bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Graft‐Host Response for Various Tissue Sources and Animal Models
Authors: WEBER COLLIN J. , SAFLEY SUSAN , HAGLER MARY , KAPP JUDITH
Published: February 2006

ABSTRACT: The efficacy of pancreatic islet transplants in correcting hyperglycemia and slowing the progression of complications in diabetics has been confirmed by many experimental and clinical studies. Unfortunately, the availability of human islets is extremely limited and, therefore, treatment of large numbers of human diabetic patients will almost certainly require either the use of islets harvested from animals (xenografts) or the use of insulin‐secreting genetically modified cells of either human or animal origin. There is currently no effective regimen which will allow long‐term survival of xenogeneic islets from widely unrelated donor‐recipient combinations, such as pig‐to‐rodent, pig‐to‐dog, or pig‐to‐primate. There is considerable interest in the development of immunoisolation techniques for protection of donor islets. However, most materials used in immunoisolation devices are relatively bio‐incompatible. Poly‐L‐lysine‐alginate microcapsules are biocompatible and provide an optimal geometry for transmembrane diffusion of insulin and nutrients. Microcapsules allow long‐term survival of xenogeneic islets in diabetic rodents or dogs with induced diabetes. However, mice and rats with spontaneous diabetes destroy encapsulated islet grafts within 2 to 3 weeks. Biopsies reveal large numbers of macrophages, immunoglobulins and limited numbers of helper and cytotoxic T‐cells in the peri‐microcapsule environment of the peritoneal cavity. Cytokines have been identified in peritoneal fluid from mice with islet grafts and may play a role in encapsulated islet destruction. Targeted immunomodulation by treatment of recipients with either anti‐helper T‐cell antibodies, or fusion proteins which block costimulatory interactions between antigen presenting cells and host T‐cells have demonstrated synergy in significant prolongation of encapsulated islet xenograft survival in NOD mice with spontaneous diabetes. Technical improvements in microcapsule design also have contributed to prolonged graft survival. “Double‐wall” microencapsulation provides a more durable microcapsule and islet pretreatment prior to encapsulation reduces the frequency of defective capsules with islets entrapped in the membrane. Long‐term durability of encapsulated islet grafts remains a concern and further improvements in microcapsule design are a prerequisite to clinical trials.
Genetically Engineered Pancreatic β‐Cell Lines for Cell Therapy of Diabetes
Authors: EFRAT SHIMON
Published: February 2006

ABSTRACT: The optimal treatment of insulin‐dependent diabetes mellitus (IDDM), which is caused by the autoimmune destruction of pancreatic islet β cells, would require the regulated delivery of insulin by transplantation of functional β cells. β‐cell transplantation has so far been restricted by the scarcity of human islet donors. This shortage would be alleviated by the development of differentiated β‐cell lines, which could provide an abundant and well‐characterized source of β cells for transplantation. Using conditional transformation approaches, our laboratory has generated continuous β‐cell lines from transgenic mice. These cells produce insulin amounts comparable to those of normal islets and release insulin in response to physiological stimuli. Cell replication in these β cells can be tightly controlled both in culture and in vivo, allowing regulation of cell number and cell differentiation. Another challenge to cell therapy of IDDM is the protection of transplanted cells from immunological rejection and recurring autoimmunity. By employing adenovirus genes which downregulate antigen presentation and increase cell resistance to cytokines, β‐cell transplantation across allogeneic barriers was achieved without immunosuppression. In principle, similar β‐cell lines can be derived from isolated human islets using viral vectors to deliver conditionally regulated transforming and immunomodulatory genes into β cells. The combination of these approaches with immunoisolation devices holds the promise of a widely available cell therapy for treatment of IDDM in the near future.
Re‐engineering the Functions of a Terminally Differentiated Epithelial Cell in Vivo
Authors: BAUM BRUCE J. , WANG SONGLIN , CUKIERMAN EDNA , DELPORTE CHRISTINE , KAGAMI HIDEAKI , MARMARY YITZHAK , FOX PHILIP C. , MOONEY DAVID J. , YAMADA KENNETH M.
Published: February 2006

ABSTRACT: Because of their easy access, and important role in oral homeostasis, mammalian salivary glands provide a unique site for addressing key issues and problems in tissue engineering. This manuscript reviews studies by us in three major directions involving re‐engineering functions of salivary epithelial cells. Using adenoviral‐mediated gene transfer in vivo, we show approaches to i) repair damaged, hypofunctional glands and ii) redesign secretory functions to include endocrine as well as exocrine pathways. The third series of studies show our general approach to develop an artificial salivary gland for clinical situations in which all glandular tissue has been lost.
Novel Bioartificial Liver Support System: Preclinical Evaluation
Authors: PATZER JOHN F. , MAZARIEGOS GEORGE V. , LOPEZ ROBERTO , MOLMENTI ERNESTO , GERBER DAVID , RIDDERVOLD FRIDTJOV , KHANNA AJAI , YIN WEN‐YAO , CHEN YONG , SCOTT VICTOR L. , AGGARWAL SHUSHMA , KRAMER DAVID J , WAGNER ROBERT A. , ZHU YUE , FULMER MELISSA L. , BLOCK GEOFFREY D. , AMIOT BRUCE P
Published: February 2006

ABSTRACT: Preclinical safety and efficacy evaluation of a novel bioartificial liver support system (BLSS) was conducted using a d‐galactosamine canine liver failure model. The BLSS houses a suspension of porcine hepatocytes in a hollow fiber cartridge with the hepatocytes on one side of the membrane and whole blood flowing on the other. Porcine hepatocytes harvested by a collagenase digestion technique were infused into the hollow fiber cartridge and incubated for 16 to 24 hours prior to use. Fifteen purpose‐bred male hounds, 1–3 years old, 25–30 kg, were administered a lethal dose, 1.5 g/kg, of d‐galactosamine. The animals were divided into three treatment groups: (1b) no BLSS treatment (
n = 6

