Retinal ganglion cell (RGC) loss in glaucoma is sectorial in nature and preceded by deficits in axonal transport. Neuroinflammation plays an important role in the pathophysiology of glaucoma in the retina, optic nerve and visual centers of the brain, where it similarly appears to be regulated spatially. In a murine model, we examined the spatial characteristics of astrocyte reactivity  in healthy retina, retina with two glaucoma-related risk factors (aging and genetic predisposition) and glaucomatous retina and established relationships between these reactivity indices and the spatial organization of astrocytes as well as RGC health. Astrocyte reactivity was quantified by morphological techniques and RGC health was determined by uptake and transport of the neural tracer cholera toxin beta subunit (CTB). We found that: (1) astrocyte reactivity occurs in microdomains throughout glaucomatous retina as well as retina with risk factors for glaucoma, (2) these astrocyte microdomains are primarily differentiated by the degree of retinal area covered by the astrocytes within them and (3) percent retinal area covered by astrocytes is highly predictive of RGC health. Our findings suggest that microdomains of astrocyte reactivity are biomarkers for functional decline of RGCs. Based on current and emerging imaging technologies, diagnostic assessment of astrocytes in the nerve fiber layer could succeed in translating axonal transport deficits to a feasible clinical application. Glaucoma is characterized by progressive neurodegeneration of retinal ganglion cells (RGCs) and their axons, which form the optic nerve. As in many neurodegenerative disorders, glaucomatous loss of RGCs is preceded by deficits in axonal transport –8. AxonaThe experimental procedures described were approved by the Vanderbilt University Medical Center Institutional Animal Care and Use Committee. Retina from young  and aged  DBA/2 mice were compared to age-matched C57BL/6 mice . The average IOP for DBA/2 mice was 15 mmHg ± 1.4 mmHg and 22 mmHg ± 5.9 mmHg for young and aged mice, respectively. IOP was monitored monthly in awake, behaving DBA/2 mice using a tonometer (Tonolab) before mice were sacrificed .Anterograde transport tracing of the retino-collicular projection was performed as previously described . BrieflyImmunohistochemistry in whole mount retina was performed as previously described ,24,25. BWhole mount retina imaging was performed through the Vanderbilt University Medical Center Cell Imaging Shared Resource Core. Staining was imaged on an upright confocal microscope  or an inverted confocal microscope  equipped with laser scanning fluorescence . Three dimensional z-series images of the retina were acquired using a digital camera and image analysis software . For each retina, 5–10 pseudorandom images through the ganglion cell and nerve fiber layers were obtained at 63× magnification.For each of the 5–10 pseudorandom retinal images the ganglion cell- and nerve fiber-layers were collapsed into two dimensional images. We used these images to examine astrocyte reactivity and CTB labeling intensities. Images were collected between the mid-peripheral to mid-central regions of the retina; thereby avoiding the optic disc and the area immediately surrounding the optic disc, which contains large bundles of RGC axons and higher density of astrocytes that would confound our studies of CTB uptake in RGC soma as well as accuracy of astrocyte measurements. All measurements were performed using imaging analysis software . Measurements were made by two different examiners that were blind to experimental parameters.2 of retina. Hand-counts were performed by an experimenter blind to the experimental groups and the data were analyzed by a second experimenter. Density data are presented as astrocytes/mm2 and CTB+ RGCs/mm2.To evaluate changes in the density of microglia, we hand-counted the number of GFAP+ astrocyte cell somas and CTB+ RGC cell somas in each micrograph from DBA/2 and C57 retina. We divided soma counts by the area of the micrograph to calculate the number of GFAP+ astrocytes and CTB+ RGCs per mm2.To evaluate hypertrophy, we measured the soma size of astrocytes by hand-tracing of each GFAP+ cell soma in each micrograph. Soma area was determined as the area contained within the soma trace. Hand-tracing was performed by an experimenter blind to the experimental groups and the data were analyzed by a second experimenter. Hypertrophy data are presented as the soma area in mmGFAP and CTB intensity was calculated as the mean pixel intensity across each image. This value represents the total intensity values used for astrocyte and RGC health studies. For astrocyte reactivity studies, this value was divided by the number astrocytes in the image to yield mean GFAP intensity/cell.To create one index that represented changes in both astrocyte density and hypertrophy, we utilized image analysis software (ImageJ) to count the number of pixels with GFAP label above background for each micrograph. The number of GFAP+ pixels was divided by the total number of pixels in the image and expressed as a percentage (% GFAP coverage).Brains were incubated in a sucrose series (10%–20%–30%), cortex removed and 50 μm-thick serial, coronal sections through the superior colliculus were obtained on a sliding microtome. Sections were mounted serially on slides CTB signal was photographed digitally on an inverted fluorescent microscope . Using layer IV as the ventral border, we outlined the boundaries of the superficial superior colliculus, which was partitioned in to 6 μm bins from medial to lateral. CTB density was calculated as the area of pixels with CTB labeling above background (pixel strength of the non-retinorecipient layers of the superior colliculus) divided by the total pixel area for sections for each 6 μm bin. Using a colorimetric scale to illustrate % CTB density in each section, we constructed a colorimetric representation of CTB density across the retinotopic colliculus map, which was based on section thickness and intersection distance. For each SC, we determined the fraction of intact retinotopic map, defined as the percent area with CTB signal ≥ 70% density. The mean% transport was used for statistical analysis.P ≤ 0.05 was considered statistically significant.All statistical tests were performed with SigmaPlot . For comparison of means between conditions, we used Kruskal-Wallace one-way analysis of variance followed by post hoc pair wise comparisons between groups using Dunn’s Method. Correlations were assessed by both Pearson Product Moment Correlation and Spearman’s Rank Order Correlation. Statistically significant correlations were verified by polynomial regression analysis. Error bars represent ± SD of the mean. For all analyses, RGC neurodegeneration, microglia reactivity and IL-6 signaling in glaucoma occur in a spatially-dependent manner ,17,19,24We first performed a qualitative analysis of astrocyte reactivity in wholemounted retina immunolabeled with the astrocyte marker glial fibrillary acidic protein GFAP; . GFAP isQualitative assessment of astrocyte morphology across 200 μm×200 μm fields of retina from young C57, aged C57, young DBA/2 and aged DBA/2 retina revealed that, in similar areas of retinal eccentricity, there is rather large variability in the morphology of astrocytes within individual retina, especially those with glaucoma or glaucoma-related stressors . Not surTo quantify the apparent variability in astrocyte morphology related to reactivity, we quantitatively measured three indices of reactivity, including astrocyte proliferation/migration (density), hypertrophy and GFAP expression. Measurements were obtained from a minimum of ten fields per retina and the mean across images was used to determine overall changes in reactivity indices, while histogram functions were used to assess the distribution  of reactivity measures for each experimental group.2  as well as in aged C57 and young DBA/2 retina p>0.05; . HoweverFinally, we quantified GFAP expression by measuring the total intensity of GFAP immunofluorescence in each confocal micrograph. To account for astrocyte density, we divided the total intensity of GFAP immunofluorescence by the number of astrocytes in each micrograph. This yielded the average GFAP expression per cell, which was then used for all subsequent analyses. We found that GFAP expression by astrocytes was approximately 60% higher in young C57 retina than in aged C57, young DBA/2 and aged DBA/2 retina  and young DBA/2 retina , where astrocyte density could accurately predict GFAP expression in 63% and 59% of microdomains, respectively  and aged DBA/2 retina , respectively  to the superior colliculus. For axon transport studies, we sectioned the superior colliculus and measured CTB intensity in the retinorecipient layers of the superficial superior colliculus. CTB density was calculated as the area of pixels with CTB labeling divided by the total pixel area for sections at 6 μm intervals from medial to lateral borders of the superior colliculus. Using a colorimetric scale to illustrate % CTB density (0–100%) in each section, we constructed a colorimetric representation of CTB density across the retinotopic colliculus map, which was based on section thickness and intersection distance. CTB transport was analyzed as % transport. For CTB uptake studies, we examined both the total intensity of CTB and density of CTB+ RGCs micrographs from the mid-central to mid-peripheral region of retina from the same animals used in the superior colliculus studies.In agreement with previous studies, we found significant deficits in anterograde transport of CTB from the retina to the superior colliculus of aged DBA/2 mice, as indicated by a primarily green-blue intensity map across the entire retinorecipient colliculus  3]. You. You3]. Qualitative assessment of spatially-coincident CTB labeling and GFAP immunoreactivity in retina revealed potential correlations between astrocyte morphology and the intensity of CTB label . As expeTo evaluate whether astrocyte microdomains could serve as a biomarker for RGC health, we first needed to develop an approach that could quickly and accurately identify astrocyte microdomains for potential translation to a clinical setting. Our data identify soma area and density as primary indicators of astrocyte microdomains for glaucomatous retina and retina with glaucoma risk factors. Both of these measures relate to the amount of retinal area covered by astrocytes. As such, we modified our astrocyte analysis to incorporate both indices into one index that addresses coverage area. To do so, we counted the number of pixels with any GFAP labeling, regardless of intensity, and divided this by the total number of pixels in each image. This provided a percentage value for coverage of retinal area by astrocytes. Since GFAP intensity was also relevant for defining astrocyte microdomains, we also measured for the total image. Using these calculations, we observed detectable differences in the percent of retinal area covered by astrocytes (% GFAP coverage) between healthy and disease-related states. As predicted by our hypertrophy and proliferation/migration analyses, aged C57 retina exhibited a 56% reduction in the mean retinal area covered by astrocytes, as compared to young C57 and young DBA/2 retina , total GFAP intensity, total CTB intensity and CTB+ RGC density in spatially-coincident samples. In young C57 retina, % GFAP coverage positively correlated with total CTB intensity  than density of CTB+ RGCs , as indicated by predictive capacities of 23% and 13%, respectively  and the density of CTB+ RGCs , which was similar to the same relationship in aged C57 retina  exhibited a tendency towards shrinkage of astrocytes (hypotrophy), suggesting that astrocyte hypotrophy may be of interest in examining the pathophysiology of glaucoma risk and onset. Finally, the mean GFAP expression per cell was greatest in healthy retina, which exhibited the least spatial variability across all astrocyte reactivity indices. Histogram analysis revealed that while some groups of astrocytes exhibited high levels of GFAP expression in retina challenged by glaucoma-related stressors, a majority of the astrocyte population had decreased levels of GFAP expression. In combination with our findings regarding astrocyte hypotrophy in response glaucoma risk factors, these data may indicate that astrocyte decline could be a relevant factor glaucoma onset and progression. This is supported by recent evidence in optic nerve and elsewhere in the central nervous system, suggesting that astrocyte decline may contribute to neurodegeneration ,34.To determine whether spatial variations in indices of astrocyte reactivity were inter-related, we performed correlation and regression analysis. We found that individual indices of astrocyte reactivity could reliably predict one another in spatially-coincident astrocytes. This indicates that astrocytes in close proximity share morphological characteristics relevant to their reactivity states, which suggests the formation of astrocyte microdomains that not only underlie increased variability noted in our morphological analyses, but also likely have distinct functional significance. These findings are in accordance with recent work describing spatial variations in both microglia reactivity and IL-6 signaling in DBA/2 retina ,17.Although the degree of predictability between reactivity indices was stressor-dependent, the most striking finding was that measures of density and hypertrophy were most consistent in their ability to predict astrocyte organization. That both density and hypertrophy are related to the amount of retinal area covered by astrocytes suggests that the degree of contact between astrocytes and the surrounding neural and vascular structures is a key defining microdomains of reactivity. When using percent of retina area covered by astrocytes as an index of astrocyte microdomains, we found a dramatic decrease in astrocyte coverage in aged C57 retina, which again suggests that astrocyte decline may be contribute to risk for neurodegeneration. We found that in aged DBA/2 mice the area of astrocyte coverage increased more than 3-fold from coverage quantified in age-matched C57 mice, which suggests a link between elevated IOP and area of astrocyte coverage. Our results in glaucomatous retina are contrary to previously published studies of astrocyte coverage in the optic nerve of aged DBA/2 mice  and in rA hallmark of the slow, progressive degeneration of RGCs in glaucoma is failure in both anterograde and retrograde axonal transport –8. TheseFrom a technical standpoint, visualization of astrocytes and measurement of astrocyte coverage area in the human retina is within the scope of current imaging technologies in the clinic ,37. In fTogether, these data suggest that: (1) astrocyte reactivity occurs in microdomains throughout glaucomatous retina as well as retina with risk factors for glaucoma, (2) these astrocyte microdomains are primarily differentiated by the degree of retinal area covered by the astrocytes within them and (3) percent retinal area covered by astrocytes has the potential to be a biomarker for decreased RGC health, which could allow practitioners to identify regions with faltering RGC health prior to overt nerve fiber layer or visual field defects.


