Srikant Rangaraju

Imaging of Effector Memory T Cells during a Delayed-Type Hypersensitivity Reaction and Suppression by Kv1.3 Channel Block

Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.

Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes.

Kv1.3 potassium channels maintain the membrane potential of effector memory (TEM) T cells that are important mediators of multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5), containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin, is a potent and selective blocker of these channels. However, a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability, ShK-192, contains a nonhydrolyzable phosphotyrosine surrogate, a methionine isostere, and a C-terminal amide. ShK-192 shows the same overall fold as ShK, and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys411 of the channel. ShK-192 blocks Kv1.3 with an IC50 of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 μg/kg, ∼100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24, 48, and 72 h after the injection. ShK-192 effectively inhibits the proliferation of TEM cells and suppresses delayed type hypersensitivity when administered at 10 or 100 μg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by TEM cells.

Kv1.3 potassium channels as a therapeutic target in multiple sclerosis

We discuss the potential use of inhibitors of Kv1.3 potassium channels in T lymphocytes as therapeutics for multiple sclerosis. Current treatment strategies target the immune system in a non-selective manner. The resulting general immunosuppression, toxic side-effects and increased risk of opportunistic infections create the need for more selective therapeutics. Autoreactive effector-memory T (TEM) cells, considered to be major mediators of autoimmunity, express large numbers of Kv1.3 channels. Selective blockers of Kv1.3 inhibit calcium signaling, cytokine production and proliferation of TEM cells in vitro, and TEM cell-motility in vivo. Kv1.3 blockers ameliorate disease in animal models of multiple sclerosis, rheumatoid arthritis, type 1 diabetes mellitus and contact dermatitis without compromising the protective immune response to acute infections. Kv1.3 blockers have a good safety profile in rodents and primates.

Potassium channel modulation by a toxin domain in matrix metalloprotease 23

Peptide toxins found in a wide array of venoms block K+ channels, causing profound physiological and pathological effects. Here we describe the first functional K+ channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23TxD) that is evolutionarily related to peptide toxins from sea anemones. MMP23TxD shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K+ channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K+ channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K+ channels by co-localizing with and trapping MMP23TxD-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.

Comparison of Final Infarct Volumes in Patients Who Received Endovascular Therapy or Intravenous Thrombolysis for Acute Intracranial Large-Vessel Occlusions

IMPORTANCE Studies comparing the efficacy of intra-arterial therapy (IAT) and medical therapy in reducing final infarct volume (FIV) in intracranial large-vessel occlusions (ILVOs) are lacking. OBJECTIVES To assess whether patients with ILVOs who received IAT have smaller FIVs than patients who received either intravenous tissue plasminogen activator therapy (IVT) or no reperfusion therapy (NRT) and to determine a National Institutes of Health Stroke Scale (NIHSS) threshold score that identifies patients most likely to benefit from IAT. DESIGN Retrospective cohort study of patients with ILVOs between 2009 and 2011. SETTING Two large-volume stroke centers. PARTICIPANTS Adults with anterior circulation ILVOs who presented within 360 minutes from the time last seen as normal. Patients with isolated extracranial occlusions were not included. EXPOSURE Intra-arterial therapy, IVT, or NRT. MAIN OUTCOMES AND MEASURES Final infarct volumes, rates of acceptable outcome defined as a modified Rankin Scale score of 0 to 3 at hospital discharge, and NIHSS threshold scores. RESULTS A total of 203 consecutive patients with ILVOs were evaluated. Baseline characteristics were similar among the 3 groups. The median infarct volume was significantly smaller for the IAT group (42 cm3) than for the IVT group (109 cm3; P = .001) or the NRT group (110 cm3; P < .01). A higher magnitude of infarct volume reduction in more proximal occlusions was noted in the IAT group compared with the IVT and NRT groups combined: internal carotid artery terminus (75 vs 190 cm3; P < .001), M1 middle cerebral artery (39 vs 109 cm3; P = .004), and M2 middle cerebral artery (33 vs 59 cm3; P = .04) occlusions. Patients were stratified based on NIHSS score at presentation (8-13, 14-19, and ≥20). For patients with an NIHSS score of 14 or higher at presentation, IAT significantly reduced FIV (46 cm3 with IAT vs 149 cm3 with IVT or NRT; P < .001) compared with patients with an NIHSS score of 8 to 13 (22 cm3 with IAT vs 44 cm3 with IVT or NRT; P = .40). Patients with an NIHSS score of 14 or higher who received IAT appear to benefit most from IAT. CONCLUSIONS AND RELEVANCE Our data suggest a greater reduction of FIV with IAT compared with either IVT or NRT. Moreover, patients with an NIHSS score of 14 or higher may be the best candidates for endovascular reperfusion therapy.

