlation between these was performed in our study, but no significant findings were obtained.Finding ways to improve neuroprotection in patients with demyelinating diseases has been a major problem. In this respect, due to the good accessibility of visual structures and established vision-related outcome measures, optic neuritis has been a good model, and several clinical trials are ongoing. –15, 29 AOur study has several limitations. First, this was a single hospital-based observational study; although this hospital is the most long-standing medical center in Taiwan, some significance values might have been underestimated due to the limited sample size, especially when we set strict criteria for recruitment. Second, the follow-up interval was not long enough to examine the correlations with long-term outcomes, such as the MS conversion rate or retinal degeneration. However, we tried our best to analyze the associations between vascular hypoperfusion and visual outcome. Third, the patient population was purely comprised of Asian individuals; therefore, the results should be carefully interpreted in light of the heterogeneity of different races/ethnicities.Nevertheless, our study also has several strengths. First, to the best of our knowledge, this is the first published study to have systemically analyzed peripapillary perfusion in acute optic neuritis. Second, we designed a new computerized program to analyze images and to maximize the information that we can obtain through non-invasive OCT imaging techniques. Third, by using strict criteria for recruitment, including disease history and refractive status, the interference of confounding factors in evaluation of PCT should have been minimized.In conclusion, vascular hypoperfusion in the peripapillary area was found in eyes exhibiting acute optic neuritis and disc swelling. This phenomenon was positively correlated with the severity of nerve swelling and was associated with poorer trough vision, which may lead to poorer final vision after the episode. This finding may correspond to the cerebral hypoperfusion that occurs in brain demyelinating diseases and may represent a new neurovascular model in this field. Hopefully, these results might also lead to new insights for future therapy in the field of neuroprotection.S1 File(DOCX)Click here for additional data file.


Sarcopenic muscular degeneration has been consistently identified as an independent risk factor for mortality in aging populations. Recent investigations have realized the quantitative potential of computed tomography (CT) image analysis to describe skeletal muscle volume and composition; however, the optimum approach to assessing these data remains debated. Current literature reports average Hounsfield unit (HU) values and/or segmented soft tissue cross-sectional areas to investigate muscle quality. However, standardized methods for CT analyses and their utility as a comorbidity index remain undefined, and no existing studies compare these methods to the assessment of entire radiodensitometric distributions. The primary aim of this study was to present a comparison of nonlinear trimodal regression analysis (NTRA) parameters of entire radiodensitometric muscle distributions against extant CT metrics and their correlation with lower extremity function (LEF) biometrics  and biochemical and nutritional parameters, such as total solubilized cholesterol (SCHOL) and body mass index (BMI). Data were obtained from 3,162 subjects, aged 66–96 years, from the population-based AGES-Reykjavik Study. 1-D k-means clustering was employed to discretize each biometric and comorbidity dataset into twelve subpopulations, in accordance with Sturges’ Formula for Class Selection. Dataset linear regressions were performed against eleven NTRA distribution parameters and standard CT analyses . Parameters from NTRA and CT standards were analogously assembled by age and sex. Analysis of specific NTRA parameters with standard CT results showed linear correlation coefficients greater than 0.85, but multiple regression analysis of correlative NTRA parameters yielded a correlation coefficient of 0.99 (P<0.005). These results highlight the specificities of each muscle quality metric to LEF biometrics, SCHOL, and BMI, and particularly highlight the value of the connective tissue regime in this regard. The progressive degeneration of aging muscle, known as Sarcopenia, has been consistently identified as an independent risk factor for mortality –7, and wAging skeletal muscle has a significantly reduced proportion of glycolytic type II muscle fibers compared to young muscle, which in-turn directly elicits diminished myofibril contractility , 18. AddStudying how these changes affect mobility is the prime motive for lower extremity function (LEF) research, which cites LEF as the main indicator for mobility as a clinical screening tool . LEF is Muscle biopsy is the standard clinical procedure used for the assessment of muscle, but the procedure is invasive and occasionally limited in relevance by the small size of excised tissue. However, recent investigations have realized the potential of X-ray computed Tomography (CT) and Magnetic Resonance Imaging (MRI) to describe muscle quality and composition. This is often performed either quasai-quantitatively, via the visual grading of muscle structure morphologies –34, or qWhile the propensity of CT imaging literature describing sarcopenia differs largely regarding analytical methodology, one metric that remains ubiquitous is the use of average HU values to characterize muscle quality. Goodpaster et al report the use of average HU values for quantifying skeletal muscle lipid content, highlighting the potential for the method to investigate the association between muscle composition and function –45. HickWhile average HU values might indeed describe general shifts in adiposity, generalizing CT image matrices in this regard risks eliminating other distribution characteristics that could explain additional subtle changes in muscle properties. We have previously shown the potential for computational modelling of entire radiodensitometric distributions using the novel nonlinear trimodal regression analysis (NTRA) method . The eleThe 3,162 volunteer subjects for this study were recruited from participants in the prospective population-based five-year follow-up investigation of the Age Gene/Environment Susceptibility Study in Reykjavík, Iceland (AGESII-Reykjavik) –53. The The follow-up group initially included 3,316 subjects whereof 3,169 participated in the acquisition of CT scans of the mid-thigh. Seven additional subjects were excluded due to their non-participation in biometric or comorbidity measurements. The remaining 3,162 subjects  participated in least one of the investigation’s data measurements, and, out