Mouse models have greatly assisted our understanding of retinal degenerations. However, the mouse retina does not have a macula, leading to the question of whether the mouse is a relevant model for macular degeneration. In the present study, a quantitative comparison between the organization of the central mouse retina and the human macula was made, focusing on some structural characteristics that have been suggested to be important in predisposing the macula to stresses leading to degeneration: photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch’s membrane. Light and electron microscopy measurements from retinas of two strains of mice, together with published data on human retinas, were used for calculations and subsequent comparisons. As in the human retina, the central region of the mouse retina possesses a higher photoreceptor cell density and a thinner Bruch’s membrane than in the periphery; however, the magnitudes of these periphery to center gradients are larger in the human. Of potentially greater relevance is the actual photoreceptor cell density, which is much greater in the mouse central retina than in the human macula, underlying a higher phagocytic load for the mouse RPE. Moreover, at eccentricities that correspond to the peripheral half of the human macula, the rod to cone ratio is similar between mouse and human. Hence, with respect to photoreceptor density and phagocytic load of the RPE, the central mouse retina models at least the more peripheral part of the macula, where macular degeneration is often first evident. More so than for other neurodegenerations, the field of retinal degeneration has been assisted by numerous mouse models that have provided close parallels with the human disease. The first inherited form of retinal degeneration to be reported in a mouse strain occurred 90 years ago . Since tper se, leading to the question of whether the mouse is a relevant model for macular degeneration.Macular retinal degenerations affect the central retina, and thus impair high acuity vision. Age-related macular degeneration (AMD) is a major cause of blindness amongst the elderly , 5. AMD The macula subtends an angle of ~20 degrees in the center of human and many non-human primate retinas; the optic disc lies at the nasal edge of the macula. The macula has a number of characteristics that distinguish it from other parts of the retina. It has been referred to as the macula lutea, meaning yellow spot, due to the presence of the carotenoids, zeaxanthin and lutein . The cenWhy the macula may be more susceptible to degeneration is not known. The concentration of zeaxanthin and lutein has been thought to be protective rather than a liability . HoweverOne suggestion has been that differences in the thickness of Bruch’s membrane may contribute to this susceptibility . Bruch’sAnother potential contributor to the vulnerability of the macula is its high density of photoreceptor cells, especially in relation to the high metabolic load this density places on the RPE cells, which must degrade the shed outer segment disk membranes of the photoreceptors . For forTo address the question of the relevance of mouse models for macular degeneration, it is important to assess the mouse retina with respect to parameters that may be related to susceptibility to macular degeneration. Given the suggestions that rod and cone photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch’s membrane may play a role in susceptibility to macular degeneration, in the present study, we have compared these structural characteristics between the mouse retina and the human macula.In all experiments two WT strains of mice, C57BL/6J and BALB/C were used. Mice were kept at a 12 h light/12 h dark cycle and were sacrificed at an age of 8 weeks. These experiments were performed in accordance to the guidelines established by the Animal Research Committee of the University of California Los Angeles and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.2 was thus calculated. Distance along the retinal arc was converted into eccentricity, based on a posterior nodal distance for the mouse eye of 1.75 mm , and 20 mMEDTA), and then 4 incisions from the periphery towards the optic nerve were made in order to flatten the eyecup. The sensory retina was gently removed with a brush, and the RPE and choroid were transferred to a wet nitrocellulose filter on a glass slide. The resulting wholemounts were oriented, fixed in 4% formaldehyde for 1h at RT, and washed in PBS and blocked with 1% BSA and 0.2% tween-20 in PBS for 1h at RT. They were then washed in PBS, incubated in FITC-phalloidin at 1 μg/ml  and DAPI at 2 μg/ml  in 1% BSA, 0.2% tween-20 in PBS for 2h at RT, and finally washed in PBS and mounted on slides. They were imaged with an Olympus Fluoview FV1000 confocal laser microscope. The surface area of each RPE cell was determined using ImageJ.Eyecups were obtained from enucleated eyes in Ringer’s solution , and, in the center, 1410 cells/2500 μm2 were counted , indicating a density that is 33% higher. Cone photoreceptor cell density, though much lower overall, changes in a similar manner across the retina. At 75° off-axis, 24 cone cells/2500 μm2 were counted , and, in the center, 32 cells/2500 μm2 were counted . Beyond 75° off-axis to the edge of the retina, at the ora serrata, photoreceptor cell density declines steeply  were examined by EM . OverallAvailable data show that Bruch’s membrane in the human retina tends to thicken with age. However, by comparing measurements from 10 to 45 year old donor eyes, as reported by Newsome et al. , with ouTabulation of mean RPE cell size, density, photoreceptor RPE cell ratio, and Bruch’s membrane and elastin layer thickness, at different eccentricities of the human and the mouse retina, is shown in The present study has addressed the question of how the central retina of the mouse compares with the human macula, in the context of some structural specializations that may make the macula more susceptible to degeneration. The specializations are photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch’s membrane. This question is important when considering the relevance of mouse models of macular degeneration, including AMD.2, among different eyes. The density reported by Osterberg [2) lies within this range, although it is towards the low end.The most obvious specialization of the macula is the sharp increase in photoreceptor cell density. Here, we used data from Osterberg , who madsterberg  . Moreover, at eccentricities that correspond to the more peripheral part of the macula in the human retina, the mouse retina contains a substantially higher density of cone photoreceptors, as well as rod photoreceptors. In this region, the rod to cone ratio is comparable between the human and mouse retinas. It is noteworthy that the peripheral parts of the macula are included in pathogenesis of macular degenerations, and rods typically degenerate before the cones , 67.In conclusion, we have illustrated that a large central area of the mouse retina possesses some of the structural characteristics, which, in the human retina, have been suggested to make the macula more susceptible to degeneration.S1 Table(1) along dorso-ventral axis.(DOCX)Click here for additional data file.S2 Table(1) Human data from [(2) Human data from [(3) Data for ratio derived from photoreceptor density and RPE cell area. (4) Human data from [ata from . (2) Humata from . (3) Datata from .(DOCX)Click here for additional data file.S1 Raw Data(XLSX)Click here for additional data file.


