bluuebunny/crossref_metadata_2025 huggingface dataset:
IFNα-stimulated neutrophils and monocytes release a soluble form of TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) displaying apoptotic activity on leukemic cells
Authors: Tecchio Cristina , Huber Veronica , Scapini Patrizia , Calzetti Federica , Margotto Daniela , Todeschini Giuseppe , Pilla Lorenzo , Martinelli Giovanni , Pizzolo Giovanni , Rivoltini Licia , Cassatella Marco A.
Published: January 2004

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily exerting cytotoxic activities toward tumor cells. Herein, we demonstrate that therapeutic concentrations of interferon α (IFNα) stimulate the expression of high levels of TRAIL mRNA and the release of elevated amounts of a soluble bioactive form of TRAIL (sTRAIL) in both human neutrophils and monocytes. Supernatants harvested from IFNα-treated neutrophils/monocytes elicited, on TRAIL-sensitive leukemic cell lines, proapoptotic activities that were significantly reduced by either a combination of TRAIL-R1/Fc and TRAIL-R2/Fc chimeras or neutralizing anti-TRAIL, anti–TRAIL-R1, and anti–TRAIL-R2 antibodies, suggesting that they were mediated by released sTRAIL acting on both TRAIL receptors. Since diseases such as chronic myeloid leukemia (CML) and melanoma are effectively treated with IFNα,we also demonstrate that CML neutrophils and peripheral blood mononuclear cells (PBMCs) cultured with IFNα at therapeutic concentrations retain the capacity of releasing sTRAIL, suggesting that CML leukocytes, in vivo, might represent an important source of sTRAIL. In this regard, we show that sTRAIL serum levels as well as leukocyte-associated TRAIL significantly increase in melanoma patients following IFNα administration. Collectively, these findings indicate that sTRAIL released by IFNα-activated neutrophils and monocytes contributes not only to the immunoregulatory actions but also to the therapeutic activities of IFNα.
Hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation: donor type matters
Authors: El-Zimaity Maha , Saliba Rima , Chan Kawah , Shahjahan Munir , Carrasco Antonio , Khorshid Ola , Caldera Humberto , Couriel Daniel , Giralt Sergio , Khouri Issa , Ippoliti Cindy , Champlin Richard , de Lima Marcos
Published: January 2004

Hemorrhagic cystitis (HC) remains a common complication of allogeneic blood and marrow transplantation. Previous analyses of risk factors for this complication were performed in heterogenous populations, with dissimilar diagnosis and conditioning regimens. We postulated that HC is more prevalent in matched unrelated donor (MUD) and unrelated cord blood (UCB) transplantations than in matched related donor (MRD) transplantations. We performed a retrospective study on 105 acute lymphocytic leukemia patients treated with 12 Gy total body irradiation-based regimens and allogeneic transplants (MUD, n = 38; UCB, n = 15; mismatched related, n = 20; MRD, n = 32). HC occurred in 16% of patients receiving MRD transplants, 30% of recipients of mismatched related, and 40% of MUD or UCB transplants (hazard ratio 2.9, 95% CI 1.0-7.9 for the comparison of MRD versus MUD). The excessive rate of HC among MUD and UCB patients became evident after the first 30 days after transplantation. Recipients younger than 26 years had a significantly higher incidence of HC (HR 2.5, 95% CI 1.1-5.8). This donor type and age effect was independent of platelet engraftment, development of graft-versus-host disease (GVHD), source of stem cells, use of anti-thymocyte globulin (ATG) or cyclophosphamide in the regimen, steroid use, or stem cell source. We concluded that HC is more prevalent in MUD and UCB transplantations. (Blood. 2004;103:4674-4680)
Adult bone marrow-derived stem cells use R-cadherin to target sites of neovascularization in the developing retina
Authors: Dorrell Michael I. , Otani Atsushi , Aguilar Edith , Moreno Stacey K. , Friedlander Martin
Published: January 2004

Adult bone marrow contains a population of hematopoietic stem cells (HSCs) that can give rise to cells capable of targeting sites of neovascularization in the peripheral or retinal vasculature. However, relatively little is known about the mechanism of targeting of these cells to sites of neovascularization. We have analyzed subpopulations of HSCs for the expression of a variety of cell surface adhesion molecules and found that R-cadherin, a calcium-dependent cell-cell adhesion molecule important for normal retinal endothelial cell guidance, was preferentially expressed by functionally targeting HSCs. Preincubation of HSCs with function-blocking anti-R-cadherin antibodies or novel R-cadherin-specific peptide antagonists effectively prevented targeting of bone marrow-derived cells to the developing retinal vasculature in vivo. Whereas control-injected HSCs targeted to all 3 normal developing retinal vascular layers, blocking R-cadherin-mediated adhesion resulted in mistargeting of the HSCs to the normally avascular outer retina. Our results suggest that vascular targeting of bone marrow-derived HSCs is dependent on mechanisms similar to those used by endogenous retinal vascular endothelial cells. Thus, R-cadherin antagonists may be useful in the treatment of neovascular diseases in which circulating HSCs contribute to abnormal angiogenesis. (Blood. 2004;103:3420-3427)
Signaling through GP Ib-IX-V activates αIIbβ3 independently of other receptors
Authors: Kasirer-Friede Ana , Cozzi Maria Rita , Mazzucato Mario , De Marco Luigi , Ruggeri Zaverio M. , Shattil Sanford J.
Published: January 2004