of this total investigation cohort, the percentage of participants in each measurement ranged from 93.9% to 98.8%. Only those whose data mas acquired for each metric were included into the present work.All participants in the project were scanned with a 4-row CT detector system at 120-kV  as previously described –53. The Fat and muscle lean areas were segmented using the fascial plane outline between muscle and subcutaneous fat as previously described . A manuaLEF biometrics were assessed as part of the aforementioned AGES-Reykjavik Study. Continuous-class measurements that were available for investigation included normal and fastest-comfortable gait speed , timed up-and-go (TUG), and isometric leg strength (STR) . Gait spThe SCHOL and BMI parameters from the AGES-Reykjavik study were included on the basis of both their availability as age-related comorbidities and their being continuous-class in nature. BMI was calculated as the subject’s weight (kilograms) divided by height (in meters) squared, as previously reported . FastingFor each subject, HU distributions were derived from summing and transforming each pixel’s CT number value according to the following linear transformation expression:Following transformation, HU values were binned into 128 bins, as typical for CT assessment protocols . ResultaN is the amplitude, μ is the location, σ is the width, and α is the skewness of each distribution–all of which are iteratively evaluated at each CT bin, x. This definition is resultant from the hypothesis that each HU distribution is trimodal, in that they consist of three separate tissue types whose linear attenuation coefficients occupy distinct HU domains: namely, fat (i = 1) [-200 to -10 HU], loose connective tissue and atrophic muscle (i = 2) [-9 to 40], and normal muscle (i = 3) [41 to 200]. Additionally, we hypothesized that the inwardly-sloping asymmetries within the fat and muscle peaks could be described by skewnesses of their probability density functions, whereas the central connective tissue distribution was assumed to be a normal, non-skewed Gaussian distribution. Utilizing this definition, a theoretical curve was generated by employing a iterative generalized reduced gradient algorithm via minimization of the sum of standard errors at each CT bin value, x, thereby generating an 11-parameter matrix of PDF variables. This algorithm iterates each function variable according to the computed variance of each step, and the selection of new trial values is guided by computing the rates of change of this variance as new inputs are generated. The minimization of the sum of standard errors at each point, and thereby the maximization of R2, was computed according to their standard definitions.The method utilized to computationally define each HU distribution was a modified methodology for nonlinear regression analysis. First, the general equation for each distribution was defined as a quasi-probability density function by summing two skewed and one standard (α = 0) Gaussian distributions:An illustration of the results of this concept is shown in With the modern adoption of electronic medical records (EMRs), the availability and breadth of clinical data for use in medical research has markedly increased . CommensFor this reason, the present work identified the discretization of continuous class LEF biometrics, SCHOL, and BMI as a strategic priority. Unsupervised methods for discretization were initially selected, as they eliminate the requirement for class labels and can eventually be utilized for multiple applications, unlike supervised discretization methods , 64. Aftvariable . Using SLinear regression models were utilized to statistically correlate the relationships of each independent variable  with respect to dependent variables . Multiple regression models were then utilized on assemblies of each dependent variable whose correlation coefficients described greater than 85% of the variance in each independent metric. One-way ANOVA and F-tests for overall significance were compiled from each multiple regression model to illustrate statistically meaningful correlation.2).As is evident in As a complimentary analysis to standard CT methods, NTRA parameters were analogously assembled. Figs 2>0.85), while others yield minimal or no correlation. It may be readily hypothesized that the amplitude parameter varies identically with cross-sectional area against these metrics, assumptively suggesting a direct relationship to the overall quantity of each soft tissue type. Indeed, this is readily apparent in fat and muscle amplitudes: increasing fat amplitudes with decreasing LEF performance can be seen, along with an inverse muscle amplitude relationship. What is clearly different is the inclusion of the loose connective/water equivalent tissue regime, which according to these results seems less tied to quantity, and instead more to overall tissue quality–with the clear exception of isometric leg strength yielding a strong relationship with connective tissue amplitude. For instance, it is apparent here how connective tissue locations clearly shift towards more negative HU values with decreasing LEF performance. In the context of the aforementioned presence of myosteatosis in aging muscle, the commensurate presence of fibrosis has been observed [What is first apparent from observed , 70, butIt is likewise possible to hypothesize that the NTRA width parameter might be directly related to the overall variation in pixel values for each tissue regime, as an increasing range of HU values would simply imply an increasing Gaussian distribution width. However, these data suggest that this parameter is minimally related to gait speed and isometric leg strength, but is instead almost singularly tied to timed up-and-go duration, as decreasing width clearly confers longer TUG times. In all LEF metrics, however, the same relationship is observed: increasing fat width and decreasing muscle width confer decreasing LEF performance. This could likewise be more related to notions of tissue quality, as myosteatosis would present itself as an increase in partial volume effect from an increase in the relative proximity of inter- and intramuscular fat and lean muscle. However, why this may be singularly apparent in the fat distribution remains unclear.In regards to the NTRA location parameter, as before, our results indicate that shifts in these values are less related to muscle and fat, but highly connected to loose connective/water equivalent tissue. The standard CT analogue, in the case of location values would be the average HU value. When comparing the linear relationships of these parameters, analogous shifts towards increasingly negative values are seen, in accordance with decreasing LEF performance.Finally, the NTRA skewness parameter may again be