Baird, Richard A.
The bullfrog saccule, a sensor of gravity and substrate-borne vibration, is a model system for hair cell transduction. Saccular hair cells also increase in number throughout adult life and rapidly recover after hair cell damage, making this organ an ideal system for studying hair cell development, repair, and regeneration. We have used of hair cell and supporting cell immunocytochemical markers to identify damaged hair cells and hair cell precursors in organotypic cultures of the bullfrog saccule. We then used an innovative combination of confocal, electron, and time-lapse microscopy to study the fate of damaged hair cells and the origin of new hair cells after gentamicin ototoxicity in normal and mitotically blocked saccular cultures. These studies have shown that gentamicin ototoxicity produces both lethal and sublethal hair cell damage. They have also shown that hair cell recovery in this organ takes place by both the repair of sublethally damaged hair cells and by the replacement of lost hair cells by mitotic regeneration. In parallel studies, we have used biophysical and molecular biological techniques to study the differentiation and innervation of developing, repairing, and regenerating hair cells. More specifically, we have used RT-PCR to obtain the bullfrog homologues of L-type voltage- gated calcium (L-VGCC) and large-conductance Ca(2+)-activated potassium (BK) channel genes. We have then obtained probes for these genes and, using in situ hybridization, begun to examine their expression in the bullfrog saccule and amphibian papilla. We have also used fluorescent-labeled channel toxins and channel toxin derivatives to determine the time of appearance of L-type voltage-gated calcium (L-VGCC) and Ca(2+)-activated potassium (BK) channels and to study dynamic changes in the number, distribution, and co-localization of these proteins in developing, repairing, and regenerating hair cells. Using time-lapse microscopy, we are also studying the dynamic relationship
Full Text Available The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23, protocadherin-15 (PCDH15 and the very large G-protein coupled receptor 1 (VLGR1 have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1-/- mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzer(av3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well.
Roux, Isabelle; Hosie, Suzanne; Johnson, Stuart L; Bahloul, Amel; Cayet, Nadège; Nouaille, Sylvie; Kros, Corné J; Petit, Christine; Safieddine, Saaid
The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca2+-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca2+ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca2+-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca2+ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.
Duncker, Susanne V; Franz, Christoph; Kuhn, Stephanie; Schulte, Uwe; Campanelli, Dario; Brandt, Niels; Hirt, Bernhard; Fakler, Bernd; Blin, Nikolaus; Ruth, Peter; Engel, Jutta; Marcotti, Walter; Zimmermann, Ulrike; Knipper, Marlies
The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.
Walters, Bradley J; Zuo, Jian
The organ of Corti in the mammalian inner ear is comprised of mechanosensory hair cells (HCs) and nonsensory supporting cells (SCs), both of which are believed to be terminally post-mitotic beyond late embryonic ages. Consequently, regeneration of HCs and SCs does not occur naturally in the adult mammalian cochlea, though recent evidence suggests that these cells may not be completely or irreversibly quiescent at earlier postnatal ages. Furthermore, regenerative processes can be induced by genetic and pharmacological manipulations, but, more and more reports suggest that regenerative potential declines as the organ of Corti continues to age. In numerous mammalian systems, such effects of aging on regenerative potential are well established. However, in the cochlea, the problem of regeneration has not been traditionally viewed as one of aging. This is an important consideration as current models are unable to elicit widespread regeneration or full recovery of function at adult ages yet regenerative therapies will need to be developed specifically for adult populations. Still, the advent of gene targeting and other genetic manipulations has established mice as critically important models for the study of cochlear development and HC regeneration and suggests that auditory HC regeneration in adult mammals may indeed be possible. Thus, this review will focus on the pursuit of regeneration in the postnatal and adult mouse cochlea and highlight processes that occur during postnatal development, maturation, and aging that could contribute to an age-related decline in regenerative potential. Second, we will draw upon the wealth of knowledge pertaining to age related senescence in tissues outside of the ear to synthesize new insights and potentially guide future research aimed at promoting HC regeneration in the adult cochlea.
The cochlea processes auditory signals over a wide range of frequencies and intensities. However, the transfer characteristics at hair cell ribbon synapses are still poorly understood at different frequency locations along the cochlea. Using recordings from mature gerbils, we report here a surprisingly strong block of exocytosis by the slow Ca2+ buffer EGTA (10 mM) in basal hair cells tuned to high frequencies (∼30 kHz). In addition, using recordings from gerbil, mouse, and bullfrog auditory organs, we find that the spatial coupling between Ca2+ influx and exocytosis changes from nanodomain in low-frequency tuned hair cells (∼2 kHz). Hair cell synapses have thus developed remarkable frequency-dependent tuning of exocytosis: accurate low-latency encoding of onset and offset of sound intensity in the cochlea's base and submillisecond encoding of membrane receptor potential fluctuations in the apex for precise phase-locking to sound signals. We also found that synaptic vesicle pool recovery from depletion was sensitive to high concentrations of EGTA, suggesting that intracellular Ca2+ buffers play an important role in vesicle recruitment in both low- and high-frequency hair cells. In conclusion, our results indicate that microdomain coupling is important for exocytosis in high-frequency hair cells, suggesting a novel hypothesis for why these cells are more susceptible to sound-induced damage than low-frequency cells; high-frequency inner hair cells must have a low Ca2+ buffer capacity to sustain exocytosis, thus making them more prone to Ca2+-induced cytotoxicity. SIGNIFICANCE STATEMENT In the inner ear, sensory hair cells signal reception of sound. They do this by converting the sound-induced movement of their hair bundles present at the top of these cells, into an electrical current. This current depolarizes the hair cell and triggers the calcium-induced release of the neurotransmitter glutamate that activates the postsynaptic auditory fibers. The speed and
Walters, Brandon J; Diao, Shiyong; Zheng, Fei; Walters, Bradley J; Layman, Wanda S; Zuo, Jian
The mammalian cochlea is a highly specialized organ within the inner ear. Sensory hair cells (HC) in the cochlea detect and transduce sound waves into electrical impulses that are sent to the brain. Studies of the molecular pathways regulating HC formation are hindered by the very sparse nature of HCs, where only ~3300 are found within an entire mouse cochlea. Current cell lines mimic certain aspects of HCs but lack terminal HC marker expression. Here we successfully "pseudo-immortalized" cochlear progenitor cells using the "conditional reprogramming" technique. These cells, termed "Conditionally Reprogrammed Otic Stem Cells" (CR-OSC), are able to bypass the senescence inherent to cochlear progenitor cells without genetic alterations, allowing for the generation of over 15 million cells from a single cochlea. These cells can be differentiated and up-regulate both early and terminal differentiation genes associated with HCs, including the terminal HC differentiation marker prestin. CR-OSCs also respond to known HC cues, including upregulation of HC genes in response to Atoh1 overexpression, and upregulation of prestin expression after thyroid hormone application. Overall, we describe the creation of a HC line capable of regulated expression of HC genes that can easily be recreated in any laboratory from any mouse of interest.
Hair cells are the mechanosensory cells thatconvert sound and motion signals into electrical i m-pulses in cochlear and vestibular end organs of innerear.Although mature mammals nor mally do notgenerate new hair cells,recentin vivoandin vitrostudies have demonstrated mitotic activity and i m-mature-looking hair cells in mammalian vestibularepithelia after exposure to ototoxic drugs[1-3],sug-gesting that vestibular hair cell regeneration inmammals may be inducible.However,the possibil-ity of auditory hair ce...
Tinevez, J.-Y.; Martin, P.; Jülicher, F.
The hair bundle is both a mechano-sensory antenna and a force generator that might help the vertebrate hair cell from the inner ear to amplify its responsiveness to small stimuli. To study active hair-bundle motility, we combined calcium iontophoresis with mechanical stimulation of single hair bundles from the bullfrog's sacculus. A hair bundle could oscillate spontaneously, or be quiescent but display non-monotonic movements in response to abrupt force steps. Extracellular calcium changes or static biases to the bundle's position at rest could affect the kinetics of bundle motion and evoke transitions between the different classes of motility. The calcium-dependent location of a bundle's operating point within its nonlinear force-displacement relation controlled the type of movements observed. A unified theoretical description, in which mechanical activity stems from myosin-based adaptation and electro-mechanical feedback by Ca2+, could account for the fast and slow manifestations of active hair-bundle motility.
Shang, Jialin; Cafaro, Jon; Nehmer, Rachel; Stone, Jennifer
In chickens, nonsensory supporting cells divide and regenerate auditory hair cells after injury. Anatomical evidence suggests that supporting cells can also transdifferentiate into hair cells without dividing. In this study, we characterized an organ culture model to study auditory hair cell regeneration, and we used these cultures to test if direct transdifferentiation alone can lead to significant hair cell regeneration. Control cultures (organs from posthatch chickens maintained without streptomycin) showed complete hair cell loss in the proximal (high-frequency) region by 5 days. In contrast, a 2-day treatment with streptomycin induced loss of hair cells from all regions by 3 days. Hair cell regeneration proceeded in culture, with the time course of supporting cell division and hair cell differentiation generally resembling in vivo patterns. The degree of supporting cell division depended upon the presence of streptomycin, the epithelial region, the type of culture media, and serum concentration. On average, 87% of the regenerated hair cells lacked the cell division marker BrdU despite its continuous presence, suggesting that most hair cells were regenerated via direct transdifferentiation. Addition of the DNA polymerase inhibitor aphidicolin to culture media prevented supporting cell division, but numerous hair cells were regenerated nonetheless. These hair cells showed signs of functional maturation, including stereociliary bundles and rapid uptake of FM1-43. These observations demonstrate that direct transdifferentiation is a significant mechanism of hair cell regeneration in the chicken auditory after streptomycin damage in vitro.
Michael E. Smith
Full Text Available Mature mammals exhibit very limited capacity for regeneration of auditory hair cells, while all non-mammalian vertebrates examined can regenerate them. In an effort to find therapeutic targets for deafness and balance disorders, scientists have examined gene expression patterns in auditory tissues under different developmental and experimental conditions. Microarray technology has allowed the large-scale study of gene expression profiles (transcriptomics at whole-genome levels, but since mRNA expression does not necessarily correlate with protein expression, other methods, such as microRNA analysis and proteomics, are needed to better understand the process of hair cell regeneration. These technologies and some of the results of them are discussed in this review. Although there is a considerable amount of variability found between studies owing to different species, tissues and treatments, there is some concordance between cellular pathways important for hair cell regeneration. Since gene expression and proteomics data is now commonly submitted to centralized online databases, meta-analyses of these data may provide a better picture of pathways that are common to the process of hair cell regeneration and lead to potential therapeutics. Indeed, some of the proteins found to be regulated in the inner ear of animal models (e.g., IGF-1 have now gone through human clinical trials.
Full Text Available Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C and PMCA2 (ATP2B2; plasma-membrane Ca(2+-ATPase isoform 2 to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.
Full Text Available Abstract Background Stem cells divide to reproduce themselves and produce differentiated progeny. A fundamental problem in human biology has been the inability to measure how often stem cells divide. Although it is impossible to observe every division directly, one method for counting divisions is to count replication errors; the greater the number of divisions, the greater the numbers of errors. Stem cells with more divisions should produce progeny with more replication errors. Methods To test this approach, epigenetic errors (methylation in CpG-rich molecular clocks were measured from human hairs. Hairs exhibit growth and replacement cycles and "new" hairs physically reappear even on "old" heads. Errors may accumulate in long-lived stem cells, or in their differentiated progeny that are eventually shed. Results Average hair errors increased until two years of age, and then were constant despite decades of replacement, consistent with new hairs arising from infrequently dividing bulge stem cells. Errors were significantly more frequent in longer hairs, consistent with long-lived but eventually shed mitotic follicle cells. Conclusion Constant average hair methylation regardless of age contrasts with the age-related methylation observed in human intestine, suggesting that error accumulation and therefore stem cell latency differs among tissues. Epigenetic molecular clocks imply similar mitotic ages for hairs on young and old human heads, consistent with a restart with each new hair, and with genealogies surreptitiously written within somatic cell genomes.
Zhang, Yiming; Xing, Yizhan; Guo, Haiying; Ma, Xiaogen; Li, Yuhong
The regulation of the periodic regeneration of hair follicles is complicated. Although Wnt10b has been reported to induce hair follicle regeneration, the characteristics of induced hair follicles, especially the target cells of Wnt10b, have not yet been clearly elucidated. Thus, we systematically evaluated the expression and proliferation patterns of Wnt10b-induced hair follicles. We found that Wnt10b promoted the proliferation of hair follicle stem cells from 24 hours after AdWnt10b injection. Seventy-two hours after AdWnt10b injection, cells outside of bulge area began to proliferate. When the induced hair follicle entered full anagen, although the hair follicle stem cells were normal, canonical Wnt signaling was maintained in the hair precortex cells. Our results reveal that the target cells that overexpressed Wnt10b included hair follicle stem cells, hair precortex cells, and matrix cells.
Miller, M R; Beck, J
The pattern of afferent and efferent innervation of two to four unidirectional (UHC) and two to nine bidirectional (BHC) hair cells of five different types of lizard auditory papillae was determined by reconstruction of serial TEM sections. The species studies were Crotaphytus wislizeni (iguanid), Podarcis (Lacerta) sicula and P. muralis (lacertids), Ameiva ameiva (teiid), Coleonyx variegatus (gekkonid), and Mabuya multifasciata (scincid). The main object was to determine in which species and in which hair cell types the nerve fibers were innervating only one (exclusive innervation), or two or more hair cells (nonexclusive innervation); how many nerve fibers were supplying each hair cell; how many synapses were made by the innervating fibers; and the total number of synapses on each hair cell. In the species studies, efferent innervation was limited to the UHC, and except for the iguanid, C. wislizeni, it was nonexclusive, each fiber supplying two or more hair cells. Afferent innervation varied both with the species and the hair cell types. In Crotaphytus, both the UHC and the BHC were exclusively innervated. In Podarcis and Ameiva, the UHC were innervated exclusively by some fibers but nonexclusively by others (mixed pattern). In Coleonyx, the UHC were exclusively innervated but the BHC were nonexclusively innervated. In Mabuya, both the UHC and BHC were nonexclusively innervated. The number of afferent nerve fibers and the number of afferent synapses were always larger in the UHC than in the BHC. In Ameiva, Podarcis, and Mabuya, groups of bidirectionally oriented hair cells occur in regions of cytologically distinct UHC, and in Ameiva, unidirectionally oriented hair cells occur in cytologically distinct BHC regions.
Shatz, L F
The relationship between size and shape of the hair bundle of a hair cell in the inner ear and its sensitivity at asymptotically high and low frequencies was determined, thereby extending the results of an analysis of hair bundle hydrodynamics in two dimensions (Freeman and Weiss, 1990. Hydrodynamic analysis of a two-dimensional model for micromechanical resonance of free-standing hair bundles. Hear. Res. 48, 37-68) to three dimensions. A hemispheroid was used to represent the hair bundle. The hemispheroid had a number of advantages: it could represent shapes that range from thin, pencil-like shapes, to wide, flat, disk-like shapes. Also analytic methods could be used in the high frequency range to obtain an exact solution to the equations of motion. In the low frequency range, where an approximate solution was found using boundary element methods, the sensitivity of the responses of hair cells was mainly proportional to the cube of the heights of their hair bundles, and at high frequencies, the sensitivity of the hair cells was mainly proportional to the inverse of their heights. An excellent match was obtained between measurements of sensitivity curves in the basillar papilla of the alligator and bobtail lizards and the model's predictions. These results also suggest why hair bundles of hair cells in vestibular organs which are sensitive to low frequencies have ranges of heights that are an order of magnitude larger than the range of heights of hair bundles of hair cells found in auditory organs.
Breuskin, Ingrid; Bodson, Morgan; Thelen, Nicolas; Thiry, Marc; Nguyen, Laurent; Belachew, Shibeshih; Lefebvre, Philippe P; Malgrange, Brigitte
Deafness commonly results from a lesion of the sensory cells and/or of the neurons of the auditory part of the inner ear. There are currently no treatments designed to halt or reverse the progression of hearing loss. A key goal in developing therapy for sensorineural deafness is the identification of strategies to replace lost hair cells. In amphibians and birds, a spontaneous post-injury regeneration of all inner ear sensory hair cells occurs. In contrast, in the mammalian cochlea, hair cells are only produced during embryogenesis. Many studies have been carried out in order to demonstrate the persistence of endogenous progenitors. The present review is first focused on the occurrence of spontaneous supernumerary hair cells and on nestin positive precursors found in the organ of Corti. A second approach to regenerating hair cells would be to find genes essential for their differentiation. This review will also focus on critical genes for embryonic hair cell formation such as the cell cycle related proteins, the Atoh1 gene and the Notch signaling pathway. Understanding mechanisms that underlie hair cell production is an essential prerequisite to defining therapeutic strategies to regenerate hair cells in the mature inner ear.
Lu, Xiaowei; Sipe, Conor W
Hearing loss is the most common and costly sensory defect in humans and genetic causes underlie a significant proportion of affected individuals. In mammals, sound is detected by hair cells (HCs) housed in the cochlea of the inner ear, whose function depends on a highly specialized mechanotransduction organelle, the hair bundle. Understanding the factors that regulate the development and functional maturation of the hair bundle is crucial for understanding the pathophysiology of human deafness. Genetic analysis of deafness genes in animal models, together with complementary forward genetic screens and conditional knock-out mutations in essential genes, have provided great insights into the molecular machinery underpinning hair-bundle development and function. In this review, we highlight recent advances in our understanding of hair-bundle morphogenesis, with an emphasis on the molecular pathways governing hair-bundle polarity and orientation. We next discuss the proteins and structural elements important for hair-cell mechanotransduction as well as hair-bundle cohesion and maintenance. In addition, developmental signals thought to regulate tonotopic features of HCs are introduced. Finally, novel approaches that complement classic genetics for studying the molecular etiology of human deafness are presented. WIREs Dev Biol 2016, 5:85-101. doi: 10.1002/wdev.202 For further resources related to this article, please visit the WIREs website.
Etournay, Raphaël; Lepelletier, Léa; Boutet de Monvel, Jacques; Michel, Vincent; Cayet, Nadège; Leibovici, Michel; Weil, Dominique; Foucher, Isabelle; Hardelin, Jean-Pierre; Petit, Christine
Epithelial cells acquire diverse shapes relating to their different functions. This is particularly relevant for the cochlear outer hair cells (OHCs), whose apical and basolateral shapes accommodate the functioning of these cells as mechano-electrical and electromechanical transducers, respectively. We uncovered a circumferential shape transition of the apical junctional complex (AJC) of OHCs, which occurs during the early postnatal period in the mouse, prior to hearing onset. Geometric analysis of the OHC apical circumference using immunostaining of the AJC protein ZO1 and Fourier-interpolated contour detection characterizes this transition as a switch from a rounded-hexagon to a non-convex circumference delineating two lateral lobes at the neural side of the cell, with a negative curvature in between. This shape tightly correlates with the 'V'-configuration of the OHC hair bundle, the apical mechanosensitive organelle that converts sound-evoked vibrations into variations in cell membrane potential. The OHC apical circumference remodeling failed or was incomplete in all the mouse mutants affected in hair bundle morphogenesis that we tested. During the normal shape transition, myosin VIIa and myosin II (A and B isoforms) displayed polarized redistributions into and out of the developing lobes, respectively, while Shroom2 and F-actin transiently accumulated in the lobes. Defects in these redistributions were observed in the mutants, paralleling their apical circumference abnormalities. Our results point to a pivotal role for actomyosin cytoskeleton tensions in the reshaping of the OHC apical circumference. We propose that this remodeling contributes to optimize the mechanical coupling between the basal and apical poles of mature OHCs.
Joseph, Leena D; Devi, M Kanmani; Sundaram, Sandhya; Rajendiran, S
A 65 year old postmenopausal female presented with left sided abdominal pain. Sonogram revealed an intra-abdominal 7.4 x 5.7 cm heterogenous mass. On laparotomy, approximately 10 X 10 cm mesenteric mass was seen adherent to the descending colon. Multiple omental tumor deposits were also noted. Gross examination showed solid and cystic tumor with sebaceous material admixed with hair. Histopathology showed mature cystic teratoma with a spectrum of well to poorly differentiated squamous cell carcinoma with omental metastasis.
LI Yi; He David Z
Cochlear outer hair cells (OHCs) are involved in a mechanical feedback loop in which the fast somatic motility of OHCs is required for cochlear amplification. Alternatively, amplification is thought to arise from active hair bundle movements ob-served in non-mammalian hair cells. We measured the voltage-evoked hair bundle motions in the gerbil cochlea to determine if such movements are also present in mammalian OHCs. The OHCs displayed a large hair bundle movement that was not based on mechanotransducer channels but based on somatic motility. Significantly, bundle movements were able to generate radial motion of the tectorial membrane in situ. This result implies that the motility-associated hair bundle motion may be part of the cochlear amplifier.
Baird, Richard A.; Schuff, N. R.; Bancroft, J.
Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not stain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type 1 hair cells while labeling, as in the bullfrog, Type 2 hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.
Full Text Available Abstract Background Initiation of the hair follicle placode and its subsequent growth, maturation and cycling in post-natal skin requires signaling interactions between epithelial cells and adjacent dermal cells and involves Shh signaling via the primary cilium. Previous reports have implicated laminins in hair follicle epithelial invagination. Results Here we use a human BCC model system and mouse mutants to re-evaluate the role of laminin-511 in epithelial invagination in the skin. Blocking laminin 511 and 332 in BCCs maintains primary cilia and Shh signalling, but prevents invagination. Similarly, in laminin-511 and dermal beta-1 integrin mutants, dermal papilla development and primary cilia formation are normal. Dermal beta-1 integrin mutants have normal hair follicle development. Conclusions Our data provides support for a primary role of laminin-511 promoting hair follicle epithelial downgrowth without affecting dermal primary cilia and Shh target gene induction.
Full Text Available The prevalence of acquired hearing loss is very high. About 10% of the total population and more than one third of the population over 65 years suffer from debilitating hearing loss. The most common type of hearing loss in adults is idiopathic sudden sensorineural hearing loss (ISSHL. In the majority of cases, ISSHL is permanent and typically associated with loss of sensory hair cells in the organ of Corti. Following the loss of sensory hair cells, the auditory neurons undergo secondary degeneration. Sensory hair cells and auditory neurons do not regenerate throughout life, and loss of these cells is irreversible and cumulative. However, recent advances in stem cell biology have gained hope that stem cell therapy comes closer to regenerating sensory hair cells in humans. A major advance in the prospects for the use of stem cells to restore normal hearing comes with the recent discovery that hair cells can be generated ex vivo from embryonic stem (ES cells, adult inner ear stem cells and neural stem cells. Furthermore, there is increasing evidence that stem cells can promote damaged cell repair in part by secreting diffusible molecules such as growth factors. These results suggest that stem-cell-based treatment regimens can be applicable to the damaged inner ear as future clinical applications.Previously we have established an animal model of cochlear ischemia in gerbils and showed progressive hair cell loss up to 4 days after ischemia. Auditory brain stem response (ABR recordings have demonstrated that this gerbil model displays severe deafness just after cochlear ischemia and gradually recovers thereafter. These pathological findings and clinical manifestations are reminiscent of ISSHL in humans. In this study, we have shown the effectiveness of stem cell therapy by using this animal model of ISSHL.
Jerry D. Monroe
Full Text Available Sensory hair cells are specialized mechanotransductive receptors required for hearing and vestibular function. Loss of hair cells in humans and other mammals is permanent and causes reduced hearing and balance. In the early 1980’s, it was shown that hair cells continue to be added to the inner ear sensory epithelia in cartilaginous and bony fishes. Soon thereafter, hair cell regeneration was documented in the chick cochlea following acoustic trauma. Since then, research using chick and other avian models has led to great insights into hair cell death and regeneration. However, with the rise of the zebrafish as a model organism for studying disease and developmental processes, there has been an increased interest in studying sensory hair cell death and regeneration in its lateral line and inner ears. Advances derived from studies in zebrafish and other fish species include understanding the effect of ototoxins on hair cells and finding otoprotectants to mitigate ototoxin damage, the role of cellular proliferation versus direct transdifferentiation during hair cell regeneration, and elucidating cellular pathways involved in the regeneration process. This review will summarize research on hair cell death and regeneration using fish models, indicate the potential strengths and weaknesses of these models, and discuss several emerging areas of future studies.
Hoffman, Robert M
The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is discussed. Hair follicle delivery systems are described such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.
The purpose of this article is to present additional information about the flow-velocity sensors described briefly in the immediately preceding article. As noted therein, these sensors can be characterized as artificial hair cells that implement an approximation of the sensory principle of flow-sensing cilia of fish: A cilium is bent by an amount proportional to the flow to which it is exposed. A nerve cell at the base of the cilium senses the flow by sensing the bending of the cilium. In an artificial hair cell, the artificial cilium is a microscopic cantilever beam, and the bending of an artificial cilium is measured by means of a strain gauge at its base (see Figure 1). Figure 2 presents cross sections of a representative sensor of this type at two different stages of its fabrication process. The process consists of relatively- low-temperature metallization, polymer-deposition, microfabrication, and surface-micromachining subprocesses, including plastic-deformation magnetic assembly (PDMA), which is described below. These subprocesses are suitable for a variety of substrate materials, including silicon, some glasses, and some polymers. Moreover, because it incorporates a polymeric supporting structure, this sensor is more robust, relative to its silicon-based counterparts.
Full Text Available In this paper, we developed a two-step-induction method of generating functional hair cells from inner ear multipotent cells. Multipotent cells from the inner ear were established and induced initially into progenitor cells committed to the inner ear cell lineage on the poly-L-lysine substratum. Subsequently, the committed progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells containing characteristic stereocilia bundles. The hair-cell-like cells exhibited rapid permeation of FM1-43FX. The whole-cell patch-clamp technique was used to measure the membrane currents of cells differentiated for 7 days on chicken utricle stromal cells and analyze the biophysical properties of the hair-cell-like cells by recording membrane properties of cells. The results suggested that the hair-cell-like cells derived from inner ear multipotent cells were functional following differentiation in an enabling environment.
Full Text Available BACKGROUND: Exposure to intense sound or high doses of aminoglycoside antibiotics can increase hearing thresholds, induce cochlear dysfunction, disrupt hair cell morphology and promote hair cell death, leading to permanent hearing loss. When the two insults are combined, synergistic ototoxicity occurs, exacerbating cochlear vulnerability to sound exposure. The underlying mechanism of this synergism remains unknown. In this study, we tested the hypothesis that sound exposure enhances the intra-cochlear trafficking of aminoglycosides, such as gentamicin, leading to increased hair cell uptake of aminoglycosides and subsequent ototoxicity. METHODS: Juvenile C57Bl/6 mice were exposed to moderate or intense sound levels, while fluorescently-conjugated or native gentamicin was administered concurrently or following sound exposure. Drug uptake was then examined in cochlear tissues by confocal microscopy. RESULTS: Prolonged sound exposure that induced temporary threshold shifts increased gentamicin uptake by cochlear hair cells, and increased gentamicin permeation across the strial blood-labyrinth barrier. Enhanced intra-cochlear trafficking and hair cell uptake of gentamicin also occurred when prolonged sound, and subsequent aminoglycoside exposure were temporally separated, confirming previous observations. Acute, concurrent sound exposure did not increase cochlear uptake of aminoglycosides. CONCLUSIONS: Prolonged, moderate sound exposures enhanced intra-cochlear aminoglycoside trafficking into the stria vascularis and hair cells. Changes in strial and/or hair cell physiology and integrity due to acoustic overstimulation could increase hair cell uptake of gentamicin, and may represent one mechanism of synergistic ototoxicity.
Muller, Mees; Heeck, Kier; Elemans, Coen P.H.
Vertebrate semicircular canals (SCC) first appeared in the vertebrates (i.e. ancestral fish) over 600 million years ago. In SCC the principal mechanoreceptors are hair cells, which as
compared to cochlear hair cells are distinctly longer (70 vs. 7 μm), 10 times more compliant to bending (4
Patrick J Atkinson
Full Text Available The degeneration of hair cells in the mammalian cochlea results in permanent sensorineural hearing loss. This study aimed to promote the regeneration of sensory hair cells in the mature cochlea and their reconnection with auditory neurons through the introduction of ATOH1, a transcription factor known to be necessary for hair cell development, and the introduction of neurotrophic factors. Adenoviral vectors containing ATOH1 alone, or with neurotrophin-3 and brain derived neurotrophic factor were injected into the lower basal scala media of guinea pig cochleae four days post ototoxic deafening. Guinea pigs treated with ATOH1 gene therapy, alone, had a significantly greater number of cells expressing hair cell markers compared to the contralateral non-treated cochlea when examined 3 weeks post-treatment. This increase, however, did not result in a commensurate improvement in hearing thresholds, nor was there an increase in synaptic ribbons, as measured by CtBP2 puncta after ATOH1 treatment alone, or when combined with neurotrophins. However, hair cell formation and synaptogenesis after co-treatment with ATOH1 and neurotrophic factors remain inconclusive as viral transduction was reduced due to the halving of viral titres when the samples were combined. Collectively, these data suggest that, whilst ATOH1 alone can drive non-sensory cells towards an immature sensory hair cell phenotype in the mature cochlea, this does not result in functional improvements after aminoglycoside-induced deafness.
Full Text Available Atonal homolog1 (Atoh1 is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO mouse line using Tg(Atoh1-cre, in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to "self-terminate" its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.
Gentile, Pietro; Scioli, Maria G; Bielli, Alessandra; Orlandi, Augusto; Cervelli, Valerio
Hair follicles are known to contain a well-characterized niche for adult stem cells: the bulge, which contains epithelial and melanocytic stem cells. Stem cells in the hair bulge, a clearly demarcated structure within the lower permanent portion of hair follicles, can generate the interfollicular epidermis, hair follicle structures, and sebaceous glands. The bulge epithelial stem cells can also reconstitute in an artificial in vivo system to a new hair follicle. In this study, we have developed a new method to isolate human adult stem cells by mechanical centrifugation of punch biopsy from human hair follicles without culture condition. Here, we used human follicle stem cells (HFSCs), to improve the hair density in 11 patients (38 to 61 years old) affected by AGA in stage 3-5 as determined by the Norwood-Hamilton classification scale. The primary outcomes were microscopic identification and counting of HFSCs. The secondary outcomes were clinical preliminary results and safety and feasibility in HFSCs-treated scalp. Each scalp tissue suspension contained about 3,728.5±664.5 cells. The percentage of hair follicle-derived mesenchymal stem cells CD44+ [from dermal papilla (DP)] was about 5%+0.7% whereas the percentage of hair follicle epithelial stem cells CD200+ (from the bulge) was about 2.6%+0.3%. In total, 23 weeks after the last treatment with HFSCs mean hair count and hair density increases over baseline values. In particular, a 29%±5% increase in hair density for the treated area and less than a 1% increase in hair density for the placebo area. We have shown that the isolated cells are capable to improve the hair density in patients affected by androgenetic alopecia (AGA). These cells appear to be located in the bulge area of human.
Smith, Michael E
Exposure to intense sound or ototoxic chemicals can damage the auditory hair cells of vertebrates, resulting in hearing loss. Although the relationship between such hair cell damage and auditory function is fairly established for terrestrial vertebrates, there are limited data available to understand this relationship in fishes. Although investigators have measured either the morphological damage of the inner ear or the functional deficits in the hearing of fishes, very few have directly measured both in an attempt to find a relationship between the two. Those studies that have examined both auditory hair cell damage in the inner ear and the resulting hearing loss in fishes are reviewed here. In general, there is a significant linear relationship between the number of hair cells lost and the severity of hearing threshold shifts, although this varies between species and different hair cell-damaging stimuli. After trauma to the fish ear, auditory hair cells are able to regenerate to control level densities. With this regeneration also comes a restoration of hearing. Thus there is also a significant relationship between hair cell recovery and hearing recovery in fishes.
Hoffman, Robert M
Nestin-expressing stem cells of the hair follicle, discovered by our laboratory, have been shown to be able to form outer-root sheaths of the follicle as well as neurons and many other non-follicle cell types. We have termed the nestin-expressing stem cells of the hair follicle as hair-follicle-associated pluripotent (HAP) stem cells. We have shown that the HAP stem cells from the hair follicle can effect the repair of peripheral nerve and spinal cord injury. The hair follicle stem cells differentiate into neuronal and glial cells after transplantation to the injured peripheral nerve and spinal cord, and enhance injury repair and locomotor recovery. When the excised hair follicle with its nerve stump was placed in Gelfoam(®) 3D histoculture, HAP stem cells grew and extended the hair follicle nerve which consisted of βIII-tubulin-positive fibers with F-actin expression at the tip. These findings indicate that βIII-tubulin-positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in HAP stem cells, which appeared to play a major role in its elongation and interaction with other nerves in 3D Gelfoam(®) histoculture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. These results suggest that a major function of the HAP stem cells in the hair follicle is for growth of the follicle sensory nerve. Recently, we have shown that HAP stem cells can differentiate into beating cardiac muscle cells. HAP stem cells have critical advantages for regenerative medicine over embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in that they are highly accessible from each patient, thereby eliminating immunological issues since they are autologous, require no genetic manipulation, are non-tumorigenic, and do not present ethical issues.
vanNetten, Sietse M.
The complex mechanical behaviour of a hair cell bundle appears to be a direct consequence of the gating forces on the individual transduction channels. The mechanical molecular interactions involved in transduction channel gating, therefore, also bear a reciprocal influence, via the hair bundles; on
The complex mechanical behaviour of a hair cell bundle appears to be a direct consequence of the gating forces on the individual transduction channels. The mechanical molecular interactions involved in transduction channel gating, therefore, also bear a reciprocal influence, via the hair bundles; on
Brugeaud, Aurore; Travo, Cécile; Demêmes, Danielle; Lenoir, Marc; Llorens, Jordi; Puel, Jean-Luc; Chabbert, Christian
International audience; In the rat utricle, synaptic contacts between hair cells and the nerve fibers arising from the vestibular primary neurons form during the first week after birth. During that period, the sodium-based excitability that characterizes neonate utricle sensory cells is switched off. To investigate whether the establishment of synaptic contacts was responsible for the modulation of the hair cell excitability, we used an organotypic culture of rat utricle in which the setting ...
Rah, Yoon Chan; Choi, June; Yoo, Myung Hoon; Yum, Gunhwee; Park, Saemi; Oh, Kyoung Ho; Lee, Seung Hoon; Kwon, Soon Young; Cho, Seung Hyun; Kim, Suhyun; Park, Hae-Chul
Ecabet sodium (ES) is currently applied to some clinical gastrointestinal disease primarily by the inhibition of the ROS production. In this study, the protective role of ES was evaluated against the neomycin-induced hair cell loss using zebrafish experimental animal model. Zebrafish larvae (5-7 dpf), were treated with each of the following concentrations of ES: 5, 10, 20, 40, and 80 μg/mL for 1 h, followed by 125 μM neomycin for 1h. The positive control group was established by 125 μM neomycin-only treatment (1h) and the negative control group with no additional chemicals was also established. Hair cells inside four neuromasts ( SO1, SO2, O1, OC1) were assessed using fluorescence microscopy (n = 10). Hair cell survival was calculated as the mean number of viable hair cells for each group. Apoptosis and mitochondrial damage were investigated using special staining (TUNEL and DASPEI assay, respectively), and compared among groups. Ultrastructural changes were evaluated using scanning electron microscopy. Pre-treatment group with ES increased the mean number of viable hair cells as a dose-dependent manner achieving almost same number of viable hair cells with 40 μM/ml ES treatment (12.98 ± 2.59 cells) comparing to that of the negative control group (14.15 ± 1.39 cells, p = 0.72) and significantly more number of viable hair cells than that of the positive control group (7.45 ± 0.91 cells, p neomycin treatment than the negative control group and significantly decreased down to 105% with the pre-treatment with 40 μM/ml ES (n = 40, p = 0.04). A significantly less number of TUNEL-positive cells (reflecting apoptosis, p neomycin-induced hair cell loss possibly by reducing apoptosis, mitochondrial damages, and the ROS generation.
Yu, Dongzhen; Ding, Dalian; Jiang, Haiyan; Stolzberg, Daniel; Salvi, Richard
Mefloquine is an effective and widely used anti-malarial drug; however, some clinical reports suggest that it can cause dizziness, balance, and vestibular disturbances. To determine if mefloquine might be toxic to the vestibular system, we applied mefloquine to organotypic cultures of the macula of the utricle from postnatal day 3 rats. The macula of the utricle was micro-dissected out as a flat surface preparation and cultured with 10, 50, 100, or 200 μM mefloquine for 24 h. Specimens were stained with TRITC-conjugated phalloidin to label the actin in hair cell stereocilia and TO-PRO-3 to visualize cell nuclei. Some utricles were also labeled with fluorogenic caspase-3, -8, or -9 indicators to evaluate the mechanism of programmed cell death. Mefloquine treatment caused a dose-dependent loss of utricular hair cells. Treatment with 10 μM caused a slight reduction, 50 μM caused a significant reduction, and 200 μM destroyed nearly all the hair cells. Hair cell nuclei in mefloquine-treated utricles were condensed and fragmented, morphological features of apoptosis. Mefloquine-treated utricles were positive for the extrinsic initiator caspase-8 and intrinsic initiator caspase-9 and downstream executioner caspase-3. These results indicate that mefloquine can induce significant hair cell degeneration in the postnatal rat utricle and that mefloquine-induced hair cell death is initiated by both caspase-8 and caspase-9.
Full Text Available Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing.
Mann, Scott E; Johnson, Matthew; Meredith, Frances L; Rennie, Katherine J
Significant ototoxicity limits the use of aminoglycoside (AG) antibiotics. Several mechanisms may contribute to the death of both auditory and vestibular hair cells. In this study the effects of gentamicin and neomycin on K(+) currents in mature and early postnatal type I vestibular hair cells (HCI) were tested directly. The whole-cell patch clamp technique was used to assess the effects of AG and KCNQ channel modulators on K(+) currents (IK) in HCI acutely isolated from gerbil semicircular canals. Extracellular neomycin (1 mM) rapidly reduced peak outward IK by 16 ± 4% (n = 9) in mature HCI (postnatal days, P, 25-66). Gentamicin (5 mM) reduced outward IK by 16 ± 3% (n = 8). A similar reduction in outward current was seen in immature HCI (P5-9) that lacked the low-voltage-activated component of IK observed in mature cells. Intracellular application of gentamicin and neomycin also reduced IK in mature HCI. Modulators of KCNQ channels were used to probe KCNQ channel involvement. The selective KCNQ antagonist XE991 did not reduce IK and the neomycin-induced reduction in IK was not reversed by the KCNQ agonist flupirtine. Application of intracellular poly-D-lysine to sequester PIP2 did not reduce IK. Application of the K(+) channel blocker 4-aminopyridine (4-AP) strongly reduced IK, and extracellular AG in the presence of 4-AP gave no further inhibition of IK. In summary, AG significantly reduce the 4-AP-sensitive IK in early postnatal and mature HCI. K(+) current inhibition differs from that seen in outer hair cells, since it does not appear to involve PIP2 sequestration or KCNQ channels.
The hair cell provides the transduction of mechanical vibrations in the balance and acoustic sense of all vertebrates that swim, walk, or fly. The current theory places hair cell transduction in a mechanically controlled ion channel. Although the theory of a mechanical input modulating the flow of ions through an ion pore has been a useful tool, it is falsified by experimental data in the literature and can be definitively falsified by a proposed experiment.
Muller, Mees; Heeck, Kier
Vertebrate semicircular canals (SCC) first appeared in the vertebrates (i.e. ancestral fish) over 600 million years ago. In SCC the principal mechanoreceptors are hair cells, which as compared to cochlear hair cells are distinctly longer (70 vs. 7 μm), 10 times more compliant to bending (44 vs. 500 nN/m), and have a 100-fold higher tip displacement threshold (mechanoreceptors of the SCC. PMID:27448330
Larouche, Danielle; Cuffley, Kristine; Paquet, Claudie; Germain, Lucie
The aim of this study was to evaluate whether tissue-engineered skin produced in vitro was able to sustain growth of hair follicles in vitro and after grafting. Different tissues were designed. Dissociated newborn mouse keratinocytes or newborn mouse hair buds (HBs) were added onto dermal constructs consisting of a tissue-engineered cell-derived matrix elaborated from either newborn mouse or adult human fibroblasts cultured with ascorbic acid. After 7-21 days of maturation at the air-liquid interface, no hair was noticed in vitro. Epidermal differentiation was observed in all tissue-engineered skin. However, human fibroblast-derived tissue-engineered dermis (hD) promoted a thicker epidermis than mouse fibroblast-derived tissue-engineered dermis (mD). In association with mD, HBs developed epithelial cyst-like inclusions presenting outer root sheath-like attributes. In contrast, epidermoid cyst-like inclusions lined by a stratified squamous epithelium were present in tissues composed of HBs and hD. After grafting, pilo-sebaceous units formed and hair grew in skin elaborated from HBs cultured 10-26 days submerged in culture medium in association with mD. However, the number of normal hair follicles decreased with longer culture time. This hair-forming capacity after grafting was not observed in tissues composed of hD overlaid with HBs. These results demonstrate that epithelial stem cells can be kept in vitro in a permissive tissue-engineered dermal environment without losing their potential to induce hair growth after grafting.
Full Text Available The LKB1 gene, which encodes a serine/threonine kinase, was discovered to play crucial roles in cell differentiation, proliferation, and the establishment of cell polarity. In our study, LKB1 conditional knockout mice (Atoh1-LKB1-/- mice were generated to investigate LKB1 function in the inner ear. Tests of auditory brainstem response and distortion product otoacoustic emissions revealed significant decreases in the hearing sensitivities of the Atoh1-LKB1-/- mice. In Atoh1-LKB1-/- mice, malformations of hair cell stereocilliary bundles were present as early as postnatal day 1 (P1, a time long before the maturation of the hair cell bundles. In addition, we also observed outer hair cell (OHC loss starting at P14. The impaired stereocilliary bundles occurred long before the presence of hair cell loss. Stereociliary cytoskeletal structure depends on the core actin-based cytoskeleton and several actin-binding proteins. By Western blot, we examined actin-binding proteins, specifically ERM (ezrin/radixin/moesin proteins involved in the regulation of the actin cytoskeleton of hair cell stereocilia. Our results revealed that the phosphorylation of ERM proteins (pERM was significantly decreased in mutant mice. Thus, we propose that the decreased pERM may be a key factor for the impaired stereocillia function, and the damaged stereocillia may induce hair cell loss and hearing impairments. Taken together, our data indicates that LKB1 is required for the development and maintenance of stereocilia in the inner ear.
van Netten, SM; Kros, CJ
We quantified the molecular energies and forces involved in opening and closing of mechanoelectrical transducer channels in hair cells using a novel generally applicable method. It relies on a thermodynamic description of the free energy of an ion channel in terms of its open probability. The
Bryan D Monesson-Olson
Full Text Available Vertebrate hair cells are responsible for the high fidelity encoding of mechanical stimuli into trains of action potentials (spikes in afferent neurons. Here, we generated a transgenic zebrafish line expressing Channelrhodopsin-2 (ChR2 under the control of the hair-cell specific myo6b promoter, in order to examine the role of the mechanoelectrical transduction (MET channel in sensory encoding in afferent neurons. We performed in vivo recordings from afferent neurons of the zebrafish lateral line while activating hair cells with either mechanical stimuli from a waterjet or optical stimuli from flashes of ∼470-nm light. Comparison of the patterns of encoded spikes during 100-ms stimuli revealed no difference in mean first spike latency between the two modes of activation. However, there was a significant increase in the variability of first spike latency during optical stimulation as well as an increase in the mean number of spikes per stimulus. Next, we compared encoding of spikes during hair-cell stimulation at 10, 20, and 40-Hz. Consistent with the increased variability of first spike latency, we saw a significant decrease in the vector strength of phase-locked spiking during optical stimulation. These in vivo results support a physiological role for the MET channel in the high fidelity of first spike latency seen during encoding of mechanical sensory stimuli. Finally, we examined whether remote activation of hair cells via ChR2 activation was sufficient to elicit escape responses in free-swimming larvae. In transgenic larvae, 100-ms flashes of ∼470-nm light resulted in escape responses that occurred concomitantly with field recordings indicating Mauthner cell activity. Altogether, the myo6b:ChR2 transgenic line provides a platform to investigate hair-cell function and sensory encoding, hair-cell sensory input to the Mauthner cell, and the ability to remotely evoke behavior in free-swimming zebrafish.
Burns, Joseph C; Burns, Joseph; Christophel, J Jared; Collado, Maria Sol; Magnus, Christopher; Carfrae, Matthew; Corwin, Jeffrey T
Debilitating hearing and balance deficits often arise through damage to the inner ear's hair cells. For humans and other mammals, such deficits are permanent, but nonmammalian vertebrates can quickly recover hearing and balance through their innate capacity to regenerate hair cells. The biological basis for this difference has remained unknown, but recent investigations in wounded balance epithelia have shown that proliferation follows cellular spreading at sites of injury. As mammalian ears mature during the first weeks after birth, the capacity for spreading and proliferation declines sharply. In seeking the basis for those declines, we investigated the circumferential bands of F-actin that bracket the apical junctions between supporting cells in the gravity-sensitive utricle. We found that those bands grow much thicker as mice and humans mature postnatally, whereas their counterparts in chickens remain thin from hatching through adulthood. When we cultured utricular epithelia from chickens, we found that cellular spreading and proliferation both continued at high levels, even in the epithelia from adults. In contrast, the substantial reinforcement of the circumferential F-actin bands in mammals coincides with the steep declines in cell spreading and production established in earlier experiments. We propose that the presence of thin F-actin bands at the junctions between avian supporting cells may contribute to the lifelong persistence of their capacity for shape change, cell proliferation, and hair cell replacement and that the postnatal reinforcement of the F-actin bands in maturing humans and other mammals may have an important role in limiting hair cell regeneration. (c) 2008 Wiley-Liss, Inc.
Basler, Tina; Brumshagen, Christina; Beineke, Andreas; Goethe, Ralph; Bäumer, Wolfgang
Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease, a chronic, granulomatous enteritis of ruminants. Dendritic cells (DC) of the gut are ideally placed to combat invading mycobacteria; however, little is known about their interaction with MAP. Here, we investigated the interaction of MAP and the closely related M. avium ssp. avium (MAA) with murine DC and the effect of infected macrophages on DC maturation. The infection of DC with MAP or MAA induced DC maturation, which differed to that of LPS as maturation was accompanied by higher production of IL-10 and lower production of IL-12. Treatment of maturing DC with supernatants from mycobacteria-infected macrophages resulted in impaired DC maturation, leading to a semi-mature, tolerogenic DC phenotype expressing low levels of MHCII, CD86 and TNF-α after LPS stimulation. Though the cells were not completely differentiated they responded with an increased IL-10 and a decreased IL-12 production. Using recombinant cytokines we provide evidence that the semi-mature DC phenotype results from a combination of secreted cytokines and released antigenic mycobacterial components of the infected macrophage. Our results indicate that MAP and MAA are able to subvert DC function directly by infecting and indirectly via the milieu created by infected macrophages.
Sienknecht, Ulrike J
Phylogenetically and ontogenetically, vertebrate development led to the generation of several inner ear sensory organs. During embryogenesis, cell fate specification determines whether each progenitor cell differentiates into a sensory hair cell or a supporting cell within the common sensory primordium. Finally, all sensory epithelia of the inner ear consist of a hair cell/supporting cell mosaic, albeit with anatomical differences depending on the sensory organ type. Hair cells develop a polarized bundle of stereovilli that is of functional importance for mechanotransduction. After initiating stereovillar development, hair cells align their bundles in a coordinated fashion, generating a characteristic hair cell orientation pattern, a process referred to as planar cell polarity (PCP). The pathway that controls PCP in the inner ear needs both to establish the development of a polarized morphology of the stereovillar bundle of the hair cell and to organize a systematic hair cell alignment. Because the hair cell orientation patterns of the various inner ear organs and vertebrate species differ fundamentally, it becomes apparent that in vertebrates, different aspects of PCP need to be independently controlled. In spite of important progress recently gained in the field of PCP research, we still need to identify the mechanisms (1) that initiate molecular asymmetries in cells, (2) that guide the transmission of polarity information from cell to cell, and (3) that consistently translate such polarity information into morphological asymmetries of hair cells.
Full Text Available It is well known that the production of free radicals is associated with sensory cell death induced by an aminoglycoside. Many researchers have reported that antioxidant reagents protect sensory cells in the inner ear, and coenzyme Q10 (CoQ10 is an antioxidant that is consumed as a health food in many countries. The purpose of this study was to investigate the role of CoQ10 in mammalian vestibular hair cell death induced by aminoglycoside. Cultured utricles of CBA/CaN mice were divided into three groups (control group, neomycin group, and neomycin + CoQ10 group. In the neomycin group, utricles were cultured with neomycin (1 mM to induce hair cell death. In the neomycin + CoQ10 group, utricles were cultured with neomycin and water-soluble CoQ10 (30-0.3 µM. Twenty-four hours after exposure to neomycin, the cultured tissues were fixed, and vestibular hair cells were labeled using an anti-calmodulin antibody. Significantly more hair cells survived in the neomycin + CoQ10 group than in the neomycin group. These data indicate that CoQ10 protects sensory hair cells against neomycin-induced death in the mammalian vestibular epithelium; therefore, CoQ10 may be useful as a protective drug in the inner ear.
Stenn, K S; Combates, N J; Eilertsen, K J; Gordon, J S; Pardinas, J R; Parimoo, S; Prouty, S M
Research in hair biology has embarked in the pursuit for molecules that control hair growth. Many molecules already have been associated with the controls of hair patterning, hair maturation, and hair cycling and differentiation. Knowing how these molecules work gives us the tools for understanding and treating patients with hair disorders.
Fu, Mingyu; Chen, Mengzi; Yang, Xueying
The cochlea converts sound vibration into electrical impulses and amplifies the low-level sound signal. Urethane, a widely used anesthetic in animal research, has been shown to reduce the neural responses to auditory stimuli. However, the effects of urethane on cochlea, especially on the function of outer hair cells, remain largely unknown. In the present study, we compared the cochlear microphonic responses between awake and urethane-anesthetized rats. The results revealed that the amplitude of the cochlear microphonic was decreased by urethane, resulting in an increase in the threshold at all of the sound frequencies examined. To deduce the possible mechanism underlying the urethane-induced decrease in cochlear sensitivity, we examined the electrical response properties of isolated outer hair cells using whole-cell patch-clamp recording. We found that urethane hyperpolarizes the outer hair cell membrane potential in a dose-dependent manner and elicits larger outward current. This urethane-induced outward current was blocked by strychnine, an antagonist of the α9 subunit of the nicotinic acetylcholine receptor. Meanwhile, the function of the outer hair cell motor protein, prestin, was not affected. These results suggest that urethane anesthesia is expected to decrease the responses of outer hair cells, whereas the frequency selectivity of cochlea remains unchanged. PMID:28050287
Full Text Available The cochlea converts sound vibration into electrical impulses and amplifies the low-level sound signal. Urethane, a widely used anesthetic in animal research, has been shown to reduce the neural responses to auditory stimuli. However, the effects of urethane on cochlea, especially on the function of outer hair cells, remain largely unknown. In the present study, we compared the cochlear microphonic responses between awake and urethane-anesthetized rats. The results revealed that the amplitude of the cochlear microphonic was decreased by urethane, resulting in an increase in the threshold at all of the sound frequencies examined. To deduce the possible mechanism underlying the urethane-induced decrease in cochlear sensitivity, we examined the electrical response properties of isolated outer hair cells using whole-cell patch-clamp recording. We found that urethane hyperpolarizes the outer hair cell membrane potential in a dose-dependent manner and elicits larger outward current. This urethane-induced outward current was blocked by strychnine, an antagonist of the α9 subunit of the nicotinic acetylcholine receptor. Meanwhile, the function of the outer hair cell motor protein, prestin, was not affected. These results suggest that urethane anesthesia is expected to decrease the responses of outer hair cells, whereas the frequency selectivity of cochlea remains unchanged.
Full Text Available Background. Emerging research revealed the essential role of mitochondria in regulating stem/progenitor cell differentiation of neural progenitor cells, mesenchymal stem cells and other stem cells through reactive oxygen species (ROS, Notch or other signaling pathway. Inhibition of mitochondrial protein synthesis results in hair loss upon injury. However, alteration of mitochondrial morphology and metabolic function during hair follicle stem cells (HFSCs differentiation and how they affect hair regeneration has not been elaborated upon. Methods. We compared the difference in mitochondrial morphology and activity between telogen bulge cells and anagen matrix cells. Expression levels of mitochondrial ROS and superoxide dismutase 2 (SOD2 were measured to evaluate redox balance. In addition, the level of pyruvate dehydrogenase kinase (PDK and pyruvate dehydrogenase (PDH were estimated to present the change in energetic metabolism during differentiation. To explore the effect of the mitochondrial metabolism on regulating hair regeneration, hair growth was observed after application of a mitochondrial respiratory inhibitor upon hair plucking. Results. During HFSCs differentiation, mitochondria became elongated with more abundant organized cristae and showed higher activity in differentiated cells. SOD2 was enhanced for redox balance with relatively stable ROS levels in differentiated cells. PDK increased in HFSCs while differentiated cells showed enhanced PDH, indicating that respiration switched from glycolysis to oxidative phosphorylation during differentiation. Inhibiting mitochondrial respiration in differentiated hair follicle cells upon hair plucking repressed hair regeneration in vivo. Conclusions. Upon HFSCs differentiation, mitochondria are elongated with more abundant cristae and show higher activity, accompanying with activated aerobic respiration in differentiated cells for higher energy supply. Also, dysfunction of mitochondrial
Burns, Joseph C; On, Doan; Baker, Wendy; Collado, M Sol; Corwin, Jeffrey T
Many non-mammalian vertebrates produce hair cells throughout life and recover from hearing and balance deficits through regeneration. In contrast, embryonic production of hair cells declines sharply in mammals where deficits from hair cell losses are typically permanent. Hair cell density estimates recently suggested that the vestibular organs of mice continue to add hair cells after birth, so we undertook comprehensive counting in murine utricles at different ages. The counts show that 51% of the hair cells in adults arise during the 2 weeks after birth. Immature hair cells are most common near the neonatal macula's peripheral edge and striola, where anti-Ki-67 labels cycling nuclei in zones that appear to contain niches for supporting-cell-like stem cells. In vivo lineage tracing in a novel reporter mouse where tamoxifen-inducible supporting cell-specific Cre expression switched tdTomato fluorescence to eGFP fluorescence showed that proteolipid-protein-1-expressing supporting cells are an important source of the new hair cells. To assess the contributions of postnatal cell divisions, we gave mice an injection of BrdU or EdU on the day of birth. The labels were restricted to supporting cells 1 day later, but by 12 days, 31% of the labeled nuclei were in myosin-VIIA-positive hair cells. Thus, hair cell populations in neonatal mouse utricles grow appreciably through two processes: the progressive differentiation of cells generated before birth and the differentiation of new cells arising from divisions of progenitors that progress through S phase soon after birth. Subsequent declines in these processes coincide with maturational changes that appear unique to mammalian supporting cells.