); (2b) BLSS treatment starting at 24–26 h post d‐galactosamine (
n = 5

); and (2c) BLSS treatment starting at 16–18 h post d‐galactosamine (
n = 4

). While maintained under isoflurane anesthesia, canine supportive care was guided by electrolyte and invasive physiologic monitoring consisting of arterial pressure, central venous pressure, extradural intracranial pressure (ICP), pulmonary artery pressure, urinary catheter, and end‐tidal CO2. All animals were treated until death or death‐equivalent (inability to sustain systolic blood pressure > 80 mmHg for 20 minutes despite massive fluid resuscitation and/or dopamine administration), or euthanized at 60 hours. All animals developed evidence of liver failure at 12–24 hours as evidenced by blood pressure lability, elevated ICP, marked hepatocellular enzyme elevation with microscopic massive hepatocyte necrosis and cerebral edema, elevated prothrombin time, and metabolic acidosis. Groups 2b and 2c marginally prolong survival compared with Group 1b (pairwise log rank censored survival time analysis,
p= 0.096

and
p= 0.064

, respectively). Since survival times for Groups 2b and 2c are not significantly different (
p= 0.694

), the groups were combined for further statistical analysis. Survival times for the combined active treatment Groups 2b and 2c are significantly prolonged versus Group 1b (
p= 0.047
). These results suggest the novel BLSS reported here can have a significant impact on the course of liver failure in the d‐galactosamine canine liver failure model. The BLSS is ready for Phase I safety evaluation in a clinical setting.
Three‐dimensional in Vitro Cell Culture Leads to a Marked Upregulation of Cell Function in Human Hepatocyte Cell Lines‐an Important Tool for the Development of a Bioartificial Liver Machine
Authors: SELDEN CLARE , SHARIAT ALI , MCCLOSKEY PASCHAL , RYDER TIM , ROBERTS EVE , HODGSON HUMPHREY
Published: February 2006

ABSTRACT: Upregulating hepatocyte function in proliferating human liver cell lines could provide cells for a bio‐artificial liver. Ideally, a means of mimicking the biological extracellular matrix with a relatively inert, bio‐compatible matrix is required. Alginate encapsulation of primary hepatocytes is biocompatible. This study aimed to characterize cells grown in a 3D configuration in alginate. A human‐derived liver cell line encapsulated in 1% alginate was assessed for synthetic and detoxification functions. Secreted proteins measured (e.g., albumin, fibrinogen, α‐1‐antitrypsin etc.) were increased in alginate compared with monolayers. Cytochrome P450 1A1 activity increased three‐ to fourfold, whilst urea synthesis, undetectable in monolayer cultures, was synthesized by cells in alginate at levels approaching in vivo production. TEM revealed good ultrastructure reminiscent of normal hepatocytes. Alginate promotes 3D colonies of proliferating cells with upregulated liver functions. Rapid recovery of function of cryopreserved cells (<18h) provides added advantages for this system to support the biological component of an artificial liver for patients with fulminant hepatic failure.
Cultivation and Characterization of a New Immortalized Human Hepatocyte Cell Line, HepZ, for Use in an Artificial Liver Support System
Authors: WERNER A. , DUVAR S. , MÜTHING J. , BÜNTEMEYER H. , KAHMANN U. , LüNSDORF H. , LEHMANN J.
Published: February 2006

ABSTRACT: The new human hepatocyte cell line HepZ was investigated with regard to use it for a mass cell cultivation. The cells were originally derived from a human liver biopsy and immortalized through lipofectamine‐mediated transfection of albumin‐promotor‐regulated antisense constructions against the negative controlling cell cycle proteins Rb and p53 (pAlb asRb, pAlb asp53). Furthermore, plasmids including genes coding for the cellular transcription factor E2F and D1 cyclin (pCMV E2F, pSV2neo D1) were cotransfected to overcome the G1‐restriction point. Cell cultivation was performed in a 2‐liter bioreactor with a working volume of 1 liter. With CultiSpher G microcarriers used in a concentration of 3 g/l a maximal density of 7.1 3 106 cells/ml was achieved in a cultivation period of 20 days. The cells exhibited a maximal specific growth rate of 1.0 per day in the first 4 days. After 9 days of cultivation the stationary growth phase was reached with an average cell density of 5.5 3 106 cells/ml. The viability status of the culture was determined indirectly by measuring of the lactate dehydrogenase activity (LDH) at 378C. During the growth phase the activity rose slightly up to a value of 200 U/l. The cells were flat after first attachment on the gelatine microcarriers and spherical after growing into the three‐dimensional inner matrix‐both of which characteristics were verified by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The liver‐specific cytochrome P450 activity was challenged with a pulse of 7 mg/ml lidocaine at a cell density of 4.5 3 106 cells/ml. After an induction period of 3 days with 50 μg/ml of phenobarbital, 26 ng/ml MEGX were generated within one day compared to 5 ng/ml without induction. The new cell line HepZ has proven to retain liver‐specific qualities and to be appropriate for mass cell cultivation for bioartificial devices.
rhBMP‐Collagen Sponges as Osteoinductive Devices: Effects of in Vitro Sponge Characteristics and Protein pI on in Vivo rhBMP Pharmacokinetics
Authors: ULUDAG H. , FRIESS W. , WILLIAMS D. , PORTER T. , TIMONY G. , D'AUGUSTA D. , BLAKE C. , PALMER R. , BIRON B. , WOZNEY J.
Published: February 2006