Although extensive research on neural plasticity resulting from hearing deprivation has been conducted, the direct influence of compromised audition on the auditory cortex and the potential impact of long durations of incomplete sensory stimulation on the adult cortex are still not fully understood. In this study, using voxel-based morphometry, we evaluated gray matter (GM) volume changes that may be associated with reduced hearing ability and the duration of hearing impairment in 42 unilateral hearing loss (UHL) patients with acoustic neuromas compared to 24 normal controls. We found significant GM volume increases in the somatosensory and motor systems and GM volume decreases in the auditory  and visual systems  in UHL patients. The GM volume decreases in the primary auditory cortex  correlated with reduced hearing ability. Meanwhile, the GM volume decreases in structures involving high-level cognitive control functions  correlated positively with hearing loss duration. Our findings demonstrated that the severity and duration of UHL may contribute to the dissociated morphology of auditory and high-level neural structures, providing insight into the brain’s plasticity related to chronic, persistent partial sensory loss. Sensory perception modulates the adaptation of cortical structures to various sensory experiences13469Unlike the congenitally deaf, most individuals with unilateral hearing loss (UHL) suffer from post-lingual hearing damage and have passed the critical period for cortical development. Therefore, any influence on the brain might induce traumatic changes in cortical morphology because of defective compensation. In particular, the ability to capture auditory information is largely preserved in UHL, but changes in auditory perception for further cognitive processing are more complicated11This study used volumetric MRI with voxel-based morphometry (VBM)1415p values > 0.06). Most UHL participants had significant hearing damage in the impaired ear (14 left UHL with pure tone audiometry (PTA) ≥ 50 dB and 7 with 50 > PTA ≥ 20 dB; 16 right UHL with PTA ≥ 50 dB and the other 5 with 50 > PTA ≥ 20 dB). UHL patients showed higher PTA values in the impaired ear than the same-side ear in NCs . However, no significant difference was found between PTA values of unaffected ears in UHL patients and the same-side ears in NCs . There were no significant differences in the duration of hearing damage between left UHL (29.7 ± 10.7 ms) and right UHL (23.4 ± 11.4 ms) . PTA values showed no significant differences between the two patient groups between compromised ears  and contralateral ears with intact hearing . t = −0.422, p = 0.676). There were no significant differences in the signal-to-noise ratio (SNR) between compromised ears of left UHL (30.9 ± 23.2 dB S/N)) and right UHL patients (38.9 ± 24.6 dB S/N)) .Significantly increased AN tumor volume was observed in left and right UHL patients compared to normal controls . We obseThe GM volume of the right perirhinal cortex in left UHL patients was greater than in right UHL patients , Fig. 3.Regression analyses with hearing ability  as the independent variable and GM volume as the dependent variable showed that PTA values  were positively correlated with the GM volume of the right superior frontal gyrus and left fusiform gyrus , Fig. 4.Regression analyses with hearing damage duration as the independent variable and GM volume as the dependent variable revealed that hearing damage duration was positively correlated to the GM volume of the right middle occipital gyrus and right middle temporal gyrus but was negatively correlated to the GM volume of the bilateral anterior cingulate cortex, extending to the supplementary motor area , Fig. 5.For better representation of the types of hearing challenges UHL individuals experience in their daily lives, a speech-recognition in noise test (MHINT) was used to reflect the hearing adaptation besides pure tone. We chose Heschl’s gyrus and the anterior cingulate cortex from differences between UHL and NC groups shown in  as the rIn this study, we examined the experience-dependent brain plasticity in UHL participants using an MRI-based morphometric analysis. While the GM volume reductions in primary sensory regions were significantly correlated with subjective hearing ability, the hearing damage duration was related to decreased GM volumes in cortical areas involved in high-order cognitive processing. These results suggest that the severity and duration of UHL respectively contribute to alterations in the GM volume of auditory and high-level functional structures.Most previous studies have focused on completely deaf individuals, and those volumetric studies revealed either preserved192023243132Additionally, UHL might disrupt more elaborate auditory information, such as musical perception, vocal communication sounds and spatial and temporal auditory processing. These high-level auditory processes involve many brain regions, particularly the insula, as shown in lesion studies3435363839403839Previous studies have shown that the anatomical and functional changes observed in deaf individuals are present in basic sensory and higher-level cognitive processing132444813None of the post-lingual UHL participants in our study were in the critical period of auditory development; thus, the sensory system may be too stable to adequately modulate plastic modality. Post-lingual UHL induces a decline in the ability to process advanced auditory processing, such as phonological analysis and phonological processing for visual communication We also observed an approximate inversion symmetry of cortical reorganization between participants with UHL on different sides. The structural changes in the perirhinal cortex/parahippocampus were specific to the laterality of UHL. The perirhinal cortex is a critical association area for the cross-modal integration of highly processed sensory informationThere are some limitations in this study. We investigated the brain morphological changes and their correlations to hearing damage in AN patients. Growth of the tumor in the cerebellopontine angle is likely to change anatomical relationships in the brain. However, the tumors grow subtentorially, thus requiring long-term growth to affect hemispheric structures. Considering patients with UHL without a growing tumor in the brain would increase the probability that the changes in morphometry are not by chance and not due to tumor growth, and further studies including data on UHL in the absence of a neurinoma would be helpful. Moreover, the reorganization of higher-level cognitive processing areas is generally convincing in the face of severe and persistent UHL, and we performed basic speech-in-noise tests to obtain more direct evidence supporting this claim. Future studies may provide detailed information by measuring the higher-order auditory processing more specifically.In conclusion, our results indicate that UHL induces plasticity in low-level sensory and high-level representation systems to adapt to the impaired auditory input. In addition, brain remodeling occurs in anatomically distinguishable substrates in response to the severity and duration of hearing damage.Patients with unilateral acoustic neuroma (AN) provided consent and were hospitalized in our neurosurgery department  between July 2014 and December 2014. We collected MRI data from 48 patients in total. All the patients were preoperatively diagnosed with UHL resulting from intracranial tumors based on MRI scanning and auditory assessment . Six of the patients were confirmed pathologically as having meningioma and were excluded from the experiment. Therefore, we reported results of the other 42 UHL patients with the same etiology. Twenty-one patients had hearing damage in the left ear, and 21 patients had hearing damage in the right ear. Twenty-four normal control (NC) participants were recruited from the local community in Beijing, with age, gender and education matched to the patients. All the controls also underwent the same MRI and auditory tests. These assessments were necessary for the patients during their clinical management, with no additional cost for them, and the control group received all the tests for free. The study was conducted on the UHL patients with AN preoperatively, and all the UHL participants had undergone the craniotomy to remove the neuromas after the MRI and auditory exam according to the clinical arrangement. All participants were right-handed and reported no previous or current psychiatric disorders  was calculated for each participant as the primary audiometric measure. In general, higher PTA values imply worse hearing ability.MHINT was conducted by using the bilateral implant test system (BLIMP) which was developed by House Ear InstituteAn MMSE2 with an in-plane resolution of 0.9375 × 0.9375 mm2.All structural images were acquired using the same 3.0 Tesla Siemens TRIO scanner  with a standard head coil in a single scanning session. Head fixation with foam pads and hearing protection with earplugs were used for each participant. T1-weighted sagittal anatomical images were obtained using the following gradient echo sequence: 176 slices, slice thickness/gap = 1/0 mm, inversion time = 900 ms, repetition time = 2300 ms, echo time = 3 ms, flip angle = 7°, number of excitations = 1, field of view = 240 × 240 mmhttp://dbm.neuro.uni-jena.de/vbm/) with a statistical parametric mapping package . The structural MRI scan of each participant was preprocessed using a modified optimized-VBM-protocolImages were analyzed using the voxel-based morphometry toolbox  and GM volume in left and right UHL groups. AFNI AlphaSim (http://afni.nimh.nih.gov/afni/doc/edu) was used for Monte Carlo simulation with 1,000 iterations to correct for multiple comparisons across the thresholded GM mask, which only included voxels with a probability higher than 0.4 in the SPM8 GM template. A cluster extent of 328 contiguous resampled voxels (1.5 × 1.5 × 1.5 mm3) was considered significant assuming an individual voxel type I error of p < 0.01, which corresponds to a corrected p < 0.05.Two-sample How to cite this article: Wang, X. et al. Alterations in gray matter volume due to unilateral hearing loss. Sci. Rep.6, 25811; doi: 10.1038/srep25811 (2016).


The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies. 4E10 is an antibody that neutralizes a broad variety of HIV strains. However, 4E10 is uncommon in infected patients and has not been successfully elicited by any vaccine approach attempted. Hurdles to re-eliciting 4E10 include the accumulation of many mutations during development, demonstrated reactivity against host proteins and significant structural flexibility. Lacking a confirmed sequence for precursors of 4E10, we studied the recognition and biophysical properties of an ensemble of eight of the likeliest candidates. Surprisingly, 4E10 gained host reactivity and structural flexibility, but lost stability during development when compared to candidate precursors. However, recognition of HIV was remarkably conserved, despite a considerable improvement in binding. Since these results run counter to those expected from conventional vaccination protocols, 4E10 is unlikely to serve as the basis of a useful HIV vaccine. H and VL genes compatible with the development of bnAbs may not be common in human or animal model vaccinee GEP repertoires; (3) some bnAbs are autoreactive, which hinders their elicitation through self-tolerance mechanisms; (4) the unusual characteristics inherent to bnAbs, such as long complementarity determining regions (CDRs), functionally-required polyspecificity, and a high degree of somatic mutation , may not be easily achieved through conventional vaccination strategies; (5) imperfect immunogens may elicit off-target  or humoral responses with limited breadth; and, finally, (6) neutralization mechanisms may involve complexities beyond simply binding a particular epitope on Env , which may be difficult to recapitulate, since selective expansion of particular B cell clones is based solely on BCR binding properties, not higher-order functionalities An effective HIV vaccine will likely need to elicit broadly-neutralizing antibodies (bnAbs) that target the viral envelope protein (Env) as part of a protective immune response 671NWFD/NIT676) immediately adjacent to the viral membrane in the Env gp41 subunit membrane proximal external region (MPER) The bnAb 4E10, the focus of our studies, has a conserved, linear epitope  and light (LCDRs) chains) comprising the combining site KD) for their cognate antigens, typically ranging from 10−6 to 10−10 M, that are orders-of-magnitude stronger than their GEPs kd), a majority of measurably favorable mutations do not directly contact antigen. These mutations indirectly optimize binding by: (1) increasing shape