Jiang, Lingling; Jin, Ran; Xu, Jincao; Ji, Yubin; Zhang, Meiguang; Zhang, Xuebo; Zhang, Xinwen; Han, Zhongming; Zeng, Shaoju
Hair cells in posthatch chickens regenerate spontaneously through mitosis or the transdifferentiation of supporting cells in response to antibiotic injury. However, how embryonic chicken cochleae respond to antibiotic treatment remains unknown. This study is the first to indicate that unlike hair cells in posthatch chickens, the auditory epithelium was free from antibiotic injury (25-250 mg gentamicin/kg) in embryonic chickens, although FITC-conjugated gentamicin actually reached embryonic hair cells. Next, we examined and counted the cells and performed labeling for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) (triple or double labeling) in the injured cochlea ducts after gentamicin treatment at 2 h (h), 15 h, 24 h, 2 days (d), 3 d and 7 d after BrdU treatment in posthatch chickens. Our results indicated that following gentamicin administration, proliferating cells (BrdU+) were labeled for Atoh1/Math1 in the damaged areas 3d after gentamicin administration, whereas hair cells (PV+) renewed through mitosis (BrdU+) or direct transdifferentiation (BrdU-) were evident only after 5 d of gentamicin administration. In addition, Sox2 expression was up-regulated in triggered supporting cells at an early stage of regeneration, but stopped at the advent of mature hair cells. Our study also indicated that p27(kip1) was expressed in both hair cells and supporting cells but was down-regulated in a subgroup of the supporting cells that gave rise to hair cells. These data and the obtained dynamic changes of the cells labeled for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) are useful for understanding supporting cell behaviors and their fate specification during hair cell regeneration.
Full Text Available Shortage of red blood cells (RBCs, erythrocytes can have potentially life-threatening consequences for rare or unusual blood type patients with massive blood loss resulting from various conditions. Erythrocytes have been derived from human pluripotent stem cells (PSCs, but the risk of potential tumorigenicity cannot be ignored, and a majority of these cells produced from PSCs express embryonic ε- and fetal γ-globins with little or no adult β-globin and remain nucleated. Here we report a method to generate erythrocytes from human hair follicle mesenchymal stem cells (hHFMSCs by enforcing OCT4 gene expression and cytokine stimulation. Cells generated from hHFMSCs expressed mainly the adult β-globin chain with minimum level of the fetal γ-globin chain. Furthermore, these cells also underwent multiple maturation events and formed enucleated erythrocytes with a biconcave disc shape. Gene expression analyses showed that OCT4 regulated the expression of genes associated with both pluripotency and erythroid development during hHFMSC transdifferentiation toward erythroid cells. These findings show that mature erythrocytes can be generated from adult somatic cells, which may serve as an alternative source of RBCs for potential autologous transfusion.
Muller, Mees; Heeck, Kier; Elemans, Coen P H
nN/m), and have a 100-fold higher tip displacement threshold (cells where the bundle is approximated as a stiff, cylindrical elastic rod subject to friction and thermal agitation. Our models suggest that the above......Vertebrate semicircular canals (SCC) first appeared in the vertebrates (i.e. ancestral fish) over 600 million years ago. In SCC the principal mechanoreceptors are hair cells, which as compared to cochlear hair cells are distinctly longer (70 vs. 7 μm), 10 times more compliant to bending (44 vs. 500...... differences aid SCC hair cells in circumventing the masking effects of Brownian motion noise of about 70 nm, and thereby permit transduction of very low frequency (
Schallreuter, K U; Beazley, W D; Hibberts, N A; Tobin, D J; Paus, R; Wood, J M
Human dermal papilla cells (HDPC) express mRNA for the key enzymes for de novo synthesis/recycling and regulation of the pterin (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4). HDPC had significantly higher enzyme activities and 6BH4 levels in a comparative study with dermal fibroblasts, epidermal melanocytes, and keratinocytes under in vitro conditions. In addition, a significantly more rapid uptake of 14C-L-phenylalanine was demonstrated in HDPC compared with fibroblasts, whereas the differences in turnover to L-tyrosine were insignificant, suggesting a pooling of L-phenylalanine in HDPC. These results suggested that HDPC driven 6BH4 synthesis could be of major functional importance in the hair cycle. In order to follow this hypothesis in vivo, expression of enzyme activities and levels of the produced cofactor during the synchronized hair cycle were determined employing the murine model C57BL/6. These data revealed a significantly increased de novo synthesis for 6BH4 via GTP-cyclohydrolase I concomitant with high levels of 6BH4, and the induction of phenylalanine hydroxylase activities during the telogen/early anagen stage (days 0-1). Pterin levels and enzyme activities fall on day 3 and plateau during the rest of the entire cycle. In addition, thioredoxin reductase and glutathione reductase activities were measured, where the latter enzyme remained constant but thioredoxin reductase activities showed a biphasic behavior. The first peak coincided with the induction of 6BH4 de novo synthesis at the beginning of the hair cycle. The second peak was observed at mid-anagen, when melanogenesis takes place. Taken together, our results show the presence of autocrine pterin synthesis/recycling in human hair follicle cells under in vitro conditions, and a possible role for 6BH4 in the synchronized murine hair cycle.
Full Text Available Vertebrate semicircular canals (SCC first appeared in the vertebrates (i.e. ancestral fish over 600 million years ago. In SCC the principal mechanoreceptors are hair cells, which as compared to cochlear hair cells are distinctly longer (70 vs. 7 μm, 10 times more compliant to bending (44 vs. 500 nN/m, and have a 100-fold higher tip displacement threshold (< 10 μm vs. <400 nm. We have developed biomechanical models of vertebrate hair cells where the bundle is approximated as a stiff, cylindrical elastic rod subject to friction and thermal agitation. Our models suggest that the above differences aid SCC hair cells in circumventing the masking effects of Brownian motion noise of about 70 nm, and thereby permit transduction of very low frequency (<10 Hz signals. We observe that very low frequency mechanoreception requires increased stimulus amplitude, and argue that this is adaptive to circumvent Brownian motion overload at the hair bundles. We suggest that the selective advantage of detecting such low frequency stimuli may have favoured the evolution of large guiding structures such as semicircular canals and otoliths to overcome Brownian Motion noise at the level of the mechanoreceptors of the SCC.
Objective To study the recovery of the outer hair cells in the bat cochlea after gentamicin exposure. Methods Bats were injected with a daily dose of gentamicin for 15 consecutive days and bromodeoxyuridine (BrdU) was given from day 16 to day 40 of this recovery phase. Hearing was assessed by overt acoustic behavior and auditory brainstem responses analysis, which was performed one day prior to the first injection and a day after the last injection (day 16). On day 40 animals were sacrificed for detection o...
Objective: To establish the method of constructing skin-equivalents (SE) using hair follicle stem cells(HFSC).Methods: K19 positive cells derived from hair were cultivated using serum-free medium KGM and seeded on dermal equivalents (DE).After the culture between the air-liquid interface for 14 days, SE were harvested and used for evaluation. Results: K19 positive cells chosen as HFSC were located in bulge of out root sheet in hair follicle. Cultivated HFSC could build a fully developed, multi-layered epidermis on the basis of DE, resembling the skin structure. Conclusion: HFSC located in out root sheet can differentiate into keratinocyte in vitro and be used for SE construction.
Abbas, Leila; Rivolta, Marcelo N.
The Mongolian gerbil, Meriones unguiculatus, has been widely employed as a model for studies of the inner ear. In spite of its established use for auditory research, no robust protocols to induce ototoxic hair cell damage have been developed for this species. In this paper, we demonstrate the development of an aminoglycoside-induced model of hair cell loss, using kanamycin potentiated by the loop diuretic furosemide. Interestingly, we show that the gerbil is relatively insensitive to gentamicin compared to kanamycin, and that bumetanide is ineffective in potentiating the ototoxicity of the drug. We also examine the pathology of the spiral ganglion after chronic, long-term hair cell damage. Remarkably, there is little or no neuronal loss following the ototoxic insult, even at 8 months post-damage. This is similar to the situation often seen in the human, where functioning neurons can persist even decades after hair cell loss, contrasting with the rapid, secondary degeneration found in rats, mice and other small mammals. We propose that the combination of these factors makes the gerbil a good model for ototoxic damage by induced hair cell loss. PMID:25783988
Lee, Sang Goo; Huang, Mingqian; Obholzer, Nikolaus D; Sun, Shan; Li, Wenyan; Petrillo, Marco; Dai, Pu; Zhou, Yi; Cotanche, Douglas A; Megason, Sean G; Li, Huawei; Chen, Zheng-Yi
Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration.
Sang Goo Lee
Full Text Available Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration.
Obholzer, Nikolaus D.; Sun, Shan; Li, Wenyan; Petrillo, Marco; Dai, Pu; Zhou, Yi; Cotanche, Douglas A.; Megason, Sean G.; Li, Huawei; Chen, Zheng-Yi
Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration. PMID:27351484
Grillet, Nicolas; Xiong, Wei; Reynolds, Anna; Kazmierczak, Piotr; Sato, Takashi; Lillo, Concepcion; Dumont, Rachel A.; Hintermann, Edith; Sczaniecka, Anna; Schwander, Martin; Williams, David; Kachar, Bechara; Gillespie, Peter G.; Müller, Ulrich
In hair cells, mechanotransduction channels are gated by tip links, the extracellular filaments that consist of cadherin 23 (CDH23) and protocadherin 15 (PCDH15) and connect the stereocilia of each hair cell. However, which molecules mediate cadherin function at tip links is not known. Here we show that the PDZ-domain protein harmonin is a component of the upper tip-link density (UTLD), where CDH23 inserts into the stereociliary membrane. Harmonin domains that mediate interactions with CDH23 and F-actin control harmonin localization in stereocilia and are necessary for normal hearing. In mice expressing a mutant harmonin protein that prevents UTLD formation, the sensitivity of hair bundles to mechanical stimulation is reduced. We conclude that harmonin is a UTLD component and contributes to establishing the sensitivity of mechanotransduction channels to displacement. PMID:19447093
Michael S. Detamore
Full Text Available Sensory neural hearing loss and vestibular dysfunction have become the most common forms of sensory defects, affecting millions of people worldwide. Developing effective therapies to restore hearing loss is challenging, owing to the limited regenerative capacity of the inner ear hair cells. With recent advances in understanding the developmental biology of mammalian and non-mammalian hair cells a variety of strategies have emerged to restore lost hair cells are being developed. Two predominant strategies have developed to restore hair cells: transfer of genes responsible for hair cell genesis and replacement of missing cells via transfer of stem cells. In this review article, we evaluate the use of several genes involved in hair cell regeneration, the advantages and disadvantages of the different viral vectors employed in inner ear gene delivery and the insights gained from the use of embryonic, adult and induced pluripotent stem cells in generating inner ear hair cells. Understanding the role of genes, vectors and stem cells in therapeutic strategies led us to explore potential solutions to overcome the limitations associated with their use in hair cell regeneration.
Levic, Snezana; Dulon, Didier
During development, synaptic exocytosis by cochlear hair cells is first initiated by patterned spontaneous Ca(2+) spikes and, at the onset of hearing, by sound-driven graded depolarizing potentials. The molecular reorganization occurring in the hair cell synaptic machinery during this developmental transition still remains elusive. We characterized the changes in biophysical properties of voltage-gated Ca(2+) currents and exocytosis in developing auditory hair cells of a precocial animal, the domestic chick. We found that immature chick hair cells (embryonic days 10-12) use two types of Ca(2+) currents to control exocytosis: low-voltage-activating, rapidly inactivating (mibefradil sensitive) T-type Ca(2+) currents and high-voltage-activating, noninactivating (nifedipine sensitive) L-type currents. Exocytosis evoked by T-type Ca(2+) current displayed a fast release component (RRP) but lacked the slow sustained release component (SRP), suggesting an inefficient recruitment of distant synaptic vesicles by this transient Ca(2+) current. With maturation, the participation of L-type Ca(2+) currents to exocytosis largely increased, inducing a highly Ca(2+) efficient recruitment of an RRP and an SRP component. Notably, L-type-driven exocytosis in immature hair cells displayed higher Ca(2+) efficiency when triggered by prerecorded native action potentials than by voltage steps, whereas similar efficiency for both protocols was found in mature hair cells. This difference likely reflects a tighter coupling between release sites and Ca(2+) channels in mature hair cells. Overall, our results suggest that the temporal characteristics of Ca(2+) entry through T-type and L-type Ca(2+) channels greatly influence synaptic release by hair cells during cochlear development.
Lewis, Julian; Davies, Alex
Sensory hair cells in the ear and lateral line have an asymmetrical hair-bundle structure, essential for their function as directional mechanotransducers. We examine four questions: (1) how does the planar asymmetry of the individual hair cell originate? (2) How are the orientations of neighboring hair cells coordinated? (3) How is the orientation of a group of hair cells controlled in relation to the ear as a whole? (4) How does the initial cell asymmetry lead to creation of the asymmetrical hair bundle? Studies of the development of hairs and bristles in Drosophila, combined with genetic data from vertebrates, suggest that the answer to questions (1) and (2) lies in asymmetries that develop at the cell cortex and at cell-cell junctions, generated by products of a set of primary planar cell polarity genes, including the transmembrane receptor Frizzled. A separate and largely independent mechanism controls asymmmetric allocation of cell fate determinants such as Numb at mitosis, in Drosophila and possibly in the ear also. Little is known about long-range signals that might orient hair cells globally in the ear, but progress has been made in identifying a set of genes responsible for read-out of the primary polarity specification. These genes, in flies and vertebrates, provide a link to assembly of the polarized cytoskeleton; myosin VIIA appears to belong in this group. The mechanism creating the staircase pattern of stereocilium lengths is unknown, but could involve regulation of stereocilium growth by Ca(2+) ions entering via transduction channels.
Liu Hui; Zhu Hongliang; Li Shengli; Yao Xiaobao; Wang Xiaoxia
Objective To establish adult rat auditory epithelial cell culture and try to find precursor cells of auditory hair cells in vitro. Methods With refinement of culture media and techniques, cochlear sensory epithelial cells of adult rat were cultured. Immunocytochemistry and Bromodeoxyuridine (BrdU)labeling were used to detect properties and mitotic status of cultured cells. Results The cultured auditory epithelial cells showed a large, flat epithelial morphotype and expressed F-actin and cytokeratin, a subset of cells generated from auditory epithelium were labeled by calretinin, a specific marker of early hair cell. Conclusion Adult rat auditory epithelium can be induced to generate hair cell-like cells by nature culture, this phenomenon suggests that progenitor cells may exist in rat cochlea and they may give birth to new hair cells. Whether these progenitor cells are tissue specific stem cells is still need more study.
Maurer, M; Paus, R; Czarnetzki, B M
While the central role of mast cells (MC) in allergy and inflammation is well-appreciated, much less is known about their physiological functions. The impressive battery of potent growth modulatory MC products, and increasing evidence of MC involvement in hyperproliferative and fibrotic disorders suggest that tissue remodelling may be one of those, namely in the skin. Here, we delineate why this may best be studied by analysing the potential role of MC in hair growth regulation. On the background of numerous, yet widely under-appreciated hints from the older literature, we summarize and discuss our recent observations from the C57BL/6 mouse model for hair research which support the concept that MC are functionally important modulators of hair follicle cycling, specifically during anagen development. This invites to exploit the murine hair cycle as a model for dissecting the physiological growth modulatory functions of MC and encourages the exploration of MC-targeting pharmaceutical strategies for the treatment of hair growth disorders.
Full Text Available BACKGROUND: Hair cells in the auditory, vestibular, and lateral-line systems respond to mechanical stimulation and transmit information to afferent nerve fibers. The sensitivity of mechanoelectrical transduction is modulated by the efferent pathway, whose activity usually reduces the responsiveness of hair cells. The basis of this effect remains unknown. METHODOLOGY AND PRINCIPAL FINDINGS: We employed immunocytological, electrophysiological, and micromechanical approaches to characterize the anatomy of efferent innervation and the effect of efferent activity on the electrical and mechanical properties of hair cells in the bullfrog's sacculus. We found that efferent fibers form extensive synaptic terminals on all macular and extramacular hair cells. Macular hair cells expressing the Ca(2+-buffering protein calretinin contain half as many synaptic ribbons and are innervated by twice as many efferent terminals as calretinin-negative hair cells. Efferent activity elicits inhibitory postsynaptic potentials in hair cells and thus inhibits their electrical resonance. In hair cells that exhibit spiking activity, efferent stimulation suppresses the generation of action potentials. Finally, efferent activity triggers a displacement of the hair bundle's resting position. CONCLUSIONS AND SIGNIFICANCE: The hair cells of the bullfrog's sacculus receive a rich efferent innervation with the heaviest projection to calretinin-containing cells. Stimulation of efferent axons desensitizes the hair cells and suppresses their spiking activity. Although efferent activation influences mechanoelectrical transduction, the mechanical effects on hair bundles are inconsistent.
Brownell, William E.; Gummer, Anthony W.
A discussion moderated by the authors on the topic "Outer Hair Cells and Prestin" was held on 18 July 2011 at the 11th International Mechanics of Hearing Workshop in Williamstown, Massachusetts. The paper provides an edited transcript of the session.
Karavitaki, K. Domenica; Ricci, Anthony J.
A discussion moderated by the authors on the topic "Hair Cells: Bundles, Tuning, Transduction" was held on 17 July 2011 at the 11th International Mechanics of Hearing Workshop in Williamstown, Massachusetts. The paper provides an edited transcript of the session.
Sadeghi, Soroush G.; Pyott, Sonja J.; Yu, Zhou; Glowatzki, Elisabeth
In the vestibular periphery a unique postsynaptic terminal, the calyx, completely covers the basolateral walls of type I hair cells and receives input from multiple ribbon synapses. To date, the functional role of this specialized synapse remains elusive. There is limited data supporting glutamaterg
Full Text Available Behavioural anomalies suggesting an inner ear disorder were observed in a colony of transgenic mice. Affected animals were profoundly deaf. Severe hair bundle defects were identified in all outer and inner hair cells (OHC, IHC in the cochlea and in hair cells of vestibular macular organs, but hair cells in cristae were essentially unaffected. Evidence suggested the disorder was likely due to gene disruption by a randomly inserted transgene construct. Whole-genome sequencing identified interruption of the SorCS2 (Sortilin-related VPS-10 domain containing protein locus. Real-time-qPCR demonstrated disrupted expression of SorCS2 RNA in cochlear tissue from affected mice and this was confirmed by SorCS2 immuno-labelling. In all affected hair cells, stereocilia were shorter than normal, but abnormalities of bundle morphology and organisation differed between hair cell types. Bundles on OHC were grossly misshapen with significantly fewer stereocilia than normal. However, stereocilia were organised in rows of increasing height. Bundles on IHC contained significantly more stereocilia than normal with some longer stereocilia towards the centre, or with minimal height differentials. In early postnatal mice, kinocilia (primary cilia of IHC and of OHC were initially located towards the lateral edge of the hair cell surface but often became surrounded by stereocilia as bundle shape and apical surface contour changed. In macular organs the kinocilium was positioned in the centre of the cell surface throughout maturation. There was disruption of the signalling pathway controlling intrinsic hair cell apical asymmetry. LGN and Gαi3 were largely absent, and atypical Protein Kinase C (aPKC lost its asymmetric distribution. The results suggest that SorCS2 plays a role upstream of the intrinsic polarity pathway and that there are differences between hair cell types in the deployment of the machinery that generates a precisely organised hair bundle.
Taylor, Ruth R.; Lovett, Michael; Jagger, Daniel J.
Behavioural anomalies suggesting an inner ear disorder were observed in a colony of transgenic mice. Affected animals were profoundly deaf. Severe hair bundle defects were identified in all outer and inner hair cells (OHC, IHC) in the cochlea and in hair cells of vestibular macular organs, but hair cells in cristae were essentially unaffected. Evidence suggested the disorder was likely due to gene disruption by a randomly inserted transgene construct. Whole-genome sequencing identified interruption of the SorCS2 (Sortilin-related VPS-10 domain containing protein) locus. Real-time-qPCR demonstrated disrupted expression of SorCS2 RNA in cochlear tissue from affected mice and this was confirmed by SorCS2 immuno-labelling. In all affected hair cells, stereocilia were shorter than normal, but abnormalities of bundle morphology and organisation differed between hair cell types. Bundles on OHC were grossly misshapen with significantly fewer stereocilia than normal. However, stereocilia were organised in rows of increasing height. Bundles on IHC contained significantly more stereocilia than normal with some longer stereocilia towards the centre, or with minimal height differentials. In early postnatal mice, kinocilia (primary cilia) of IHC and of OHC were initially located towards the lateral edge of the hair cell surface but often became surrounded by stereocilia as bundle shape and apical surface contour changed. In macular organs the kinocilium was positioned in the centre of the cell surface throughout maturation. There was disruption of the signalling pathway controlling intrinsic hair cell apical asymmetry. LGN and Gαi3 were largely absent, and atypical Protein Kinase C (aPKC) lost its asymmetric distribution. The results suggest that SorCS2 plays a role upstream of the intrinsic polarity pathway and that there are differences between hair cell types in the deployment of the machinery that generates a precisely organised hair bundle. PMID:28346477
Shirokova, Vera; Biggs, Leah C.; Jussila, Maria; Ohyama, Takahiro; Groves, Andrew K.; Mikkola, Marja L.
The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ. Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary hair germ marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary hair germ activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. PMID:26992132
Full Text Available Background & objectives: Adult stem cells are undifferentiated cells that replace dead or injured cells. There are adult stem cells in some regions of human tissues and hair follicle is one of the tissues that have adult stem cell source and these cells have an important role in hair life cycle. In this study, we investigated the isolation of hair follicle stem cells (HFSCs and expression of mesenchymal stem cell markers on the isolated cells. Methods : Human hair follicles obtained from men scalp tissue by micro punch technique. Hair follicles isolated and cultured in culture flasks in DMEM-F12 + FBS. After outgrowth of stem cells from hair bulges, they analyzed by flow cytometry for detection of stem cell markers. Results: 23 to 27 days after isolation and culture of HFSCs in uncoated cell culture flasks, cell surface markers expression studied by flow cytometry. Flow cytometric analysis showed 25.26% Stro-1, 50.85% CD90, 45.24% CD105, 61.20% CD44, 8.20% CD45, 11.86% CD146, 2.72% CD106, 7.21% CD166 and 26.74% CD19 expression in HFSCs. Conclusion: In this study, isolated stem cells significantly expressed some of the mesenchymal stem cell markers higher than other markers. These markers give certain characteristics to HFSCs, and introduce the cells as an alternative option for cell therapy, tissue engineering and regenerative medicine.
Crawford, A C; Fettiplace, R
1. Intracellular recordings were made from single cochlear hair cells in the isolated half-head of the turtle. The electrical responses of the cells were recorded under two conditions: (a) when the ear was stimulated with low-intensity tones of different frequencies and (b) when current steps were injected through the intracellular electrode. The aim of the experiments was to evaluate the extent to which the cochlea's frequency selectivity could be accounted for by the electrical properties of the hair cells.2. At low levels of acoustic stimulation, the amplitude of the hair cell's receptor potential was proportional to sound pressure. The linear tuning curve, which is defined as the sensitivity of the cell as a function of frequency when the cell is operating in its linear range, was measured for a number of hair cells with characteristic frequencies from 86 Hz to 425 Hz.3. A rectangular current passed into a hair cell elicited a membrane potential change consisting of a damped oscillation superimposed on a step. Small currents produced symmetrical oscillations at the beginning and end of the pulse. Larger currents increased the initial ringing frequency if depolarizing and decreased it if hyperpolarizing.4. For small currents the frequency of the oscillations and the quality factor (Q) of the electrical resonance derived from the decay of the oscillations were close to the characteristic frequency and Q of the hair-cell linear tuning curve obtained from sound presentations.5. The hair cell's membrane potential change to small-current pulses or low-intensity tone bursts could be largely described by representing the hair cell as a simple electrical resonator consisting of an inductance, resistor and capacitor.6. When step displacements of 29-250 nm were applied to a micropipette, placed just outside a hair cell in the basilar papilla, an initial periodic firing of impulses could be recorded from single fibres in the auditory nerve. Currents of up to 1 nA, injected
Plikus, Maksim V; Vollmers, Christopher; de la Cruz, Damon; Chaix, Amandine; Ramos, Raul; Panda, Satchidananda; Chuong, Cheng-Ming
Regenerative cycling of hair follicles offers an unique opportunity to explore the role of circadian clock in physiological tissue regeneration. We focused on the role of circadian clock in actively proliferating transient amplifying cells, as opposed to quiescent stem cells. We identified two key sites of peripheral circadian clock activity specific to regenerating anagen hair follicles, namely epithelial matrix and mesenchymal dermal papilla. We showed that peripheral circadian clock in epithelial matrix cells generates prominent daily mitotic rhythm. As a consequence of this mitotic rhythmicity, hairs grow faster in the morning than in the evening. Because cells are the most susceptible to DNA damage during mitosis, this cycle leads to a remarkable time-of-day-dependent sensitivity of growing hair follicles to genotoxic stress. Same doses of γ-radiation caused dramatic hair loss in wild-type mice when administered in the morning, during mitotic peak, compared with the evening, when hair loss is minimal. This diurnal radioprotective effect becomes lost in circadian mutants, consistent with asynchronous mitoses in their hair follicles. Clock coordinates cell cycle progression with genotoxic stress responses by synchronizing Cdc2/Cyclin B-mediated G2/M checkpoint. Our results uncover diurnal mitotic gating as the essential protective mechanism in highly proliferative hair follicles and offer strategies for minimizing or maximizing cytotoxicity of radiation therapies.
Kachar, Bechara; Brownell, William E.; Altschuler, Richard; Fex, Jörgen
Rapid mechanical changes have been associated with electrical activity in a variety of non-muscle excitable cells1-5. Recently, mechanical changes have been reported in cochlear hair cells6-8. Here we describe electrically evoked mechanical changes in isolated cochlear outer hair cells (OHCs) with characteristics which suggest that direct electrokinetic phenomena are implicated in the response. OHCs make up one of two mechanosensitive hair cell populations in the mammalian cochlea; their role may be to modulate the micromechanical properties of the hearing organ through mechanical feedback mechanisms6-10. In the experiments described here, we applied sinusoidally modulated electrical potentials across isolated OHCs; this produced oscillatory elongation and shortening of the cells and oscillatory displacements of intracellular organdies. The movements were a function of the direction and strength of the electrical field, were inversely related to the ionic concentration of the medium, and occurred in the presence of metabolic uncouplers. The cylindrical shape of the OHCs and the presence of a system of membranes within the cytoplasm-laminated cisternae11-may provide the anatomical substrate for electrokinetic phenomena such as electro-osmosis12,13.
Will J. McLean
Full Text Available Death of cochlear hair cells, which do not regenerate, is a cause of hearing loss in a high percentage of the population. Currently, no approach exists to obtain large numbers of cochlear hair cells. Here, using a small-molecule approach, we show significant expansion (>2,000-fold of cochlear supporting cells expressing and maintaining Lgr5, an epithelial stem cell marker, in response to stimulation of Wnt signaling by a GSK3β inhibitor and transcriptional activation by a histone deacetylase inhibitor. The Lgr5-expressing cells differentiate into hair cells in high yield. From a single mouse cochlea, we obtained over 11,500 hair cells, compared to less than 200 in the absence of induction. The newly generated hair cells have bundles and molecular machinery for transduction, synapse formation, and specialized hair cell activity. Targeting supporting cells capable of proliferation and cochlear hair cell replacement could lead to the discovery of hearing loss treatments.
Kandyba, Eve; Kobielak, Krzysztof
The hair follicle (HF) is an exceptional mini-organ to study the mechanisms which regulate HF morphogenesis, cycling, hair follicle stem cell (hfSCs) homeostasis and progeny differentiation. During morphogenesis, Wnt signaling is well characterized in the initiation of HF patterning but less is known about which particular Wnt ligands are required and whether individual Wnt ligands act in an indispensable or redundant manner during postnatal hfSCs anagen onset and HF cycle progression. Previo...
Hoffman, Robert M
The hair follicle is a skin appendage with a complex structure containing many cell types that produce highly specialised proteins. The hair follicle is in a continuous cycle: anagen is the hair growth phase, catagen the involution phase and telogen is the resting phase. The follicle offers many potential therapeutic targets. Hoffman and colleagues have pioneered hair-follicle-specific targeting using liposomes to deliver small and large molecules, including genes. They have also pioneered ex vivo hair-follicle targeting with continued expression of the introduced gene following transplantation. Recently, it has been discovered that hair follicle stem cells are highly pluripotent and can form neurons, glial cells and other cell types, and this has suggested that hair follicle stem cells may serve as gene therapy targets for regenerative medicine.
Sensory hair cells in auditory and vestibular organs rely on active mechanisms to achieve high sensitivity and frequency selectivity. Recent experimental studies have documented self-sustained oscillations in hair cells of lower vertebrates on two distinct levels. First, the hair bundle can undergo spontaneous mechanical oscillations. Second, somatic electric voltage oscillations across the baso-lateral membrane of the hair cell have been observed. We develop a biophysical model of the bullfrog's saccular hair cell consisting of two compartments, mechanical and electrical, to study how the mechanical and the voltage oscillations interact to produce coherent self-sustained oscillations and how this interaction contributes to the overall sensitivity and selectivity of the hair cell. The model incorporates nonlinear mechanical stochastic hair bundle system coupled bi-directionally to a Hodgkin-Huxley type system describing somatic ionic currents. We isolate regions of coherent spontaneous oscillations in the par...
Full Text Available Animals have evolved two general strategies to counter injury and maintain physiological function. The most prevalent is protection by isolating vital organs into body cavities. However, protection is not optimal for sensory systems because their external components need to be exposed to the environment to fulfill their receptive function. Thus, a common strategy to maintain sensory abilities against persistent environmental insult involves repair and regeneration. However, whether age or frequent injuries affect the regenerative capacity of sensory organs remains unknown. We have found that neuromasts of the zebrafish lateral line regenerate mechanosensory hair cells after recurrent severe injuries and in adulthood. Moreover, neuromasts can reverse transient imbalances of Notch signaling that result in defective organ proportions during repair. Our results reveal inextinguishable hair-cell regeneration in the lateral line, and suggest that the neuromast epithelium is formed by plastic territories that are maintained by continuous intercellular communication.
Wichmann, C; Moser, T
In the mammalian cochlea, sound is encoded at synapses between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs). Each SGN receives input from a single IHC ribbon-type active zone (AZ) and yet SGNs indefatigably spike up to hundreds of Hz to encode acoustic stimuli with submillisecond precision. Accumulating evidence indicates a highly specialized molecular composition and structure of the presynapse, adapted to suit these high functional demands. However, we are only beginning to understand key features such as stimulus-secretion coupling, exocytosis mechanisms, exo-endocytosis coupling, modes of endocytosis and vesicle reformation, as well as replenishment of the readily releasable pool. Relating structure and function has become an important avenue in addressing these points and has been applied to normal and genetically manipulated hair cell synapses. Here, we review some of the exciting new insights gained from recent studies of the molecular anatomy and physiology of IHC ribbon synapses.
Huth, M. E.; Ricci, A. J.; Cheng, A. G.
Aminoglycosides are commonly prescribed antibiotics with deleterious side effects to the inner ear. Due to their popular application as a result of their potent antimicrobial activities, many efforts have been undertaken to prevent aminoglycoside ototoxicity. Over the years, understanding of the antimicrobial as well as ototoxic mechanisms of aminoglycosides has increased. These mechanisms are reviewed in regard to established and potential future targets of hair cell protection. PMID:22121370
Rabbitt, R. D.; Boyle, R.; Holstein, G. R.; Highstein, S. M.
The time course and extent of adaptation in semicircular canal hair cells was compared to adaptation in primary afferent neurons for physiological stimuli in vivo to study the origins of the neural code transmitted to the brain. The oyster toadfish, Opsanus tau, was used as the experimental model. Afferent firing-rate adaptation followed a double-exponential time course in response to step cupula displacements. The dominant adaptation time constant varied considerably among afferent fibers an...
Frank, T. C.; Dye, B. J.; Newlands, S. D.; Dickman, J. D.
OBJECTIVE: The purpose of this study was to develop a technique to investigate the regeneration of utricular hair cells in the adult pigeon (Columba livia) following complete hair cell loss through administration of streptomycin. STUDY DESIGN: Experimental animal study. METHODS: Animals were divided into four groups. Group 1 received 10 to 15 days of systemic streptomycin injections. Animals in Groups 2 and 3 received a single direct placement of a 1-, 2-, 4-, or 8-mg streptomycin dose into the perilymphatic space. Animals in Groups 1 and 2 were analyzed within 1 week from injection to investigate hair cell destruction, whereas Group 3 was investigated at later dates to study hair cell recovery. Group 4 animals received a control injection of saline into the perilymphatic space. Damage and recovery were quantified by counting hair cells in isolated utricles using scanning electron microscopy. RESULTS: Although systemic injections failed to reliably achieve complete utricular hair cell destruction, a single direct placement of a 2-, 4-, or 8-mg streptomycin dose caused complete destruction within the first week. Incomplete hair cell loss was observed with the 1-mg dose. Over the long term, regeneration of the hair cells was seen with the 2-mg dose but not the 8-mg dose. Control injections of saline into the perilymphatic space caused no measurable hair cell loss. CONCLUSIONS: Direct placement of streptomycin into the perilymph is an effective, reliable method for complete destruction of utricular hair cells while preserving the regenerative potential of the neuroepithelium.
Khamesian, Mahvand; Neiman, Alexander B.
Hair cells mediating the senses of hearing and balance rely on active mechanisms for amplification of mechanical signals. In amphibians, hair cells exhibit spontaneous self-sustained mechanical oscillations of their hair bundles. We study the response of the mechanical oscillations to perturbation of the cell's membrane potential in a model for hair bundle of bullfrog saccular hair cells. We identify bifurcation mechanism leading to mechanical oscillations using the membrane potential and the strength of fast adaptation as control parameters and then compute static and dynamic sensitivity of mechanical oscillations to voltage variations. We show that fast adaptation results in the static sensitivity of oscillating hair bundles in the range 0.1-0.2 nm/mV, consistent with recent experimental work. Predicted dynamic response of oscillating hair bundle to voltage variations is characterized by the values of sensitivity of up to 2 nm/mV, enhanced by the presence of fast adaptation.
Ramdasi, Sushilkumar; Tiwari, Shashi Kant
Hair loss can have major psychological impact on affected population belonging to varied ethnic background. Hair is a mini organ in itself and serves many distinguishing functions ranging from maintaining body temperature to promoting social interactions. Major cause of hair loss is androgenic alopecia. Hair follicles possess receptor for androgen. However, DHT (Dihydrotestosterone) in excess results into shrinkage of hair follicle affecting hair growth adversely. The present review is focused on etiology of hair loss, traditional treatment approach and their limitations with side effects with special emphasis on unique properties of stem cells, favourable growth factors secreted by stem cells and strategies to enhance favourable growth factor/cytokine production for hair loss therapeutics. We discussed in details the present available treatment options for hair loss like drugs (Finasteride and Minoxidil), follicular hair transplant, laser therapy and serum therapy. These treatment options have their own disadvantages and side effects with appropriate alerts from regulatory authorities. The side effects of these modalities cannot be ignored and demands alternate therapy approach with less or no side effects. We feel that the stem cell therapy is advancing and is a promising modality in near future owing to its advantages and promising outcomes. This review article discusses possible stem cell therapy for hair regrowth and its advantages. We focused on use of conditioned media derived from stem cells instead of using stem cells directly for the therapy.
Smotherman, M S; Narins, P M
The whole-cell patch-clamp technique was used to identify and characterize ionic currents in isolated hair cells of the leopard frog basilar papilla (BP). This end organ is responsible for encoding the upper limits of a frog's spectral sensitivity (1.25-2.0 kHz in the leopard frog). Isolated BP hair cells are the smallest hair cells in the frog auditory system, with spherical cell bodies typically less than 20 microm in diameter and exhibiting whole-cell capacitances of 4-7 pF. Hair cell zero-current resting potentials (Vz) varied around a mean of -65 mV. All hair cells possessed a non-inactivating, voltage-dependent calcium current (I(Ca)) that activates above a threshold of -55 mV. Similarly all hair cells possessed a rapidly activating, outward, calcium-dependent potassium current (I(K)(Ca)). Most hair cells also possessed a slowly activating, outward, voltage-dependent potassium current (I(K)), which is approximately 80% inactive at the hair cell Vz, and a fast-activating, inward-rectifying potassium current (I(K1)) which actively contributes to setting Vz. In a small subset of cells I(K) was replaced by a fast-inactivating, voltage-dependent potassium current (I(A)), which strongly resembled the A-current observed in hair cells of the frog sacculus and amphibian papilla. Most cells have very similar ionic currents, suggesting that the BP consists largely of one homogeneous population of hair cells. The kinetic properties of the ionic currents present (in particular the very slow I(K)) argue against electrical tuning, a specialized spectral filtering mechanism reported in the hair cells of birds, reptiles, and amphibians, as a contributor to frequency selectivity of this organ. Instead BP hair cells reflect a generalized strategy for the encoding of high-frequency auditory information in a primitive, mechanically tuned, terrestrial vertebrate auditory organ.
Chi, Woo; Wu, Eleanor; Morgan, Bruce A
Although the hair shaft is derived from the progeny of keratinocyte stem cells in the follicular epithelium, the growth and differentiation of follicular keratinocytes is guided by a specialized mesenchymal population, the dermal papilla (DP), that is embedded in the hair bulb. Here we show that the number of DP cells in the follicle correlates with the size and shape of the hair produced in the mouse pelage. The same stem cell pool gives rise to hairs of different sizes or types in successive hair cycles, and this shift is accompanied by a corresponding change in DP cell number. Using a mouse model that allows selective ablation of DP cells in vivo, we show that DP cell number dictates the size and shape of the hair. Furthermore, we confirm the hypothesis that the DP plays a crucial role in activating stem cells to initiate the formation of a new hair shaft. When DP cell number falls below a critical threshold, hair follicles with a normal keratinocyte compartment fail to generate new hairs. However, neighbouring follicles with a few more DP cells can re-enter the growth phase, and those that do exploit an intrinsic mechanism to restore both DP cell number and normal hair growth. These results demonstrate that the mesenchymal niche directs stem and progenitor cell behaviour to initiate regeneration and specify hair morphology. Degeneration of the DP population in mice leads to the types of hair thinning and loss observed during human aging, and the results reported here suggest novel approaches to reversing hair loss.
Lü Zhong-fa; CAI Sui-qing; WU Jin-jin; ZHENG Min
Background Dermal papilla cells (DPC) are a group of mesenchyme-derived cells at the base of the hair follicle, where they regulate and control hair follicle growth through the expression and secretion of cytokines. Nevertheless, the role of DPC derived chemokines and other cytokines in the hair follicle biology remain speculative. In this study, we investigated the expression of basic fibroblast growth factor (bFGF), endothelin-1 (ET-1) and stem cell factor (SCF) in different passages of cultured DPC and their effects on the biological behaviour of DPC.Methods The expression of bFGF, ET-1 and SCF in different passages of cultured DPC and their possible effects on the biological behavior of DPC are investigated using in situ hybridization and immunochemistry. In addition, we performed transplantation of hair follicle cells into nude mice. The cultured DPC, dermal sheath cells and fibroblast of human scalp, respectively, were mixed with cells of the hair follicle epithelium in different ratios, and then were cultured in hair follicle organotypic cultures or implanted into the subcutis of nude mice.Results The expression of ET-1 and SCF in early passages of cultured DPC became stronger, but turned weaker and even negative in late passages (>6 passages). Hair follicle-like structures were formed after DPC combined with the cells of hair follicle epithelium cells in hair follicle organotypic cultures. When hair follicle organotypic cultures were implanted into the subcutis of nude mice, the relative intact hair follicles were formed. After the transplantation of hair follicle cells into the nude mice, the hair follicle-like structure was formed in the group that contained DPC mixed with hair follicle epithelium cells. However, no hair follicles were formed in the other two groups. It was found that the higher the expression of ET-1 and SCF in DPC, the stronger the ability of DPC to induce hair follicle regeneration.Conclusions The cultured DPC can induce hair follicle
Garcia-Vergara, Mauricio; Gomez-Correa, Gilberto; Ramirez-Santiago, Guillermo
Epithelia cells are polarized along an axis perpendicular to the apical-basal axis, --``Planar cell polarization'' (PCP)--. In Drosophila adult cuticle cells are hexagonally packed and the PCP gives rise to the elaboration of an actin-rich hair that develops from one of the hexagon vertex and pointing distally. Genetic analyses have identified a group of proteins whose activities are required to polarize each cell and produce the phenomenon of PCP. To describe the PCP in the epithelia some quantitative models intended to explain this phenomenon by invoking diffusion of several proteins and all their interactions. Here we propose a simpler model consisting of two reaction-diffusion equations that describe the redistribution process of two chemical agents inside a cell. This redistribution occurs as a response to an external gradient of a quimio-attractor. We emulate the collective cell polarization by introducing ``interactions'' between neighboring cells that propagate trough the epithelia. This collective polarization gives rise to an orientational pattern in the actin-rich hairs.
Full Text Available Alopecia is an exceedingly prevalent problem effecting men and women of all ages. The standard of care for alopecia involves either transplanting existing hair follicles to bald areas or attempting to stimulate existing follicles with topical and/or oral medication. Yet, these treatment options are fraught with problems of cost, side effects, and, most importantly, inadequate long-term hair coverage. Innovative cell-based therapies have focused on the dermal papilla cell as a way to grow new hair in previously bald areas. However, despite this attention, many obstacles exist, including retention of dermal papilla inducing ability and maintenance of dermal papilla productivity after several passages of culture. The use of adipocyte lineage cells, including adipose-derived stem cells, has shown promise as a cell-based solution to regulate hair regeneration and may help in maintaining or increasing dermal papilla cells inducing hair ability. In this review, we highlight recent advances in the understanding of the cellular contribution and regulation of dermal papilla cells and summarize adipocyte lineage cells in hair regeneration.
Lu, Na [Otology Skull Base Surgery Department, Hearing Research Institute, Eye and ENT Hospital of Shanghai Medical School, Fudan University, Shanghai 200031 (China); Department of Otolaryngology and Program in Neuroscience, Harvard Medical School and Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114 (United States); Chen, Yan [Central Laboratory, Hearing Research Institute, Eye and ENT Hospital of Shanghai Medical School, Fudan University, Shanghai 200031 (China); Wang, Zhengmin [Otology Skull Base Surgery Department, Hearing Research Institute, Eye and ENT Hospital of Shanghai Medical School, Fudan University, Shanghai 200031 (China); Institute of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Chen, Guoling [Otology Skull Base Surgery Department, Hearing Research Institute, Eye and ENT Hospital of Shanghai Medical School, Fudan University, Shanghai 200031 (China); Lin, Qin [Otology Skull Base Surgery Department, Hearing Research Institute, Eye and ENT Hospital of Shanghai Medical School, Fudan University, Shanghai 200031 (China); Department of Otolaryngology, First Affiliated Hospital of Fujian Medical University, Otolaryngology Institute of Fujian Province, Fuzhou (China); Chen, Zheng-Yi, E-mail: Zhengfirstname.lastname@example.org [Department of Otolaryngology and Program in Neuroscience, Harvard Medical School and Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114 (United States); Li, Huawei, E-mail: email@example.com [Otology Skull Base Surgery Department, Hearing Research Institute, Eye and ENT Hospital of Shanghai Medical School, Fudan University, Shanghai 200031 (China); Institute of Biomedical Sciences, Fudan University, Shanghai 200032 (China)
Highlights: Black-Right-Pointing-Pointer Shh activation in neonatal cochleae enhances sensory cell proliferation. Black-Right-Pointing-Pointer Proliferating supporting cells can transdifferentiate into hair cells. Black-Right-Pointing-Pointer Shh promotes proliferation by transiently modulating pRb activity. Black-Right-Pointing-Pointer Shh inhibits pRb by inhibiting transcription and increasing phosphorylation of pRb. -- Abstract: Cell cycle re-entry by cochlear supporting cells and/or hair cells is considered one of the best approaches for restoring hearing loss as a result of hair cell damage. To identify mechanisms that can be modulated to initiate cell cycle re-entry and hair cell regeneration, we studied the effect of activating the sonic hedgehog (Shh) pathway. We show that Shh signaling in postnatal rat cochleae damaged by neomycin leads to renewed proliferation of supporting cells and hair cells. Further, proliferating supporting cells are likely to transdifferentiate into hair cells. Shh treatment leads to inhibition of retinoblastoma protein (pRb) by increasing phosphorylated pRb and reducing retinoblastoma gene transcription. This results in upregulation of cyclins B1, D2, and D3, and CDK1. These results suggest that Shh signaling induces cell cycle re-entry in cochlear sensory epithelium and the production of new hair cells, in part by attenuating pRb function. This study provides an additional route to modulate pRb function with important implications in mammalian hair cell regeneration.
Takeda, Seiji; Gapper, Catherine; Kaya, Hidetaka; Bell, Elizabeth; Kuchitsu, Kazuyuki; Dolan, Liam
The specification and maintenance of growth sites are tightly regulated during cell morphogenesis in all organisms. ROOT HAIR DEFECTIVE 2 reduced nicotinamide adenine dinucleotide phosphate (RHD2 NADPH) oxidase-derived reactive oxygen species (ROS) stimulate a Ca2+ influx into the cytoplasm that is required for root hair growth in Arabidopsis thaliana. We found that Ca2+, in turn, activated the RHD2 NADPH oxidase to produce ROS at the growing point in the root hair. Together, these components could establish a means of positive feedback regulation that maintains an active growth site in expanding root hair cells. Because the location and stability of growth sites predict the ultimate form of a plant cell, our findings demonstrate how a positive feedback mechanism involving RHD2, ROS, and Ca2+ can determine cell shape.
Full Text Available Hair cells (HCs are the sensory preceptor cells in the inner ear, which play an important role in hearing and balance. The HCs of organ of Corti are susceptible to noise, ototoxic drugs, and infections, thus resulting in permanent hearing loss. Recent approaches of HCs regeneration provide new directions for finding the treatment of sensor neural deafness. To have normal hearing function, the regenerated HCs must be reinnervated by nerve fibers and reform ribbon synapse with the dendrite of spiral ganglion neuron through nerve regeneration. In this review, we discuss the research progress in HC regeneration, the synaptic plasticity, and the reinnervation of new regenerated HCs in mammalian inner ear.
Shatz, Lisa F
The effect of the size and the shape of the hair bundle of a hair cell in the inner ear of non-mammals on its motion for the full range of frequencies is determined thereby extending the results of a previous analysis of hair bundle motion for high and low frequencies [Hear Res. 141 (2000) 39-50]. A hemispheroid is used to represent the hair bundle because it can represent a full range of shapes, from thin, pencil-like shapes to wide, flat, disk-like shapes. Boundary element methods are used to approximate the solution for the hydrodynamics. For physiologically relevant parameters, an excellent match is obtained between the model's predictions and measurements of hair bundle motion in the free-standing region of the basilar papilla of the alligator lizard [Aranyosi, Measuring sound-induced motions of the alligator lizard cochlea. Massachusetts Institute of Technology, PhD Thesis, 2002]. Neither in the model's predictions nor in experimental measurements is sharp tuning observed. The model predicted the low frequency region of neural tuning curves for the alligator lizard and bobtail lizard, but could not predict the sharp tuning or the high frequency region. An element that represents an active mechanism is added to the hair bundle model to predict neural tuning curves, which are sharply tuned, and an excellent match is obtained for all the characteristics of neural tuning curves for the alligator lizard, and for the low and high frequency regions for the bobtail lizard. The model does not predict well the sharp tuning of the shorter hair bundles of the bobtail lizard, possibly because it does not represent tectorial sallets.
Johnson, Stuart L.; Ceriani, Federico; Houston, Oliver; Polishchuk, Roman; Polishchuk, Elena; Crispino, Giulia; Zorzi, Veronica
Mutations in the genes encoding for gap junction proteins connexin 26 (Cx26) and connexin 30 (Cx30) have been linked to syndromic and nonsyndromic hearing loss in mice and humans. The release of ATP from connexin hemichannels in cochlear nonsensory cells has been proposed to be the main trigger for action potential activity in immature sensory inner hair cells (IHCs), which is crucial for the refinement of the developing auditory circuitry. Using connexin knock-out mice, we show that IHCs fire spontaneous action potentials even in the absence of ATP-dependent intercellular Ca2+ signaling in the nonsensory cells. However, this signaling from nonsensory cells was able to increase the intrinsic IHC firing frequency. We also found that connexin expression is key to IHC functional maturation. In Cx26 conditional knock-out mice (Cx26Sox10-Cre), the maturation of IHCs, which normally occurs at approximately postnatal day 12, was partially prevented. Although Cx30 has been shown not to be required for hearing in young adult mice, IHCs from Cx30 knock-out mice exhibited a comprehensive brake in their development, such that their basolateral membrane currents and synaptic machinery retain a prehearing phenotype. We propose that IHC functional differentiation into mature sensory receptors is initiated in the prehearing cochlea provided that the expression of either connexin reaches a threshold level. As such, connexins regulate one of the most crucial functional refinements in the mammalian cochlea, the disruption of which contributes to the deafness phenotype observed in mice and DFNB1 patients. SIGNIFICANCE STATEMENT The correct development and function of the mammalian cochlea relies not only on the sensory hair cells, but also on the surrounding nonsensory cells. Although the nonsensory cells have been largely implicated in the general homeostasis in the mature cochlea, their involvement in the initial functional differentiation of the sensory inner hair cells is less
Full Text Available The innervation of the inner ear critically depends on the two neurotrophins Ntf3 and Bdnf. In contrast to this molecularly well established dependency, evidence regarding the need of innervation for long-term maintenance of inner ear hair cells is inconclusive due to experimental variability. Mutant mice that lack both neurotrophins could shed light on the long-term consequences of innervation loss on hair cells without introducing experimental variability, but do not survive after birth. Mutant mice with conditional deletion of both neurotrophins lose almost all innervation by postnatal day 10 and show an initially normal development of hair cells by this stage. No innervation remains after three weeks and complete loss of all innervation results in near complete loss of outer and many inner hair cells of the organ of Corti within 4 months. Mutants that retain one allele of either neurotrophin have only partial loss of innervation of the organ of Corti and show a longer viability of cochlear hair cells with more profound loss of inner hair cells. Ultimately, however, hair cells disappear with a base to apex progression, proportional to the residual density of innervation and similar to carboplatin ototoxicity. Similar to reports of hair cell loss after aminoglycoside treatment, blobbing of stereocilia of apparently dying hair cells protrude into the cochlear duct. Denervation of vestibular sensory epithelia for several months also resulted in variable results, ranging from unusual hair cells resembling the aberrations found in the organ of Corti, to near normal hair cells in the canal cristae. Fusion and/or resorption of stereocilia and loss of hair cells follows a pattern reminiscent of Myo6 and Cdc42 null mice. Our data support a role of innervation for long-term maintenance but with a remarkable local variation that needs to be taken into account when attempting regeneration of the organ of Corti.
SHU Wei-ning; ZHAO Li-dong; ZHANG Xiao-bing; YANG Shi-ming
@@ Hair cells in the mammalian inner ear are very fragile and are often injured as a result of acoustic trauma or exposure to ototoxic drugs (cisplatin, aminoglycosides, etc). In amphibians and birds, spontaneous post-injury regeneration of all inner ear sensory hair cell occurs, while in the mammalian cochlea, such hearing loss is usually permanent as there are currently no treatments that can lead to post-injury hair cell regeneration.
Trapani, Josef G.; Nikolaus Obholzer; Weike Mo; Brockerhoff, Susan E.; Teresa Nicolson
To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed bas...
Esterberg, Robert; Hailey, Dale W.; Rubel, Edwin W; Raible, David W.
Mechanosensory hair cells are vulnerable to environmental insult, resulting in hearing and balance disorders. We demonstrate that directional compartmental flow of intracellular Ca2+ underlies death in zebrafish lateral line hair cells after exposure to aminoglycoside antibiotics, a well characterized hair cell toxin. Ca2+ is mobilized from the ER and transferred to mitochondria via IP3 channels with little cytoplasmic leakage. Pharmacological agents that shunt ER-derived Ca2+ directly to cyt...
Yang, Qianqian; Sun, Gaoying; Cao, Zhixin; Yin, Haiyan; Qi, Qi; Wang, Jinghan; Liu, Wenwen; Bai, Xiaohui; Wang, Haibo; Li, Jianfeng
Nucleotide-binding domain and leucine-rich-repeat-containing family member X1 (NLRX1) is a cytoplasmic pattern recognition receptor that is predominantly located in mitochondria, which is tightly related to mitochondrial damage, reactive oxygen species (ROS) production, inflammation and apoptosis. The present study was designed to explore whether NLRX1 expresses in C57BL/6 mice cochlear hair cells and, if so, to investigate the possible correlations between NLRX1 and hearing. The location and dynamic expression of NLRX1 were investigated by immunofluorescence, real-time PCR and Western blotting. Hearing thresholds of C57BL/6 mice were measured by auditory brainstem response (ABR). Moreover, the downstream inflammatory and apoptotic pathways regulated by NLRX1 were examined in age-related and neomycin-induced hair cell damage. Data showed that NLRX1 expressed in cytoplasm of C57BL/6 cochlear hair cells, especially in the cilia, which were essential for sound sensation. The expression of NLRX1 in hair cells increased as the mice grew up, and, decreased as they aged. Additionally, the activated apoptotic JNK pathway was detected in 9-month old mice with worse-hearing and 3-month old mice treated with neomycin. Overall, results indicate that NLRX1 may relate to hair cell maturity, hearing formation and maintenance, and promote hair cell apoptosis through JNK pathway induced by aging and neomycin.