ABSTRACT: Osteoinductive devices, comprised of biodegradable collagen scaffolds and recombinant human Bone Morphogenetic Proteins (rhBMPs), are being currently pursued for local bone induction. To better understand the biological performance of such devices, we have carried out a series of studies to investigate the effects of sponge properties and protein structural features on the pharmacokinetics of implanted rhBMPs. The results indicated little dependence of the rhBMP‐2 pharmacokinetics on the in vitro determined sponge properties. The protein isoelectric point (pI), on the other hand, was found to significantly affect the initial implant retention of rhBMPs, but not the subsequent pharmacokinetics. A 100‐fold difference in the implant‐retained dose could be observed depending on the type of rhBMP implanted. We conclude that protein structural features are important variables controlling in vivo pharmacokinetics of rhBMPs, and possibly the osteoinductive potency of the devices.
Computer Controlled Bioreactor for Large‐scale Production of Cultured Skin Grafts
Authors: PRENOSIL J. E. , KINO‐OKA M.
Published: February 2006

ABSTRACT: KERATOR‐an automated membrane bioreactor‐was developed to produce Autologous Wound Dressing (AWD) at significantly reduced cost and time of transplantation down to two weeks time. At the same time, the risk of human error is largely eliminated. The computer‐controlled reactor is modular, allowing the production of up to 0.5 m2 AWD at one time. A special feature of the reactor is a hydrophilic polymeric support membrane on which the human keratinocytes attach and proliferate. Recently developed serum‐free medium is used to culture keratinocytes as a monolayer without a feeder layer of murine fibroblasts. The use of composite skin grafts consisting of a subconfluent keratinocyte layer on a polymeric support film is a very promising method for skin transplantation owing to the high activity of non‐differentiated keratinocyte cells and reduction of the time needed to prepare the skin grafts. A microscopic video system with image analysis was developed for on‐line monitoring of the cell growth and morphology in the KERATOR. The computer uses the obtained information to control medium change and to predict the end of cultivation.
A Method for Tissue Engineering of Cartilage by Cell Seeding on Bioresorbable Scaffolds
Authors: SCHREIBER RONDA E. , DUNKELMAN NOUSHIN S. , NAUGHTON GAIL , RATCLIFFE ANTHONY
Published: February 2006

ABSTRACT: This chapter describes a method for the in vitro generation of a 3‐dimensional cartilage matrix from articular chondrocytes seeded onto a bioresorbable polymeric scaffold. This particular growth system was chosen for the subject of this chapter owing to the relative simplicity of the methods required and the ease with which the necessary materials can be obtained. The tissue produced using this protocol is a cellular, metabolically active hyaline‐like matrix, containing the major cartilage constituents: sulfated proteoglycan, collagen type II, and water. It serves as a useful in vitro tool for studying the influence of various mechanical and chemical factors on cartilage metabolism, as well as providing an implantable material for in vivo cartilage repair studies.
Historical Perspective of Defining Charcot‐Marie‐Tooth Type 1B
Authors: BIRD THOMAS D.
Published: February 2006

ABSTRACT: A single family (1521) with CMT has been followed for 36 years (1962‐1998) at Children's Hospital and the University of Washington in Seattle. The family was initially called peroneal muscular atrophy with severely slowed motor nerve conduction velocities (5–15 m/sec). In the late 1970s the family was part of several genetic studies of CMT and in 1980 represented linkage of CMT to the Duffy (Fy) locus on chromosome 1q. This finding was confirmed in an Indiana CMT family by Stebbins and Conneally (1982). This subtype of CMT was designated 1B. These investgations represented some of the last successful linkage studies in the now seemingly “ancient” pre‐DNA marker era. In 1993 Hayasaka and colleagues found a point mutation in the myelin P0 gene (Asp 90 Glu) in this family, giving CMT1B a molecular basis. The historical development of this “defining” of a neurogenetic disorder reveals interesting insights into the workings of clinical genetics over the past 3 decades.
Charcot‐Marie‐Tooth Disease Type 2
Authors: VANCE JEFFERY M.
Published: February 2006

ABSTRACT: No unique genes have yet been found for CMT2, but both Cx32 and P0 appear to contribute to the phenotype. Not surprisingly, CMT2 is likely to display much more genetic heterogeneity than CMT1. However, it is also likely continue to challenge previous concepts on classification and relationship of traditional inherited phenotypes in neurology. Future work on CMT2 should produce insight not only into the cellular interactions of the peripheral nerve especially Schwann cell and axon relationships, but also into idiopathic neuropathy.
Autosomal Recessive Hereditary Motor and Sensory Neuropathy with Focally Folded Myelin Sheaths (CMT4B)
Authors: GAMBARDELLA ANTONIO , BONO FRANCESCO , MUGLIA MARIELLA , VALENTINO PAOLA , QUATTRONE ALDO
Published: February 2006

ABSTRACT: Hereditary motor and sensory neuropathy with focally folded myelin sheaths, or Charcot‐Marie‐Tooth neuropathy type 4B (CMT4B), is a distinct clinical and genetic entity belonging to the heterogeneous group of autosomal recessive demyelinating neuropathies. We first described a large inbred pedigree with 10 patients affected by CMT4B, which enabled us to uncover the genetic findings, clinical spectrum, and natural history of such a disorder. The clinical picture was characterized by infantile onset with progressive symmetric distal and proximal muscular weakness. Using homozygosity mapping and haplotype sharing analysis, we found evidence of linkage to chromosome 11q23. We then identified a second unrelated family in which two individuals were affected with CMT4B. Although the clinical findings were similar to those previously reported, we excluded the disease locus segregating in this smaller pedigree from the 11q23 region. We thus provided evidence for a second locus causing the CMT4B phenotype. All these findings indicate that CMT4B seems to be phenotypically quite homogeneous, but is genetically heterogeneous.
Distal Hereditary Motor Neuropathy Type II (Distal HMN Type II): Phenotype and Molecular Genetics
Authors: TIMMERMAN V. , BEUTEN J. , IROBI J. , De JONGHE P. , MARTIN J.‐J. , VAN BROECKHOVEN C.
Published: February 2006