complementarity between paratopes and epitopes through more global effects on structure; (2) increasing antibody stability, typically measured as solution thermostabilities (Tm), thus compensating for deleterious effects of other mutations that improve affinity; and (3) structurally rigidifying the combining site conformer optimal for binding antigen from the accessible ensemble. Rigidifying the combining site can affect measured interaction parameters in different ways depending on the mechanism of binding. The two mechanistic extremes are known as “conformer-selection” and “instructive-encounter”, or “induced-fit”, binding. In conformer-selection mode, binding does not occur until the compatible conformer is adopted. Rigidification of the binding site through mutation then typically improves entropies of interaction and on-rates (ka). In instructive-encounter mode, initial binding occurs to sub-optimal conformers which affects the rate of interchange with the optimal conformer. Rigidification of the binding site through mutation then typically improves affinity through changes distributed over kd and ka. However, the consensus of studies of binding proteins and enzymes suggests that conformer selection is the preeminent recognition mechanism Affinity-matured Abs display univalent equilibrium binding constants  Surprisingly, comparisons of the bound and unbound structures of 4E10 revealed that this affinity-matured bnAb incorporates considerable HCDR conformational flexibility Lacking access to the original donor, identification of a single GEP sequence with high confidence for many bnAbs, including 4E10, is complicated by extensive editing and TdT N-nucleotide insertion during rearrangement H+VL) constructs to ease expression, analysis, and crystallization and to prevent monobody-diabody interchange, following protocols developed for 4E10 in vitro with what in our experience were exceptionally high efficiencies of 20 to 40%. GEP 5 was not included in subsequent experiments because its extremely poor in vitro refolding efficiency suggested that this was not a viable pairing. GEP constructs were stable and monodisperse in solution, running exclusively as monomers by size exclusion chromatography (SEC). Reduced/non-reduced PAGE analysis of GEPs confirmed purity and proper disulfide bond formation. GEP Tm values .GEPs were engineered as cleavable, single-chain, variable domain cassette  soluble Env gp140 trimers (gp1403) KD of 12 nM, SF162 gp1403 with a KD of 98 nM, and ESs with KDs of either ≤10 picomolar (T117) or ∼100 picomolar  K160N) gp1403 and T72, T93, and T344 in qualitative SPR analyses, with KDs all estimated to be well above the ∼micromolar B cell activation threshold. Quantitative SPR analyses of GEPs binding to T117 showed KDs ranging from the low nanomolar to low micromolar range. 4E10 interactions with ESs ranged from 100- to 10,000-fold stronger than GEPs, which was qualitatively consistent with the observed difference in 4E10 versus GEP interactions with gp1403. Since the GEP/gp1403 interactions were too weak to quantitate, peptide binding studies were not performed on the expectation that they would also be too weak to measure accurately. Five GEPs  bind T117 with nearly identical behavior, including GEP 1 and GEP 7, which differ only by alternate J segment utilization, showing that the two incorporated mutations did not affect binding, so no further IGHJ1*01 GEPs were produced for study. GEP 4 and GEP 6, which both incorporate differences from G96H in 4E10 (A or V), showed approximately 10-fold (GEP 4) or 100-fold (GEP 6) reductions in affinity relative to the cluster of other GEPs. Kinetically, the five clustering GEPs  showed affinity reductions for T117 relative to 4E10 overwhelmingly through faster off-rates (kds). GEPs 4 and 6, in addition to comparable increases in kd, also showed progressive reductions in on-rates (kas).In order to characterize the change in binding properties during 4E10 ontogeny, the binding of 4E10 and GEP Fvs to three  did not affect Tm, only marginally increased affinity by about three-fold, and did not improve neutralization potency  showed that GEPs retained higher degrees of structural conservation than 4E10 between the bound and unbound states. These analyses need to be interpreted cognizant of the constraints imposed by crystallization, which involved varying non-physiological solution conditions and variable crystal contacts between structures. However, these caveats were minimized by comparing multiple structures with multiple copies per asymmetric unit.In order to determine whether the 4E10 combining site rigidified during affinity maturation, undergoing binding site preconfiguration, the crystal structures of GEP 7 and GEP 1 were determined in the unbound state at resolution values of 1.9 Å and 1.7 Å respectively, rebuilt and refined with good statistics Table 2,2)  specificities. Nevertheless, the T117 scaffold is highly complementary to 4E10 (Sc for GEP2/T117 = 0.69), which may help explain the increased affinity of T117 for 4E10 and GEPs, and may be ideal for preferentially targeting GEPs through the F54H interaction . The additional T117 contacts appear to have had the effect of increasing the affinity for T117 over other ESs by two orders of magnitude for 4E10 and by one to three orders of magnitude for the GEPs. However, structural superpositions show that these extra Ab/scaffold interactions do not affect Ab/epitope interactions, or the structure of the epitope in the complexes, which are highly conserved  Fig. 11.3R), matches the top hit from the previous PhIP-Seq analysis of IgG 4E10 10P-values for the top ten scoring peptides of 35.3 to 255 for IgG versus 4.35 to 12.3 for Fv), likely due to decreased accessibility of coupled Fv relative to IgG, an increased chance of inactivating Fv versus IgG during chemical coupling, and the inherent increase in local avidity of bivalent IgG versus univalent Fv on potentially sparsely-coupled beads. Given these caveats, overall scoring behavior was very similar across the Fvs tested, with 4E10 and 1C6, the affinity-matured Abs, showing the highest average scores. GEP 4 and 1C6 showed the largest number of high-scoring hits, with 61 and 194 peptides with replicate-averaged −Log10P-values of ≥4.0 respectively; 4E10 and GEP2 had 12 and 20 peptides, respectively, scoring ≥4.0. Qualitatively, the results were not dramatically different, but with GEP 4 and 1C6 showing nominally greater spreads of top-scoring peptides rising above the bulk responses. None of the top-scoring three-dozen 4E10 peptides appeared in the top three-dozen hits from either GEP; however two of the top-scoring dozen GEP peptides  were in common between GEP 2 and GEP 4, scoring 1 and 12 (GEP 2) and 4 and 2 (GEP 4), respectively. However, no common peptide motifs could be identified within or between Fv results. None of the IP3R peptides scored in the top 65,000 GEP hits. While 1C6 showed the highest scoring spread of top hits , the top ten scoring peptides displayed a considerably higher average hydrophobic character (Φ), with average Φ values of: 4E10 = 0.37; GEP 2 = 0.42; GEP 4 = 0.45; 1C6 = 0.71 . Using relative hydrophobicity of the top-scoring PhIP-Seq peptides as a surrogate measure of the overall hydrophobicity of the combining site was consistent with the structures of the Abs  was used to assess the polyspecificity and autoantigen recognition profiles of GEPs in comparison to 4E10 3 as part of a higher-order neutralization mechanism 3 registering at distant sites. Since b12 dissociation kinetics were unaffected, 4E10 did not preclude achieving a similar b12 bound-state conformation.In order to test the hypothesis that 4E10 may induce global conformational changes in gp1403 proteins and engineered ESs. This gain in affinity is potentially sufficient to account for the concurrent gain in neutralization potency over GEPs. Previous results showing minimal contributions from FWR mutations e.g. 32L and 95H, that add or improve direct contacts to epitope. This limited number of mutations is presumably readily achievable during conventional vaccination. Based on PhIP-Seq peptidome binding results , one VH gene segment also confidently assigned (IGHV1-69*06), plus one of six likely D segments  and either of two likely J segments . The CDR3 junctions and presumed N-nucleotide insertions in mature 4E10 were retained in the candidate GEPs. Sequences are numbered following Kabat E. coli BL21(DE3) RIL cells (Invitrogen), refolded, purified, and validated by SEC and PAGE as previously described 2HPO4/KH2PO4 (pH = 7.4). Temperature melts were performed at 210 nM with a temperature ramp from 25°C to 95°C at a slope of 2°C/minute, data pitch of 2°C, and delay time of 10 s . Fabs of 447-52D and 4E10 were prepared by digestion of IgG with papain (Pierce), affinity chromatography with protein A (Pierce) and preparative SEC ; IgG 4E10 was purchased from Polymun Scientific, and IgG 447-52D and Fab b12 were kindly provided by Pamela Bjorkman .The 4E10 heavy and light chain nucleotide sequences were analyzed using JoinSolver  of 10 s Fig. 2. Neutralization assays Fig. 2B ,3 (3 at 30 µg/mL in 10 mM sodium acetate (pH = 5.0) were direct amine coupled at a density of ∼2200 RUs on a CM5 sensor chip ; a reference surface was prepared by activating and deactivating a flow cell without the addition of protein; gp1403 were prepared as previously described 3 in the presence or absence of 4E10 Fv  with injection 1 at 5 minutes, injection 2 at 2 minutes and a final dissociation time of 5 minutes. Fab-alone curves were generated by injecting HBS-EP+ followed by an injection of 10 nM Fab b12 or 447-52D and double referenced by subtracting a dual injection of HBS-EP+ followed by HBS-EP+. Fab with 4E10 Fv curves were generated by injecting 3 µM 4E10 Fv (∼90 RUs bound) followed by an injection of 10 nM Fab b12 or 447-52D in the presence of 3 µM 4E10 Fv and double referenced by subtracting a dual injection of 3 µM 4E10 Fv followed by 3 µM 4E10 Fv. Optimal regeneration was achieved by injection of 10 mM glycine (pH = 1.5) at a flow rate of 50 µL/minute for 5 seconds followed by a 6 minute buffer stabilization phase. Sensorgrams were corrected by the double-subtraction method All SPR experiments were performed at 25°C on a Biacore T100 instrument with the T200 sensitivity enhancement . For analyses of the binding of 4E10 and GEP Fv analytes to chip-captured ES ligands ,Table 1Crystals of GEPs and 4E10 Fv were grown by the hanging-drop vapor diffusion method at 25°C with the following well solutions:GEP 1: 25% w/v PEG 3350, 0.2 M NaCl, 0.1 M Tris (pH = 8.5)GEP 1/T117: unbuffered 12% w/v PEG 8000, 0.1 M KCl, 5% v/v glycerol2SO4, 0.1 M sodium acetate (pH = 5.0)GEP 2/T117: 1.6 M LiGEP 7: 20% w/v PEG 2000, 0.2 M Trimethylamine N-oxide, 0.1 M Tris (pH = 9.0)4OAc, 0.1 M Bis-Tris (pH = 5.5)GEP 7/T117: 10% w/v PEG 10000, 0.1 M NH1C6: 3.5 M sodium formate, 0.1 M Bis-Tris Propane (pH = 7.0)Crystals were cryopreserved in mother liquor containing 15% v/v glycerol , mother liquor containing 10% (1C6) or 20% (GEP 7) v/v glycerol, or 20% w/w sucrose (GEP 2/T117). Diffraction data for GEP 1, 4E10, and 1C6 were collected at the Advanced Light Source beamline 5.0.2  and reduced using HKL-2000 4 (pH = 7.4). Beads were rocked at ambient temperature for 24 hrs with 60 µg of each Fv in 1 M (NH4)2SO4 and then washed with 10 mM glycine in PBS to cap unreacted epoxy groups. Activity of Fv coupled beads was confirmed by epitope-scaffold binding prior to PhIP-Seq analyses. PhIP-Seq analyses were performed as previously described 10P-values; highly discordant, and therefore spurious, hits falling near the axes were excluded from analysis. 241 peptides were also discarded because they displayed nonspecific binding to all Abs tested 4E10, GEP and 1C6 Fvs were coupled to beads and analyzed in duplicate. For each Fv analyzed, 3 mg of magnetic beads (Invitrogen M-270 Epoxy Dynabeads) were resuspended in 60 µL 0.1 M NaPOCoordinates and structure factor amplitudes have been deposited in the Protein Data Bank Movie S1Superposition of epitope binding site contacts from bound and unbound forms of 4E10. Residues from epitope binding site contacts are represented as mesh, pink for bound and yellow for unbound. The heavy chain CDR1 and CDR3 loops are shown in cartoon representations with residues W100H, W100bH, and F29H shown in licorice stick representations.(M4V)Click here for additional data file.Movie S2Superposition of epitope binding site contacts from bound and unbound forms of GEP 7. Residues from epitope binding site contacts are represented as mesh, pink for bound and yellow for unbound. The heavy chain CDR1 and CDR3 loops are shown in cartoon representations with residues W100H, W100bH, and F29H shown in licorice stick representations.(M4V)Click here for additional data file.