Kathryn D Breneman
Full Text Available Microvilli (stereocilia projecting from the apex of hair cells in the inner ear are actively motile structures that feed energy into the vibration of the inner ear and enhance sensitivity to sound. The biophysical mechanism underlying the hair bundle motor is unknown. In this study, we examined a membrane flexoelectric origin for active movements in stereocilia and conclude that it is likely to be an important contributor to mechanical power output by hair bundles. We formulated a realistic biophysical model of stereocilia incorporating stereocilia dimensions, the known flexoelectric coefficient of lipid membranes, mechanical compliance, and fluid drag. Electrical power enters the stereocilia through displacement sensitive ion channels and, due to the small diameter of stereocilia, is converted to useful mechanical power output by flexoelectricity. This motor augments molecular motors associated with the mechanosensitive apparatus itself that have been described previously. The model reveals stereocilia to be highly efficient and fast flexoelectric motors that capture the energy in the extracellular electro-chemical potential of the inner ear to generate mechanical power output. The power analysis provides an explanation for the correlation between stereocilia height and the tonotopic organization of hearing organs. Further, results suggest that flexoelectricity may be essential to the exquisite sensitivity and frequency selectivity of non-mammalian hearing organs at high auditory frequencies, and may contribute to the "cochlear amplifier" in mammals.
Zallocchi, Marisa; Delimont, Duane; Meehan, Daniel T; Cosgrove, Dominic
Usher syndrome is a genetically heterogeneous disorder characterized by hearing and balance dysfunction and progressive retinitis pigmentosa. Mouse models carrying mutations for the nine Usher-associated genes have splayed stereocilia, and some show delayed maturation of ribbon synapses suggesting these proteins may play different roles in terminal differentiation of auditory hair cells. The presence of the Usher proteins at the basal and apical aspects of the neurosensory epithelia suggests the existence of regulated trafficking through specific transport proteins and routes. Immature mouse cochleae and UB/OC-1 cells were used in this work to address whether specific variants of PCDH15 and VLGR1 are being selectively transported to opposite poles of the hair cells. Confocal colocalization studies between apical and basal vesicular markers and the different PCDH15 and VLGR1 variants along with sucrose density gradients and the use of vesicle trafficking inhibitors show the existence of Usher protein complexes in at least two vesicular subpools. The apically trafficked pool colocalized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associated with membrane microdomains and SNAP25. Moreover, coimmunoprecipitation experiments between SNAP25 and VLGR1 show a physical interaction of these two proteins in organ of Corti and brain. Collectively, these findings establish the existence of a differential vesicular trafficking mechanism for specific Usher protein variants in mouse cochlear hair cells, with the apical variants playing a potential role in endosomal recycling and stereocilia development/maintenance, and the basolateral variants involved in vesicle docking and/or fusion through SNAP25-mediated interactions.
Cohen, G. M.; Reschke, M.; Homick, J.
The bullfrog's saccule were examined using light and scanning electron microscopy. No evidence of a striola was found. Type A hair cells were not only distributed peripherally, but also throughout the central macula, though far less frequently than the dominant type D. Two primary hair cell types were distinguished, which corresponded to the ciliary patterns: type A cilia are associated with short, conical hair cells, and type D cilia are associated with long, cylindrical hair cells. Each displays at least one subtype, which may represent developmental precursors. The otolithic membrane is crisscrossed with tunnels and topped with statoconia.
Grierson, C.; Nielsen, E.; Ketelaar, T.; Schiefelbein, J.
Roots hairs are cylindrical extensions of root epidermal cells that are important for acquisition of nutrients, microbe interactions, and plant anchorage. The molecular mechanisms involved in the specification, differentiation, and physiology of root hairs in Arabidopsis are reviewed here. Root hair
The outer hair cell (OHC) of the mammalian inner ear is a highly partitioned neuroepithelial cell whose lateral membrane is devoted to electromotility, a fast mechanical length change owing to the motor protein, prestin. Spatially restricted measures of prestin-derived nonlinear capacitance or gating charge, using either electrical amputation or discrete membrane mechanical deformation, were used to determine that functional variation exists within the extensive lateral membrane of the cell. This was evidenced by variation in the motor's operating voltage range and sensitivity among microdomains within the lateral membrane. That is, localized regions of the membrane evidenced Boltzmann distributions of motor charge whose midpoint voltage and slope differed from those obtained for the whole cell. These data highlight the functional independence of microdomains and imply that measured whole cell characteristics may differ from the microscopic characteristics of elementary motors.
Siegel, J H; Brownell, W E
Membrane recycling in the mechanoreceptive sensory cells of the mammalian cochlea was studied by observing membrane-bound horseradish peroxidase (HRP) reaction product following brief in vivo exposure to the enzyme. In the inner hair cell (IHC), peroxidase was taken up into coated vesicles and became incorporated into synaptic vesicles surrounding presynaptic bodies, but much HRP was also transported to the apical zone where reaction product appeared in all components of the Golgi complex. Neither the subsurface cisternae nor a tubular network associated with clusters of mitochondria were labelled. Outer hair cells (OHCs) showed considerably less membrane-bound reaction product than IHCs, indicating less rapid plasmalemmal recycling. Most membrane-bound reaction product was contained in coated vesicles and small vacuoles in the synaptic zone, but was occasionally seen in multivesicular bodies in the most apical zone. No labelled organelles were detected in the large central region of the OHC. A diffuse staining of the cytoplasm, particularly pronounced in OHCs, often interfered with the evaluation of membrane-bound reaction product in OHCs. This staining pattern could be qualitatively reproduced in both IHCs and OHCs by incubating fixed segments of the organ of Corti in oxidized diaminobenzidine. The presence of labelled synaptic vesicles associated with presynaptic bodies of IHCs and OHCs suggests that they are formed from membrane retrieved from the plasmalemma. We found no evidence that the subsurface cisternae of IHCs or the laminated cisternae of OHCs are derived from the cell surface as they never contained reaction product.
Song, Lei; Santos-Sacchi, Joseph
Nonlinear capacitance (NLC) measures are often used as surrogate measures of outer hair cell (OHC) electromotility (eM), since the two are commonly thought to share many biophysical features. The measurement of NLC is simpler than direct measurements of eM and, therefore, many investigators have adopted it. A standard patch-clamp hardware configuration is sufficient for recording NLC, given the proper software interface. Thus, the approach is cost effective. We use the software jClamp since it is tailored to capacitance measurement. Here we detail steps that we use to measure NLC. The walk through includes isolation of guinea pig OHCs, building voltage commands, recording, and analysis.
Lepelletier, Léa; de Monvel, Jacques Boutet; Buisson, Johanna; Desdouets, Chantal; Petit, Christine
Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements o...
Peng, A.W.; Effertz, T.; Ricci, A.J.
Adaptation is a hallmark of hair cell mechanotransduction, extending the sensory hair bundle dynamic range while providing mechanical filtering of incoming sound. In hair cells responsive to low frequencies, two distinct adaptation mechanisms exist, a fast component of debatable origin and a slow myosin-based component. It is generally believed that Ca2+ entry through mechano-electric transducer channels is required for both forms of adaptation. This study investigates the calcium dependence ...
Herget, Meike; Scheibinger, Mirko; Guo, Zhaohua; Jan, Taha A; Adams, Christopher M; Cheng, Alan G; Heller, Stefan
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.
Sun, Shaoyang; Wang, Xu; Li, Wenyan; Li, Huawei
The c-Jun amino-terminal kinase (JNK) proteins are a subgroup of the mitogen-activated protein kinase family. They play a complex role in cell proliferation, survival, and apoptosis. Here, we report a novel role of JNK signalling in hair cell regeneration. We eliminated hair cells of 5-day post-fertilization zebrafish larvae using neomycin followed by JNK inhibition with SP600125. JNK inhibition strongly decreased the number of regenerated hair cells in response to neomycin damage. These changes were associated with reduced proliferation. JNK inhibition also increased cleaved caspase-3 activity and induced apoptosis in regenerating neuromasts. Finally, JNK inhibition with SP600125 decreased the expression of genes related to Wnt. Over-activation of the Wnt signalling pathway partly rescued the hair cell regeneration defects induced by JNK inhibition. Together, our findings provide novel insights into the function of JNK and show that JNK inhibition blocks hair cell regeneration by controlling the Wnt signalling pathway. PMID:27438150
Hirvonen, Timo P; Minor, Lloyd B; Hullar, Timothy E; Carey, John P
Gentamicin is toxic to vestibular hair cells, but its effects on vestibular afferents have not been defined. We treated anesthetized chinchillas with one injection of gentamicin (26.7 mg/ml) into the middle ear and made extracellular recordings from afferents after 5-25 (early) or 90-115 days (late). The relative proportions of regular, intermediate, and irregular afferents did not change after treatment. The spontaneous firing rate of regular afferents was lower (P galvanic currents was unaffected for all afferents. Intratympanic gentamicin treatment reduced the histological density of all hair cells by 57% (P = 0.04). The density of hair cells with calyx endings was reduced by 99% (P = 0.03), although some remaining hair cells had other features suggestive of type I morphology. Type II hair cell density was not significantly reduced. These findings suggest that a single intratympanic gentamicin injection causes partial damage and loss of vestibular hair cells, particularly type I hair cells or their calyceal afferent endings, does not damage the afferent spike initiation zones, and preserves enough hair cell synaptic activity to drive the spontaneous activity of vestibular afferents.
Full Text Available Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20-30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment.
Root hairs are single tubular cells formed from the differentiation of epidermal cells on roots. They are involved in water and nutrient uptake, and represent the infection site on leguminous roots by rhizobia, soil bacteria that establish a nitrogen fixing symbiosis. Root hairs develop by polar cel...
Kawashima, Yoshiyuki; Geleoc, Gwenaelle S. G.; Kurima, Kiyoto; Labay, Valentina; Lelli, Andrea; Asai, Yukako; Makishima, Tomoko; Wu, Doris K.; Della Santina, Charles C.; Holt, Jeffrey R.; Griffith, Andrew J.
Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in
Pollock, Lana M; Chou, Shih-Wei; McDermott, Brian M
The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509017) reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle's morphology and hearing.
van Netten, Sietse M.; Meulenberg, Cecil J. W.; Lennan, George W. T.; Kros, Corne J.
Hair cells in the inner ear provide the basis for the exquisite hearing capabilities of mammals. These cells transduce sound-induced displacements of their mechanosensitive hair bundle into electrical currents within a fraction of a millisecond and with nanometer fidelity. Excitatory displacements
Yuan Yasheng; Wang Yang; Chi Fanglu
Background Replacement of spiral ganglion neurons would be one prioritized step in an attempt to restore sensory neuronal hearing loss.However,the possibility that transplanted neurons could regenerate new synaptic connections to hair cells has not been explored.The objective of this study was to test whether neural stem cell (NSC)-derived neurons can form synaptic connections with hair cells in vitro.Methods NSCs were mechanically separated from the hippocampus in SD rat embryos (E12-E14) and cultured in a serum-free medium containing basic fibroblast growth factor and epidermal growth factor.Rat NSCs were co-cultured with explants of cochlea sensory epithelia obtained from postnatal Day 3 rats under transway filter membrane.Results At Day 3,the NSCs began to show chemotactic differentiation and grew toward cochlea sensory epithelia.After 9-day co-culture,neurites of NSC-derived neurons predominantly elongated toward hair cells.Immunohistochemical analyses revealed the fibers overlapped with synapsin and hair cells,indicating the formation of new synaptic connections.After 14-day culture,triple staining revealed the fibers overlapped with PSD95 (postsynaptic density) which is juxtaposed with CtBP2 (presynaptic vesicle),indicating the formation of new ribbon synapse.Conclusions NSC-derived neurons can make synaptic connections with hair cells and provide a model for studying synaptic plasticity and regeneration.Whether the newly forming synapse is functional merits further electrophysiological study.
Yuan, Yasheng; Wang, Yang; Chi, Fanglu
Replacement of spiral ganglion neurons would be one prioritized step in an attempt to restore sensory neuronal hearing loss. However, the possibility that transplanted neurons could regenerate new synaptic connections to hair cells has not been explored. The objective of this study was to test whether neural stem cell (NSC)-derived neurons can form synaptic connections with hair cells in vitro. NSCs were mechanically separated from the hippocampus in SD rat embryos (E12-E14) and cultured in a serum-free medium containing basic fibroblast growth factor and epidermal growth factor. Rat NSCs were co-cultured with explants of cochlea sensory epithelia obtained from postnatal Day 3 rats under transway filter membrane. At Day 3, the NSCs began to show chemotactic differentiation and grew toward cochlea sensory epithelia. After 9-day co-culture, neurites of NSC-derived neurons predominantly elongated toward hair cells. Immunohistochemical analyses revealed the fibers overlapped with synapsin and hair cells, indicating the formation of new synaptic connections. After 14-day culture, triple staining revealed the fibers overlapped with PSD95 (postsynaptic density) which is juxtaposed with CtBP2 (presynaptic vesicle), indicating the formation of new ribbon synapse. NSC-derived neurons can make synaptic connections with hair cells and provide a model for studying synaptic plasticity and regeneration. Whether the newly forming synapse is functional merits further electrophysiological study.
LIN Chang-min; LI Yu; JI Ying-chang; HUANG Keng; CAI Xiang-na; LI Guo-qiang
Objective: To induce hair follicle regeneration in rat ear by microencapsulated dermal papillae (DP) cells.Methods: Intact dermal papillae were obtained from human scalp follicles which were digested with collagenase I. The human hair DP cells were encapsulated with alginate-polylysine-alginate (APA) by a high-voltage electric field droplet generator. The diameters of the DP cell microcapsules were optimized by regulating the voltage, the distance be-tween the needle head and the solution surface and the injection speed. Then DP cell microencapsulations were xenotransplanted into ears of 20 SD rats with a novel method. One rat was killed every week at the postoperative 2-12 weeks and the implantation sites were biopsied for histo-logical observation.Results: The DP cell microencapsulations were found in a group of round, smooth and transparent microcapsules under a phase-contrast microscope. The optimal combina-tion of parameters to obtain 0.4 mm DP cell microcapsules was voltage 7.0 kV, injection speed 55 mm/h, and distance 10mm. After 4-12 weeks, 18 of 20 DP cell microcapsule implan-tations had produced high-density hair. Histological obser-vation indicated that both large follicles and sebaceous gland structures were formed in the rat ear within 3-12 weeks.Conclusions: These findings show that the DP cell microencapsulation maintain the capacity for initiating the follicle regeneration and can be considered as a substitute for fresh isolated dermal papillae.
Togarrati, Padma Priya; Suknuntha, Kran
A number of malignant and non-malignant hematological disorders are associated with the abnormal production of mature blood cells or primitive hematopoietic precursors. Their capacity for continuous self-renewal without loss of pluripotency and the ability to differentiate into adult cell types from all three primitive germ layers make human embryonic stem cells and induced pluripotent stem cells (hiPSCs) attractive complementary cell sources for large-scale production of transfusable mature blood cell components in cell replacement therapies. The generation of patient-specific hematopoietic stem/precursor cells from iPSCs by the regulated manipulation of various factors involved in reprograming to ensure complete pluripotency, and developing innovative differentiation strategies for generating unlimited supply of clinically safe, transplantable, HLA-matched cells from hiPSCs to outnumber the inadequate source of hematopoietic stem cells obtained from cord blood, bone marrow and peripheral blood, would have a major impact on the field of regenerative and personalized medicine leading to translation of these results from bench to bedside.
Xia, Anping; Liu, Xiaofang; Raphael, Patrick D; Applegate, Brian E; Oghalai, John S
Frequency tuning within the auditory papilla of most non-mammalian species is electrical, deriving from ion-channel resonance within their sensory hair cells. In contrast, tuning within the mammalian cochlea is mechanical, stemming from active mechanisms within outer hair cells that amplify the basilar membrane travelling wave. Interestingly, hair cells in the avian basilar papilla demonstrate both electrical resonance and force-generation, making it unclear which mechanism creates sharp frequency tuning. Here, we measured sound-induced vibrations within the apical half of the chicken basilar papilla in vivo and found broadly-tuned travelling waves that were not amplified. However, distortion products were found in live but not dead chickens. These findings support the idea that avian hair cells do produce force, but that their effects on vibration are small and do not sharpen tuning. Therefore, frequency tuning within the apical avian basilar papilla is not mechanical, and likely derives from hair cell electrical resonance.
Dominique J Wiener
Full Text Available Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii the lower isthmus (comprising the bulge harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients.
Levy, Michael; Molzon, Adrian; Lee, Jae-Hyun; Kim, Ji-Wook; Cheon, Jinwoo; Bozovic, Dolores
Auditory and vestibular hair cell bundles exhibit active mechanical oscillations at natural frequencies that are typically lower than the detection range of the corresponding end organs. We explore how these noisy nonlinear oscillators mode-lock to frequencies higher than their internal clocks. A nanomagnetic technique is used to stimulate the bundles without an imposed mechanical load. The evoked response shows regimes of high-order mode-locking. Exploring a broad range of stimulus frequencies and intensities, we observe regions of high-order synchronization, analogous to Arnold Tongues in dynamical systems literature. Significant areas of overlap occur between synchronization regimes, with the bundle intermittently flickering between different winding numbers. We demonstrate how an ensemble of these noisy spontaneous oscillators could be entrained to efficiently detect signals significantly above the characteristic frequencies of the individual cells.
Iwasa, Kuni H
Electromotility of outer hair cells (OHCs) has been extensively studied with in vitro experiments because of its physiological significance in the cochlear amplifier, which provides the exquisite sensitivity and frequency selectivity of the mammalian ear. However, these studies have been performed largely under load-free conditions or with static load, while these cells function in vivo in a dynamic environment, receiving electrical energy to enhance mechanical oscillation in the inner ear. This gap leaves uncertainties in addressing a key issue, how much mechanical energy an OHC provides. The present report is an attempt of bridging the gap by introducing a simple one-dimensional model for electromotility of OHC in a dynamic environment. This model incorporates a feedback loop involving the receptor potential and the mechanical load on OHC, and leads to an analytical expression for the membrane capacitance, which explicitly describes the dependence on the elastic load, viscous drag, and the mass. The derived...
Behra, Martine; Bradsher, John; Sougrat, Rachid; Gallardo, Viviana; Allende, Miguel L; Burgess, Shawn M
In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.
Full Text Available In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho. Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.
Severinsen, Stig Åvall; Kirkegaard, Mette; Nyengaard, Jens Randel
injection. Total volume of the utricle, as well as total number of hair and supporting cells, were estimated on light microscopic sections. Total volume and mean volume of hair cell types I and II and supporting cells were estimated on digital transmission electron micrographs. Total volume of the utricular...... macula, hair cell type I and supporting cells decreased significantly in animals injected with kanamycin but not in animals co-treated with DHB. Hair and supporting cell numbers remained unchanged in all three groups. In conclusion, the kanamycin-induced volume reduction of type I hair cells...
Full Text Available Tissue engineering as a rapidly developing branch of science offers hope for the use of its products in medical practice. Among the components of tissue substitutes are different types of cells, especially stem cells. A promising source of adult stem cells is hair follicles. Development of follicles in the skin takes place even during fetal life. They arise due to the impact of epidermal and mesenchymal cells. The next steps in the formation of hair follicles are under the control of many factors. Hair follicles are the niche of various stem cell populations and are a major source of cells responsible for regeneration of the hair, sebaceous glands and epidermis. The term „hair follicle stem cells” is most often used in relation to the epithelial cell population. Hair follicle stem cell studies are complicated by the fact that these stem cells divide relatively rarely.The aim of this study is to present the characteristics of cells isolated from the hair follicle in the light of recent research.
Bu, Zhang-Yu; Wu, Li-Min; Yu, Xiao-Hong; Zhong, Jian-Bo; Yang, Ping; Chen, Jian
The present investigation explored the in vitro culture, isolation and characterization of hair follicle cell differentiation from umbilical cord blood mesenchymal stem cells (MSCs). Flow cytometry was used to obtain MSCs from the isolation and purification of human umbilical cord blood MSCs. Culture suspension of hair follicle organ was centrifuged and the supernatant used in the culture medium of MSCs, and the entire process of induced differentiation was recorded by photomicroscopy. The expression level of surface marker CK15 of hair follicle cells obtained from induced differentiation was detected with immunofluorescence. RT-PCR method was used to further detect the difference in expression of CK15 between hair follicle cells and umbilical cord blood MSCs, and statistical analysis was carried out. CD44(+)CD29(+) double-labeled cells accounted for 50.8% of all the samples of umbilical cord blood MSCs in this study. The diameter of hair follicle cells differentiated from umbilical cord blood stem cells reached 800×10(-3) mm after 3 weeks of cell culture. Based on the detection and colocalization of CK15 expression in induced hair follicle cells, the overlap ratio between CK15 and nuclei reached 83% in hair follicle cells, which was obviously higher than that in umbilical cord blood stem cells. The difference had statistical significance (Pumbilical cord blood stem cells by using the supernatant from hair follicle cells. This method can be used for high-speed induced differentiation with high purity, which is promising for clinical application.
Edri, Yuval; Yochelis, Arik
The auditory system displays remarkable mechanical sensitivity and frequency discrimination. These attributes have been shown to rely on an amplification process, which requires biochemical feedback loops. In some systems, the active process was shown to lead to spontaneous oscillations of hair cell bundles. In the last decade, models that display proximity to an oscillatory onset (a.k.a. Hopf bifurcation) have gained increasing support due to many advantages in explaining the hearing phenomenology. Particularly, they exhibit resonant responses to distinct frequencies of incoming sound waves. Unlike previous studies, two types of driving forces are being examined: additive, in which the external forcing term does not couple directly on the systems observable (passive coupling), and parametric, in which the forcing term directly affects the observable and thus intrinsically modifies the systems properties (active coupling). By applying universal principles near the Hopf bifurcation onset, we find several funda...
Uribe, Phillip M; Mueller, Melissa A; Gleichman, Julia S; Kramer, Matthew D; Wang, Qi; Sibrian-Vazquez, Martha; Strongin, Robert M; Steyger, Peter S; Cotanche, Douglas A; Matsui, Jonathan I
Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.
Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.
Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.
Matsui, Jonathan I.; Haque, Asim; Huss, David; Messana, Elizabeth P.; Alosi, Julie A.; Roberson, David W.; Cotanche, Douglas A.; Dickman, J. David; Warchol, Mark E.
The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.
Wang, Changquan; Zhong, Zhenmin; Sun, Peng
The zebrafish has become an established model organism for the study of hearing and balance systems in the past two decades. The classical approach to examine hair cells is to use dye to conduct selective staining, which shows the number and morphology of hair cells but does not reveal their function. Startle response is a behavior closely related to the auditory function of hair cells; therefore it can be used to measure the function of hair cells. In this study, we developed a device to measure the startle response of zebrafish larvae. By applying various levels of stimulus, it showed that the system can discern a 10 dB difference. The hair cell in zebrafish can regenerate after damage due to noise exposure or drug treatment. With this device, we measured the startle response of zebrafish larvae during and after drug treatment. The results show a similar trend to the classical hair cell staining method. The startle response was reduced with drug treatment and recovered after removal of the drug. Together it demonstrated the capability of this behavioral assay in evaluating the hair cell functions of fish larvae and its potential as a high-throughput screening tool for auditory-related gene and drug discovery.
Full Text Available The zebrafish has become an established model organism for the study of hearing and balance systems in the past two decades. The classical approach to examine hair cells is to use dye to conduct selective staining, which shows the number and morphology of hair cells but does not reveal their function. Startle response is a behavior closely related to the auditory function of hair cells; therefore it can be used to measure the function of hair cells. In this study, we developed a device to measure the startle response of zebrafish larvae. By applying various levels of stimulus, it showed that the system can discern a 10 dB difference. The hair cell in zebrafish can regenerate after damage due to noise exposure or drug treatment. With this device, we measured the startle response of zebrafish larvae during and after drug treatment. The results show a similar trend to the classical hair cell staining method. The startle response was reduced with drug treatment and recovered after removal of the drug. Together it demonstrated the capability of this behavioral assay in evaluating the hair cell functions of fish larvae and its potential as a high-throughput screening tool for auditory-related gene and drug discovery.
Janusz Joachim Jadasz
Full Text Available Oligodendroglial progenitor/precursor cells (OPCs represent the main cellular source for the generation of new myelinating oligodendrocytes in the adult central nervous system (CNS. In demyelinating diseases such as multiple sclerosis (MS myelin repair activities based on recruitment, activation and differentiation of resident OPCs can be observed. However, the overall degree of successful remyelination is limited and the existence of an MS-derived anti-oligodendrogenic milieu prevents OPCs from contributing to myelin repair. It is therefore of considerable interest to understand oligodendroglial homeostasis and maturation processes in order to enable the development of remyelination therapies. Mesenchymal stem cells (MSC have been shown to exert positive immunomodulatory effects, reduce demyelination, increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. We here addressed whether MSC secreted factors can boost the OPC's oligodendrogenic capacity in a myelin non-permissive environment. To this end, we analyzed cellular morphologies, expression and regulation of key factors involved in oligodendroglial fate and maturation of primary rat cells upon incubation with MSC-conditioned medium. This demonstrated that MSC-derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. Accelerated maturation resulted in elevated levels of myelin expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as Id2 and Id4. We thus conclude that apart from their suggested application as potential anti-inflammatory and immunomodulatory MS treatment, these cells might also be exploited to support endogenous myelin repair activities.
Full Text Available Aminoglycosides (AGs are widely used antibiotics because of their low cost and high efficacy against gram-negative bacterial infection. However, AGs are ototoxic, causing the death of sensory hair cells in the inner ear. Strategies aimed at developing or discovering agents that protect against aminoglycoside ototoxicity have focused on inhibiting apoptosis or more recently, on preventing antibiotic uptake by the hair cells. Recent screens for ototoprotective compounds using the larval zebrafish lateral line identified phenoxybenzamine as a potential protectant for aminoglycoside-induced hair cell death. Here we used live imaging of FM1-43 uptake as a proxy for aminoglycoside entry, combined with hair-cell death assays to evaluate whether phenoxybenzamine can protect mammalian cochlear hair cells from the deleterious effects of the aminoglycoside antibiotic neomycin. We show that phenoxybenzamine can block FM1-43 entry into mammalian hair cells in a reversible and dose-dependent manner, but pre-incubation is required for maximal inhibition of entry. We observed differential effects of phenoxybenzamine on FM1-43 uptake in the two different types of cochlear hair cell in mammals, the outer hair cells (OHCs and inner hair cells (IHCs. The requirement for pre-incubation and reversibility suggests an intracellular rather than an extracellular site of action for phenoxybenzamine. We also tested the efficacy of phenoxybenzamine as an otoprotective agent. In mouse cochlear explants the hair cell death resulting from 24 h exposure to neomycin was steeply dose-dependent, with 50% cell death occurring at ~230 μM for both IHC and OHC. We used 250 μM neomycin in subsequent hair-cell death assays. At 100 μM with 1 h pre-incubation, phenoxybenzamine conferred significant protection to both IHCs and OHCs, however at higher concentrations phenoxybenzamine itself showed clear signs of ototoxicity and an additive toxic effect when combined with neomycin. These
Xiao, Shun-e; Hu, Zhi-qi; Feng, Chuan-bo; Liu, Ge; Miao, Yong
To construct a convenient, reliable and visual model of hair follicle development to test the hair-inductive potential of follicular cells and investigate the molecular mechanism regulating hair follicle morphogenesis and cycling. An open chamber was transplanted into the nude mice dorsal skin, dermal and epidermal cells isolated from newborn C57BL/6 mice skin were mixed at a specific ratio and then injected into the chamber together, 1 week after transplantation, the chamber was removed, and then, hair formation and regeneration after hair plucking was observed. 1 week after cells implantation, the wound was moist without apparent contraction and among that pink and translucent tissue was formed. 2 weeks after implantation, the wound healed completely. 3 weeks after implantation, black hair grew from the skin was observed. 4 weeks after implantation, thick and black hair grew from the skin vertically. Completely developed structure of hair follicle was observed with paraffin section and HE staining. 1 week after plucking, new hair had regrown. The ratio of cell component was varied, whereas the other component was fixed at 1 x 10(7) cells. When the number of epidermal cells was reduced to 1 x 10(6) cells, the efficiency of hair follicle reconstitution was mostly unchanged. On the other hand, the density of newly formed hair was diminished considerably by reducing the number of dermal cells to 5 x 10(6) cells or lower. Neither epidermal cells nor dermal cells transplanted alone formed hair follicle. Newborn mice skin cells transplanted by chamber method can construct a complete model of hair follicle development, which can be used to test the hair-inductive potential of follicular cells and investigate the molecular mechanism regulating hair follicle morphogenesis and cycling.
Trapani, Josef G; Obholzer, Nikolaus; Mo, Weike; Brockerhoff, Susan E; Nicolson, Teresa
To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses.
Josef G Trapani
Full Text Available To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1. Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses.
Cecola, R P; Bobbin, R P
In general, increasing external K+ concentration, as well as exposure to hypotonic medium, induces a shortening of outer hair cells (OHCs) accompanied by an increase in width and volume. One possible mechanism suggested for these changes is a movement of Cl- and/or water across the cell membrane. We therefore examined the role of Cl- in OHC volume maintenance by testing the effect of decreasing extracellular Cl- concentration on OHC length and shape. In addition, the effect of hypotonic medium was examined. OHCs were isolated from guinea pig cochleae, mechanically dissociated and dispersed, and placed in a modified Hanks balanced salt solution (HBS). Exposing the cells to a Cl(-)-free HBS produced an initial shortening, which was rapidly followed by an increase in length. After about 9 min of exposure to Cl(-)-free HBS, the cells appeared to lose all water and collapsed. Upon return to normal HBS, the OHCs returned to their normal shape. We speculate that the collapse of the OHCs may be due to the loss of intracellular Cl-, which, in turn, resulted in the loss of intracellular K+ and water. The results indicate that Cl- contributes greatly to the maintenance of OHC volume. In addition, we confirmed that isolated OHCs swell in hypotonic medium and maintain their swollen state until returned to normal medium. The mechanism for maintenance of the swollen state is unknown.
Full Text Available Objective: Advances in melanocyte culture techniques have not yet led to reliable clinical methods for treating hypopigmentation disorders. We hypothesized that melanocytes harvested from plucked hair follicles may provide a renewable source of melanocytes for the treatment of hypopigmentation. Methods: Hairs with attached cells from the follicles were plucked from Yucatan pigs and implanted in a collagen-glycosaminoglycan matrix for either immediate or delayed implantation into full-thickness excisional porcine wounds. Wounds were allowed to heal and were biopsied at 2 and 4 weeks, respectively. Results: Fully healed wounds with transplanted hair follicles showed central areas of dark pigmentation corresponding to the location of implanted hair follicles. Corresponding collagen-glycosaminoglycan matrix wounds showed no central areas of pigmentation. Conclusions: Hair follicle--derived melanocytes may potentially serve as a renewable source of pigment-producing cells for treating hypopigmentation disorders.
Severinsen, Stig A; Raarup, Merete Krog; Ulfendahl, Mats
Waltzing guinea pigs are an inbred guinea pig strain with a congenital and progressive balance and hearing disorder. A unique rod-shaped structure is found in the type I vestibular hair cells, that traverses the cell in an axial direction, extending towards the basement membrane. The present study...... estimates the total number of utricular hair cells and supporting cells in waltzing guinea pigs and age-matched control animals using the optical fractionator method. Animals were divided into four age groups (1, 7, 49 and 343 day-old). The number of type I hair cells decreased by 20% in the 343 day......-old waltzing guinea pigs compared to age-matched controls and younger animals. Two-photon confocal laser scanning microscopy using antibodies against fimbrin and betaIII-tubulin showed that the rods were exclusive to type I hair cells. There was no significant change in the length of the filament rods with age...
von Essen, Marina Rode; Kongsbak, Martin; Geisler, Carsten
During an immune response antigen-primed B-cells increase their antigen responsiveness by affinity maturation mediated by somatic hypermutation of the genes encoding the antigen-specific B-cell receptor (BCR) and by selection of higher-affinity B cell clones. Unlike the BCR, the T-cell receptor...
Ketelaar, T.; Emons, A.M.C.
The actin cytoskeleton plays an important role in root hair development. It is involved in both the delivery of growth materials to the expanding tip of root hairs and the regulation of the area of tip growth. This review starts with a discussion of the techniques that are available to visualize the
Ketelaar, T.; Emons, A.M.C.
The actin cytoskeleton plays an important role in root hair development. It is involved in both the delivery of growth materials to the expanding tip of root hairs and the regulation of the area of tip growth. This review starts with a discussion of the techniques that are available to visualize the
Driskell, Ryan R; Giangreco, Adam; Jensen, Kim B; Mulder, Klaas W; Watt, Fiona M
The dermal papilla comprises the specialised mesenchymal cells at the base of the hair follicle. Communication between dermal papilla cells and the overlying epithelium is essential for differentiation of the hair follicle lineages. We report that Sox2 is expressed in all dermal papillae at E16.5, but from E18.5 onwards expression is confined to a subset of dermal papillae. In postnatal skin, Sox2 is only expressed in the dermal papillae of guard/awl/auchene follicles, whereas CD133 is expressed both in guard/awl/auchene and in zigzag dermal papillae. Using transgenic mice that express GFP under the control of the Sox2 promoter, we isolated Sox2(+) (GFP(+)) CD133(+) cells and compared them with Sox2(-) (GFP(-)) CD133(+) dermal papilla cells. In addition to the 'core' dermal papilla gene signature, each subpopulation expressed distinct sets of genes. GFP(+) CD133(+) cells had upregulated Wnt, FGF and BMP pathways and expressed neural crest markers. In GFP(-) CD133(+) cells, the hedgehog, IGF, Notch and integrin pathways were prominent. In skin reconstitution assays, hair follicles failed to form when dermis was depleted of both GFP(+) CD133(+) and GFP(-) CD133(+) cells. In the absence of GFP(+) CD133(+) cells, awl/auchene hairs failed to form and only zigzag hairs were found. We have thus demonstrated a previously unrecognised heterogeneity in dermal papilla cells and shown that Sox2-positive cells specify particular hair follicle types.
Philp, Deborah; St-Surin, Sharleen; Cha, Hee-Jae; Moon, Hye-Sung; Kleinman, Hynda K; Elkin, Michael
Thymosin beta 4 is a small 43-amino-acid molecule that has multiple biological activities, including promotion of cell migration angiogenesis, cell survival, protease production, and wound healing. We have found that thymosin beta 4 promotes hair growth in various rat and mice models including a transgenic thymosin beta 4 overexpressing mouse. We have also determined the mechanism by which thymosin beta 4 acts to promote hair growth by examining its effects on follicle stem cell growth, migration, differentiation, and protease production.
Kil, J; Warchol, M E; Corwin, J T
We have examined the level of on-going cell death in the chick vestibular epithelia using the TUNEL method and compared this to the rate of on-going cell proliferation. Utricles contained 22.6 +/- 6.8 TUNEL-labeled cells (mean +/- s.e.m.) while saccules contained 15.1 +/- 4.0, with approximately 90% being labeled hair cells. In separate experiments, chicks were given a single injection of BrdU and killed 2 h later. Utricles contained 116.9 +/- 6.5 BrdU-labeled cells (mean +/- s.e.m.) and saccules contained 41.0 +/- 2.2. After 24 h in culture, utricles treated with 1 mM neomycin contained 115.5 +/- 38.9 TUNEL-labeled cells, an increase of 270% over controls. After 48 h, neomycin-treated saccules contained 40.9 +/- 7.8, an increase of 152% over controls. The majority of labeled cells were in the hair cell layer. Thus, neomycin exposure results in an apoptotic death of hair cells. The in vivo data measured here were used to estimate that the average life span of utricular hair cells in young chickens is approximately 20 days, in sharp contrast to the life spans assumed for hair cells in humans.
Yang, Xiao-Yu; Jin, Kai; Ma, Rui; Yang, Juan-Mei; Luo, Wen-Wei; Han, Zhao; Cong, Ning; Ren, Dong-Dong; Chi, Fang-Lu
Planar cell polarity (PCP) signaling regulates cochlear extension and coordinates orientation of sensory hair cells in the inner ear. Retroviral-mediated introduction of the Math1 transcription factor leads to the transdifferentiation of some mature supporting cells into hair cells. Testosterone, a gonadal sex steroid hormone, is associated with neuroprotection and regeneration in Central Nervous System (CNS) development. Experiments were performed in vitro using Ad5-EGFP-Math1/Ad5-Math1 in neonatal mouse cochleas. Establishment of ectopic hair-cell like cell(HCLC) polarity in the lesser epithelial ridge (LER) with or without testosterone-3-(O-carboxymethyl) oxime bovine serum albumin (testosterone-BSA) treatment was investigated to determine the role of the PCP pathway in regulating ectopic regenerated (HCLCs) through induction by Math1 and testosterone treatment. After Math1 infection, new ectopic regenerated HCLCs were detected in the LER. After the HCLCs developed actin-rich stereocilia, the basal bodies moved from the center to the distal side. Moreover, the narrower, non-sensory LER region meant that the convergent extension (CE) was also established after transfection with Math1. After 9 days of in vitro testosterone-BSA treatment, more Edu(+), Sox2(+), and HCLC cells were observed in the LER with an accompanying downregulation of E-cadherin. Interestingly, the CE of the Ad5-EGFP-math1 treated LER is altered, but the intrinsic cellular polarity of the HCLCs is not obviously changed. In summary, our results indicate that PCP signaling is involved in the development of ectopic HCLCs and the CE of the ectopic sensory region is altered by testosterone-BSA through downregulation of cell-cell adhesion. Testosterone-BSA and Math1 treatment could promote an increase in HCLCs in the LER through proliferation and transdifferentiation.
Song Eun Lee
Full Text Available Quiescent hair follicle (HF bulge stem cells (SCs differentiate to early progenitor (EP hair germ (HG cells, which divide to produce transit-amplifying matrix cells. EPs can revert to SCs upon injury, but whether this dedifferentiation occurs in normal HF homeostasis (hair cycle and the mechanisms regulating both differentiation and dedifferentiation are unclear. Here, we use lineage tracing, gain of function, transcriptional profiling, and functional assays to examine the role of observed endogenous Runx1 level changes in the hair cycle. We find that forced Runx1 expression induces hair degeneration (catagen and simultaneously promotes changes in the quiescent bulge SC transcriptome toward a cell state resembling the EP HG fate. This cell-state transition is functionally reversible. We propose that SC differentiation and dedifferentiation are likely to occur during normal HF degeneration and niche restructuring in response to changes in endogenous Runx1 levels associated with SC location with respect to the niche.
Full Text Available Hair follicle stem cells (HFSCs in the bugle circularly generate outer root sheath (ORS through linear proliferation within limited cycles during anagen phases. However, the mechanisms controlling the pace of HFSC proliferation remain unclear. Here we revealed that Foxp1, a transcriptional factor, was dynamically relocated from the nucleus to the cytoplasm of HFSCs in phase transitions from anagen to catagen, coupled with the rise of oxidative stress. Mass spectrum analyses revealed that the S468 phosphorylation of Foxp1 protein was responsive to oxidative stress and affected its nucleocytoplasmic translocation. Foxp1 deficiency in hair follicles led to compromised ROS accrual and increased HFSC proliferation. And more, NAC treatment profoundly elongated the anagen duration and HFSC proliferation in Foxp1-deficient background. Molecularly, Foxp1 augmented ROS levels through suppression of Trx1-mediated reductive function, thereafter imposing the cell cycle arrest by modulating the activity of p19/p53 pathway. Our findings identify a novel role for Foxp1 in controlling HFSC proliferation with cellular dynamic location in response to oxidative stress during hair cycling.
López-Schier, Hernán; Hudspeth, A J
The restoration of planar cell polarity is an essential but poorly understood step toward physiological recovery during sensory-organ regeneration. Investigating this issue in the lateral line of the zebrafish, we found that hair cells regenerate in pairs along a single axis established by the restricted localization and oriented division of their progenitors. By analyzing mutants lacking the planar-polarity determinant Vangl2, we ascertained that the uniaxial production of hair cells and the subsequent orientation of their hair bundles are controlled by distinct pathways, whose combination underlies the establishment of hair-cell orientation during development and regeneration. This mechanism may represent a general principle governing the long-term maintenance of planar cell polarity in remodeling epithelia.
Gaboyard-Niay, Sophie; Calin-Jageman, Irina; Chidavaenzi, Robstein L.; Venteo, Stephanie; Desmadryl, Gilles; Goldberg, Jay M.; Lysakowski, Anna; Chabbert, Christian
Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements. PMID:23049999
Full Text Available Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST, a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.
Full Text Available In humans, auditory hair cells are not replaced when injured. Thus, cochlear hair cell loss causes progressive and permanent hearing loss. Conversely, nonmammalian vertebrates are capable of regenerating lost sensory hair cells. The zebrafish lateral line has numerous qualities that make it well suited for studying hair cell development and regeneration. Histone deacetylase (HDAC activity has been shown to have an important role in regenerative processes in vertebrates, but its function in hair cell regeneration in vivo is not fully understood. Here, we have examined the role of HDAC activity in hair cell regeneration in the zebrafish lateral line. We eliminated lateral line hair cells of 5-day post-fertilization larvae using neomycin and then treated the larvae with HDAC inhibitors. To assess hair cell regeneration, we used 5-bromo-2-deoxyuridine (BrdU incorporation in zebrafish larvae to label mitotic cells after hair cell loss. We found that pharmacological inhibition of HDACs using trichostatin A (TSA or valproic acid (VPA increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage. Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21Cip1 and p27Kip1 expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos. Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis. Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line.
Matsumoto, Taro; Kano, Koichiro; Kondo, Daisuke; Fukuda, Noboru; Iribe, Yuji; Tanaka, Nobuaki; Matsubara, Yoshiyuki; Sakuma, Takahiro; Satomi, Aya; Otaki, Munenori; Ryu, Jyunnosuke; Mugishima, Hideo
When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. In the present study, we examined the multilineage differentiation potential of DFAT cells. DFAT cells obtained from adipose tissues of 18 donors exhibited a fibroblast-like morphology and sustained high proliferative activity. Flow cytometric analysis revealed that DFAT cells comprised a highly homogeneous cell population compared with that of adipose-derived stem/stromal cells (ASCs), although the cell-surface antigen profile of DFAT cells was very similar to that of ASCs. DFAT cells lost expression of mature adipocytes marker genes but retained or gained expression of mesenchymal lineage-committed marker genes such as peroxisome proliferator-activated receptor gamma (PPARgamma), RUNX2, and SOX9. In vitro differentiation analysis revealed that DFAT cells could differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate culture conditions. DFAT cells also formed osteoid matrix when implanted subcutaneously into nude mice. In addition, clonally expanded porcine DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages. These results indicate that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application for cell-based therapies.
Ding, Dalian; Roth, Jerome; Salvi, Richard
Occupational exposure to high atmospheric levels of Mn produces a severe and debilitating disorder known as manganism characterized by extrapyramidal disturbances similar to that seen in Parkinson's disease. Epidemiological and case studies suggest that persistent exposures to Mn may have deleterious effects on other organs including the auditory system and hearing. Mn accumulates in the inner ear following acute exposure raising the possibility that it can damage the sensory hair cells that convert sound into neural activity or spiral ganglion neurons (SGN) that transmit acoustic information from the hair cells to the brain via the auditory nerve. In this paper we demonstrate for first time that Mn causes significant damage to the sensory hair cells, peripheral auditory nerve fibers (ANF) and SGN in cochlear organotypic cultures isolated from postnatal day three rats. The peripheral ANF that make synaptic contact with the sensory hair cells were particularly vulnerable to Mn toxicity; damage occurred at concentrations as low 0.01 mM and increased with dose and duration of Mn exposure. Sensory hair cells, in contrast, were slightly more resistant to Mn toxicity than the ANF. Mn induced an atypical pattern of sensory cell damage; Mn was more toxic to inner hair cells (IHC) than outer hair cells (OHC) and in addition, IHC loss was relatively uniform along the length of the cochlea. Mn also caused significant loss and shrinkage of SGN soma. These findings are the first to demonstrate that Mn can produce severe lesions to both neurons and hair cells in the postnatal inner ear.
Conclusion: The present findings confirmed the protective effects of C1P in the cisplatin ototoxicity. The balance between ceramide and C1P may play a critical role in the determination of hair cell fate in cisplatin ototoxicity.
This paper discusses how ion transport proteins in the hair cells of the mammalian cochlea work to produce a sensitive but stable hearing organ. The transport proteins in the inner and outer hair cells are summarized (including their current voltage characteristics), and the roles of these proteins in determining intracellular Ca(2+), membrane potential, and ultimately cochlear sensitivity are discussed. The paper also discusses the role of the Ca(2+) sequestration sacs in outer hair cells in the autoregulation of hair cell membrane potential and cochlear gain, and how the underdamped control of Ca(2+) within these sacs may produce the observed slow oscillations in cochlear sensitivity and otoacoustic emissions after cochlear perturbations, including perilymphatic perfusions and prolonged low-frequency tones. The relative insensitivity of cochlear gain to short-term changes in the endocochlear potential is also discussed.
Peng, Anthony W; Effertz, Thomas; Ricci, Anthony J
Adaptation is a hallmark of hair cell mechanotransduction, extending the sensory hair bundle dynamic range while providing mechanical filtering of incoming sound. In hair cells responsive to low frequencies, two distinct adaptation mechanisms exist, a fast component of debatable origin and a slow myosin-based component. It is generally believed that Ca(2+) entry through mechano-electric transducer channels is required for both forms of adaptation. This study investigates the calcium dependence of adaptation in the mammalian auditory system. Recordings from rat cochlear hair cells demonstrate that altering Ca(2+) entry or internal Ca(2+) buffering has little effect on either adaptation kinetics or steady-state adaptation responses. Two additional findings include a voltage-dependent process and an extracellular Ca(2+) binding site, both modulating the resting open probability independent of adaptation. These data suggest that slow motor adaptation is negligible in mammalian auditory cells and that the remaining adaptation process is independent of calcium entry.
Makoto Akashi; Haruhiko Soma; Takuro Yamamoto; Asuka Tsugitomi; Shiko Yamashita; Takuya Yamamoto; Eisuke Nishida; Akio Yasuda; James K. Liao; Koichi Node; Joseph S. Takahashi
.... This limitation has greatly hampered our understanding of human circadian rhythm. Here we report a convenient, reliable, and less invasive method for detecting human clock gene expression using biopsy samples of hair follicle cells from the head or chin...
Le, Quang; Tabuchi, Keiji; Hara, Akira
Ceramide-1-phosphate (C1P) is a phosphorylated form of ceramide. While ceramide is known to be an inducer of apoptosis of cochlear hair cells in cisplatin ototoxicity, little is known about the function of C1P in cochlear diseases. The present study was designed to examine whether C1P could protect cochlear hair cells against cisplatin ototoxicity. Explants of cochlear basal turns collected from C57BL/6J mice at postnatal days 3-5 were used in all experiments. Cochlear explants were exposed to 5 or 10 μM cisplatin for 48 h to assess the effects of C1P, NVP-231 (a ceramide kinase inhibitor), or ceramide. Western blotting of pAkt/Akt and pMAPK/MAPK was examined to check whether this pathway was modulated by C1P. C1P activated the Akt and MAPK pathway and significantly reduced cochlear cell death induced by cisplatin. Coadministration of cisplatin and ceramide significantly increased cochlear hair cell death. In addition, when treating cochlear hair cells with NVP-231 in the presence of cisplatin or ceramide, a remarkable increase in apoptosis of hair cells was observed. The present findings confirmed the protective effects of C1P in the cisplatin ototoxicity. The balance between ceramide and C1P may play a critical role in the determination of hair cell fate in cisplatin ototoxicity.
Lou, Xiangxin; Yuan, Huihua; Xie, Jing; Wang, Xianliu; Yang, Liangliang; Zhang, Yanzhong
We have demonstrated that selected growth factors are involved in regulating survival and proliferation of progenitor cells derived from the neonatal rat organ of Corti (OC). The protective and regenerative effects of these defined growth factors on the injured organ of Corti were therefore investigated. The organ of Corti dissected from the Wistar rat pups (P3-P5) was split into apical, middle, and basal parts, explanted and cultured with or without neomycin and growth factors. Insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and epidermal growth factor (EGF) protected the inner hair cells (IHCs) and outer hair cells (OHCs) from neomycin ototoxicity. Using EGF, IGF-1, and FGF-2 alone induced no protective effect on the survival of auditory hair cells. Combining 2 growth factors (EGF + IGF-1, EGF + FGF-2, or IGF-1 + FGF-2) gave statistically protective effects. Similarly, combining all three growth factors effectively protected auditory hair cells from the ototoxic insult. None of the growth factors induced regeneration of hair cells in the explants injured with neomycin. Thus various combinations of the three defined factors (IGF-1, FGF-2, and EGF) can protect the auditory hair cells from the neomycin-induced ototoxic damage, but no regeneration was seen. This offers a possible novel approach to the treatment of hearing loss.
Köppl, Christine; Forge, Andrew; Manley, Geoffrey A
Hair cells are the mechanoreceptive cells of the vertebrate lateral line and inner ear. In addition to their sensory function, hair cells display motility and thus themselves generate mechanical energy, which is thought to enhance sensitivity. Two principal cellular mechanism are known that can mediate hair-cell motility in vitro. One of these is based on voltage-dependent changes of an intramembrane protein and has so far been demonstrated only in outer hair cells of the mammalian cochlea. Correlated with this, the cell membranes of outer hair cells carry an extreme density of embedded particles, as revealed by freeze fracturing. The present study explored the possibility of membrane-based motility in hair cells of nonmammals, by determining their density of intramembrane particles. Replicas of freeze-fractured membrane were prepared from auditory hair cells of a lizard, the Tokay gecko, and a bird, the barn owl. These species were chosen because of independent evidence for active cochlear mechanics, in the form of spontaneous otoacoustic emissions. For quantitative comparison, mammalian inner and outer hair cells, as well as vestibular hair, cells were reevaluated. Lizard and bird hair cells displayed median densities of 2,360 and 1,880 intramembrane particles/microm2, respectively. This was not significantly different from the densities in vestibular and mammalian inner hair cells; however, it was about half the density in of mammalian outer hair cells. This suggests that nonmammalian hair cells do not possess high densities of motor protein in their membranes and are thus unlikely to be capable of somatic motility. 2004 Wiley-Liss, Inc.
Huifang Sun; Chia-Hui Lin; Smith, Michael E.
BACKGROUND: Previous microarray analysis showed that growth hormone (GH) was significantly upregulated following acoustic trauma in the zebrafish (Danio rerio) ear suggesting that GH may play an important role in the process of auditory hair cell regeneration. Our objective was to examine the effects of exogenous and endogenous GH on zebrafish inner ear epithelia following acoustic trauma. METHODOLOGY/PRINCIPAL FINDINGS: We induced auditory hair cell damage by exposing zebrafish to acoustic o...
Full Text Available The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60-70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.
The neural processing of gravitational-produced sensory stimulation of statocyst hair cells in the nudibranch mollusk Hermissenda was studied. The goal in these studies was to understand how: gravireceptor neurons sense or transduce gravitational forces, gravitational stimulation is integrated so as to produce a graded receptor potential, and ultimately the generation of an action potential, and various neural adaptation phenomena which hair cells exhibit arise. The approach to these problems was primarily electrophysical.
Kandyba, Eve; Kobielak, Krzysztof
The hair follicle (HF) is an exceptional mini-organ to study the mechanisms which regulate HF morphogenesis, cycling, hair follicle stem cell (hfSCs) homeostasis, and progeny differentiation. During morphogenesis, Wnt signaling is well-characterized in the initiation of HF patterning but less is known about which particular Wnt ligands are required and whether individual Wnt ligands act in an indispensable or redundant manner during postnatal hfSCs anagen onset and HF cycle progression. Previously, we described the function of the bone morphogenetic protein (BMP) signaling target gene WNT7a in intrinsic regulation of hfSCs homeostasis in vivo. Here, we investigated the role of Wnt7b, which was also intrinsically upregulated in hfSCs during physiological and precocious anagen after BMP inhibition in vivo. We demonstrated Wnt7b to be a direct target of canonical BMP signaling in hfSCs and using Wnt7b conditional gene targeting during HF morphogenesis revealed disrupted HF cycling including a shorter anagen, premature catagen onset with overall shorter hair production, and diminished HF differentiation marker expression. Additionally, we observed that postnatal ablation of Wnt7b resulted in delayed HF activation, affecting both the hair germ and bulge hfSCs but still maintaining a two-step sequence of HF stimulation. Interestingly, Wnt7b cKO hfSCs participated in reformation of the new HF bulge, but with slower self-renewal. These findings demonstrate the importance of intrinsic Wnt7b expression in hfSCs regulation and normal HF cycling and surprisingly reveal a nonredundant role for Wnt7b in the control of HF anagen length and catagen entry which was not compensated by other Wnt ligands.
Maass, Juan C.; Gu, Rende; Cai, Tiantian; Wan, Ying-Wooi; Cantellano, Silvia C.; Asprer, Joanna S. T.; Zhang, Hongyuan; Jen, Hsin-I; Edlund, Renée K.; Liu, Zhandong; Groves, Andrew K.