ABSTRACT: The distal hereditary motor neuropathies (distal HMN) are clinically and genetically heterogeneous and are subdivided in seven subtypes according to the mode of inheritance, age at onset and clinical evolution. We studied a multigenerational Belgian pedigree with autosomal dominant distal HMN type II. The clinical phenotype closely resembles classical Charcot‐Marie‐Tooth (CMT) disease with an age at onset between 15 and 25 years. Linkage studies have shown that distal HMN II is not linked to the known CMT1 and CMT2 loci. A genome‐wide search was performed and significant linkage was obtained between markers D12S86 and D12S340, suggesting that a gene causing distal HMN II is located on chromosome 12q24.3. The gene encoding the human pancreatic phospholipase A2 (PLA2A), which is expressed in peripheral nerves during degeneration, is a positional candidate gene. Because no disease‐specific mutations were detected in the coding region, however, PLA2A is most likely not the disease causing gene. A yeast artificial chromosome (YAC) contig map spanning the candidate region has been constructed to isolate the gene responsible for distal HMN II. Positional and functional candidate genes are currently being screened for the presence of mutations in distal HMN II patients.
Distal Hereditary Motor Neuronopathy of the Jerash Type
Authors: MIDDLETON L. T. , CHRISTODOULOU K. , MUBAIDIN A. , ZAMBA E. , TSINGIS M. , KYRIACOU K. , ABU‐SHEIKH S. , KYRIAKIDES T. , NEOCLEOUS V. , GEORGIOU D. M. , EL‐KHATEEB M. , AL‐QUDAN A. , HORANY K.
Published: February 2006

ABSTRACT: A novel form of autosomal recessive distal hereditary motor neuronopathy (distal HMN) is reported. The presence of pyramidal signs within the early stages of the disease with persistence of knee hyperreflexia form distinctive clinical features. We have mapped the HMN‐J gene to chromosome 9p21.1‐p12, within an estimated interval of 1.2‐Mb.
Why Do Schwann Cells Survive in the Absence of Axons?
Authors: JESSEN K. R. , MIRSKY R.
Published: February 2006

Schwann cell precursors in embryonic nerves rely for survival on signals from the axons they associate with. A major component of this signal is β neuregulin. While it can be argued that such paracrine axonal regulation makes biological sense in embryonic nerves, such an arrangement would be problematic postnatally, since nerve damage would then lead to Schwann cell death with adverse consequences for regeneration; in fact, transection of older nerves is not accompanied by a detectable increase in Schwann cell death. Our evidence indicates that this is, at least in part, due to the ability of Schwann cells to support their own survival by autocrine circuits. These circuits are not present in Schwann cell precursors. We have identified insulin‐like growth factor, neurotrophin‐3 and platelet‐derived growth factor‐BB as components of the autocrine Schwann cell survival signal.
P0‐Cre Transgenic Mice for Inactivation of Adhesion Molecules in Schwann Cells
Authors: FELTRI MARIA LAURA , D'ANTONIO MAURIZIO , PREVITALI STEFANO , FASOLINI MARINA , MESSING ALBEE , WRABETZ LAWRENCE
Published: February 2006

ABSTRACT: Normal peripheral nerve myelination depends on Schwann cell‐basal lamina interactions. An important component of Schwann cell basal lamina is laminin‐predominantly laminins 2 and 4. Mutations in the alpha 2 chain common to these two isoforms are associated with dysmyelination in mouse (dy) and man (congenital muscular dystrophy). Thus, laminin 2 and 4 receptors are also likely to be important for myelin formation. Several laminin 2/4 receptors are detected at the basal lamina surface of myelin‐forming Schwann cells, namely, α6β4 and α6β1 integrins and dystroglycan. The evidence linking these receptors to myelination is suggestive, but not conclusive. Genetic studies have not yet confirmed a role for these molecules in myelin formation. Natural or targeted inactivation of α6, β4, and β1 integrins and of dystroglycan have profound effects on other tissues causing embryonic or perinatal death before myelination. Therefore, to conditionally inactivate these receptors specifically in myelin‐forming Schwann cells, we have constructed and initially characterized a P0‐Cre transgene that activates Cre‐mediated recombination of loxP‐containing genes in peripheral nerve.
IGF‐I Promotes Peripheral Nervous System Myelination
Authors: CHENG HSIN‐LIN , RUSSELL JAMES W. , FELDMAN EVA L.
Published: February 2006

ABSTRACT: Insulin‐like growth factor‐I (IGF‐I) promotes the proliferation and differentiation of Schwann cells (SC). We use SC/dorsal root ganglion neuron (DRG) cocultures to examine the effects of IGF‐I on the interaction between axons and SC. As SC extend processes toward the axon in the presence of IGF‐I, these processes attach to and ensheath axons. Continued IGF‐I exposure leads to enhanced P0 expression and long‐term myelination. No myelination occurs in the absence of IGF‐I. These data imply that IGF‐I is critical not only for SC attachment and ensheathment of axons but also for long‐term myelination.
New Vistas on the Pathomechanism of Charcot‐Marie‐Tooth and Related Peripheral Neuropathies
Authors: MÜLLER H. W.
Published: February 2006