To examine the consequences of early-onset hearing loss on several social and health measures and any related gender differences in Japanese populations.Data from a 2007 nationally representative cross-sectional household survey of 136,849 men and women aged 20 to 39 years were obtained . We focused particularly on four social and health measures: employment status (employed/unemployed), marital status (married/unmarried), smoking behavior (yes/no), and psychological distress (K6 instrument: ≥ 5 or not). We examined the association of hearing loss for each measure using generalized estimating equations to account for correlated individuals within households.p = 0.447). Men with hearing loss were more likely to be married, whereas women with hearing loss were less likely to be married (p < 0.001 for interaction). Although hearing loss was not associated with a current smoking status in men, women with hearing loss were more likely to be current smokers (p < 0.001 for interaction). Moreover, hearing loss was associated with psychological distress in men and women (both p < 0.001).There was no significant association with employment status (These findings suggest that hearing loss is related to social and health issues in daily life, including a lower likelihood of marriage, more frequent smoking, and poorer mental health, especially in women. These issues may reflect a gap between the actual needs of women with hearing loss and the formal support received as a result of existing public health policies in Japan. Hearing loss (or hearing impairment) is one of the most frequent types of disability . CurrentThe United Nations adopted the Convention on the Rights of Persons with Disabilities (CRPD) in 2006, which came into effect in 2008 . The conJapan was the 140th country worldwide to ratify the CRPD in January 2014 (27th among the 34 OECD countries) –15. Althn = 350,000) had some degree of hearing loss  v.s. 19.0% [without hearing loss]), but not among men (50.0% [with hearing loss] v.s. 51.6% [without hearing loss]) in 2007 , smokers, and psychologically distressed than women without early-onset hearing loss. This may be because they have a larger number of unmet needs in their daily lives, and because those with mild to moderate hearing loss have been overlooked in public and health policies and even in public health research. Although the implications of this study cannot be easily stated, the general aims of the CRPD may not be achieved only by legislation.Future initiatives can include recruitment of professional female counseling staffs in the local government offices, training of women with hearing loss themselves as counselors (peer support), and construction of strong networks of young working-age women with/without hearing loss to get together and share their experiences . This study illustrates that many aspects of people with disabilities—job, marriage, smoking, and mental health—require special attention and multidimensional approach by policy makers and all involved. Such attention and approach will accelerate the social change of Japan, and also contribute to realizing the aims of CRPD.


Muckle-Wells syndrome (MWS), a phenotype within the spectrum of cryopyrine-associated periodic syndrome (CAPS) is characterized by excessive IL-1 release resulting in chronic systemic and organ-specific inflammation including sensorineural hearing loss. During continuous anti-IL-1-therapy clinical symptoms are controlled and hearing loss remains stable. Limited data exists about discontinuation of IL-1-inhibition during the course of disease.To report the case of a MWS patient, with sudden deterioration of hearing loss due to discontinuation of anti-IL-1-therapy and improvement of hearing after re-therapy.A 29 year old female patient was diagnosed with MWS at age 17. Her left ear had been deaf since early childhood. The patient experienced arthralgia, exanthema, fatigue and progressive hearing loss of the right ear during childhood. Anti-IL-1-therapy with Anakinra was started 4 years after diagnosis. In 2007, she was switched to Canakinumab resulting in complete resolution of exanthema and arthralgia and improved fatigue. Hearing loss was stable during therapy as documented by frequent high frequency pure tone assessments (HFPTA).When she became pregnant, MWS treatment was changed from Canakinumab to Anakinra as suggested by the safety profile. The patient gave birth to a healthy boy, who has a CAPS mutation.The patient decided to discontinue IL-1-inhibition while breast-feeding. After four months off anti-IL-1-therapy, her hearing had markedly deteriorated: HFPTA demonstrated a decrease of 20-30 dB in frequencies most relevant for speech discrimination, a substantially impairment for a unilateral deaf patient. The patient decided to stop breast-feeding and Canakinumab therapy was immediately re-initiated. Two months later improved hearing was documented with 5-10 dB but still 10-25 dB less than before discontinuation.Long-term IL-1-inhibition prevents decline of hearing ability in CAPS. Withdrawal of treatment may result in rapid and marked hearing loss. An early restart of anti-IL-1-therapy may partially reverse hearing loss. This indicates a window of opportunity for reversal of hearing loss by IL-1-inhibition. To our knowledge, this is the first case in which such a close connection between IL-1-inhibition and hearing ability in MWS has been documented. This case report shows the importance of continuation of anti-IL-1-therapy during pregnancy and breast-feeding to reduce the risk of sequel.Written informed consent for publication of their clinical details was obtained from the patient/parent/guardian/relative of the patient.


The genetic basis of hearing loss in humans is relatively poorly understood. In recent years, experimental approaches including laboratory studies of early onset hearing loss in inbred mouse strains, or proteomic analyses of hair cells or hair bundles, have suggested new candidate molecules involved in hearing function. However, the relevance of these genes/gene products to hearing function in humans remains unknown. We investigated whether single nucleotide polymorphisms (SNPs) in the human orthologues of genes of interest arising from the above-mentioned studies correlate with hearing function in children.p-values were then investigated in 3417 adults in the G-EAR study of hearing.577 SNPs from 13 genes were each analysed by linear regression against averaged high  or low frequency  audiometry data from 4970 children in the Avon Longitudinal Study of Parents and Children (ALSPAC) birth-cohort at age eleven years. Genes found to contain SNPs with low p = 0.00014): rs12959910 in CBP80/20-dependent translation initiation factor (CTIF) for averaged high frequency hearing ; and rs10492452 in L-plastin (LCP1) for averaged low frequency hearing . For low frequencies, rs9567638 in LCP1 also enhanced hearing in females . SNPs in LCP1 and CTIF were then examined against low and high frequency hearing data for adults in G-EAR. Although the ALSPAC results were not replicated, a SNP in LCP1, rs17601960, is in strong LD with rs9967638, and was associated with enhanced low frequency hearing in adult females in G-EAR (p = 0.00084).Genotypic data were available in ALSPAC for a total of 577 SNPs from 13 genes of interest. Two SNPs approached sample-wide significance  contains supplementary material, which is available to authorized users. Hearing impairment has a major impact on quality of life. The difficulties associated with progressive hearing loss are particularly apparent with regard to the growing population of older individuals , 2. Neve2+ ion movements; in inner ear hair cells this initiates intracellular signals and second messengers that activate impulses in adjacent nerve cells *[the number of independent tests]); however, it is noted that this may still be conservative because the high and low frequency PTAs might be associated, making them non-independent tests. All analyses were carried out in Stata version 13 . In the reporting of the effect sizes of the minor (SNP) alleles, a positive value represents reduced hearing and a negative value represents enhanced hearing.Linear regression was used to determine if any of the SNPs correlated with an effect on the high- or low-frequency PTAs or the broadband OAE results, using the 10 principal components as co-variables. The analyses were also stratified by gender, because evidence from our validation study (see below) suggested that the observed effects were gender specific. The p-values and directions of effects. The full G-EAR study is described elsewhere . The effect size (β) of the regression represents the decibel (dB) change in the hearing threshold for the averaged frequencies for each copy of the minor allele; an increase indicates a detrimental effect on hearing function.Haploview’s solid spine of LD computed 118 groups of LD, which gave a sample-wide SNP threshold CTIF for the high frequency PTA  and rs10492452 in LCP1 for the low frequency PTA  .To our knowledge, a GWAS of hearing function in children has not been conducted. Therefore, we attempted validation of our findings from ALSPAC within the G-EAR cohort of adults over 18 years of age in isolated European populations, which had been designed to assess the hearing function and thresholds of isolated European populations within the international G-EAR consortium . The 10 CTIF, showed a relationship with broadband OAE in the non-stratified and sub-group analyses of the ALSPAC cohort. The effect was specific to females and correlated with smaller amplitude OAE, i.e., poorer hearing function    , effect in girls  . There were no differences in the proportion of minor (SNP) alleles between the genders  under investigation are in 8/577 undCTIF and LCP1 were identified in dbSNP and examined with regard to the human genome reference assembly. All five SNPs of interest: rs12959910 and rs7233521 in CTIF and rs10492452, rs17601960 and rs9567638 in LCP1, are intronic variants. The two SNPs in CTIF have distinct locations within the gene  hearing loss in BXD recombinant inbred mice [Ctif was demonstrated to be expressed in the mouse cochlea with an expression level that is influenced by the parental allele, but the exact sites of expression have yet to be established [CTIF levels or functionality could potentially affect many target mRNAs. Further research will be needed to establish the expression patterns and functional significance of CTIF in the inner ear.In mice, red mice : mice heablished . The CTIablished . Thus, aLCP1, designated plastin-2 or L-plastin, is a member of the plastin family of actin-binding proteins. The mammalian family also includes plastin-1, also known as I-plastin or fimbrin, which is highly expressed in intestine and kidney, and plastin-3 or T-plastin, which is expressed in most solid tissues [Lcp1−/− mice are viable but show defects in B cell development and immune responses [LCP1 has been correlated with gender- and tumour-stage specific prognostic significance in colorectal cancer recurrence [Pls1) gene knockout mice were found to have moderate, progressive hearing loss across all frequencies that correlated with morphological abnormalities of stereocilia in mature hair cells [Lcp1 gene knockout mice have focused on immunological functions.The protein product of  tissues , 35. Plaesponses , 37. Up-currence , 39. Thecurrence –42. Subscurrence , 32. To ir cells . InvestiLCP1 appeared to be sex-specific, were protective and had low minor allele frequencies, which could possibly represent a relatively new set of mutations. Because these are intronic SNPs, it is possible that their effects relate to mRNA stability. It would be helpful to conduct audiometry testing on Lcp1 knockout mice to determine if there is a causal relationship between Lcp1 and hearing function.In our study, the SNPs in CTIF had an influence on high frequency but not low frequency hearing, whereas LCP1 affected only low frequency hearing; these differential frequency effects will require further investigation. It is interesting that the associations were observed with audiometry but not with otoacoustic emissions. Otoacoustic emissions are sensitive to the cochlear amplification function of the outer hair cells [CTIF in females.ir cells . In thisCDH23 or PCDH15 were included in our study set of genes due to their known causal roles in inherited forms of deafness [PCDH15 were amongst those with the lowest p values for effects on high or low frequency hearing in ALSPAC . Due to the above-mentioned scarcity of genome-wide association studies that have examined hearing in children, replication of our results could not be attempted in a dataset that tested children. The G-EAR dataset used for replication comprised adults with varying degrees of hearing loss, likely to be caused by age and environmental effects. The ALSPAC cohort at age 11 mostly had hearing thresholds within the normal range. The effect sizes of the G-EAR dataset could not be compared to ALSPAC due to differences in analysis methods. It is possible that effects on hearing of other genes studied were not detected due to: small effect sizes; the effect appearing after 11 years of age; the demographics of our study; or that functionally important regions of the genes were not covered by the SNPs examined.Several limitations are recognised. Due to the genotyping chip used, some of the selected genes were poorly represented by SNPs in ALSPAC to draw information from (e.g., CTIF and LCP1 with effects on hearing function in children at age 11. Although these findings were not validated against an independent adult population, there was evidence of a sex-specific locus in LCP1 being correlated with enhanced low frequency hearing function in girls and adult women. Further studies of CTIF and LCP1 in relation to hearing function and hair cell physiology would be warranted.This study demonstrates, for the first time, suggestive associations of SNPs in