Neonatal mouse cochlear supporting cells have a limited ability to divide and trans-differentiate into hair cells, but this ability declines rapidly in the two weeks after birth. This decline is concomitant with the morphological and functional maturation of the organ of Corti prior to the onset of hearing. However, despite this association between maturation and loss of regenerative potential, little is known of the molecular changes that underlie these events. To identify these changes, we used RNA-seq to generate transcriptional profiles of purified cochlear supporting cells from 1- and 6-day-old mice. We found many significant changes in gene expression during this period, many of which were related to regulation of proliferation, differentiation of inner ear components and the maturation of the organ of Corti prior to the onset of hearing. One example of a change in regenerative potential of supporting cells is their robust production of hair cells in response to a blockade of the Notch signaling pathway at the time of birth, but a complete lack of response to such blockade just a few days later. By comparing our supporting cell transcriptomes to those of supporting cells cultured in the presence of Notch pathway inhibitors, we show that the transcriptional response to Notch blockade disappears almost completely in the first postnatal week. Our results offer some of the first molecular insights into the failure of hair cell regeneration in the mammalian cochlea. PMID:27918591
He, Yingzi; Yu, Huiqian; Cai, Chengfu; Sun, Shan; Chai, Renjie; Li, Huawei
Aminoglycoside-induced hair cell loss is a major cause of hearing impairment in children and deserves more attention in medical research. Epigenetic mechanisms have been shown to protect hair cells from ototoxic drugs. In this study, we focused on the role of dimethylated histone H3K4 (H3K4me2) in hair cell survival. To investigate the effects of lysine-specific demethylase 1 (LSD1)--the histone demethylase primarily responsible for demethylating H3K4me2--on neomycin-induced hair cell loss, isolated cochleae were pretreated with LSD1 inhibitors followed by neomycin exposure. There was a severe loss of hair cells in the organ of Corti after neomycin exposure, and inhibition of LSD1 significantly protected against neomycin-induced hair cell loss. H3K4me2 expression in the nuclei of hair cells decreased after exposure to neomycin, and blocking the decreased expression of H3K4me2 with LSD1 inhibitors prevented hair cell loss. Local delivery of these inhibitors in vivo also protected hair cells from neomycin-induced ototoxicity and maintained the hearing threshold in mice as determined by auditory brain stem response. This inhibition of neomycin-induced apoptosis occurs via reduced caspase-3 activation. Together, our findings demonstrate the protective role for H3K4me2 against neomycin-induced hair cell loss and hearing loss.
Shin, Hyoseung; Won, Chong Hyun; Chung, Woon-Kyung; Park, Byung-Soon
The primary roles of mesenchymal stem cells (MSCs) are to maintain the stem cell niche, facilitate recovery after injury, and ensure healthy aging and the homeostasis of organ and tissues. MSCs have recently emerged as a new therapeutic option for hair loss. Since adipose-derived stem cells (ADSCs) are the most accessible sources of MSCs, ADSC-based hair regeneration is currently under investigation. Besides replacing degenerated cells in affected organs, ADSCs exhibit their beneficial effects through the paracrine actions of various cytokines and growth factors. Several laboratory experiments and animal studies have shown that ADSC-related proteins can stimulate hair growth. In this paper, we introduce our clinical pilot studies using conditioned media of ADSCs for pattern hair loss in men and women. We also discuss practical therapeutic challenges and the direction of future research. Copyright© Bentham Science Publishers; For any queries, please email at firstname.lastname@example.org.
YU Ning; ZHAI Suo-qiang; YANG Shi-ming; HAN Dong-yi; ZHAO Hong-bo
Prestin has been identified as a motor protein responsible for outer hair cell (OHC) electromotility and is expressed on the OHC surface. Previous studies revealed that OHC eleetromotility and its associated nonlinear capacitance were mainly located at the OHC lateral wall and absent at the apical cutieular plate and the basal nucleus region. Immunofluorescent staining for prestin also failed to demonstrate prestin expression at the OHC basal ends in whole-mount preparation of the organ of Corti. However, there lacks a definitive demonstration of the pattern of prestin distribution. The OHC lateral wall has a trilaminate organization and is composed of the plasma membrane, cortical lattice, and subsurface cisternae. In this study, the location of prestin proteins in dissociated OHCs was examined using immunofluorescent staining and confocal microscopy. We found that prestin was uniformly expressed on the basolateral surface, including the basal pole. No staining was seen on the cuticular plate and stereocilia. When co-stained with a membrane marker di-8-ANEPPS, prestin-labeling was found to be in the outer layer of the OHC lateral wall. After separating the plasma membrane from the underlying subsurface eisternae using a hypotonic extracellular solution, prestin-labeling was found to be in the plasma membrane, not the subsurface cisternae. The data show that prestin is expressed in the plasma membrane on the entire OHC basolateral surface.
Ana Maria Abreu Velez
Full Text Available Context: Hair loss is one of the most striking clinical features of active systemic lupus erythematosus (SLE, however, very few studies have investigated the immunological features of this process. Case report: We describe a 33 years old female who presented with scalp hair loss and arthralgias. Physical examination revealed erythematous plaques on the nose and scalp, with bitemporal hair loss. Scalp biopsies revealed epidermal hyperkeratosis, with a mild interface infiltrate of lymphocytes and histiocytes and a superficial and deep, perivascular and periadnexal infiltrate of mostly CD4 positive cells. Antibodies to HAM 56, CD68, CD1a, S-100, mast cell tryptase and c-kit/CD117 were strongly positive around the hair follicles, and in the adjacent sebaceous glands. Conclusion : We present the first report showing a significant presence of several antigen presenting cells around the hair follicular units in a patient with alopecia in active SLE. Today, antigen presenting cells and dendritic cells (DC are modeled as the master regulators of human immunity. One aspect that has become clearly appreciated is the great diversity of DC subtypes, each with considerable functional differences. Thus, we suggest that APC and DCs are equipped with Pattern Recognition Receptors (PRRs to some hair follicular unit antigens; that these innate sensors recognize conserved molecular patterns on self- tissue, and play a significant role in the pathophysiology of alopecia in SLE patients
Full Text Available Wnt signaling is a highly conserved pathway crucial for development and homeostasis of multicellular organisms. Secreted Wnt ligands bind Frizzled receptors to regulate diverse processes such as axis patterning, cell division, and cell fate specification. They also serve to govern self-renewal of somatic stem cells in several adult tissues. The complexity of the pathway can be attributed to the myriad of Wnt and Frizzled combinations as well as its diverse context-dependent functions. In the developing mouse inner ear, Wnt signaling plays diverse roles, including specification of the otic placode and patterning of the otic vesicle. At later stages, its activity governs sensory hair cell specification, cell cycle regulation, and hair cell orientation. In regenerating sensory organs from non-mammalian species, Wnt signaling can also regulate the extent of proliferative hair cell regeneration. This review describes the current knowledge of the roles of Wnt signaling and Wnt-responsive cells in hair cell development and regeneration. We also discuss possible future directions and the potential application and limitation of Wnt signaling in augmenting hair cell regeneration.
Mahmoodian Sani, Mohammad Reza; Hashemzadeh-Chaleshtori, Morteza; Saidijam, Massoud; Jami, Mohammad-Saeid; Ghasemi-Dehkordi, Payam
miRNAs are essential factors of an extensively conserved post-transcriptional process controlling gene expression at mRNA level. Varoius biological processes such as growth and differentiation are regulated by miRNAs. Web of Science and PubMed databases were searched using the Endnote software for the publications about the role miRNA-183 family in inner ear: hair cell development and deafness published from 2000 to 2016. A triplet of these miRNAs particularly the miR-183 family is highly expressed in vertebrate hair cells, as with some of the peripheral neurosensory cells. Point mutations in one member of this family, miR-96, underlie DFNA50 autosomal deafness in humans and lead to abnormal hair cell development and survival in mice. In zebrafish, overexpression of the miR-183 family induces extra and ectopic hair cells, while knockdown decreases the number of hair cell. The miR-183 family (miR-183, miR-96 and miR-182) is expressed abundantly in some types of sensory cell in the eye, nose and inner ear. In the inner ear, mechanosensory hair cells have a robust expression level. Despite much similarity of these miRs sequences, small differences lead to distinct targeting of messenger RNAs targets. In the near future, miRNAs are likely to be explored as potential therapeutic agents to repair or regenerate hair cells, cell reprogramming and regenerative medicine applications in animal models because they can simultaneously down-regulate dozens or even hundreds of transcripts.
Plikus, Maksim V
The timing mechanism of the hair cycle remains poorly understood. However, it has become increasingly clear that the telogen-to-anagen transition is controlled jointly by at least the bone morphogenic protein (BMP), WNT, fibroblast growth factor (FGF), and transforming growth factor (TGF)-β signaling pathways. New research shows that Fgf18 signaling in hair follicle stem cells synergizes BMP-mediated refractivity, whereas Tgf-β2 signaling counterbalances it. Loss of Fgf18 signaling markedly accelerates anagen initiation, whereas loss of Tgf-β2 signaling significantly delays it, supporting key roles for these pathways in hair cycle timekeeping.
ZHANG Yuan; ZHAI Suo-qiang; SONG Wei; GUO Wei; ZHENG Gui-liang; HU Yin-yan
Objective To present an experimental method that allows isolation of greater epithelial ridge (GER) and lesser epithelial ridge(LER) cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation and to investigate a retrovirus-mediated gene transfer technique for its possibl utility in immortalization of the GER and LER cell lines, in an effort to establish an in vitro model system of hair cell differentiation. Methods GER and LER cells were dissected from postnatal rat cochleae and immortalized by transferring the SV40 large T antigen using a retrovirus. The established cell lines were confirmed through morphology observation, immunnocytochemical staining and RT-PCR analysis. The Hathl gene was transferred into the cell lines using adenovirus-mediated techniques to explore their potential to differentiate into hair cells. Results The established cell lines were stably maintained for more than 20 passages and displayed many features similar to primary GER and LER cells. They grew in patches and assumed a polygonal morphology. Immunostaining showed labeling by SV40 large T antigen and Islet1 (a specific marker for GER and LER). All passages of the cell lines expressed SV40 large T antigen on RT-PCR analysis. The cells also showed the capability to differenti-ate into hair cell-like cells when forced to express Hathl. Conclusion Retrovirus-mediated gene transfer can be used in establishing immortalized progenitor hair cell lines in newborn rat, which may provide an invaluable system for studying hair cell differentiation and regeneration for new treatment of sensory hearing loss caused by hair cell loss.
Kateri J Spinelli
Full Text Available We optimized Fluo-4 AM loading of chicken cochlea to report hair-bundle Ca(2+ signals in populations of hair cells. The bundle Ca(2+ signal reported the physiological state of the bundle and cell; extruding cells had very high bundle Fluo-4 fluorescence, cells with intact bundles and tip links had intermediate fluorescence, and damaged cells with broken tip links had low fluorescence. Moreover, Fluo-4 fluorescence in the bundle correlated with Ca(2+ entry through transduction channels; mechanically activating transduction channels increased the Fluo-4 signal, while breaking tip links with Ca(2+ chelators or blocking Ca(2+ entry through transduction channels each caused bundle and cell-body Fluo-4 fluorescence to decrease. These results show that when tip links break, bundle and soma Ca(2+ decrease, which could serve to stimulate the hair cell's tip-link regeneration process. Measurement of bundle Ca(2+ with Fluo-4 AM is therefore a simple method for assessing mechanotransduction in hair cells and permits an increased understanding of the interplay of tip links, transduction channels, and Ca(2+ signaling in the hair cell.
Spinelli, Kateri J; Gillespie, Peter G
We optimized Fluo-4 AM loading of chicken cochlea to report hair-bundle Ca(2+) signals in populations of hair cells. The bundle Ca(2+) signal reported the physiological state of the bundle and cell; extruding cells had very high bundle Fluo-4 fluorescence, cells with intact bundles and tip links had intermediate fluorescence, and damaged cells with broken tip links had low fluorescence. Moreover, Fluo-4 fluorescence in the bundle correlated with Ca(2+) entry through transduction channels; mechanically activating transduction channels increased the Fluo-4 signal, while breaking tip links with Ca(2+) chelators or blocking Ca(2+) entry through transduction channels each caused bundle and cell-body Fluo-4 fluorescence to decrease. These results show that when tip links break, bundle and soma Ca(2+) decrease, which could serve to stimulate the hair cell's tip-link regeneration process. Measurement of bundle Ca(2+) with Fluo-4 AM is therefore a simple method for assessing mechanotransduction in hair cells and permits an increased understanding of the interplay of tip links, transduction channels, and Ca(2+) signaling in the hair cell.
Phillip M Uribe
Full Text Available Children born to mothers with substantial alcohol consumption during pregnancy can present a number of morphological, cognitive, and sensory abnormalities, including hearing deficits, collectively known as fetal alcohol syndrome (FAS. The goal of this study was to determine if the zebrafish lateral line could be used to study sensory hair cell abnormalities caused by exposure to ethanol during embryogenesis. Some lateral line sensory hair cells are present at 2 days post-fertilization (dpf and are functional by 5 dpf. Zebrafish embryos were raised in fish water supplemented with varying concentrations of ethanol (0.75%-1.75% by volume from 2 dpf through 5 dpf. Ethanol treatment during development resulted in many physical abnormalities characteristic of FAS in humans. Also, the number of sensory hair cells decreased as the concentration of ethanol increased in a dose-dependent manner. The dye FM 1-43FX was used to detect the presence of functional mechanotransduction channels. The percentage of FM 1-43-labeled hair cells decreased as the concentration of ethanol increased. Methanol treatment did not affect the development of hair cells. The cell cycle markers proliferating cell nuclear antigen (PCNA and bromodeoxyuridine (BrdU demonstrated that ethanol reduced the number of sensory hair cells, as a consequence of decreased cellular proliferation. There was also a significant increase in the rate of apoptosis, as determined by TUNEL-labeling, in neuromasts following ethanol treatment during larval development. Therefore, zebrafish are a useful animal model to study the effects of hair cell developmental disorders associated with FAS.
Volgger, Michael; Lang, Ingeborg; Ovecka, Miroslav; Lichtscheidl, Irene
We analysed cell wall formation in rapidly growing root hairs of Triticum aestivum under reduced turgor pressure by application of iso- and hypertonic mannitol solutions. Our experimental series revealed an osmotic value of wheat root hairs of 150 mOsm. In higher concentrations (200-650 mOsm), exocytosis of wall material and its deposition, as well as callose synthesis, still occurred, but the elongation of root hairs was stopped. Even after strong plasmolysis when the protoplast retreated from the cell wall, deposits of wall components were observed. Labelling with DiOC(6)(3) and FM1-43 revealed numerous Hechtian strands that spanned the plasmolytic space. Interestingly, the Hechtian strands also led towards the very tip of the root hair suggesting strong anchoring sites that are readily incorporated into the new cell wall. Long-term treatments of over 24 h in mannitol solutions (150-450 mOsm) resulted in reduced growth and concentration-dependent shortening of root hairs. However, the formation of new root hairs does occur in all concentrations used. This reflects the extraordinary potential of wheat root cells to adapt to environmental stress situations.
Tominaga-Wada, Rumi; Wada, Takuji
CAPRICE (CPC) encodes a small protein with an R3 MYB motif and regulates root hair and trichome cell differentiation in Arabidopsis thaliana. Six additional CPC-like MYB proteins including TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ENHANCER OF TRY AND CPC2 (ETC2), ENHANCER OF TRY AND CPC3/CPC-LIKE MYB3 (ETC3/CPL3), TRICHOMELESS1 (TCL1), and TRICHOMELESS2/CPC-LIKE MYB4 (TCL2/CPL4) also have the ability to regulate root hair and/or trichome cell differentiation in Arabidopsis. In this review, we describe our latest findings on how CPC-like MYB transcription factors regulate root hair cell differentiation. Recently, we identified the tomato SlTRY gene as an ortholog of the Arabidopsis TRY gene. Transgenic Arabidopsis plants harboring SlTRY produced more root hairs, a phenotype similar to that of 35S::CPC transgenic plants. CPC is also known to be involved in anthocyanin biosynthesis. Anthocyanin accumulation was repressed in the SlTRY transgenic plants, suggesting that SlTRY can also influence anthocyanin biosynthesis. We concluded that tomato and Arabidopsis partially use similar transcription factors for root hair cell differentiation, and that a CPC-like R3 MYB may be a key common regulator of plant root-hair development.
Full Text Available CAPRICE (CPC encodes a small protein with an R3 MYB motif and regulates root hair and trichome cell differentiation in Arabidopsis thaliana. Six additional CPC-like MYB proteins including TRIPTYCHON (TRY, ENHANCER OF TRY AND CPC1 (ETC1, ENHANCER OF TRY AND CPC2 (ETC2, ENHANCER OF TRY AND CPC3/ CPC-LIKE MYB3 (ETC3/CPL3, TRICHOMELESS1 (TCL1 and TRICHOMELESS2/CPC-LIKE MYB4 (TCL2/CPL4 also have the ability to regulate root hair and/or trichome cell differentiation in Arabidopsis. In this review, we describe our latest findings on how CPC-like MYB transcription factors regulate root hair cell differentiation. Recently, we identified the tomato SlTRY gene as an ortholog of the Arabidopsis TRY gene. Transgenic Arabidopsis plants harboring SlTRY produced more root hairs, a phenotype similar to that of 35S::CPC transgenic plants. CPC is also known to be involved in anthocyanin biosynthesis. Anthocyanin accumulation was repressed in the SlTRY transgenic plants, suggesting that SlTRY can also influence anthocyanin biosynthesis. We concluded that tomato and Arabidopsis partially use similar transcription factors for root hair cell differentiation, and that a CPC-like R3 MYB may be a key common regulator of plant root-hair development.
Furmanski, Anna L; O'Shaughnessy, Ryan F L; Saldana, Jose Ignacio; Blundell, Michael P; Thrasher, Adrian J; Sebire, Neil J; Davies, E Graham; Crompton, Tessa
Here we present a mouse model for T-cell targeting of hair follicles, linking the pathogenesis of alopecia to that of depigmentation disorders. Clinically, thymus transplantation has been successfully used to treat T-cell immunodeficiency in congenital athymia, but is associated with autoimmunity. We established a mouse model of thymus transplantation by subcutaneously implanting human thymus tissue into athymic C57BL/6 nude mice. These xenografts supported mouse T-cell development. Surprisingly, we did not detect multiorgan autoimmune disease. However, in all transplanted mice, we noted a striking depigmentation and loss of hair follicles. Transfer of T cells from transplanted nudes to syngeneic black-coated RAG(-/-) recipients caused progressive, persistent coat-hair whitening, which preceded patchy hair loss in depigmented areas. Further transfer experiments revealed that these phenomena could be induced by CD4+ T cells alone. Immunofluorescent analysis suggested that Trp2+ melanocyte-lineage cells were decreased in depigmented hair follicles, and pathogenic T cells upregulated activation markers when exposed to C57BL/6 melanocytes in vitro, suggesting that these T cells are not tolerant to self-melanocyte antigens. Our data raise interesting questions about the mechanisms underlying tissue-specific tolerance to skin antigens.
Full Text Available Loss of sensory hair cells of the inner ear due to aminoglycoside exposure is a major cause of hearing loss. Using an immortalized multipotent otic progenitor (iMOP cell line, specific signaling pathways that promote otic cell survival were identified. Of the signaling pathways identified, the PI3K pathway emerged as a strong candidate for promoting hair cell survival. In aging animals, components for active PI3K signaling are present but decrease in hair cells. In this study, we determined whether activated PI3K signaling in hair cells promotes survival. To activate PI3K signaling in hair cells, we used a small molecule inhibitor of PTEN or genetically ablated PTEN using a conditional knockout animal. Hair cell survival was challenged by addition of gentamicin to cochlear cultures. Hair cells with activated PI3K signaling were more resistant to aminoglycoside-induced hair cell death. These results indicate that increased PI3K signaling in hair cells promote survival and the PI3K signaling pathway is a target for preventing aminoglycoside-induced hearing loss.
Matthias, Patrick; Rolink, Antonius G
The development of B cells from haematopoietic stem cells proceeds along a highly ordered, yet flexible, pathway. At multiple steps along this pathway, cells are instructed by transcription factors on how to further differentiate, and several check-points have been identified. These check-points are initial commitment to lymphocytic progenitors, specification of pre-B cells, entry to the peripheral B-cell pool, maturation of B cells and differentiation into plasma cells. At each of these regulatory nodes, there are transcriptional networks that control the outcome, and much progress has recently been made in dissecting these networks. This article reviews our current understanding of this exciting field.
Accumulation of excess glutamate plays a central role in eliciting the pathological events that follow intensely loud noise exposures and ischemia-reperfusion injury. Glutamate excitotoxicity has been characterized in cochlear nerve terminals, but much less is known about whether excess glutamate signaling also contributes to pathological changes in sensory hair cells. I therefore examined whether glutamate excitotoxicity damages hair cells in zebrafish larvae exposed to drugs that mimic excitotoxic trauma. Exposure to ionotropic glutamate receptor (iGluR) agonists, kainic acid (KA) or N-methyl-D-aspartate (NMDA), contributed to significant, progressive hair cell loss in zebrafish lateral-line organs. To examine whether hair-cell loss was a secondary effect of excitotoxic damage to innervating neurons, I exposed neurog1a morphants—fish whose hair-cell organs are devoid of afferent and efferent innervation—to KA or NMDA. Significant, dose-dependent hair-cell loss occurred in neurog1a morphants exposed to either agonist, and the loss was comparable to wild-type siblings. A survey of iGluR gene expression revealed AMPA-, Kainate-, and NMDA-type subunits are expressed in zebrafish hair cells. Finally, hair cells exposed to KA or NMDA appear to undergo apoptotic cell death. Cumulatively, these data reveal that excess glutamate signaling through iGluRs induces hair-cell death independent of damage to postsynaptic terminals. PMID:28112265
Full Text Available Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 µM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa’s protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa’s robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.
Zorrilla de San Martín, Javier; Ballestero, Jimena; Katz, Eleonora; Elgoyhen, A Belén; Fuchs, Paul A
The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include 'ryanodine receptors', whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the alpha9alpha10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the alpha9alpha10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 microM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken 'short' hair cells likewise was enhanced in the presence of 100 microM ryanodine. This facilitatory effect on current through the AChR could enhance brief ( approximately 1 s) activation of associated calcium-dependent K(+) (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate alpha9alpha10-mediated responses and for potential inner ear therapeutics based on this interaction.
Full Text Available The dermal sheath (DS of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres. The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.
Mercati, F.; Pascucci, L.; Ceccarelli, P.; Dall’Aglio, C.; Pedini, V.; Gargiulo, A.M.
The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.
Geoffrey C Horwitz
Full Text Available The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were originally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as If, IQ or Ih. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50-70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.
Chen, Yan; Yu, Huiqian; Zhang, Yanping; Li, Wen; Lu, Na; Ni, Wenli; He, Yingzi; Li, Jin; Sun, Shan; Wang, Zhengmin; Li, Huawei
The ideal strategy for hair cell regeneration is promoting residual supporting cell proliferation followed by induction of hair cell differentiation. In this study, cultured neonatal mouse organs of Corti were treated with neomycin to eliminate hair cells followed by incubation with recombined adenovirus expressing Pax2 and/or Math1. Results showed that overexpression of Pax2 significantly promoted proliferation of supporting cells. The number of BrdU⁺/myosin VIIA⁺ cells increased significantly in hair cell pre-existing region two weeks after adenovirus infection in Ad-Pax2-IRES-Math1 group compared to Ad-Pax2 and Ad-Math1 groups. This indicated that cotransfection of Pax2 and Math1 induced supporting cells to proliferate and differentiate into hair cells in situ. Most new hair cells were labeled by FM1-43 suggesting they acquired certain function. The results also suggest that inducing proliferating cells rather than quiescent cells to differentiate into hair cells by forced expression of Math1 is feasible for mammalian hair cell regeneration.
Liu, Yaping; Lyle, Stephen; Yang, Zaixin; Cotsarelis, George
Putative epithelial stem cells in the hair follicle bulge are thought to play pivotal roles in the homeostasis, aging, and carcinogenesis of the cutaneous epithelium. Elucidating the role of bulge cells in these processes has been hampered by the lack of gene promoters that target this area with specificity. Here we describe the isolation of the mouse keratin 15 (K15) promoter and demonstrate its utility for preferentially targeting hair follicle bulge cells in adult K15/lacZ transgenic mice. We found that patterns of K15 expression and promoter activity changed with age and correlated with levels of differentiation within the cutaneous epithelium; less differentiated keratinocytes in the epidermis of the neonatal mouse and in the bulge area of the adult mouse preferentially expressed K15. These findings demonstrate the utility of the K15 promoter for targeting epithelial stem cells in the hair follicle bulge and set the stage for elucidating the role of bulge cells in skin biology.
It is widely thought that organisms detect sound by sensing the deflection of hair-like projections, the stereocilia, at the apex of hair cells. In the case of mammals, the standard interpretation is that hair cells in the cochlea respond to deflection of stereocilia induced by motion generated by a hydrodynamic travelling wave. But in the light of persistent anomalies, an alternative hypothesis seems to have some merit: that sensing cells (in particular the outer hair cells) may, at least at low intensities, be reacting to a different stimulus – the rapid pressure wave that sweeps through the cochlear fluids at the speed of sound in water. This would explain why fast responses are sometimes seen before the peak of the travelling wave. Yet how could cells directly sense fluid pressure? Here, a model is constructed of the outer hair cell as a pressure vessel able to sense pressure variations across its cuticular pore, and this ‘fontanelle’ model, based on the sensing action of the basal body at this compliant spot, could explain the observed anomalies. Moreover, the fontanelle model can be applied to a wide range of other organisms, suggesting that direct pressure detection is a general mode of sensing complementary to stereociliar displacement.
Matthews, T M; Duncan, R K; Zidanic, M; Michael, T H; Fuchs, P A
In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74+/-0.17 microM. The expressed channels were blocked by apamin (IC(50)=73.3+/-5.0 pM) and d-tubocurarine (IC(50)=7.6+/-1.0 microM), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.
Wit, HP; vanDijk, P; Segenhout, HM
Frequency and impulse responses were determined for isolated guinea pig outer hair cells by electrically stimulating the cells between two wire electrodes with white noise. Cells were attached to the bottom of a small culture dish at one end while the other end was freely moving. Results have the ch
Full Text Available Although STAT3 signaling is demonstrated to regulate sensory cell differentiation and regeneration in the zebrafish, its exact role is still unclear in mammalian cochleae. Here, we report that STAT3 and its activated form are specifically expressed in hair cells during mouse cochlear development. Importantly, conditional cochlear deletion of Stat3 leads to an inhibition on hair cell differentiation in mice in vivo and in vitro. By cell fate analysis, inactivation of STAT3 signaling shifts the cell division modes from asymmetric to symmetric divisions from supporting cells. Moreover, inhibition of Notch signaling stimulates STAT3 phosphorylation, and inactivation of STAT3 signaling attenuates production of supernumerary hair cells induced by a Notch pathway inhibitor. Our findings highlight an important role of the STAT3 signaling during mouse cochlear hair cell differentiation and may have clinical implications for the recovery of hair cell loss-induced hearing impairment.
Berk, L.C.J. van den; Roelofs, H.; Huijs, T.; Siebers-Vermeulen, K.G.C.; Raymakers, R.A.P.; Kogler, G.; Figdor, C.G.; Torensma, R.
Pathogen-derived entities force the tissue-resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord
Surguchev, Alexei; Bai, Jun-Ping; Joshi, Powrnima; Navaratnam, Dhasakumar
Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contrib...
Hakizimana, Pierre; Fridberger, Anders
Hearing depends on sound-evoked deflections of the stereocilia that protrude from the sensory hair cells in the inner ear. Although sound provides an important force driving stereocilia, forces generated through mechanically sensitive ion channels and through the motor protein prestin have been shown to influence stereocilia motion in solitary hair cells. While a possible influence of prestin on mechanically sensitive ion channels has not been systematically investigated, a decrease in transducer currents is evident in solitary hair cells when prestin is blocked with salicylate, raising the question of whether a reduced prestin activity or salicylate itself affected the mechanotransduction apparatus. We used two- and three-dimensional time-resolved confocal imaging to visualize outer hair cell stereocilia during sound stimulation in the apical turn of cochlear explant preparations from the guinea pig. Surprisingly, following application of salicylate, outer hair cell stereocilia deflections increased, while cochlear microphonic potentials decreased. However, when prestin activity was altered with the chloride ionophore tributyltin, both the cochlear microphonic potential and the stereocilia deflection amplitude decreased. Neither positive nor negative current stimulation abolished the bundle movements in the presence of salicylate, indicating that the observed effects did not depend on the endocochlear potential. These data suggest that salicylate may alter the mechanical properties of stereocilia, decreasing their bending stiffness.
Smith, Michael E; Monroe, J David
Sensory hair cells are the mechanotransductive receptors that detect gravity, sound, and vibration in all vertebrates. Damage to these sensitive receptors often results in deficits in vestibular function and hearing. There are currently two main reasons for studying the process of hair cell loss in fishes. First, fishes, like other non-mammalian vertebrates, have the ability to regenerate hair cells that have been damaged or lost via exposure to ototoxic chemicals or acoustic overstimulation. Thus, they are used as a biomedical model to understand the process of hair cell death and regeneration and find therapeutics that treat or prevent human hearing loss. Secondly, scientists and governmental natural resource managers are concerned about the potential effects of intense anthropogenic sounds on aquatic organisms, including fishes. Dr. Arthur N. Popper and his students, postdocs and research associates have performed pioneering experiments in both of these lines of fish hearing research. This review will discuss the current knowledge regarding the causes and consequences of both lateral line and inner ear hair cell damage in teleost fishes.
The amplification of acoustic stimuli is a feature of hair cells that evolved early on in vertebrates. Though standard stereocilia mechanisms to promote such amplification may persist in the mammal, an additional mechanism evolved to enhance high frequency sensation. Only in mammals, a special cell type, the outer hair cell, arose that possesses a remarkably fast somatic mechanical response, which probably endows the passive cochlea with a boost in sensitivity by a factor of 100 (40dB), at least. Experiments conducted over the past few years have shed light on many aspects of outer hair cell electromotility, including the molecular identification of the motor, the effects of a knockout, and underlying mechanisms of action. A review of this remarkable progress is attempted.
Nowruz Najafzadeh; Maliheh Nobakht; Bagher Pourheydar; Mohammad Ghasem Golmohammadi
Emerging studies of treating spinal cord injury (SCI) with adult stem cells led us to evaluate the effects of transplantation of hair fol icle stem cells in rats with a compression-induced spinal cord lesion. Here, we proposed a hypothesis that rat hair fol icle stem celltransplantation can promote the recovery of injured spinal cord. Compression-induced spinal cord injury was induced in Wistar rats in this study. The bulge area of the rat vibrissa fol icles was isolated, cultivated and characterized with nestin as a stem cellmarker. 5-Bromo-2′-deoxyuridine (BrdU) labeled bulge stem cells were transplanted into rats with spinal cord injury. Immunohistochemical staining results showed that some of the grafted cells could survive and differentiate into oligodendrocytes (receptor-interacting protein positive cells) and neuronal-like cells (βIII-tubulin positive cells) at 3 weeks after transplantation. In addition, recovery of hind limb locomotor function in spinal cord injury rats at 8 weeks fol owing celltransplantation was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale. The results demon-strate that the grafted hair fol icle stem cells can survive for a long time period in vivo and differentiate into neuronal- and glial-like cells. These results suggest that hair fol icle stem cells can promote the recovery of spinal cord injury.
Surguchev, Alexei; Bai, Jun-Ping; Joshi, Powrnima; Navaratnam, Dhasakumar
Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.
Stolovich-Rain, Miri; Enk, Jonatan; Vikesa, Jonas;
Because tissue regeneration deteriorates with age, it is generally assumed that the younger the animal, the better it compensates for tissue damage. We have examined the effect of young age on compensatory proliferation of pancreatic β cells in vivo. Surprisingly, β cells in suckling mice fail...... to enter the cell division cycle in response to a diabetogenic injury or increased glycolysis. The potential of β cells for compensatory proliferation is acquired following premature weaning to normal chow, but not to a diet mimicking maternal milk. In addition, weaning coincides with enhanced glucose...... a discrete maturation step of β cells, elevating both the mitogenic and secretory response to glucose....
Michler, Jule K; Hillmann, Aline; Savkovic, Vuk; Mülling, Christoph K W
The easily accessible niche represented by skin and its appendages may serve as a promising source to complement modern regenerative medicine for horses. In humans and in animal models for human medicine, the hair follicle and its stem cell niches are well characterized. Since literature in this field of equine research is scarce, we sought to analyze cells of the dermal stem cell niche of the equine hair follicle morphologically and for a subset of markers useful for cell characterization via immunolabeling. We cultured equine forelock skin explants to obtain cultures with cells migrating from the hair follicles. Isolation of cells revealed typical fibroblast morphology with a strong tendency to aggregate and form spheroids. For immunofluorescent characterization of primary isolations, we tested an antibody panel consisting of lineage makers for the dermal compartment of the hair follicle, markers associated with an undifferentiated cell status and markers for epithelial cell types as negative controls. All antibodies used were also tested on equine skin sections. The isolated cells displayed clear profiles of dermal and undifferentiated cells. To substantiate our findings, we tested our primary isolations for established equine multipotent mesenchymal stromal cell antigen expression markers in flow cytometry experiments yielding strong convergence. The data presented here provide insights to a stem cell source in horses almost unnoticed to date. The basic investigations of the equine dermal hair follicle stem cell niche confirm the expression of standard markers used in other species and lay the foundation for future studies on this easily available adult stem cell source. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.
Piérard-Franchimont, C; Piérard, G E
Hair is influenced by the effects of the daily environment. Some toxic xenobiotics slow down or block the cell renewal of the hair matrix, thus inhibiting hair growth. The ultraviolet light obviously influences the physical structure and physiology of the hair follicle. Tobacco is similarly responsible for negative influences on the evolution of various alopecias. Several cosmetic procedures for maintaining and making hair more attractive are not always harmless, and they occasionally represent a possible origin for alopecia.
van den Berk, Lieke C J; Roelofs, Helene; Huijs, Tonnie; Siebers-Vermeulen, Kim G C; Raymakers, Reinier A; Kögler, Gesine; Figdor, Carl G; Torensma, Ruurd
Pathogen-derived entities force the tissue-resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord blood an immature MSC population was identified. Remarkably, these immature stem cells modulated DCs in a different way. Marker expression was unchanged during the differentiation of monocytes towards immature DCs (iDCs) when cocultured with cord blood MSC [unrestricted somatic stem cells (USSCs)]. The maturation to mature DCs (mDCs) was enhanced when DCs were co-cultured with USSC, as evidenced by the up-regulation of costimulatory molecules. Endocytosis of dextran by iDCs was hampered in the presence of USSCs, which is indicative for the maturation of iDCs. Despite this maturation, the migration of iDCs cocultured with USSCs appeared to be identical to iDCs cultured alone. However, USSCs increased the migration of mDCs towards CCL21 and boosted interleukin-12 production. So, USSCs mature iDCs, thereby redirecting the antigen-uptake phenotype towards a mature phenotype. Furthermore, DC maturation by lipopolysaccharide (LPS) or USSCs reflects two distinct pathways because migration was unaffected when iDCs were matured by coculture with USSCs, while it was strongly enhanced in the presence of LPS. DCs are able to discriminate the different MSC subtypes, resulting in diverse differentiation programmes.
Full Text Available BACKGROUND: Mammalian hearing is refined by amplification of the sound-evoked vibration of the cochlear partition. This amplification is at least partly due to forces produced by protein motors residing in the cylindrical body of the outer hair cell. To transmit power to the cochlear partition, it is required that the outer hair cells dynamically change their length, in addition to generating force. These length changes, which have not previously been measured in vivo, must be correctly timed with the acoustic stimulus to produce amplification. METHODOLOGY/PRINCIPAL FINDINGS: Using in vivo optical coherence tomography, we demonstrate that outer hair cells in living guinea pigs have length changes with unexpected timing and magnitudes that depend on the stimulus level in the sensitive cochlea. CONCLUSIONS/SIGNIFICANCE: The level-dependent length change is a necessary condition for directly validating that power is expended by the active process presumed to underlie normal hearing.
Han, Jun Sae; Oh, Keun Ha; Moon, Won Kyu; Kim, Kyungseop; Joh, Cheeyoung; Seo, Hee Seon; Bollina, Ravi; Park, Seong Jin
A piezoelectric artificial hair cell sensor was fabricated by the powder injection molding process in order to make an acoustic vector hydrophone. The entire process of powder injection molding was developed and optimized for PMN-PZT ceramic powder. The artificial hair cell sensor, which consists of high aspect ratio hair cell and three rectangular mechanoreceptors, was precisely fabricated through the developed powder injection molding process. The density and the dielectric property of the fabricated sensor shows 98% of the theoretical density and 85% of reference dielectric property of PMN-PZT ceramic powder. With regard to homogeneity, three rectangular mechanoreceptors have the same dimensions, with 3 μm of tolerance with 8% of deviation of dielectric property. Packaged vector hydrophones measure the underwater acoustic signals from 500 to 800 Hz with -212 dB of sensitivity. Directivity of vector hydrophone was acquired at 600 Hz as analyzing phase differences of electric signals.
Füllgrabe, Anja; Joost, Simon; Are, Alexandra; Jacob, Tina; Sivan, Unnikrishnan; Haegebarth, Andrea; Linnarsson, Sten; Simons, Benjamin D; Clevers, Hans; Toftgård, Rune; Kasper, Maria
The dynamics and interactions between stem cell pools in the hair follicle (HF), sebaceous gland (SG), and interfollicular epidermis (IFE) of murine skin are still poorly understood. In this study, we used multicolor lineage tracing to mark Lgr6-expressing basal cells in the HF isthmus, SG, and IFE.
Yuan Yasheng; Chi Fanglu
Background Previous studies have suggested that primary degeneration of hair cells causes secondary degeneration of spiral ganglion neurons (SGNs),but the effect of SGN degeneration on hair cells has not been studied.In the adult mouse inner ear ouabain can selectively and permanently induce the degeneration of type 1 SGNs while leaving type 2 SGNs,efferent fibers,and sensory hair cells relatively intact.This study aimed to investigate the dynamic changes in hair cell ribbon synapse induced by loss of SGNs using ouabain application to the round window niche of adult mice.Methods In the analysis,24 CBA/CAJ mice aged 8-10 weeks,were used,of which 6 normal mice were used as the control group.After ouabain application in the round window niche 6 times in an hour,ABR threshold shifts at least 30 dB in the three experimental groups which had six mice for 1-week group,six for 1-month group,and six for 3-month group.All 24 animals underwent function test at 1 week and then immunostaining at 1 week,1 month,and 3 months.Results The loss of neurons was followed by degeneration of postsynaptic specializations at the afferent synapse with hair cells.One week after ouabain treatment,the nerve endings of type 1 SGNs and postsynaptic densities,as measured by Na/K ATPase and PSD-95,were affected but not entirely missing,but their partial loss had consequences for synaptic ribbons that form the presynaptic specialization at the synapse between hair cells and primary afferent neurons.Ribbon numbers in inner hair cells decreased (some of them broken and the ribbon number much decreased),and the arrangement of the synaptic ribbons had undergone a dynamic reorganization:ribbons with or without associated postsynaptic densities moved from their normal location in the basal membrane of the cell to a more apical location and the neural endings alone were also found at more apical locations without associated ribbons.After 1 month,when the neural postsynaptic densities had completed their
Tominaga-Wada, Rumi; Wada, Takuji
CAPRICE (CPC) encodes a small protein with an R3 MYB motif and regulates root hair and trichome cell differentiation in Arabidopsis thaliana. Six additional CPC-like MYB proteins including TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ENHANCER OF TRY AND CPC2 (ETC2), ENHANCER OF TRY AND CPC3/CPC-LIKE MYB3 (ETC3/CPL3), TRICHOMELESS1 (TCL1), and TRICHOMELESS2/CPC-LIKE MYB4 (TCL2/CPL4) also have the ability to regulate root hair and/or trichome cell differentiation in Arabidopsis. In this r...
Sengupta, Soma; George, Manju; Miller, Katharine K.; Naik, Khurram; Chou, Jonathan; Cheatham, Mary Ann; Dallos, Peter; Naramura, Mayumi; Band, Hamid; Zheng, Jing
Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. ...
Marcotti, W; van Netten, SM; Kros, CJ
The most serious side-effect of the widely used aminoglycoside antibiotics is irreversible intracellular damage to the auditory and vestibular hair cells of the inner ear. The mechanism of entry into the hair cells has not been unequivocally resolved. Here we report that extracellular
Marcotti, Walter; Johnson, Stuart L; Rusch, Alfons; Kros, Corne J
Before the onset of hearing at postnatal day 12, mouse inner hair cells (IHCs) produce spontaneous and evoked action potentials. These spikes are likely to induce neurotransmitter release onto auditory nerve fibres. Since immature IHCs express both alpha1D (Cav1.3) Ca2+ and Na+ currents that activate near the resting potential, we examined whether these two conductances are involved in shaping the action potentials. Both had extremely rapid activation kinetics, followed by fast and complete voltage-dependent inactivation for the Na+ current, and slower, partially Ca2+-dependent inactivation for the Ca2+ current. Only the Ca2+ current is necessary for spontaneous and induced action potentials, and 29 % of cells lacked a Na+ current. The Na+ current does, however, shorten the time to reach the action-potential threshold, whereas the Ca2+ current is mainly involved, together with the K+ currents, in determining the speed and size of the spikes. Both currents increased in size up to the end of the first postnatal week. After this, the Ca2+ current reduced to about 30 % of its maximum size and persisted in mature IHCs. The Na+ current was downregulated around the onset of hearing, when the spiking is also known to disappear. Although the Na+ current was observed as early as embryonic day 16.5, its role in action-potential generation was only evident from just after birth, when the resting membrane potential became sufficiently negative to remove a sizeable fraction of the inactivation (half inactivation was at -71 mV). The size of both currents was positively correlated with the developmental change in action-potential frequency.
Garcin, Clare L; Ansell, David M
The hair follicle has an established role in wound re-epithelialisation, a phenomenon that has been appreciated since at least the first half of the last century. The bulge niche, one location of hair follicle epithelial stem cells has been of particular interest to researchers over recent years, with numerous studies showing its ability to directly contribute to epidermal repair. However, recent work has highlighted other progenitor regions of the hair follicle that appear to act as stem cells during epidermal repair. In addition, several studies within the last 12 months have questioned the importance of the bulge during re-epithelialisation, producing conflicting literature. Here we provide a new model to demonstrate how several important differences in experimental design between studies could account for these seemingly opposing findings, which may have implications for how future studies are conducted.
Farahbakhsh, Nasser A; Narins, Peter M
We investigated the process of slow motility in non-mammalian auditory hair cells by recording the time course of shape change in hair cells of the frog amphibian papilla. The tall hair cells in the rostral segment of this organ, reported to be the sole recipients of efferent innervation, were found to shorten in response to an increase in the concentration of the intracellular free calcium. These shortenings are composed of two partially-overlapping phases: an initial rapid iso-volumetric contraction, followed by a slower length decrease accompanied with swelling. It is possible to unmask the iso-volumetric contraction by delaying the cell swelling with the help of K+ or Cl- channel inhibitors, quinine or furosemide. Furthermore, it appears that the longitudinal contraction in these cells is Ca2+-calmodulin-dependent: in the presence of W-7, a calmodulin inhibitor, only a slow, swelling phase could be observed. These findings suggest that amphibian rostral AP hair cells resemble their mammalian counterparts in expressing both a Ca2+-calmodulin-dependent contractile structure and an "osmotic" mechanism capable of mediating length change in response to extracellular stimuli. Such a mechanism might be utilized by the efferent neurotransmitters for adaptive modulation of mechano-electrical transduction, sensitivity enhancement, frequency selectivity, and protection against over-stimulation.
Zahide Eriş Eken
Full Text Available Hair is composed of a mixture of trace elements in small quantities, proteins, lipids and water. Proteins consist of helical polypeptide amino acid molecules. In the hair cells; polypeptide chains of keratin protein would be organized in filaments. In recent years, hair cosmetics showed a significant change and development. The content of shampoos which is used to cleanse the hair has enhanced significantly. Hair conditioner, hair styling products, pomades, brilliantine, and gloss sprays, hair protective products, camouflage products are most commonly used hair cosmetics. Hair shaping procedures are frequently applied.
Kirjavainen, Anna; Laos, Maarja; Anttonen, Tommi; Pirvola, Ulla
Hair cells of the organ of Corti (OC) of the cochlea exhibit distinct planar polarity, both at the tissue and cellular level. Planar polarity at tissue level is manifested as uniform orientation of the hair cell stereociliary bundles. Hair cell intrinsic polarity is defined as structural hair bundle asymmetry; positioning of the kinocilium/basal body complex at the vertex of the V-shaped bundle. Consistent with strong apical polarity, the hair cell apex displays prominent actin and microtubule cytoskeletons. The Rho GTPase Cdc42 regulates cytoskeletal dynamics and polarization of various cell types, and, thus, serves as a candidate regulator of hair cell polarity. We have here induced Cdc42 inactivation in the late-embryonic OC. We show the role of Cdc42 in the establishment of planar polarity of hair cells and in cellular patterning. Abnormal planar polarity was displayed as disturbances in hair bundle orientation and morphology and in kinocilium/basal body positioning. These defects were accompanied by a disorganized cell-surface microtubule network. Atypical protein kinase C (aPKC), a putative Cdc42 effector, colocalized with Cdc42 at the hair cell apex, and aPKC expression was altered upon Cdc42 depletion. Our data suggest that Cdc42 together with aPKC is part of the machinery establishing hair cell planar polarity and that Cdc42 acts on polarity through the cell-surface microtubule network. The data also suggest that defects in apical polarization are influenced by disturbed cellular patterning in the OC. In addition, our data demonstrates that Cdc42 is required for stereociliogenesis in the immature cochlea.
Full Text Available Cell fates are determined by specific transcriptional programs. Here we provide evidence that the transcriptional coactivator, Mediator 1 (Med1, is essential for the cell fate determination of ectodermal epithelia. Conditional deletion of Med1 in vivo converted dental epithelia into epidermal epithelia, causing defects in enamel organ development while promoting hair formation in the incisors. We identified multiple processes by which hairs are generated in Med1 deficient incisors: 1 dental epithelial stem cells lacking Med 1 fail to commit to the dental lineage, 2 Sox2-expressing stem cells extend into the differentiation zone and remain multi-potent due to reduced Notch1 signaling, and 3 epidermal fate is induced by calcium as demonstrated in dental epithelial cell cultures. These results demonstrate that Med1 is a master regulator in adult stem cells to govern epithelial cell fate.
Koh Hui Yee
Full Text Available Background: Hairdresser is one of the fastest growing occupations in today’s society. Hairdresser help styling, cutting, colouring, perming, curling, straightening hair and various treatment to customer. Somehow, hairdresser are constantly exposed to chemical substances such as aromatic amines, hydrogen peroxide, thioglycolic acid, formaldehyde in hair products which can cause damage to human’s genome. Micronucleus is one of the effective biomarker for processes associated with the induction of DNA damage. Purpose: The aim of this study was to determine the micronucleus frequencies in buccal mucosa epithelial cells of hairdresser who were exposed to chemical of hair products. Method: This study was conducted on twenty female subjects, who were divided into 2 groups: exposed and non-exposed (control group. All subjects recruited were working in the same beauty salon. Buccal cells were obtained from each individual by using cytobrush. The cells were stained with modified Feulgen-Ronssenback method and counting of micronucleus per 1000 cell was done under light microscope. The data were analyzed using independent t-test and one-way Anova (p<0.05. Result: The result showed a significant difference in micronucleus frequency between 2 groups. There were a significantly increase of micronucleus frequency in hairdressers and increase of micronucleus frequency with the longer duration of exposure. Conclusion: It concluded that the chemical substances of hair products had affected the micronucleus frequency ofthe epithelial cells in buccal mucosa of hairdressers.
SHI Xiao-rui; Alfred Nuttall
Apical membrane recycling has been proposed to be important for normal hair cell function. The current study reports an in vitro work that demonstrates the presence of phosphatidylserine (PS) and PS-positive vesicles labeled by Annexin V in the apical portion of hair cells. The following characteristics of the PS-positive vesicles were noticed using scanning confocal fluorescence microscopy: (1) variable sizes around 200 nm; (2)variable distribution patterns (either uniformly along individual stereocilia in the hair bundle or irregular) in the stereocilia from cell to cell; (3) variable sizes and numbers at locations along the border of the cuticular plate (CP),with a large number of them located at the vestigal kinocilial location; (4) motility with some of the vesicles during the observation period; (5) increase in PS labeling and the number of PS-positive vesicles after loud sound stimulation; and (6) decreased PS labeling and PS-positive vesicle numbers following treatment with LY-294002, a PI3 -kinase inhibitor. These results suggest that the presence of PS-positive vesicles at the apical area of hair cells may be indicative of vesicle shedding or transportation of a protein or rafts.
Jarboe, J K; Hallworth, R
Quinine intoxication causes a well-described syndrome that includes tinnitus, sensorineural hearing loss and vertigo. The pathophysiology of quinine's effects on hearing is unknown, but may include a peripheral component. The cochlear outer hair cell is known to be motile and to contribute force to amplify the vibration pattern of the organ of Corti. The outer hair cell is also a target of diseases involving tinnitus and sensorineural hearing loss, including salicylate intoxication. These effects may be mediated through changes either in motile force or in mechanical properties. Quinine's effects on outer hair cell motility and mechanical properties have therefore been examined in vitro. Quinine at 5.0 mM substantially decreased active force generation in isolated guinea pig cochlear outer hair cells. Isolated cells also elongated and dilated in diameter when exposed to 5.0 mM quinine. No consistent changes in mechanical properties were observed. 1.0 mM quinine was ineffective in either force reduction or elongation. Trifluoperazine, a calmodulin inhibitor, and ML-9, a blocker of myosin light chain kinases, were ineffective in blocking quinine-induced force reduction or elongation. Deferoxamine, a hydroxyl free radical scavenger, also failed to block either the force decrease or the elongation.
Full Text Available Introduction. Malignant transformation in a mature cystic teratoma of the ovary is a rare complication. Squamous cell carcinoma is the most common transformation. We describe a new case of squamous cell carcinoma arising in a mature cystic teratoma. Case Report. A premenopausal 52-year-old female patient is diagnosed with vaginal bleeding. According to examination made on the women and the pelvic scanning, 7 cm mass is found on the right adnexa of the patient. Total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, pelvic lymph node dissection, and debulking were the treatments completed on the patient. According to histopathological diagnosis, squamous cell carcinoma arising in a mature cystic teratoma is diagnosed as a reason for the mass in the right adnexa of the patient. Conclusion. The prognosis of the malign transformation of MCT depends on surgery stage; however it is extremely poor. The patient should receive chemotherapy regardless of stage. We have decided to administer second cycle carboplatin and paclitaxel treatments on the patient.
Full Text Available Tissue-specific microenvironments shape the fate of mononuclear phagocytes [1–3]. Interstitial osmolarity is a tissue biophysical parameter which considerably modulates the phenotype and function of dendritic cells . In the present report we provide a detailed description of our experimental workflow and bioinformatic analysis applied to our gene expression dataset (GSE72174, aiming to investigate the influence of different osmolarity conditions on the gene expression signature of bone marrow-derived dendritic cells. We established a cell culture system involving murine bone marrow cells, cultured under different NaCl-induced osmolarity conditions in the presence of the dendritic cell growth factor GM-CSF. Gene expression analysis was applied to mature dendritic cells (day 7 developed in different osmolarities, with and without prior stimulation with the TLR2/4 ligand LPS.
Schneider, Marie; Dieckmann, Christina; Rabe, Katrin; Simon, Jan-Christoph; Savkovic, Vuk
Bench-to-Bedside concepts for regenerative therapy place significant weight on noninvasive approaches, with harvesting of the starting material as a header. This is particularly important in autologous treatments, which use one's bodily constituents for therapy. Precisely the stretch between obtaining therapeutic elements invasively and noninvasively places non-intrusive "sampling" rather than "biopsy" in the center of the road map of developing an autologous regenerative therapy. We focus on such a noninvasively available source of adult stem cells that we carry with us throughout our life, available at our fingertips-or shall we say hair roots, by a simple plucking of hair: the human hair follicle. This chapter describes an explant procedure for cultivating melanocytes differentiated from the stem cell pool of the hair follicle Outer Root Sheath (ORS). In vivo, the most abundant derivatives of the heterogeneous ORS stem cell pool are epidermal cells-melanocytes and keratinocytes which complete their differentiation-either spontaneously or upon picking up regenerative cues from damaged skin-and migrate from the ORS towards the adjacent regenerating area of the epidermis. We have taken advantage of the ORS developmental potential by optimizing explant primary culture, expansion and melanogenic differentiation of resident ORS stem cells towards end-stage melanocytes in order to obtain functional melanocytes noninvasively for the purposes of transplantation and use them for the treatment of depigmentation disorders. Our protocol specifies sampling of hair with their ORS, follicle medium-air interface primary culture, stimulation of cell outgrowth, adherent culture and differentiation of ORS stem cells and precursors towards fully functional melanocytes. Along with cultivation, we describe selection techniques for establishing and maintaining a pure melanocyte population and methods suitable for determining melanocyte identity.
