Publications by authors named "Kristian Pfaller"

58 Publications

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

J Anat 2018 06 27;232(6):908-918. Epub 2018 Feb 27.

Department of Trauma Surgery, Medical University Innsbruck, Innsbruck, Austria.

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.
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http://dx.doi.org/10.1111/joa.12795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979627PMC
June 2018

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

Biochim Biophys Acta Biomembr 2018 May 6;1860(5):1152-1161. Epub 2018 Jan 6.

Institute of General Physiology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.

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.
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http://dx.doi.org/10.1016/j.bbamem.2018.01.010DOI Listing
May 2018

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

Ups J Med Sci 2017 Aug 17;122(3):160-170. Epub 2017 May 17.

a Department of Surgical Sciences, Head and Neck Surgery, Section of Otolaryngology, Department of Otolaryngology , Uppsala University Hospital , Uppsala , Sweden.

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.
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http://dx.doi.org/10.1080/03009734.2017.1322645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5649321PMC
August 2017

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

Ups J Med Sci 2017 Mar 1;122(1):11-19. Epub 2017 Feb 1.

g Department of Histology and Molecular Cell Biology , Institute of Anatomy and Histology, Medical University of Innsbruck , Innsbruck , Austria.

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.
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http://dx.doi.org/10.1080/03009734.2016.1271843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5361427PMC
March 2017

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

PLoS One 2016 15;11(9):e0163160. Epub 2016 Sep 15.

Institute of Botany, University of Innsbruck, Innsbruck, Austria.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163160PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025027PMC
August 2017

Towards understanding microvillus inclusion disease.

Mol Cell Pediatr 2016 Dec 29;3(1). Epub 2016 Jan 29.

Department of Paediatrics I, Medical University of Innsbruck, Anichstrasse 35, 6020, Innsbruck, Austria.

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.
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http://dx.doi.org/10.1186/s40348-016-0031-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733813PMC
December 2016

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

Front Cell Neurosci 2015 23;9:454. Epub 2015 Nov 23.

Division of Neurobiochemistry, Biocenter, Innsbruck Medical University Innsbruck, Austria.

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.
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http://dx.doi.org/10.3389/fncel.2015.00454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655273PMC
December 2015

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

Eur Spine J 2016 Jan 19;25(1):80-95. Epub 2015 Jul 19.

Department of Traumatology and Sports Medicine, Sankt Vinzenz Krankenhaus Zams, Zams, Austria.

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.
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http://dx.doi.org/10.1007/s00586-015-4089-5DOI Listing
January 2016

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

J Infect Dis 2015 Oct 25;212(7):1140-9. Epub 2015 Mar 25.

Division of Hygiene and Medical Microbiology.

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 β-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.
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http://dx.doi.org/10.1093/infdis/jiv191DOI Listing
October 2015

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

Cell Tissue Res 2015 May 7;360(2):245-62. Epub 2015 Feb 7.

Department of Surgical Sciences, Head and Neck Surgery, section of Otolaryngology, Uppsala University Hospital, 751 85, Uppsala, Sweden,

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-β2 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.
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http://dx.doi.org/10.1007/s00441-014-2098-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4412841PMC
May 2015

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

Mycologia 2015 Jan-Feb;107(1):227-38. Epub 2014 Nov 6.

Institute of Microbiology, Leopold-Franzens University, Technikerstraβe 25, 6020 Innsbruck, Austria.

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).
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http://dx.doi.org/10.3852/14-188DOI Listing
March 2015

The viral make-up makes a world of difference.

AIDS Res Hum Retroviruses 2014 Jul;30(7):642-3

1 Division of Hygiene and Medical Microbiology, Innsbruck Medical University , Innsbruck, Austria .

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http://dx.doi.org/10.1089/aid.2014.0061DOI Listing
July 2014

Hydrogen Surface Reactions and Adsorption Studied on YO, YSZ, and ZrO

J Phys Chem C Nanomater Interfaces 2014 Apr 2;118(16):8435-8444. Epub 2014 Apr 2.

Institute of Physical Chemistry, University of Innsbruck , Innrain 52a, A-6020 Innsbruck, Austria.

The surface reactivity of YO, YSZ, and ZrO polycrystalline powder samples toward H 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 H was proven on the basis of molecular H 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.
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http://dx.doi.org/10.1021/jp5008472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4001439PMC
April 2014

Loss of syntaxin 3 causes variant microvillus inclusion disease.

Gastroenterology 2014 Jul 12;147(1):65-68.e10. Epub 2014 Apr 12.