With great interest we read the recently published article by Bianchin et al. describing a case report of a child with Van Maldergem Syndrome causing severe craniofacial dysmorphism and bilateral congenital conductive hearing loss due to microtia and external auditory canal atresia . AuditorIn the case report, clinical evaluation at five years of age showed a speech development delay, despite the application of traditional bone-conduction hearing aids from the age of twenty-two months. During preoperative pure-tone audiometry, good functional gain with hearing aids was detected, but good compliance was lacking and the device was not used correctly. This can be explained by the fact that steel spring headband was being used. Zarowski et al. stated that this type of head band should not be used for more than 1-2 hours, because of unpleasant pressure and pain caused by the tight fitting –5. This Without proper comfort during testing, the conventional skin-drive bone-conduction device is not representative for a percutaneous BI. Therefore, using steel spring headband for prolonged period, that is, longer than a few days or maximum 2-3 weeks of preoperative testing phase requires, is considered obsolete.Skin dampening must also be considered during patient counseling. Differences in audiometric threshold and speech reception thresholds (SRT) found between preoperative testing with both the softband or steel spring headband and the final postoperative results are significant at high frequencies . These aAn important issue, lacking in the manuscript, to consider when choosing between a bone implant and VSB is MRI safety. MRI is being used more frequently as diagnostic tool. Between 1996 and 2010 the use of MRI has quadrupled . In 2014Another key issue in which the VSB differs from BI is duration of surgery and therefore airway management. In both surgeries, general anesthesia is traditionally used. It must be noted that BI implantation in children younger than 10 years is generally provided as a 2-stage procedure . AlthougFurther issues are the inherent risks of the surgical procedure. BI implantation carries significantly less surgical risk with low morbidity when compared to VSB implantation and it is essentially a “reversible” procedure. The percutaneous abutment can simply be removed when the child is older and so will not compromise future implant choices. This is an extremely important discussion to have with the carer and parents of any child with the hearing loss and a complex medical condition described as in this article.We believe that in this time of rapidly developing technology in the field of implantation otology, it is even more essential to make a shared and informed decision with the patient and caretakers. The long term implications of all the possible hearing solutions offered must be considered within the multidisciplinary assessment and discussed along with the risk and benefits of treatments with the parents.In our tertiary referral centres we have gained a large amount of expertise in order to establish the benefits of bilateral application of bone-conduction devices. The current generation of bone implants shows improved skin tolerance and good survival rates in children . It commFinally, we would question whether all risks and benefits were discussed in this reported case. Furthermore this decision was made with suboptimal presurgical audiological assessment. We were very concerned that such a young child with complex medical conditions was implanted with a VSB despite the higher anesthetic/surgical risks, huge implications for future imaging, implications for future craniofacial surgery/microtia surgery, and of course the inevitable compromise this will now have on future hearing choices.


TCs) are a newly discovered type of cell with numerous functions. They have been found in a large variety of organs: heart ; digestive tract and annex glands ; respiratory system (trachea and lungs); urinary system ; female reproductive system ; vasculature ; serous membranes (mesentery and pleura); and other organs . Likewise, TCs are widely distributed in vertebrates . This review summarizes particular features of TCs in the female reproductive system, emphasizing their involvement in physiological and pathophysiological processes.Telocytes ( Telocytes (TCs) were discovered in 2005 when L.M. Popescu's group from Bucharest, Romania, described a new type of cell that resides in the stroma of several organs, which became known as interstitial Cajal‐like cells (ICLC). This group named these cells as ICLC because of their apparent similarity with the canonical gastrointestinal cells of Cajal (ICC), the gut pacemaker cells. A few years later, in 2008, M.S. Faussone‐Pellegrini and her team from Florence, Italy, described ICLC in the muscle coat of the human gut and noticed they consistently differed from the ICC in both ultrastructure and immunophenotype. In 2010, after confirming the presence of this particular cell type in the stroma of many organs and characterized it by immunohistochemistry and electron microscopy, the two groups agreed they were describing a ‘novel’ cell type and that the name ICLC should be changed to a more appropriate one. From then on, this novel cell type became known as the TCs (using the Greek affix ‘Telos’) Telocytes have been shown to differ from fibroblasts and mesenchymal stem cells in terms of gene profile and proteomics, and in microRNA expression. They have the principal morphological characteristics contrasted with other types of cells The TC has a small, oval‐shaped cellular body, containing a nucleus, surrounded by a small amount of cytoplasm. The cellular body average dimensions are, as measured on TEM images, 9.39 μm ± 3.26 μm . The nucleus occupies about 25% of the cell volume and contains clusters of heterochromatin attached to the nuclear envelope.The perinuclear cytoplasm is rich in mitochondria (which occupy about 5% of the cell body) particularly in podoms, which contain a small Golgi complex, as well as the elements of rough and smooth endoplasmic reticulum and cytoskeletal elements (thin and intermediate filaments) The cell periphery is represented by the usual plasmalema, with no (or thin and discontinuous) basal lamina, and some caveolae .etc. Their spatial appearance is that of a polyhedron with a different number of vertices, depending on their TP number The shape of the TCs depends on the number of their telopodes Tps: piriform for one prolongation, spindle for two Tps, triangular for three, stellate, Number: 1–5, frequently only 2–3 Tps are observed on a single section, depending on site and angle of section, since their 3D convolutions impede their observation in a very thin 2D section along the entire length of the TP their full length.Length: from tens to hundreds of μm, as measured on EM images. However, under favourable condition in cell cultures, their entire length can be captured.Thickness: an uneven caliber, mostly below 0.2 μm (resolving power of light microscopy), visible under electron microscopy, only 0.10 μm ± 0.05 μm Moniliform aspect, with many dilations along their length;2+‐release units’ at the level of the dilations, accommodating mitochondria, elements of endoplasmic reticulum and caveolae;Presence of ‘CaBranching, with a dichotomous pattern;Organization in a network—forming a labyrinthine system by tridimensional convolution and overlapping, communicating through gap junctions.Telocytes have a variable number of Tps (very long cellular extensions), which are probably the longest cellular prolongations in the human body. Tps are made by an alternation of dilated portions, named podoms (250–300 nm), containing mitochondria and endoplasmic reticulum and podomers (~80 nm) with thin segments. The main characteristics of Tps: These characteristic features make Tps clearly different from neuronal dendrites, the processes of antigen‐presenting dendritic cells or fibroblasts and myofibroblasts versus 75 nm), and the podoms were thicker in pregnant myometrium (~316 versus 269 nm) In uterine tissue, TCs can influence contractile activity of smooth muscle cells. They differed with pregnant states in telopodal width and podomic thickness, which can be considered to be related to their function Popescu and Enciu experimentally demonstrated that TCs morphology changes with ageing and by modifying the redox balance of the cell culture environment. Oxidative stress impairs the ability of TCs to form Tps and lessens the migration pathway length. Ageing further aggravated this effect Furthermore, TCs demonstrate specific direct (homocellular and heterocellular junctions) and/or indirect  contacts with various surrounding cells Telopodes are connected to each other by homocellular junctions and appear to form a 3D network in the interstitial space at the border of smooth muscle cell bundles. They also contain cytoskeleton elements. Connections between TCs‐exosomes‐intercellular junctions‐cytoskeleton form the equivalent of a primitive nervous system Heterocellular nanocontactsTelocytes also surrounded stem cell niches with Tps and heterocellular contacts. In addition, they establish physical contacts with nerve endings, blood vessels and different types of progenitor cells. Accumulating studies have shown that TCs play an indisputable role in neo‐angiogenesis. As TCs have cytoskeleton elements , this predicts that they could be responsible for detecting smooth muscle cell stretch during the enlargement of the uterus in pregnancy Telocytes release at least three types of extracellular vesicles: exosomes 45 ± 8 nm), ectosomes (128 ± 28 nm) and multivesicular cargos (1 ± 0.4 μm) from their Tps and, occasionally, from the cell body 5 ± 8 nm,Nowadays, increasing knowledge in the chromosomal gene expression profile of TCs can be a further step in research. Several studies have shown interconnections between TCs and genes of chromosomes 1, 2, 3, 4, 17 and 18. Three genes, Capn2, Fhl2 and Qsox1, were overexpressed in pulmonary TCs compared with the other cells. TCs enriched with Capn2 and Fhl2 may be associated with the regulation of tissue inflammation, injury, repair, immune responses or cell movement. Besides, as Capn2 is important for embryogenesis, it explains the involvement of TCs in morphogenesis and tissue homeostasis. The lower expression of chromosome 1 genes in TCs, as compared with other cells, was found for Ifi203 (interferon‐activated gene 203) and Tcea1 (transcription elongation factor A SII‐1) e.g. Pltp, Gzf1, Polr1b, Tasp1, Zbtb34 and Zfp120, down‐expressed most in TCs, differentially from other cells. In chromosome 3, there were 13 up‐ and 59 down‐regulated genes in TCs In chromosome 2, 26 genes were overexpressed in TCs. One gene Myl9  was