Platelet adhesion to von Willebrand factor (VWF) activates αIIbβ3, a prerequisite for thrombus formation. However, it is unclear whether the primary VWF receptor, glycoprotein (GP) Ib-IX-V, mediates αIIbβ3 activation directly or through other signaling proteins physically associated with it (eg, FcR γ-chain), possibly with the contribution of other agonist receptors and of VWF signaling through αIIbβ3. To resolve this question, human and GP Ibα transgenic mouse platelets were plated on dimeric VWF A1 domain (dA1VWF), which engages only GP Ib-IX-V, in the presence of inhibitors of other agonist receptors. Platelet adhesion to dA1VWF induced Src kinase-dependent tyrosine phosphorylation of the FcR γ-chain and the adapter molecule, ADAP, and triggered intracellular Ca2+ oscillations and αIIbβ3 activation. Inhibition of Ca2+ oscillations with BAPTA-AM prevented αIIbβ3 activation but not tyrosine phosphorylation. Pharmacologic inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI 3-kinase) prevented αIIbβ3 activation but not Ca2+ oscillations. Inhibition of Src with 2 distinct compounds blocked all responses downstream of GP Ib-IX-V under static or flow conditions. However, dA1VWF-induced responses were reduced only slightly in GP Ibα transgenic platelets lacking FcR γ-chain. These data establish that GP Ib-IX-V itself can signal to activate αIIbβ3, through sequential actions of Src kinases, Ca2+ oscillations, and PI 3-kinase/PKC. (Blood. 2004;103:3403-3411)
TEL-AML1 promotes development of specific hematopoietic lineages consistent with preleukemic activity
Authors: Morrow Michelle , Horton Sarah , Kioussis Dimitris , Brady Hugh J. M. , Williams Owen
Published: January 2004

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis, fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro, preferentially promoting B-lymphocyte development, enhancing self-renewal of B-cell precursors, and leading to the establishment of long-term growth factor–dependent pre–B-cell lines. However, it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineage-specific activity in vivo. TEL-AML1–expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis, TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion.
Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia
Authors: Smith B. Douglas , Levis Mark , Beran Miloslav , Giles Francis , Kantarjian Hagop , Berg Karin , Murphy Kathleen M. , Dauses Tianna , Allebach Jeffrey , Small Donald
Published: January 2004

Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in approximately 30% of patients with de novo acute myeloid leukemia (AML) and are associated with lower cure rates from standard chemotherapy-based treatment. Targeting the mutation by inhibiting the tyrosine kinase activity of FLT3 is cytotoxic to cell lines and primary AML cells harboring FLT3 mutations. Successful FLT3 inhibition can also improve survival in mouse models of FLT3-activated leukemia. CEP-701 is an orally available, novel, receptor tyrosine kinase inhibitor that selectively inhibits FLT3 autophosphorylation. We undertook a phase 1/2 trial to determine the in vivo hematologic effects of single-agent CEP-701 as salvage treatment for patients with refractory, relapsed, or poor-risk AML expressing FLT3-activating mutations. Fourteen heavily pretreated AML patients were treated with CEP-701 at an initial dose of 60 mg orally twice daily. CEP-701–related toxicities were minimal. Five patients had clinical evidence of biologic activity and measurable clinical response, including significant reductions in bone marrow and peripheral blood blasts. Laboratory data confirmed that clinical responses correlated with sustained FLT3 inhibition to CEP-701. Our results show that FLT3 inhibition is associated with clinical activity in AML patients harboring FLT3-activating mutations and indicate that CEP-701 holds promise as a novel, molecularly targeted therapy for this disease.
Occult hypoperfusion is associated with increased mortality in hemodynamically stable, high-risk, surgical patients
Authors: Meregalli André , Oliveira Roselaine P , Friedman Gilberto
Published: January 2004

Background

Our aim was to examine whether serial blood lactate levels could be used as predictors of outcome.

Methods

We prospectively studied 44 high-risk, hemodynamically stable, surgical patients. Blood lactate values, mean arterial pressure, heart rate and urine output were obtained at patient admission to the study, at 12, 24 and 48 hours.

Results

The nonsurvivors (n = 7) had similar blood lactate levels initially (3.1 ± 2.3 mmol/l versus 2.2 ± 1.0 mmol/l, P = not significant [NS]), but had higher levels after 12 hours (2.9 ± 1.7 mmol/l versus 1.6 ± 0.9 mmol/l, P = 0.012), after 24 hours (2.1 ± 0.6 mmol/l versus 1.5 ± 0.7 mmol/l, P = NS) and after 48 hours (2.7 ± 1.8 mmol/l versus 1.9 ± 1.4 mmol/l, P = NS) as compared with the survivors (n = 37). Arterial bicarbonate concentrations increased significantly in survivors and were higher than in nonsurvivors after 24 hours (22.9 ± 5.2 mEq/l versus 16.7 ± 3.9 mEq/l, P = 0.01) and after 48 hours (23.1 ± 4.1 mEq/l versus 17.6 ± 7.1 mEq/l, P = NS). The PaO2/FiO2 ratio was higher in survivors initially (334 ± 121 mmHg versus 241 ± 133 mmHg, P = 0.03) and remained elevated for 48 hours. There were no significant differences in mean arterial pressure, heart rate, and arterial blood oxygenation at any time between survivors and nonsurvivors. The intensive care unit stay (40 ± 42 hours versus 142 ± 143 hours, P < 0.001) and the hospital stay (12 ± 11 days versus 24 ± 17 days, P = 0.022) were longer for nonsurvivors than for survivors. The Simplified Acute Physiology Score II score was higher for nonsurvivors than for survivors (34 ± 9 versus 25 ± 14, P = NS). The urine output was slightly lower in the nonsurvivor group (P = NS). The areas under the receiving operating characteristic curves were larger for initial values of Simplified Acute Physiology Score II and blood lactate for predicting death.