Division of Pediatrics, Department of Pediatric Gastroenterology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1053/j.gastro.2014.04.002DOI Listing
July 2014

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

Front Zool 2014 Feb 12;11(1):12. Epub 2014 Feb 12.

Institute of Zoology and Center of Molecular Bioscience Innsbruck, University of Innsbruck, Technikerstr, 25, Innsbruck A-6020, Austria.

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.
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http://dx.doi.org/10.1186/1742-9994-11-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4016567PMC
February 2014

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

Traffic 2014 Jan 19;15(1):22-42. Epub 2013 Nov 19.

Division of Cell Biology, Biocenter Innsbruck, Medical University Innsbruck, Innsbruck, Austria; Division of Pathology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Canada.

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.
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http://dx.doi.org/10.1111/tra.12131DOI Listing
January 2014

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

Spine (Phila Pa 1976) 2013 Sep;38(19):1672-80

*Department of Traumatology †Division of Histology and Embryology, and ‡Department for Medical Statistics, Informatics and Health Economics, Medical University of Innsbruck, Austria §Department of Traumatology and Sports Medicine, Sankt Vinzenz Krankenhaus Zams, Austria; and ¶German Scoliosis Center, Werner-Wicker-Clinic, Bad Wildungen, Germany.

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.
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http://dx.doi.org/10.1097/BRS.0b013e31829e0956DOI Listing
September 2013

Virulence and thrombocyte affectation of two Aspergillus terreus isolates differing in amphotericin B susceptibility.

Med Microbiol Immunol 2013 Oct 31;202(5):379-89. Epub 2013 May 31.

Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Fritz-Pregl-Straße 3, 6020, Innsbruck, Austria.

Aspergillus terreus-induced invasive infections exhibit high lethality, partly due to the intrinsic resistance for amphotericin B (AmB). We compared the virulence and pathogenesis of an AmB-resistant isolate of A. terreus (ATR) with that of a rare variant showing enhanced sensitivity for AMB (ATS). The modifications that result in enhanced AmB sensitivity of isolates are not associated with reduced virulence in vivo; instead, the ATS-infected mice died even faster than the ATR-infected animals. Since A. terreus enters the blood stream in most patients and frequently induces thrombosis, we studied a putative correlation between virulence of the two A. terreus isolates and their effect on thrombocytes. Those mice infected with the more virulent ATS isolate had lower thrombocyte numbers and more phosphatidylserine exposure on platelets than ATR-infected mice. In vitro experiments confirmed that ATS and ATR differ in their effect on thrombocytes. Conidia, aleurioconidia and hyphae of ATS were more potent than ATR to trigger thrombocyte stimulation, and thrombocytes adhered better to ATS than to ATR fungal structures. Furthermore, ATS secreted more soluble factors that triggered platelet stimulation than ATR. Thus, it might be suggested that the capacity of a fungal isolate to modulate thrombocyte parameters contributes to its virulence in vivo.
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http://dx.doi.org/10.1007/s00430-013-0300-7DOI Listing
October 2013

Cryo-immunoelectron microscopy of adherent cells improved by the use of electrospun cell culture substrates.

Traffic 2013 Aug 23;14(8):886-94. Epub 2013 May 23.

Department of Therapeutic Radiology and Oncology, Innsbruck Medical University, Anichstrasse 35, A-6020, Innsbruck, Austria.

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non-disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute-long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze-substitution, sample rehydration and cryosection-immunolabelling or with freeze-fracture replica-immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.
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http://dx.doi.org/10.1111/tra.12080DOI Listing
August 2013

Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth) families.

PLoS One 2012 27;7(12):e52278. Epub 2012 Dec 27.

Department Biologie II, Ludwig-Maximilians-University, Munich, Germany.

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052278PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531485PMC
July 2013

Human cochlea: anatomical characteristics and their relevance for cochlear implantation.

Anat Rec (Hoboken) 2012 Nov 8;295(11):1791-811. Epub 2012 Oct 8.

Department of Otolaryngology, Uppsala University Hospital, 75185 Uppsala, Sweden.