ABSTRACT: A gene‐dosage mechanism in CMT1A and HNPP has been postulated previously. Here, recent findings are discussed concerning (i) the functional consequences of altered PMP22 expression on Schwann cell growth regulation and on the capacity of genetically modified Schwann cells to myelinate peripheral axons, (ii) the cell physiological effects caused by the expression of certain disease‐related missense mutations of PMP22 that are known to alter the Schwann cell phenotype and impair myelination in vivo, and (iii) the pathomechanism of CMT1 in light of findings on a novel association between PMP22 and P0 in PNS myelin.
Ultrastructural Immunocytochemical Abnormalities of Peripheral Myelin Proteins in Hereditary Sensory‐Motor Neuropathies: 12 cases
Authors: ANANI THIERRY , SINDOU PHILIPPE , RICHARD LAURENCE , DIOT MARTINE , VALLAT JEAN‐MICHEL
Published: February 2006

ABSTRACT: Hereditary sensorimotor neuropathies form a heterogeneous group of genetically determined diseases, of which Charcot‐Marie‐Tooth (CMT) disease is the most common. In order to establish relations between genotype and the expression of peripheral myelin proteins, we carried out a quantitative study by ultrastructural immunocytochemistry of several myelin proteins (PMP22, P0, MBP) on sural nerve biopsy samples from 12 unrelated CMT patients. The diagnosis of CMT was based on the clinical, electrophysiological, and histological findings along with those of molecular biological studies. CMT X diagnoses were not included in this study. The expression of myelin proteins was well correlated with the molecular biological findings in these patients. The results also provided evidence for interference between different myelin proteins. Our findings are in line with the results from animal studies (trembler and knock‐out mice), which may provide insights into the pathogenesis of these human conditions.
Schwann Cell‐Derived Desert Hedgehog Signals Nerve Sheath Formation
Authors: MIRSKY RHONA , PARMANTIER ERIC , MCMAHON ANDREW P. , JESSEN KRISTJAN R.
Published: February 2006

ABSTRACT: Reciprocal signaling between axons and Schwann cells during development is well established. The contribution of Schwann cells to the formation and maintenance of the protective nerve sheaths (endo‐, peri‐, and epineurium) has been less studied. Although mesenchymal cells contribute to all these structures, only perineurial cells contribute to the diffusion barrier between nerves and surrounding tissues. During development, prospective perineurial cells shift from a mesenchymal to epithelial phenotype, forming concentric layers of cells around the nerve fascicles that collectively form a barrier against unwanted molecules and cellular infiltration. We have studied the role of Schwann cells in the formation and maintenance of this barrier. The signaling molecule Desert hedgehog is expressed in Schwann cell precursors, and in Schwann cells until at least postnatal day 10, while its receptor patched is seen in mesenchymal cells surrounding the developing nerve at embryo day 15. In Desert hedgehog knockout mice, the connective tissue sheaths in adult nerves appear highly abnormal by electron microscopy. There is almost no epineurium, and the perineurium is thin and highly abnormal. In addition, perineurial‐like cells invade the endoneurial space, forming mini‐fascicles around small bundles of nerve fibers similar to those seen in regenerating nerves. Functional tests reveal that the diffusion and cellular infiltration barrier is compromised, demonstrating that Desert hedgehog signaling from Schwann cells to the mesenchyme is involved in the formation of a morphologically and functionally normal perineurium.
Neurofibromin, the Neurofibromatosis Type 1 Ras‐GAP, Is Required for Appropriate P0 Expression and Myelination
Authors: ROSENBAUM THORSTEN , KIM HAESUN A. , BOISSY YING L. , LING BO , RATNER NANCY
Published: February 2006

ABSTRACT: The neurofibromatosis type 1 (NF1) gene product, neurofibromin, regulates activation of the Ras intracellular signaling pathway in Schwann cells. Schwann cells purified from mouse embryos with null mutations in the Nf1 gene increase expression of the major myelin glycoprotein P0. v‐Ras expression in cultured Schwann cells partially mimics loss of Nf1, suggesting a role for Ras in upregulation of P0 expression in Nf1‐deficient cells. We tested whether loss of Nf1 alters the ability of Schwann cells to form myelin. No significant changes in myelin formation resulted when Nf1‐deficient or v‐Ras‐expressing Schwann cells were cultured with normal neurons. Yet, in organotypic cultures of neurons, Schwann cells, and fibroblasts without neurofibromin, myelination was dramatically reduced. We suggest that Nf1‐dependent signaling cascades in neurons and/or fibroblasts, as well as Schwann cells, are required for normal myelination.
The Biology of Chronically Denervated Schwann Cells
Authors: HALL SUSAN M.
Published: February 2006

ABSTRACT: This paper is divided into two sections, both concerned with the effects of chronic denervation on Schwann cells. The first group of experiments explore the proposal that lengthy denervation produces changes in Schwann cells in vivo that compromise axonal regeneration. The second group of experiments compare the molecular phenotypes of acutely and chronically denervated Schwann cells in vitro, and examine their responses to an exogenously applied neuregulin.
Transgenic Mouse Models of CMT1A and HNPP
Authors: SUTER UELI , NAVE KLAUS‐ARMIN
Published: February 2006

ABSTRACT: We have generated several PMP22 animal mutants with altered PMP22 gene dosage. A moderate increase in the number of PMP22 genes led to hypomyelination comparable to CMT1A, whereas high copy numbers of transgenic PMP22 resulted in phenotypes resembling more severe forms of hereditary motor and sensory neuropathies. In contrast, eliminating one of the two normal PMP22 genes by gene targeting caused unstable focal hypermyelination (tomacula) similar to the pathology in HNPP. A related but more severe phenotype was observed in mice that lack PMP22 completely. Detailed analysis of the different PMP22 mutants revealed, in addition to the obvious myelinopathy, distal axonopathy as a characteristic feature. We conclude that the maintenance of axons might be a promising target for therapeutic interventions in these demyelinating hereditary neuropathies. Furthermore, our results strongly support the concept that PMP22‐related neuropathies (and most likely also other forms of inherited motor and sensory neuropathies) should be viewed as the consequence of impaired neuron‐Schwann cell interactions that are likely already to be operative during development. Such considerations should be taken into account in the design of potential novel treatment strategies.
The “CMT Rat”: Peripheral Neuropathy and Dysmyelination Caused by Transgenic Overexpression of PMP22
Authors: NIEMANN STEPHAN , SEREDA MICHAEL W. , ROSSNER MORITZ , STEWART HELEN , SUTER UELI , MEINCK HANS‐MICHAEL , GRIFFITHS IAN R. , NAVE KLAUS‐ARMIN
Published: February 2006

