A CRISPR screen in intestinal epithelial cells identifies novel factors for polarity and apical transport.

Epithelial polarization and polarized cargo transport are highly coordinated and interdependent processes. In our search for novel regulators of epithelial polarization and protein secretion, we used a genome-wide CRISPR/Cas9 screen and combined it with an assay based on fluorescence-activated cell sorting (FACS) to measure the secretion of the apical brush-border hydrolase dipeptidyl peptidase 4 (DPP4). In this way, we performed the first CRISPR screen to date in human polarized epithelial cells. Using high-resolution microscopy, we detected polarization defects and mislocalization of DPP4 to late endosomes/lysosomes after knockout of TM9SF4, anoctamin 8, and ARHGAP33, confirming the identification of novel factors for epithelial polarization and apical cargo secretion. Thus, we provide a powerful tool suitable for studying polarization and cargo secretion in epithelial cells. In addition, we provide a dataset that serves as a resource for the study of novel mechanisms for epithelial polarization and polarized transport and facilitates the investigation of novel congenital diseases associated with these processes.

Low kindlin-3 levels in osteoclasts of kindlin-3 hypomorphic mice result in osteopetrosis due to leaky sealing zones.

Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist of densely packed podosomes tightly interconnected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3 (also known as Fermt3). In this study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5-10% of the kindlin-3 level of wild-type mice. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro-generated kindlin-3-hypomorphic osteoclast-like cells arrange their podosomes into adhesion patches and belts, but their podosome and actin organization is abnormal. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays, immunohistochemical staining and electron micrographs of bone sections showed that they fail to seal the resorption lacunae properly, which is required for secreted proteinases to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrin-mediated adhesion structures, such as sealing zones.

The SZT2 Interactome Unravels New Functions of the KICSTOR Complex.

Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.

Time-dependent changes in bone healing capacity of scaphoid fractures and non-unions.

The scaphoid is the most frequently fractured carpal bone and prone to non-union due to mechanical and biological factors. Whereas the importance of stability is well documented, the evaluation of biological activity is mostly limited to the assessment of vascularity. The purpose of this study was to select histological and immunocytochemical parameters that could be used to assess healing potential after scaphoid fractures and to correlate these findings with time intervals after fracture for the three parts of the scaphoid (distal, gap and proximal). Samples were taken during operative intervention in 33 patients with delayed or non-union of the scaphoid. Haematoxylin and Eosin (HE), Azan, Toluidine, von Kossa and Tartrate-resistant acid phosphatase (TRAP) staining were used to characterise the samples histologically. We determined distribution of collagen 1 and 2 by immunocytochemistry, and scanning electron microscopy (SEM) was used to investigate the ultrastructure. To analyse the samples, parameters for biological healing status were defined and grouped according to healing capacity in parameters with high, partial and little biological activity. These findings allowed scoring of biological healing capacity, and the ensuing results were correlated with different time intervals after fracture. The results showed reduced healing capacity over time, but not all parts of the scaphoid were affected in the same way. For the distal fragment, regression analysis showed a statistically significant correlation between summarised healing activity scores and time from initial fracture (r = -0.427, P = 0.026) and decreasing healing activity for the gap region (r = -0.339, P = 0.090). In contrast, the analyses of the proximal parts for all patients did not show a correlation (r = 0.008, P = 0.969) or a decrease in healing capacity, with reduced healing capacity already at early stages. The histological and immunocytochemical characterisation of scaphoid non-unions (SNUs) and the scoring of healing parameters make it possible to analyse the healing capacity of SNUs at certain time points. This information is important as it can assist the surgeon in the selection of the most appropriate SNU treatment.

Polarized light microscopy reveals physiological and drug-induced changes in surfactant membrane assembly in alveolar type II pneumocytes.