Conclusion

Elevated blood lactate levels are associated with a higher mortality rate and postoperative complications in hemodynamically stable surgical patients.
Regulation of TLR signaling and inflammation by SOCS family proteins
Authors: Yoshimura Akihiko , Ohishi Hiroyuki Mori Masanobu , Aki Daisuke , Hanada Toshikatsu
Published: January 2004

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins and interferons. These cytokines exert their biological functions through Janus tyrosine kinases and signal transducer and activator of transcription factors. The cytokine-inducible Src homology 2 protein (CIS) and suppressors of cytokine signaling (SOCS) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine responses, including those that regulate the inflammatory systems. In this short review, we focused on the molecular mechanism of the action of CIS/SOCS family proteins and their roles in Toll-like receptor signal regulation and inflammatory diseases.
The mast cell: an antenna of the microenvironment that directs the immune response
Authors: Frossi Barbara , De Carli Marco , Pucillo Carlo
Published: January 2004

Mast cells (MCs) have long been considered as critical effector cells during immunoglobulin (Ig)E-mediated allergic disease and immune response to parasites. Recent studies, however, suggest that this understanding of MC function is incomplete and does not consider the complex roles that MCs play in adaptive and innate immunity. The added function gives an innovative vision of regulation of immune responses and the development of autoimmune diseases. It had been assumed that the aggregation of Fc ɛ receptor I with IgE and specific antigen is the main stimulus able to induce the MC activation, degranulation, release, and generation of mediators of the allergic reaction. However, MCs exhibit an array of molecules involved in cell–cell and cell–extracellular matrix adhesion, mediating delivery of costimulatory signals that empower those cells with an ability to react to multiple nonspecific and specific stimuli. Their tissue distribution and their capability to release many cytokines after stimulation indicate MCs as potential regulatory linkers between innate and acquired immunity. In this review, we will summarize some findings on the roles of MCs in innate and acquired immunity, on the molecular mechanism and signaling pathways, and on selective signals that induce discrete MC response and its ability to polarize adaptive-immune response.
PTX3 function as an opsonin for the dectin-1-dependent internalization of zymosan by macrophages
Authors: Diniz S N , Nomizo R , Cisalpino P S , Teixeira M M , Brown G D , Mantovani A , Gordon S , Reis L F L , Dias A A M
Published: January 2004

Pentraxin 3 (PTX3) is a tumor necrosis factor and interleukin-1β-stimulated gene that encodes a long PTX with proinflammatory activity. Here, we show that peritoneal macrophages derived from PTX3 transgenic (Tg) mice express higher levels of PTX3 mRNA than macrophages from wild-type (WT) mice, at basal level as well as upon stimulation with zymosan (Zy). Macrophages from Tg mice also showed improved opsonin-independent phagocytosis of Zy particles and the yeast form of the fungus Paracoccidioides brasiliensis. In the case of P. brasiliensis, an enhanced microbicidal activity accompanied by higher production of nitric oxide was also observed in macrophages from Tg mice. Using fluorescein-activated cell sorter analysis and reverse transcriptase-polymerase chain reaction, we demonstrated that basal level of Toll-like receptor-6 and Zy-induced dectin-1 expression was slightly but consistently higher in macrophages from Tg mice than in macrophages from WT mice. Recombinant (r)PTX3 protein binds to Zy particles as well as to yeast cells of P. brasiliensis and addition of rPTX3, to a culture of WT-derived macrophages containing Zy leads to an increase in the phagocytic index, which parallels that of Tg-derived macrophages, demonstrating the opsonin-like activity of PTX3. It is important that blockade of dectin-1 receptor inhibited the phagocytosis of Zy particles by WT and PTX3 Tg macrophages, pointing out the relevant role of dectin-1 as the main receptor involved in Zy uptake. Our results provide evidence for a role of PTX3 as an important component of the innate-immune response and as part of the host mechanisms that control fungal recognition and phagocytosis.
Leukocyte fluid shear response in the presence of glucocorticoid
Authors: Fukuda Shunichi , Mitsuoka Hiroshi , Schmid-Schönbein Geert W
Published: January 2004

Leukocytes respond to physiological fluid shear stress (∼1.5 dyn/cm2) by cytoplasmic reorganization. The cytoplasm is also influenced, however, by glucocorticoids. In this study, we explore how glucocorticoids may affect the leukocyte fluid shear response. Normal leukocytes, exposed to fluid shear in vitro during active migration, retract pseudopods accompanied by modestly decreasing intracellular calcium ions. In contrast, dexamethasone (DX)-treated leukocytes project pseudopods after shear exposure with a significant rise in intracellular calcium ions, an effect that can be blocked by voltage-dependent calcium channel blockers. Although a cyclic adenine monophosphate analog blocks calcium influx and pseudopod projection by DX, inhibition of A-kinase induces reversal of the shear response, as seen with DX treatment. DX also reverses the leukocyte shear response in vivo in the rat circulation. Leukocytes that adhere to the endothelium in postcapillary venules of control rats return into the circulation only after pseudopod retraction, and in DX-treated rats, adherent leukocytes return into the circulation still with projecting pseudopods. The fraction of circulating leukocytes with pseudopods in DX-treated rats is higher than in controls. Thus, the reversal of leukocyte shear response by glucocorticoids may contribute to an enhanced incidence of circulating leukocytes with pseudopods, a process that affects the kinetics of these cells in the microcirculation.
Controlling the Toll road to dendritic cell polarization
Authors: Mazzoni Alessandra , Segal David M
Published: January 2004