Relationship Between Lesion Topology and Clinical Outcome in Anterior Circulation Large Vessel Occlusions

Background and Purpose— Diffusion-weighted imaging (DWI) Alberta Stroke Program Early CT Score (ASPECTS), a surrogate of infarct volume, predicts outcome in anterior large vessel occlusion strokes. We aim to determine whether topological information captured by DWI ASPECTS contributes additional prognostic value. Methods— Adults with intracranial internal carotid artery, M1 or M2 middle carotid artery occlusions who underwent endovascular therapy were included. The primary outcome measure was poor clinical outcome (3-month modified Rankin Scale score, 3–6). Prognostic value of the 10 DWI ASPECTS regions in predicting poor outcome was determined by multivariable logistic regression, controlling for final infarct volume, age, and laterality. Results— Two hundred and thirteen patients (mean age, 66.1±14.5 years; median National Institutes of Health Stroke Scale, 15) were included. Inter-rater reliability was good for DWI ASPECTS (deep regions, &kgr;=0.72; cortical regions, &kgr;=0.63). All DWI ASPECTS regions with the exception of the putamen were significant predictors (P<0.05) of poor outcome in univariate analyses. Statistical collinearity among ASPECTS regions was not observed. Using penalized multivariable logistic regression, only M4 (odds ratio, 2.82; 95% confidence interval, 1.39–5.76) and M6 (odds ratio, 2.45; 95% confidence interval, 1.15–5.3) involvement were associated with poor outcome. M6 involvement independently predicted poor outcome in right hemispheric strokes (odds ratio, 5.8; 95% confidence interval, 1.9–20.3), whereas M4 (odds ratio, 4.3; 95% confidence interval, 1.3–15.0) involvement predicted poor outcome in left hemispheric strokes adjusting for infarct volume. Topologic information modestly improved the predictive ability of a prognostic score that incorporates age, infarct volume, and hemorrhagic transformation. Conclusions— Involvement of the right parieto-occipital (M6) and left superior frontal (M4) regions affect clinical outcome in anterior large vessel occlusions over and above the effect of infarct volume and should be considered during prognostication.

Potassium Channel Kv1.3 Is Highly Expressed by Microglia in Human Alzheimer's Disease

Recent genetic studies suggest a central role for innate immunity in Alzheimers disease (AD) pathogenesis, wherein microglia orchestrate neuroinflammation. Kv1.3, a voltage-gated potassium channel of therapeutic relevance in autoimmunity, is upregulated by activated microglia and mediates amyloid-mediated microglial priming and reactive oxygen species production in vitro. We hypothesized that Kv1.3 channel expression is increased in human AD brain tissue. In a blinded postmortem immunohistochemical semi-quantitative analysis performed on ten AD patients and ten non-disease controls, we observed a significantly higher Kv1.3 staining intensity (p = 0.03) and Kv1.3-positive cell density (p = 0.03) in the frontal cortex of AD brains, compared to controls. This paralleled an increased number of Iba1-positive microglia in AD brains. Kv1.3-positive cells had microglial morphology and were associated with amyloid-β plaques. In immunofluorescence studies, Kv1.3 channels co-localized primarily with Iba1 but not with astrocyte marker GFAP, confirming that elevated Kv1.3 expression is limited to microglia. Higher Kv1.3 expression in AD brains was also confirmed by western blot analysis. Our findings support that Kv1.3 channels are biologically relevant and microglia-specific targets in human AD.

Pittsburgh Outcomes After Stroke Thrombectomy Score Predicts Outcomes After Endovascular Therapy for Anterior Circulation Large Vessel Occlusions