In alveolar type II (AT II) cells, pulmonary surfactant (PS) is synthetized, stored and exocytosed from lamellar bodies (LBs), specialized large secretory organelles. By applying polarization microscopy (PM), we confirm a specific optical anisotropy of LBs, which indicates a liquid-crystalline mesophase of the stored surfactant phospholipids (PL) and an unusual case of a radiation-symmetric, spherocrystalline organelle. Evidence is shown that the degree of anisotropy is dependent on the amount of lipid layers and their degree of hydration, but unaffected by acutely modulating vital cell parameters like intravesicular pH or cellular energy supply. In contrast, physiological factors that perturb this structure include osmotic cell volume changes and LB exocytosis. In addition, we found two pharmaceuticals, Amiodarone and Ambroxol, both of which severely affect the liquid-crystalline order. Our study shows that PM is an easy, very sensitive, but foremost non-invasive and label-free method able to collect important structural information of PS assembly in live AT II cells which otherwise would be accessible by destructive or labor intense techniques only. This may open new approaches to dynamically investigate LB biosynthesis - the incorporation, folding and packing of lipid membranes - or the initiation of pathological states that manifest in altered LB structures. Due to the observed drug effects, we further suggest that PM provides an appropriate way to study unspecific drug interactions with alveolar cells and even drug-membrane interactions in general.

Molecular composition and distribution of gap junctions in the sensory epithelium of the human cochlea-a super-resolution structured illumination microscopy (SR-SIM) study.

BACKGROUND: Mutations in the GJB2 gene, which encodes the Connexin26 (Cx26) protein, are the most common cause of childhood hearing loss in American and European populations. The cochlea contains a gap junction (GJ) network in the sensory epithelium and two connective tissue networks in the lateral wall and spiral limbus. The syncytia contain the GJ proteins beta 2 (GJB2/Cx26) and beta 6 (GJB6/Cx30). Our knowledge of their expression in humans is insufficient due to the limited availability of tissue. Here, we sought to establish the molecular arrangement of GJs in the epithelial network of the human cochlea using surgically obtained samples. METHODS: We analyzed Cx26 and Cx30 expression in GJ networks in well-preserved adult human auditory sensory epithelium using confocal, electron, and super-resolution structured illumination microscopy (SR-SIM). RESULTS: Cx30 plaques (<5 µm) dominated, while Cx26 plaques were subtle and appeared as 'mini-junctions' (2-300 nm). 3-D volume rendering of Z-stacks and orthogonal projections from single optical sections suggested that the GJs are homomeric/homotypic and consist of assemblies of identical GJs composed of either Cx26 or Cx30. Occasionally, the two protein types were co-expressed, suggesting functional cooperation. CONCLUSIONS: Establishing the molecular composition and distribution of the GJ networks in the human cochlea may increase our understanding of the pathophysiology of Cx-related hearing loss. This information may also assist in developing future strategies to treat genetic hearing loss.

Supernumerary human hair cells-signs of regeneration or impaired development? A field emission scanning electron microscopy study.

BACKGROUND: Current attempts to regenerate cochlear sensorineural structures motivate further inspection of the human organ of hearing. Here, we analyzed the supernumerary inner hair cell (sIHC), a possible sign of regeneration and cell replacement. METHODS: Human cochleae were studied using field emission scanning electron microscopy (FESEM; maximum resolution 2 nm) obtained from individuals aged 44, 48, and 58 years with normal sensorineural pure-tone average (PTA) thresholds (PTA <20 dB). The wasted tissue was harvested during trans-cochlear approaches and immediately fixed for ultrastructural analysis. RESULTS: All specimens exhibited sIHCs at all turns except at the extreme lower basal turn. In one specimen, it was possible to image and count the inner hair cells (IHCs) along the cochlea representing the 0.2 kHz-8 kHz region according to the Greenwood place/frequency scale. In a region with 2,321 IHCs, there were 120 scattered one-cell losses or 'gaps' (5%). Forty-two sIHCs were present facing the modiolus. Thirty-eight percent of the sIHCs were located near a 'gap' in the IHC row (±6 IHCs). CONCLUSIONS: The prevalence of ectopic inner hair cells was higher than expected. The morphology and placement could reflect a certain ongoing regeneration. Further molecular studies are needed to verify if the regenerative capacity of the human auditory periphery might have been underestimated.

Persistent Supercooling of Reproductive Shoots Is Enabled by Structural Ice Barriers Being Active Despite an Intact Xylem Connection.

