Electrophysiological and pharmacological studies coupled with molecular identification have revealed a unique network of ion channels—Kv1.3, KCa3.1, CRAC (Orai1 + Stim1), TRPM7, Clswell—in ...lymphocytes that initiates and maintains the calcium signaling cascade required for activation. The expression pattern of these channels changes during lymphocyte activation and differentiation, allowing the functional network to adapt during an immune response. The Kv1.3 channel is of interest because it plays a critical role in subsets of T and B lymphocytes implicated in autoimmune disorders. The ShK toxin from the sea anemone Stichodactyla helianthus is a potent blocker of Kv1.3. ShK-186, a synthetic analog of ShK, is being developed as a therapeutic for autoimmune diseases, and is scheduled to begin first-in-man phase-1 trials in 2011. This review describes the journey that has led to the development of ShK-186.
► ShK-186 is a 37-residue synthetic derivative of ShK, a peptide toxin from the sea anemone Stichodactyla helianthus. ► ShK-186 blocks Kv1.3 potassium channels with picomolar affinity and with a high degree of specificity. ► Kv1.3 potassium channels play a critical role in regulating the function of effector-memory T cells and class-switched memory B cells that are implicated in autoimmune diseases. ► ShK-186 has a good safety profile in rats and it ameliorates disease in rat models of multiple sclerosis and rheumatoid arthritis. ► ShK-186 is being developed as a therapeutic for autoimmune diseases and phase 1 human trials are planned for the near future.
Non-alcoholic fatty liver disease (NAFLD) has been on a global rise. While animal models have rendered valuable insights to the pathogenesis of NAFLD, discrepancy with patient data still exists. ...Since non-alcoholic steatohepatitis (NASH) involves chronic inflammation, and CD4
T cell infiltration of the liver is characteristic of NASH patients, we established and characterized a humanized mouse model to identify human-specific immune response(s) associated with NAFLD progression. Immunodeficient mice engrafted with human immune cells (HIL mice) were fed with high fat and high calorie (HFHC) or chow diet for 20 weeks. Liver histology and immune profile of HIL mice were analyzed and compared with patient data. HIL mice on HFHC diet developed steatosis, inflammation and fibrosis of the liver. Human CD4
central and effector memory T cells increased within the liver and in the peripheral blood of our HIL mice, accompanied by marked up-regulation of pro-inflammatory cytokines (IL-17A and IFNγ).
depletion of human CD4
T cells in HIL mice reduced liver inflammation and fibrosis, but not steatosis. Our results highlight CD4
memory T cell subsets as important drivers of NAFLD progression from steatosis to fibrosis and provides a humanized mouse model for pre-clinical evaluation of potential therapeutics.
MMP23 is a member of the matrix metalloprotease family of zinc- and calcium-dependent endopeptidases, which are involved in a wide variety of cellular functions. Its catalytic domain displays a high ...degree of structural homology with those of other metalloproteases, but its atypical domain architecture suggests that it may possess unique functional properties. The
N
-terminal MMP23 pro-domain contains a type-II transmembrane domain that anchors the protein to the plasma membrane and lacks the cysteine-switch motif that is required to maintain other MMPs in a latent state during passage to the cell surface. Instead of the
C
-terminal hemopexin domain common to other MMPs, MMP23 contains a small toxin-like domain (TxD) and an immunoglobulin-like cell adhesion molecule (IgCAM) domain. The MMP23 pro-domain can trap Kv1.3 but not closely-related Kv1.2 channels in the endoplasmic reticulum, preventing their passage to the cell surface, while the TxD can bind to the channel pore and block the passage of potassium ions. The MMP23
C
-terminal IgCAM domain displays some similarity to Ig-like C2-type domains found in IgCAMs of the immunoglobulin superfamily, which are known to mediate protein–protein and protein–lipid interactions. MMP23 and Kv1.3 are co-expressed in a variety of tissues and together are implicated in diseases including cancer and inflammatory disorders. Further studies are required to elucidate the mechanism of action of this unique member of the MMP family.
Pharmacologically induced electroencephalogram (EEG) silence increases tolerance of ischemic period by reducing cerebral metabolism. We hypothesized that sevoflurane, a cerebral vasodilator, will ...maintain cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO2) better than propofol, a cerebral vasoconstrictor, during EEG silence. To validate this, we compared the effect of sevoflurane and propofol on CBF and CMRO2 during surgical plane of anasthesia (SP) and burst suppression on EEG (BS).
We conducted a prospective, double-blinded trial where patients undergoing neurosurgery were randomized to receive propofol or sevoflurane. Mean velocity (MV) and pulsatility index (PI) of bilateral middle cerebral arteries (MCA) were measured as surrogate of CBF. Jugular venous oxygen saturation (SjvO2) and arteriovenous oxygen difference (AjvDO2) were obtained to assess CMRO2. The values were compared between groups using Student t test and within the group with analysis of variance at SP and BS.
BS decreased MV and increased PI in propofol group (P < 0.001 and P < 0.02 on normal side, P < 0.004 and P < 0.001 on tumor side). There was no significant change in sevoflurane group. BS with sevoflurane increased SjvO2 (P < 0.001) and decreased AjvDO2 (P < 0.001). Change in SjvO2 and AjvDO2 with propofol at SP and BS was variable.
In our study, sevoflurane had a safer profile on cerebral oxygenation during BS while not altering the CBF, suggesting increased availability of oxygen. Propofol, on the other hand, produced cerebral vasoconstriction with BS. The effect of propofol on oxygenation was unpredictable, with low SjvO2 and high AjvDO2 even at surgical plane of anesthesia.