potentially thought of as a direct descriptor for muscle quality, as intermuscular adiposity may incur the migration of lean tissue pixels towards the center of the overall distribution, resulting in an increasingly-skewed PDF shape. Intriguingly, all LEF parameters present the same trend: increasing muscle skewness and less-negative fat skewness values inversely correlate with LEF performance. However, this dependency is more tied to muscle than fat–an explanation for which, again, remains unclear.Finally, Multiple regression analyses were performed on all standard CT analyses and NTRA parameters with individual linear regression coefficients of determination over 0.85 for each LEF and SCHOL/BMI metric. Results from these analyses produced multiple regression models involving each parameter, and all models yielded multiple correlation coefficients greater than 0.99 with ANOVA and F-test results with P<0.005. These results indicate the high fidelity of multivariate correlation and robust statistical significance when all highly-linear LEF biometrics and SCHOL/BMI values are included. Additionally, such strong multivariate correlation suggests, at the very least, that these NTRA parameters should be included alongside extant standard methods for muscle quality quantification.As an additional investigation, NTRA parameters and standard CT metrics were assessed for linear correlation with age, stratified by subject sex. As previously mentioned, partial volume effects can affect HU distributions, which can lead to errors in parameter estimation, but such errors would be systematic throughout the whole dataset and therefore not affect the overall results. The strength of this study is the large patient cohort evaluated and the trends that show high correlation with the lower extremity functions as well as the biochemical and nutritional parameters. However, since age and sex may be possible confounding factors in muscle quality analysis due to their occasionally-strong linear dependencies on NTRA and standard CT analysis metrics. While this suggests the necessity for model correction in this regard, age and sex correlation coefficients were much lower overall compared to those of LEF biometrics and SCHOL/BMI parameters. It would be a valuable future step in the direction of this work to rigorously evaluate these potential dependencies. Furthermore, as is true in any such study, the use of more subjects and aging comorbidities will be essential to reinforcing any of the physiological interpretations reported here, and further discussion regarding potential applications and adjustments to the reported model will be requisite.The present study illustrates the notion that rigorous quantification of entire HU distributions can elicit many unique assessment parameters and therein provide additional information regarding muscle quality alongside extant current standard CT analysis methods. Parameters obtained by the NTRA method demonstrated higher correlation with lower extremity functions and biochemical variables than parameters obtained from standardized CT analysis. These results highlight the methodological importance of including entire radiodensitometric distributions in accordance with the NTRA assessment method and provide further insight into how muscle degeneration in sarcopenia can be optimally diagnosed and quantified.


A 60-year-old woman was diagnosed with metastatic pulmonary adenocarcinoma (c-stage IV) with an L858R point mutation in the gene encoding epidermal growth factor receptor (EGFR). Serum amylase levels were elevated  with the salivary-type enzyme dominating. First-line chemotherapy using carboplatin plus paclitaxel reduced serum amylase levels, although second-line gefitinib eventually failed to control tumor growth and hyperamylasemia after 4.5 months of treatment. The cancer cells harbored a positive EGFR mutation and secreted amylase. The number of amylase-producing cancer cells and the immunochemical staining intensity for amylase were significantly reduced after gefitinib treatment. This was a rare case of a lung cancer that expressed amylase and harbored a positive EGFR mutation. Since Weiss et al presented the first case of a bronchogenic carcinoma associated with elevated serum amylase levels in 1951 -6. The iActivation of the somatic mutations of the gene encoding epidermal growth factor receptor (EGFR) is the major determinant of the clinical response to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, in patients with non-small cell lung cancer , 9. MoreHere, we report on a patient with an amylase-producing lung adenocarcinoma with an activating EGFR mutation.4N3M1, stage IV). The serum amylase level was elevated , and amylase isozyme patterns identified the salivary type as the most abundant (94%). The pancreas and salivary glands were unlikely to have had any clinical involvement in the development of hyperamylasemia. At that time, we failed to immunohistochemically demonstrate amylase production in cancer tissue because of small number of cancer cells in the bronchoscopical sample. The level of carcinogenic embryonic antigen (CEA) was within the normal range. An EGFR L858R point mutation was detected in exon 21 from the bronchial lavage specimen using the peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp method.A 60-year-old Japanese woman who had never smoked was referred to our hospital in March 2010 because of a persistent cough and left subclavicular lymphadenopathy. A chest X-ray  and a co2) as the first-line regimen. Her amylase levels rapidly decreased after the introduction of chemotherapy and eventually reached the normal range  plus weekly paclitaxel  as the second-line regimen induced a remarkable tumor reduction seen on chest X-ray film within the initial 1.5 months of gefitinib treatment , but treAlthough several cases of amylase-producing lung cancer have been reported, there has been only one case report regarding lung cancer with hyperamylasemia and a positive EGFR mutation. Ko et al described a patient who had lung adenocarcinoma and hyperamylasemia with an in-frame deletion of EGFR exon 19, and achieved a good response to gefitinib; however, this study only detected hyperamylasemia, but did not demonstrate amylase production in the lung cancer cells . In contThe clinical usefulness of hyperamylasemia as a tumor marker is controversial. The amylase levels detected in tumor tissues of some patients did not correlate with hyperamylasemia , 13; howDespite practical difficulties in performing a second lung cancer biopsy, we fortunately obtained sufficient cancer tissue before and after gefitinib treatment. The metastatic cervical lymph node obtained before gefitinib treatment stained positively for both amylase and a mutated form of EGFR, while the endotracheal metastatic