Extracellular ice nucleation usually occurs at mild subzero temperatures in most plants. For persistent supercooling of certain plant parts ice barriers are necessary to prevent the entry of ice from already frozen tissues. The reproductive shoot of Calluna vulgaris is able to supercool down to below -22°C throughout all developmental stages (shoot elongation, flowering, fruiting) despite an established xylem conductivity. After localization of the persistent ice barrier between the reproductive and vegetative shoot at the base of the pedicel by infrared differential thermal analysis, the currently unknown structural features of the ice barrier tissue were anatomically analyzed on cross and longitudinal sections. The ice barrier tissue was recognized as a 250 µm long constriction zone at the base of the pedicel that lacked pith tissue and intercellular spaces. Most cell walls in this region were thickened and contained hydrophobic substances (lignin, suberin, and cutin). A few cell walls had what appeared to be thicker cellulose inclusions. In the ice barrier tissue, the area of the xylem was as much as 5.7 times smaller than in vegetative shoots and consisted of tracheids only. The mean number of conducting units in the xylem per cross section was reduced to 3.5% of that in vegetative shoots. Diameter of conducting units and tracheid length were 70% and 60% (respectively) of that in vegetative shoots. From vegetative shoots water transport into the ice barrier must pass pit membranes that are likely impermeable to ice. Pit apertures were about 1.9 µm x 0.7 µm, which was significantly smaller than in the vegetative shoot. The peculiar anatomical features of the xylem at the base of the pedicel suggest that the diameter of pores in pit membranes could be the critical constriction for ice propagation into the persistently supercooled reproductive shoots of C. vulgaris.

Towards understanding microvillus inclusion disease.

Microvillus inclusion disease (MVID) is characterised by onset of intractable life-threatening watery diarrhoea during infancy. Transmission electron microscopy demonstrates shortening or absence of apical microvilli, pathognomonic microvillus inclusions in mature enterocytes and subapical accumulation of periodic acid-Schiff-positive granules or vesicles confirming diagnosis. Mutations in MYO5B have been found to cause MVID. In two patients with MVID, whole-exome sequencing of DNA revealed homozygous truncating mutations in STX3. Mutations in these genes disrupt trafficking between apical cargo vesicles and the apical plasma membrane. Thus, disturbed delivery of certain brush border membrane proteins is a common defect in MVID.

Morphological changes in the human cervical intervertebral disc post trauma: response to fracture-type and degeneration grade over time.

PURPOSE: In the first 24 h post-intervertebral disc (IVD) trauma, up to 75 % cell death has been reported. In addition, burst fractures cause post-traumatic disc degeneration by elevated pro-apoptotic and pro-inflammatory gene transcription. Moreover, some patients have pre-trauma degenerative disc disease. The aim of the study was to assess histological changes and cell-death over a time period of up to 1 year caused by mechanical and structural factors. METHODS: 116 anterior portions of IVDs of the cervical spine were studied histologically by light microscopy and ultrastructurally by transmission electron microscopy (TEM). The group was investigated with regard to three main parameters: fracture mechanism (compressive vs. tensile/shear loads), degeneration grade (low vs. high) and endplate fracture (with vs. without). Disc architecture (e.g. ruptures) was studied histologically. Cell morphology was examined ultrastructurally to quantify cell-death, healthy and balloon cells. According to ultrastructural observations, two time-groups (up to 6 days vs. later) were established. Statistical analyses were carried out within and between time-groups. RESULTS: Histological changes were obvious in the annulus fibrosus where ruptures with haematoma were replaced by granulation tissue. Significant differences in cell-death were seen in the first few days due to different loads. In contrast to the more degenerated segments, low degenerated ones revealed significantly less cell death with time post-trauma. Interestingly, no difference was found between groups after the sixth day. Cell-death (mean 44 % for all investigated groups) remained high after day 6 post-trauma. CONCLUSION: IVDs retrieved from low grade degenerated segments revealed a significant recovery, with less cell-death and a partially restored disc matrix, although cell-death remained high. Long-term clinical studies of stabilized segments arising from different fracture mechanisms are required.

Schwann Cell Expressed Nogo-B Modulates Axonal Branching of Adult Sensory Neurons Through the Nogo-B Receptor NgBR.