The molecular targets and neural circuits that underlie general anesthesia are not fully elucidated. Here, we directly demonstrate that Kv1-family (Shaker-related) delayed rectifier K(+) channels in ...the central medial thalamic nucleus (CMT) are important targets for volatile anesthetics. The modulation of Kv1 channels by volatiles is network specific as microinfusion of ShK, a potent inhibitor of Kv1.1, Kv1.3, and Kv1.6 channels, into the CMT awakened sevoflurane-anesthetized rodents. In heterologous expression systems, sevoflurane, isoflurane, and desflurane at subsurgical concentrations potentiated delayed rectifier Kv1 channels at low depolarizing potentials. In mouse thalamic brain slices, sevoflurane inhibited firing frequency and delayed the onset of action potentials in CMT neurons, and ShK-186, a Kv1.3-selective inhibitor, prevented these effects. Our findings demonstrate the exquisite sensitivity of delayed rectifier Kv1 channels to modulation by volatile anesthetics and highlight an arousal suppressing role of Kv1 channels in CMT neurons during the process of anesthesia.
How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice, N-glycan branching by the ...Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity, cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis, spontaneous inflammatory demyelination and neurodegeneration, the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D(3), including genetic variants in interleukin-7 receptor-α (IL7RA*C), interleukin-2 receptor-α (IL2RA*T), MGAT1 (IV(A)V(T-T)) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IV(A)V(T-T)) and vitamin D(3), optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway, namely N-glycosylation.
Macrophages are abundant immune cells in the microenvironment of diffuse large B-cell lymphoma (DLBCL). Macrophage estimation by immunohistochemistry shows varying prognostic significance across ...studies in DLBCL, and does not provide a comprehensive analysis of macrophage subtypes. Here, using digital spatial profiling with whole transcriptome analysis of CD68+ cells, we characterize macrophages in distinct spatial niches of reactive lymphoid tissues (RLTs) and DLBCL. We reveal transcriptomic differences between macrophages within RLTs (light zone /dark zone, germinal center/ interfollicular), and between disease states (RLTs/ DLBCL), which we then use to generate six spatially-derived macrophage signatures (MacroSigs). We proceed to interrogate these MacroSigs in macrophage and DLBCL single-cell RNA-sequencing datasets, and in gene-expression data from multiple DLBCL cohorts. We show that specific MacroSigs are associated with cell-of-origin subtypes and overall survival in DLBCL. This study provides a spatially-resolved whole-transcriptome atlas of macrophages in reactive and malignant lymphoid tissues, showing biological and clinical significance.
The voltage-gated Kv1.3 K(+) channel is a novel target for immunomodulation of autoreactive effector memory T (T(EM)) cells that play a major role in the pathogenesis of autoimmune diseases. We ...describe the characterization of the novel peptide ShK(L5) that contains l-phosphotyrosine linked via a nine-atom hydrophilic linker to the N terminus of the ShK peptide from the sea anemone Stichodactyla helianthus. ShK(L5) is a highly specific Kv1.3 blocker that exhibits 100-fold selectivity for Kv1.3 (K(d) = 69 pM) over Kv1.1 and greater than 250-fold selectivity over all other channels tested. ShK(L5) suppresses the proliferation of human and rat T(EM) cells and inhibits interleukin-2 production at picomolar concentrations. Naive and central memory human T cells are initially 60-fold less sensitive than T(EM) cells to ShK(L5) and then become resistant to the peptide during activation by up-regulating the calcium-activated K(Ca)3.1 channel. ShK(L5) does not exhibit in vitro cytotoxicity on mammalian cell lines and is negative in the Ames test. It is stable in plasma and when administered once daily by subcutaneous injection (10 mug/kg) attains "steady state" blood levels of approximately 300 pM. This regimen does not cause cardiac toxicity assessed by continuous EKG monitoring and does not alter clinical chemistry and hematological parameters after 2-week therapy. ShK(L5) prevents and treats experimental autoimmune encephalomyelitis and suppresses delayed type hypersensitivity in rats. ShK(L5) might prove useful for therapy of autoimmune disorders.
Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel ...KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.
Obesity is an epidemic, calling for innovative and reliable pharmacological strategies. Here, we show that ShK-186, a selective and potent blocker of the voltage-gated Kv1.3 channel, counteracts the ...negative effects of increased caloric intake in mice fed a diet rich in fat and fructose. ShK-186 reduced weight gain, adiposity, and fatty liver; decreased blood levels of cholesterol, sugar, HbA1c, insulin, and leptin; and enhanced peripheral insulin sensitivity. These changes mimic the effects of Kv1.3 gene deletion. ShK-186 did not alter weight gain in mice on a chow diet, suggesting that the obesity-inducing diet enhances sensitivity to Kv1.3 blockade. Several mechanisms may contribute to the therapeutic benefits of ShK-186. ShK-186 therapy activated brown adipose tissue as evidenced by a doubling of glucose uptake, and increased β-oxidation of fatty acids, glycolysis, fatty acid synthesis, and uncoupling protein 1 expression. Activation of brown adipose tissue manifested as augmented oxygen consumption and energy expenditure, with no change in caloric intake, locomotor activity, or thyroid hormone levels. The obesity diet induced Kv1.3 expression in the liver, and ShK-186 caused profound alterations in energy and lipid metabolism in the liver. This action on the liver may underlie the differential effectiveness of ShK-186 in mice fed a chow vs. an obesity diet. Our results highlight the potential use of Kv1.3 blockers for the treatment of obesity and insulin resistance.
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