polyp showed positive staining for a mutated form of EGFR after gefitinib treatment, but negative to slightly positive for amylase. Thus, staining intensities for amylase were remarkably reduced before and after gefitinib treatment, despite the difficulty in comparison of the different sites of biopsy. In contrast to reduction of staining intensities for amylase, serum amylase level was increasing gradually during gefitinib treatment. Serum amylase levels correlated with tumor activity through the clinical course during the first- and second-line chemotherapies. The discrepancy between immunohistochemical staining and serum levels of amylase can be regarded as the result of continuous response of EGFR-TKI even beyond PD, because EGFR-TKI in general, gradually develops resistance. We doubted another mechanism other than amylase production in the lung cancer cells because of the following two reasons. First, both at the start of gefitinib treatment, May 2011, and at the documented progression, August 2011, we did not detect any metastatic lesions in the pancreas and salivary glands and also confirmed the dominant secretion of salivary-type amylase in the serum. Second, despite of tiny bronchoscopical samples, we did not detect anywhere other than cancer cells stained by amylase. Thus, we considered that salivary-type amylase was produced exclusively by the cancer cells throughout the clinical course.Relationship between amylase secretion and EGFR downstream signaling is unknown. Amylase secretion from experimental rat pancreatic acinar cells was regulated through EGFR downstream signaling via the inositol triphosphate pathway and was stimulated by a high concentration of EGF . HoweverThere were several limitations regarding our case study. First, biopsy sites differed before and after gefitinib treatment. We only detected amylase-producing cancer cells with an activating EGFR mutation before gefitinib treatment, and there were cancer cells that produced less amylase but still maintained an activating EGFR mutation after gefitinib treatment. We could not directly demonstrate that the cancer cells reduced or lost their own amylase production during the first- and second-line chemotherapies. Second, our immunohistochemical study did not quantitatively measure amylase production levels in cancer tissues. Although the endotracheal metastatic specimen suggested reduced or lost expression of amylase in cancer cells after gefitinib treatment, we could not directly compare amylase production levels between the different metastatic specimens before and after gefitinib treatment. Thus, we have to be careful in interpreting the immunohistochemical differences before and after gefitinib treatment.In conclusion, we have presented a rare case of lung adenocarcinoma that produced amylase and harbored an EGFR-activating mutation. A detailed trace of cancer cells with two unique propensities may help clarify the mechanisms of recurrence or acquired drug resistance in cancer cells.


In humans, brain-derived neurotrophic factor (BDNF) has been shown to play a pivotal role in neurocognition, and its gene contains a functional polymorphism  that may explain individual differences in brain volume and memory-related activity.n = 45), Val/Met (n = 86) and Val/Val (n = 55). Seed-based analysis was performed focusing on four SCN networks. Neurobehavioral scores served as the major outcome factor.In this study, we enrolled 186 Alzheimer's disease (AD) patients who underwent 3D T1 magnetic resonance imaging, and explored the gray matter (GM) structural covariance networks (SCN). The patients were divided into three groups according to their genotype: Met/Met (Only peak cluster volumes of default mode medial temporal lobe network showed significant genotype interactions, of which the interconnected peak clusters showed dose-dependent genotype effects. There were also significant correlations between the cognitive test scores and interconnected-cluster volumes, especially in the orbitofrontal cortex.These findings support the hypothesis that BDNF rs6265 polymorphisms modulate entorhinal cortex-interconnected clusters and the valine allele was associated with stronger structural covariance patterns that determined the cognitive outcomes. Neurotrophins are a group of molecules which play a key role in regulating neural survival, development and maintenance. The most abundant neurotrophic factor is brain-derived neurotrophic factor (BDNF) , 2 and hvs. Val/Val), recessive  and additive  carriers. Another meta-analysis reported that the Met66 allele conferred susceptibility to AD in women but not in men [In humans, the BDNF gene has been mapped to chromosome 11p14.1. A common single nucleotide polymorphisms (SNP) consisting of a missense change of the coding exon at position 66  has been shown to produce non-conservative amino acid changes . The Met allele carriers have been shown to have poorer episodic memory, and this may be reflected in lower activities of BDNF with impaired neuronal processing and trafficking . A recenThe influence of BDNF polymorphisms on human cognition has been linked with its impact on changes in brain structure. However, whether the Met allele leads to worse outcomes is controversial. Smaller brain volumes have been reported in Met carriers in the hippocampal and dorsolateral prefrontal areas , hippocaRecent research has suggested that highly related regions show covariance in morphometric characteristics, so called structural covariance. Structural covariance networks (SCNs) can be used to test the influence of the genotype with careful control of other factors. Three SCNs have been reported to be relevant to patients with AD: default mode network (DMN) –22, saliThe potential mechanisms of genetic-based neurobiology are still under investigation, however a number of studies have highlighted how genetic variations –30, epigThe demographic characteristics and neuropsychiatric test results of the three genotype groups are listed in Table Among the three BDNF genotypes and four seed regions Figure , there wIn the entorhinal seed-based DMN networks, the Met/Met homozygotes showed no clusters with a voxel size > 100 , while tFor the frontoinsular seed, the Met/Met homozygotes   Figure .The clinical significance of each peak cluster linking to the entorhinal seed showing a genotype effect was evaluated by correlation analysis Figure . Among tvia modulation of the strength of structural covariance between spatially scattered but functionally coherent regions rather than having a direct impact on the seed or peak cluster volume. Third, while the volumes of the seed and peak clusters of the DMN medial temporal subsystem correlated significantly with cognitive test scores, only the orbitofrontal peak cluster volume predicted cognitive test