This is a review of the anatomical characteristics of human cochlea and the importance of variations in this anatomy to the process of cochlear implantation (CI). Studies of the human cochlea are essential to better comprehend the physiology and pathology of man's hearing. The human cochlea is difficult to explore due to its vulnerability and bordering capsule. Inner ear tissue undergoes quick autolytic changes making investigations of autopsy material difficult, even though excellent results have been presented over time. Important issues today are novel inner ear therapies including CI and new approaches for inner ear pharmacological treatments. Inner ear surgery is now a reality, and technical advancements in the design of electrode arrays and surgical approaches allow preservation of remaining structure/function in most cases. Surgeons should aim to conserve cochlear structures for future potential stem cell and gene therapies. Renewal interest of round window approaches necessitates further acquaintance of this complex anatomy and its variations. Rough cochleostomy drilling at the intricate "hook" region can generate intracochlear bone-dust-inducing fibrosis and new bone formation, which could negatively influence auditory nerve responses at a later time point. Here, we present macro- and microanatomic investigations of the human cochlea viewing the extensive anatomic variations that influence electrode insertion. In addition, electron microscopic (TEM and SEM) and immunohistochemical results, based on specimens removed at surgeries for life-threatening petroclival meningioma and some well-preserved postmortal tissues, are displayed. These give us new information about structure as well as protein and molecular expression in man. Our aim was not to formulate a complete description of the complex human anatomy but to focus on aspects clinically relevant for electric stimulation, predominantly, the sensory targets, and how surgical atraumaticity best could be reached.
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http://dx.doi.org/10.1002/ar.22599DOI Listing
November 2012

Morphological changes in disc herniation in the lower cervical spine: an ultrastructural study.

Eur Spine J 2012 Jul 10;21(7):1396-409. Epub 2012 Mar 10.

Department of Traumatology, Medical University of Innsbruck, Anichstr 35, 6020 Innsbruck, Austria.

Introduction: The basis of disc degeneration is still unknown, but is believed to be a cell-mediated process. Apoptosis might play a major role in degenerative disc disease (DDD). The aim of this study was to correlate the viability of disc cells with the radiological degeneration grades (rDG) in disc herniation.

Materials And Methods: Forty anterior IVD's (C4-C7) from 39 patients with DDD were studied histologically and ultrastructurally to quantify healthy, "balloon", chondroptotic, apoptotic and necrotic cells. Patients were classified to their rDG, as having either prolapse (P: DGII + III) and/or osteochondrosis (O: DGIV + V). Similar studies were undertaken on eight control discs.

Results: Cell death by necrosis (mean 35%) was common but differed not significantly in both groups. All patients with a disc prolapse DGII + III revealed balloon cells (iAF: mean 32%). All appeared alive and sometimes were hypertrophic. However, significantly less balloon cells were found in the O-Group. Control samples revealed no evidence of "balloon" cells in DGII and only a minor rate in DGIII.

Conclusion: According to the different rDG, quantitative changes were obvious in healthy and "balloon" cells, but not for cell death. At the moment it can only be hypothesized if "balloon" cells are part of a repair strategy and/or cause of disc herniation.
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http://dx.doi.org/10.1007/s00586-012-2212-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3389114PMC
July 2012

Evaluation of MBEC™-HTP biofilm model for studies of implant associated infections.

J Orthop Res 2012 Jul 6;30(7):1176-80. Epub 2012 Jan 6.

Experimental Orthopaedics, Medical University Innsbruck, Innsbruck, Austria.

The bacteria in implant-related infections can evade host defenses by forming biofilms. The more we understand biofilm behavior, the better we can fight against then clinically. In vitro models for biofilms allow tests simulating in vivo conditions. In this study we evaluated the Minimum Biofilm Eradication Concentration-High Throughput Plates (MBEC™-HTP) as biofilm in vitro model for studies of implant associated infections. Staphylococcus aureus and Staphylococcus epidermidis biofilms were grown on MBEC™-HTP. To ensure the biofilm formation, antibiotic susceptibility tests and scanning electron microscopy (SEM) was carried out. Susceptibility tests were carried out using gentamicin, vancomycin, rifampicin, fosfomycin, clindamycin, and linezolid. Colony forming units counting were carried out. Minimal inhibitory concentration (MIC) and biofilm inhibitory concentration (BIC) were estimated. The CFU counting showed potency of rifampicin and daptomycin against S. epidermidis biofilms and rifampicin against S. aureus biofilms. SEM images showed proteic material in contact with cells. The differences between BIC and MIC demonstrated the biofilm formation as well as the SEM images. Rifampicin and daptomycin are good choices against biofilm related infections. Moreover, after suggested modifications, the model used in this study is eligible to further studies of implant associated infections.
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http://dx.doi.org/10.1002/jor.22065DOI Listing
July 2012

Complement activating antibodies to myelin oligodendrocyte glycoprotein in neuromyelitis optica and related disorders.