The activation of dendritic cells (DC) via Toll-like receptors (TLRs) plays a decisive role in shaping the outcome of primary immune responses. Following TLR engagement by microbial products, DC migrate from peripheral tissues to lymphoid organs and up-regulate major histocompatibility complex and costimulatory molecules, acquiring the unique capacity to prime pathogen-specific, naïve T cells. In addition, DC determine the character of the ensuing immune response by secreting cytokines that drive the development of T cells into T helper cell type 1 (Th1), Th2, or T regulatory effector cells. Three major factors influence the pattern of cytokines released by DC and accordingly, the Th balance: the lineage to which DC belong; the maturation stimulus; and inflammatory mediators present at the site of infection. A major focus of this review is the capacity of DC to integrate these factors and elicit distinct classes of immune responses.
Neurophysiology of the Neuromuscular Junction: Overview
Authors: RUFF ROBERT L.
Published: January 2004

Abstract: This manuscript reviews the physiology of neuromuscular transmission with emphasis on four features: (1) the structure of the neuromuscular junction, (2) release of vesicles of acetylcholine from the nerve terminal, (3) the roles of postsynaptic Na+ channels and acetylcholine receptors in converting the chemical signal from the nerve terminal into a propagated action potential on the muscle fiber, and (4) the safety factor for neuromuscular transmission. It also introduces new information about how the neuromuscular junction maintains physiological integrity during muscle fiber stretch and contraction.
Ca2+ Channels and Synaptic Transmission at the Adult, Neonatal, and P/Q‐Type Deficient Neuromuscular Junction
Authors: NUDLER SILVANA , PIRIZ JOAQUIN , URBANO FRANCISCO J. , ROSATO‐SIRI MARCELO D. , RENTERIA ERIKA S. PIEDRAS , UCHITEL OSVALDO D.
Published: January 2004

Abstract: Different types of voltage‐activated Ca2+ channels have been established based on their molecular structure and pharmacological and biophysical properties. One of them, the P/Q‐type, is the main channel involved in nerve‐evoked neurotransmitter release at neuromuscular junctions and the immunological target in Eaton‐Lambert Syndrome. At adult neuromuscular junctions, L‐ and N‐type Ca2+ channels become involved in transmitter release only under certain experimental or pathological conditions. In contrast, at neonatal rat neuromuscular junctions, nerve‐evoked synaptic transmission depends jointly on both N‐ and P/Q‐type channels. Synaptic transmission at neuromuscular junctions of the ataxic P/Q‐type Ca2+ channel knockout mice is also dependent on two different types of channels, N‐ and R‐type. At both neonatal and P/Q knockout junctions, the K+‐evoked increase in miniature endplate potential frequency was not affected by N‐type channel blockers, but strongly reduced by both P/Q‐ and R‐type channel blockers. These differences could be accounted for by a differential location of the channels at the release site, being either P/Q‐ or R‐type Ca2+ channels located closer to the release site than N‐type Ca2+ channels. Thus, Ca2+ channels may be recruited to mediate neurotransmitter release where P/Q‐type channels seem to be the most suited type of Ca2+ channel to mediate exocytosis at neuromuscular junctions.
Effect of Inherited Abnormalities of Calcium Regulation on Human Neuromuscular Transmission
Authors: MASELLI RICARDO A. , BOOKS WENDY , DUNNE VANESSA
Published: January 2004

Abstract: Synaptotagmins are abundant synaptic proteins that represent the best candidate for the calcium sensor at the nerve terminal. The pore‐forming, voltage‐sensing transmembrane alpha‐1 subunit of the P/Q voltage‐gated calcium channel (or Cav2.1) encoded by the CACNA1A gene is another major component of the process of action potential‐evoked exocytosis at the adult mammalian neuromuscular junction. Defects of these proteins, in nonhuman species, result in severe disruption of rapid synaptic transmission. This paper investigates the molecular bases of inherited presynaptic deficits of neuromuscular transmission in humans. Patients with congenital presynaptic failure, including two patients with episodic ataxia type 2 (EA‐2) due to CACNA1A mutations, were studied with muscle biopsy, microelectrode studies, electron microscopy, DNA amplification, and sequencing. All patients, including EA‐2 patients, showed selective failure of the action potential‐dependent release without reduction of the spontaneous release of neurotransmitter. In addition, patients with EA‐2 showed partial blockade of neuromuscular transmission with the N‐type blocker ω‐conotoxin not seen in controls. The EM showed a varied degree of increased complexity of postsynaptic folds. Mutational analysis in candidate genes, including human synaptotagmin II, syntaxin 1A, synaptobrevin I, SNAP 25, CACNA1A, CACNB2, and Rab3A, was unrevealing. Although no mutations in candidate genes were found in patients with inborn presynaptic failure, functional and structural similarities between this group and patients with EA‐2 due to CACNA1A mutations suggest a common pathogenic mechanism.
Protein Ubiquitylation and Synaptic Function
Authors: CREMONA OTTAVIO , COLLESI CHIARA , RAITERI ELISABETTA
Published: January 2004

Abstract: Conjugation of ubiquitin to proteins is a well‐established signal to regulate an ever expanding range of cellular processes. Here, we discuss recent findings that deeply link ubiquitin signaling to synaptic activity.
Nicotinic Acetylcholine Receptors of Muscles and Nerves
Authors: LINDSTROM JON M.
Published: January 2004