UNLABELLED: In contrast to the central nervous system (CNS) nerve fibers do regenerate in the peripheral nervous system (PNS) although in a clinically unsatisfying manner. A major problem is excessive sprouting of regenerating axons which results in aberrant reinnervation of target tissue and impaired functional recovery. In the CNS, the reticulon protein Nogo-A has been identified as a prominent oligodendrocyte expressed inhibitor of long-distance growth of regenerating axons. We show here that the related isoform Nogo-B is abundantly expressed in Schwann cells in the PNS. Other than Nogo-A in oligodendrocytes, Nogo-B does not localize to the myelin sheath but is detected in the ER and the plasma membrane of Schwann cells. Adult sensory neurons that are cultured on nogo-a/b deficient Schwann cells form significantly fewer axonal branches vs. those on wildtype Schwann cells, while their maximal axonal extension is unaffected. We demonstrate that this effect of Nogo-B on neuronal morphology is restricted to undifferentiated Schwann cells and is mediated by direct physical contact between these two cell types. Moreover, we show that blocking the Nogo-B specific receptor NgBR, which we find expressed on sensory neurons and to interact with Schwann cell expressed Nogo-B, produces the same branching phenotype as observed after deletion of Nogo-B. These data provide evidence for a novel function of the nogo gene that is implemented by the Nogo-B isoform. The remarkably specific effects of Nogo-B/NgBR on axonal branching, while leaving axonal extension unaffected, are of potential clinical relevance in the context of excessive axonal sprouting after peripheral nerve injury. MAIN POINTS: Nogo-B is prominently expressed in Schwann cells and localizes to the ER and plasma membrane. It distributes to the external cytoplasmic compartment of Schwann cells in vivo, but is absent from the myelin sheath.Genetic deletion of Nogo-B in Schwann cells reduces axonal branching, but not long-distance growth, of co-cultured adult sensory neurons.Schwann cell expressed Nogo-B interacts with neuronal NgBR. Blockade of NgBR mimics the loss-of-nogo branching phenotype.

Identification of Aspergillus fumigatus Surface Components That Mediate Interaction of Conidia and Hyphae With Human Platelets.

BACKGROUND: Platelets were recently identified as a part of innate immunity. They are activated by contact with Aspergillus fumigatus; putative consequences include antifungal defense but also thrombosis, excessive inflammation, and thrombocytopenia. We aimed to identify those fungal surface structures that mediate interaction with platelets. METHODS: Human platelets were incubated with Aspergillus conidia and hyphae, isolated wall components, or fungal surface mutants. Interaction was visualized microscopically; activation was quantified by flow cytometry of specific markers. RESULTS: The capacity of A. fumigatus conidia to activate platelets is at least partly due to melanin, because this effect can be mimicked with "melanin ghosts"; a mutant lacking melanin showed reduced platelet stimulating potency. In contrast, conidial hydrophobin masks relevant structures, because an A. fumigatus mutant lacking the hydrophobin protein induced stronger platelet activation than wild-type conidia. A. fumigatus hyphae also contain surface structures that interact with platelets. Wall proteins, galactomannan, chitin, and ß-glucan are not the relevant hyphal components; instead, the recently identified fungal polysaccharide galactosaminogalactan potently triggered platelet activation. CONCLUSIONS: Conidial melanin and hydrophobin as well as hyphal galactosaminogalactan represent important pathogenicity factors that modulate platelet activity and thus might influence immune responses, inflammation, and thrombosis in infected patients.

Macromolecular organization and fine structure of the human basilar membrane - RELEVANCE for cochlear implantation.

INTRODUCTION: Cochlear micromechanics and frequency tuning depend on the macromolecular organization of the basilar membrane (BM), which is still unclear in man. Novel techniques in cochlear implantation (CI) motivate further analyses of the BM. MATERIALS AND METHODS: Normal cochleae from patients undergoing removal of life-threatening petro-clival meningioma and an autopsy specimen from a normal human were used. Laser-confocal microscopy, high resolution scanning (SEM) and transmission electron microscopy (TEM) were carried out in combination. In addition, one human temporal bone was decellularized and investigated by SEM. RESULTS: The human BM consisted in four separate layers: (1) epithelial basement membrane positive for laminin-ß2 and collagen IV, (2) BM "proper" composed of radial fibers expressing collagen II and XI, (3) layer of collagen IV and (4) tympanic covering layer (TCL) expressing collagen IV, fibronectin and integrin. BM thickness varied both radially and longitudinally (mean 0.55-1.16 µm). BM was thinnest near the OHC region and laterally. CONCLUSIONS: There are several important similarities and differences between the morphology of the BM in humans and animals. Unlike in animals, it does not contain a distinct pars tecta (arcuate) and pectinata. Its width increases and thickness decreases as it travels apically in the cochlea. Findings show that the human BM is thinnest and probably most vibration-sensitive at the outer pillar feet/Deiter cells at the OHCs. The inner pillar and IHCs seem situated on a fairly rigid part of the BM. The gradient design of the BM suggests that its vulnerability increases apical wards when performing hearing preservation CI surgery.