J Neuroinflammation 2011 Dec 28;8:184. Epub 2011 Dec 28.

Clinical Department of Neurology, Innsbruck Medical University, Innsbruck, Austria.

Background: Serum autoantibodies against the water channel aquaporin-4 (AQP4) are important diagnostic biomarkers and pathogenic factors for neuromyelitis optica (NMO). However, AQP4-IgG are absent in 5-40% of all NMO patients and the target of the autoimmune response in these patients is unknown. Since recent studies indicate that autoimmune responses to myelin oligodendrocyte glycoprotein (MOG) can induce an NMO-like disease in experimental animal models, we speculate that MOG might be an autoantigen in AQP4-IgG seronegative NMO. Although high-titer autoantibodies to human native MOG were mainly detected in a subgroup of pediatric acute disseminated encephalomyelitis (ADEM) and multiple sclerosis (MS) patients, their role in NMO and High-risk NMO (HR-NMO; recurrent optic neuritis-rON or longitudinally extensive transverse myelitis-LETM) remains unresolved.

Results: We analyzed patients with definite NMO (n = 45), HR-NMO (n = 53), ADEM (n = 33), clinically isolated syndromes presenting with myelitis or optic neuritis (CIS, n = 32), MS (n = 71) and controls (n = 101; 24 other neurological diseases-OND, 27 systemic lupus erythematosus-SLE and 50 healthy subjects) for serum IgG to MOG and AQP4. Furthermore, we investigated whether these antibodies can mediate complement dependent cytotoxicity (CDC). AQP4-IgG was found in patients with NMO (n = 43, 96%), HR-NMO (n = 32, 60%) and in one CIS patient (3%), but was absent in ADEM, MS and controls. High-titer MOG-IgG was found in patients with ADEM (n = 14, 42%), NMO (n = 3, 7%), HR-NMO (n = 7, 13%, 5 rON and 2 LETM), CIS (n = 2, 6%), MS (n = 2, 3%) and controls (n = 3, 3%, two SLE and one OND). Two of the three MOG-IgG positive NMO patients and all seven MOG-IgG positive HR-NMO patients were negative for AQP4-IgG. Thus, MOG-IgG were found in both AQP4-IgG seronegative NMO patients and seven of 21 (33%) AQP4-IgG negative HR-NMO patients. Antibodies to MOG and AQP4 were predominantly of the IgG1 subtype, and were able to mediate CDC at high-titer levels.

Conclusions: We could show for the first time that a subset of AQP4-IgG seronegative patients with NMO and HR-NMO exhibit a MOG-IgG mediated immune response, whereas MOG is not a target antigen in cases with an AQP4-directed humoral immune response.
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http://dx.doi.org/10.1186/1742-2094-8-184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278385PMC
December 2011

Cyclin-dependent kinase 16/PCTAIRE kinase 1 is activated by cyclin Y and is essential for spermatogenesis.

Mol Cell Biol 2012 Feb 19;32(4):868-79. Epub 2011 Dec 19.

Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Innsbruck, Austria.

Cyclin-dependent kinase 16 (CDK16, PCTK1) is a poorly characterized protein kinase, highly expressed in the testis and the brain. Here, we report that CDK16 is activated by membrane-associated cyclin Y (CCNY). Treatment of transfected human cells with the protein kinase A (PKA) activator forskolin blocked, while kinase inhibition promoted, CCNY-dependent targeting of CDK16-green fluorescent protein (GFP) to the cell membrane. CCNY binding to CDK16 required a region upstream of the kinase domain and was found to be inhibited by phosphorylation of serine 153, a potential PKA phosphorylation site. Thus, in contrast to other CDKs, CDK16 is regulated by phosphorylation-controlled cyclin binding. CDK16 isolated from murine testis was unphosphorylated, interacted with CCNY, and exhibited kinase activity. To investigate the function of CDK16 in vivo, we established a conditional knockout allele. Mice lacking CDK16 developed normally, but male mice were infertile. Spermatozoa isolated from their epididymis displayed thinning and elongation of the annulus region, adopted a bent shape, and showed impaired motility. Moreover, CDK16-deficient spermatozoa had malformed heads and excess residual cytoplasm, suggesting a role of CDK16 in spermiation. Thus, CDK16 is a membrane-targeted CDK essential for spermatogenesis.
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http://dx.doi.org/10.1128/MCB.06261-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272973PMC
February 2012

Nogo-A expression in the brain of mice with cerebral malaria.

PLoS One 2011 29;6(9):e25728. Epub 2011 Sep 29.

