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1.
Transplant Proc ; 41(6): 2601-6, 2009.
Article in English | MEDLINE | ID: mdl-19715983

ABSTRACT

Currently, there is an unmet clinical need for novel immunosuppressive agents for long-term prevention of kidney transplant rejection as alternatives to the nephrotoxic calcineurin inhibitor cyclosporine (CsA). Recent studies have shown that K(+) channels have a crucial role in T-lymphocyte activity. We investigated whether combined blockade of the T-cell K(+) channels K(Ca)3.1 and K(v)1.3, both of which regulate calcium signaling during lymphocyte activation, is effective in prevention of rejection of kidney allografts from Fisher rats to Lewis rats. All recipients were initially treated with CsA (5 mg/kg d) for 7 days. In rats with intact allograft function, treatment was continued for 10 days with either CsA (5 mg/kg d), or a combination of TRAM-34 (K(Ca)3.1 inhibitor; 120 mg/kg d) plus Stichodactyla helianthus toxin (ShK, K(v)1.3 inhibitor; 80 microg/kg 3 times daily), or vehicle alone. Kidney sections were stained with periodic acid-Schiff or hematoxylin-eosin and histochemically for markers of macrophages (CD68), T-lymphocytes (CD43), or cytotoxic T-cells (CD8). Our results showed that treatment with TRAM-34 and ShK reduced total interstitial mononuclear cell infiltration (-42%) and the number of CD43+ T-cells (-32%), cytotoxic CD8+ T-cells (-32%), and CD68+ macrophages (-26%) in allografts when compared to vehicle treatment alone. Efficacy of TRAM-34/ShK treatment was comparable with that of CsA. In addition, no visible organ damage or other discernible adverse effects were observed with this treatment. Thus, selective blockade of T-lymphocyte K(Ca)3.1 and K(v)1.3 channels may represent a novel alternative therapy for prevention of kidney allograft rejection.


Subject(s)
Graft Rejection/prevention & control , Immunosuppression Therapy/methods , Intermediate-Conductance Calcium-Activated Potassium Channels/immunology , Kidney Transplantation/immunology , Kv1.1 Potassium Channel/immunology , T-Lymphocytes/immunology , Animals , Cnidarian Venoms/pharmacology , Intermediate-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors , Kv1.1 Potassium Channel/antagonists & inhibitors , Lymphocyte Culture Test, Mixed , Male , Pyrazoles/pharmacology , Rats , Rats, Inbred F344 , Rats, Inbred Lew , Spleen/cytology , Spleen/drug effects , Spleen/immunology
2.
Neuroscience ; 158(4): 1500-8, 2009 Feb 18.
Article in English | MEDLINE | ID: mdl-19118603

ABSTRACT

Potassium channels play an important role in microglial activation but their involvement in main functions of microglia including secretion of proinflammatory cytokines has remained uncertain. This study has revealed the specific expression of Kv1.1 in microglia both in vivo and in vitro. Kv1.1 immunoreactivity was localized in the amoeboid microglia in the rat brain between postnatal (P) day 1 (P1) and day 10 (P10); it was, however, progressively reduced with age and was hardly detected at P14 and P21 in ramified microglia, a derivative cell of amoeboid microglia. Following hypoxic exposure, Kv1.1 expression in amoeboid microglia was enhanced or induced in ramified microglia in more mature brain at P21 when compared with their matching controls. RT-PCR and Western blot analysis confirmed Kv1.1 mRNA and protein expression in murine BV-2 cells which was up-regulated by hypoxia or lipopolysaccharide (LPS) treatment; it was reduced significantly by dexamethasone. Neutralization with Kv1.1 antibody suppressed the expression and release of tumor necrosis factor-alpha, interleukin-1beta, endothelins and nitric oxide (NO) in LPS-activated BV-2 cells. It is concluded that Kv1.1, constitutively expressed by microglia, is elicited by hypoxia and LPS and this may be linked to production of proinflammatory cytokines, endothelins and NO.