Application of micro-computed tomography to microstructure studies of the medicinal fungus Hericium coralloides.

The potential of 3-D nondestructive imaging techniques such as micro-computed tomography (micro-CT) was evaluated to study morphological patterns of the potential medicinal fungus Hericium coralloides (Basidiomycota). Micro-CT results were correlated with histological information gained from scanning electron microscopy (SEM) and light microscopy (LM). It is demonstrated that the combination of these imaging methods results in a more distinct picture of the morphology of the edible and potentially medicinal Hericium coralloides basidiomata. In addition we have created 3-D reconstructions and visualizations based on micro-CT imagery from a randomly selected part of the upper region of a fresh H. coralloides basidioma: Analyses for the first time allowed an approximation of the evolutionary effectiveness of this bizarrely formed basidioma type in terms of the investment of tissue biomass and its reproductive output (production of basidiospores).

Hydrogen Surface Reactions and Adsorption Studied on Y2O3, YSZ, and ZrO2.

The surface reactivity of Y2O3, YSZ, and ZrO2 polycrystalline powder samples toward H2 has been comparatively studied by a pool of complementary experimental techniques, comprising volumetric methods (temperature-programmed volumetric adsorption/oxidation and thermal desorption spectrometry), spectroscopic techniques (in situ electric impedance and in situ Fourier-transform infrared spectroscopy), and eventually structural characterization methods (X-ray diffraction and scanning electron microscopy). Reduction has been observed on all three oxides to most likely follow a surface or near-surface-limited mechanism involving removal of surface OH-groups and associated formation of water without formation of a significant number of anionic oxygen vacancies. Partly reversible adsorption of H2 was proven on the basis of molecular H2 desorption. Dictated by the specific hydrophilicity of the oxide, readsorption of water eventually takes place. The inference of this surface-restricted mechanism is further corroborated by the fact that no bulk structural and/or morphological changes were observed upon reduction even at the highest reduction temperatures (1173 K). We anticipate relevant implications for the use of especially YSZ in fuel cell research, since in particular the chemical state and structure of the surface under typical reducing high-temperature conditions affects the operation of the entire cell.

Loss of syntaxin 3 causes variant microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a disorder of intestinal epithelial differentiation characterized by life-threatening intractable diarrhea. MVID can be diagnosed based on loss of microvilli, microvillus inclusions, and accumulation of subapical vesicles. Most patients with MVID have mutations in myosin Vb that cause defects in recycling of apical vesicles. Whole-exome sequencing of DNA from patients with variant MVID showed homozygous truncating mutations in syntaxin 3 (STX3). STX3 is an apical receptor involved in membrane fusion of apical vesicles in enterocytes. Patient-derived organoid cultures and overexpression of truncated STX3 in Caco-2 cells recapitulated most characteristics of variant MVID. We conclude that loss of STX3 function causes variant MVID.

Biological adhesion of the flatworm Macrostomum lignano relies on a duo-gland system and is mediated by a cell type-specific intermediate filament protein.