Department of Neurology, Innsbruck Medical University, Innsbruck, Austria.

Cerebral malaria (CM) is associated with a high rate of transient or persistent neurological sequelae. Nogo-A, a protein that is highly expressed in the endoplasmic reticulum (ER) of the mammalian central nervous system (CNS), is involved in neuronal regeneration and synaptic plasticity in the injured CNS. The current study investigates the role of Nogo-A in the course of experimental CM. C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. Brain homogenates of mice with different clinical severity levels of CM, infected animals without CM and control animals were analyzed for Nogo-A up-regulation by Western blotting and immunohistochemistry. Brain regions with Nogo-A upregulation were evaluated by transmission electron microscopy. Densitometric analysis of Western blots yielded a statistically significant upregulation of Nogo-A in mice showing moderate to severe CM. The number of neurons and oligodendrocytes positive for Nogo-A did not differ significantly between the studied groups. However, mice with severe CM showed a significantly higher number of cells with intense Nogo-A staining in the brain stem. In this region ultrastructural alterations of the ER were regularly observed. Nogo-A is upregulated during the early course of experimental CM. In the brain stem of severely affected animals increased Nogo-A expression and ultrastructural changes of the ER were observed. These data indicate a role of Nogo-A in neuronal stress response during experimental CM.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025728PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183069PMC
January 2012

Gliotoxin as putative virulence factor and immunotherapeutic target in a cell culture model of cerebral aspergillosis.

Mol Immunol 2011 Sep 30;48(15-16):2122-9. Epub 2011 Jul 30.

Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Innsbruck, Austria.

The mycotoxin gliotoxin is an important metabolite produced by Aspergillus fumigatus, but its precise role in the pathogenesis of cerebral aspergillosis is not yet determined. We could demonstrate that growth in cerebrospinal fluid (CSF) induced the production and secretion of significant amounts of gliotoxin by A. fumigatus. These concentrations of 590-720nM were sufficient to reduce the viability of astrocytes and neurons, as well as of primary microglia, already after few hours of incubation. Annexin staining and electron microscopy revealed the induction of apoptosis rather than necrosis as the relevant mode of gliotoxin action in the brain. Furthermore, even a low gliotoxin concentration of 100nM, which was subtoxic for astrocytes, was able to significantly down-modulate the phagocytic capacity of astrocytes. In order to improve the current antimycotic therapy of cerebral aspergillosis by supporting innate immunity in the fight against Aspergillus, we aimed to neutralize the toxic potency of gliotoxin towards different brain cell types. Compounds such as dithiothreitol (DTT) or glutathione that reduce the internal disulfide bond of gliotoxin were shown here to be able to interfere with the gliotoxin-induced decrease of cell viability and to save the cells from induction of apoptosis. Thus, exploration of these substances may lead to novel approaches for adjunctive treatment of cerebral aspergillosis.
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http://dx.doi.org/10.1016/j.molimm.2011.07.005DOI Listing
September 2011

Non-toxic cadmium concentrations induce vascular inflammation and promote atherosclerosis.

Circ J 2011 28;75(10):2491-5. Epub 2011 Jul 28.

Department of Neurology, Innsbruck Medical University, Innsbruck, Austria.

Background: Cadmium is a potential new risk factor for early atherosclerosis and cardiovascular diseases in humans, yet pathogenetic mechanisms are still a matter of debate.

Methods And Results: In-depth histological analysis of 18 sections taken from 6 cadmium-fed ApoE-/- mice and 12 sections from 5 litter-mates not exposed to cadmium by light and scanning electron microscopy was performed. Cadmium-fed mice showed a marked increase in lesion load (plaque area) and severity as classified according to the American Heart Association vascular lesion grading. All inflammatory markers studied (CD68, CD3, CD25, vascular cell adhesion molecule 1 (VCAM-1), and heat shock protein 60 (Hsp60)) yielded a higher expression in cadmium-fed mice. Statistical difference was achieved for VCAM-1 and Hsp60 (P=0.03 and P=0.02). The shoulder region of atherosclerotic plaques in cadmium-fed mice showed a prominent retraction of endothelial cells on electron microscopy.

Conclusions: Our data indicate that cadmium exposure amplifies the development of vessel pathology in atherosclerosis susceptible ApoE-/- mice and suggests upregulation of VCAM-1 and Hsp60 and endothelial leakage as potential pathomechanisms.
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http://dx.doi.org/10.1253/circj.cj-11-0196DOI Listing
March 2012

SidL, an Aspergillus fumigatus transacetylase involved in biosynthesis of the siderophores ferricrocin and hydroxyferricrocin.