Subject(s)
Brain/cytology , Cytokines/metabolism , Endothelins/metabolism , Gene Expression Regulation, Developmental/physiology , Kv1.1 Potassium Channel/metabolism , Microglia/metabolism , Nitric Oxide/metabolism , Age Factors , Animals , Animals, Newborn , Antibodies/pharmacology , Brain/growth & development , Brain/metabolism , Cell Line, Transformed , Colorimetry/methods , Enzyme-Linked Immunosorbent Assay/methods , Gene Expression Regulation, Developmental/drug effects , Hypoxia/metabolism , Hypoxia/pathology , Kv1.1 Potassium Channel/genetics , Kv1.1 Potassium Channel/immunology , Lipopolysaccharides/pharmacology , Mice , Microglia/drug effects , Nitric Oxide/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar
3.
Neurology ; 70(20): 1883-90, 2008 May 13.
Article in English | MEDLINE | ID: mdl-18474843

ABSTRACT

OBJECTIVE: To document neurologic, oncologic, and serologic associations of patients in whom voltage-gated potassium channel (VGKC) autoantibodies were detected in the course of serologic evaluation for neuronal, glial, and muscle autoantibodies. METHODS: Indirect immunofluorescence screening of sera from 130,000 patients performed on a service basis for markers of paraneoplastic neurologic autoimmunity identified 80 patients whose IgG bound to the synapse-rich molecular layer of mouse cerebellar cortex in a pattern consistent with VGKC immunoreactivity. Antibody specificity was confirmed in all cases by immunoprecipitation of detergent-solubilized brain synaptic proteins complexed with (125)I-alpha-dendrotoxin. RESULTS: Clinical information was available for 72 patients: 51% women, median age at symptom onset 65 years, and median follow-up period 14 months. Neurologic manifestations were acute to subacute in onset in 71% and multifocal in 46%; 71% had cognitive impairment, 58% seizures, 33% dysautonomia, 29% myoclonus, 26% dyssomnia, 25% peripheral nerve dysfunction, 21% extrapyramidal dysfunction, and 19% brainstem/cranial nerve dysfunction. Creutzfeldt-Jakob disease was a common misdiagnosis (14%). Neoplasms encountered (confirmed histologically in 33%) included 18 carcinomas, 5 adenomas, 1 thymoma, and 3 hematologic malignancies. Hyponatremia was documented in 36%, other organ-specific autoantibodies in 49%, and a co-existing autoimmune disorder in 33% (including thyroiditis 21%, type 1 diabetes mellitus 11%). Benefit was reported for 34 of 38 patients (89%) receiving immunotherapy and was marked in 50%. CONCLUSIONS: The spectrum of neurologic manifestations and neoplasms associated with voltage-gated potassium channel (VGKC) autoimmunity is broader than previously recognized. Evaluation for VGKC antibodies is recommended in the comprehensive autoimmune serologic testing of subacute idiopathic neurologic disorders.


Subject(s)
Autoantibodies/blood , Paraneoplastic Syndromes/immunology , Peripheral Nervous System Diseases/immunology , Shaker Superfamily of Potassium Channels/immunology , Adenoma/complications , Adolescent , Adult , Aged , Aged, 80 and over , Autonomic Nervous System Diseases/etiology , Autonomic Nervous System Diseases/immunology , Basal Ganglia Diseases/etiology , Basal Ganglia Diseases/immunology , Child , Cranial Nerve Diseases/etiology , Cranial Nerve Diseases/immunology , Female , Fluorescent Antibody Technique, Indirect , Hematologic Neoplasms/complications , Humans , Kv1.1 Potassium Channel/immunology , Kv1.2 Potassium Channel/immunology , Kv1.6 Potassium Channel , Male , Middle Aged , Myoclonus/etiology , Myoclonus/immunology , Paraneoplastic Syndromes/complications , Peripheral Nervous System Diseases/etiology , Thymoma/complications , Thymus Neoplasms/complications
4.
Brain ; 129(Pt 6): 1570-84, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16613892