ABSTRACT: We have generated a transgenic rat model of Charcot‐Marie‐Tooth disease type 1A (CMT1A) providing formal proof that this neuropathy can be caused by increased expression of peripheral myelin protein‐22 (PMP22). Heterozygous PMP22‐transgenic rats develop muscle weakness and gait abnormalities as well as reduced nerve conduction velocities and EMG abnormalities, which closely resemble recordings in patients with CMT1A. Dys‐ and demyelination, Schwann cell hypertrophy, and “onion bulb” formation are also similar to findings in humans. When bred to homozygosity, transgenic rats completely fail to elaborate myelin, but all myelin‐forming Schwann cells segregate with axons in the normal one‐to‐one ratio. Although arrested at this “promyelin” stage, differentiation proceeds in homozygous rats at the molecular level, as demonstrated by high‐level expression of myelin structural genes. Intracellular trafficking of the wild‐type protein is not visibly impaired, even when strongly overexpressed, suggesting that PMP22 blocks myelin assembly in a late Golgi/cell membrane compartment of the affected Schwann cell.
The Absence of Myelin P0 Protein Produces a Novel Molecular Phenotype in Schwann Cell
Authors: MENICHELLA DANIELA MARIA , XU WENBO , JIANG HUIYUAN , SOHI JASLOVELEEN , VALLAT JEAN‐MICHAEL , BARON PIERLUIGI , KAMHOLZ JOHN , SHY MICHAEL
Published: February 2006

ABSTRACT: In order to better understand the pathogenesis of demyelination in P0 knockout (P0−/−) mice, we analyzed the myelin gene expression and the localization of myelin proteins in P0 null mouse sciatic nerve. We have demonstrated that the severe demyelinating neuropathy of P0‐knockout mouse is associated with changes in the program of myelin gene expression. Some changes in myelin gene expression occur early, others occur during adulthood. We also provide evidence that the absence of P0 is associated with changes in the localization of specific paranodal proteins in the peripheral nerve. These data suggest that P0 plays an important role, either directly or indirectly, in the program of Schwann cell gene expression and in the specific distribution of peripheral myelin proteins. Furthermore, myelin gene dysregulation and improper localization of paranodal proteins may account, in part, for the pathogenesis of demyelination in P0‐knockout mice, as well as in human demyelinating peripheral neuropathy associated with mutations in the P0 gene.
Nerve Conduction Abnormalities and Neuromyotonia in Genetically Engineered Mouse Models of Human Hereditary Neuropathies
Authors: ZIELASEK JÜRGEN , TOYKA KLAUS V.
Published: February 2006

ABSTRACT: We performed electrophysiological studies in myelin protein mutant mice in order to characterize nerve conduction changes. We performed neurographic studies on the facial and sciatic nerves and needle electromyography (EMG). Mice homozygously deficient for the peripheral myelin protein 22 gene (Pmp22−/−) exhibited increased motor latencies, reduced nerve conduction velocities, and polyphasia of the M‐response, which are the typical electrophysiological signs of dysmyelination. PMP22 +/− mice developed only mild conduction slowing at an old age and a mild reduction of the M‐amplitude, which indicates mild axonal dysfunction. Mice overexpressing Pmp22 developed severe electrophysiological signs of dysmyelination. In myelin protein zero‐deficient mice (P0−/−), we found alterations similar to those found in Pmp22−/− mice, whereas P0+/− mice developed mildly increased sciatic nerve F‐wave latencies only late in life, which indicates only mild dysmyelination. Connexin 32‐deficient mice showed electrophysiological evidence of mild axonal damage. By EMG, we found the clinical and electrophysiological signs of neuromyotonia, that is, continuous spontaneous motor unit discharges, often in rhythmic patterns (myokymia), in P0−/−, Pmp22−/−, Trembler, Trembler‐J, and Pmp22‐overexpressing mice. This indicates abnormal impulse generation in these dysmyelinated nerves. In summary, our studies demonstrate nerve conduction changes in mice with myelin protein gene defects that are similar to those found in patients with Charcot‐Marie‐Tooth disorders. In addition, we identified new mouse models of hereditary neuromyotonia.
Peripheral Neuropathy Caused by Proteolipid Protein Gene Mutations
Authors: GARBERN JAMES Y. , CAMBI FRANCA , LEWIS RICHARD , SHY MICHAEL , SIMA ANDERS , KRAFT GEORGE , VALLAT J. M. , BOSCH E. P. , HODES M. E. , DLOUHY STEPHEN , RASKIND WENDY , BIRD THOMAS , MACKLIN WENDY , KAMHOLZ JOHN
Published: February 2006