翟所强; 王大君; 王嘉陵
The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons (SGNs) prepared from P3 mice were exposed to 20mM glutamate for 2 hours before the culture medium was replaced with fresh medium containing 0, 25, 50, and 100 ng/ml bFGF, respectively. Fourteen days later, all cultures were fixed with 4% paraformaldehyde, and stained with 1% toluidine blue. The number of surviving SGNs were counted and the length of SGNs neurites were measured. Exposure to 20 mM glutamate for 24 hours resulted in an inhibition on neurite outgrowth of SGNs and elevated cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and elevated number of surviving SGNs. Effects of bFGF were dose dependent with the highest potency at 100 ng/ml. In experiment Ⅱ, in vivo studies were carried out with guinea pigs in which bFGF or artificial perilymph was perfused into the cochlea to assess possible protective effects of bFGF on cochlear hair cells and compound action potentials(CAP). The CAPs were measured before, immediatly and 48 hours after exposure to noise. Significant differences in CAP were observed (p＜0. 05 ) among the bFGF perfused group, control group(t =3. 896 ) and artificial perilymph perfused group (t =2. 520) at 48 hours after noise exposure, Cochleae were removed and hair cell Loss was analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 651 and 687 inner hair cells in the control and artificial perilymph perfused group, respectively. In sharp contrast, only 31 inner hair cells were lost in the bFGF perfused ears. Similarly, more outer hair cells died in the control and perilymph perfuesed group (41830 and 41968, respectively) than in the group treated with bFGF (34258). Our results demonstrate that bFGF protected SGNs against glutmate
Kim, Kyung-Joong; Ahn, Kang-Hun
We investigate the significance of the inclined angle of a hair bundle at equilibrium. We find that, while the angle gives a geometrical conversion factor between the bundle deflection and the ion channel displacement, it also controls the dynamics of the bundle. We show that a Hopf bifurcation, which enhances sensitivity, can be driven by the geometrical factor. However, existing experimental data indicate that mammalian auditory hair-cell bundles are located far away from the Hopf bifurcation point, suggesting that the high sensitivity of mammalian hearing might come from other mechanisms.
Full Text Available The Meddis inner hair cell model is a widely accepted, but computationally intensive computer model of mammalian inner hair cell function. We have produced an analogue VLSI implementation of this model that operates in real time in the current domain by using translinear and log-domain circuits. The circuit has been fabricated on a chip and tested against the Meddis model for (a rate level functions for onset and steady-state response, (b recovery after masking, (c additivity, (d two-component adaptation, (e phase locking, (f recovery of spontaneous activity, and (g computational efficiency. The advantage of this circuit, over other electronic inner hair cell models, is its nearly exact implementation of the Meddis model which can be tuned to behave similarly to the biological inner hair cell. This has important implications on our ability to simulate the auditory system in real time. Furthermore, the technique of mapping a mathematical model of first-order differential equations to a circuit of log-domain filters allows us to implement real-time neuromorphic signal processors for a host of models using the same approach.
Déborah I. Scheffer
Full Text Available Hair cells of the inner ear are mechanoreceptors for hearing and balance, and proteins highly enriched in hair cells may have specific roles in the development and maintenance of the mechanotransduction apparatus. We identified XIRP2/mXinβ as an enriched protein likely to be essential for hair cells. We found that different isoforms of this protein are expressed and differentially located: short splice forms (also called XEPLIN are targeted more to stereocilia, whereas two long isoforms containing a XIN-repeat domain are in both stereocilia and cuticular plates. Mice lacking the Xirp2 gene developed normal stereocilia bundles, but these degenerated with time: stereocilia were lost and long membranous protrusions emanated from the nearby apical surfaces. At an ultrastructural level, the paracrystalline actin filaments became disorganized. XIRP2 is apparently involved in the maintenance of actin structures in stereocilia and cuticular plates of hair cells, and perhaps in other organs where it is expressed.
Shin, Masashi; Larsson, Lars-Inge; Hougaard, David M.
The anthracycline antibiotic daunomycin (DM) is useful for the treatment of leukemia but has side-effects such as alopecia. Using immunocytochemistry, we show that, after a single i.v. injection, DM accumulates in the nuclei of matrix cells and in the outer root sheath of hair follicles. DM...
Auer, Manfred; Koster, Bram; Ziese, Ulrike; Bajaj, Chandrajit; Volkmann, Niels; Wang, Da Neng; Hudspeth, A. James
The senses of hearing and balance rest upon mechanoelectrical transduction by the hair bundles of hair cells in the inner ear. Located at the apical cellular surface, each hair bundle comprises several tens of stereocilia and a single kinocilium that are interconnected by extracellular proteinaceous links. Using electron-microscopic tomography of bullfrog saccular sensory epithelia, we examined the three-dimensional structures of ankle or basal links, kinociliary links, and tip links. We observed clear differences in the dimensions and appearances of the three links. We found two distinct populations of tip links suggestive of the involvement of two proteins or splice variants. We noted auxiliary links connecting the upper portions of tip links to the taller stereocilia. Tip links and auxiliary links show a tendency to adopt a globular conformation when disconnected from the membrane surface.
Ma, Xianghui; Tian, Yuhua; Song, Yongli; Shi, Jianyun; Xu, Jiuzhi; Xiong, Kai; Li, Jia; Xu, Wenjie; Zhao, Yiqiang; Shuai, Jianwei; Chen, Lei; Plikus, Maksim V; Lengner, Christopher J; Ren, Fazheng; Xue, Lixiang; Yu, Zhengquan
Hair follicles (HFs) undergo precisely regulated cycles of active regeneration (anagen), involution (catagen), and relative quiescence (telogen). Hair follicle stem cells (HFSCs) play important roles in regenerative cycling. Elucidating mechanisms that govern HFSC behavior can help uncover the underlying principles of hair development, hair growth disorders, and skin cancers. RNA-binding proteins of the Musashi (Msi) have been implicated in the biology of different stem cell types, yet they have not been studied in HFSCs. Here we utilized gain- and loss-of-function mouse models to demonstrate that forced MSI2 expression retards anagen entry and consequently delays hair growth, whereas loss of Msi2 enhances hair regrowth. Furthermore, our findings show that Msi2 maintains quiescent state of HFSCs in the process of the telogen-to-anagen transition. At the molecular level, our unbiased transcriptome profiling shows that Msi2 represses Hedgehog signaling activity and that Shh is its direct target in the hair follicle. Taken together, our findings reveal the importance of Msi2 in suppressing hair regeneration and maintaining HFSC quiescence. The previously unreported Msi2-Shh-Gli1 pathway adds to the growing understanding of the complex network governing cyclic hair growth. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Kao, Eddy; Villalon, Rosalina; Ribeiro, Salustiano; Berger, Trish
Reducing prepubertal endogenous estrogens led to increased numbers of Sertoli cells and the associated increased testicular size and testicular sperm production capacity in boars. The increased number of Sertoli cells might be due to a longer time for proliferation; delayed differentiation of Sertoli cells during suppressed endogenous estrogens would be consistent with this hypothesized, prolonged proliferation interval. This study used immunohistochemical detection of anti-Müllerian hormone (AMH), a marker of immature Sertoli cells, and of CDKN1B, a cell cycle inhibitor associated with more mature Sertoli cells, to determine if suppressing endogenous estrogens detectably delayed "differentiation" of porcine Sertoli cells. Testes were from littermate pairs of boars previously treated with Letrozole, an aromatase inhibitor, or vehicle, from the first week of age until tissue collection at 2, 3, 4, 5 or 6 months of age. Four animals were examined at each age following Letrozole treatment and their corresponding littermates evaluated following treatment with vehicle. Amount of AMH protein in Sertoli cells decreased with age of boar and could not be detected at 6 months of age. The AMH labeling was greater in the Letrozole-treated boars compared with littermate vehicle controls at 4 months of age (P=0.03). The percentage of CDKN1B-labeled Sertoli cells apparently increased with age through 5 months of age. At 4 and 5 months of age, the mean percentage of CDKN1B-labeled Sertoli cells was less in the Letrozole-treated animals than in the vehicle control animals (P = 0.03 and 0.04, respectively). These results are consistent with the hypothesis that continual inhibition of aromatase (and concomitatant reduced estrogen synthesis) causes a delay in Sertoli cell maturation in boars.
Buchen, B; Hensel, D; Sievers, A
Both the apical and the basal cell poles of the sensory cells in trigger hairs of Dionaea muscipula are structured identically. A complex of concentrically arranged endoplasmic reticulum cisternae occupies each of the poles. One to four vacuoles are enclosed within the central cisterna and contain polyphenols (deposits of "tannin"). Structural polarity, whether symmetric or asymmetric, as well as the occurrence of abundant endoplasmic reticulum and numerous mitochondria are characteristics of the perception cells of most animals and plants.
Scarfone, E; Ulfendahl, M; Figueroa, L; Flock, A
Type I hair cells isolated from animals anaesthetised with barbiturates or ether were found to be shorter and to lack a prominent 'neck' region when compared to cells isolated from non-anaesthetised animals. Ketamine did not have this effect. The changes observed could have important implications for the physiology of inner ear receptors. These findings infer that care should be taken in the choice of anaesthetics used in studies on cells from the inner ear.
Tai, Yu-Tzu; Anderson, Kenneth C
Novel effective immunotherapies are needed for patients with multiple myeloma (MM), since disease recurrence remains a major obstacle. B-cell maturation antigen (BCMA), a cell surface protein universally expressed on malignant plasma cells , has emerged as a very selective antigen to be targeted in novel treatments for MM. We here first review BCMA-related biology, and then highlight the recent clinical development of a novel afucosylated anti-BCMA monoclonal antibody conjugated with monomethyl auristatin F via noncleavable linker (GSK2857916). Chimeric antigen receptor-expressing T cells targeting BCMA may also induce specific and durable anti-MM responses by patients’ own effector cells. Clinical trials testing these two approaches (NCT02064387, NCT02215967) are currently ongoing in relapsed and refractory MM patients. PMID:26370838
Wakame, Koji; Okawa, Hiroshi; Komatsu, Ken-Ich; Nakata, Akifumi; Sato, Keisuke; Ingawa, Hiroyuki; Kohchi, Chie; Nishizawa, Takashi; Soma, Gen-Ichiro
The lipopolysaccharide (LPS)-like compound derived from Pantoea agglomerans (immunopotentiator from Pantoea agglomerans 1 (IP-PA1)) has been used not only as dietary supplement or cosmetic for humans, but also by Japanese veterinarians as an anti-tumor, anti-allergy, "keep a fine coat of fur" and hair growth-promoting functional food for dogs and cats. In the present study, we focused on the hair growth-promoting effects of IP-PA1 on a hair-shaved animal model and its mechanism of action. We also investigated its potential on gene expression after stimulating human dermal papilla cells with IP-PA1. The hair on the back of a C3H/HeN mouse was shaved and IP-PA1 was orally administered or applied to the skin. The status of hair growth was observed and recorded for 14 days. Skin was collected and histological tissue examination was performed with respect to hair growth status using hematoxylin and eosin staining. After IP-PA1 administration (2 and 10 μg/ml) to human dermal papilla cell culture system for 24 h, fibroblast growth factor-7 (FGF-7) and vascular endothelial growth factor (VEGF) mRNA expression were measured using real-time polymerase chain reaction (PCR) analysis. IP-PA1, when given orally, showed a tendency to promote hair growth in mice. In addition, skin application also significantly promoted hair growth, while histopathological examinations further demonstrated hair elongation from dermal papilla cells. In the human dermal papilla cell culture system, significant FGF-7 and VEGF mRNA expressions were observed (p<0.05). An underlying mechanism of gene expression by which IP-PA1 promotes hair growth was suggested to be different from that of medicine and traditional hair tonics, such as minoxidil and adenosine. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
Pietrella, Donatella; Corbucci, Cristina; Perito, Stefano; Bistoni, Giovanni; Vecchiarelli, Anna
Our previous data show that mannoproteins (MPs) from Cryptococcus neoformans are able to induce protective responses against both C. neoformans and Candida albicans. Here we provide evidence that MPs foster maturation and activation of human dendritic cells (DCs). Maturation was evaluated by the ability of MPs to facilitate expression of costimulatory molecules such as CD40, CD86, CD83, and major histocompatibility complex classes I and II and to inhibit receptors such as CD14, CD16, and CD32. Activation of DCs was measured by the capacity of MPs to promote interleukin-12 and tumor necrosis factor alpha secretion. DC-induced maturation and interleukin-12 induction are largely mediated by engagement of mannose receptors and presume MP internalization and degradation. DC activation leads to IkappaBalpha phosphorylation, which is necessary for nuclear factor kappaB transmigration into the nucleus. MP-loaded DCs are efficient stimulators of T cells and show a remarkable capacity to promote CD4 and CD8 proliferation. In conclusion, we have evidenced a novel regulatory role of MPs that promotes their candidacy as a vaccine against fungi.
Dallos, P; Evans, B N; Hallworth, R
It is the prevailing notion that cochlear outer hair cells function as mechanical effectors as well as sensory receptors. Electrically induced changes in the shape of mammalian outer hair cells, studied in vitro, are commonly assumed to represent an aspect of their effector process that may occur in vivo. The nature of the motile process is obscure, even though none of the established cellular motors can be involved. Although it is known that the motile response is under voltage control, it is uncertain whether the stimulus is a drop in the voltage along the long axis of the cell or variation in the transmembrane potential. We have now performed experiments with cells partitioned in differing degrees between two chambers. Applied voltage stimulates the cell membrane segments in opposite polarity to an amount dependent on the partitioning. The findings show, in accordance with previous suggestions, that the driving stimulus is a local transmembrane voltage drop and that the cellular motor consists of many independent elements, distributed along the cell membrane and its associated cortical structures. We further show that the primary action of the motor elements is along the longitudinal dimension of the cell without necessarily involving changes in intracellular hydrostatic pressure. This establishes the outer hair cell motor as unique among mechanisms that control cell shape.
In addition to their ubiquitous apical-basal polarity, many epithelia are also polarized along an orthogonal axis, a phenomenon termed planar cell polarity (PCP. In the mammalian inner ear and the zebrafish lateral line, PCP is revealed through the orientation of mechanosensitive hair cells relative to each other and to the body axes. In each neuromast, the receptor organ of the lateral line, hair bundles are arranged in a mirror-symmetrical fashion. Here we show that the establishment of mirror symmetry is preceded by rotational rearrangements between hair-cell pairs, a behavior consistently associated with the division of hair-cell precursors. Time-lapse imaging of trilobite mutants, which lack the core PCP constituent Vang-like protein 2 (Vangl2, shows that their misoriented hair cells correlate with misaligned divisions of hair-cell precursors and an inability to complete rearrangements accurately. Vangl2 is asymmetrically localized in the cells of the neuromast, a configuration required for accurate completion of rearrangements. Manipulation of Vangl2 expression or of Notch signaling results in a uniform hair-cell polarity, indicating that rearrangements refine neuromast polarity with respect to the body axes.
Chunxia Cao; Wei Yang; Yonglie Chu; Qingguang Liu; Liang Yu; Cheng'en Pan
Objective: Heat shock protein (HSP) has the promiscuous abilities to chaperone and present a broad repertoire of tumor antigens to antigen presenting cells including DCs. In this report, we analyzed the modulation of immature DC by HSP 96 (gp96).Method: Murine bone marrow-derived DC was induced by GM-CSF plus IL-4, which aped the immunostimulatory effects of DC.Cocultured DC and gp96-peptide complexes (gp96-PC) or inactivated H22 cells, the expression of MHC class Ⅱ, CD40, CD80 was quantified by flow cytometry. The concentration of IL-12 and TNF- in culture supernatants were determined by ELISA. Cr release assay was used to test specific cytotoxic T cell. Results: Our study demonstrated that the extent of DC maturation induced by gp96-PC, which was reflected in surface density of costimulatory and MHC Ⅱ molecules, was correlated with the secretion of IL-12 and with the T cellactivating potential in vitro. Conclusion: Heat shock protein 96 could be isolated and purified from H22 cells and could induce maturation of dendritic cell. Our findings might be relevance to the use of DC vaccine in therapy of human tumors.
Chen, Chih-Chiang; Chuong, Cheng Ming
Stem cells are fascinating because of their potential in regenerative medicine. Stem cell homeostasis has been thought to be mainly regulated by signals from their adjacent micro-environment named the “stem cell niche”. However, recent studies reveal that there can be multiple layers of environmental controls. Here we review these environmental controls using the paradigm of hair stem cells, because to observe and analyze the growth of hair is easier due to their characteristic cyclic regener...
Zajic, G; Schacht, J
Shape changes can be induced in isolated outer hair cells by various stimuli and quantified from digitized video-images. While overall changes in length between base and apex are easily measured, changes in defined segments of the cell require fixed landmarks on the cell body. The problem of locating such landmarks makes it difficult to assess if a change in length is uniform or largely confined to a particular segment of the cell. This information is important in identifying the location of a contractile apparatus and the elucidation of mechanisms of motility. We demonstrate here that microspheres can serve as reference points for such measurements. By attaching microspheres to cells we determined that, when outer hair cells increased their volume upon K(+)-depolarization, their middle segment shortened more significantly (14 +/- 6%) than either the basal (10 +/- 5%) or apical section (7 +/- 6%; P less than 0.01). In contrast, when cortical contractions were induced by elevating intracellular Ca2+, the elongation of the cells was more pronounced in their basal (8 +/- 2%) than their apical (6 +/- 2%; P = 0.06) or middle region (6 +/- 3%). This study provides further insight into the mechanisms of shape changes in isolated outer hair cells and illustrates a method to analyze localized changes in the absence of internal landmarks.
Full Text Available Hair cells and spiral ganglion neurons of the mammalian auditory system do not regenerate, and their loss leads to irreversible hearing loss. Aminoglycosides induce auditory hair cell death in vitro, and evidence suggests that phosphatidylinositol-3-kinase/Akt signaling opposes gentamicin toxicity via its downstream target, the protein kinase Akt. We previously demonstrated that somatostatin-a peptide with hormone/neurotransmitter properties-can protect hair cells from gentamicin-induced hair cell death in vitro, and that somatostatin receptors are expressed in the mammalian inner ear. However, it remains unknown how this protective effect is mediated. In the present study, we show a highly significant protective effect of octreotide (a drug that mimics and is more potent than somatostatin on gentamicin-induced hair cell death, and increased Akt phosphorylation in octreotide-treated organ of Corti explants in vitro. Moreover, we demonstrate that somatostatin receptor-1 knockout mice overexpress somatostatin receptor-2 in the organ of Corti, and are less susceptible to gentamicin-induced hair cell loss than wild-type or somatostatin-1/somatostatin-2 double-knockout mice. Finally, we show that octreotide affects auditory hair cells, enhances spiral ganglion neurite number, and decreases spiral ganglion neurite length.
Defourny, Jean; Poirrier, Anne-Lise; Lallemend, François; Mateo Sánchez, Susana; Neef, Jakob; Vanderhaeghen, Pierre; Soriano, Eduardo; Peuckert, Christiane; Kullander, Klas; Fritzsch, Bernd; Nguyen, Laurent; Moonen, Gustave; Moser, Tobias; Malgrange, Brigitte
Hearing requires an optimal afferent innervation of sensory hair cells by spiral ganglion neurons in the cochlea. Here we report that complementary expression of ephrin-A5 in hair cells and EphA4 receptor among spiral ganglion neuron populations controls the targeting of type I and type II afferent fibres to inner and outer hair cells, respectively. In the absence of ephrin-A5 or EphA4 forward signalling, a subset of type I projections aberrantly overshoot the inner hair cell layer and invade the outer hair cell area. Lack of type I afferent synapses impairs neurotransmission from inner hair cells to the auditory nerve. By contrast, radial shift of type I projections coincides with a gain of presynaptic ribbons that could enhance the afferent signalling from outer hair cells. Ephexin-1, cofilin and myosin light chain kinase act downstream of EphA4 to induce type I spiral ganglion neuron growth cone collapse. Our findings constitute the first identification of an Eph/ephrin-mediated mutual repulsion mechanism responsible for specific sorting of auditory projections in the cochlea.
Zhang, Duan-Sun; Piazza, Valeria; Perrin, Benjamin J; Rzadzinska, Agnieszka K; Poczatek, J Collin; Wang, Mei; Prosser, Haydn M; Ervasti, James M; Corey, David P; Lechene, Claude P
Hair cells of the inner ear are not normally replaced during an animal's life, and must continually renew components of their various organelles. Among these are the stereocilia, each with a core of several hundred actin filaments that arise from their apical surfaces and that bear the mechanotransduction apparatus at their tips. Actin turnover in stereocilia has previously been studied by transfecting neonatal rat hair cells in culture with a β-actin-GFP fusion, and evidence was found that actin is replaced, from the top down, in 2-3 days. Overexpression of the actin-binding protein espin causes elongation of stereocilia within 12-24 hours, also suggesting rapid regulation of stereocilia lengths. Similarly, the mechanosensory 'tip links' are replaced in 5-10 hours after cleavage in chicken and mammalian hair cells. In contrast, turnover in chick stereocilia in vivo is much slower. It might be that only certain components of stereocilia turn over quickly, that rapid turnover occurs only in neonatal animals, only in culture, or only in response to a challenge like breakage or actin overexpression. Here we quantify protein turnover by feeding animals with a (15)N-labelled precursor amino acid and using multi-isotope imaging mass spectrometry to measure appearance of new protein. Surprisingly, in adult frogs and mice and in neonatal mice, in vivo and in vitro, the stereocilia were remarkably stable, incorporating newly synthesized protein at hair cells expressing β-actin-GFP we bleached fiducial lines across hair bundles, but they did not move in 6 days. When we stopped expression of β- or γ-actin with tamoxifen-inducible recombination, neither actin isoform left the stereocilia, except at the tips. Thus, rapid turnover in stereocilia occurs only at the tips and not by a treadmilling process.
Slepecky, N; Ulfendahl, M
Individual isolated outer hair cells (OHCs) from the cochlea were maintained in a collagen gel and viewed in the light microscope. They were observed during fixation and processing for transmission electron microscopy and individual cells were selected for observation in the electron microscope. Application of glutaraldehyde at several concentrations caused OHCs to become shorter. Shrinkage occurred during dehydration but there was no further change during infiltration with the epoxy resin. Ultrastructural analysis of isolated cells fixed with glutaraldehyde and postfixed with osmium tetroxide showed that these cells were similar to cells fixed in the intact cochlea. The glutaraldehyde-induced cell shape change is similar to the shortening seen in intact OHCs in response to the application of solutions containing high potassium or caffeine. Application of glutaraldehyde to cells pretreated with potassium or caffeine caused further shortening. Glutaraldehyde-induced cell shape change was not blocked by the application of tetracaine, which did prevent potassium-induced and caffeine-induced shortening. Glutaraldehyde-induced cell shape change was not stopped by short treatment with N-ethylmaleimide, which did inhibit potassium-induced shortening. Results from these experiments suggest that the glutaraldehyde-induced OHC shape change is not caused by an effect on the membrane or by calcium activation of a contractile response. Shortening may be caused by shrinkage due to cross-linking of proteins.
Holley, M C; Ashmore, J F
A two-dimensional cortical cytoskeletal lattice associated with the lateral plasma membranes of mammalian outer hair cells maintains cell shape and provides a restoring force to oppose active changes in cell length. The lattice is composed of two morphologically distinct filaments which are arranged to reinforce the cell circumferentially whilst allowing limited changes in cell length and diameter. This function can only be fulfilled if intracellular pressure is high enough to put the lattice under tension.
C. R. Nascimento
Full Text Available Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua. Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days. To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.
Beurg, Maryline; Goldring, Adam C; Fettiplace, Robert
Sound stimuli are converted into electrical signals via gating of mechano-electrical transducer (MT) channels in the hair cell stereociliary bundle. The molecular composition of the MT channel is still not fully established, although transmembrane channel-like protein isoform 1 (TMC1) may be one component. We found that in outer hair cells of Beethoven mice containing a M412K point mutation in TMC1, MT channels had a similar unitary conductance to that of wild-type channels but a reduced selectivity for Ca(2+). The Ca(2+)-dependent adaptation that adjusts the operating range of the channel was also impaired in Beethoven mutants, with reduced shifts in the relationship between MT current and hair bundle displacement for adapting steps or after lowering extracellular Ca(2+); these effects may be attributed to the channel's reduced Ca(2+) permeability. Moreover, the density of stereociliary CaATPase pumps for Ca(2+) extrusion was decreased in the mutant. The results suggest that a major component of channel adaptation is regulated by changes in intracellular Ca(2+). Consistent with this idea, the adaptive shift in the current-displacement relationship when hair bundles were bathed in endolymph-like Ca(2+) saline was usually abolished by raising the intracellular Ca(2+) concentration.
Full Text Available Immunosuppressive drugs successfully prevent rejection of islet allografts in the treatment of type I diabetes. However, the drugs also suppress systemic immunity increasing the risk of opportunistic infection and cancer development in allograft recipients. In this study, we investigated a new treatment for autoimmune diabetes using naturally immune privileged, hair follicle derived, autologous cells to provide localized immune protection of islet allotransplants. Islets from Balb/c mouse donors were cotransplanted with syngeneic hair follicle dermal sheath cup cells (DSCC, group 1 or fibroblasts (FB, group 2 under the kidney capsule of immune-competent, streptozotocin induced, diabetic C57BL/6 recipients. Group 1 allografts survived significantly longer than group 2 (32.2 ± 12.2 versus 14.1 ± 3.3 days, P<0.001 without administration of any systemic immunosuppressive agents. DSCC reduced T cell activation in the renal lymph node, prevented graft infiltrates, modulated inflammatory chemokine and cytokine profiles, and preserved better beta cell function in the islet allografts, but no systemic immunosuppression was observed. In summary, DSCC prolong islet allograft survival without systemic immunosuppression by local modulation of alloimmune responses, enhancing of beta cell survival, and promoting of graft revascularization. This novel finding demonstrates the capacity of easily accessible hair follicle cells to be used as local immunosuppression agents in islet transplantation.
Song, Jae-Jun; Chang, Jiwon; Choi, Jungim; Im, Gi Jung; Chae, Sung Won; Lee, Seung Hoon; Kwon, Soon-Young; Jung, Hak Hyun; Chung, Ah-Young; Park, Hae-Chul; Choi, June
NecroX-5, one of the derivatives of NecroX series compounds, is a mitochondrial reactive oxygen species and reactive nitrogen species scavenger that inhibits cell death against various kinds of oxidative stresses. The objective of the present study was to evaluate the effects of NecroX-5 on neomycin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five days post-fertilization, zebrafish larvae were exposed to 125 μM neomycin and one of the following NecroX-5 concentrations for 1 h: 10, 25, 50, and 75 μM. Hair cells within the neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed using fluorescence microscopy (n = 10). The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and 2-[4-(dimethylamino) styryl]-N-ethylpyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. Ultrastructural changes were evaluated using scanning electron microscopy. NecroX-5 decreased neomycin-induced hair cell loss in the neuromasts (NecroX-5 50 μM: 13.4 ± 2.0 cells, 125 μM neomycin only: 8.1 ± 1.2 cells; n = 10, P neomycin and 50 μM NecroX-5. NecroX-5 decreased apoptosis and mitochondrial damage. In conclusion, NecroX-5 attenuated neomycin-induced hair cell loss in zebrafish.
Wang, Xiaojie; Hao, Jianqiang; Leung, Gigi; Breitkopf, Trisia; Wang, Eddy; Kwong, Nicole; Akhoundsadegh, Noushin; Warnock, Garth L; Shapiro, Jerry; McElwee, Kevin J
Immunosuppressive drugs successfully prevent rejection of islet allografts in the treatment of type I diabetes. However, the drugs also suppress systemic immunity increasing the risk of opportunistic infection and cancer development in allograft recipients. In this study, we investigated a new treatment for autoimmune diabetes using naturally immune privileged, hair follicle derived, autologous cells to provide localized immune protection of islet allotransplants. Islets from Balb/c mouse donors were cotransplanted with syngeneic hair follicle dermal sheath cup cells (DSCC, group 1) or fibroblasts (FB, group 2) under the kidney capsule of immune-competent, streptozotocin induced, diabetic C57BL/6 recipients. Group 1 allografts survived significantly longer than group 2 (32.2 ± 12.2 versus 14.1 ± 3.3 days, P < 0.001) without administration of any systemic immunosuppressive agents. DSCC reduced T cell activation in the renal lymph node, prevented graft infiltrates, modulated inflammatory chemokine and cytokine profiles, and preserved better beta cell function in the islet allografts, but no systemic immunosuppression was observed. In summary, DSCC prolong islet allograft survival without systemic immunosuppression by local modulation of alloimmune responses, enhancing of beta cell survival, and promoting of graft revascularization. This novel finding demonstrates the capacity of easily accessible hair follicle cells to be used as local immunosuppression agents in islet transplantation.
Hung, Sandy S C; Pébay, Alice; Wong, Raymond C B
Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.
Full Text Available The sensory organs of the inner ear possess resident populations of macrophages, but the function of those cells is poorly understood. In many tissues, macrophages participate in the removal of cellular debris after injury and can also promote tissue repair. The present study examined injury-evoked macrophage activity in the mouse utricle. Experiments used transgenic mice in which the gene for the human diphtheria toxin receptor (huDTR was inserted under regulation of the Pou4f3 promoter. Hair cells in such mice can be selectively lesioned by systemic treatment with diphtheria toxin (DT. In order to visualize macrophages, Pou4f3-huDTR mice were crossed with a second transgenic line, in which one or both copies of the gene for the fractalkine receptor CX3CR1 were replaced with a gene for GFP. Such mice expressed GFP in all macrophages, and mice that were CX3CR1GFP/GFP lacked the necessary receptor for fractalkine signaling. Treatment with DT resulted in the death of ~70% of utricular hair cells within seven days, which was accompanied by increased numbers of macrophages within the utricular sensory epithelium. Many of these macrophages appeared to be actively engulfing hair cell debris, indicating that macrophages participate in the process of ‘corpse removal’ in the mammalian vestibular organs. However, we observed no apparent differences in injury-evoked macrophage numbers in the utricles of CX3CR1+/GFP mice vs. CX3CR1GFP/GFP mice, suggesting that fractalkine signaling is not necessary for macrophage recruitment in these sensory organs. Finally, we found that repair of sensory epithelia at short times after DT-induced hair cell lesions was mediated by relatively thin cables of F-actin. After 56 days recovery, however, all cell-cell junctions were characterized by very thick actin cables.
Goldberg, Ethan M; Jeong, Hyo-Young; Kruglikov, Ilya; Tremblay, Robin; Lazarenko, Roman M; Rudy, Bernardo
Fast-spiking (FS) cells are a prominent subtype of neocortical γ-aminobutyric acidergic interneurons that mediate feed-forward inhibition and the temporal sculpting of information transfer in neural circuits, maintain excitation/inhibition balance, and contribute to network oscillations. FS cell dysfunction may be involved in the pathogenesis of disorders such as epilepsy, autism, and schizophrenia. Mature FS cells exhibit coordinated molecular and cellular specializations that facilitate rapid responsiveness, including brief spikes and sustained high-frequency discharge. We show that these features appear during the second and third postnatal weeks driven by upregulation of K(+) channel subunits of the Kv3 subfamily. The low membrane resistance and fast time constant characteristic of FS cells also appears during this time, driven by expression of a K(+) leak current mediated by K(ir)2 subfamily inward rectifier K(+) channels and TASK subfamily 2-pore K(+) channels. Blockade of this leak produces dramatic depolarization of FS cells suggesting the possibility for potent neuromodulation. Finally, the frequency of FS cell membrane potential oscillations increases during development and is markedly slower in TASK-1/3 knockout mice, suggesting that TASK channels regulate FS cell rhythmogenesis. Our findings imply that some of the effects of acidosis and/or anesthetics on brain function may be due to blockade of TASK channels in FS cells.
Issa, N P; Hudspeth, A J
The presynaptic active zone is the critical region of a chemical synapse at which Ca2+ entry provokes neurotransmitter release by exocytotic fusion of synaptic vesicles. To facilitate investigations of synaptic function, we have identified a group of fluorescent substances that label individual active zones in living hair cells. The Ca2+ indicator fluo-3, the compound studied in most detail, binds to the presynaptic dense bodies that are characteristic of active zones in hair cells and other cells that tonically release transmitter. The indicator's binding is reversible, with a dissociation constant of approximately 350 microM. Because fluo-3 that is bound to a presynaptic dense body continues to detect Ca2+ with an unaltered dissociation constant, the binding of this substance provides a valuable tool for exploration of the Ca2+ concentration at the site of vesicle fusion.
Cárdenas, Luis; Quinto, Carmen
Reactive oxygen species (ROS) are involved in supporting polar growth in pollen tubes, fucoid cells and root hair cells. However, there is limited evidence showing ROS changes during the earliest stages of the interaction between legume roots and rhizobia. We recently reported using Phaseolus vulgaris as a model system, the occurrence of a transient increase of ROS, within seconds, at the tip of actively growing root hair cells after treatment with Nod factors (NFs).1 This transient response is NFs-specific, and clearly distinct from the ROS changes induced by a fungal elicitor, with which sustained increases in ROS signal, is observed. Since ROS levels are transiently elevated after NFs perception, we propose that this ROS response is specific of the symbiotic interaction. Furthermore, the observed ROS changes correlate spatially and temporarily with the reported transient increases in calcium levels suggesting key roles for calcium and ROS during the early NF perception.
Chan, E; Ulfendahl, M
The mechanical properties of outer hair cells are of importance for normal hearing, and it has been shown that damage of the cells can lead to a reduction in the hearing sensitivity. In this study, we measured the stiffness of isolated outer hair cells in hyper- and hypotonic conditions, and examined the change in stiffness in relation to the corresponding changes in internal cell pressure and cell shape. The results showed that the axial stiffness of isolated outer hair cells (30-90 microns in length, 8-12 microns in diameter), ranging from 0.13-5.39 mN m-1, was inversely related to cell length. Exposure to hyper- and hypotonic external media with a small percentage change in osmolality caused a similar magnitude of change in cell length and cell diameter, but an average 60% change in cell stiffness. Therefore, a moderate osmotic change in the external medium can lead to a significant alteration in cell stiffness. The findings thus indicate an important contribution of internal cell pressure to cell stiffness.
Phillip M Uribe
Full Text Available Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction, and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.
Chen, Chih-Chiang; Chuong, Cheng Ming
Stem cells are fascinating because of their potential in regenerative medicine. Stem cell homeostasis has been thought to be mainly regulated by signals from their adjacent micro-environment named the “stem cell niche”. However, recent studies reveal that there can be multiple layers of environmental controls. Here we review these environmental controls using the paradigm of hair stem cells, because to observe and analyze the growth of hair is easier due to their characteristic cyclic regeneration pattern. The length of hair fibers is regulated by the duration of the growth period. In the hair follicles, hair stem cells located in the follicle bulge interact with signals from the dermal papilla. Outside of the follicle, activation of hair stem cells has been shown to be modulated by molecules released from the intra-dermal adipose tissue as well as body hormone status, immune function, neural activities, and aging. The general physiological status of an individual is further influenced by circadian rhythms and changing seasons. The interactive networks of these environmental factors provide new understanding on how stem cell homeostasis is regulated, inspiring new insights for regenerative medicine. Therapies do not necessarily have to be achieved by using stem cells themselves which may constitute a higher risk but by modulating stem cell activity through targeting one or multiple layers of their micro- and macro-environments. PMID:22391240
Chen, Chih-Chiang; Chuong, Cheng Ming
Stem cells are fascinating because of their potential in regenerative medicine. Stem cell homeostasis has been thought to be mainly regulated by signals from their adjacent micro-environment named the "stem cell niche". However, recent studies reveal that there can be multiple layers of environmental controls. Here we review these environmental controls using the paradigm of hair stem cells, because to observe and analyze the growth of hair is easier due to their characteristic cyclic regeneration pattern. The length of hair fibers is regulated by the duration of the growth period. In the hair follicles, hair stem cells located in the follicle bulge interact with signals from the dermal papilla. Outside of the follicle, activation of hair stem cells has been shown to be modulated by molecules released from the intra-dermal adipose tissue as well as body hormone status, immune function, neural activities, and aging. The general physiological status of an individual is further influenced by circadian rhythms and changing seasons. The interactive networks of these environmental factors provide new understanding on how stem cell homeostasis is regulated, inspiring new insights for regenerative medicine. Therapies do not necessarily have to be achieved by using stem cells themselves which may constitute a higher risk but by modulating stem cell activity through targeting one or multiple layers of their micro- and macro-environments.
acceptance. Tanner staging assesses pubic hair growth and increase in genital size, the latter factor being best reflected by ..... Research Methodology with Statistics for Health and Social. Sciences. ... observation in one practice. Ann Trop ...
... Surgery? Choosing the Right Sport for You Shyness Hair Removal KidsHealth > For Teens > Hair Removal Print A ... you need any of them? Different Types of Hair Before removing hair, it helps to know about ...
Hair - oily ... are some tips for preventing and treating oily hair: Shampoo your hair every day. Leaving the shampoo on your head ... minutes before rinsing may help. Avoid brushing your hair too often or too vigorously, since the brushing ...
Full Text Available Sound and head movements are perceived through sensory hair cells in the inner ear. Mounting evidence indicates that this process is initiated by the opening of mechanically sensitive calcium-permeable channels, also referred to as the mechanoelectrical transducer (MET channels, reported to be around the tips of all but the tallest stereocilia. However, the identity of MET channel remains elusive. Literature suggests that the MET channel is a non-selective cation channel with a high Ca(2+ permeability and ~100 picosiemens conductance. These characteristics make members of the transient receptor potential (TRP superfamily likely candidates for this role. One of these candidates is the transient receptor potential melastatin 1 protein (TRPM1, which is expressed in various cells types within the cochlea of the mouse including the hair cells. Recent studies demonstrate that mutations in the TRPM1 gene underlie the inherited retinal disease complete congenital stationary night blindness in humans and depolarizing bipolar cell dysfunction in the mouse retina, but auditory function was not assessed. Here we investigate the role of Trpm1 in hearing and as a possible hair cell MET channel using mice homozygous for the null allele of Trpm1 (Trpm1(-/- or a missense mutation in the pore domain of TRPM1 (Trpm1(tvrm27/tvrm27. Hearing thresholds were evaluated in adult (4-5 months old mice with auditory-evoked brain stem responses. Our data shows no statistically significant difference in hearing thresholds in Trpm1(-/- or Trpm1(tvrm27/tvrm27 mutants compared to littermate controls. Further, none of the mutant mice showed any sign of balance disorder, such as head bobbing or circling. These data suggest that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is a component of the hair cell MET channel.
Lue, June-Horng; Day, An-Shiou; Cheng, Po-Wen; Young, Yi-Ho
This study applied the vestibular evoked myogenic potential (VEMP) test to guinea pigs coupled with electronic microscopic examination to determine whether VEMPs are dependent on type I or II hair cell activity of the saccular macula. An amount of 0.05 ml of gentamicin (40 mg/ml) was injected directly overlaying, but not through, the round window membrane of the left ear in guinea pigs.One week after surgery, auditory brainstem response test revealed normal responses in 12 animals (80%), and elevated thresholds in 3 animals (20%). The VEMP test using click stimulation showed absent responses in all 15 animals (100%). Another 6 gentamicin-treated animals underwent the VEMP test using galvanic stimulation and all 6 also displayed absent responses. Ultrathin sections of the saccular macula in the gentamicin-treated ears displayed morphologic alterations in type I or II hair cells, including shrinkage and/or vacuolization in the cytoplasm, increased electron density of the cytoplasm and nuclear chromatin, and cellular lucency. However, extrusion degeneration was rare and only present in type II hair cells. Quantitative analysis demonstrated that the histological density of intact type I hair cells was 1.1 +/- 1.2/4000 microm(2) in the gentamicin-treated ears, showing significantly less than that in control ears (4.5 +/- 1.8/4000 microm(2)). However, no significant difference was observed in the densities of intact type II hair cells and supporting cells between treated and control ears. Furthermore, the calyx terminals surrounding the damaged type I hair cells were swollen and disrupted, while the button afferents contacting the damaged type II hair cells were not obviously deformed. Based on the above results, we therefore conclude that VEMPs are heavily dependent on type I hair cell activity of the saccular macula in guinea pigs.
Robert J H Payne
Full Text Available Local activation of Rho GTPases is important for many functions including cell polarity, morphology, movement, and growth. Although a number of molecules affecting Rho-of-Plants small GTPase (ROP signalling are known, it remains unclear how ROP activity becomes spatially organised. Arabidopsis root hair cells produce patches of ROP at consistent and predictable subcellular locations, where root hair growth subsequently occurs.We present a mathematical model to show how interaction of the plant hormone auxin with ROPs could spontaneously lead to localised patches of active ROP via a Turing or Turing-like mechanism. Our results suggest that correct positioning of the ROP patch depends on the cell length, low diffusion of active ROP, a gradient in auxin concentration, and ROP levels. Our theory provides a unique explanation linking the molecular biology to the root hair phenotypes of multiple mutants and transgenic lines, including OX-ROP, CA-rop, aux1, axr3, tip1, eto1, etr1, and the triple mutant aux1 ein2 gnom(eb.We show how interactions between Rho GTPases (in this case ROPs and regulatory molecules (in this case auxin could produce characteristic subcellular patterning that subsequently affects cell shape. This has important implications for research on the morphogenesis of plants and other eukaryotes. Our results also illustrate how gradient-regulated Turing systems provide a particularly robust and flexible mechanism for pattern formation.
Sengupta, Soma; George, Manju; Miller, Katharine K; Naik, Khurram; Chou, Jonathan; Cheatham, Mary Ann; Dallos, Peter; Naramura, Mayumi; Band, Hamid; Zheng, Jing
Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. To confirm the interaction, we first demonstrated the EHD4 mRNA expression in hair cells using in situ hybridization. Next, we showed that EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells. Interestingly, the co-immunoprecipitation was found to be calcium-sensitive. To investigate the role of EHD4 in hearing, compound action potentials were measured in EHD4 knock-out (KO) mice. Although EHD4 KO mice have normal hearing sensitivity, analysis of mouse cochlear lysates revealed a 2-fold increase in EHD1, but no increase in EHD2 or EHD3, in EHD4 KO cochleae compared with wild type, suggesting that a compensatory increase in EHD1 levels may account for the absence of a hearing defect in EHD4 KO mice. Taken together, these data indicate that EHD4 is a novel CDH23-interacting protein that could regulate CDH23 trafficking/localization in a calcium-sensitive manner.
Chang, Jiwon; Choi, June; Rah, Yoon Chan; Yoo, Myung Hoon; Oh, Kyoung Ho; Im, Gi Jung; Lee, Seung Hoon; Kwon, Soon Young; Park, Hae-Chul; Chae, Sung Won; Jung, Hak Hyun
Sodium selenite is a trace element essential for many physiological functions in the body. It is involved in various biological processes; it acts as a cofactor for antioxidant enzymes that protect against free radicals and is reported to limit metal-mediated oxidative DNA damage. In the present study, we investigated the effect of sodium selenite on neomycin ototoxicity in wild-type and transgenic zebrafish (Brn3C: EGFP). Five or six days post-fertilization, zebrafish larvae were co-exposed to 125 μM neomycin and various concentrations (10 μM, 100 μM, 250 μM, and 500 μM) of sodium selenite for 1 h. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed by fluorescence microscopy (n = 10 fish per treatment). Hair cell survival was estimated as the ratio of the hair cell numbers in each group compared to those of the control group that were not exposed to neomycin. Apoptosis and hair cell damage of neuromasts were evaluated using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay and 2-[4-(dimethylamino) styryl]-N-ethylpyridinium iodide (DASPEI) assay, respectively. Ultrastructural changes were evaluated using scanning electron microscopy and transmission electron microscopy. Neuromast hair cells were preserved in zebrafish exposed to 125 μM neomycin and 500 μM sodium selenite for 1 h. Sodium selenite protected against neomycin-induced hair cell loss of neuromasts, reduced apoptosis, and prevented zebrafish ultrastructural changes. We propose that sodium selenite protects against neomycin-induced hair cell damage by inhibiting apoptosis, decreasing the disarray of stereocilia, and preventing ultrastructural changes in the neuromast hair cells of the zebrafish.
Nakamagoe, Mariko; Tabuchi, Keiji; Nishimura, Bungo; Hara, Akira
As neuroactive steroids, sex steroid hormones have non-reproductive effects. We previously reported that 17β-estradiol (βE2) had protective effects against gentamicin (GM) ototoxicity in the cochlea. In the present study, we examined whether the protective action of βE2 on GM ototoxicity is mediated by the estrogen receptor (ER) and whether other estrogens (17α-estradiol (αE2), estrone (E1), and estriol (E3)) and other neuroactive steroids, dehydroepiandrosterone (DHEA) and progesterone (P), have similar protective effects. The basal turn of the organ of Corti was dissected from Sprague-Dawley rats and cultured in a medium containing 100 μM GM for 48h. The effects of βE2 and ICI 182,780, a selective ER antagonist, were examined. In addition, the effects of other estrogens, DHEA and P were tested using this culture system. Loss of outer hair cells induced by GM exposure was compared among groups. βE2 exhibited a protective effect against GM ototoxicity, but its protective effect was antagonized by ICI 182,780. αE2, E1, and E3 also protected hair cells against gentamicin ototoxicity. DHEA showed a protective effect; however, the addition of ICI 182,780 did not affect hair cell loss. P did not have any effect on GM-induced outer hair cell death. The present findings suggest that estrogens and DHEA are protective agents against GM ototoxicity. The results of the ER antagonist study also suggest that the protective action of βE2 is mediated via ER but that of DHEA is not related to its conversion to estrogen and binding to ER. Further studies on neuroactive steroids may lead to new insights regarding cochlear protection.
Ding, Dalian; Roth, Jerome; Salvi, Richard
Occupational exposure to high atmospheric levels of Mn produces a severe and debilitating disorder known as manganism characterized by extrapyramidal disturbances similar to that seen in Parkinson’s disease. Epidemiological and case studies suggest that persistent exposures to Mn may have deleterious effects on other organs including the auditory system and hearing. Mn accumulates in the inner ear following acute exposure raising the possibility that it can damage the sensory hair cells that ...
Usher syndrome type IIA (USH2A), characterized by progressive photoreceptor degeneration and congenital moderate hearing loss, is the most common subtype of Usher syndrome. In this article, we show that the USH2A protein, also known as usherin, is an exceptionally large (≈600-kDa) matrix protein expressed specifically in retinal photoreceptors and developing cochlear hair cells. In mammalian photoreceptors, usherin is localized to a spatially restricted membrane microdomain at the apical inne...
Duruflé, Harold; Clemente, Hélène San; Balliau, Thierry; Zivy, Michel; Dunand, Christophe; Jamet, Elisabeth
Plant stems carry flowers necessary for species propagation and need to be adapted to mechanical disturbance and environmental factors. The stem cell walls are different from other organs and can modify their rigidity or viscoelastic properties for the integrity and the robustness required to withstand mechanical impacts and environmental stresses. Plant cell wall is composed of complex polysaccharide networks also containing cell wall proteins (CWPs) crucial to perceive and limit the environmental effects. The CWPs are fundamental players in cell wall remodeling processes, and today, only 86 have been identified from the mature stems of the model plant Arabidopsis thaliana. With a destructive method, this study has enlarged its coverage to 302 CWPs. This new proteome is mainly composed of 27.5% proteins acting on polysaccharides, 16% proteases, 11.6% oxido-reductases, 11% possibly related to lipid metabolism and 11% of proteins with interacting domains with proteins or polysaccharides. Compared to stem cell wall proteomes already available (Brachypodium distachyon, Sacharum officinarum, Linum usitatissimum, Medicago sativa), that of A. thaliana stems has a higher proportion of proteins acting on polysaccharides and of proteases, but a lower proportion of oxido-reductases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Kelly N Owens
Full Text Available Inner ear sensory hair cell death is observed in the majority of hearing and balance disorders, affecting the health of more than 600 million people worldwide. While normal aging is the single greatest contributor, exposure to environmental toxins and therapeutic drugs such as aminoglycoside antibiotics and antineoplastic agents are significant contributors. Genetic variation contributes markedly to differences in normal disease progression during aging and in susceptibility to ototoxic agents. Using the lateral line system of larval zebrafish, we developed an in vivo drug toxicity interaction screen to uncover genetic modulators of antibiotic-induced hair cell death and to identify compounds that confer protection. We have identified 5 mutations that modulate aminoglycoside susceptibility. Further characterization and identification of one protective mutant, sentinel (snl, revealed a novel conserved vertebrate gene. A similar screen identified a new class of drug-like small molecules, benzothiophene carboxamides, that prevent aminoglycoside-induced hair cell death in zebrafish and in mammals. Testing for interaction with the sentinel mutation suggests that the gene and compounds may operate in different pathways. The combination of chemical screening with traditional genetic approaches is a new strategy for identifying drugs and drug targets to attenuate hearing and balance disorders.
Hu, J; Xu, M; Yuan, J; Li, B; Entenman, S; Yu, H; Zheng, Q Y
Sensorineural hearing loss has long been the subject of experimental and clinical research for many years. The recently identified novel mutation of the Cadherin23 (Cdh23) gene, Cdh23(erl/erl), was proven to be a mouse model of human autosomal recessive nonsyndromic deafness (DFNB12). Tauroursodeoxycholic acid (TUDCA), a taurine-conjugated bile acid, has been used in experimental research and clinical applications related to liver disease, diabetes, neurodegenerative diseases, and other diseases associated with apoptosis. Because hair cell apoptosis was implied to be the cellular mechanism leading to hearing loss in Cdh23(erl/erl) mice (erl mice), this study investigated TUDCA's otoprotective effects in erl mice: preventing hearing impairment and protecting against hair cell death. Our results showed that systemic treatment with TUDCA significantly alleviated hearing loss and suppressed hair cell death in erl mice. Additionally, TUDCA inhibited apoptotic genes and caspase-3 activation in erl mouse cochleae. The data suggest that TUDCA could be a potential therapeutic agent for human DFNB12.
Altschuler, Richard A.; Wys, Noel; Prieskorn, Diane; Martin, Cathy; DeRemer, Susan; Bledsoe, Sanford; Miller, Josef M.
Noise overstimulation can induce loss of synaptic ribbons associated with loss of Inner Hair Cell – Auditory Nerve synaptic connections. This study examined if systemic administration of Piribedil, a dopamine agonist that reduces the sound evoked auditory nerve compound action potential and/or Memantine, an NMDA receptor open channel blocker, would reduce noise-induced loss of Inner Hair Cell ribbons. Rats received systemic Memantine and/or Piribedil for 3 days before and 3 days after a 3 hour 4 kHz octave band noise at 117 dB (SPL). At 21 days following the noise there was a 26% and 38% loss of synaptic ribbons in regions 5.5 and 6.5 mm from apex, respectively, elevations in 4-, 8- and 20 kHz tonal ABR thresholds and reduced dynamic output at higher intensities of stimulation. Combined treatment with Piribedil and Memantine produced a significant reduction in the noise-induced loss of ribbons in both regions and changes in ABR sensitivity and dynamic responsiveness. Piribedil alone gave significant reduction in only the 5.5 mm region and Memantine alone did not reach significance in either region. Results identify treatments that could prevent the hearing loss and hearing disorders that result from noise-induced loss of Inner Hair Cell – Auditory Nerve synaptic connections. PMID:27686418
Kikkawa, Yayoi S; Nakagawa, Takayuki; Taniguchi, Mirei; Ito, Juichi
Cisplatin is a widely used chemotherapeutic agent for the treatment of various malignancies. However, its maximum dose is often limited by severe ototoxicity. Cisplatin ototoxicity may require the production of reactive oxygen species (ROS) in the inner ear by activating enzymes specific to the cochlea. Molecular hydrogen was recently established as an antioxidant that selectively reduces ROS, and has been reported to protect the central nervous system, liver, kidney and cochlea from oxidative stress. The purpose of this study was to evaluate the potential of molecular hydrogen to protect cochleae against cisplatin. We cultured mouse cochlear explants in medium containing various concentrations of cisplatin and examined the effects of hydrogen gas dissolved directly into the media. Following 48-h incubation, the presence of intact auditory hair cells was assayed by phalloidin staining. Cisplatin caused hair cell loss in a dose-dependent manner, whereas the addition of hydrogen gas significantly increased the numbers of remaining auditory hair cells. Additionally, hydroxyphenyl fluorescein (HPF) staining of the spiral ganglion showed that formation of hydroxyl radicals was successfully reduced in hydrogen-treated cochleae. These data suggest that molecular hydrogen can protect auditory tissues against cisplatin toxicity, thus providing an additional strategy to protect against drug-induced inner ear damage.
Y. Sandberg (Yorick)
textabstractDuring T-cell development, thymocytes undergo a sequence of immunophenotypic and immunogenotypic changes resulting in the formation of mature T cells with receptors that recognize antigens with high specificity: the T-cell receptor (TCR). Recombination processes underlie the generatio
Fung, KM; Peringa, J; Venkatachalam, S; Lee, VMY; Trojanowski, JQ
We investigated cell proliferation and cell death in the olfactory epithelium (OE) of mice from birth to maturity using bromodeoxyuridine and terminal deoxynucleotidyl transferase nick end labeling. We show that cell death events and proliferative activity diminish concomitantly with age in the OE.
Fung, KM; Peringa, J; Venkatachalam, S; Lee, VMY; Trojanowski, JQ
We investigated cell proliferation and cell death in the olfactory epithelium (OE) of mice from birth to maturity using bromodeoxyuridine and terminal deoxynucleotidyl transferase nick end labeling. We show that cell death events and proliferative activity diminish concomitantly with age in the OE.
Rhee, Chung-Ku; Kim, Young Hoon; Kim, Se Hyung; He, Peijie; Ahn, Jin Chul
Aim: To investigate effects of low level laser therapy (LLLT) on hair cell regeneration following gentamicin ototoxicity in organotypic culture of rat cochlea. Methods: Organotypic cultures of cochlea in culture medium were allowed to grow for 17 days (C group). The organotypic cultures were irradiated daily with 808 nm LD laser, at 28.8 J/ cm2(L group). The organotypic culture were exposed to 1 mM of gentamicin for 48 hr and allowed to recover (G group) or allowed to recover in the culture medium with daily LLLT at 28.8 J/ cm2 (GL group) for 17 days. The cochleae were stained with FM1-43. The number of hair cells was counted in each group serially for 17 days. Results: While the C group kept on losing hair cells in vitro culture, the hair cells remained rather stationary in the L group. The number of hair cells revealed significantly larger number of hair cells in the L group compared to the C group (p=0.05). And the group × time interaction was also significant (p=0.04). That is, the number of hair cells in the C group showed decreasing tendency which was significantly different from the L group. In G group, the initial number of hair cells decreased to 37.2% of that of the gentamicin non-exposed groups. While the G group kept on losing hair cells, the number of hair cells increased in the GL group. The number of hair cells revealed significantly larger in the GL group (p=0.01) compared to G group. And the group × time interaction was also significant (p=0.01). Also, the number of hair cells in the GL group showed increasing tendency which was significantly different from the G group. Conclusion: These results suggest that LLLT promotes hair cell regeneration following gentamicin damage in cochlear explants.
van Netten, S M; Khanna, S M
Cupular vibration in the lateral-line canal of fish was measured in response to motion of the fluid in the canal by laser-heterodyne interferometry. The results show that the mechanical output/input ratio of the cupula depends on the stimulus amplitude; the cupula thus behaves nonlinearly. The nonlinearity is due to the hair bundles, since it disappears when the cupula is uncoupled from the underlying hair cells. A model of cupular dynamics in which the behavior of the gating springs of the hair cells is incorporated predicts nonlinear responses that are similar to the measurements, suggesting that the nonlinear behavior of the cupula may be attributed to the opening and closing of the transduction channels of the hair cells.