Abstract: There are fetal and adult subtypes of muscle nicotinic receptors (AChRs), whose structures and functional roles are reasonably well known. Mutations of their subunits cause congenital myasthenic syndromes. An autoimmune response to them causes myasthenia gravis (MG). The main immunogenic region (MIR) on muscle AChRs accounts for many aspects of the pathological mechanisms by which the autoimmune response impairs neuromuscular transmission. There are many other AChR subtypes, each defined by a different combination of subunits, some of which are transiently expressed in muscle during development, others of which are expressed in keratinocytes, vascular and bronchial epithelia, and other nonneuronal cells, as well as in a wide variety of neurons. Their varied structures and functional roles are much less well known. Mutations in subunits of some of these AChRs have thus far been associated with rare forms of epilepsy and dysautonomia, but other genetic diseases associated with them probably remain to be discovered. Autoimmune responses to some of these subunits are associated with rare dysautonomias and a skin disease. The pathological mechanisms by which these autoimmune responses impair function are much less well known than in the case of MG. AChRs may provide useful drug targets in several neurological diseases. By far, the biggest direct medical impact of AChRs is addiction to tobacco, which is mediated by nicotine acting on a variety of neuronal AChRs.
Structure and Function of AChBP, Homologue of the Ligand‐Binding Domain of the Nicotinic Acetylcholine Receptor
Authors: SMIT AUGUST B. , BREJC KATJUŠA , SYED NAWEED , SIXMA TITIA K.
Published: January 2004

Abstract: Acetylcholine‐binding protein (AChBP) is a novel protein with high similarity to the extracellular domain of the nicotinic acetylcholine receptor. AChBP lacks the transmembrane domains and intracellular loops typical for the nAChRs. AChBP is secreted from glia cells in the central nervous system of the freshwater snail, Lymnaea stagnalis, where it modulates synaptic transmission. AChBP forms homopentamers with pharmacology that resembles the α7‐type of nicotinic receptors. As such, AChBP is a good model for the ligand‐binding domain of the nAChRs. In the crystal structure of AChBP at 2.7 Å, each protomer has a modified immunoglobulin fold. Almost all residues previously shown to be involved in ligand binding in the nicotinic receptor are found in a pocket at the subunit interface, which is lined with aromatic residues. The AChBP crystal structure explains many of the biochemical studies on the nicotinic acetylcholine receptors. Surprisingly, the interface between protomers is relatively weakly conserved between families in the superfamily of pentameric ligand‐gated ion channels. The lack of conservation has implications for the mechanism of gating of the ion channels.
The Binding Site of Acetylcholine Receptor
Authors: FUCHS SARA , KASHER RONI , BALASS MOSHE , SCHERF TALI , HAREL MICHAL , FRIDKIN MATI , SUSSMAN JOEL L. , KATCHALSKI‐KATZIR EPHRAIM
Published: January 2004

Abstract: Our group has been employing short synthetic peptides, encompassing sequences from the acetylcholine receptor (AChR) α‐subunit for the analysis of the binding site of the AChR. A 13‐mer peptide mimotope, with similar structural motifs to the AChR binding region, was selected by α‐bungarotoxin (α‐BTX) from a phage‐display peptide library. The solution structure of a complex between this library‐lead peptide and α‐BTX was solved by NMR spectroscopy. On the basis of this NMR study and on structure‐function analysis of the AChR binding site, and in order to obtain peptides with higher affinity to α‐BTX, additional peptides resulting from systematic residue replacement in the lead peptide were designed and characterized. Of these, four peptides, designated high‐affinity peptides (HAPs), homologous to the binding region of the AChR, inhibited the binding of α‐BTX to the AChR with an IC50 of 2 nM. The solution and crystal structures of complexes of α‐BTX with HAP were solved, demonstrating that the HAP fits snugly to α‐BTX and adopts a β‐hairpin conformation. The X‐ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP) are remarkably similar. Their superposition results in a model indicating that α‐BTX wraps around the receptor binding‐site loop and, in addition, binds tightly at the interface of two of the receptor subunits, where it inserts a finger into the ligand‐binding site. Our proposed model explains the strong antagonistic activity of α‐BTX and accommodates much of the biochemical data on the mode of interaction of α‐BTX with the AChR.
Agonist‐Induced Transitions of the Acetylcholine Receptor
Authors: FAIRCLOUGH ROBERT H. , AGIUS MARK A. , GUDIPATI ESWARI , SILVIAN LAURA , HAMAOKA BRENT , BELTZNER CHRISTOPHER C. , LIN MIKE Y. , CHUANG ANTHONY R. , RICHMAN DAVID P.
Published: January 2004

Abstract: Anti‐acetylcholine receptor (AChR) monoclonal antibody 383C binds to the β‐hairpin loop α(187‐199) of only one of the two Torpedo AChR α subunits. The loop recognized is associated with the α subunit corresponding to the high‐affinity d‐tubocurarine (dTC) binding site. Desensitization of the receptor with carbamylcholine completely blocks the binding of 383C. Mild reduction of AChR α subunit cys 192‐193 disulfide with DTT and subsequent reaction with 5‐iodoacetamidofluorescein label only the high‐affinity dTC α subunit. Rhodamine‐labeled α‐bungarotoxin (R‐Btx) binds to the unlabeled AChR α subunit as monitored by fluorescence resonance energy transfer between the fluorescein and rhodamine dyes. A 10‐Å contraction of the distance between the dyes is observed following the addition of carbamylcholine. In a small angle X‐ray diffraction experiment exploiting anomalous X‐ray scattering from Tb(III) ions titrated into AChR Ca(II) binding sites, we find evidence for a change in the Tb(III) ion distribution in the region of the ion channel following addition of carbamylcholine to the AChR. The carbamylcholine‐induced loss of the 383C epitope, the 10‐Å contraction of the β‐hairpin loop, and the loss of multivalent cations from the channel likely represent the first molecular transitions leading to AChR channel opening.
Structural Abnormalities of the AChR Caused by Mutations Underlying Congenital Myasthenic Syndromes
Authors: BEESON DAVID , WEBSTER RICHARD , EALING JOHN , CROXEN REBECCA , BROWNLOW SHARON , BRYDSON MARTIN , NEWSOM‐DAVIS JOHN , SLATER CLARKE , HATTON CHRIS , SHELLEY CHRIS , COLQUHOUN DAVID , VINCENT ANGELA
Published: January 2004