ABSTRACT: Pelizaeus‐Merzbacher disease (PMD) is a dysmyelinating disorder of the central nervous system typically caused by duplications or missense mutations of the proteolipid protein (PLP) gene. Most investigators have found that peripheral nerve function and structure is normal in PMD patients. We have found that null mutations of the PLP gene cause demyelinating peripheral neuropathy, whereas duplications and a proline 14 to leucine mutation do not affect nerve function. A family with a nonsense mutation at position 144, which affects only PLP but not the alternatively spliced gene product DM20, has a very mild syndrome, including normal peripheral nerve function. Our findings suggest that DM20 alone is sufficient to maintain normal nerve function and that there may be domains of PLP/DM20 that have a relatively more active role in the peripheral nervous system compared with that in the central nervous system.
Genotype/Phenotype Correlations in X‐Linked Dominant Charcot‐Marie‐Tooth Disease
Authors: HAHN ANGELIKA F. , BOLTON CHARLES F. , WHITE CHRISTOPHER M. , BROWN WILLIAM F. , TUUHA SASCHA E. , TAN CHARLES C. , AINSWORTH PETER J.
Published: February 2006

ABSTRACT: We have studied the relationship between genotype, clinical phenotype, and pathology in 13 families with dominant X‐linked Charcot‐Marie‐Tooth (CMT) neuropathy. Connexin32 (Cx32) gene mutations were spread throughout the coding region and included eight missense mutations, one 8‐bp deletion/4‐bp insertion frame shifting mutation, two nonsense mutations, and one deletion of the entire coding sequence. One hundred sixteen affected CMTX patients (53 males and 63 females) and 63 unaffected, at‐risk individuals were compared by neurological and electrophysiological examinations and analyzed by gender; nerve biopsies were available from seven index cases. It was found that mutations within all regions of the Cx32 gene coding sequence caused an identical clinical phenotype. Male CMTX patients were affected more severely and showed an age‐dependent progression of clinical signs and of the pathology; there was, however, variability in the severity of disease expression, irrespective of age, among males within families of defined genotype. All but 10% of female CMTX patients had only mild signs. Motor nerve conduction velocities were moderately slowed (median nerve MNCV: males 34.5 ± 6.2 m/sec; females 45.8 ± 7.3 m/sec), and motor and sensory nerve amplitudes were reduced (median nerve CMAP: males 3.7 ± 3.7 mV; females 7.8 ± 3.4 mV), with electromyographic evidence of chronic denervation. Differences were significant between gender and between affected and unaffected individuals. In agreement with the electrophysiological observations, pathological studies showed evidence of paranodal demyelination and of a length‐related axonal degeneration in motor and sensory nerve fibers. Correlations between genotype and clinical phenotype suggested that missense mutations located within the second transmembrane domain and/or cytoplasmic loop might be associated with milder clinical phenotype, and therefore might be less disruptive of Connexin32 gap junction function. Missense, chain‐terminating, or deletion mutations in all other locations of the Connexin32 protein caused severe forms of CMTX and disease onset in the first decade. Observed variability of disease severity among males within kinships suggests the influence of other modifying factors.
Mutation Testing in Charcot‐Marie‐Tooth Neuropathy
Authors: NICHOLSON GARTH A.
Published: February 2006

ABSTRACT: In order to determine the optimal approach for mutation testing in the form of Charcot‐Marie‐Tooth (CMT) neuropathy, consecutive patients with a CMT phenotype, available family history information on at least first‐degree relatives, and median motor conduction velocities of less than 50 m/sec were tested for the CMT1A duplication and for connexin32, peripheral myelin protein 22 (PMP22) and myelin protein zero (P0) point mutations. A cutoff value for median motor conduction velocity of less than 50 m/sec was adopted to include all CMTX families.

All of the connexin32 mutations, except for one sporadic case, were found by first selecting families with no male‐to‐male inheritance of CMT and neurophysiological indicators of CMTX. All PMP22 and P0 mutations were found by selecting Dejerine‐Sottas cases or dominantly inherited CMT1 with a very severe phenotype. It is concluded that “blind” testing of CMT1 families for connexin32, P0, and PMP22 mutations is of limited value.
Molecular Mechanisms of Cell Death in the Ear
Authors: WATERS CATHERINE
Published: February 2006

ABSTRACT: Damage to sensory cells and neurones of the inner ear, induced by ototoxic drugs, physical stimulation, or even normal aging, occurs by mechanisms that are not yet fully elucidated. However, for the individual the consequent loss of hearing can be devastating. Thus the concept that, in some instances, this brain cell death may be preventable has provided an exciting and novel route to thwart the degenerative process. Signals that trigger cell death can be as diverse as the removal of essential growth factors, damage by exogenous toxins (including ototoxic drugs), free radicals, and excitotoxins. An important facet to the newly discovered death cascade is that it can be halted, and such interventions may rescue the dying cell. The question now remains whether any of the cell death observed on ototoxic or noise‐induced hearing loss occurs by an “active” programmed mechanism (apoptosis), as physiological cell death does in the developing ear, and if so, whether it can be prevented.
Calpain Inhibitors as Therapeutic Agents in Nerve and Muscle Degeneration
Authors: STRACHER ALFRED
Published: February 2006

ABSTRACT: It seems plausible to hypothesize that in all forms of neurodegeneration or other forms of tissue degeneration, a common pathway exists that, when deciphered, could lead to our understanding of a variety of diseases that result in tissue necrosis, as well as offer potential for therapeutic intervention. In recent years progress toward elucidating this common pathway has been accelerated through the studies of a number of laboratories, including our own, on the role of the protease calpain in this process. Thus, in a variety of disorders, such as stroke, spinal cord injury, traumatic nerve injury, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, muscular dystrophy, cataract formation, unregulated calpain proteolysis, initiated via dysregulation of calcium ion homeostasis, participates in the pathogenesis and is a potentially unifying mechanistic event.