scores in the three BDNF genotype groups.The results of this study provide data on the network-specific genetic influence of BDNF Val66Met in the early stages of AD. There were three main findings. First, the seed-based SCN pattern validated the hypothesis that the BDNF functional polymorphism targets large-scale brain networks rather than selectively in a focal brain region. Second, a genetic dosage effect of the BDNF Val allele was found only in the DMN medial temporal subsystem with cortical hubs consisting of entorhinal, PCC and prefrontal regions. The presence of the genetic dosage effect suggests that the mechanism related to this functional polymorphism may be via synaptic connections, and BDNF has been reported to attenuate the neurotoxicity [The salient episodic memory impairment in AD has been reported to be strongly associated with hippocampal/entorhinal volume , 36, whitoxicity , 8. The While our results suggest that there was no direct effect of BDNF genotype on hippocampal or entorhinal volume, we found that the BDNF genotype may specifically modulate the DMN medial temporal lobe subsystem structural covariance patterns, of which the increased correlations between entorhinal seed and peak clusters demonstrated the genetic dosage effect of the Val allele. While early amyloid burden or GM atrophy has been reported to involve the DMN medial temporal lobe subsystem in the early stage of AD , lower BThese alterations can be caused by several factors, and the disease- and genetic-related changes in structural covariance can be difficult to interpret. As the cortical hubs of the DMN are highly functionally anchored, the spatially scattered DMN-related clusters that show structural covariance may validate the notion that BDNF Val66Met polymorphisms only affect activity-dependent pathways . AttenuaIn contrast, stronger correlations modulated by increased BDNF activity may suggest greater regional connectivity and synchronized GM loss in regions targeted by the pathological process. Although the pair-wise correlations did not necessarily influence the global organizational properties of structural covariance, as shown by the lack of differences in regional volumes among genotypes, the significant correlations between cognitive outcomes and seed  volumes within the DMN hubs still address the pathological-genetic alterations of local clustering. The findings of the DMN still reflect segregated and less integrated components in AD networks that could predict cognitive outcomes.Among the four seed-based SCNs, the Val  carriers had more extended voxels of structural covariance in the DMN medial temporal lobe subsystem. In contrast, the Met  carriers showed stronger structural associations in the DMN midline core system, salience and executive control networks. As BDNF activity was different among the genotypes, the SCN patterns may reflect different strengths of structural covariance between cortical GM and seed region. The SCN patterns may therefore provide evidence that BDNF genotypes target specific large-scale brain networks in patients with AD that may modulate distinct degenerative patterns. In normal human cortical morphology, Met carriers have been reported to have smaller hippocampal and prefrontal cortical volumes , 43. HowStudies on the genetic risk of developing AD , 11 or tvia BDNF gene expression [Possible explanations why the BDNF genotypes showed inconsistent results on brain structures may be related to epistasis , variabipression , 53. In pression .As such,Despite our observations that BDNF Val66Met polymorphisms may affect structural covariance patterns, there were no significant differences in cognitive measurements in our genotype groups. On the basis of the none-significance in the PCC and dorsolateral prefrontal seed volumes among the three genotype groups, the volumes of both seeds were still significantly correlated with the cognitive scores. The finding supports the hypothesis that brain structure may be considered to be an endophenotype that is more sensitive to a genetic effect than behavioral level. The PCC is an early target of amyloid deposition in both patients with AD  and in cAn important limitation of this study is that we did not include a control group. The effects of BDNF Val66Met genetic variations on regional GM volume and functional state in healthy subjects have been reported , 47, 50.In summary, our structural covariance analysis supports the role of BDNF functional polymorphisms in modulating the GM degenerative scaffold in the early stages of AD. Our results suggest that the Val66Met functional polymorphism carries different weightings on the DMN, salience and executive control networks. Within the DMN medial temporal lobe subsystem, the genetic dosage effect of the Val allele on entorhinal seed-based SCN was validated by correlation strength gradients between seed and peak clusters, thus supporting the importance of BDNF in AD degenerative network.This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Chang Gung Memorial Hospital. The study participants were treated at the Cognition and Aging Center, Department of General Neurology, Kaohsiung Chang Gung Memorial Hospital. A total of 186 subjects  were included after the consensus of a panel composed of neurologists, neuropsychologists, neuroradiologists and experts in nuclear medicine . AD was n = 45), Met/Val (n = 86) and Val/Val homozygotes (n = 55). To avoid possible confounding, the three genotype groups were matched for gender, age, educational level, clinical dementia rating [The patients were classified into three groups based on the genotype: Met/Met homozygotes , followed by genotyping for BDNF Val66Met polymorphisms using the polymerase chain reaction (PCR)-restriction fragment length polymorphism method. Genotyping was conducted with the operator blinded to the clinical data. The apolipoprotein E4 genotype was also determined using a PCR-restriction fragment length polymorphism assay and restriction enzyme HhaI .It has been reported that factors such as oxidative stress, deregulated neuroinflammation and an elevated blood sugar level are related to changes in BDNF expression –69. We i3.MR images were acquired using a 3.0T MRI scanner . Structural images were acquired for structural covariance analysis using the following protocols: a T1-weighted, inversion-recovery-prepared, three-dimensional, gradient-recalled acquisition in a steady-state sequence with a repetition time/echo time/inversion time of 8,600 ms/minimal/450 ms, a 256 × 256 mm field of view, and a 1-mm slice sagittal thickness with a resolution of 0.5 × 0.5 × 1 mmhttp://www.fil.ion.ucl.ac.uk/spm/). The T1 images were reoriented, realigned, and normalized using the standard Montreal Neurological Institute (MNI) space. The images were then segmented into GM