Buhl, A E; Kawabe, T T; MacCallum, D K; Waldon, D J; Knight, K A; Johnson, G A
To identify minoxidil target cells in hair follicles we followed the uptake of radiolabeled drug in mouse vibrissae follicles both in vitro and in vivo. Autoradiography showed that both 3H-minoxidil and 3H-minoxidil sulfate accumulated in the differentiating epithelial matrix cells superior to the dermal papilla, a distribution similar to that of pigment. Minoxidil localized in melanocytes, melanocyte processes, and areas of greater melanin concentrations within the epithelial cells. Although uptake of minoxidil was significantly less in unpigmented follicles, the drug stimulated proliferation and differentiation of both pigmented and unpigmented follicles. Labeled minoxidil bound to Sepia melanin and was displaced with unlabeled minoxidil and other electron donor drugs. This interaction with melanin acts as a targeting mechanism of minoxidil to pigmented hair follicles but has no apparent functional significance in hair growth. This work illustrates how measurement of drugs in hair may be biased by pigmentation.
Fridberger, A; Ulfendahl, M
Impaired auditory function following acoustic overstimulation, or noise, is mainly reported to be accompanied by cellular changes such as damage to the sensory hair bundles, but changes in the cell bodies of the outer hair cells have also been described. To investigate more closely the immediate cellular responses to overstimulation, isolated guinea pig outer hair cells were subjected to a 200 Hz oscillating water jet producing intense mechanical stimulation. The water jet was aimed at the cell body of the isolated outer hair cell. Cell shape changes were studied using video microscopy, and intracellular calcium concentration changes were monitored by means of the fluorescent calcium indicator Fluo-3. Cells exposed to a high-intensity stimulus showed surprisingly small light-microscopical alterations. The cytoplasmic calcium concentration increased in most cells, although some cells appeared very resistant to the mechanical stress. No correlation could be found be tween the calcium concentration changes and the cell length. The changes in calcium concentration reported here are suggested to be involved in the long-term pathogenesis of noise-induced hair cell damage.
Mistriotis, Panagiotis; Andreadis, Stelios T
The adult body harbors powerful reservoirs of stem cells that enable tissue regeneration under homeostatic conditions or in response to disease or injury. The hair follicle (HF) is a readily accessible mini organ within the skin and contains stem cells from diverse developmental origins that were shown to have surprisingly broad differentiation potential. In this review, we discuss the biology of the HF with particular emphasis on the various stem cell populations residing within the tissue. We summarize the existing knowledge on putative HF stem cell markers, the differentiation potential, and technologies to isolate and expand distinct stem cell populations. We also discuss the potential of HF stem cells for drug and gene delivery, tissue engineering, and regenerative medicine. We propose that the abundance of stem cells with broad differentiation potential and the ease of accessibility makes the HF an ideal source of stem cells for gene and cell therapies.
The adult body harbors powerful reservoirs of stem cells that enable tissue regeneration under homeostatic conditions or in response to disease or injury. The hair follicle (HF) is a readily accessible mini organ within the skin and contains stem cells from diverse developmental origins that were shown to have surprisingly broad differentiation potential. In this review, we discuss the biology of the HF with particular emphasis on the various stem cell populations residing within the tissue. We summarize the existing knowledge on putative HF stem cell markers, the differentiation potential, and technologies to isolate and expand distinct stem cell populations. We also discuss the potential of HF stem cells for drug and gene delivery, tissue engineering, and regenerative medicine. We propose that the abundance of stem cells with broad differentiation potential and the ease of accessibility makes the HF an ideal source of stem cells for gene and cell therapies. PMID:23157470
Smotherman, M S; Narins, P M
Leopard frog saccular hair cells exhibit an electrical resonance in response to a depolarizing stimulus that has been proposed to contribute to the tuning properties of the frog sacculus by acting as an electrical band-pass filter. With the whole cell patch-clamp technique, we have investigated the effect of temperature on electrical resonances in isolated saccular hair cells, and we have described the effects of temperature on the currents and channel kinetics underlying electrical resonance. A hair cell's onset resonant frequency in response to a constant depolarizing current pulse increases linearly with temperature at a rate of 11 Hz/1 degrees C, exhibiting a mean Q10 of 1.7 between 15 and 35 degrees C. However, offset resonant frequencies continue to double every 10 degrees C, exhibiting a mean Q10 of 2.1. If steady-state voltage during the stimulus is held constant, all oscillatory frequencies increase with a mean Q10 of 2.1. The average level of steady-state depolarization during a +150-pA depolarizing current pulse decreases with increasing temperature (-6 mV from 15 to 25 degrees C). This temperature-dependent reduction of the steady-state membrane potential causes a shift in the voltage-dependent channel kinetics to slower rates, thus reducing the apparent Q10 for onset resonant frequencies. The peak outward tail current and net steady-state outward current, which is the sum of a voltage-dependent inward calcium current (ICa) and an outward calcium-dependent potassium current (IK(Ca)), increase with temperature, exhibiting a mean Q10 of 1.7 between 15 and 25 degrees C. The activation rate (T1/2) of the outward current exhibits a mean Q10 of 2.3 between 15 and 25 degrees C, while the deactivation rate (taurel) exhibits a mean Q10 of 2.9 over the same temperature range. These results support previous models of the molecular determination of resonant frequency, which have proposed that a combination of IK(Ca) channel kinetics and the overall magnitude of the
Izquierdo-Useros, Nuria; Esteban, Olga; Rodriguez-Plata, Maria T; Erkizia, Itziar; Prado, Julia G; Blanco, Julià; García-Parajo, Maria F; Martinez-Picado, Javier
Dendritic cells (DCs) capture human immunodeficiency virus (HIV) through a non-fusogenic mechanism that enables viral transmission to CD4(+) T cells, contributing to in vivo viral dissemination. Although previous studies have provided important clues to cell-free viral capture by mature DCs (mDCs), dynamic and kinetic insight on this process is still missing. Here, we used three-dimensional video microscopy and single-particle tracking approaches to dynamically dissect both cell-free and cell-associated viral capture by living mDCs. We show that cell-free virus capture by mDCs operates through three sequential phases: virus binding through specific determinants expressed in the viral particle, polarized or directional movements toward concrete regions of the cell membrane and virus accumulation in a sac-like structure where trapped viral particles display a hindered diffusive behavior. Moreover, real-time imaging of cell-associated viral transfer to mDCs showed a similar dynamics to that exhibited by cell-free virus endocytosis leading to viral accumulation in compartments. However, cell-associated HIV type 1 transfer to mDCs was the most effective pathway, boosted throughout enhanced cellular contacts with infected CD4(+) T cells. Our results suggest that in lymphoid tissues, mDC viral uptake could occur either by encountering cell-free or cell-associated virus produced by infected cells generating the perfect scenario to promote HIV pathogenesis and impact disease progression.
Wanbao YANG,Qinqun LI,Bo SU,Mei YU
Full Text Available MicroRNAs (miRNAs, small non-coding RNAs, are involved in many aspects of biological processes. Previous studies have indicated that miRNAs are important for hair follicle development and growth. In our study, we found by qRT-PCR that miR-148b was significantly upregulated in sheep wool follicle bulbs in anagen phase compared with the telogen phase of the hair follicle cycle. Overexpression of miR-148b promoted proliferation of both HHDPC and HHGMC. By using the TOPFlash system we demonstrated that miR-148b could activate Wnt/β-catenin pathway and b-catenin, cycD, c-jun and PPARD were consistently upregulated accordingly. Furthermore, transcript factor nuclear factor of activated T cells type 5 (NFAT5 and Wnt10b were predicted to be the target of miR-148b and this was substantiated using a Dual-Luciferase reporter system. Subsequently NFAT5 was further identified as the target of miR-148b using western blotting. These results were considered to indicate that miR-148b could activate the Wnt/β-catenin signal pathway by targeting NFAT5 to promote the proliferation of human hair follicle cells.
Full Text Available BACKGROUND: The bulge region of the hair follicle contains resident epithelial stem cells (SCs that are activated and mobilized during hair growth and after epidermal wounding. However, little is known about the signals that modulate these processes. Clinical and experimental observations show that a reduced supply of sensory innervation is associated with delayed wound healing. Since axon terminals of sensory neurons are among the components of the bulge SC niche, we investigated whether these neurons are involved in the activation and mobilization of the hair stem cells during wound healing. METHODOLOGY/PRINCIPAL FINDINGS: We used neonatal capsaicin treatment to reduce sensory terminals in the rat skin and performed morphometric analyses using design-based stereological methods. Epithelial proliferation was analyzed by quantifying the number of bromodeoxyuridine-labeled (BrdU(+ nuclei in the epidermis and hair follicles. After wounding, the epidermis of capsaicin-treated rats presented fewer BrdU(+ nuclei than in control rats. To assess SC progeny migration, we employed a double labeling protocol with iododeoxyuridine and chlorodeoxyuridine (IdU(+/CldU(+. The proportion of double-labeled cells was similar in the hair follicles of both groups at 32 h postwounding. IdU(+/CldU(+ cell proportion increased in the epidermis of control rats and decreased in treated rats at 61 h postwounding. The epidermal volume immunostained for keratin 6 was greater in treated rats at 61 h. Confocal microscopy analysis revealed that substance P (SP and calcitonin gene-related peptide (CGRP receptor immunoreactivity were both present in CD34(+ and BrdU-retaining cells of the hair follicles. CONCLUSIONS/SIGNIFICANCE: Our results suggest that capsaicin denervation impairs SC progeny egress from the hair follicles, a circumstance associated with a greater epidermal activation. Altogether, these phenomena would explain the longer times for healing in denervated skin
Shin, Hyoseung; Choi, Soon-Jin; Cho, A-Ri; Kim, Dong Young; Kim, Kyu Han
Background Stress is a known cause of hair loss in many species. Objective In this study, we investigated the role of acute stress on hair growth using a rat model. Methods Rats were immobilized for 24 hours and blood samples, and skin biopsies were taken. The effect of stress-serum on the in vitro proliferation of rat and human dermal papilla cells (hDPCs), as well as serum cortisol and corticotropin-releasing hormone levels, were measured. Mast cell staining was performed on the biopsied tissue. In addition, Western blot and quantitative real time polymerase chain reaction were used to assess mast cell tryptase and cytokine expression, respectively in rat skin biopsies. Results Stress-serum treatment reduced significantly the number of viable hDPCs and arrested the cell cycle in the G1 phase, compared to serum from unrestrained rats (p<0.05, respectively). Moreover, restrained rats had significantly higher levels of cortisol in serum than unrestrained rats (p<0.01). Acute stress serum increased mast cell numbers and mast cell tryptase expression, as well as inducing interleukin (IL)-6 and IL-1β up-regulation. Conclusion These results suggest that acute stress also has an inhibitory effect on hair growth via cortisol release in addition to substance P-mast cell pathway. PMID:27746640
Dew, L A; Owen, R G; Mulroy, M J
In this study we describe changes in the size and shape of auditory hair cells of the alligator lizard in vivo during noise-induced temporary threshold shift. These changes consist of a decrease in cell volume, a decrease in cell length and an increase in cell width. We speculate that these changes are due to relaxation of cytoskeletal contractile elements and osmotic loss of intracellular water. We also describe a decrease in the surface area of the hair cell plasmalemma, and speculate that it is related to the endocytosis and intracellular accumulation of cell membrane during synaptic vesicle recycling. Finally we describe an increase in the endolymphatic surface area of the hair cell, and speculate that this could alter the micromechanics of the stereociliary tuft to attenuate the effective stimulus.
Integral hair lipid (IHL) is bound to the keratinized cell surface to make an environmentally resistant lipid envelope. It is mainly positioned on the hair cuticle and inner root sheath. IHL in the hair follicle may regard as hair barrier to be similar to the epidermal lipid layer functioning as skin barrier. Major constituents of IHL are fatty acid, phytosphingosine, ceramide in decreasing order. Minor constituents of IHL are cholesterol, cholesterol sulfate and cholesterol oleate. Cuticle or cortical cell surface in hair are abundant in fatty acids unlike the keratinized area of epidermis or sebaceous gland, and about 30-40% of such fatty acids are composed of 18-methyl-eicosanoic acid which is known to be bound to proteins by ester or thioester bond. Various factors including moisture, solvent, oxidative damage during bleaching or permanent waving affect IHL. Photochemical changes also can occur in IHL as well as in hair protein and hair pigment. Lipid metabolism is thought to play an essential role in lipid envelope of hair, but also involvement in hair development and function. Copyright Â© 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Dichlberger, Andrea; Schlager, Stefanie; Lappalainen, Jani; Käkelä, Reijo; Hattula, Katarina; Butcher, Sarah J; Schneider, Wolfgang J; Kovanen, Petri T
Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.
Wang, Xiaoxiao; Wang, Xusheng; Liu, Jianjun; Cai, Ting; Guo, Ling; Wang, Shujuan; Wang, Jinmei; Cao, Yanpei; Ge, Jianfeng; Jiang, Yuyang; Tredget, Edward E; Cao, Mengjun; Wu, Yaojiong
: Stem cell-based organ regeneration is purported to enable the replacement of impaired organs in the foreseeable future. Here, we demonstrated that a combination of cultured epidermal stem cells (Epi-SCs) derived from the epidermis and skin-derived precursors (SKPs) was capable of reconstituting functional hair follicles and sebaceous glands (SG). When Epi-SCs and SKPs were mixed in a hydrogel and implanted into an excisional wound in nude mice, the Epi-SCs formed de novo epidermis along with hair follicles, and SKPs contributed to dermal papilla in the neogenic hair follicles. Notably, a combination of culture-expanded Epi-SCs and SKPs derived from the adult human scalp were sufficient to generate hair follicles and hair. Bone morphogenetic protein 4, but not Wnts, sustained the expression of alkaline phosphatase in SKPs in vitro and the hair follicle-inductive property in vivo when SKPs were engrafted with neonatal epidermal cells into excisional wounds. In addition, Epi-SCs were capable of differentiating into sebocytes and formed de novo SGs, which excreted lipids as do normal SGs. Thus our results indicate that cultured Epi-SCs and SKPs are sufficient to generate de novo hair follicles and SGs, implying great potential to develop novel bioengineered skin substitutes with appendage genesis capacity. In postpartum humans, skin appendages lost in injury are not regenerated, despite the considerable achievement made in skin bioengineering. In this study, transplantation of a combination of culture-expanded epidermal stem cells and skin-derived progenitors from mice and adult humans led to de novo regeneration of functional hair follicles and sebaceous glands. The data provide transferable knowledge for the development of novel bioengineered skin substitutes with epidermal appendage regeneration capacity. ©AlphaMed Press.
Diseases and Conditions Ingrown hair By Mayo Clinic Staff An ingrown hair occurs when a shaved or tweezed hair grows back into the skin. It can cause ... and tiny bumps in the area where the hair was removed. Ingrown hair is a common condition ...
Kasica, Natalia; Podlasz, Piotr; Sundvik, Maria; Tamas, Andrea; Reglodi, Dora; Kaleczyc, Jerzy
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs-neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H2O2 for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.
Full Text Available Synaptic ribbons are presynaptic structures formed by the self-association of RIBEYE-the main structural component of ribbon synapses. RIBEYE consists of two domains: a unique N-terminal A-domain and a C-terminal B-domain that is identical to the transcription co-repressor C-terminal binding protein 2 (CtBP2. Previous studies in cell lines have shown that RIBEYE A-domain alone is sufficient to form ribbon-like aggregates and that both A- and B- domains form homo-and heterotypic interactions. As these interactions are likely the basis for synaptic-ribbon assembly and structural plasticity, we wanted to examine how zebrafish Ribeye A- and B- domains interact with synaptic ribbons in vivo. To that end, we characterized the localization of exogenously expressed Ribeye A- and B- domains and the closely related protein, CtBP1, in the hair cells of transgenic zebrafish larvae. Unexpectedly, exogenously expressed Ribeye A-domain showed variable patterns of localization in hair cells; one zebrafish paralog of A-domain failed to self-associate or localize to synaptic ribbons, while the other self-assembled but sometimes failed to localize to synaptic ribbons. By contrast, Ribeye B-domain/CtBP2 was robustly localized to synaptic ribbons. Moreover, both exogenously expressed B-domain/CtBP2 and CtBP1 were preferentially localized to the basal end of ribbons adjacent to the postsynaptic density. Overexpression of B-domain/CtBP2 also appeared to affect synaptic-ribbon composition; endogenous levels of ribbon-localized Ribeye were significantly reduced as hair cells matured in B-domain/CtBP2 transgenic larvae compared to wild-type. These results reveal how exogenously expressed Ribeye domains interact with synaptic ribbons, and suggest a potential organization of elements within the ribbon body.
Clark Elliott Strimbu
Full Text Available Active hair bundle motility has been proposed to underlie the amplification mechanism in the auditory endorgans of non-mammals and in the vestibular systems of all vertebrates, and to constitute a crucial component of cochlear amplification in mammals. We used semi-intact in vitro preparations of the bullfrog sacculus to study the effects of elastic mechanical loading on both natively coupled and freely oscillating hair bundles. For the latter, we attached glass fibers of different stiffness to the stereocilia and observed the induced changes in the spontaneous bundle movement. When driven with sinusoidal deflections, hair bundles displayed phase-locked response indicative of an Arnold Tongue, with the frequency selectivity highest at low amplitudes and decreasing under stronger stimulation. A striking broadening of the mode-locked response was seen with increasing stiffness of the load, until approximate impedance matching, where the phase-locked response remained flat over the physiological range of frequencies. When the otolithic membrane was left intact atop the preparation, the natural loading of the bundles likewise decreased their frequency selectivity with respect to that observed in freely oscillating bundles. To probe for signatures of the active process under natural loading and coupling conditions, we applied transient mechanical stimuli to the otolithic membrane. Following the pulses, the underlying bundles displayed active movement in the opposite direction, analogous to the twitches observed in individual cells. Tracking features in the otolithic membrane indicated that it moved in phase with the bundles. Hence, synchronous active motility evoked in the system of coupled hair bundles by external input is sufficient to displace large overlying structures.
Yuxin Ni; Kaizhi Zhang; Xuejuan Liu; Tingting Yang; Baixiang Wang; Li Fu; Lan A; Yanmin Zhou
Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair folli-cles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA (miR)-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist (agomir-21), the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist (antagomir-21), the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3′-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regu-lating SOX protein expression by binding to the 3′-UTR of SOX2 mRNA.
Full Text Available Background and Aim: N-acetylcysteine, a glutathione precursor and reactive oxygen species scavenger, is reported to be effective in reducing noise-induced hearing loss. Many workers in industry are exposed simultaneously to noise and chemical pollutants such as carbon monoxide. We investigated effectiveness of N-acetylcysteine in protecting the cochlea from simultaneous noise and carbon monoxide damages.Methods: Twelve rabbits were exposed simeltaneously to 100 dB sound pressure level of broad band noise and carbon monoxide 8 hours a day for 5 days. One hour before exposure, experimental group received 325 mg/kg of N-acetylcysteine while normal saline was administered for the control group. The protective effect of N-acetylcysteine was evaluated 3 weeks after exposure by histological assessment of the hair cells.Results: Simultaneous exposure to noise and carbon monoxide resulted in a considerable damage to the outer hair cells; however, the inner hair cells and the pillar cells remained intact. Use of N-acetylcysteine in the experimental group significantly reduced the extent of outer hair cell loss.Conclusion: N-acetylcysteine attenuates simultaneous noise and carbon monoxide induced hair cell damage in rabbits.
Rhee, Chung-Ku; He, Peijie; Jung, Jae Yun; Ahn, Jin-Chul; Chung, Phil-Sang; Lee, Min Young; Suh, Myung-Whan
The primary cause of hearing loss includes damage to cochlear hair cells. Low-level laser therapy (LLLT) has become a popular treatment for damaged nervous systems. Based on the idea that cochlea hair cells and neural cells are from same developmental origin, the effect of LLLT on hearing loss in animal models is evaluated. Hearing loss animal models were established, and the animals were irradiated by 830-nm diode laser once a day for 10 days. Power density of the laser treatment was 900 mW/cm2, and the fluence was 162 to 194 J. The tympanic membrane was evaluated after LLLT. Thresholds of auditory brainstem responses were evaluated before treatment, after gentamicin, and after 10 days of LLLT. Quantitative scanning electron microscopic (SEM) observations were done by counting remaining hair cells. Tympanic membranes were intact at the end of the experiment. No adverse tissue reaction was found. On SEM images, LLLT significantly increased the number of hair cells in middle and basal turns. Hearing was significantly improved by laser irradiation. After LLLT treatment, both the hearing threshold and hair-cell count significantly improved.
Full Text Available Loud sound exposure exacerbates aminoglycoside ototoxicity, increasing the risk of permanent hearing loss and degrading the quality of life in affected individuals. We previously reported that loud sound exposure induces temporary threshold shifts (TTS and enhances uptake of aminoglycosides, like gentamicin, by cochlear outer hair cells (OHCs. Here, we explore mechanisms by which loud sound exposure and TTS could increase aminoglycoside uptake by OHCs that may underlie this form of ototoxic synergy.Mice were exposed to loud sound levels to induce TTS, and received fluorescently-tagged gentamicin (GTTR for 30 minutes prior to fixation. The degree of TTS was assessed by comparing auditory brainstem responses before and after loud sound exposure. The number of tip links, which gate the GTTR-permeant mechanoelectrical transducer (MET channels, was determined in OHC bundles, with or without exposure to loud sound, using scanning electron microscopy.We found wide-band noise (WBN levels that induce TTS also enhance OHC uptake of GTTR compared to OHCs in control cochleae. In cochlear regions with TTS, the increase in OHC uptake of GTTR was significantly greater than in adjacent pillar cells. In control mice, we identified stereociliary tip links at ~50% of potential positions in OHC bundles. However, the number of OHC tip links was significantly reduced in mice that received WBN at levels capable of inducing TTS.These data suggest that GTTR uptake by OHCs during TTS occurs by increased permeation of surviving, mechanically-gated MET channels, and/or non-MET aminoglycoside-permeant channels activated following loud sound exposure. Loss of tip links would hyperpolarize hair cells and potentially increase drug uptake via aminoglycoside-permeant channels expressed by hair cells. The effect of TTS on aminoglycoside-permeant channel kinetics will shed new light on the mechanisms of loud sound-enhanced aminoglycoside uptake, and consequently on ototoxic
Full Text Available BACKGROUND: The hearing of tetrapods including humans is enhanced by an active process that amplifies the mechanical inputs associated with sound, sharpens frequency selectivity, and compresses the range of responsiveness. The most striking manifestation of the active process is spontaneous otoacoustic emission, the unprovoked emergence of sound from an ear. Hair cells, the sensory receptors of the inner ear, are known to provide the energy for such emissions; it is unclear, though, how ensembles of such cells collude to power observable emissions. METHODOLOGY AND PRINCIPAL FINDINGS: We have measured and modeled spontaneous otoacoustic emissions from the ear of the tokay gecko, a convenient experimental subject that produces robust emissions. Using a van der Pol formulation to represent each cluster of hair cells within a tonotopic array, we have examined the factors that influence the cooperative interaction between oscillators. CONCLUSIONS AND SIGNIFICANCE: A model that includes viscous interactions between adjacent hair cells fails to produce emissions similar to those observed experimentally. In contrast, elastic coupling yields realistic results, especially if the oscillators near the ends of the array are weakened so as to minimize boundary effects. Introducing stochastic irregularity in the strength of oscillators stabilizes peaks in the spectrum of modeled emissions, further increasing the similarity to the responses of actual ears. Finally, and again in agreement with experimental findings, the inclusion of a pure-tone external stimulus repels the spectral peaks of spontaneous emissions. Our results suggest that elastic coupling between oscillators of slightly differing strength explains several properties of the spontaneous otoacoustic emissions in the gecko.
... normally style your hair? Do you use a hair dryer? What type? How often? What other symptoms are also present? Diagnostic tests that may be performed include: Examination of the hair under a microscope Blood tests
... Loss Surgery? A Week of Healthy Breakfasts Shyness Hair Removal KidsHealth > For Teens > Hair Removal A A A ... recommend an electrologist with the proper credentials. Laser Hair Removal How It Works: A laser is directed through ...
Holmes, William R; Huwe, Janice A; Williams, Barbara; Rowe, Michael H; Peterson, Ellengene H
Vestibular bouton afferent terminals in turtle utricle can be categorized into four types depending on their location and terminal arbor structure: lateral extrastriolar (LES), striolar, juxtastriolar, and medial extrastriolar (MES). The terminal arbors of these afferents differ in surface area, total length, collecting area, number of boutons, number of bouton contacts per hair cell, and axon diameter (Huwe JA, Logan CJ, Williams B, Rowe MH, Peterson EH. J Neurophysiol 113: 2420-2433, 2015). To understand how differences in terminal morphology and the resulting hair cell inputs might affect afferent response properties, we modeled representative afferents from each region, using reconstructed bouton afferents. Collecting area and hair cell density were used to estimate hair cell-to-afferent convergence. Nonmorphological features were held constant to isolate effects of afferent structure and connectivity. The models suggest that all four bouton afferent types are electrotonically compact and that excitatory postsynaptic potentials are two to four times larger in MES afferents than in other afferents, making MES afferents more responsive to low input levels. The models also predict that MES and LES terminal structures permit higher spontaneous firing rates than those in striola and juxtastriola. We found that differences in spike train regularity are not a consequence of differences in peripheral terminal structure, per se, but that a higher proportion of multiple contacts between afferents and individual hair cells increases afferent firing irregularity. The prediction that afferents having primarily one bouton contact per hair cell will fire more regularly than afferents making multiple bouton contacts per hair cell has implications for spike train regularity in dimorphic and calyx afferents.NEW & NOTEWORTHY Bouton afferents in different regions of turtle utricle have very different morphologies and afferent-hair cell connectivities. Highly detailed computational
Full Text Available Reactive oxygen species are important elements in ototoxic damage to hair cells (HCs, appearing early in the damage process. Higher levels of natural antioxidants are positively correlated with resistance to ototoxins and many studies have shown that exogenous antioxidants can protect HCs from damage. While a very wide variety of antioxidants with different characteristics and intracellular targets exist, most ototoxicity studies have focused upon one or a few well-characterized compounds. Relatively little research has attempted to determine the comparative efficacy of large variety of different antioxidants. This has been in part due to the lack of translation between cell culture and in vivo measures of efficacy. To circumvent this limitation, we used an in vitro assay based on micro-explants from the basal and middle turns of the neonatal mouse organ of Corti to screen a commercial redox library of diverse antioxidant compounds for their ability to protect mammalian HCs from a high dose of the ototoxic antibiotic gentamicin. The library included several antioxidants that have previously been studied as potential treatments for HC damage, as well as many antioxidants that have never been applied to ototoxicity. The micro-explants were treated with 200 μM gentamicin alone, gentamicin plus one of three dosages of a redox compound, the highest dosage of compound alone, or were untreated. HC counts were determined before the gentamicin insult and at 1, 2, and 3 days afterward to evaluate the HC survival. From a total of 81 antioxidant compounds, 13 exhibited significant protection of HCs. These included members of a variety of antioxidant classes with several novel antioxidants, not previously tested on HCs, appearing to alleviate the damaging gentamicin effect. Some compounds previously shown to be protective of HCs were correspondingly protective in this in vitro screen, while others were not. Finally, one of the three pro-oxidant compounds
Ito, Mayumi; Liu, Yaping; Yang, Zaixin; Nguyen, Jane; Liang, Fan; Morris, Rebecca J; Cotsarelis, George
The discovery of long-lived epithelial stem cells in the bulge region of the hair follicle led to the hypothesis that epidermal renewal and epidermal repair after wounding both depend on these cells. To determine whether bulge cells are necessary for epidermal renewal, here we have ablated these cells by targeting them with a suicide gene encoding herpes simplex virus thymidine kinase (HSV-TK) using a Keratin 1-15 (Krt1-15) promoter. We show that ablation leads to complete loss of hair follicles but survival of the epidermis. Through fate-mapping experiments, we find that stem cells in the hair follicle bulge do not normally contribute cells to the epidermis which is organized into epidermal proliferative units, as previously predicted. After epidermal injury, however, cells from the bulge are recruited into the epidermis and migrate in a linear manner toward the center of the wound, ultimately forming a marked radial pattern. Notably, although the bulge-derived cells acquire an epidermal phenotype, most are eliminated from the epidermis over several weeks, indicating that bulge stem cells respond rapidly to epidermal wounding by generating short-lived 'transient amplifying' cells responsible for acute wound repair. Our findings have implications for both gene therapy and developing treatments for wounds because it will be necessary to consider epidermal and hair follicle stem cells as distinct populations.
Shah, Aarti N; Marfatia, Ritu K; Saikia, Siddhartha S
Context: Vitiligo surgeries have come a long way from tissue grafts to cultured and non cultured cell transplantation. Extracted hair follicle outer root sheath cell transplantation (EHF ORS) suspension is more enriched with melanocyte. In a hair bulb, there is one melanocyte for every five keratinocytes which is much higher than the epidermal melanin unit. Aims: To analyse the effectiveness of cultured EHF ORS and to perform objective evaluation based on clinical improvement & photographic evidence. To observe any untoward events or side effects. Settings and Design: The study was open and uncontrolled. All the patients were screened at preliminary visit. Reviews were done every two weeks. The endpoint selected was six months post procedure. Materials and Methods: Twenty five patients of stable Vitiligo were included in the study and follicular unit were harvested by Follicular Unit Extraction method. Outer root sheath cells were extracted by trypsinization. The solution was transplanted over dermabraded recipient site. Pressure dressing was given. Patients were followed up regularly. Statistical Analysis Used: Descriptive Statistics, Chi-Square. Results: Mean ± SD repigmentation was 80.15% ± 22.9% with excellent repigmentation (90-100%) in 60% of patients. Conclusions: This method is safe, effective, and simpler than the other methods involving cell culturing and requiring a laboratory set-up but selection of patients is crucial for the success of the outcome. PMID:27601859
Full Text Available Direct videomicroscopic visualization of organ formation and regeneration in toto is a powerful strategy to study cellular processes that often cannot be replicated in vitro. Intravital imaging aims at quantifying changes in tissue architecture or subcellular organization over time during organ development, regeneration or degeneration. A general feature of this approach is its reliance on the optical isolation of defined cell types in the whole animals by transgenic expression of fluorescent markers. Here we describe a simple and robust method to analyze sensory hair-cell development and regeneration in the zebrafish lateral line by high-resolution intravital imaging using laser-scanning confocal microscopy (LSCM and selective plane illumination microscopy (SPIM. The main advantage of studying hair-cell regeneration in the lateral line is that it occurs throughout the life of the animal, which allows its study in the most natural context. We detail protocols to achieve continuous videomicroscopy for up to 68 hours, enabling direct observation of cellular behavior, which can provide a sensitive assay for the quantitative classification of cellular phenotypes and cell-lineage reconstruction. Modifications to this protocol should facilitate pharmacogenetic assays to identify or validate otoprotective or reparative drugs for future clinical strategies aimed at preserving aural function in humans.
Fujii, Shin-Ichiro; Shimizu, Kanako; Hemmi, Hiroaki; Steinman, Ralph M
The observation that the glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent stimulator of natural killer T (NKT) cells has provided an important means for investigating NKT cell biology. alpha-GalCer is presented on CD1d to the invariant NKT receptor, leading to interleukin-12 (IL-12) production by dendritic cells (DCs) and to NK cell activation. We review our research on the tumor-protective properties of alpha-GalCer, particularly the major role played by DCs. We compared administration of alpha-GalCer on mature DCs with soluble glycolipid and found that DCs induced more prolonged interferon-gamma (IFN-gamma) production by NKT cells and better protection against B16 melanoma. Human alpha-GalCer-loaded DCs also expanded NKT cell numbers in cancer patients. alpha-GalCer-activated NKT cells were then found to induce DC maturation in vivo. The maturing DCs produced IL-12, upregulated co-stimulatory molecules, and induced adaptive immunity to captured cellular antigens, including prolonged, combined CD4(+)/CD8(+) T-cell immunity to dying tumor cells. Surprisingly, co-stimulator-poor tumor cells, if directly loaded with alpha-GalCer ('tumor/Gal') and injected intravenously, also induced strong NKT- and NK-cell responses. The latter killed the tumor/Gal, which were subsequently cross presented by CD1d on DCs to elicit DC maturation and prolonged adaptive T-cell immunity, which lasted 6-12 months. These findings help explain tumor protection via alpha-GalCer and urge development of the DC-NKT axis to provide innate and adaptive immunity to human cancers.
Flores, A; Manilla, S; Huidobro, N; De la Torre-Valdovinos, B; Kristeva, R; Mendez-Balbuena, I; Galindo, F; Treviño, M; Manjarrez, E
The stochastic resonance (SR) is a phenomenon of nonlinear systems in which the addition of an intermediate level of noise improves the response of such system. Although SR has been studied in isolated hair cells and in the bullfrog sacculus, the occurrence of this phenomenon in the vestibular system in development is unknown. The purpose of the present study was to explore for the existence of SR via natural mechanical-stimulation in the hair cell-vestibular primary afferent transmission. In vitro experiments were performed on the posterior semicircular canal of the chicken inner ear during development. Our experiments showed that the signal-to-noise ratio of the afferent multiunit activity from E15 to P5 stages of development exhibited the SR phenomenon, which was characterized by an inverted U-like response as a function of the input noise level. The inverted U-like graphs of SR acquired their higher amplitude after the post-hatching stage of development. Blockage of the synaptic transmission with selective antagonists of the NMDA and AMPA/Kainate receptors abolished the SR of the afferent multiunit activity. Furthermore, computer simulations on a model of the hair cell - primary afferent synapse qualitatively reproduced this SR behavior and provided a possible explanation of how and where the SR could occur. These results demonstrate that a particular level of mechanical noise on the semicircular canals can improve the performance of the vestibular system in their peripheral sensory processing even during embryonic stages of development. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.
Hamblet, Corinne E.; Makowski, Stefanie L.; Tritapoe, Julia M.; Pomerantz, Joel L.
NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. Here, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC Class I-deficient tumors in vivo, and cytotoxicity against tumor cells i...
Severinsen, Stig Åvall; Kirkegaard, Mette; Nyengaard, Jens Randel
The aminoglycoside kanamycin is a commonly used antibiotic, but unfortunately it is oto- and nephrotoxic in large doses. The negative effects are thought to be due to the formation of free radicals which is why strong antioxidants and iron chelators like 2,3-dihydroxybenzoic acid (DHB) are of great...... interest. This study estimates cellular quantitative changes in the utricular macula of mice following systemic treatment with kanamycin alone or in combination with DHB. The animals were injected with either saline, kanamycin or kanamycin+DHB for 15 days and perfusion fixed three weeks after last...... macula, hair cell type I and supporting cells decreased significantly in animals injected with kanamycin but not in animals co-treated with DHB. Hair and supporting cell numbers remained unchanged in all three groups. In conclusion, the kanamycin-induced volume reduction of type I hair cells...
Fridberger, Anders; Guinan, John J.
The following is an edited transcript of a recorded discussion session on the topic of "What Shapes the Stimulus to the Inner Hair Cell?". The discussion, moderated by the authors, took place at the 12th International Workshop on the Mechanics of Hearing held at Cape Sounio, Greece, in June 2014. All participants knew that the session was being recorded. In view of both the spontaneous nature of the discussion and the editing, however, this transcript may not represent the considered or final views of the participants, and may not represent a consensus of experts in the field. The reader is advised to consult additional independent publications.
Cochran, S. L.
The concentrations of inorganic cations (K+, Na+, and Ca2+) bathing the isolated frog labyrinth were varied in order to assess their role in influencing and mediating synaptic transmission at the hair cell-afferent fiber synapse. Experiments employed intracellular recordings of synaptic activity from VIIIth nerve afferents. Recordings were digitized continuously at 50 kHz, and excitatory postsynaptic potentials were detected and parameters quantified by computer algorithms. Particular attention was focused on cationic effects upon excitatory postsynaptic potential frequency of occurrence and excitatory postsynaptic potential amplitude, in order to discriminate between pre- and postsynaptic actions. Because the small size of afferents preclude long term stable recordings, alterations in cationic concentrations were applied transiently and their peak effects on synaptic activity were assessed. Increases in extracellular K+ concentration of a few millimolar produced a large increase in the frequency of occurrence of excitatory postsynaptic potentials with little change in amplitude, indicating that release of transmitter from the hair cell is tightly coupled to its membrane potential. Increasing extracellular Na+ concentration resulted in an increase in excitatory postsynaptic potential amplitude with no significant change in excitatory postsynaptic potential frequency of occurrence, suggesting that the transmitter-gated subsynaptic channel conducts Na+ ions. Decreases in extracellular Ca2+ concentration had little effect upon excitatory postsynaptic potential frequency, but increased excitatory postsynaptic potential frequency and amplitude. These findings suggest that at higher concentrations Ca2+ act presynaptically to prevent transmitter release and postsynaptically to prevent Na+ influx during the generation of the excitatory postsynaptic potential. The influences of these ions on synaptic activity at this synapse are remarkably similar to those reported at the
Full Text Available Abstract Background T cell development occurs within the highly specialized thymus. Cytotoxic CD8 T cells are critical in adaptive immunity by targeting virally infected or tumor cells. In this study, we addressed whether functional CD8 T cells can be generated fully in vitro using human umbilical cord blood (UCB hematopoietic stem cells (HSCs in coculture with OP9-DL1 cells. Results HSC/OP9-DL1 cocultures supported the differentiation of CD8 T cells, which were TCR/CD3hi CD27hi CD1aneg and thus phenotypically resembled mature functional CD8 single positive thymocytes. These in vitro-generated T cells also appeared to be conventional CD8 cells, as they expressed high levels of Eomes and low levels of Plzf, albeit not identical to ex vivo UCB CD8 T cells. Consistent with the phenotypic and molecular characterization, upon TCR-stimulation, in vitro-generated CD8 T cells proliferated, expressed activation markers (MHC-II, CD25, CD38, secreted IFN-γ and expressed Granzyme B, a cytotoxic T-cell effector molecule. Conclusion Taken together, the ability to direct human hematopoietic stem cell or T-progenitor cells towards a mature functional phenotype raises the possibility of establishing cell-based treatments for T-immunodeficiencies by rapidly restoring CD8 effector function, thereby mitigating the risks associated with opportunistic infections.
Full Text Available Neuropeptide- and hormone-containing secretory granules (SGs are synthesized at the trans-Golgi network (TGN as immature secretory granules (ISGs and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs. Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs.
Spicer, S S; Schulte, B A
K+ effluxed from outer hair cells and their nerves is thought to flow laterally to strial marginal cells for recycling into scala media. Observations reported here provide evidence that K+ effluxed from inner hair cells and inner radial nerves travels medially through border cells, inner sulcus cells (ISCs), limbal fibrocytes and interdental cells (IDCs) for return to endolymph. Morphologic features of ISCs in the medial route resembled those of Hensen and Claudius cells in the lateral indicating an ion transport role for ISCs like that of Hensen and Claudius cells. Na,K-ATPase in plasmalemma of IDCs testified to their capacity to resorb and transport K+ through their known gap junctions. IDCs were differentiated into three subgroups. The most lateral IDCs formed short and long columns. Long columns contacted the medialmost ISC inferiorly and the undersurface of the tectorial membrane superiorly providing thereby a potential transcellular route for K+ transit from ISCs to endolymph. Short columns faced inner sulcus below and tectorial membrane above and accordingly possessed cells with opposite polarity at the bottom and top of the column. Short columns thus appeared situated to resorb electrolytes from limbal stroma for release into inner sulcus and beneath tectorial membrane at opposite ends of the column. The central IDCs were positioned for resorbing and transporting K+ effluxing from the Na,K-ATPase-rich stellate fibrocytes which spread toward the IDCs from near the inner sulcus. The most medial IDCs lined cuplike invaginations near the attachment of Reissner's membrane and lay apposed to light fibrocytes located between supralimbal fibrocytes and the medial IDCs. Content of Na,K-ATPase and position in the K+ transport route likened the limbal stellate fibrocytes to the spiral ligament type II fibrocytes and supralimbal fibrocytes to suprastrial fibrocytes in the lateral wall. From content of creatine kinase and position in the transport path, limbal light
... For Consumers Consumer Information by Audience For Women Hair Dye and Hair Relaxers Share Tweet Linkedin Pin it More sharing ... products. If you have a bad reaction to hair dyes and relaxers, you should: Stop using the ...
Coffin, Allison B; Rubel, Edwin W; Raible, David W
Sensorineural hearing loss is a normal consequence of aging and results from a variety of extrinsic challenges such as excessive noise exposure and certain therapeutic drugs, including the aminoglycoside antibiotics. The proximal cause of hearing loss is often death of inner ear hair cells. The signaling pathways necessary for hair cell death are not fully understood and may be specific for each type of insult. In the lateral line, the closely related aminoglycoside antibiotics neomycin and gentamicin appear to kill hair cells by activating a partially overlapping suite of cell death pathways. The lateral line is a system of hair cell-containing sense organs found on the head and body of aquatic vertebrates. In the present study, we use a combination of pharmacologic and genetic manipulations to assess the contributions of p53, Bax, and Bcl2 in the death of zebrafish lateral line hair cells. Bax inhibition significantly protects hair cells from neomycin but not from gentamicin toxicity. Conversely, transgenic overexpression of Bcl2 attenuates hair cell death due to gentamicin but not neomycin, suggesting a complex interplay of pro-death and pro-survival proteins in drug-treated hair cells. p53 inhibition protects hair cells from damage due to either aminoglycoside, with more robust protection seen against gentamicin. Further experiments evaluating p53 suggest that inhibition of mitochondrial-specific p53 activity confers significant hair cell protection from either aminoglycoside. These results suggest a role for mitochondrial p53 activity in promoting hair cell death due to aminoglycosides, likely upstream of Bax and Bcl2.
Marc Libault; Andrew Farmer; Laurent Brechenmacher; Jenny Drnevich; Raymond J. Langley; Damla D. Bilgin; Osman Radwan; David J. Neece; Steven J. Clough; Gregory D. May; Gary Stacey
...] and Bradyrhizobium japonicum) initiated by the infection of plant root hair cells by the symbiont. Fewer than 20 plant genes involved in the nodulation process have been functionally characterized...
Draelos, Z K
Alterations in the cuticle, cortex, and medulla are necessary to modify the hair cosmetically. The hair can be modified externally by the use of shampoos to remove excess sebum, conditioners to restore shine, and styling aids to increase manageability. Several different formulations of all these products exist, depending on the needs of the patient. Furthermore, the hair can be modified both externally and internally through the use of hair dyes, permanent waving lotions, and hair straighteners. Use of these products causes external damage to the hair shaft by disrupting the overlapping cuticular scales, rendering the hair susceptible to static electricity and the effects of humidity while decreasing manageability and shine. Internal damage created by these products decreases the hair shaft's elastic properties, allowing increased hair breakage. The dermatologist can better aid the patient with hair difficulties if he or she has an understanding of the formulation and effects of products designed to cleanse, beautify, and modify the hair.
Dallos, P; Hallworth, R; Evans, B N
1. A theory of cochlear outer hair cell electromotility is developed and specifically applied to somatic shape changes elicited in a microchamber. The microchamber permits the arbitrary electrical and mechanical partitioning of the outer hair cell along its length. This means that the two partitioned segments are stimulated with different input voltages and undergo different shape changes. Consequently, by imposing more constraints than other methods, experiments in the microchamber are particularly suitable for testing different theories of outer hair cell motility. 2. The present model is based on simple hypotheses. They include a distributed motor associated with the cell membrane or cortex and the assumption that the displacement generated by the motor is related to the transmembrane voltage across the associated membrane element. It is expected that the force generated by the motor is counterbalanced by an elastic restoring force indigenous to the cell membrane and cortex, and a tensile force due to intracellular pressure. It is assumed that all changes take place while total cell volume is conserved. The above elements of the theory taken together permit the development of qualitative and quantitative predictions about the expected motile responses of both partitioned segments of the cell. Only a DC treatment is offered here. 3. Both a linear motor and an expanded treatment that incorporates a stochastic molecular motor model are considered. The latter is represented by a two-state Boltzmann process. We show that the linear motor treatment is an appropriate extrapolation of the stochastic motor theory for the case of small voltage driving signals. Comparison of experimental results with model responses permits the estimation of model parameters. Good match of data is obtained if it is assumed that the molecular motors undergo conformational length changes of 0.7-1.0 nm, that they have an effective displacement vector at approximately -20 degrees with the long
Griguer, C; Kros, C J; Sans, A; Lehouelleur, J
Type II vestibular hair cells were isolated from cristae ampullares of guinea-pig and maintained in vitro for 2-3 h. Outward membrane currents were studied under whole-cell voltage-clamp conditions. Type II hair cells had resting potentials of about -45 mV. Depolarizing voltage steps from a holding potential of -80 or -90 mV induced time- and voltage-dependent outward currents which slowly decayed to a sustained level. Tail currents reversed at about -70 mV, indicating that the outward currents were mainly carried by potassium ions. The currents had an activation threshold around -50 mV. The transient component was completely removed by a depolarizing pre-pulse positive to -10 mV. While bath application of 4-aminopyridine (5 mM) reduced both components, extracellular tetraethylammonium (10 mM) or zero calcium preferentially diminished the sustained current. We conclude that at least two potassium conductances are present, a delayed rectifier with a relatively fast inactivation and a calcium-dependent potassium current. Depolarizing current injections induced an electrical resonance in the voltage responses, with a frequency of 25-100 Hz, larger currents causing higher frequencies.
Heather C Jensen-Smith
Full Text Available Aminoglycosides (AG, including gentamicin (GM, are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs preferentially succumb to multiple HL pathologies while inner hair cells (IHCs are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH fluorescence during acute (1 h GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies.
Suzuki, Jun; Hashimoto, Ken; Xiao, Ru; Vandenberghe, Luk H.; Liberman, M. Charles
The use of viral vectors for inner ear gene therapy is receiving increased attention for treatment of genetic hearing disorders. Most animal studies to date have injected viral suspensions into neonatal ears, via the round window membrane. Achieving transduction of hair cells, or sensory neurons, throughout the cochlea has proven difficult, and no studies have been able to efficiently transduce sensory cells in adult ears while maintaining normal cochlear function. Here, we show, for the first time, successful transduction of all inner hair cells and the majority of outer hair cells in an adult cochlea via virus injection into the posterior semicircular canal. We used a “designer” AAV, AAV2/Anc80L65, in which the main capsid proteins approximate the ancestral sequence state of AAV1, 2, 8, and 9. Our injections also transduced ~10% of spiral ganglion cells and a much larger fraction of their satellite cells. In the vestibular sensory epithelia, the virus transduced large numbers of hair cells and virtually all the supporting cells, along with close to half of the vestibular ganglion cells. We conclude that this viral vector and this delivery route hold great promise for gene therapy applications in both cochlear and vestibular sense organs. PMID:28367981
Lee, Soung-Hoon; Yoon, Juyong; Shin, Seung Ho; Zahoor, Muhamad; Kim, Hyoung Jun; Park, Phil June; Park, Won-Seok; Min, Do Sik; Kim, Hyun-Yi; Choi, Kang-Yell
Background Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA), a GSK3β inhibitor that activates the Wnt/β-catenin pathway, to promote hair re-growth in vitro and in vivo. Methodology/ Principal Findings Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP). VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/β-catenin pathway, whereas minoxidil (MNX), a drug commonly used to treat alopecia, did not significantly affect the Wnt/β-catenin pathway. VPA analogs and other GSK3β inhibitors that activate the Wnt/β-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl2 also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice. Conclusions/ Significance Our findings indicate that small molecules that activate the Wnt/β-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth. PMID:22506014
Full Text Available BACKGROUND: Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA, a GSK3β inhibitor that activates the Wnt/β-catenin pathway, to promote hair re-growth in vitro and in vivo. METHODOLOGY/ PRINCIPAL FINDINGS: Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP. VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/β-catenin pathway, whereas minoxidil (MNX, a drug commonly used to treat alopecia, did not significantly affect the Wnt/β-catenin pathway. VPA analogs and other GSK3β inhibitors that activate the Wnt/β-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl(2 also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice. CONCLUSIONS/ SIGNIFICANCE: Our findings indicate that small molecules that activate the Wnt/β-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth.
Invariant Natural Killer (iNK)T cells play opposite immune functions. They participate in the innate immune response to promote anti-microbial and anti-tumor immunity and they are crucial to maintain T cell tolerance and prevent autoimmune diseases. While it is well known that the adjuvant function of iNKT cells is mediated through maturation of dendritic cells (DC), the mechanism underlying the tolerogenic function of iNKT cells remains unclear. We performed co-culture experiments with immat...
Haugen, A; Laerum, O D; Bock, E
The effect of partially purified extracts from adult pig brains containing a glia maturation protein factor (BE) has been investigated on neural cells during carcinogenesis. Pregnant BD IX-rats were given a single transplacental dose of the carcinogen ethylnitrosourea (EtNU) on the 18th day of ge...... on GFA-content was seen any longer, although some few weakly GFA positive cells could be observed in all permanent cell lines. Fetal rat brain cells therefore seem to become less responsive to this differentiation inducer during neoplastic transformation in cell culture....
... to support hair growth. Some teens who are vegetarians also lose their hair if they don't get enough protein from non-meat sources. And some athletes are at higher risk for hair loss because they may be more likely to develop iron-deficiency anemia. Disruption of the hair growth cycle. Some ...
Karabourniotis, G; Kofidis, G; Fasseas, C; Liakoura, V; Drossopoulos, I
The subcellular localization (cytoplasm, vacuoles, cell walls) of polyphenol compounds during the development of the multicellular nonglandular leaf hairs of Olea europaea (scales) and Quercus ilex (stellates), was investigated. Hairs of all developmental stages were treated with specific inducers of polyphenol fluorescence, and the bright yellow-green fluorescence of individual hairs was monitored with epifluorescence microscopy. During the early ontogenetic stages, bright fluorescence was emitted from the cytoplasm of the cells composing the multicellular shield of the scales of O. europaea. Transmission electron micrographs of the same stages showed that these cells possessed poor vacuolation and thin cell walls. The nucleus of these cells may be protected against ultraviolet-B radiation damage. The progressive vacuolation that occurred during maturation was followed by a shifting of the bright green-yellow fluorescence from the perinuclear region and the cytoplasm to the cell walls. The same trends were observed during the development of the nonglandular stellate hairs of Quercus ilex, in which maturation was also accompanied by a considerable secondary thickening of the cell walls. Despite the differences in morphology, high concentrations of polyphenol compounds are initially located mainly in the cytoplasm of the developing nonglandular hairs, and their deposition on the cell walls takes place during the secondary cell wall thickening. These structural changes during the development of the leaf hairs make them a very effective barrier against abiotic (uv-B radiation) and probably biotic (pathogenic) stresses.
Kim, Chi Yeon; Lee, Hyun Sup; Cho, Yo Han; Joh, Cheeyoung; Choi, Pyung; Park, Seong Jin
The aim of this work is to design and fabricate a flow sensor using an artificial hair cell (AHC) inspired by biological hair cells of fish. The sensor consists of a single cilium structure with high aspect ratio and a mechanoreceptor using force sensitive resistor (FSR). The cilium structure is designed for capturing a drag force with direction due to flow field around the sensor and the mechanoreceptor is designed for sensing the drag force with direction from the cilium structure and converting it into an electric signal. The mechanoreceptor has a symmetric four electrodes to sense the drag force and its direction. To fabricate the single cilium structure with high aspect ratio, we have proposed a new design concept using a separated micro mold system (SMS) fabricated by the LIGA process. For a successful replication of the cilium structure, we used the hot embossing process with the help of a double-sided mold system. We used a composite of multiwall carbon nanotube and polydimethylsiloxane (MWCNT-PDMS). The performance of the mechanoreceptors was measured by a computer-controlled nanoindenter. We carried out several experiments with the sensor in the different flow rate and direction using the experimental test apparatus. To calibrate the sensor and calculate the velocity with direction based the signal from the sensor, we analyzed the coupled phenomena between flow field and the cilium structure to calculate the deflection of the cilium structure and the drag force applying to the cilium structure due to the flow field around sensor.
Rubin, I M C; Dabelsteen, S; Nielsen, M M;
We have recently shown that commercial p-phenylenediamine (PPD)-containing hair dyes are potent immune activators that lead to severe contact hypersensitivity in an animal model. However, only a minority of people exposed to permanent hair dyes develops symptomatic contact hypersensitivity....... This suggests that the majority of people exposed to hair dyes does not become sensitized or develop immunological tolerance....
Svane, I M; Nikolajsen, K; Walter, M R;
Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). In order to obtain full functional capacity, maturation is required......, maturation with IFN-alpha has only a small effect on induction of autologous T-cell stimulatory capacity of the DC. However, an increase in DC allogeneic T-cell stimulatory capacity was observed. These data suggest that IFN-alpha has a potential as a maturation agent used in DC-based cancer vaccine trials...