Abstract: The objective was to define the molecular mechanisms underlying congenital myasthenic syndromes (CMS) by studying mutations within genes encoding the acetylcholine receptor (AChR) and related proteins at the neuromuscular junction. It was found that mutations within muscle AChRs are the most common cause of CMS. The majority are located within the ε‐subunit gene and result in AChR deficiency.
Mechanistic Diversity Underlying Fast Channel Congenital Myasthenic Syndromes
Authors: SINE STEVEN M. , WANG HAI‐LONG , OHNO KINJI , SHEN XIN‐MING , LEE WON YONG , ENGEL ANDREW G.
Published: January 2004

Abstract: A host of missense mutations in muscle nicotinic receptor subunits have been identified as the cause of congenital myasthenic syndromes (CMS). Two classes of CMS phenotypes have been identified: slow channel myasthenic syndromes (SCCMSs) and fast channel myasthenic syndromes (FCCMSs). Although both have similar phenotypic consequences, they are physiologic opposites. Expression of the FCCMS phenotype requires the missense mutation to be accompanied by a second mutation, either a null or a missense mutation, in the second allele encoding the same receptor subunit. This seemingly rare scenario has arisen with surprisingly high incidence over the past few years, and analyses of the syndromes have revealed a diverse array of mechanisms underlying the pathology. This review focuses on new mechanisms underlying the FCCMS.
Congenital Myasthenic Syndromes: Multiple Molecular Targets at the Neuromuscular Junction
Authors: ENGEL ANDREW G. , OHNO KINJI , SHEN XIN‐MING , SINE STEVEN M.
Published: January 2004

Abstract:Congenital myasthenic syndromes (CMS) stem from defects in presynaptic, synaptic, and postsynaptic proteins. The presynaptic CMS are associated with defects that curtail the evoked release of acetylcholine (ACh) quanta or ACh resynthesis. Defects in ACh resynthesis have now been traced to mutations in choline acetyltransferase. A synaptic CMS is caused by mutations in the collagenic tail subunit (ColQ) of the endplate species of acetylcholinesterase that prevent the tail subunit from associating with catalytic subunits or from becoming inserted into the synaptic basal lamina. Most postsynaptic CMS are caused by mutations in subunits of the acetylcholine receptor (AChR) that alter the kinetic properties or decrease the expression of AChR. The kinetic mutations increase or decrease the synaptic response to ACh and result in slow‐ and fast‐channel syndromes, respectively. Most low‐expressor mutations reside in the AChR ε subunit and are partially compensated by residual expression of the fetal‐type γ subunit. In a subset of CMS patients, endplate AChR deficiency is caused by mutations in rapsyn, a molecule that plays a critical role in concentrating AChR in the postsynaptic membrane.
Autoimmune Diseases as the Loss of Active “Self‐Control”
Authors: BACH JEAN‐FRANÇOIS
Published: January 2004

Abstract: Converging experimental evidence indicates that the clinical expression of autoimmunity is under the control of T cell‐mediated immunoregulatory circuits. Several types of suppressor T cells have been described. Some of them are closely dependent upon cytokines such as TH2 cells and Tr1 cells. Others appear to rely more on cell‐cell contact (such as CD25+ CD62L+ T cells), although some cytokines, notably TGF‐β, may be involved in their growth or their mode of action. It is tempting to separate suppressor cells that appear spontaneously, such as CD25+ T cells and NKT cells (innate immunoregulation), from those that are only observed after antigen administration, such as TH2 cells and Tr1 cells (adaptive immunoregulation). The role of these diverse cell types in the control of the onset or the progression of autoimmune diseases is likely, but still a matter of debate. A central question is to determine whether immune dysregulation precedes the burst of pathogenic autoimmunity.
Immunology of Paraneoplastic Syndromes
Authors: POSNER JEROME B.
Published: January 2004

Abstract: Cancers that cause disturbances of organs or tissues remote from the site of the tumor or its metastases are called paraneoplastic syndromes. The nervous system can be affected at virtually any site, including the neuromuscular junction (e.g., Lambert‐Eaton myasthenic syndrome, myasthenia gravis). Paraneoplastic syndromes affecting the central nervous system are characterized by (1) high titers of antibodies that react with both the cancer and the affected portion of the nervous system, (2) specifically reacting T cells in the blood and cerebrospinal fluid, and (3) autopsy evidence of neuronal destruction, inflammatory infiltrates, and antibody penetration. Clinically, paraneoplastic syndromes affecting the central nervous system are usually subacute in onset, rapid in evolution, and cause severe damage, but generally stabilize after several months with or without treatment. Immune suppression does not appear to be particularly effective in treating these disorders. Treatment of the underlying cancer sometimes ameliorates symptoms.
Pathogenic Autoantibodies in the Lambert‐Eaton Myasthenic Syndrome
Authors: LANG BETHAN , PINTO ASHWIN , GIOVANNINI FEDERICA , NEWSOM‐DAVIS JOHN , VINCENT ANGELA
Published: January 2004