In order to demonstrate the feasibility of the approach we have taken in using the calpain inhibitor leupeptin as a therapeutic agent, I will describe two areas of research in which we have been engaged over the past 20 years. One is our long‐standing interest in muscular dystrophy. The other is of more recent vintage, and involves the use of calpain inhibitors to protect sensory hair cells and spiral ganglion neurons from damage associated with acoustic trauma, this latter in collaboration with Dr. R. Salvi at SUNY‐Buffalo and Dr. A. Shulman at SUNY‐Downstate.
Acute and Chronic Effects of Aminoglycosides on Cochlear Hair Cells
Authors: ARAN JEAN‐MARIE , ERRE JEAN‐PAUL , DA COSTA DEISE LIMA , DEBBARH IKRAM , DULON DIDIER
Published: February 2006

ABSTRACT: The first detectable effect on the auditory system after a single high‐dose injection of an aminoglycosidic antibiotic (AA) like gentamicin (GM) is the reversible blockade of medial efferent function, probably via blockade of calcium channels at the base of the outer hair cells (OHC). The kinetics of this effect are compatible with that of the molecule in perilymph. In the course of chronic treatment with lower doses, however, ototoxicity develops only after several days of treatment. Still GM can be observed inside the OHCs as soon as 24 hours after the first injection, and will be still present in some OHCs as long as 11 months after a chronic, nonototoxic 6‐day treatment.

In vitro, the short‐term viability of isolated OHCs is not affected by exposure to AAs, but their transduction channels and their response to acetylcholine are reversibly blocked. However, developing organs of Corti in culture are highly and rapidly affected by exposure to AAs. Yet during direct intracochlear perilymphatic perfusion of GM, 2‐mM solutions are not ototoxic, and with perfusion with a 20‐mM solution ototoxicity develops only after several days of perfusion.

From these various observations one can describe some aspects of the mechanisms of ototoxicity of AAs, from their access to perilymph and endolymph, to penetration in the hair cells, likely via endocytosis at their apical pole, and intracellular cytotoxic events.
Normal and Altered Cytoarchitecture of the Inner Ear
Authors: SERRA A. , GRASSO D. L. , COCUZZA S. , TROMBETTA L. , LA MANTIA I.
Published: February 2006

ABSTRACT: Scanning electron microscope gave new insights into real morphology of the inner ear and has enhanced basic research. In fact, during the last twenty years, the cellular system that composes the organ of Corti and the vestibular apparatus has been clearly described by many authors. We now have a better understanding of each cell type and that they have a physiological functioning as ear and vestibular system.

In addition, new insights into the damage caused by many drugs, such as aminoglucosides, can be investigated by studying the morphological changes and the reparative process following the injury. Animals such as guinea pigs can be used as ideal models to investigate the alterations in the sensory epithelium of the cochlear and veastibular receptors following ototoxic poisoning.
An Animal Model Based on the Sprague Dawley Rat for the Evaluation of Ototoxicity
Authors: MARTINI A. , HATZOPOULOS S. , RUBINI R. , DI STEFANO M. , ALBERTIN A. , GOVONI E.
Published: February 2006

ABSTRACT: Various methodological approaches that can be used to detect ototoxic effects caused by the administration of various substances are presented, using the Sprague‐Dawley rat as an animal model. Electrophysiological data are also presented to show how the model behaves with potentially ototoxic (hyaluronic acid) and initially inert (hydroxy‐propyl‐methyl‐cellulose) substances.
Evaluation of Cisplatin Ototoxicity in a Rat Animal Model
Authors: HATZOPOULOS S. , DI STEFANO M. , ALBERTIN A. , MARTINI A.
Published: February 2006

ABSTRACT: The ototoxic effects of cisplatin were evaluated by otoacoustic emissions and evoked auditory responses. A transient otoacoustic emissions protocol indicated no significant ototoxic effects in rats treated intravenously with 7.5 mg/kg/week for 2‐weeks. A chronic 6‐week treatment (2.5 mg/kg/week) monitored by 2F1‐F2 distortion product emissions presented significant SNR alterations in a narrow range of frequencies (5.04–5.66 kHz). An acute treatment of 15 mg/kg, using slow 30‐min intraperitoneal infusion, presented the highest DP and ABR alterations. The SNR at the 2F1‐F2 frequencies 6.34, 7.13, and 7.56 kHz was found significantly decreased, and ABR latency measurements from 8‐kHz burst stimuli verified these alterations.
Excitotoxicity, Synaptic Repair, and Functional Recovery in the Mammalian Cochlea: A Review of Recent Findings
Authors: PUJOL RÉMY , PUEL JEAN‐LUC
Published: February 2006

ABSTRACT: Besides its fast excitatory properties, glutamate is known to have neurotoxic properties when released in large amounts or when incompletely recycled. This so‐called excitotoxicity is involved in a number of acute and/or degenerative forms of neuropathology such as epilepsy, Alzheimer's, Parkinson's, stroke, and retinal ischemia. In the cochlea, excitotoxicity may occur in two pathological conditions: anoxia and noise trauma. It is characterized by a two‐step mechanism: (1) An acute swelling, which primarily depends on the AMPA/kainate type of receptors, together with a disruption of the postsynaptic structures (type I afferent dendrites) resulting in a loss of function. Within the next 5 days, synaptic repair may be observed with a full or a partial (acoustic trauma) recovery of cochlear potentials. (2) The second phase of excitotoxicity, which may develop after strong and/or repetitive injury, consists of a cascade of metabolic events triggered by the entry of Ca2+, which leads to neuronal death in the spiral ganglion. Ongoing experiments in animals, tracking the molecular basis of both these processes, presages the development of new pharmacological strategies to help neurites to regrow and reconnect properly to the IHCs, and to prevent or delay neuronal death in the spiral ganglion. Human applications should follow, and a local (transtympanic) strategy against cochlear excitotoxicity may, in the near future, prove to be helpful in ischemic‐ or noise‐induced sudden deafness, as well as in the related tinnitus.