overexpressed most in TCs, different from other cells. Myl9 regulatory gene encodes the regulatory light chains of myosin II molecule, known to play a central role in cell adhesion, migration and division. There were six genes, Approximately 15% and 12% genes in chromosomes 17 and 18 were identified as TCs‐specific genes. Sixteen and 10 of TCs‐specific genes were up‐regulated, and 68 and 22 were down‐regulated in chromosome 17 and 18, as compared with other cells. Most of the observed genes in both chromosomes were immune‐associated, which supported the role of TCs in immune surveillance and immune homeostasis In chromosome 4, 17 genes were up‐ and 56 down‐regulated. Among up‐regulated genes were Akap2, Gpr153, Sdc3, Tbc1d2, which encode proteins involved in cell signalling pathways and cytoskeleton organization. It seems that TCs could integrate signals and autoregulate its own fate, integrating autophagy with endocytic trafficking As any type of cells, TCs have own immunohistochemical profile. Current studies demonstrate expressions or (co‐) expression of different markers in TCs, which at the same time not are peculiar to a given organ. TCs are immunohistochemically positive for CD34 Table , CD117/cIt is important to note that TCs might have subpopulations. The expression of c‐kit receptors differs between TCs populations (possible site dependant). For instance, rat uterus tissue contains different types of immune positive TCs: c‐kit (−)/vimentin (+), c‐kit (+)/vimentin (+), c‐kit (+)/CD34 (+). This range might be basis of region‐specific TCs roles In the chorial villi, some TCs are c‐kit‐positive; and some CD34 positive A of the CD34 positive TC express vimentin and calveolin‐1, and some of them also c‐kit. In cultured cells from the same placental villi, some TCs are double positive for c‐kit and iNOS and others for c‐kit and VEGF Telocytes are also involved in the electrical modulation of excitable tissue, such as the smooth muscle of the gut and uterus. They can spontaneously initiate electrical activity and modulate of glandular and immune activity T‐type calcium channels are present in TCs from human myometrium, which, in pregnancy and labour, could participate in the generation of endogenous bioelectric signals responsible for the regulation of the surrounding cell behaviour. Surprisingly, this might be the missing link for describing the molecular mechanisms by which TCs are involved in mechanical stretching during uterine enlargement in pregnancy. The expression of α‐subunit of T‐type calcium channels in TCs is less intense in the case of non‐pregnant myometrium Steroid hormones and oxytocin might mediate the higher expression of T‐type calcium channels in TCs derived from pregnant myometrium. As TCs have steroid hormone receptors, this might lead to frequent and sustained contractions able to trigger birth et al. and Allix et al., a possible mechanism could be via a tyrosine‐kinase independent signalling pathway There is the opposite situation in the expression of small‐conductance calcium‐activated potassium channels isoform 3 (SK3). This type of ion channel has been identified in the myometrium of several species including human, mice and rats, but with great interspecies variation in the pattern of expression and regulation In the myometrium, TCs contain only outward potassium‐rectifier channels, but no inward currents have been evidence so far. There also is a lack of regular slow waves of depolarization in TCs from myometrium.The low‐level laser stimulation (LLLS) of TCs produces a higher growth rate of lateral telopodal extension in pregnant myometrium primary cultures as compared to non‐pregnant ones. The angel of deviation is more accentuated in TCs from human pregnant myometrium than in TCs from non‐pregnant myometrium. Therefore, some implications are emerging for low‐level laser therapy in uterine regenerative medicine. Telopodes from the pregnant uterus are more prone to extend upon LLLS compared to those from the non‐pregnant uterus. The required number of stimulations is sometimes three times higher than in pregnant myometrium. In both preparations, Tps seem to accumulate a big part of their resources near the stimulation area. Finger‐like structures were probed with the laser beam. In some experiments, the TP appears to be is breaking off its old connection, maintaining only thin ‘anchors’ beyond the point of stimulation.Twenty‐five percent of TCs from pregnant uterus present a local thickening of the TP upon LLLS. The local thickening phenomenon was directly correlated with a delayed telopodal response to stimulation Some experiments have been carried out on the action of imatinib (c‐kit receptor antagonist) on the spontaneous and oxytocin‐induced contractions in human non‐pregnant myometrium and the myometrium of pregnant rabbits. C‐kit inhibition led to a reduction in both the amplitude and frequency of myometrial contraction in a dose‐dependent manner. Results show that TCs might be players in the coordination of uterine activity in a kit‐independent manner. This may be under hormonal influences, since express oestrogen and PRs. Also TCs could be modulators of the electrical excitability of underlying contractions, but are not necessarily pacemakers Numerous experiments have shown that the amount of TCs in endometrium and myometrium in rats changes depending on its reproductive state. In pregnant uteri endometrial TCs increase, compared with non‐pregnant, in spite of a significant decline in the number of myometrial TCs. Postpartum uteri show the highest significant count of myometrial TCs and non‐significant difference in endometrial TC count, as compared with the adult non‐pregnant group. The lowest count of both types of TCs occurs in the immature rat uteri, whereas the highest amount of endometrial TCs was detected in the pregnant uteri and the highest amount of myometrial TCs in the postpartum uteri. Telocytes produce electric slow waves that trigger and coordinate smooth muscle contractions; the decrease in myometrial TCs numbers in the pregnant rat uterus may explain the prevention of preterm delivery (uterine contractility before term). The increase after delivery may be connected with the involution of the uterus accompanied by myometrial contractions et al. has suggested that TCs are frequently present in tissues that have a low cell density and significant space between neighbouring cells. Moreover, TCs express connexin 43, a gap junction protein, which most likely has a vital role in decidual maturation, as its decrease is associated with recurrent pregnancy loss In one further example of involving TCs in pregnancy is their increase during that condition. Hence, Hatta In a literature study, we have estimated the average number of TCs in human uterus. Telocytes constituted about 7% of the total cell number in non‐pregnant myometrial cell culture and about 3% of the entire cell population in the myometrium of adult non‐pregnant humans In addition, the number of CD117‐positive cells is reduced during pregnancy under normal condition. This prevents myogenic contractions and early delivery, as CD‐117‐positive cells within the myometrium are involved in myogenic contractile mechanisms during pregnancy A study on the role of murine TCs pregnancy‐induced liver growth demonstrated the potential involvements of TCs in hepatic proliferative adaptations to pregnancy. An increase in CD34/PDGFR‐α positive TCs in pregnant liver accompanied by high level of hepatocyte proliferation In the human endometrium, the location of TCs is contoured to the shape of the adjacent epithelial architecture. They may support the structure of the gland and nearby stroma as a functional unit by forming a scaffold around them. In addition, they may take part in loosening the matrix or secreting factors during decidual formation Telocytes, which were observed from rat's endometrium, stained positive for connexin 43, a protein involved in gap junctions. Connexin 43 is involved in maturation of decidua and its decreasing connection with recurrent pregnancy loss. Endometrial TCs may play the role of gap junctions and have impact on normal developing of pregnancy In the human Fallopian tube, TCs are located mainly in mucosa and muscular layer among smooth muscle fibres Damage and loss of TCs can change the activity of TCs‐stem cells and decrease tissue reparation or renewal capacity, subsequently inducing the development of tissue fibrosis within endometriosis‐affected oviduct. This will then cause Fallopian tube dysfunction and infertility. On the other hand, the reconstruction TCs network might be of great value for structural and functional reparation of fibrous disease of Fallopian tube via connexin43‐mediated mechanism In addition, oviduct TCs express oestrogen receptor alpha and PR‐A, and thus might act as ‘hormonal sensors’, and their function may be in part under hormonal control. These receptors are specific for TCs localization. For example, TCs of gallbladder are negative for both types of receptors and gallbladder muscular contractions are not regulated by sex steroids. TCs were more often tested with anti PR‐A antibody. The PR‐A expression is recognized by most anti‐PR antibodies, whereas the PR‐B expression was not detected. It is possible that sex steroid levels could impact on TCs The placenta also contains functional TCs. They were discovered in the large stem villi, with their long, slender process surrounding the blood vessel wall, or interposed between arterioles and the trophoblast basement membrane in small stem villi. The TCs were connected by gap junctions in a network extending into the placental stroma between concentric layers formed by smooth muscle cells and myofibroblasts around large blood vessels. The TCs had close contacts with mast cells, Hofbauer cells,In addition, TCs in human term placenta are positive for c‐kit, CD34, vimentin, caveolin‐1, VEGF and iNOS. Presumptive roles of TCs in term placenta are partly related to their immunologic profile. Firstly, as TCs are located near vessels and, as the placenta is a non‐innervated organ, they might be involved in blood flow regulation. Secondly, TCs express VEGF and might have impact on tissue remodelling. Finally, TCs appear frequently cited as an ‘intercellular bridge’ between two HBCs. They might play the role of immune surveillance. Moreover, TCs express steroid hormone receptors. This could have an impact on the development of connections between the placenta and uterus in vitro and in vivoTelocytes are identified close to small vessels and express angiogenesis‐related factors  and pro‐angiogenic microRNAs . Moreover, it has been shown that CD34 positive cells secrete vesicles (exosomes) that have an independent angiogenic activity, both Numerous studies have been carried out in order to detect the properties and functions of TCs in female reproductive organs over the past 10 years. Apparently, these new cells play significant roles in normal physiological processes (and their pathologies) like myometrial contractility, immune responses, the development and secretions of the placenta, regulation of adaptive response to overcome vascular resistance during normal pregnancy.The list also includes the role of TCs on angiogenesis and tissue development, which can be crucial in cancerogenesis, uterine leiomyoma formation, ectopic tubal pregnancy, endometriosis and salpingitis.Telocytes are located in the neuromuscular spindles and participate in the control of muscle tone and motor activity. Telocytes form homo‐ and heterocellular contacts with adjacent cells and might even be able to control and regulate their activity, participating in tissue remodelling/renewal Telocytes could serve as the ‘hormonal sensors’ in human uterus, since there is an evidence of some uterine stromal cells that play a role in endometrial growth and differentiation in a hormone‐dependent manner Telocytes, myocytes and leucocytes could work together in pregnancy myometrium or in the onset of labour Genetic and immunologic profiles of TCs give us an opportunity to explore its cooperation with immune cells. Applications in regenerative medicine may extend the therapeutic role of TCs in pathological conditions. We are only at the beginning of our explorations of the powers and the versatility of TCs.The authors confirm that there are no conflicts of interest.