Abstract: Lambert‐Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of neuromuscular transmission in which antibodies are directed against voltage‐gated calcium channels (VGCCs). We studied the action of LEMS immunoglobulin G (IgG) on cloned human VGCCs stably transfected into human embryonic kidney cells (HEK293). All LEMS IgGs tested bound to the surface of the P/Q‐type VGCC cell line and caused a significant reduction in whole‐cell calcium currents in these cells. By contrast, only 2 out of 6 IgGs bound extracellularly to the N‐type VGCC cell line, and none of the LEMS IgGs tested was able to reduce whole‐cell calcium currents in these cells. We used this apparent specificity of LEMS IgG for the P/Q‐type VGCC to investigate the action of these IgGs on model systems where a number of different VGCC populations exist in equilibrium. LEMS IgG caused a significant downregulation in the ω‐agatoxin IVA‐sensitive P/Q‐type VGCCs of cultured rat cerebellar neurons, but this was accompanied by a concomitant rise in the “resistant” R‐type VGCCs. By using the passive transfer model of LEMS, similar results were observed at the mouse neuromuscular junction, where a significant reduction in P/Q‐type VGCCs was paralleled by an increase in L‐ and R‐type VGCCs. These results demonstrate an unexpected plasticity in the expression of VGCCs in mammalian neurons and may represent a mechanism by which the pathogenic effects of LEMS IgG are reduced.
Autoimmune Disorders of Neuronal Potassium Channels
Authors: NEWSOM‐DAVIS JOHN , BUCKLEY CAMILLA , CLOVER LINDA , HART IAN , MADDISON PAUL , TÜZÜM ERDEM , VINCENT ANGELA
Published: January 2004

Abstract: Antibodies to voltage‐gated potassium channels (VGKCs) appear likely to be the effector mechanisms in many patients with acquired peripheral nerve hyperexcitability (APNH) syndromes, a group of disorders that include neuromyotonia, cramp‐fasciculation syndrome, and Isaacs' syndrome. They may contribute to the associated autonomic changes. Through a central action, they may also be the effector mechanism in those with Morvan's syndrome and in some patients with limbic encephalitis. Evidence supporting this hypothesis includes the increased association of APNH with autoimmune diseases (in particular, myasthenia gravis and thymoma), the response to plasmapheresis, passive transfer of APNH to experimental animals by patients' plasma or immunoglobulins, the action of their serum on VGKC currents studied in vitro, and the presence in many patients of IgG antibodies to VGKCs.
Neuronal Ganglionic Acetylcholine Receptor Autoimmunity
Authors: VERNINO STEVEN , LENNON VANDA A.
Published: January 2004

Abstract: We have developed and validated an assay to detect serum antibodies specific for the ganglionic AChR. The assay uses ganglionic AChR solubilized from membranes of the human neuroblastoma cell line (IMR‐32) as antigen. The ganglionic AChR is radiolabeled by complexing with 125I‐epibatidine. Among patients with acquired dysautonomia, seropositivity is highly associated with the diagnosis of idiopathic or paraneoplastic autonomic neuropathy and can distinguish these disorders from other forms of autonomic dysfunction. Ganglionic AChR binding antibodies are detectable in 50% of patients with subacute autoimmune autonomic neuropathy (AAN). These patients often have a high antibody value (>0.2 nmol/L). Lower values (0.05–0.20 nmol/L) are found in ∼10% of patients with limited AAN.
Humoral and Cellular Autoimmune Responses in Stiff Person Syndrome
Authors: LOHMANN TOBIAS , LONDEI MARCO , HAWA MOHAMMED , LESLIE R. DAVID G.
Published: January 2004

Abstract: Stiff person syndrome (SPS) is a chronic autoimmune disease associated with humoral and cellular immune responses to glutamic acid decarboxylase (GAD) 65. Another chronic autoimmune disease, type 1 diabetes (T1D), is also associated with autoimmune responses to this antigen, but T1D patients develop SPS only extremely rarely and only a third of SPS patients develop T1D (mostly mild manifestations in adulthood). In a previous study, we described important differences between T1D and SPS in the autoimmune response to GAD 65: (1) T cells of SPS patients recognize epitopes in the middle of GAD 65 (amino residues 81–171 and 313–403), whereas patients with T1D preferentially recognize another middle (161–243) and a C‐terminal region (473–555); and (2) GAD antibodies (Abs) were nearly exclusively of the Th1‐associated IgG1 type in T1D, whereas SPS patients had both Th1‐ and Th2‐associated IgG4 and IgE GAD Abs. These differences were not simply related to different HLA alleles. Fine epitope mapping revealed further distinct T cell epitopes in both diseases despite similar HLA background. Therefore, a single autoantigen can elicit different immune responses causing distinct chronic autoimmune diseases possibly related to a Th1 or Th2 bias of the disease.
The Role of Thymomas in the Development of Myasthenia Gravis
Authors: MARX ALEXANDER , MÜLLER‐HERMELINK HANS KONRAD , STRÖBEL PHILIPP
Published: January 2004

Abstract:Thymic pathology occurs in 80–90% of myasthenia gravis patients. Significant associations between different thymic alterations and clinical findings are discussed. To highlight peculiarities in thymoma‐associated myasthenia gravis, we briefly review myasthenia gravis associated with thymic lymphofollicular hyperplasia (TFH) and thymic atrophy.
Scenarios for Autoimmunization of T and B Cells in Myasthenia Gravis
Authors: SHIONO H. , ROXANIS I. , ZHANG W. , SIMS G. P. , MEAGER A. , JACOBSON L. W. , LIU J‐L. , MATTHEWS I. , WONG Y‐L. , BONIFATI M. , MICKLEM K. , STOTT D. I. , TODD J. A. , BEESON D. , VINCENT A. , WILLCOX N.
Published: January 2004