and white matter. Related tissue segments were used to create a custom template using the diffeomorphic anatomical registration using exponentiated lie algebra (DARTEL) approach. The DARTEL approach is one of the highest ranking registration methods in patients with AD [p < 0.05, corrected for a false discovery rate (FDR) with a cluster size > 100 voxels.Image preprocessing and statistical analysis were performed using SPM8 . Statistical significance was set at p < 0.05.Clinical and laboratory data were expressed as mean ± standard deviation. Analysis of variance with Bonferroni correction for multiple comparisons was used to compare levels of cerebrovascular risk biomarkers or continuous variables among the Val/Val homozygotes, and Met/Val and Met/Met homozygotes. All statistical analyses were conducted using SPSS software  were extracted from the 186 preprocessed images. The seed ROI included the right entorhinal cortex , left posterior cingulate cortex , right frontoinsular cortex , and right dorsolateral prefrontal cortex  Figure . Accordip < 0.01, corrected for FDR with a cluster size > 100 voxels.From the modified GM images, the GM volumes of a 4-mm radius sphere around the seed ROI coordinates were calculated, followed by four separate correlation analyses using the extracted GM volumes as the covariates of interest. The three BDNF genotype groups were modeled separately. For each BDNF genotype group, specific contrasts were set to identify voxels that showed positive correlations for each seed ROI. The results reflected the SCNs of each ROI and the threshold was set at in vivo study in which the expression of BDNF was found to be highest in Val/Val, followed by Val/Met and Met/Met [p < 0.001 (uncorrected) with a cluster size > 100 voxels. In addition, for the peak clusters showing significant between-group differences, a 4-mm radius sphere was placed on the peak voxel, and the GM volumes were then calculated for regression analysis. The seed ROI was considered to be the predictive variable for the extracted SCN peak voxel, and the threshold was set at p < 0.05 with multiple corrections. To evaluate the clinical significance of the seed or identified peak voxel, we used a linear regression model with the cognitive test scores serving as the dependent variable. The threshold was set at p < 0.05 with multiple corrections.In addition, to investigate how genetic variance may interfere with structural covariance patterns, voxels showing significant differences in the regression slopes in each seed-peak cluster correlations were compared, pointing to possible interactions between Val/Val > Met/Met or Val/Met > Met/Met. The genetic dosage model was based on an  Met/Met . Specifi


Novel pharmacotherapies introduce additional options to providers and patients in how to best treat chronic obstructive pulmonary disease (COPD). Emerging data question the role of inhaled corticosteroids in COPD treatment, particularly as combination dual bronchodilator pharmacotherapies demonstrate robust results. For those maximized on pharmacotherapy with continued dyspnea or exacerbations or both, emerging bronchoscopic procedures may offer additional therapy in select patients. This review focuses on data supporting the use of novel ultra bronchodilators, particularly in combination, and on the role for inhaled corticosteroid withdrawal and new bronchoscopic procedures. Acute exacerbations of COPD are associated with worsening symptoms, including breathlessness, decreased quality of life (QOL)4, and an accelerated loss of lung function5. Those hospitalized for acute exacerbations of COPD are at an increased risk of one-year mortality of at least 18%6. The majority of an estimated $50 billion cost associated with COPD care in the United States is spent treating acute exacerbations7. An array of emerging pharmacotherapies challenges the traditional way COPD has been managed. This review will focus on the current evidence for use of combined long-acting muscarinic antagonists (LAMAs) with long-acting beta-2 agonists (LABAs), withdrawal of inhaled corticosteroids (ICSs), and emerging data on bronchoscopic interventions in COPD.Chronic obstructive pulmonary disease (COPD) is projected to become the third most common cause of death worldwide by 203010. Long-acting bronchodilators show similar efficacy in patients with moderate compared with more severe COPD11, indicating that forced expiratory volume in one second (FEV1) does not predict bronchodilator treatment response. Several once-daily LABAs have become available over the past several years, and indacaterol, olodaterol, and vilanterol are the newest. The existing drug classes (beta-2 agonists and muscarinic receptor antagonists) work by relaxing airway smooth muscle tone, leading to reduced respiratory muscle activity and subsequent reduction in airway resistance and making it easier for patients to breathe. Bronchodilation aims at alleviating bronchial obstruction and airflow limitation, reducing hyperinflation, improving emptying of the lung and exercise performance13, thus improving dyspnea. This explains why all current COPD practice recommendations highlight that inhaled bronchodilators are the mainstay of current management regardless of disease severity16.Long-acting bronchodilators improve lung function, thereby improving symptoms and exercise performance, and prevent exacerbations18, and therefore exhalation may not be completed prior to the onset of the next breath20  LABAs formoterol, salmeterol, and arformoterol and are similar to those of the LAMA tiotropium26.The efficacy of ultra LABAs is well established. Among others, two randomized, double-blind, placebo-controlled, parallel-group phase 3 studies have shown the long-term efficacy and safety of once-daily olodaterol 5 and 10 μg in patients with moderate to severe COPD continuing usual-care maintenance therapy15. Although a randomized, placebo-controlled 6-month trial of tiotropium versus salmeterol was conducted27, it is debatable whether such a direct LABA-versus-LAMA comparison will be performed, particularly given the increasing number of options available28.The debate as to which class of inhaled bronchodilator should be the first-line agent in COPD continues. Guidelines do not distinguish which long-acting bronchodilator agent, LABA or LAMA, should be considered first line, but rather only that the use of a long-acting bronchodilator agent is advised29, although LAMAs also impact favorably on both outcomes28. By contrast, LAMAs appear to be more effective than LABAs if exacerbations are the expected primary outcome, regardless of whether LABAs are administered on a twice-daily30 or once-daily31 basis. The outcome of interest may largely determine which bronchodilator to start with in a patient with COPD32. In the