Jintao Yu; Dalian Ding; Fengjun Wang; Haiyan Jiang; Hong Sun; Richard Salvi
To gain insights into the ototoxic effects of aminoglycoside antibiotics (AmAn) and delayed peripheral ganglion neuron death in the inner ear, experimental animal models were widely used with several different approaches including AmAn systemic injections, combination treat-ment of AmAn and diuretics, or local application of AmAn. In these approaches, systemic AmAn treatment alone usually causes incomplete damage to hair cells in the inner ear. Co-administration of diuretic and AmAn can completely destroy the cochlear hair cells, but it is impossible to damage the vestibular system. Only the approach of AmAn local application can selectively eliminate most sensory hair cells in the inner ear. Therefore, AmAn local application is more suitable for studies for complete hair cell destructions in cochlear and vestibular system and the following delayed peripheral ganglion neuron death. In current studies, guinea pigs were unilaterally treated with a high concentration of gentamicin (GM, 40 mg/ml) through the tympanic membrane into the middle ear cavity. Auditory functions and vestibular functions were measured before and after GM treatment. The loss of hair cells and delayed degeneration of ganglion neurons in both cochlear and vestibular system were quantified 30 days or 60 days after treatment. The results showed that both auditory and vestibular functions were completely abolished after GM treatment. The sensory hair cells were totally missing in the cochlea, and severely destroyed in vestibular end-organs. The delayed spiral ganglion neuron death 60 days after the deafening procedure was over 50%. However, no obvious pathological changes were observed in vestibular ganglion neurons 60 days post-treatment. These results indicated that a high concentration of gentamycin delivered to the middle ear cavity can destroy most sensory hair cells in the inner ear that subsequently causes the delayed spiral ganglion neuron degeneration. This model might be useful for studies
Full Text Available Retrograde degeneration of spiral ganglion cells in the cochlea following hair cell loss is similar to dying back in pathology. The EFR3A gene has recently been discovered to be involved in the pathogenesis of dying back. The relationship of EFR3A and spiral ganglion degeneration, however, was rarely investigated. In this study, we destroyed the hair cells of the mouse cochlea by co-administration of kanamycin and furosemide and then investigated the EFR3A expression during the induced spiral ganglion cell degeneration. Our results revealed that co-administration of kanamycin and furosemide quickly induced hair cell loss in the C57BL/6J mice and then resulted in progressive degeneration of the spiral ganglion beginning at day 5 following drug administration. The number of the spiral ganglion cells began to decrease at day 15. The expression of EFR3A increased remarkably in the spiral ganglion at day 5 and then decreased to near normal level within the next 10 days. Our study suggested that the change of EFR3A expression in the spiral ganglion was coincident with the time of the spiral ganglion degeneration, which implied that high expression of EFR3A may be important to prompt initiation of spiral ganglion degeneration following hair cell loss.
Rohmann, Kevin N; Wersinger, Eric; Braude, Jeremy P; Pyott, Sonja J; Fuchs, Paul Albert
Cholinergic neurons of the brainstem olivary complex project to and inhibit outer hair cells (OHCs), refining acoustic sensitivity of the mammalian cochlea. In all vertebrate hair cells studied to date, cholinergic inhibition results from the combined action of ionotropic acetylcholine receptors and
Wiersinga-Post, JEC; van Netten, SM
Amiloride is a known blocker of the mechano-electrical transduction current in sensory hair cells. Measurements of cupular motion in the lateral line organ of fish now show that amiloride concurrently changes the micromechanical properties of the hair cell bundles. The effects of amiloride on the me
Wiersinga-Post, JEC; van Netten, SM
Amiloride is a known blocker of the mechano-electrical transduction current in sensory hair cells. Measurements of cupular motion in the lateral line organ of fish now show that amiloride concurrently changes the micromechanical properties of the hair cell bundles. The effects of amiloride on the
Full Text Available Background of the study: Mature and functional Sertoli cells are essential for the survival of germ cells in testes. In Sertoli cell only syndrome (SCOS, there is no germ cells. Then, question arises whether absence of germ cells in SCOS secondary to Sertoli cells immaturity or mal function. Sertoli cells maturational and functional status is unclear in SCOS. This study investigated status of maturation and function of Sertoli cells in patients with SCOS. Materials and Methods: The present study was comprised of 37 cases of SCOS and 50 normal control males. Detailed clinical examination and investigation were carried out as per pre-determined proforma. Semen analysis, hormonal analysis (FSH, LH, testosterone, etc., and fine needle aspiration cytology (FNAC of testes (bilateral were performed. Fluorescence in situ hybridization (FISH with XY probes was carried out in addition to conventional chromosome analysis to find out chromosomal abnormalities, in particular sex chromosome aneuploidy, including mosaicism. Yq microdeletion status was also investigated. The anti-mullerian hormone (AMH, inhibin B, and seminal lactate were estimated by ELISA methods. Results: The study did not find any case of high AMH. About 78% cases had low inhibin B, and 60% had low AMH. FSH was high in about 78% cases. Low level of lactate was found in 49% cases. There was one case of high level of inhibin B. There were 6 (16.2% cases of chromosomal abnormality (2 mosaic Klinefelter and 4 Klinefelter syndrome and 4 (10.8% cases of Yq microdeletion. Conclusion: We conclude that Sertoli cell immaturity does not play any role in SCOS (no case of high AMH. It seems, in majority cases, Sertoli cells are functionally- and/or numerically-deficient (low inhibin B, AMH and lactate. However, in about 22% cases, Sertoli cell function and/or number remains normal (normal inhibin B, AMH. Inhibin B and FSH seems best predictor/marker of Sertoli cell function.
Stuart, Lynda M; Lucas, Mark; Simpson, Cathy; Lamb, Jonathan; Savill, John; Lacy-Hulbert, Adam
Dendritic cells (DCs) are the sentinels of the immune system, able to interact with both naive and memory T cells. The recent observation that DCs can ingest cells dying by apoptosis has raised the possibility that DCs may, in fact, present self-derived Ags, initiating both autoimmunity and tumor-specific responses, especially if associated with appropriate danger signals. Although the process of ingestion of apoptotic cells has not been shown to induce DC maturation, the exact fate of these phagocytosing DCs remains unclear. In this paper we demonstrate that DCs that ingest apoptotic cells are able to produce TNF-alpha but have a diminished ability to produce IL-12 in response to external stimuli, a property that corresponds to a failure to up-regulate CD86. By single-cell analysis we demonstrate that these inhibitory effects are restricted to those DCs that have engulfed apoptotic cells, with bystander DCs remaining unaffected. These changes were independent of the production of anti-inflammatory cytokines TGF-beta1 and IL-10 and corresponded with a diminished capacity to stimulate naive T cells. Thus, the ingestion of apoptotic cells is not an immunologically null event but is capable of modulating DC maturation. These results have important implications for our understanding of the role of clearance of dying cells by DCs not only in the normal resolution of inflammation but also in control of subsequent immune responses to apoptotic cell-derived Ags.
Boisvert, William A; Yu, Miri; Choi, Youngbin; Jeong, Gi Hee; Zhang, Yi-Lin; Cho, Sunghun; Choi, Changsun; Lee, Sanghyun; Lee, Bog-Hieu
Geranium sibiricum L. has been used as a medicinal plant to treat diarrhea, bacterial infection, and cancer in Bulgaria, Peru, and Korea. However, its hair growth-promoting effect was not investigated so far. This study examined the effects of Geranium sibiricum L. extract (GSE) on hair growth, using in vitro and in vivo models. Antioxidant, proliferation and migration assay of GSE was performed with human dermal papilla cells (hDPCs). Hair-growth promoting effect was measured in animal model. Relative expression of interleukin-1, vascular endothelial growth factor, hepatocyte growth factor, and transforming growth factor beta 1 was determined by real time RT-PCR. Expression of Ki-67 and stem cell factor were analyzed by immunohistochemistry. GSE treatment proliferated and migrated human dermal papilla cells (hDPCs) more than treatment of 10 μM minoxidil. GSE significantly stimulated the expression of Ki-67 protein and the mRNA levels of hepatocyte growth factor and vascular endothelial growth factor in hDPCs. Topical application of 1,000 ppm GSE for 3 weeks promoted more significant hair growth on shaved C57BL/6 mice than did 5% minoxidil. The histological morphology of hair follicles demonstrated an active anagen phase with the induction of stem cell factor. GSE treatment significantly reduced the number of mast cells and the expression of transforming growth factor beta 1 in mouse skin tissues. These results demonstrated that GSE promotes hair growth in vitro and in vivo by regulating growth factors and the cellular response.
Full Text Available Experiments utilizing the Looptail mutant mouse, which harbors a missense mutation in the vangl2 gene, have been essential for studies of planar polarity and linking the function of the core planar cell polarity proteins to other developmental signals. Originally described as having dominant phenotypic traits, the molecular interactions underlying the Looptail mutant phenotype are unclear because Vangl2 protein levels are significantly reduced or absent from mutant tissues. Here we introduce a vangl2 knockout mouse and directly compare the severity of the knockout and Looptail mutant phenotypes by intercrossing the two lines and assaying the planar polarity of inner ear hair cells. Overall the vangl2 knockout phenotype is milder than the phenotype of compound mutants carrying both the Looptail and vangl2 knockout alleles. In compound mutants a greater number of hair cells are affected and changes in the orientation of individual hair cells are greater when quantified. We further demonstrate in a heterologous cell system that the protein encoded by the Looptail mutation (Vangl2(S464N disrupts delivery of Vangl1 and Vangl2 proteins to the cell surface as a result of oligomer formation between Vangl1 and Vangl2(S464N, or Vangl2 and Vangl2(S464N, coupled to the intracellular retention of Vangl2(S464N. As a result, Vangl1 protein is missing from the apical cell surface of vestibular hair cells in Looptail mutants, but is retained at the apical cell surface of hair cells in vangl2 knockouts. Similarly the distribution of Prickle-like2, a putative Vangl2 interacting protein, is differentially affected in the two mutant lines. In summary, we provide evidence for a direct physical interaction between Vangl1 and Vangl2 through a combination of in vitro and in vivo approaches and propose that this interaction underlies the dominant phenotypic traits associated with the Looptail mutation.
Mravec, Jozef; Guo, Xiaoyuan; Hansen, Aleksander Riise
The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases though, plant cells are programmed to detach and root cap-derived border cells are examples of this....... Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we...... undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immuno-carbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy (FT-IR), quantitative RT-PCR of cell wall biosynthetic genes, analysis...
Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma; Recurrent Mature T- and NK-Cell Non-Hodgkin Lymphoma; Refractory Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma; Recurrent Cutaneous T-Cell Non-Hodgkin Lymphoma; Refractory Cutaneous T-Cell Non-Hodgkin Lymphoma
... treatments include hair coloring, hair curling (permanents), hair bleaching, and hair straightening (relaxers) agents. For this fact sheet, hair ... permanent wave chemicals include ammonium thioglycolate and ammonia. Hair bleaching chemicals include hydrogen peroxide. Hair straighteners (relaxers) use ...
Full Text Available Background: Nowadays, dendritic cells (DCs have a special place in cancer treatment strategies and they have been used for tumor immunotherapy as they can induce immune response against tumor cells. Researchers have been trying to generate efficient dendritic cells in vitro; therefore, this research was done to generate them for use in research and tumor immunotherapy. Methods: This study took place at Urmia University in 2010-2011 years. In this study plastic adherent monocytes were incubated with granulocyte-macrophage colony stimulating factor (GM-CSF and interleukin-4 (IL-4 for five days. Finally, fully matured and stable DCs were generated by 48 hours of incubation in a monocyte conditioned medium (MCM containing tumor necrosis factor-α (TNF-α and epithelial cells. Phenotypic and functional analysis were carried out by using anti-CD14, anti-CD80, anti-CD86, and anti-CD83 monoclonal antibodies, and by determining their phagocytic activity, mixed lymphocyte reaction (MLR and cytokine production, respectively. Results: Dendritic cells were produced with high levels of surface molecule, i.e. of CD80, CD83, CD86, HLA-DR, expression and low levels of CD14 expression. Dendritic cells showed efficient phagocytosis and ability to stimulate T-lymphocytes. Moreover, dendritic cells could secrete high levels of interleukin-12 (IL-12 cytokine which was depictive of their full maturation. Measurement of the produced cytokines showed the generation of type-1 dendritic cells (DC1. Conclusion: Our study showed that skin epithelial cells could induce maturation of monocyte-derived dendritic cells (DCs. This feeder layer led to the production of efficient dendritic cells with the ability to be used for tumor immunotherapy.
Jia, Yan-Jun; Liu, Jiang; Zhang, Ke-Ying; Shang, Xiao-Yan; Li, Wei; Wang, Li-Jun; Liu, Na; Wang, Lin; Cui, Shuang; Ni, Lei; Zhao, Bo-Tao; Wang, Dong-Mei; Gao, Song-Ming; Zhang, Zhi-Xin
Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.
Ikeda, Ryo; Gu, Jianguo G
We have recently shown that Merkel cells transduce tactile stimuli via Piezo2 channels to initiate the sense of touch. Here we performed patch-clamp recordings to assess single channel activity on the membranes of Merkel cells in whisker hair follicles. Under the cell-attached configuration, most Merkel cell membrane patches showed large outward unitary currents with single channel conductance being ∼200pS. The outward unitary currents were not affected by negative pressures up to 150mmHg when applied to the membrane patches. The application of negative pressures up to 190mmHg also could not directly elicit any inward unitary current in the membrane patches. However, after establishing the whole-cell configuration, mechanically activated currents (MA) that resembled Piezo2 currents could be elicited by membrane displacements in every Merkel cell tested. While the MA current decayed rapidly, a small steady-state current component with significant channel noise could be observed. Applications of stationary and non-stationary fluctuation analyses to the MA currents yielded single channel conductance of 32.5±3.8 and 54.0±5.3pS, respectively. The lack of mechanical responses under the cell-attached configuration and the existence of Piezo2 MA currents under the whole-cell configuration raised a possibility that Piezo2 channels are preferentially located on Merkel cell processes, the membrane domains inaccessible by recording electrodes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
Full Text Available ABSTRACT Background: Nitric oxide (NO have a dual action in mouse oocyte meiotic maturation which depends on its concentration, but the mechanisms by which it influences oocyte maturation has not been exactly clarified. In this study different signaling mechanisms which exist for in vitro maturation of meiosis was examined in cumulus cell-enclosed oocytes (CEOs after injection of pregnant mare's serum gonadotropin (PMSG to immature female mice. Methods: The CEOs were cultured in spontaneous maturation and hypoxanthine (HX arrested model. Results: Sodium nitroprusside (SNP, an NO donor, 10mM delayed germinal vesicle breakdown (GVBD significantly during the first 5 hrs of incubation and inhibited the formation of first polar body (PB1 at the end of 24 hrs of incubation. SNP (10-5M stimulated the meiotic maturation of oocytes significantly by overcoming the inhibition of HX. Sildenafil (a cGMP stimulator, 100 nM, had a significant inhibitory effects on both spontaneous meiotic maturation and HX-arrested meiotic maturation. Forskolin (an adenylate cyclase stimulator, 6µM and SNP (10mM had the same effects on GVBD. Forskolin reversed the SNP (10-5M stimulated meiotic maturation. Conclusion: These results suggest that differences in pathways are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation in mouse oocytes
Full Text Available Doping with anabolic agents is a topic in sports where strength is crucial, e.g. sprinting, weight lifting and many more. Testosterone and its functional analogs are the drugs of choice taken as pills, creams, tape or injections to increase muscle mass and body performance, and to reduce body fat. Stanozolol (17β-hydroxy-17α-methyl-5α-androst- 2-eno[3,2c]pyrazol is a testosterone analogue with the same anabolic effect like testosterone but its ring structure makes it possible to take it orally. Therefore, stanozolol is one of the most frequently used anabolic steroids. Common verification methods for anabolic drugs exist, identifying the chemicals in tissues, like hair or blood samples. The idea of this feasibility study was to search for specific gene expression regulations induced by stanozolol to identify the possible influence of the synthetically hormone on different metabolic pathways. Finding biomarkers for anabolic drugs could be supportive of the existing methods and an additional proof for illegal drug abuse. In two separate cell cultures, human HFDPC (hair follicle dermal papilla cells from a female and a male donor were treated with stanozolol. In the female cell culture treatment concentrations of 0 nM (control, 1 nM, 10 nM and 100 nM were chosen. Cells were taken 0 h, 6 h, 24 h and 48 h after stimulation and totalRNA was extracted. Learning from the results of the pilot experiment, the male cell culture was treated in 10 nM and 100 nM concentrations and taken after 0 h, 6 h, 24 h and 72 h. Using quantitative real-time RT-PCR expression of characteristics of different target genes were analysed. Totally 13 genes were selected according to their functionality by screening the actual literature and composed to functional groups: factors of apoptosis regulation were Fas Ligand (FasL, its receptor (FasR, Caspase 8 and Bcl-2. Androgen receptor (AR and both estrogen receptors (ERα, ERβ were summarized in the steroid receptor group
Tierney, Emily P; Goldberg, David J
A number of lasers and light devices are now available for the treatment of unwanted hair. The goal of laser hair removal is to damage stem cells in the bulge of the follicle through the targeting of melanin, the endogenous chromophore for laser and light devices utilized to remove hair. The competing chromophores in the skin and hair, oxyhemoglobin and water, have a decreased absorption between 690 nm and 1000 nm, thus making this an ideal range for laser and light sources. Pearls of laser hair removal are presented in this review, focusing on four areas of recent development: 1 treatment of blond, white and gray hair; 2 paradoxical hypertrichosis; 3 laser hair removal in children; and 4 comparison of lasers and IPL. Laser and light-based technologies to remove hair represents one of the most exciting areas where discoveries by dermatologists have led to novel treatment approaches. It is likely that in the next decade, continued advancements in this field will bring us closer to the development of a more permanent and painless form of hair removal.
Photoaggravation of hair aging includes various chemical and physical changes in fiber properties which lead to an increase in fiber porosity, loss of mechanical strength and an increase in surface roughness. These changes come from lipid oxidation, disulfide bond cleavage, tryptophan degradation and cysteic acid formation. Hair exposed to sunlight is claimed to be more brittle, stiffer and drier than before irradiation and exhibits a reduced water-absorption capacity. Hair pigments function to provide photochemical protection to hair proteins. Hair pigments accomplish this protection by absorbing and filtering the impinging radiation and subsequently dissipating this energy as heat. However, in the process of protecting the hair proteins from light, the pigments are degraded or bleached. Dark hair is more resistant to photodegradation than light hair, because of the higher photostability of eumelanin compared to pheomelanin. Integral lipids of hair fibers are degraded by ultraviolet light, as well as by visible light, helping to explain the weakening of the cell membrane complex exposed to light radiation. PMID:20927230
Wang, Xiong; Shi, Ying; Zhou, Qiong; Liu, Xiaoming; Xu, Shizheng; Lei, Tiechi
In the bulge region of the hair follicle, a densely and concentrically packed cell mass is encircled by the arrector pili muscle (APM), which offers a specilized microenvironment (niche) for housing heterogeneous adult stem cells. However, the detailed histological architecture and the cellular composition of the bulge region warrants intensive study and may have implications for the regulation of hair follicle growth regulation. This study was designed to define the gene-expression profiles of putative stem cells and lineage-specific precursors in the mid-portions of plucked hair follicles prepared according to the presence of detectable autofluorescence. The structure was also characterized by using a consecutive sectioning technique. The bulge region of the hair follicle with autofluorescence was precisely excised by employing a micro-dissection procedure. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to identify the gene expression profiles specific for epithelial, melanocyte and stromal stem cells in the bulge region of the hair follicle visualized by autofluorescence. The morphology and its age-dependent changes of bulge region of the hair follicles with autofluorescence segment were also examined in 9 scalp skin specimens collected from patients aged 30 weeks to 75 years, by serial sectioning and immuno-staining. Gene expression profile analysis revealed that there were cells with mRNA transcripts of Dct(Hi)Tyrase(Lo)-Tyrp1(Lo)MC1R(Lo)MITF(Lo)/K15(Hi)/NPNT(Hi) in the bulge region of the hair follicle with autofluorescence segments, which differed from the patterns in hair bulbs. Small cell-protrusions that sprouted from the outer root sheath (ORS) were clearly observed at the APM inserting level in serial sections of hair follicles by immunohistological staining, which were characteristically replete with K15+/K19+expressing cells. Likewise, the muscle bundles of APM positive for smooth muscle actin intimately
Choi, Kyung-Dal; Vodyanik, Maxim; Slukvin, Igor I.
Here we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin-CD34+CD43+CD45+ multipotent progenitors. The protocol is comprised of three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells, (ii) short-term expansion of multipotent myeloid progenitors with a high dose of GM-CSF, and ...
Clerici, W J; Chertoff, M E; Brownell, W E; Fechter, L D
Trimethyltin (TMT) and triethyltin (TET) disrupt auditory function at doses far below those shown to be neurotoxic. In vivo studies suggest that the initial effect of TMT on hearing occurs at the inner hair cell/spiral ganglion cell synapse, while later, the outer hair cell (OHC) undergoes structural and functional damage. TET produces acute effects upon afferent neurotransmission similar to those observed following TMT, but TET's effects on OHC structure and function have not been examined. OHCs are motile elements within the cochlea, believed to modulate the sensitivity and tuning within the inner ear. Changes in OHC length may alter hearing function, and length changes have been reported following exposure to various ototoxic agents in vitro. In the present study, 77 OHCs from 45 pigmented male guinea pigs were isolated in primary culture and exposed for 90 min to concentrations between 30 microM and 1.0 mM of TMT or TET and then to bathing medium for 30 min to remove the toxicant. Significant shortening of the OHC cell body occurred at all doses to both organotins, with a mean reduction in length of 15.1 and 20.2% for 1.0 mM TMT and TET, respectively, at the end of testing; control cells were only 3.4% shorter at the end of 90 min of perfusion with bathing medium. The effect of organotin exposure on OHC volume was not consistently related to either TMT or TET concentration or altered cell length. In addition, disruption of the plasma membrane characterized by bleb formation, the forceful ejection of cytoplasm, or bursting was seen in 80% of cells exposed to 1.0 mM TET, although not TMT; lower concentrations of both organotins disrupted the cell membrane in 10-30% of cells. Membrane rupture was not reliably associated with either increased cell volume or decreased length, implicating a weakening of the plasma membrane or cortical lattice as the basis for this effect. Consistent with the irreversible structural weakening of the lateral wall, resorption of
Lee, Andrew W; Hertel, Laura; Louie, Ryan K; Burster, Timo; Lacaille, Vashti; Pashine, Achal; Abate, Davide A; Mocarski, Edward S; Mellins, Elizabeth D
Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.
Kros, CJ; Marcotti, W; van Netten, SM; Self, TJ; Libby, RT; Brown, SDM; Richardson, GP; Steel, KP
Mutations in Myo7a cause hereditary deafness in mice and humans. We describe the effects of two mutations, Myo7a(6J) and Myo7a(4626SB). on mechano-electrical transduction in cochlear hair cells. Both mutations result in two major functional abnormalities that would interfere with sound transduction.
Dinklo, Theo; Meulenberg, Cecil J. W.; van Netten, Sietse M.
The investigation of small physiological mechanosensory systems, such as hair cells or their accessory structures in the inner ear or lateral line organ, requires mechanical stimulus equipment that allows spatial manipulation with micrometer precision and stimulation with amplitudes down to the nano
O'Keeffe, Mary G; Thorne, Peter R; Housley, Gary D; Robson, Simon C; Vlajkovic, Srdjan M
A complex extracellular nucleotide signalling system acting on P2 receptors is involved in regulation of cochlear function in the mammalian inner ear. Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are ectonucleotidases that regulate P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from the CD39/ENTPD family (NTPDase1-8) are expressed in the adult rat cochlea, but their expression and distribution in the vestibular end organ is unknown. This report demonstrates selective expression of NTPDase6 by rat vestibular hair cells. Hair cells transducing both angular acceleration (crista ampullaris) and static head position (maculae of the utricle and saccule) exhibited strong immunolabelling with a bias towards the sensory pole and in particular, the hair cell bundle. NTPDase6 is an intracellular enzyme that can be released in a soluble form from cell cultures and shows an enzymatic preference for nucleoside 5'-diphosphates, such as guanosine 5'-diphosphate (GDP) and uridine 5'-diphosphate (UDP). The main function of NTPDase6 may be the regulation of nucleotide levels in cellular organelles by regulating the conversion of nucleotides to nucleosides. NTPDase6 immunolocalisation in the vestibular end organ could be linked to the regulation of P2 receptor signalling and sensory transduction, including maintenance of vestibular hair bundles.
Wiersinga-Post, JEC; van Netten, SM
The mechanical frequency selectivity of the cupula located in the supraorbital lateral line canal and the frequency selectivity of the hair cells driven by the cupula were measured simultaneously in vivo. Laser interferometry was used to measure cupular mechanics and extracellular receptor potential
Givi, Masoumeh Ezzati; Folkerts, Gert; Wagenaar, Gerry T M; Redegeld, Frank A; Mortaz, Esmaeil
BACKGROUND: Dendritic cells (DCs) as professional antigen presenting cells (APCs) play a critical role in the regulation of host immune responses. DCs evolve from immature, antigen-capturing cells, to mature antigen-presenting cells. The relative contribution of DCs to cigarette smoke-induced inflam
Bax, Marieke; Garcia-Vallejo, Juan J.; Jang-Lee, Jihye; North, Simon J.; Gilmartin, Tim J.; Hernandez, Gilberto; Crocker, Paul R.; Leffler, Hakon; Head, Steven R.; Haslam, Stuart M.; Dell, Anne; van Kooyk, Yvette
Dendritic cells (DC) are the most potent APC in the organism. Immature dendritic cells (iDC) reside in the tissue where they capture pathogens whereas mature dendritic cells (mDC) are able to activate T cells in the lymph node. This dramatic functional change is mediated by an important genetic repr
Full Text Available Inner hair cells (IHCs, the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca(2+ action potentials before the onset of hearing. Although this firing activity is mainly sustained by a depolarizing L-type (Ca(V1.3 Ca(2+ current (I(Ca, IHCs also transiently express a large Na(+ current (I(Na. We aimed to investigate the specific contribution of I(Na to the action potentials, the nature of the channels carrying the current and whether the biophysical properties of I(Na differ between low- and high-frequency IHCs. We show that I(Na is highly temperature-dependent and activates at around -60 mV, close to the action potential threshold. Its size was larger in apical than in basal IHCs and between 5% and 20% should be available at around the resting membrane potential (-55 mV/-60 mV. However, in vivo the availability of I(Na could potentially increase to >60% during inhibitory postsynaptic potential activity, which transiently hyperpolarize IHCs down to as far as -70 mV. When IHCs were held at -60 mV and I(Na elicited using a simulated action potential as a voltage command, we found that I(Na contributed to the subthreshold depolarization and upstroke of an action potential. We also found that I(Na is likely to be carried by the TTX-sensitive channel subunits Na(V1.1 and Na(V1.6 in both apical and basal IHCs. The results provide insight into how the biophysical properties of I(Na in mammalian cochlear IHCs could contribute to the spontaneous physiological activity during cochlear maturation in vivo.
Full Text Available Abstract Background Dendritic cells (DCs are often produced by granulocyte-macrophage colony-stimulating factor (GM-CSF and interleukin-4 (IL-4 stimulation of monocytes. To improve the effectiveness of DC adoptive immune cancer therapy, many different agents have been used to mature DCs. We analyzed the kinetics of DC maturation by lipopolysaccharide (LPS and interferon-γ (IFN-γ induction in order to characterize the usefulness of mature DCs (mDCs for immune therapy and to identify biomarkers for assessing the quality of mDCs. Methods Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-γ and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA expression analysis. Results After 24 hours of LPS and IFN-γ stimulation, DC surface expression of CD80, CD83, CD86, and HLA Class II antigens were up-regulated. Th1 attractant genes such as CXCL9, CXCL10, CXCL11 and CCL5 were up-regulated during maturation but not Treg attractants such as CCL22 and CXCL12. The expression of classical mDC biomarker genes CD83, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1 and HES4 were up-regulated throughout maturation while MTIB, MTIE, MTIG, MTIH, GADD45A and LAMP3 were only up-regulated late in maturation. The expression of miR-155 was up-regulated 8-fold in mDCs. Conclusion DCs, matured with LPS and IFN-γ, were characterized by increased levels of Th1 attractants as opposed to Treg attractants and may be particularly effective for adoptive immune cancer therapy.
The ontogeny of lectin-positive epithelial cell types and the maturation of polarized expression of the glycocalyx of the collecting ducts (CD) of the rat kidney were studied from samples of 18th-day fetal and neonatal kidneys of various ages. Lectins from Dolichos biflorus (DBA) and Vicia villosa (VVA), with preferential affinity to principal cells, stained virtually all CD cells of the fetal kidneys. However, within two days postnatally, the number of cells positive for DBA and VVA decreased to amounts found in the adult kidneys. Moreover, a characteristic change occurred rapidly after birth in the intracellular polarization of the reactive glycoconjugates, from a uniform plasmalemmal to a preferentially apical staining. In contrast, lectins from Arachis hypogaea (PNA), Maclura pomifera (MPA) and Lotus tetragonolobus (LTA), reacting indiscriminatively with principal and intercalated cells of adult kidneys, stained most CD cells in the fetal kidneys, and failed to show any postnatal change in the amount of positive cells or in the intracellular polarization. The immunocytochemical tests for (Na + K)-ATPase and carbonic anhydrase (CA II) revealed the characteristic postnatal decrease in the amount of principal cells and simultaneous increase in the amount of CA II rich intercalated cells. DBA and VVA reactive cells also decreased postnatally, paralleling the changes observed in the (Na + K)-ATPase positive principal cells. The present results suggest that the expression of the cell type-specific glycocalyx of principal and intercalated cells is developmentally regulated, undergoes profound changes during maturation, and is most likely associated with electrolyte transport phenomena.
Ghermezi, Michael; Li, Mingjie; Vardanyan, Suzie; Harutyunyan, Nika Manik; Gottlieb, Jillian; Berenson, Ariana; Spektor, Tanya M; Andreu-Vieyra, Claudia; Petraki, Sophia; Sanchez, Eric; Udd, Kyle; Wang, Cathy S; Swift, Regina A; Chen, Haiming; Berenson, James R
B-cell maturation antigen is expressed on plasma cells. In this study, we have identified serum B-cell maturation antigen as a novel biomarker that can monitor and predict outcomes for multiple myeloma patients. Compared to healthy donors, patients with multiple myeloma showed elevated serum B-cell maturation antigen levels (Pmultiple myeloma patients (n=243). Specifically, patients with serum B-cell maturation antigen levels above the median level at the time of starting front-line (P=0.0043) or a new salvage therapy (P=0.0044) were found to have shorter progression-free survival. Importantly, serum B-cell maturation antigen levels did not show any dependence on renal function and maintained independent significance when tested against other known prognostic markers for multiple myeloma such as age, serum β2 microglobulin, hemoglobin, and bone disease. These data identify serum B-cell maturation antigen as a new biomarker to manage multiple myeloma patients. Copyright© Ferrata Storti Foundation.
Donghui Zhang; Wei Jiang; Meng Liu; Xin Sui; Xiaolei Yin; Song Chen; Yan Shi; Hongkui Deng
Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDX1-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insulin-producing cells. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.
Md Shakhawat eHossain
Full Text Available Our current understanding of plant functional genomics derives primarily from measurements of gene, protein and/or metabolite levels averaged over the whole plant or multicellular tissues. These approaches risk diluting the response of specific cells that might respond strongly to the treatment but whose signal is diluted by the larger proportion of non-responding cells. For example, if a gene is expressed at a low level, does this mean that it is indeed lowly expressed or is it highly expressed, but only in a few cells? In order to avoid these issues, we adopted the soybean root hair cell, derived from a single, differentiated root epidermal cell, as a single-cell model for functional genomics. Root hair cells are intrinsically interesting since they are major conduits for root water and nutrient uptake and are also the preferred site of infection by nitrogen-fixing rhizobium bacteria. Although a variety of other approaches have been used to study single plant cells or single cell types, the root hair system is perhaps unique in allowing application of the full repertoire of functional genomic and biochemical approaches. In this mini review, we summarize our published work and place this within the broader context of root biology, with a significant focus on understanding the initial events in the soybean-rhizobium interaction.
Full Text Available The continual exposure of outer hair cells (OHCs to thermal noise causes vibrations in resonant frequency. As these vibrations are backprojected, they should be recordable as audiofrequencies in the outer ear canal. But even though they are likely to be amplified in some areas by clustering in terms of the chaos theory, they cannot be picked up in the outer ear canal by currently available recording technologies. Conditions change in the presence of pathology, e.g. loss of OHCs and fibrous replacement: Clusters grow in size and amplitudes become larger so that the vibrations can be picked up as spontaneous oto-acoustic emissions (SOAEs in the outer ear canal. Efforts are needed to demonstrate the presence of physiological OHC vibrations (emission by incessant vibration, EIV by processing auditory recordings with statistical methods.
Pangrsic, Tina; Lasarow, Livia; Reuter, Kirsten; Takago, Hideki; Schwander, Martin; Riedel, Dietmar; Frank, Thomas; Tarantino, Lisa M; Bailey, Janice S; Strenzke, Nicola; Brose, Nils; Müller, Ulrich; Reisinger, Ellen; Moser, Tobias
Inner hair cell ribbon synapses indefatigably transmit acoustic information. The proteins mediating their fast vesicle replenishment (hundreds of vesicles per s) are unknown. We found that an aspartate to glycine substitution in the C(2)F domain of the synaptic vesicle protein otoferlin impaired hearing by reducing vesicle replenishment in the pachanga mouse model of human deafness DFNB9. In vitro estimates of vesicle docking, the readily releasable vesicle pool (RRP), Ca(2+) signaling and vesicle fusion were normal. Moreover, we observed postsynaptic excitatory currents of variable size and spike generation. However, mutant active zones replenished vesicles at lower rates than wild-type ones and sound-evoked spiking in auditory neurons was sparse and only partially improved during longer interstimulus intervals. We conclude that replenishment does not match the release of vesicles at mutant active zones in vivo and a sufficient standing RRP therefore cannot be maintained. We propose that otoferlin is involved in replenishing synaptic vesicles.
Harasztosi, Csaba; Harasztosi, Emese; Gummer, Anthony W.
Rapid endocytic activity of outer hair cells (OHCs) in the guinea-pig cochlea has been already studied using the fluorescent membrane marker FM1-43. It was demonstrated that vesicles were endocytosed at the apical pole of OHCs and transcytosed to the basolateral membrane and through a central strand towards the nucleus. The significance of endocytic activity in the infranuclear region is still not clear. Therefore, in this study endocytic activity at the synaptic pole of OHCs was investigated. Confocal laser scanning microscopy was used to visualize dye uptake of OHCs isolated from the guinea-pig cochlea. Signal intensity changes were quantified in the apical and basal poles relative to the signal at the membrane. Data showed no significant difference in fluorescent signal intensity changes between the opposite poles of the OHC. These results suggest that endocytic activities in both the basal and the apical poles contribute equally to the membrane recycling of OHCs.
Choudhary, S; Elsaie, M L; Nouri, K
A number of lasers and light devices are now available for the treatment of unwanted hair. The goal of laser hair removal is to damage stem cells in the bulge of the hair follicle by targeting melanin, the endogenous chromophore for laser and light devices utilized to remove hair. The competing chromophores in the skin and hair, oxyhemoglobin and water, have a decreased absorption between 690 nm and 1000 nm, thus making this an ideal range for laser and light sources. Laser hair removal is achieved through follicular unit destruction based on selective photothermolysis. The principle of selective photothermolysis predicts that the thermal injury will be restricted to a given target if there is sufficient selective absorption of light and the pulse duration is shorter than the thermal relaxation time of the target. This review will focus on the mechanisms of laser assisted hair removal and provide an update on the newer technologies emerging in the field of lasers assisted hair removal.
Lee, Bo Yon, E-mail: email@example.com [Department of Obstetrics and Gynecology, Kyung Hee University Hospital, Kyung Hee University, School of Medicine, Seoul (Korea, Republic of); Shim, Sang Woo; Kim, Young Sun; Kim, Seung Bo [Department of Obstetrics and Gynecology, Kyung Hee University Hospital, Kyung Hee University, School of Medicine, Seoul (Korea, Republic of)
Highlights: Black-Right-Pointing-Pointer The sperm centriole is the progenitor of centrosomes in all somatic cells. Black-Right-Pointing-Pointer Centrioles and centrosomes exist in parthenogenetic ovarian teratoma cells. Black-Right-Pointing-Pointer Without a sperm centriole, parthenogenetic oocytes produce centrioles and centrosomes. Black-Right-Pointing-Pointer Parthenogenetic human oocytes can develop and differentiate into mature cells. -- Abstract: In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue.
Averbeck, Marco; Braun, Thorsten; Pfeifer, Gunther; Sleeman, Jonathan; Dudda, Jan; Martin, Stefan F; Kremer, Bernhard; Aktories, Klaus; Simon, Jan C; Termeer, Christian
The interplay between dendritic cells (DC) and T cells is a dynamic process critically depending on DC maturation. Ca(2+) influx is one of the initial events occurring during DC/T cell contacts. To determine how DC maturation influences DC/T cell contacts, time-lapse video microscopy was established using TCR-transgenic CD8(+) T cells from P14 mice. DC maturation shifted DC/T cell contacts from short-lived interactions with transient Ca(2+) influx in T cells to long-lasting interactions and sustained Ca(2+) influx of 30 min and more. Follow-up of DC/T cell interactions after 2 h using confocal microscopy revealed that long-lasting Ca(2+) responses in T cells were preferentially associated with the formation of an immunological synapse involving CD54 and H2-K(b) at the DC/T cell interface. Such synapse formation preceded MHC or B7 up-regulation, since DC developed into potent Ca(2+) stimulators 7 h after initiation of maturation. Instead, the enhanced capacity of 7 h-matured DC to induce sustained Ca(2+) responses in CD8(+) T cells is critically dependent on the polarization and rearrangement of the cytoskeleton, as shown by Clostridium difficile toxin B inhibitor experiments. These data indicate that already very early after receiving a maturation stimulus, DC display enhanced cytoskeletal activity resulting in the rapid formation of immunological synapses and effective CD8(+) T cell stimulation.
Zhang, Guojin; McMullen, Roger L; Kulcsar, Lidia
Color fastness is a major concern for consumers and manufacturers of oxidative hair dye products. Hair dye loss results from multiple wash cycles in which the hair dye is dissolved by water and leaches from the hair shaft. In this study, we carried out a series of measurements to help us better understand the kinetics of the leaching process and pathways associated with its escape from the fiber. Hair dye leaching kinetics was measured by suspending hair in a dissolution apparatus and monitoring the dye concentration in solution (leached dye) with an ultraviolet-visible spectrophotometer. The physical state of dye deposited in hair fibers was evaluated by a reflectance light microscopy technique, based on image stacking, allowing enhanced depth of field imaging. The dye distribution within the fiber was monitored by infrared spectroscopic imaging of hair fiber cross sections. Damage to the ultrafine structure of the hair cuticle (surface, endocuticle, and cell membrane complex) and cortex (cell membrane complex) was determined in hair cross sections and on the hair fiber surface with atomic force microscopy. Using differential scanning calorimetry, we investigated how consecutive coloring and leaching processes affect the internal proteins of hair. Further, to probe the surface properties of hair we utilized contact angle measurements. This study was conducted on both pigmented and nonpigmented hair to gain insight into the influence of melanin on the hair dye deposition and leaching processes. Both types of hair were colored utilizing a commercial oxidative hair dye product based on pyrazole chemistry.
HAN Wei-ju; SHI Xiao-rui; Alfred Nuttall
Background Modern research has provided new insights into the biological mechanisms of noise-induced hearing loss,and a number of studies showed the appearance of increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) during and after noise exposure.This study was designed to investigate the noise exposure induced nitrotyrosine change and the mechanism of outer hair cells death in guinea pig cochlea.Method Thirty guinea pigs were used in this study.The experimental animals were either exposed for 4 hours per day to broadband noise at 122 dB SPL (A-weighted) for 2 consecutive days or perfused cochleae with 5 mg/ml of the SIN1 solutions,an exogenous NO and superoxide donor,for 30 minutes.Then the cochleae of the animals were dissected.Propidium iodide (PI),a DNA intercalating fluorescent probe,was used to trace morphological changes in OHC nuclei.The distribution of nitrotyrosine (NT) in the organ of Corti and the cochlear lateral wall tissue from the guinea pigs were examined using fluorescence immunohistochemistry method.Whole mounts of organ of Corti were prepared.Morphological and fluorescent changes were examined under a confocal microscope.Results Either after noise exposure or after SIN1 perfusion,outer hair cells (OHCs) death with characteristics of both apoptotic and necrotic degradation appeared.Nitrotyrosine immunolabeling could be observed in the OHCs from the control animals.After noise exposure,NT immunostaining became much greater than the control animals in OHCs.The apoptotic OHC has significant increase of nitrotyrosine in and around the nucleus following noise exposure.In the normal later wall of cochleae,relatively weak nitrotyrosine immunolabeling could be observed.After noise exposure,nitrotyrosine immunoactivity became stronger in stria vascularis.Conclusion Noise exposure induced increase of nitrotyrosine production is associated with OHCs death suggesting reactive nitrogen species participation in the cochlear
Mankelow, Tosti J; Griffiths, Rebecca E; Trompeter, Sara; Flatt, Joanna F; Cogan, Nicola M; Massey, Edwin J; Anstee, David J
During maturation to an erythrocyte, a reticulocyte must eliminate any residual organelles and reduce its surface area and volume. Here we show this involves a novel process whereby large, intact, inside-out phosphatidylserine (PS)-exposed autophagic vesicles are extruded. Cell surface PS is a well-characterized apoptotic signal initiating phagocytosis. In peripheral blood from patients after splenectomy or in patients with sickle cell disease (SCD), the number of circulating red cells exposing PS on their surface is elevated. We show that in these patients PS is present on the cell surface of red cells in large (∼1.4 µm) discrete areas corresponding to autophagic vesicles. The autophagic vesicles found on reticulocytes are identical to those observed on red cells from splenectomized individuals and patients with SCD. Our data suggest the increased thrombotic risk associated with splenectomy, and patients with hemoglobinopathies is a possible consequence of increased levels of circulating mature reticulocytes expressing inside-out PS-exposed autophagic vesicles because of asplenia.
Francisco Santaolalla; Carlos Salvador; Agustn Martnez; Jose Mara Snchez; Ana Snchez del Rey
Most recent studies on regeneration of inner ear hair cel s focus on use of stem cel s, gene therapy and neurotrophic factors. Cochlear gene therapy has been successful y used in the treatment of neu-rosensory hearing loss. This suggests that cochlear hair cel regeneration is possible. The objective of this paper is to review research and clinical application of inner near hair cel regeneration.
In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO- Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex- pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans- genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and
GUO XuDong; YANG DongShan; Ao XuDong; WU Xia; LI GuangPeng; WANG LingLing; BAO MingTao; XUE Lian; BOU ShorGan
In the present study, cashmere goat fetal flbroblasta were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1(IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasta cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h.Parthenogenetic ooctyes were used as a model to Investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% va 83.9%, P>0.05 and 23.1% vs 17.2%, P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05).After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex-pressing red fluorescence. Two of the red fluorescent blastocysta were randomly selected to identify transgene by polymeraee chain reaction. Both were positive. These results showed that: (i) RFP and Neo genes were correctly expressed indicating that transgenlc somatic cell lines and positive trans-genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and
Full Text Available Neurosensory responses of hearing and balance are mediated by receptors in specialized neuroepithelial sensory cells. Any disruption of the biochemical and molecular pathways that facilitate these responses can result in severe deficits, including hearing loss and vestibular dysfunction. Hearing is affected by both environmental and genetic factors, with impairment of auditory function being the most common neurosensory disorder affecting 1 in 500 newborns, as well as having an impact on the majority of elderly population. Damage to auditory sensory cells is not reversible, and if sufficient damage and cell death have taken place, the resultant deficit may lead to permanent deafness. Cochlear implants are considered to be one of the most successful and consistent treatments for deaf patients, but only offer limited recovery at the expense of loss of residual hearing. Recently there has been an increased interest in the auditory research community to explore the regeneration of mammalian auditory hair cells and restoration of their function. In this review article, we examine a variety of recent therapies, including genetic, stem cell and molecular therapies as well as discussing progress being made in genome editing strategies as applied to the restoration of hearing function.
Wei-jia KONG; Hua-mao CHENG; Paul van CAUWENBERGE
Aim: To explore the cell specific existence of α9 AChR in the vestibular type Ⅱ hair cells (VHC Ⅱ) of rats. Methods: To detect the expression of α9 AChR messenger RNA (mRNA) in the vestibular endorgans and single VHC Ⅱ of rats by using the reverse transcription polymerase chain reaction (RT-PCR) technique and the single cell RT-PCR technique, respectively. Results: It was shown that α9 AChR mRNA was detected in the vestibular endorgans. By using single-cell RT-PCR, mRNA encoding α9 AChR was also detected in the VHC Ⅱ of the rats. Sequence analysis of the PCR products confirmed identity to corresponding cDNA sequence in the predicted region. Conclusion: We established a method which could effectively detect the cell specific expression of mRNA in an individual VHC. Present data confirm that α9 AChR mRNA is expressed in the VHC Ⅱ of rats and indicates that α9 AChR may function as a mediator of efferent cholinergic signaling in mammalian VHC.
Full Text Available The dynamics and interactions between stem cell pools in the hair follicle (HF, sebaceous gland (SG, and interfollicular epidermis (IFE of murine skin are still poorly understood. In this study, we used multicolor lineage tracing to mark Lgr6-expressing basal cells in the HF isthmus, SG, and IFE. We show that these Lgr6+ cells constitute long-term self-renewing populations within each compartment in adult skin. Quantitative analysis of clonal dynamics revealed that the Lgr6+ progenitor cells compete neutrally in the IFE, isthmus, and SG, indicating population asymmetry as the underlying mode of tissue renewal. Transcriptional profiling of Lgr6+ and Lgr6− cells did not reveal a distinct Lgr6-associated gene expression signature, raising the question of whether Lgr6 expression requires extrinsic niche signals. Our results elucidate the interrelation and behavior of Lgr6+ populations in the IFE, HF, and SG and suggest population asymmetry as a common mechanism for homeostasis in several epithelial skin compartments.
Song, Lei; Santos-Sacchi, Joseph
The cylindrical outer hair cell (OHC) of Corti's organ drives cochlear amplification by a voltage-dependent activation of the molecular motor, prestin (SLC26a5), in the cell's lateral membrane. The voltage-dependent nature of this process leads to the troublesome observation that the membrane resistor-capacitor filter could limit high-frequency acoustic activation of the motor. Based on cable theory, the unique 30 nm width compartment (the extracisternal space, ECS) formed between the cell's lateral membrane and adjacent subsurface cisternae (SSC) could further limit the influence of receptor currents on lateral membrane voltage. Here, we use dual perforated/whole-cell and loose patch clamp on isolated OHCs to sequentially record currents resulting from excitation at apical, middle, and basal loose patch sites before and after perforated patch rupture. We find that timing of currents is fast and uniform before whole-cell pipette washout, suggesting little voltage attenuation along the length of the lateral membrane. Prior treatment with salicylate, a disrupter of the SSC, confirms the influence of the SSC on current spread. Finally, a cable model of the OHC, which can match our data, indicates that the SSC poses a minimal barrier to current flow across it, thereby facilitating rapid delivery of voltage excitation to the prestin-embedded lateral membrane. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Clerici, W J; DiMartino, D L; Prasad, M R
Reactive oxygen species (ROS) have been implicated in the ototoxicity of various agents. This study examines the effects of superoxide anion (O2), hydroxyl radical (OH.) and hydrogen peroxide (H2O2), on isolated cochlear outer hair cell (OHC) morphology. OHCs were superfused with artificial perilymph (AP) or AP containing a specific ROS scavenger, and then with AP, ROS system or scavenger plus ROS system for 90 min. The generation of ROS as well as the scavenging properties of other agents were confirmed by specific biochemical assays. Control cells decreased 4.8% in mean length, and showed no obvious membrane damage. Generation of O2. or OH. resulted in high rates (85.7 and 42.9%, respectively) of bleb formation at the synaptic pole, and decreased (O2., 15.2%; OH., 17.3%) mean cell length. Length change and bleb formation rate were H2O2 concentration-dependent. 20 mM H2O2 led to 33.3% decreased mean cell length, and only 20% bleb formation; 0.1 mM H2O2 led to 83.3% bleb formation, with no length decrease. Superoxide dismutase, deferoxamine and catalase protected against O2., OH. and H2O2 effects, respectively. Bleb formation and diminished cell length likely represent differential lipid peroxidative outcomes at supra- and infranuclear membranes, and are consistent with effects of certain ototoxicants.
Chen, Chih-Chiang; Plikus, Maksim V; Tang, Pin-Chi; Widelitz, Randall B; Chuong, Cheng Ming
Hair and feathers are unique because (1) their stem cells are contained within a follicle structure, (2) they undergo cyclic regeneration repetitively throughout life, (3) regeneration occurs physiologically in healthy individuals and (4) regeneration is also induced in response to injury. Precise control of this cyclic regeneration process is essential for maintaining the homeostasis of living organisms. While stem cells are regulated by the intra-follicle-adjacent micro-environmental niche, this niche is also modulated dynamically by extra-follicular macro-environmental signals, allowing stem cells to adapt to a larger changing environment and physiological needs. Here we review several examples of macro-environments that communicate with the follicles: intradermal adipose tissue, innate immune system, sex hormones, aging, circadian rhythm and seasonal rhythms. Related diseases are also discussed. Unveiling the mechanisms of how stem cell niches are modulated provides clues for regenerative medicine. Given that stem cells are hard to manipulate, focusing translational therapeutic applications at the environments appears to be a more practical approach.
... loss at the scarred areas. These conditions include lichen planus, some types of lupus and sarcoidosis. Hair- ... increase your risk of hair loss, including: Family history Age Poor nutrition Certain medical conditions, such as ...
... or some other combination? Hair color comes from melanin (say: MEL-uh-nun), the substance that gives ... its pigment. The lighter someone's hair, the less melanin there is. A person with brown or black ...
... To avoid spreading infections, don’t share razors. Hair removal creams, gels, and liquids (depilatories) These use chemicals ... electrologist with a current license or certification. Laser hair removal Light is beamed through the skin to stop ...
... Skin Scars Skin Growths Skin Lesions Spider Veins Stretch Marks Sun-damaged Skin Unwanted Hair Unwanted Tattoos Varicose ... Skin Scars Skin Growths Skin Lesions Spider Veins Stretch Marks Sun-damaged Skin Unwanted Hair Unwanted Tattoos Varicose ...
Hamblet, Corinne E.; Makowski, Stefanie L.; Tritapoe, Julia M.; Pomerantz, Joel L.
NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. Here, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC Class I-deficient tumors in vivo, and cytotoxicity against tumor cells in vitro. This phenotype is concomitant with reduced numbers of CD27+CD11b+ and CD27−CD11b+ NK cells, indicating a requirement for SPPL3 in efficient NK cell maturation. NK cell-specific deletion of SPPL3 results in the same deficiencies, revealing a cell-autonomous role for SPPL3 in these processes. CRISPR/Cas9 genomic editing in murine zygotes was used to generate knock-in mice with a catalytically compromised SPPL3 D271A allele. Mice engineered to express only SPPL3 D271A in NK cells phenocopy mice deleted for SPPL3, indicating a requirement for SPPL3 protease activity in NK cell biology. Our results identify SPPL3 as a cell-autonomous molecular determinant of NK cell maturation and expand the role of intramembrane aspartyl proteases in innate immunity. PMID:26851218
Hamblet, Corinne E; Makowski, Stefanie L; Tritapoe, Julia M; Pomerantz, Joel L
NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. In this article, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC class I-deficient tumors in vivo, and cytotoxicity against tumor cells in vitro. This phenotype is concomitant with reduced numbers of CD27(+)CD11b(+) and CD27(-)CD11b(+) NK cells, indicating a requirement for SPPL3 in efficient NK cell maturation. NK cell-specific deletion of SPPL3 results in the same deficiencies, revealing a cell-autonomous role for SPPL3 in these processes. CRISPR/Cas9 genomic editing in murine zygotes was used to generate knockin mice with a catalytically compromised SPPL3 D271A allele. Mice engineered to express only SPPL3 D271A in NK cells phenocopy mice deleted for SPPL3, indicating a requirement for SPPL3 protease activity in NK cell biology. Our results identify SPPL3 as a cell-autonomous molecular determinant of NK cell maturation and expand the role of intramembrane aspartyl proteases in innate immunity. Copyright © 2016 by The American Association of Immunologists, Inc.
Irmak, M Kemal
Merkel cells are located in glabrous and hairy skin and in some mucosa. They are characterized by dense-core secretory granules and cytoskeletal filaments. They are attached to neighboring keratinocytes by desmosomes and contain melanosomes similar to keratinocytes. They are excitable cells in close contact with sensory nerve endings but their function is still unclear. In this review, following roles are attributed for the first time to the Merkel cells: (1) melanosomes in Merkel cells may be involved in mammalian magnetoreception. In this model melanosome as a biological magnetite is connected by cytoskeletal filaments to mechanically gated ion channels embedded in the Merkel cell membrane. The movement of melanosome with the changing electromagnetic field may open ion channels directly producing a receptor potential that can be transmitted to brain via sensory neurons. (2) Merkel cells may be involved in finger-print formation: Merkel cells in glabrous skin are located at the base of the epidermal ridges the type of which defines the finger-print pattern. Finger-print formation starts at the 10th week of pregnancy after the arrival of Merkel cells. Keratinocyte proliferation and the buckling process observed in the basal layer of epidermis resulting in the epidermal ridges may be controlled and formed by Merkel cells. (3) Brain-Merkel cell connection is bi-directional and Merkel cells not only absorb but also radiate the electromagnetic frequencies. Hence, efferent aspects of the palmar and plantar Merkel nerve endings may form the basis of the biofield modalities such as Reiki, therapeutic touch and telekinesis. (4) Adaptive geographic variations such as skin color, craniofacial morphology and hair form result from interactions between environmental factors and epigenetic inheritance system. While environmental factors produce modifications in the body, they simultaneously induce epigenetic modifications in the oocytes and in this way adaptive changes could be
Kraft, David C E; Bindslev, Dorthe A; Melsen, Birte
For engineering bone tissue, mechanosensitive cells are needed for bone (re)modelling. Local bone mass and architecture are affected by mechanical loading, which provokes a cellular response via loading-induced interstitial fluid flow. We studied whether human dental pulp-derived mesenchymal stem...... cells (PDSCs) portraying mature (PDSC-mature) or immature (PDSC-immature) bone cell characteristics are responsive to pulsating fluid flow (PFF) in vitro. We also assessed bone formation by PDSCs on hydroxyapatite-tricalcium phosphate granules after subcutaneous implantation in mice. Cultured PDSC...... expression was higher than in PDSC-immature. Implantation of PDSC-mature resulted in more osteoid deposition and lamellar bone formation than PDSC-immature. We conclude that PDSCs with a mature osteogenic phenotype are more responsive to pulsating fluid shear stress than osteogenically immature PDSCs...