Abstract: We have studied responses in thymoma patients to interferon‐α and to the acetylcholine receptor (AChR) in early‐onset myasthenia gravis (EOMG), seeking clues to autoimmunizing mechanisms. Our new evidence implicates a two‐step process: (step 1) professional antigen‐presenting cells and thymic epithelial cells prime AChR‐specific T cells; then (step 2) thymic myoid cells subsequently provoke germinal center formation in EOMG. Our unifying hypothesis proposes that AChR epitopes expressed by neoplastic or hyperplastic thymic epithelial cells aberrantly prime helper T cells, whether generated locally or infiltrating from the circulation. These helper T cells then induce antibody responses against linear epitopes that cross‐react with whole AChR and attack myoid cells in the EOMG thymus. The resulting antigen‐antibody complexes and the recruitment of professional antigen‐presenting cells increase the exposure of thymic cells to the infiltrates and provoke local germinal center formation and determinant spreading. Both these and the consequently enhanced heterogeneity and pathogenicity of the autoantibodies should be minimized by early thymectomy.
Analysis of CD4+CD25+ Cell Population in the Thymus from Myasthenia Gravis Patients
Authors: BALANDINA A. , SAOUDI A. , DARTEVELLE P. , BERRIH‐AKNIN S.
Published: January 2004

Abstract: The present study is aimed at exploring the regulatory CD4+CD25+ T cells in the thymus from myasthenia gravis (MG) patients. In early‐onset MG, the thymus is hyperplastic and contains autoreactive activated T cells. Preliminary studies indicate that these CD4+CD25+ cells include activated autoreactive T cells. Studies to characterize the phenotype and suppressive capacity of these cells will be discussed.
T Cells and Cytokines in the Pathogenesis of Acquired Myasthenia Gravis
Authors: MILANI MONICA , OSTLIE NORMA , WANG WEI , CONTI‐FINE BIANCA M.
Published: January 2004

Abstract: Although the symptoms of myasthenia gravis (MG) and experimental MG (EAMG) are caused by autoantibodies, CD4+ T cells specific for the target antigen, the nicotinic acetylcholine receptor, and the cytokines they secrete, have an important role in these diseases. CD4+ T cells have a pathogenic role, by permitting and facilitating the synthesis of high‐affinity anti‐AChR antibodies. Th1 CD4+ cells are especially important because they drive the synthesis of anti‐AChR complement‐fixing IgG subclasses. Binding of those antibodies to the muscle AChR at the neuromuscular junction will trigger the complement‐mediated destruction of the postsynaptic membrane. Thus, IL‐12, a crucial cytokine for differentiation of Th1 cells, is necessary for development of EAMG. Th2 cells secrete different cytokines, with different effects on the pathogenesis of EAMG. Among them, IL‐10, which is a potent growth and differentiation factor for B cells, facilitates the development of EAMG. In contrast, IL‐4 appears to be involved in the differentiation of AChR‐specific regulatory CD4+ T cells, which can prevent the development of EAMG and its progression to a self‐maintaining, chronic autoimmune disease. Studies on the AChR‐specific CD4+ cells commonly present in the blood of MG patients support a crucial role of CD4+ T cells in the development of MG. Circumstantial evidence supports a pathogenic role of IL‐10 also in human MG. On the other hand, there is no direct or circumstantial evidence yet indicating a role of IL‐4 in the modulatory or immunosuppressive circuits in MG.
Immunoregulation in Experimental Autoimmune Myasthenia Gravis—about T Cells, Antibodies, and Endplates
Authors: De BAETS M. , STASSEN M. , LOSEN M. , ZHANG X. , MACHIELS B.
Published: January 2004

Abstract: Experimental autoimmune myasthenia gravis (EAMG) can be induced in a large number of animal species by active immunization (AI) AChR, by passive transfer (PT) of anti‐AChR antibodies, by autologous bone marrow transplantation and cyclosporin (BMT‐Cy), or spontaneously. Depending on the model used, different immunological mechanisms are operational. In the AI model, the T cell is pivotal in directing the anti‐AChR antibody production towards pathogenic, that is, cross‐linking and complement‐fixing antibodies. Injection of anti‐AChR antibodies alone suffices to induce EAMG, excluding the role of specific cell‐mediated immune responses in the effector phase of the disease. Aged animals are resistant to the induction of AI and PT EAMG. This resistance is localized at the postsynaptic membrane containing more AChR‐anchoring proteins, including S‐laminin and rapsyn in aged animals. In BMT‐CyA EAMG, a dysregulation of the immune system in the absence of immunization is capable of inducing myasthenia. The role of these animal models in relation to pathogenesis and immunotherapy is discussed.
Pathogenesis of Myositis and Myasthenia Associated with Titin and Ryanodine Receptor Antibodies
Authors: SKEIE GEIR OLVE , ROMI FREDRIK , AARLI JOHAN A. , BENTSEN PÅL TORE , GILHUS NILS ERIK
Published: January 2004

Abstract: Some myasthenia gravis (MG) patients have antibodies against skeletal muscle antigens in addition to the acetylcholine receptor (AChR). Two major antigens for these antibodies are the Ca2+ release channel of the sarcoplasmic reticulum, the ryanodine receptor (RyR), and titin, a gigantic filamentous muscle protein essential for muscle structure, function, and development. RyR and titin antibodies are found in MG patients with a thymoma and in a proportion of late‐onset MG, and they correlate with severe MG disease. The RyR antibodies recognize a region near the N‐terminus important for channel regulation. They inhibit Ca2+ release from sarcoplasmic reticulum in vitro. There is electrophysiological evidence for a disordered excitation‐contraction coupling in MG patients. The presence of titin antibodies, which bind to key regions near the A/I junction and in the central I‐band, correlates with myopathy in MG patients. However, so far, there is no direct evidence that antibodies against the intracellular antigens RyR and titin are pathogenic in vivo.