symptomatic patient, there is no substantial difference between LABAs or LAMAs, whereas in frequent exacerbators it seems preferable to use a LAMA.Cautious indirect comparisons may be made from the existing clinical trial database about how each class of drug impacts COPD outcomes, although the limitations of this approach are obvious. Nonetheless, limitations aside, LABAs are more effective than LAMAs if we consider symptoms or health-related quality of life (HRQOL) as the primary outcome33. Over the past few years, data have emerged documenting the efficacy of other drugs in the LAMA class  scores35. Umeclidinium significantly improved trough FEV1, dyspnea, and QOL scores36. The GEM (Glycopyrrolate Effect on Symptoms and Lung Function) 1 and 2 studies of glycopyrronium versus placebo show improvements in FEV1, dyspnea, QOL scores, and rescue medication use in patients with moderate to severe airflow limitation37. In recent studies of a novel soluble glycopyrrolate solution delivered via the investigational eFlow® nebulizer, the nebulized LAMA formulation was reported to be safe and well tolerated, and there were no significant changes in cardiovascular signs and electrocardiography parameters38. There was a dose-related and clinically significant improvement in FEV1 following nebulized glycopyrrolate, providing support for its development as a convenient nebulized LAMA bronchodilator for patients with COPD38. US Food and Drug Administration (FDA) approval would bring forth a novel nebulized ultra LAMA option available to patients with COPD. Availability of a nebulized LAMA would greatly complement the currently available nebulized LABA medications (formoterol and arformoterol). Of 400 caregivers and patients with COPD randomly surveyed via phone, the overwhelming majority were satisfied with traditional nebulization therapy, reporting benefits in symptom relief, ease of use, and improved QOL39.Until recently, tiotropium was the only globally available ultra LAMA, and it has a rich database of efficacy outcomes in COPDA class . The ACC14. Studies showing benefit of combination LABA and LAMA in separate devices with both short- and long-acting components42 prompted the development of a single component with multiple long-acting bronchodilators. The first of the ultra LAMA/LABA combination inhalers approved was for umeclidinium/vilanterol. Umeclidinium/vilanterol appears to be safe, produces greater improvements in lung function compared with monocomponents, and in some studies reduces the risk of exacerbations46. However, umeclidinium/vilanterol combination, compared with tiotropium or the monocomponents umeclidinium or vilanterol, has not shown dramatic improvements in dyspnea or HRQOL. The bulk of the data for the aclidinium/formoterol combination is from two 24-week randomized, placebo-controlled studies—AUGMENT and ACLIFORM studies—showing improved lung function, dyspnea, and HRQOL48. Combining data from these two studies showed reduced exacerbations49 and similar cardiovascular events over 6 months for the twice-daily-administered LAMA/LABA compared with placebo50.For patients with COPD whose disease is not well controlled—whether in terms of symptoms or exacerbation frequency as recommended by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) COPD statement—with a single long-acting bronchodilator, the most recent guidelines depart from prior use of ICSs and recommend the use of dual long-acting bronchodilators, unless a recurrent or severe exacerbator51. These studies were not powered for exacerbation reduction, but other LAMA/LABA combinations have been shown to reproducibly reduce exacerbations. In a multicenter trial, glycopyrronium/indacaterol once daily was compared with fluticasone/salmeterol twice daily in 3,362 patients with moderate to severe COPD with a history of at least one moderate to severe exacerbation in the previous year52. Glycopyrronium/indacaterol reduced the rate of mild to severe COPD exacerbations by 11% compared with fluticasone/salmeterol over the 52-week trial52. Patients with a history of two or more moderate exacerbations or one hospitalization in the previous year had similar exacerbation rates between the two treatment arms. Of note, glycopyrronium/indacaterol was associated with slightly fewer episodes of pneumonia (3.2%) compared with fluticasone/salmeterol (4.8%). The combination of a long-acting anticholinergic plus an inhaled glucocorticoid has not been compared with a long-acting anticholinergic alone.The FDA approved tiotropium/olodaterol in a soft mist inhaler in 2015. In a 6-week crossover study, it improved lung function, and a combined analysis of the TOnado 1 and 2 studies documented improved dyspnea and HRQOL54. Of note, the primary outcome of these studies was lung function, not patient-reported outcomes (PROs). Statistically significant differences have been shown for subsequent studies with PROs as the primary endpoint56 as well as a pooled analysis49. They provide a signal but are below the MCID thresholds. An associated reduction in reliever medication use suggests clinical relevance55, although overall the clinical impact of LAMA/LABA combination versus its monocomponents is unclear. Evidence of excessive ICS/LABA prescribing, coupled with emerging data (discussed below) of safe steroid withdrawal, makes LAMA/LABA combination more reasonable, particularly with the aim of maximizing bronchodilation in those with persistent dyspnea.Patients with dyspnea despite the use of either a LAMA or LABA may have improvement in HRQOL, dyspnea, and reduced rescue medication use with LAMA/LABA combination, and some agents have reported improvement in exacerbations. However, the degree of symptom improvement along with lung function improvement remains a clinical question of extreme importance. Early studies of LAMA/LABA combination describe transition dyspnea index and Saint George’s Respiratory Questionnaire meeting the minimal clinically important difference (MCID) for breathlessness versus placebo whereas individual monocomponents did not59. There has remained equipoise around this issue, however, and a meta-analysis of three of these older trials, the only trials deemed to be acceptable in terms of quality and level of bias, determined that withdrawal of ICS was not actually associated with any statistically significant increase in the exacerbation rate and that the effects on other outcomes, such as lung function and health status, were inconclusive60. Methodological limitations marred these studies, and the contradictory findings may be due to differences in heterogeneity in patient characteristics, disease severity, and outcome definitions among other factors60.As new clinical trial data emerge, a debate regarding the appropriate role of ICS withdrawal in COPD has formed. Several