Background and Purpose— Prognostication tools that predict good outcome in patients with anterior circulation large vessel occlusions after endovascular therapy are lacking. We aim to develop a tool that incorporates clinical and imaging data to predict outcomes after endovascular therapy. Methods— In a derivation cohort of anterior circulation large vessel occlusion stroke patients treated with endovascular therapy within 8 hours from time last seen well (n=247), we performed logistic regression to identify independent predictors of good outcome (90-day modified Rankin Scale, 0–2). Factors were weighted based on &bgr;-coefficients to derive the Pittsburgh Outcomes After Stroke Thrombectomy (POST) score. POST was validated in an institutional endovascular database (University of Pittsburgh Medical Center, n=393) and the Diffusion-Weighted Imaging Evaluation for Understanding Stroke Evolution Study-2 (DEFUSE-2) data set (n=105), as well as in patients treated beyond 8 hours (n=194) and in octogenarians (n=111). Results— In the derivation cohort, independent predictors (P<0.1) of good outcome included 24- to 72-hour final infarct volume (in cm3, P<0.001), age (years, P<0.001), and parenchymal hematoma types 1 and 2 (H, P=0.01). POST was calculated as age+0.5×final infarct volume+15×H. Patients with POST score <60 had a 91% chance of good outcome compared with 4% with POST score ≥120. POST accurately predicted good outcomes in the derivation (area under the curve [AUC]=0.85) and validation cohorts (University of Pittsburgh Medical Center, AUC=0.81; DEFUSE-2, AUC=0.86), as well as in patients treated beyond 8 hours (AUC, 0.85) and octogenarians (AUC=0.76). POST had better predictive accuracy for good and poor outcome than the ischemic stroke predictive risk score (iSCORE). Conclusions— POST score is a validated predictor of outcome in patients with anterior circulation large vessel occlusions after endovascular therapy.

Performance and Predictive Value of a User-Independent Platform for CT Perfusion Analysis: Threshold-Derived Automated Systems Outperform Examiner-Driven Approaches in Outcome Prediction of Acute Ischemic Stroke

BACKGROUND AND PURPOSE: Treatment strategies in acute ischemic stroke aim to curtail ischemic progression. Emerging paradigms propose patient subselection using imaging biomarkers derived from CT, CTA, and CT perfusion. We evaluated the performance of a fully-automated computational tool, hypothesizing enhancements compared with qualitative approaches. The correlation between imaging variables and clinical outcomes in a cohort of patients with acute ischemic stroke is reported. MATERIALS AND METHODS: Sixty-two patients with acute ischemic stroke and MCA or ICA occlusion undergoing multidetector CT, CTA, and CTP were retrospectively evaluated. CTP was processed on a fully operator-independent platform (RApid processing of PerfusIon and Diffusion [RAPID]) computing automated core estimates based on relative cerebral blood flow and relative cerebral blood volume and hypoperfused tissue volumes at varying thresholds of time-to-maximum. Qualitative analysis was assigned by 2 independent reviewers for each variable, including CT-ASPECTS, CBV-ASPECTS, CBF-ASPECTS, CTA collateral score, and CTA clot burden score. Performance as predictors of favorable clinical outcome and final infarct volume was established for each variable. RESULTS: Both RAPID core estimates, CT-ASPECTS, CBV-ASPECTS, and clot burden score correlated with favorable clinical outcome (P < .05); CBF-ASPECTS and collateral score were not significantly associated with favorable outcome, while hypoperfusion estimates were variably associated, depending on the selected time-to-maximum thresholds. Receiver operating characteristic analysis demonstrated disparities among tested variables, with RAPID core and hypoperfusion estimates outperforming all qualitative approaches (area under the curve, relative CBV = 0.86, relative CBF = 0.81; P < .001). CONCLUSIONS: Qualitative approaches to acute ischemic stroke imaging are subject to limitations due to their subjective nature and lack of physiologic information. These findings support the benefits of high-speed automated analysis, outperforming conventional methodologies while limiting delays in clinical management.

Activation of microglial P2Y12 receptor is required for outward potassium currents in response to neuronal injury

Microglia, the resident immune cells in the central nervous system (CNS), constantly survey the surrounding neural parenchyma and promptly respond to brain injury. Activation of purinergic receptors such as P2Y12 receptors (P2Y12R) in microglia has been implicated in chemotaxis toward ATP that is released by injured neurons and astrocytes. Activation of microglial P2Y12R elicits outward potassium current that is associated with microglial chemotaxis in response to injury. This study aimed at investigating the identity of the potassium channel implicated in microglial P2Y12R-mediated chemotaxis following neuronal injury and understanding the purinergic signaling pathway coupled to the channel. Using a combination of two-photon imaging, electrophysiology and genetic tools, we found the ATP-induced outward current to be largely dependent on P2Y12R activation and mediated by G-proteins. Similarly, P2Y12R-coupled outward current was also evoked in response to laser-induced single neuron injury. This current was abolished in microglia obtained from mice lacking P2Y12R. Dissecting the properties of the P2Y12R-mediated current using a pharmacological approach revealed that both the ATP and neuronal injury-induced outward current in microglia was sensitive to quinine (1mM) and bupivacaine (400μM), but not tetraethylammonium (TEA) (10mM) and 4-aminopyridine (4-AP) (5mM). These results suggest that the quinine/bupivacaine-sensitive potassium channels are the functional effectors of the P2Y12R-mediated signaling in microglia activation following neuronal injury.