BACKGROUND: Free-living flatworms, in both marine and freshwater environments, are able to adhere to and release from a substrate several times within a second. This reversible adhesion relies on adhesive organs comprised of three cell types: an adhesive gland cell, a releasing gland cell, and an anchor cell, which is a modified epidermal cell responsible for structural support. However, nothing is currently known about the molecules that are involved in this adhesion process. RESULTS: In this study we present the detailed morphology of the adhesive organs of the free-living marine flatworm Macrostomum lignano. About 130 adhesive organs are located in a horse-shoe-shaped arc along the ventral side of the tail plate. Each organ consists of exactly three cells, an adhesive gland cell, a releasing gland cell, and an anchor cell. The necks of the two gland cells penetrate the anchor cell through a common pore. Modified microvilli of the anchor cell form a collar surrounding the necks of the adhesive- and releasing glands, jointly forming the papilla, the outer visible part of the adhesive organs. Next, we identified an intermediate filament (IF) gene, macif1, which is expressed in the anchor cells. RNA interference mediated knock-down resulted in the first experimentally induced non-adhesion phenotype in any marine animal. Specifically, the absence of intermediate filaments in the anchor cells led to papillae with open tips, a reduction of the cytoskeleton network, a decline in hemidesmosomal connections, and to shortened microvilli containing less actin. CONCLUSION: Our findings reveal an elaborate biological adhesion system in a free-living flatworm, which permits impressively rapid temporary adhesion-release performance in the marine environment. We demonstrate that the structural integrity of the supportive cell, the anchor cell, is essential for this adhesion process: the knock-down of the anchor cell-specific intermediate filament gene resulted in the inability of the animals to adhere. The RNAi mediated changes of the anchor cell morphology are comparable to situations observed in human gut epithelia. Therefore, our current findings and future investigations using this powerful flatworm model system might contribute to a better understanding of the function of intermediate filaments and their associated human diseases.

Microvillus inclusion disease: loss of Myosin vb disrupts intracellular traffic and cell polarity.

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by loss of apical microvilli and formation of cytoplasmic inclusions lined by microvilli in enterocytes. MVID is caused by mutations in the MYO5B gene, coding for the myosin Vb motor protein. Although myosin Vb is implicated in the organization of intracellular transport and cell surface polarity in epithelial cells, its precise role in the pathogenesis of MVID is unknown. We performed correlative immunohistochemistry analyses of sections from duodenal biopsies of a MVID patient, compound heterozygous for two novel MYO5B mutations, predicting loss of function of myosin Vb in duodenal enterocytes together with a stable MYO5B CaCo2 RNAi cell system. Our findings show that myosin Vb-deficient enterocytes display disruption of cell polarity as reflected by mislocalized apical and basolateral transporter proteins, altered distribution of certain endosomal/lysosomal constituents including Rab GTPases. Together, this severe disturbance of epithelial cell function could shed light on the pathology and symptoms of MVID.

Morphological differences in adolescent idiopathic scoliosis: a histological and ultrastructural investigation.

STUDY DESIGN: Histological and ultrastructural evaluation of cell morphologies at the concave and convex side of apical intervertebral discs (IVD) of adolescent idiopathic scoliosis (AIS). OBJECTIVE: To determine changes in cell morphology, viability, and cell death after asymmetric disc loading in AIS and to compare the findings with the tilt angles. SUMMARY OF BACKGROUND DATA: The reaction of cells to loading stimuli in the IVD seems to be specific. Although dynamic loads are more beneficial to the disc cells and maintain the matrix biosynthesis, static compressive loads suppress gene expression. METHODS: Apical IVDs (Th8-Th9 to L1-L2) from 10 patients with AIS were studied histologically (including TUNEL [TdT-mediated dUTP-biotin nick end labeling] staining to identify disc cell death by apoptosis) and ultrastructurally for matrix evaluations and to quantify healthy, balloon, chondroptotic, apoptotic, and necrotic cells on the concave and convex sides. Patients' spines were classified according to the Lenke classification. Degeneration was assessed according to the Pfirrmann grading system. Two groups were established; group 1 (G1) with a tilt of 5° to 9° and group 2 (G2) with a tilt of 10° to 19°. RESULTS: Balloon cells were found in significantly higher numbers at the concave side (G1-annulus fibrosus [AF]: mean 16%), with almost none found at the convex side. Mean numbers of healthy cells did not show differences comparing both sides. Significantly higher numbers of healthy cells were found with increasing tilt angle at the concave side. Necrosis (mean, 47%) increased toward the center of the disc but did not differ between the sides of the IVDs. The fibrils found in the outer AF on the convex side were 30% thinner. CONCLUSION: This study was able to show significant differences in cell morphologies in the AF on both sides and in correlation to the different tilt angles. The type and magnitude of load seem to influence disc cells. Further studies are required to provide more information on disc and cell changes in scoliosis.