Objective. This cross-sectional study analyzes the pattern of ultrasound peripheral nerve alterations in patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and multifocal motor neuropathy (MMN) at different stages of functional disability. Material and Methods. 22 CIDP and 10 MMN patients and a group of 70 healthy controls were evaluated with an ultrasound scan of the median, ulnar, peroneal, tibial, and sural nerves. Results were correlated with clinical disability scales and nerve conduction studies. Results. Patients with intermediate functional impairment showed relatively larger cross-sectional areas than subjects with either a milder (p < 0.05) or more severe impairment (p < 0.05), both in CIDP and in MMN. In addition, MMN was associated with greater side-to-side intranerve variability (p < 0.05), while higher cross-sectional areas were observed in CIDP (p < 0.05) and in nerve segments with predominantly demyelinating features (p < 0.05). Higher CSA values were observed in nerves with demyelinating features versus axonal damage . Discussion and Conclusions. Greater extent of quantitative and qualitative US alterations was observed in patients at intermediate versus higher functional disability and in nerves with demyelinating versus axonal damage. CIDP and MMN showed differential US aspects, with greater side-to-side intranerve variability in MMN and higher cross-sectional areas in CIDP.  Multifocal motor neuropathy (MMN) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are acquired immune-mediated peripheral neuropathies (PN). MMN is a pure motor neuropathy syndrome usually beginning in one or both hands and principally affecting the upper extremities, characterized by a chronic or stepwise progressive asymmetrical limb weakness and muscle atrophy . CIDP isThe neurophysiological hallmark of MMN is conduction blocks (CB) outside the usual sites of nerve compression , 3, whilNeuromuscular ultrasound (US) is a noninvasive, painless, and radiation-free complementary imaging technique for the diagnostic work-up of PN , 8. FocaThe aim of this study is to analyze US findings in patients with CIDP and MMN at different functional disability, in order to correlate US qualitative and quantitative measures with clinical and neurophysiological features.This cross-sectional observational study includes 22 CIDP  and 10 MMN  patients recruited from the Neuromuscular Unit of Turin University Hospital between May 2014 and May 2015 and 70 healthy controls (43 men and 27 women).AOU San Giovanni Battista di Torino) approval were obtained. The authors acted in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.Patients were evaluated by means of a clinical, US, electrophysiological assessment, and a structured clinical interview. All subjects fulfilled the European Federation of Neurological Society/Peripheral Nerve Society (EFNS/PNS) criteria for CIDP or MMN , 19 and,A complete neurological examination was performed by means of the inflammatory neuropathy cause and treatment (INCAT) disability scale , the MedAccording to the INCAT disability scale, upper limb activities were scored as “no/minimal impairment” (not affected), “moderate impairment” (affected but not prevented), or “severe impairment” (prevented), while the evaluation of walking difficulties was based on the use of aids: “no/minimal impairment” ; “moderate impairment” ; “severe impairment” .The US assessment was performed by means of a SonoSite M-Turbo Ultrasound Machine equipped with a broadband linear transducer (frequency band 6–15 MHz). The US scan was performed the same day of the neurological assessment by an evaluator (Michela Rosso), who was blinded to the clinical and neurophysiological data.The following quantitative and qualitative US parameters were collected for the median, ulnar, peroneal, tibial, and sural nerves bilaterally.Nerve Cross-Sectional Area (CSA). CSA was measured by tracing the nerve just inside the hyperechoic rims with the “ellipse method” when applicable  or alternatively tracing the nerve shape (when the nerve had an irregular shape) ; MMN = 78.3% versus 20% [p: 0.010]). US abnormal features were additionally observed in 14.4% of CIDP and 10.1% of MMN nerve segments without significant neurophysiological alterations.A total of 320 nerves (220 CIDP and 100 MMN) were evaluated by means of nerve conduction studies and US assessments: neurophysiological alterations were found in 78.0% of CIDP nerve segments  and in 62.5% of MMN nerve segments . Quantitative and/or qualitative US alterations were observed in 43.2% (95/220) of CIPD nerve segments and in 40.0% (40/100) of MMN nerve segments. In both cases these alterations were found more frequently in nerves with predominantly myelin versus axonal damage (CIDP = 74.6% versus 25%  in CIDP and tau = 0.213 [p: 0.045] in MMN; ulnar nerve: tau = 0.260 [p: 0.049] in CIDP and tau = 0.271 [p: 0.048] in MMN), as was for the correlation between altered fascicles and CSA, both in CIDP  and in MMN subjects . Qualitative US analyses showed an inverse pattern compared to that observed at the lower limbs, with a moderately higher prevalence of altered fascicles in CIDP than in MMN nerve segments . As observed in the lower limbs, the CSA values were higher in nerve segments with predominantly demyelinating features versus axonal damage (p: 0.001 for CIDP and p: 0.049 for MMN)  .r = 0.456 [p: 0.002]; MMN: r = 0.450 [p: 0.036]) and between axonal damage and functional disability at the upper limbs .There was a direct correlation between axonal damage and gait impairment at the lower limbs  or between US data and ONLS score , while an inverse correlation was observed in CIDP patients between MRC scores of muscles innervated by median and ulnar nerves and the corresponding intranerve variability , disease duration , treatment duration , or IVIg dose .CSA values were not influenced by age  and/or demyelinating damage.These data are in accordance with the findings reported by Di Pasquale et al. , who obsIn addition, we found some differential aspects between MMN and CIDP: greater side-to-side intranerve variability was observed in MMN, in line with the pattern of heterogeneous and multifocal involvement characteristic of the disease; patients with CIDP showed higher CSA values, potentially indicating more prominent demyelinating processes; qualitative US analyses revealed a different distribution of abnormal fascicles in the upper and lower limbs, with more prominent US alterations in district affected by predominantly demyelinating damage  compared to district affected by secondary axonal degeneration.The majority of literature data reported increased CSA values in CIDP, with a possible association between intranerve variability and functional scores , 24. LesOur data support the complementary role of US in the assessment of CIDP and MMN, suggesting a different pattern in nerves with demyelinating versus axonal damage and in CIDP versus MMN patients, in possible relationship with the different pathological processes involved.Previous studies reported a correlation between disease duration and CSA values , 22, 27,Other factors, such as IVIg pharmacological treatment and/or individual inflammatory response, might also be implicated in the morphological modifications of peripheral nerves . HoweverTaken together, these findings suggest variable applications for US in the field of immune-mediated peripheral neuropathies, ranging from the more accurate clinicopathophysiologic phenotyping to the early detection of morphological changes associated with critical disease milestones. In addition innovative US score, such as the Bochum Ultrasound Score , will liOur findings suggest that CIDP and MMN patients with an intermediate functional disability may present more pronounced quantitative and qualitative US alterations than patients with higher disability. Moreover, some differential aspects can be recognized in CIDP versus MMN and greater US alterations might be observed in nerve segments with demyelinating versus axonal damage.The strength of our findings is partially limited by the relatively small sample size and the lack of serial prospective follow-up assessments. In addition, two aspects should be considered in the interpretation of data: (a) the “U-shaped” relationship between US findings and functional impairment, which might result in a similar US pattern in patients with either minimal or severe disability; (b) the association of CSA reduction with two different factors , indicating the need of further prospective studies to analyze which of the two features primarily correlates with nerve size reduction.


Gluconacetobacter hansenii strain NQ5 (ATCC 53582). This strain was isolated by R. Malcolm Brown, Jr. in a sugar mill in North Queensland, Australia, and is an efficient producer of bacterial cellulose. The elucidation of the genome will contribute to the study of the molecular mechanisms necessary for cellulose biosynthesis.This study reports the release of the complete nucleotide sequence of  Gluconacetobacter hansenii (formerly Acetobacter xylinum) is a particularly efficient producer of pure, highly crystalline cellulose known as bacterial cellulose (BC) (––Gluconacetobacter genus.The Gram-negative bacterium ose (BC) –3. Because (BC) ––7. TheseG. hansenii strain NQ5 (ATCC 53582). This strain is a highly efficient producer of BC and has been used for a range of studies on the effects of growth conditions on cellulose biosynthesis . The reads were assembled into contigs using Velvet version 1/2/02  has a 99% sequence similarity to all of the regions coding for cellulose biosynthesis . No other strain of Gluconacetobacter sp. shared this sequence similarity or contained three cellulose synthase-coding regions. It is important to note that the acsABCD operon is flanked by genes coding for proteins that have been determined to be essential for proper cellulose biosynthesis to occur: cmcAx, ccpAx, and bglAx (–Preliminary annotation indicated that nd bglAx –17. AddiFurther studies into third-region coding for cellulose biosynthesis and how it may or may not impact cellulose production could provide valuable insight into the important biological systems needed for cellulose biosynthesis.LUCJ00000000. The version described in this paper is the first version, LUCJ01000000.This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number