Full Text Available The hair cosmetic industry has undergone a revolutionary change over the last two decades. The focus has dramatically veered from merely cleaning to repair, increasing the tensile strength, reducing oxidative damage, and stimulating growth. Newer shorter procedures to make hair look naturally more lustrous, smooth, and manageable have evolved. Specialized grooming products have been formulated to cleanse, calm, and condition the hair, and are tailored for different hair-types, for example, dry, dry-damaged, oily, colored, and gray hair. Other products are formulated to alter the color or structure of the hair shaft, for example, hair dyes, perming/relaxing. Hair sprays and waxes/gels, can alter the ′lift′ of the hair-shaft. Although dermatologists are experts in managing scalp and hair diseases, the esthetic applications of newer cosmetic therapies still remain elusive. This article attempts to fill the lacunae in our knowledge of hair cosmetics and esthetic procedures relevant in today′s rapidly changing beauty-enhancing industry, with special emphasis on the Indian scenario for chemical and ′natural′ hair products.
Madnani, Nina; Khan, Kaleem
The hair cosmetic industry has undergone a revolutionary change over the last two decades. The focus has dramatically veered from merely cleaning to repair, increasing the tensile strength, reducing oxidative damage, and stimulating growth. Newer shorter procedures to make hair look naturally more lustrous, smooth, and manageable have evolved. Specialized grooming products have been formulated to cleanse, calm, and condition the hair, and are tailored for different hair-types, for example, dry, dry-damaged, oily, colored, and gray hair. Other products are formulated to alter the color or structure of the hair shaft, for example, hair dyes, perming/relaxing. Hair sprays and waxes/gels, can alter the 'lift' of the hair-shaft. Although dermatologists are experts in managing scalp and hair diseases, the esthetic applications of newer cosmetic therapies still remain elusive. This article attempts to fill the lacunae in our knowledge of hair cosmetics and esthetic procedures relevant in today's rapidly changing beauty-enhancing industry, with special emphasis on the Indian scenario for chemical and 'natural' hair products.
Asakawa, Kyosuke; Toyoshima, Koh-ei; Ishibashi, Naoko; Tobe, Hirofumi; Iwadate, Ayako; Kanayama, Tatsuya; Hasegawa, Tomoko; Nakao, Kazuhisa; Toki, Hiroshi; Noguchi, Shotaro; Ogawa, Miho; Sato, Akio; Tsuji, Takashi
Organ regenerative therapy aims to reproduce fully functional organs to replace organs that have been lost or damaged as a result of disease, injury, or aging. For the fully functional regeneration of ectodermal organs, a concept has been proposed in which a bioengineered organ is developed by reproducing the embryonic processes of organogenesis. Here, we show that a bioengineered hair follicle germ, which was reconstituted with embryonic skin-derived epithelial and mesenchymal cells and ectopically transplanted, was able to develop histologically correct hair follicles. The bioengineered hair follicles properly connected to the host skin epithelium by intracutaneous transplantation and reproduced the stem cell niche and hair cycles. The bioengineered hair follicles also autonomously connected with nerves and the arrector pili muscle at the permanent region and exhibited piloerection ability. Our findings indicate that the bioengineered hair follicles could restore physiological hair functions and could be applicable to surgical treatments for alopecia. PMID:22645640
He, Zuhong; Sun, Shan; Waqas, Muhammad; Zhang, Xiaoli; Qian, Fuping; Cheng, Cheng; Zhang, Mingshu; Zhang, Shasha; Wang, Yongming; Tang, Mingliang; Li, Huawei; Chai, Renjie
Aminoglycosides are ototoxic to the cochlear hair cells, and mitochondrial dysfunction is one of the major mechanisms behind ototoxic drug-induced hair cell death. TRMU (tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase) is a mitochondrial protein that participates in mitochondrial tRNA modifications, but the role of TRMU in aminoglycoside-induced ototoxicity remains to be elucidated. In this study, we took advantage of the HEI-OC-1 cell line to investigate the role of TRMU in aminoglycoside-induced cell death. We found that TRMU is expressed in both hair cells and HEI-OC-1 cells, and its expression is significantly decreased after 24 h neomycin treatment. We then downregulated TRMU expression with siRNA and found that cell death and apoptosis were significantly increased after neomycin injury. Furthermore, when we down-regulated TRMU expression, we observed significantly increased mitochondrial dysfunction and increased levels of reactive oxygen species (ROS) after neomycin injury, suggesting that TRMU regulates mitochondrial function and ROS levels. Lastly, the antioxidant N-acetylcysteine rescued the mitochondrial dysfunction and cell apoptosis that was induced by TRMU downregulation, suggesting that ROS accumulation contributed to the increased aminoglycosides sensitivity of HEI-OC-1 cells after TRMU downregulation. This study provides evidence that TRMU might be a new therapeutic target for the prevention of aminoglycoside-induced hair cell death. PMID:27405449
XIE Ding-hua; XIAO Zi-an; YANG Shu
Abstract Objective This study is to explore the relationship between acetylcholine (ACh)-induced calcium release from intracellular Ca2+ stores and function of outer hair cell (OHC) motors, in an attempt to elucidate the mechanism of OHC electromotility at resting state. Methods OHCs were isolated from adult guinea pig (200-300 g) cochlea and loaded with Fluo-3/AM. The cells were treated with ACh/dHBSS, ACh/HBSS, dHBSS only or HBSS only. Intracellular [Ca2+]i variations in cells under the four treatments were observed using an Ar-Kr laser scan confocal microscope. Results [Ca2+]i oscillations were recorded in five OHCs treated with ACh/dHBSS but not in other cells. This is the first time that Ach-excited [Ca2+]i oscillations are reported in guinea pig OHCs independent of extracellular calcium. Conclusions ACh-excited [Ca2+]i oscillations in OHCs originates from intracellular calcium release and may play a crucial role in maintaining active mechanical motility of the OHC at resting and modulating OHC electromotility.
Vahl, J Christoph; Heger, Klaus; Knies, Nathalie; Hein, Marco Y; Boon, Louis; Yagita, Hideo; Polic, Bojan; Schmidt-Supprian, Marc
Natural killer T (NKT) cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR. However, to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood. To address this issue, we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus. We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs, but they do not differentiate into NKT cells. On the other hand, induced TCR ablation on mature NKT cells did not affect their lineage identity, homeostasis, or innate rapid cytokine secretion abilities. We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR.
J Christoph Vahl
Full Text Available Natural killer T (NKT cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR. However, to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood. To address this issue, we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus. We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs, but they do not differentiate into NKT cells. On the other hand, induced TCR ablation on mature NKT cells did not affect their lineage identity, homeostasis, or innate rapid cytokine secretion abilities. We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR.
Nguyen, Jennifer V
Eyebrow hair serves many important biologic and aesthetic functions. This article reviews the structure and function of the hair follicle, as well as hair follicle morphogenesis and cycling. Eyebrow hair follicles share the same basic structure as hair follicles elsewhere on the body, but are distinguished by their shorter anagen (growing) phase. Knowledge of the hair follicle structure and cycle is important for understanding the pathophysiology of alopecia, as diseases affecting the stem cell portion of the hair follicle in the bulge region may cause permanent hair loss. Furthermore, therapeutic agents that target distinct phases and hormones involved in the hair cycle may be useful for promoting hair growth.
Peng, Anthony W; Gnanasambandam, Radhakrishnan; Sachs, Frederick; Ricci, Anthony J
The auditory system is able to detect movement down to atomic dimensions. This sensitivity comes in part from mechanisms associated with gating of hair cell mechanoelectric transduction (MET) channels. MET channels, located at the tops of stereocilia, are poised to detect tension induced by hair bundle deflection. Hair bundle deflection generates a force by pulling on tip-link proteins connecting adjacent stereocilia. The resting open probability (P(open)) of MET channels determines the linearity and sensitivity to mechanical stimulation. Classically, P(open) is regulated by a calcium-sensitive adaptation mechanism in which lowering extracellular calcium or depolarization increases P(open). Recent data demonstrated that the fast component of adaptation is independent of both calcium and voltage, thus requiring an alternative explanation for the sensitivity of P(open) to calcium and voltage. Using rat auditory hair cells, we characterize a mechanism, separate from fast adaptation, whereby divalent ions interacting with the local lipid environment modulate resting P(open). The specificity of this effect for different divalent ions suggests binding sites that are not an EF-hand or calmodulin model. GsMTx4, a lipid-mediated modifier of cationic stretch-activated channels, eliminated the voltage and divalent sensitivity with minimal effects on adaptation. We hypothesize that the dual mechanisms (lipid modulation and adaptation) extend the dynamic range of the system while maintaining adaptation kinetics at their maximal rates.
Zimmermann, U.; Fermin, C.
Cochlear outer hair cells (OHC) are commonly assumed to function as mechanical effectors as well as sensory receptors in the organ of Corti (OC) of the inner ear. OHC in vitro and in organ explants exhibit mechanical responses to electrical, chemical or mechanical stimulation which may represent an aspect of their effector process that is expected in vivo. A detailed description, however, of an OHC effector operation in situ is still missing. Specifically, little is known as to how OHC movements influence the geometry of the OC in situ. Previous work has demonstrated that the motility of isolated OHCs in response to electrical stimulation and to K(+)-gluconate is probably under voltage control and causes depolarisation (shortening) and hyperpolarization (elongation). This work was undertaken to investigate if the movements that were observed in isolated OHC, and which are induced by ionic stimulation, could change the geometry of the OC. A synchronized depolarization of OHC was induced in guinea pig cochleae by exposing the entire OC to artificial endolymph (K+). Subsequent morphometry of mid-modiolar sections from these cochleae revealed that the distance between the basilar membrane (BM) and the reticular lamina (RL) had decreased considerably. Furthermore, in the three upper turns OHC had significantly shortened in all rows. The results suggest that OHC can change their length in the organ of Corti (OC) thus deforming the geometry of the OC. The experiments reveal a tonic force generation within the OC that may change the position of RL and/or BM, contribute to damping, modulate the BM-RL-distance and control the operating points of RL and sensory hair bundles. Thus, the results suggest active self-adjustments of cochlear mechanics by slow OHC length changes. Such mechanical adjustments have recently been postulated to correspond to timing elements of animal communication, speech or music.
Zimmermann, U.; Fermin, C.
Cochlear outer hair cells (OHC) are commonly assumed to function as mechanical effectors as well as sensory receptors in the organ of Corti (OC) of the inner ear. OHC in vitro and in organ explants exhibit mechanical responses to electrical, chemical or mechanical stimulation which may represent an aspect of their effector process that is expected in vivo. A detailed description, however, of an OHC effector operation in situ is still missing. Specifically, little is known as to how OHC movements influence the geometry of the OC in situ. Previous work has demonstrated that the motility of isolated OHCs in response to electrical stimulation and to K(+)-gluconate is probably under voltage control and causes depolarisation (shortening) and hyperpolarization (elongation). This work was undertaken to investigate if the movements that were observed in isolated OHC, and which are induced by ionic stimulation, could change the geometry of the OC. A synchronized depolarization of OHC was induced in guinea pig cochleae by exposing the entire OC to artificial endolymph (K+). Subsequent morphometry of mid-modiolar sections from these cochleae revealed that the distance between the basilar membrane (BM) and the reticular lamina (RL) had decreased considerably. Furthermore, in the three upper turns OHC had significantly shortened in all rows. The results suggest that OHC can change their length in the organ of Corti (OC) thus deforming the geometry of the OC. The experiments reveal a tonic force generation within the OC that may change the position of RL and/or BM, contribute to damping, modulate the BM-RL-distance and control the operating points of RL and sensory hair bundles. Thus, the results suggest active self-adjustments of cochlear mechanics by slow OHC length changes. Such mechanical adjustments have recently been postulated to correspond to timing elements of animal communication, speech or music.
Lock-Andersen, J; Drzewiecki, K T; Wulf, H C
the present hair colour and eye colour, and the constitutive skin pigmentation was measured objectively by skin reflectance of UV unexposed buttock skin. There were no differences between basal cell carcinoma cases and controls in hair colour or eye colour or constitutive skin pigmentation, but more cases......To assess the importance of hair and eye colour, skin type and constitutive skin pigmentation as risk factors for basal cell carcinoma and cutaneous malignant melanoma in fair-skinned Caucasians, we conducted two identical case-control studies in Denmark. We studied 145 cases with basal cell...
Weiler, Simon; Krinner, Stefanie; Wong, Aaron B; Moser, Tobias; Pangršič, Tina
Sound encoding is mediated by Ca(2+) influx-evoked release of glutamate at the ribbon synapse of inner hair cells. Here we studied the role of ATP in this process focusing on Ca(2+) current through CaV1.3 channels and Ca(2+) homeostasis in mouse inner hair cells. Patch-clamp recordings and Ca(2+) imaging demonstrate that hydrolyzable ATP is essential to maintain synaptic Ca(2+) influx in inner hair cells via fueling Ca(2+)-ATPases to avoid an increase in cytosolic [Ca(2+)] and subsequent Ca(2+)/calmodulin-dependent inactivation of CaV1.3 channels.
Full Text Available Mast cells play an important role in the pathogenesis of allergic diseases. Immature mast cells migrate into peripheral tissues from the bone marrow and undergo complete maturation. Interestingly, mast cells have characteristics similar to hematopoietic stem cells (HSCs, such as self-renewal and c-kit expression. In HSCs, Wnt signaling is involved in their maintenance and differentiation. On the other hand, the relation between Wnt signaling and mast cell differentiation is poorly understood. To study whether Wnt signals play a role in the maturation of mast cells, we studied the effect of Wnt proteins on mast cell maturation of bone marrow-derived mast cells (BMMCs. The expression levels of CD81 protein and histidine decarboxylase mRNA and activity of mast cell-specific protease were all elevated in BMMCs treated with Wnt5a. In addition, Wnt5a induced the expression of Axin2 and TCF mRNA in BMMCs. These results showed that Wnt5a could promote the maturation of mast cells via the canonical Wnt signaling pathway and provide important insights into the molecular mechanisms underlying the differentiation of mast cells.
Yang, Huansheng; Wang, Xiaocheng; Xiong, Xia; Yin, Yulong
Intestinal epithelial cells continuously migrate and mature along crypt-villus axis (CVA), while the changes in energy metabolism during maturation are unclear in neonates. The present study was conducted to test the hypothesis that the energy metabolism in intestinal epithelial cells would be changed during maturation along CVA in neonates. Eight 21-day-old suckling piglets were used. Intestinal epithelial cells were isolated sequentially along CVA, and proteomics was used to analyze the changes in proteins expression in epithelial cells along CVA. The identified differentially expressed proteins were mainly involved in cellular process, metabolic process, biological regulation, pigmentation, multicellular organizational process and so on. The energy metabolism in intestinal epithelial cells of piglets was increased from the bottom of crypt to the top of villi. Moreover, the expression of proteins related to the metabolism of glucose, most of amino acids, and fatty acids was increased in intestinal epithelial cells during maturation along CVA, while the expression of proteins related to glutamine metabolism was decreased from crypt to villus tip. The expression of proteins involved in citrate cycle was also increased intestinal epithelial cells during maturation along CVA. Moreover, dietary supplementation with different energy sources had different effects on intestinal structure of weaned piglets. PMID:27558220
Kazama, Tomohiko; Fujie, Masaki; Endo, Tuyoshi; Kano, Koichiro
We have previously reported the establishment of preadipocyte cell lines, termed dedifferentiated fat (DFAT) cells, from mature adipocytes of various animals. DFAT cells possess long-term viability and can redifferentiate into adipocytes both in vivo and in vitro. Furthermore, DFAT cells can transdifferentiate into osteoblasts and chondrocytes under appropriate culture conditions. However, it is unclear whether DFAT cells are capable of transdifferentiating into skeletal myocytes, which is common in the mesodermal lineage. Here, we show that DFAT cells can be induced to transdifferentiate into skeletal myocytes in vitro. Myogenic induction of DFAT cells resulted in the expression of MyoD and myogenin, followed by cell fusion and formation of multinucleated cells expressing sarcomeric myosin heavy chain. These results indicate that DFAT cells derived from mature adipocytes can transdifferentiate into skeletal myocytes in vitro.
Full Text Available Newborn granule cells become functionally integrated into the synaptic circuitry of the adult dentate gyrus after a morphological and electrophysiological maturation process. The molecular mechanisms by which immature neurons and the neurites extending from them find their appropriate position and target area remain largely unknown. Here we show that single-cell-specific knockdown of cyclin-dependent kinase 5 (cdk5 activity in newborn cells using a retrovirus-based strategy leads to aberrant growth of dendritic processes, which is associated with an altered migration pattern of newborn cells. Even though spine formation and maturation are reduced in cdk5-deficient cells, aberrant dendrites form ectopic synapses onto hilar neurons. These observations identify cdk5 to be critically involved in the maturation and dendrite extension of newborn neurons in the course of adult neurogenesis. The data presented here also suggest a mechanistic dissociation between accurate dendritic targeting and subsequent synapse formation.
Coffin, Allison B.; Mohr, Robert A.; Sisneros, Joseph A.
The plainfin midshipman fish, Porichthys notatus, is a seasonal breeding teleost fish for which vocal-acoustic communication is essential for its reproductive success. Female midshipman use the saccule as the primary end organ for hearing to detect and locate “singing” males that produce multiharmonic advertisement calls during the summer breeding season. Previous work showed that female auditory sensitivity changes seasonally with reproductive state; summer reproductive females become better suited than winter nonreproductive females to detect and encode the dominant higher harmonic components in the male’s advertisement call, which are potentially critical for mate selection and localization. Here, we test the hypothesis that these seasonal changes in female auditory sensitivity are concurrent with seasonal increases in saccular hair cell receptors. We show that there is increased hair cell density in reproductive females and that this increase is not dependent on body size since similar changes in hair cell density were not found in the other inner ear end organs. We also observed an increase in the number of small, potentially immature saccular hair bundles in reproductive females. The seasonal increase in saccular hair cell density and smaller hair bundles in reproductive females was paralleled by a dramatic increase in the magnitude of the evoked saccular potentials and a corresponding decrease in the auditory thresholds recorded from the saccule. This demonstration of correlated seasonal plasticity of hair cell addition and auditory sensitivity may in part facilitate the adaptive auditory plasticity of this species to enhance mate detection and localization during breeding. PMID:22279221
Hair dyes resorcinol and lawsone reduce production of melanin in melanoma cells by tyrosinase activity inhibition and decreasing tyrosinase and microphthalmia-associated transcription factor (MITF) expression.
Lee, Shu-Mei; Chen, Yi-Shyan; Lin, Chih-Chien; Chen, Kuan-Hung
Hair coloring products are one of the most important cosmetics for modern people; there are three major types of hair dyes, including the temporary, semi-permanent and permanent hair dyes. The selected hair dyes (such as ammonium persulfate, sodium persulfate, resorcinol and lawsone) are the important components for hair coloring products. Therefore, we analyzed the effects of these compounds on melanogenesis in B16-F10 melanoma cells. The results proved that hair dyes resorcinol and lawsone can reduce the production of melanin. The results also confirmed that resorcinol and lawsone inhibit mushroom and cellular tyrosinase activities in vitro. Resorcinol and lawsone can also downregulate the protein levels of tyrosinase and microphthalmia-associated transcription factor (MITF) in B16-F10 cells. Thus, we suggest that frequent use of hair dyes may have the risk of reducing natural melanin production in hair follicles. Moreover, resorcinol and lawsone may also be used as hypopigmenting agents to food, agricultural and cosmetic industry in the future.
Hair Dyes Resorcinol and Lawsone Reduce Production of Melanin in Melanoma Cells by Tyrosinase Activity Inhibition and Decreasing Tyrosinase and Microphthalmia-Associated Transcription Factor (MITF Expression
Full Text Available Hair coloring products are one of the most important cosmetics for modern people; there are three major types of hair dyes, including the temporary, semi-permanent and permanent hair dyes. The selected hair dyes (such as ammonium persulfate, sodium persulfate, resorcinol and lawsone are the important components for hair coloring products. Therefore, we analyzed the effects of these compounds on melanogenesis in B16-F10 melanoma cells. The results proved that hair dyes resorcinol and lawsone can reduce the production of melanin. The results also confirmed that resorcinol and lawsone inhibit mushroom and cellular tyrosinase activities in vitro. Resorcinol and lawsone can also downregulate the protein levels of tyrosinase and microphthalmia-associated transcription factor (MITF in B16-F10 cells. Thus, we suggest that frequent use of hair dyes may have the risk of reducing natural melanin production in hair follicles. Moreover, resorcinol and lawsone may also be used as hypopigmenting agents to food, agricultural and cosmetic industry in the future.
Santos-Sacchi, Joseph; Navarrete, Enrique; Song, Lei
Outer hair cells provide amplification within the mammalian cochlea to enhance audition. The mechanism is believed to reside within the lateral membrane of the cell that houses an expansive array of molecular motors, identified as prestin, which drives somatic electromotility. By measuring nonlinear capacitance, the electrical signature of electromotility, at kilohertz rates we have uncovered new details of the early molecular events that arise from voltage perturbations of prestin. We show that dynamic changes in motor state probability occur within the kilohertz range, and signify an amplificatory event. Additionally, we show a lack of effect of Cl driving force, an absence of cell length effect (indicating that the kinetics does not vary across auditory frequency), and the first demonstration of the time dependence of tension induced amplificatory shifts. The process we have identified, where the stimulus-response function shifts in time along the stimulus axis in a multi-exponential manner, bears similarities to those components of adaptation found in the OHC stereociliar transducer identified recently. As with the forward transducer, the speed of the reverse transducer amplificatory event consequently impacts on high frequency peripheral auditory processing.
Knipper, M; Zimmermann, U; Köpschall, I; Rohbock, K; Jüngling, S; Zenner, H P
By employing immunological methods, it has been demonstrated that myosin, myosin light chain (MLC) and myosin light chain kinase (MLCK) proteins in outer hair cells (OHC) are immunologically different from isoforms in platelets, smooth muscle and heart muscle, and are probably more related to isoforms found in red blood cells (RBC). Moreover, proteins related to band 3 protein (b3p) and protein 4.1 (p 4.1), ankyrin as well as fodrin and spectrin, but not glycophorin, have been identified in isolated OHCs. Both OHCs and RBC differ from other motile non-muscle cells in their lack of smooth muscle isoforms of actin, their common high levels of spectrin-, ankyrin- and band 3-like proteins, as well as the expression of the 80 kDa protein 4.1 isoform. The data support the notion that motility of OHC may be based upon regulation of the b3p/p 4.1/ankyrin complex, and thus may be reminiscent to the active shape changes in RBC.
Lin, Chang-min; Yuan, Yan-ping; Chen, Xian-cai; Li, Hai-hong; Cai, Bo-zhi; Liu, Yang; Zhang, Huan; Li, Yu; Huang, Keng
The rat whisker hair follicle (HF) is a model for studying the reconstruction of the HF or dermal papilla (DP), and involves the Wnt/β-catenin signaling pathway, which is a key pathway in HF development and HF cycling after birth. It has been reported that Wnt/catenin signaling plays an indispensable role in human or rat pelages development and postnatal growth. However, the distribution of some Wnt/β-catenin signaling pathway factors and their relationship with the epithelial stem cell markers in whisker follicles has not been characterized. In this study, we investigated the immunolocalization of Wnt/catenin signaling pathway members, including Wnt10b, Wnt10a, Wnt5a, β-catenin, and downstream lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 3 (TCF3), as well as, HF stem-cell markers CD34, CK15 and proliferating cell nuclear antigen (PCNA) protein, in rat anagen phase whisker follicles. β-catenin, Wnt5a, Wnt10b, Wnt10a, LEF1, and TCF3 were expressed in the outer root sheath (ORS), inner root sheath, matrix and hair shaft of anagen follicles. β-catenin, Wnt10b, LEF1, and TCF3 were highly expressed and Wnt5a and Wnt10a weakly expressed in DP and dermal sheath (DS) regions. The expression of α-smooth muscle actin was strong in the lower DS and it was also detected in some DP cells. CD34, CK15 and PCNA were all expressed in the ORS; and CD34 and PCNA were also detected in the matrix, however CD34 was extensively expressed in DP and DS regions. Our studies located the position of Wnts, downstream LEF1 and TCF3 and stem cell marker proteins, which provide new information in understanding the role of the Wnt singaling pathway in whisker follicles' growth.
Fan, Chunxin; Zou, Sha; Wang, Jian; Zhang, Bo; Song, Jiakun
The lateral line found in some amphibians and fishes has two distinctive classes of sensory organs: mechanoreceptors (neuromasts) and electroreceptors (ampullary organs). Hair cells in neuromasts can be damaged by aminoglycoside antibiotics and they will regenerate rapidly afterward. Aminoglycoside sensitivity and the capacity for regeneration have not been investigated in ampullary organs. We treated Siberian sturgeon (Acipenser baerii) larvae with neomycin and observed loss and regeneration of sensory hair cells in both organs by labeling with DASPEI and scanning electron microscopy (SEM). The numbers of sensory hair cells in both organs were reduced to the lowest levels at 6 hours posttreatment (hpt). New sensory hair cells began to appear at 12 hpt and were regenerated completely in 7 days. To reveal the possible mechanism for ampullary hair cell regeneration, we analyzed cell proliferation and the expression of neural placodal gene eya1 during regeneration. Both cell proliferation and eya1 expression were concentrated in peripheral mantle cells and both increased to the highest level at 12 hpt, which is consistent with the time course for regeneration of the ampullary hair cells. Furthermore, we used Texas Red-conjugated gentamicin in an uptake assay following pretreatment with a cation channel blocker (amiloride) and found that entry of the antibiotic was suppressed in both organs. Together, our results indicate that ampullary hair cells in Siberian sturgeon larvae can be damaged by neomycin exposure and they can regenerate rapidly. We suggest that the mechanisms for aminoglycoside uptake and hair cell regeneration are conserved for mechanoreceptors and electroreceptors. J. Comp. Neurol. 524:1443-1456, 2016. © 2015 Wiley Periodicals, Inc.
Martin-Granados, Cristina; Prescott, Alan R; Le Sommer, Samantha; Klaska, Izabela P; Yu, Tian; Muckersie, Elizabeth; Giuraniuc, Claudiu V; Grant, Louise; Delibegovic, Mirela; Forrester, John V
Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity, a function they perform by converting quiescent DC to active, mature DC with the capacity to activate naïve T cells. They do this by migrating from the tissues to the T cell area of the secondary lymphoid tissues. Here, we demonstrate that myeloid cell-specific genetic deletion of PTP1B (LysM PTP1B) leads to defects in lipopolysaccharide-driven bone marrow-derived DC (BMDC) activation associated with increased levels of phosphorylated Stat3. We show that myeloid cell-specific PTP1B deletion also causes decreased migratory capacity of epidermal DC, as well as reduced CCR7 expression and chemotaxis to CCL19 by BMDC. PTP1B deficiency in BMDC also impairs their migration in vivo. Further, immature LysM PTP1B BMDC display fewer podosomes, increased levels of phosphorylated Src at tyrosine 527, and loss of Src localization to podosome puncta. In co-culture with T cells, LysM PTP1B BMDC establish fewer and shorter contacts than control BMDC. Finally, LysM PTP1B BMDC fail to present antigen to T cells as efficiently as control BMDC. These data provide first evidence for a key regulatory role for PTP1B in mediating a central DC function of initiating adaptive immune responses in response to innate immune cell activation.
Woo, Wei-Meng; Zhen, Hanson H; Oro, Anthony E
During hair follicle morphogenesis, dermal papillae (DPs) function as mesenchymal signaling centers that cross-talk with overlying epithelium to regulate morphogenesis. While the DP regulates hair follicle formation, relatively little is known about the molecular basis of DP formation. The morphogen Sonic hedgehog (Shh) is known for regulating hair follicle epithelial growth, with excessive signaling resulting in basal cell carcinomas. Here, we investigate how dermal-specific Shh signaling contributes to DP formation and hair growth. Using a Cre-lox genetic model and RNAi in hair follicle reconstitution assays, we demonstrate that dermal Smoothened (Smo) loss of function results in the loss of the DP precursor, the dermal condensate, and a stage 2 hair follicle arrest phenotype reminiscent of Shh(-/-) skin. Surprisingly, dermal Smo does not regulate cell survival or epithelial proliferation. Rather, molecular screening and immunostaining studies reveal that dermal Shh signaling controls the expression of a subset of DP-specific signature genes. Using a hairpin/cDNA lentiviral system, we show that overexpression of the Shh-dependent gene Noggin, but not Sox2 or Sox18, can partially rescue the dermal Smo knockdown hair follicle phenotype by increasing the expression of epithelial Shh. Our findings suggest that dermal Shh signaling regulates specific DP signatures to maintain DP maturation while maintaining a reciprocal Shh-Noggin signaling loop to drive hair follicle morphogenesis.
Kou, Liang; Lu, Xiao-Wen; Wu, Min-Ke; Wang, Hang; Zhang, Yu-Jiao; Sato, Soh; Shen, Jie-Fei
Dedifferentiated fat (DFAT) cells derived from mature adipocytes have been considered to be a homogeneous group of multipotent cells, which present to be an alternative source of adult stem cells for regenerative medicine. However, many aspects of the cellular nature about DFAT cells remained unclarified. This study aimed to elucidate the basic characteristics of DFAT cells underlying their functions and differentiation potentials. By modified ceiling culture technique, DFAT cells were converted from human mature adipocytes from the human buccal fat pads. Flow cytometry analysis revealed that those derived cells were a homogeneous population of CD13(+) CD29(+) CD105(+) CD44(+) CD31(-) CD34(-) CD309(-) α-SMA(-) cells. DFAT cells in this study demonstrated tissue-specific differentiation properties with strong adipogenic but much weaker osteogenic capacity. Neither did they express endothelial markers under angiogenic induction.
Miseon Lee; Kunsoo Rhee
Background Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis. Methods We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles. ...
Miseon Lee; Kunsoo Rhee
Background Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis. Methods We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles. ...
Larraona-Puy, M.; Ghita, A.; Zoladek, A.; Perkins, W.; Varma, S.; Leach, I. H.; Koloydenko, A. A.; Williams, H.; Notingher, I.
Skin cancer is the most common human malignancy and basal cell carcinoma (BCC) represents approximately 80% of the non-melanoma cases. Current methods of treatment require histopathological evaluation of the tissues by qualified personnel. However, this method is subjective and in some cases BCC can be confused with other structures in healthy skin, including hair follicles. In this preliminary study, we investigated the potential of Raman micro-spectroscopy (RMS) to discriminate between hair follicles and BCC in skin tissue sections excised during Mohs micrographic surgery (MMS). Imaging and diagnosis of skin sections was automatically generated using ' a priori'-built spectral model based on LDA. This model had 90 ± 9% sensitivity and 85 ± 9% specificity for discrimination of BCC from dermis and epidermis. The model used selected Raman bands corresponding to the largest spectral differences between the Raman spectra of BCC and the normal skin regions, associated mainly with nucleic acids and collagen type I. Raman spectra corresponding to the epidermis regions of the hair follicles were found to be closer to those of healthy epidermis rather than BCC. Comparison between Raman spectral images and the gold standard haematoxylin and eosin (H&E) histopathology diagnosis showed good agreement. Some hair follicle regions were misclassified as BCC; regions corresponded mainly to the outermost layer of hair follicle (basal cells) which are expected to have higher nucleic acid concentration. This preliminary study shows the ability of RMS to distinguish between BCC and other tissue structures associated to healthy skin which can be confused with BCC due to their similar morphology.
Lei, Mingxing; Schumacher, Linus J; Lai, Yung-Chih; Juan, Wen-Tau; Yeh, Chao-Yuan; Wu, Ping; Jiang, Ting-Xin; Baker, Ruth E; Widelitz, Randall Bruce; Yang, Li; Chuong, Cheng-Ming
Organoids made from dissociated progenitor cells undergo tissue-like organization. This in vitro self-organization process is not identical to embryonic organ formation, but it achieves a similar phenotype in vivo. This implies genetic codes do not specify morphology directly; instead, complex tissue architectures may be achieved through several intermediate layers of cross talk between genetic information and biophysical processes. Here we use newborn and adult skin organoids for analyses. Dissociated cells from newborn mouse skin form hair primordia-bearing organoids that grow hairs robustly in vivo after transplantation to nude mice. Detailed time-lapse imaging of 3D cultures revealed unexpected morphological transitions between six distinct phases: dissociated cells, cell aggregates, polarized cysts, cyst coalescence, planar skin, and hair-bearing skin. Transcriptome profiling reveals the sequential expression of adhesion molecules, growth factors, Wnts, and matrix metalloproteinases (MMPs). Functional perturbations at different times discern their roles in regulating the switch from one phase to another. In contrast, adult cells form small aggregates, but then development stalls in vitro. Comparative transcriptome analyses suggest suppressing epidermal differentiation in adult cells is critical. These results inspire a strategy that can restore morphological transitions and rescue the hair-forming ability of adult organoids: (i) continuous PKC inhibition and (ii) timely supply of growth factors (IGF, VEGF), Wnts, and MMPs. This comprehensive study demonstrates that alternating molecular events and physical processes are in action during organoid morphogenesis and that the self-organizing processes can be restored via environmental reprogramming. This tissue-level phase transition could drive self-organization behavior in organoid morphogenies beyond the skin.
Full Text Available Abstract Background Airway smooth muscle (ASM contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells. Methods Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured. Results Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of α2, β1 and γ1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype. Conclusion While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the
Ravi Shankar Akundi
Full Text Available BACKGROUND: Periventricular white matter injury (PWMI is a common form of brain injury sustained by preterm infants. A major factor that predisposes to PWMI is hypoxia. Because oligodendrocytes (OLs are responsible for myelination of axons, abnormal OL development or function may affect brain myelination. At present our understanding of the influences of hypoxia on OL development is limited. To examine isolated effects of hypoxia on OLs, we examined the influences of hypoxia on OL development in vitro. METHODOLOGY/FINDINGS: Cultures of oligodendrocyte precursor cells (OPCs were prepared from mixed glial cultures and were 99% pure. OPCs were maintained at 21% O(2 or hypoxia (1% or 4% O(2 for up to 7 days. We observed that 1% O(2 lead to an increase in the proportion of myelin basic protein (MBP-positive OLs after 1 week in culture, and a decrease in the proportion of platelet-derived growth factor receptor alpha (PDGFRalpha-positive cells suggesting premature OL maturation. Increased expression of the cell cycle regulatory proteins p27(Kip1 and phospho-cdc2, which play a role in OL differentiation, was seen as well. CONCLUSIONS: These results show that hypoxia interferes with the normal process of OL differentiation by inducing premature OPC maturation.
Anayama, Hisashi; Fukuda, Ryo; Yamate, Jyoji
The precise localization and biological characteristics of the adipose progenitor cells are still a focus of debate. In this study, the localization of the adipose progenitor cells was determined using an organotypic culture system of adipose tissue slices. The tissue slices of subcutaneous white adipose tissue from rats were placed on a porous membrane and cultured at the interface between air and the culture medium for up to 5 days with or without adipogenic stimulation. The structure of adipose tissue components was sufficiently preserved during the culture and, following adipogenic stimulation with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine, numerous multilocular adipocytes appeared in the interstitium among the mature adipocytes. Histomorphological 3-D observation using confocal laser microscopy revealed the presence of small mesenchymal cells containing little or no fat residing in the perivascular region and on the mature adipocytes and differentiation from the pre-existing mesenchymal cells to multilocular adipocytes. Immunohistochemistry demonstrated that these cells were initially present within the fibronectin-positive extracellular matrix (ECM). The adipose differentiation of the mesenchymal cells was confirmed by the enhanced expression of C/EBP-β suggesting adipose differentiation and the concurrent advent of CD105-expressing mesenchymal cells within the interstitium of the mature adipocytes. Based on the above, the mesenchymal cells embedded in the ECM around the mature adipocytes were confirmed to be responsible for adipogenesis because the transition of the mesenchymal cells to the stem state contributed to the increase in the number of adipocytes in rat adipose tissue.
Daisuke eAkita; Koichiro eKano; Yoko eSaito-Tamura; Takayuki eMashimo; Momoko eSato-Shionome; Niina eTsurumachi; Katsuyuki eYamanaka; Tadashi eKaneko; Taku eToriumi; Yoshinori eArai; Naoki eTsukimura; Taro eMatsumoto; Tomohiko eIshigami; Keitaro eIsokawa; Masaki eHonda
Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cellsWe obtained DF...
LIU Yang; LIN Zi-hao; ZHU Xiao-hai; LIU Shan-rong
Objective: To study whether mouse sertoli cells can induce the apoptosis of matured T lymphocytes in cocultures in vitro. Methods: With TUNEL, DNA electrophoresis, eleetro-mierography and flow cytometry, we examined the apoptosis and its rates of mouse matured T lymphocytes in control group (T lymphocytes only), group A (T lymphocytes + culture medium of sertoli cells), group B (T lymphocytes + sertoli cells). Results: Under electro-micrography, chromatin condensation, karyopyknosis, karyorhexis and apoptotic body were observed in some T lymphocytes in 3 groups; some nucleuses were stained dark blue with TUNEL; a typical DNA ladder was found with DNA electrophoresis. The apoptotic rates of T lymphocytes in group A and B were significantly higher than that in control group (P＜0.01). The apoptotic rate of T lymphocytes in group B was significantly higher than that in group A (P＜0.01). Conclusion: In coculture condition in vitro,mouse sertoli cells can induce the apoptosis of matured T lymphocytes.
Kim, Eun Young; Kim, Ji Won; Kim, Won Kon; Han, Baek Soo; Park, Sung Goo; Chung, Bong Hyun; Lee, Sang Chul; Bae, Kwang-Hee
Background Adipose tissue, mainly composed of adipocytes, plays an important role in metabolism by regulating energy homeostasis. Obesity is primarily caused by an abundance of adipose tissue. Therefore, specific targeting of adipose tissue is critical during the treatment of obesity, and plays a major role in overcoming it. However, the knowledge of cell-surface markers specific to adipocytes is limited. Methods and Results We applied the CELL SELEX (Systematic Evolution of Ligands by EXponential enrichment) method using flow cytometry to isolate molecular probes for specific recognition of adipocytes. The aptamer library, a mixture of FITC-tagged single-stranded random DNAs, is used as a source for acquiring molecular probes. With the increasing number of selection cycles, there was a steady increase in the fluorescence intensity toward mature adipocytes. Through 12 rounds of SELEX, enriched aptamers showing specific recognition toward mature 3T3-L1 adipocyte cells were isolated. Among these, two aptamers (MA-33 and 91) were able to selectively bind to mature adipocytes with an equilibrium dissociation constant (Kd) in the nanomolar range. These aptamers did not bind to preadipocytes or other cell lines (such as HeLa, HEK-293, or C2C12 cells). Additionally, it was confirmed that MA-33 and 91 can distinguish between mature primary white and primary brown adipocytes. Conclusions These selected aptamers have the potential to be applied as markers for detecting mature white adipocytes and monitoring adipogenesis, and could emerge as an important tool in the treatment of obesity. PMID:24844710
Vogl, Christian; Cooper, Benjamin H; Neef, Jakob; Wojcik, Sonja M; Reim, Kerstin; Reisinger, Ellen; Brose, Nils; Rhee, Jeong-Seop; Moser, Tobias; Wichmann, Carolin
Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca(2+)-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.
Johnson, Stuart L; Beurg, Maryline; Marcotti, Walter; Fettiplace, Robert
Outer hair cells (OHCs) provide amplification in the mammalian cochlea using somatic force generation underpinned by voltage-dependent conformational changes of the motor protein prestin. However, prestin must be gated by changes in membrane potential on a cycle-by-cycle basis and the periodic component of the receptor potential may be greatly attenuated by low-pass filtering due to the OHC time constant (τ(m)), questioning the functional relevance of this mechanism. Here, we measured τ(m) from OHCs with a range of characteristic frequencies (CF) and found that, at physiological endolymphatic calcium concentrations, approximately half of the mechanotransducer (MT) channels are opened at rest, depolarizing the membrane potential to near -40 mV. The depolarized resting potential activates a voltage-dependent K+ conductance, thus minimizing τ(m) and expanding the membrane filter so there is little receptor potential attenuation at the cell's CF. These data suggest that minimal τ(m) filtering in vivo ensures optimal activation of prestin.
Full Text Available The goals of this study were to investigate the effects of hypoxia on cochlear hair cell damage, and to explore the role of sirtuin1 in hypoxia-induced hair cell damage. Cochlear organotypic cultures from postnatal day 4 rats were used in this study. Hypoxia was induced by treating cochlear explants with CoCl2. Cochlear cultures were treated with CoCl2 alone or in combination with the sirtuin1 activator resveratrol and the sirtuin1 inhibitor sirtinol. Hair cell damage was identified by phalloidin staining and imaged using scanning electron microscopy. RT-PCR and Western blot analyses were used to detect the expression of sirtuin1 and acetylated nuclear factor-κB (NF-κB. Low concentrations of CoCl2 (100-200 μM did not cause an obvious change in the number and morphology of hair cells, whereas higher concentrations of CoCl2 (300-400 μM induced swelling of hair cells, accompanied by cell loss. Increased sirtuin1 expression was induced by CoCl2 at 100 to 200 μM, but not at 400 μM. NF-κB acetylation was significantly increased in explants treated with 400 μM CoCl2. Pretreatment with resveratrol prevented CoCl2-induced hair cell loss and acetylation of NF-κB. The protective effect of resveratrol was significantly reduced by sirtinol. CoCl2 induces hair cell damage in organotypic cochleae cultures. Resveratrol attenuates CoCl2-induced cochlear hair cell damage possibly via activation of sirtuin1, which deacetylates NF-κB.
Noguchi, Yoshihiro; Kurima, Kiyoto; Makishima, Tomoko; de Angelis, Martin Hrabé; Fuchs, Helmut; Frolenkov, Gregory; Kitamura, Ken; Griffith, Andrew J
Dominant mutations of transmembrane channel-like gene 1 (TMC1) cause progressive sensorineural hearing loss in humans and Beethoven (Tmc1Bth/+) mice. Here we show that Tmc1Bth/+ mice on a C3HeB/FeJ strain background have selective degeneration of inner hair cells while outer hair cells remain structurally and functionally intact. Inner hair cells primarily function as afferent sensory cells, whereas outer hair cells are electromotile amplifiers of auditory stimuli that can be functionally assessed by distortion product otoacoustic emission (DPOAE) analysis. When C3H-Tmc1Bth/Bth is crossed with either C57BL/6J or DBA/2J wild-type mice, F1 hybrid Tmc1Bth/+ progeny have increased hearing loss associated with increased degeneration of outer hair cells and diminution of DPOAE amplitudes but no difference in degeneration of inner hair cells. We mapped at least one quantitative trait locus (QTL), Tmc1m1, for DPOAE amplitude on chromosome 2 in [(C/B)F1xC]N2-Tmc1Bth/+ backcross progeny, and three other QTL on chromosomes 11 (Tmc1m2), 12 (Tmc1m3), and 5 (Tmc1m4) in [(C/D)F1xC]N2-Tmc1Bth/+ progeny. The polygenic basis of outer hair cell degeneration in Beethoven mice provides a model system for the dissection of common, complex hearing loss phenotypes, such as presbycusis, that involve outer hair cell degeneration in humans.
Yadav, Mukesh Kumar; Choi, June; Song, Jae-Jun
Gentamicin (GM) is a commonly used aminoglycoside antibiotic that generates free oxygen radicals within the inner ear, which can cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. L. (PL...
The National Institutes of Health (NIH) closure of the agency's Center for Regenerative Medicine (CRM), which focused on therapeutic development of induced pluripotent stem cells (iPS), was caused by the lack of progress in practical development of the iPSs for use in human therapies. As the NIH evaluates priorities in future stem cell therapeutic development, adult stem cell processes in the human body need to be prioritized for a number of key reasons, including (1) adult stem cells release many types of molecules that provide much of the therapeutic benefit of stem cells and (2) adult stem cells and somatic cells exist in a state of dynamic transition between different potency levels and can be naturally driven by the microenvironment to a state of pluripotency. Thus, the study and development of adult stems for therapeutic use can include naturally induced pluripotent stem cells (NiPSs) that lack the problematic genetic and epigenetic reprogramming errors found in iPSs.
le Viseur, Christoph; Hotfilder, Marc; Bomken, Simon; Wilson, Kerrie; Roettgers, Silja; Schrauder, Andre; Rosemann, Annegret; Irving, Julie; Stam, Ronald W.; Shultz, Leonard D.; Harbott, Jochen; Juergens, Heribert; Schrappe, Martin; Pieters, Rob; Vormoor, Josef
We examined the leukemic stem cell potential of blasts at different stages of maturation in childhood acute lymphoblastic leukemia (ALL). Human leukemic bone marrow was transplanted intrafemorally into NOD/scid mice. Cells sorted using the B precursor differentiation markers CD19, CD20, and CD34
Farahbakhsh, Nasser A; Narins, Peter M
Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca(2+)/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca(2+)/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter phase 1 response. However, they appear to counter effects of the inhibitors of Ca(2+)/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells.
Ferreira, Gb; Overbergh, L; Hansen, Kasper Lage
Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen-capturing cell towards a professional antigen presenting cells. In this study, a 2-D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expressi...
Adler, Paul N; Sobala, Lukasz F; Thom, Desean; Nagaraj, Ranganayaki
The cuticular hairs and sensory bristles that decorate the adult Drosophila epidermis and the denticles found on the embryo have been used in studies on planar cell polarity and as models for the cytoskeletal mediated morphogenesis of cellular extensions. ZP domain proteins have recently been found to be important for the morphogenesis of both denticles and bristles. Here we show that the ZP domain protein Dusky-like is a key player in hair morphogenesis. As is the case in bristles, in hairs dyl mutants display a dramatic phenotype that is the consequence of a failure to maintain the integrity of the extension after outgrowth. Hairs lacking dyl function are split, thinned, multipled and often very short. dyl is required for normal chitin deposition in hairs, but chitin is not required for the normal accumulation of Dyl, hence dyl acts upstream of chitin. A lack of chitin however, does not mimic the dyl hair phenotype, thus Dyl must have other targets in hair morphogenesis. One of these appears to be the actin cytoskeleton. Interestingly, dyl mutants also display a unique planar cell polarity phenotype that is distinct from that seen with mutations in the frizzled/starry night or dachsous/fat pathway genes. Rab11 was previously found to be essential for Dyl plasma membrane localization in bristles. Here we found that the expression of a dominant negative Rab11 can mimic the dyl hair morphology phenotype consistent with Rab11 also being required for Dyl function in hairs. We carried out a small directed screen to identify genes that might function with dyl and identified Chitinase 6 (Cht6) as a strong candidate, as knocking down Cht6 function led to weak versions of all of the dyl hair phenotypes.
Freedman, David S; Cohen, Howard I; Deligeorges, Socrates; Karl, Christian; Hubbard, Allyn E
An analog inner hair cell and auditory nerve circuit using a dual AGC model has been implemented using 0.35 micron mixed-signal technology. A fully-differential current-mode architecture is used and the ability to correct channel mismatch is evaluated with matched layouts as well as with digital current tuning. The Meddis test paradigm is used to examine the analog implementation's auditory processing capabilities and investigate the circuit's ability to correct DC mismatch. The correction techniques used demonstrate the analog inner hair cell and auditory nerve circuit's potential use in low-power, multiple-sensor analog biomimetic systems with highly reproducible signal processing blocks on a single massively parallel integrated circuit.
Cárdenas, Luis; Martínez, Adán; Sánchez, Federico; Quinto, Carmen
The role of reactive oxygen species (ROS) in root-nodule development and metabolism has been extensively studied. However, there is limited evidence showing ROS changes during the earliest stages of the interaction between legumes and rhizobia. Herein, using ratio-imaging analysis, increasing and transient ROS levels were detected at the tips of actively growing root hair cells within seconds after addition of Nod factors (NFs). This transient response (which lasted up to 3 min) was Nod-factor-specific, as chitin oligomers (pentamers) failed to induce a similar response. When chitosan, a fungal elicitor, or ATP was used instead, a sustained increasing signal was observed. As ROS levels are transiently elevated after the perception of NFs, we propose that this ROS response is characteristic of the symbiotic interaction. Furthermore, we discuss the remarkable spatial and temporal coincidences between ROS and transiently increased calcium levels observed in root hair cells immediately after the detection of NFs.
Beisel, K. W.; Fritzsch, B.
Both qualitative and quantitative differences in ion-channel conductances are observed along the tonotopic axis of the mammalian cochlea. We have used a molecular approach to characterize these longitudinal expression patterns of voltage-gated ion-channel (VgCN) superfamily members in the peripheral auditory system. Initially RT-PCR and sequence analyses identified the VgCN α and accessory subunits of the cochlear hair cell (HC). Next, whole mount in situ hybridizations demonstrated at least seven common longitudinal expression patterns with the apex tip and basal hook region having the greatest in disparity. These data suggest potential topological variations in hair-cell electrophysiological signatures and these gradients may contribute to cochlear HC's ability to function as efficient frequency analyzers.
Shehata, W E; Brownell, W E; Dieler, R
A reversible tinnitus and hearing loss have long been known to result from large doses of salicylate. Cochlear electrophysiology and otoacoustic emission studies suggest that the drug may interfere with outer hair cell electromotility. Exposure of isolated outer hair cells to sodium salicylate concentrations ranging from 0.05 to 10 mM reveals a dose dependent, reversible loss of turgidity and dimunition of electromotility. There was also a change in membrane conductance with salicylate superfusion that occurred later in time from the onset of shape and electromotility changes. There was no evidence of dose dependence for the change in membrane conductance, nor was the change reversible. The changes in shape and electromotility that we observe in vitro may impair cochlear partition movements in vivo and could account, at least in part, for the salicylate-induced hearing loss and effects on otoacoustic emissions.
Chong Hyun Won
Full Text Available Keratinocyte stem/progenitor cells (KSCs reside in the bulge region of the hair follicles and may be involved in hair growth. Hair follicle dermal papilla cells (HFDPCs and outer root sheath (ORS cells were treated with conditioned medium (CM of KSCs. Moreover, the effects of KSC-CM on hair growth were examined ex vivo and in vivo. A human growth factor chip array and RT-PCR were employed to identify enriched proteins in KSC-CM as compared with CM from keratinocytes. KSC-CM significantly increased the proliferation of HFDPCs and ORS cells, and increased the S-phase of the cell cycle in HFDPCs. KSC-CM led to the phosphorylation of ATK and ERK1/2 in both cell types. After subcutaneous injection of KSC-CM in C3H/HeN mice, a significant increase in hair growth and increased proliferation of hair matrix keratinocytes ex vivo was observed. We identified six proteins enriched in KSC-CM (amphiregulin, insulin-like growth factor binding protein-2, insulin-like growth factor binding protein-5, granulocyte macrophage-colony stimulating factor, Platelet-derived growth factor-AA, and vascular endothelial growth factor. A growth-factor cocktail that contains these six recombinant growth factors significantly increased the proliferation of HFDPCs and ORS cells and enhanced the hair growth of mouse models. These results collectively indicate that KSC-CM has the potential to increase hair growth via the proliferative capacity of HFDPCs and ORS cells.
Xing, Feiyue; Wang, Jiongkun; Hu, Mingqian; Yu, Yu; Chen, Guoliang; Liu, Jing
A comparative study of immature and mature bone marrow-derived dendritic cells (BMDCs) was first performed through an atomic force microscope (AFM) to clarify differences of their nanostructure and adhesion force. AFM images revealed that the immature BMDCs treated by granulocyte macrophage-colony stimulating factor plus IL-4 mainly appeared round with smooth surface, whereas the mature BMDCs induced by lipopolysaccharide displayed an irregular shape with numerous pseudopodia or lamellapodia and ruffles on the cell membrane besides becoming larger, flatter, and longer. AFM quantitative analysis further showed that the surface roughness of the mature BMDCs greatly increased and that the adhesion force of them was fourfold more than that of the immature BMDCs. The nano-features of the mature BMDCs were supported by a high level of IL-12 produced from the mature BMDCs and high expression of MHC-II on the surface of them. These findings provide a new insight into the nanostructure of the immature and mature BMDCs.
Liu, Shuzhen; Jiang, Ligang; Zhong, Tao; Kong, Shuhui; Zheng, Rongbin; Kong, Fengyun; Zhang, Cong; Zhang, Lei; An, Liguo
Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro. PMID:26275143
Full Text Available Acrylamide (ACR is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus-oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus-oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.
Full Text Available Fully mature dendritic cells (DCs play pivotal role in inducing immune responses and converting naïve T lymphocytes into functional Th1 cells. We aimed to evaluate Listeria Monocytogenes-derived protein fractions to induce DC maturation and stimulating T helper (Th1 immune responses.In the present study, we fractionated Listeria Monocytogenes-derived proteins by adding of ammonium sulfate in a stepwise manner. DCs were also generated from C57BL/6 mice bone marrow precursor cells. Then, the effects of protein fractions on bone marrow derived DC (BMDC maturation were evaluated. In addition, we assessed the capacity of activated DCs to induce cytokine production and proliferation of lymphocytes.Listeria-derived protein fractions induced fully mature DCs expressing high costimulatory molecules such as CD80, CD86 and CD40. DCs that were activated by selected F3 fraction had low capacity to uptake exogenous antigens while secreted high levels of Interleukine (IL-12. Moreover, lymphocytes cul