Appl Environ Microbiol 2011 Jul 27;77(14):4959-66. Epub 2011 May 27.

Division of Molecular Biology, Biocenter, Fritz-Pregl-Str. 3, Innsbruck Medical University, A-6020 Innsbruck, Austria.

The opportunistic fungal pathogen Aspergillus fumigatus produces four types of siderophores, low-molecular-mass iron chelators: it excretes fusarinine C (FsC) and triacetylfusarinine C (TAFC) for iron uptake and accumulates ferricrocin (FC) for hyphal and hydroxyferricrocin (HFC) for conidial iron distribution and storage. Siderophore biosynthesis has recently been shown to be crucial for fungal virulence. Here we identified a new component of the fungal siderophore biosynthetic machinery: AFUA_1G04450, termed SidL. SidL is conserved only in siderophore-producing ascomycetes and shows similarity to transacylases involved in bacterial siderophore biosynthesis and the N(5)-hydroxyornithine:anhydromevalonyl coenzyme A-N(5)-transacylase SidF, which is essential for TAFC biosynthesis. Inactivation of SidL in A. fumigatus decreased FC biosynthesis during iron starvation and completely blocked FC biosynthesis during iron-replete growth. In agreement with these findings, SidL deficiency blocked conidial accumulation of FC-derived HFC under iron-replete conditions, which delayed germination and decreased the size of conidia and their resistance to oxidative stress. Remarkably, the sidL gene is not clustered with other siderophore-biosynthetic genes, and its expression is not affected by iron availability. Tagging of SidL with enhanced green fluorescent protein suggested a cytosolic localization of the FC-biosynthetic machinery. Taken together, these data suggest that SidL is a constitutively active N(5)-hydroxyornithine-acetylase required for FC biosynthesis, in particular under iron-replete conditions. Moreover, this study revealed the unexpected complexity of siderophore biosynthesis, indicating the existence of an additional, iron-repressed N(5)-hydroxyornithine-acetylase.
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http://dx.doi.org/10.1128/AEM.00182-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147410PMC
July 2011

Development of articular cartilage and the metaphyseal growth plate: the localization of TRAP cells, VEGF, and endostatin.

J Anat 2011 Jun 3;218(6):608-18. Epub 2011 Apr 3.

Division of Clinical and Functional Anatomy, Department of Anatomy, Histology and Embryology, Innsbruck Medical University, Innsbruck, Austria.

During long bone development the original cartilaginous model in mammals is replaced by bone, but at the long bone endings the avascular articular cartilage remains. Before the articular cartilage attains structural maturity it undergoes reorganization, and molecules such as vascular endothelial growth factor (VEGF) and endostatin could be involved in this process. VEGF attracts blood vessels, whereas endostatin blocks their formation. The present study therefore focused on the spatio-temporal localization of these two molecules during the development of the articular cartilage. Furthermore, we investigated the distribution of the chondro/osteoclasts at the chondro-osseous junction of the articular cartilage with the subchondral bone. Mice served as our animal model, and we examined several postnatal stages of the femur starting with week (W) 4. Our results indicated that during the formation of the articular cartilage, VEGF and endostatin had an overlapping localization. The former molecule was, however, down-regulated, whereas the latter was uniformly intensely localized until W12. At the chondro-osseous junction, the number of tartrate-resistant acid phosphatase (TRAP)-positive chondro/osteoclasts declined with increasing age. Until W3 the articular cartilage was not well organized but at W8 it appeared structurally mature. At that time only a few TRAP cells were present, indicative of a low resorptive activity at the chondro-osseous junction. Subsequently, we examined the metaphyseal growth plate that is closed when skeletal maturity is attained. Within its hypertrophic zone, localization of endostatin and VEGF was observed until W6 and W8, respectively. At the chondro-osseous junction of the growth plate, chondro/osteoclasts remained numerous until W12 to allow for its complete resorption. According to former findings, VEGF is critical for a normal skeleton development, whereas endostatin has almost no effect on this process. In conclusion, our findings suggest that both VEGF and endostatin play a role in the structural reorganization of the articular cartilage and endostatin may be involved in the maintenance of its avascularity. In the growth plate, however, endostatin does not appear to counteract VEGF, allowing vascular invasion of hypertrophic cartilage and bone growth.
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http://dx.doi.org/10.1111/j.1469-7580.2011.01377.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125894PMC
June 2011