ABSTRACT

Autoantibodies to Shaker-type (Kv1) K+ channels are now known to be associated with three syndromes. Peripheral nerve hyperexcitability is the chief manifestation of acquired neuromyotonia; the combination of neuromyotonia with autonomic and CNS involvement is called Morvan's syndrome (MoS); and CNS manifestations without peripheral involvement is called limbic encephalitis (LE). To determine the cellular basis of these clinical manifestations, we immunostained mouse neural tissues with sera from patients with neuromyotonia (n = 10), MoS (n = 2) or LE (n = 5), comparing with specific antibodies to relevant K+ channel subunits. Fourteen of 17 patients' sera were positive for Kv1.1, Kv1.2 or Kv1.6 antibodies by immunoprecipitation of 125I-alpha-dendrotoxin-labelled rabbit brain K+ channels. Most sera (11 out of 17) labelled juxtaparanodes of peripheral myelinated axons, co-localizing with Kv1.1 and Kv1.2. In the CNS, all sera tested (n = 12) co-localized with one or more areas of high Kv1.1, Kv1.2 or Kv1.6 channel expression: 10 out of 12 sera co-localized with Kv1.1 and Kv1.2 at spinal cord juxtaparanodes or cerebellar layers, while 3 out of 12 sera co-localized additionally (n = 2) or exclusively (n = 1) with Kv1.6 subunits in Purkinje cells, motor and hippocampal neurons. However, only sera from LE patients labelled the hippocampal areas that are enriched in excitatory, Kv1.1-positive axon terminals. All sera (17 out of 17) labelled one or more of these Kv1 subunits when expressed at the cell membrane of transfected HeLa cells, but not when they were retained in the endoplasmic reticulum. Again, LE sera labelled Kv1.1 subunits more prominently than did MoS or neuromyotonia sera, suggesting an association between higher Kv1.1 specificity and limbic manifestations. In contrast, neuromyotonia sera bound more strongly to Kv1.2 subunits than to Kv1.1 or Kv1.6. These studies support the hypothesis that antibodies to mature surface membrane-expressed Shaker-type K+ channels cause acquired neuromyotonia, MoS and LE, and suggest that future assays based on immunofluorescence of cells expressing individual Kv1 subunits will prove more sensitive than the immunoprecipitation assay. Although more than one type of antibody is often detectable in individual sera, higher affinity for certain subunits or subunit combinations may determine the range of clinical manifestations.


Subject(s)
Autoantibodies/blood , Autoimmune Diseases/immunology , Isaacs Syndrome/immunology , Limbic Encephalitis/immunology , Shaker Superfamily of Potassium Channels/immunology , Adolescent , Adult , Aged , Animals , Antibody Specificity , Biomarkers/blood , Brain/immunology , Female , HeLa Cells , Hippocampus/immunology , Humans , Kv1.1 Potassium Channel/immunology , Kv1.2 Potassium Channel/immunology , Kv1.6 Potassium Channel , Male , Mice , Middle Aged , Peripheral Nerves/immunology , Presynaptic Terminals/immunology , Spinal Cord/immunology , Syringomyelia/immunology , Transfection
5.
BMC Neurosci ; 6: 65, 2005 Nov 23.
Article in English | MEDLINE | ID: mdl-16305740

ABSTRACT

BACKGROUND: The megencephaly mouse, mceph/mceph, is epileptic and displays a dramatically increased brain volume and neuronal count. The responsible mutation was recently revealed to be an eleven base pair deletion, leading to a frame shift, in the gene encoding the potassium channel Kv1.1. The predicted MCEPH protein is truncated at amino acid 230 out of 495. Truncated proteins are usually not expressed since nonsense mRNAs are most often degraded. However, high Kv1.1 mRNA levels in mceph/mceph brain indicated that it escaped this control mechanism. Therefore, we hypothesized that the truncated Kv1.1 would be expressed and dysregulate other Kv1 subunits in the mceph/mceph mice. RESULTS: We found that the MCEPH protein is expressed in the brain of mceph/mceph mice. MCEPH was found to lack mature (Golgi) glycosylation, but to be core glycosylated and trapped in the endoplasmic reticulum (ER). Interactions between MCEPH and other Kv1 subunits were studied in cell culture, Xenopus oocytes and the brain. MCEPH can form tetramers with Kv1.1 in cell culture and has a dominant negative effect on Kv1.2 and Kv1.3 currents in oocytes. However, it does not retain Kv1.2 in the ER of neurons. CONCLUSION: The megencephaly mice express a truncated Kv1.1 in the brain, and constitute a unique tool to study Kv1.1 trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth.


Subject(s)
Brain/abnormalities , Frameshift Mutation , Gene Expression Regulation/genetics , Kv1.1 Potassium Channel/genetics , Kv1.1 Potassium Channel/metabolism , Animals , Blotting, Western/methods , Brain/pathology , Cell Line , Dose-Response Relationship, Radiation , Electric Stimulation/methods , Glycosylation , Humans , Immunohistochemistry/methods , Immunoprecipitation/methods , Kv1.1 Potassium Channel/immunology , Kv1.2 Potassium Channel/metabolism , Membrane Potentials/genetics , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Mutant Strains , Oocytes/physiology , Patch-Clamp Techniques/methods , Peptide Fragments/immunology , Peptide Fragments/metabolism , Protein Structure, Tertiary/genetics , Protein Transport/genetics , Transfection/methods , Xenopus
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