earlier studies reported that an abrupt withdrawal of ICS precipitates exacerbations and results in a deterioration in lung function and symptoms14. However, a large proportion of patients are already initiated on ICS-containing regimens63. Recent studies evaluating GOLD groups A and B  have shown no consequences associated with ICS withdrawal. It was prospectively demonstrated that withdrawal of ICS in patients with symptomatic, moderate COPD with fewer than two exacerbations a year was not associated with any deterioration in lung function, symptoms, and exacerbation rate over a 6-month observation period64.Recently, two randomized controlled trials and a prospective study revealed that ICS can be safely withdrawn in certain patients. Those with COPD and a low risk of exacerbations should not be prescribed ICS-containing regimens, according to the latest (2017) GOLD guidelines65. These studies support the current GOLD recommendations that groups A and B do not benefit from ICS-containing regimens. Furthermore, they suggest that ICS therapy can be safely withdrawn from patients with moderate COPD and a low risk of exacerbations who continue taking long-acting bronchodilators. A growing armamentarium of novel, ultra LABA, ultra LAMA, and LABA/LAMA combinations can be considered to optimize bronchodilation and permit ICS withdrawal.A subsequent randomized controlled trial of those with moderate COPD and no prior exacerbation history found that switching from a fixed-dose combination of ICS/LABA to a LABA was not associated with any differences in lung function, symptoms, health status, and exacerbations66. The WISDOM trial findings indicate that not all patients benefit from including ICS in their treatment regimen despite current guidelines. Whether a subset of patients will benefit from continuing an ICS-containing regimen and how to identify such a population has not been studied, although subgroup analyses of the WISDOM trial did not show differences in exacerbation occurrence with respect to age, sex, smoking status, body mass index, ICS or beta-blocker therapy at screening, chronic bronchitis, GOLD stage and group, and prior therapy with antibiotics or systemic glucocorticoids66. This has led to our practice of withdrawing ICS in patients with stable COPD. This practice is further supported by a recent landmark study where a combination LAMA/LABA was superior to a combination ICS/LABA in preventing exacerbations52.The WISDOM trial was the largest and first to examine stepwise withdrawal of ICS in patients with COPD receiving maintenance therapy of long-acting bronchodilators, including those at risk for exacerbations. The stepwise withdrawal of glucocorticoids was non-inferior to the continuation of such therapy with respect to the risk of moderate or severe exacerbations67. However, only a particular subset of patients, those with known upper lobe predominant emphysema and low post-rehabilitation exercise capacity, derive a mortality benefit. The opportunity to expand the population with severe emphysema who may benefit from intervention by less invasive means has driven the development of potential bronchoscopic interventions for severe emphysema. A recent trial evaluated the efficacy, safety, cost, and cost-effectiveness of nitinol coils versus usual care in patients with severe emphysema68. The trial randomly assigned patients to a usual-care arm consisting of rehabilitation and bronchodilators with or without ICS and oxygen or to the treatment arm where patients received usual care plus additional therapy of approximately 10 coils per lobe placed in two bilateral lobes in two procedures. The study resulted in improved exercise capacity with high short-term costs. A second study of lung volume reduction coils showed a wide range of clinical outcomes among study participants, and some experienced important improvements in exercise tolerance and lung function whereas others had a less robust result. Although the primary endpoint of 6-minute walk distance between the treatment and control groups was statistically significant, it did not appear to be clinically meaningful69.Lung volume reduction surgery is the only surgical procedure to prolong life in COPD70. A subsequent study of 64 patients randomly assigned to the endobronchial valve group or the control group and intention-to-treat analyses showed greater improvements in the pulmonary function and exercise capacity in those treated with endobronchial valves71. Further investigation is warranted to determine whether this will be a potential approved therapy in patients with severe COPD and intact fissures.Bronchoscopic lung volume reduction with the use of one-way endobronchial valves is another potential treatment for patients with severe emphysema. To date, the benefits have been modest but have been hypothesized to be much larger in patients without interlobar collateral ventilation than in those with collateral ventilation. A single-center, double-blind, sham-controlled trial in patients with severe COPD, significant hyperinflation, and restricted exercise tolerance with a target lobe with intact interlobar fissures on chest computed tomography showed significant improvement in lung function at 3 months14. These comorbidities are so common that they are now part of the GOLD definition14 and can occur in patients with mild, moderate, or severe airflow limitation72. Comorbidities in COPD influence mortality and hospitalizations independently73. COPD itself has significant systemic effects, including skeletal muscle dysfunction which may be characterized by loss of muscle cells or abnormal function of remaining cells or both74. Although skeletal muscle dysfunction may be caused by inactivity, poor diet, inflammation, and hypoxia, it is a remediable source of exercise intolerance75. Bronchoscopic therapies aim to widen the population to whom non-pharmacologic therapies are available. However, comorbidities may present challenges of candidacy and tolerance of procedures in addition to impacting the optimization of pulmonary rehabilitation after lung volume reduction procedures have been performed.A characteristic of COPD is a disproportionately high prevalence of common comorbidities such as cardiovascular disease, diabetes, lung cancer, depression, metabolic syndrome, skeletal muscle dysfunction, and osteoporosisEmerging evidence that withdrawal of ICS is safe in some patients makes combination LAMA/LABA pharmacotherapy a reasonable option for many, particularly those with persistent dyspnea on a single long-acting bronchodilator. The growth of novel dual-agent long-acting bronchodilator inhalers may decrease the excessive over-prescription of combination ICS/LABAs. Further evidence is needed to better understand the role of bronchoscopic lung volume reduction.