Citations

The AML14 cell line and its AML14.3D10 subclone was established as a model cell system that exhibits phenotypic characteristics similar to that of human eosinophils. We investigated the effects of purinergic receptor stimulation on the physiological response of the AML 14.3D10 cell line to better evaluate its role as a suitable model for eosinophils using both molecular and functional approaches. Ca 2+ imaging experiments using Fura‐2 demonstrated the existence of purinergic receptors responsive to UTP (EC 50 of 8.9 x 10 −8 ), ATP γ‐s (EC 50 of 1.87 x 10 −7 ), and UDP (EC 50 of 7.44x 10 −6 ). Molecular identification of the purinergic receptor subtypes was confirmed by QRT‐PCR which demonstrated the presence of P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 receptors. Ratio imaging experiments using the membrane potential sensitive indicator DiBAC‐3 showed that stimulation with UTP produced a membrane hyperpolarization of approximately 10 mV that returned to the original resting potential within 10 minutes after stimulation. Whole cell perforated patch clamp experiments using amphotericin B revealed the presence of voltage‐gated potassium channel currents that were stimulated following treatment with UTP. Analysis of mRNA by QRT‐PCR indicated the existence of several voltage‐gated potassium channel α subunits including, Kv1.2, Kv1.3, Kv1.4, and Kv1.6. Previous studies of Kv1.3 channel function in T‐lymphocytes showed that the channel was involved in activation and proliferation, suggesting the possibility of a similar role related to tumorogenesis in the AML 14.3D10 cell line.

Alternaria aeroallergens stimulate allergic inflammation and exacerbation of respiratory distress in patients with asthma. Our previous studies have demonstrated that release of ATP and subsequent increases in intracellular [Ca 2+ ] was the result of Alternaria induced oxidative stress. Stimulation of Human Bronchial Epithelial Cells (HBECs) with Alternaria extract for 12 hours induced the expression of several phase II antioxidant enzymes, including cyclooxygenase 2 (COX2) which exhibited a 19 fold increase in mRNA expression. HBE cells loaded with CellRox™ Orange, a fluorogenic probe used for detecting oxidative stress, revealed that blocking COX2 inhibited resveratrol mediated inhibition of Alternaria induced oxidative stress. Furthermore induction of COX2 following Alternaria exposure was associated with an increase in expression of prostaglandin D 2 synthase and PGD 2 secretion into the extracellular media. Moreover, 15‐deoxy Δ 12,14 ‐protaglandin J 2 (15d‐PGJ 2 ) a metabolic product of PGD 2 was also shown to decrease Alternaria induced oxidative stress. These results suggest that upregulation of COX2 following sustained exposure to Alternaria induces PGD 2 synthesis and subsequent formation of 15d‐PGJ 2 , a known activator of Nrf2 signaling and ultimately, increased expression of phase II antioxidant enzymes.

Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to Alternaria alternata extract evoked rapid and sustained ATP release with greater efficacy observed in cells from asthmatic subjects. Previously, Alternaria aeroallergens were shown to elicit sustained increases in intracellular [Ca 2+ ] i that depended on coordinated activation of P2Y 2 and P2X 7 receptors. In this study, pretreatment with BAPTA‐AM inhibited ATP release, indicating dependency on increases in [Ca 2+ ] i . Alternaria ‐evoked ATP release exhibited a greater peak response and higher EC 50 value in cells obtained from asthmatic subjects compared to normal controls. Furthermore, the maximum increase in [Ca 2+ ] i was greater in cells from asthmatic subjects. The vesicle transport inhibitor brefeldin A blocked Alternaria ‐stimulated incorporation of fluorescent lipid (FM1–43) labeled vesicles into the plasma membrane and ATP release. Additionally, inhibiting vesicular ATP uptake with bafilomycin also blocked ATP release similar to brefeldin A. These results indicated that a primary mechanism for Alternaria ‐induced ATP release involved Ca 2+ ‐dependent vesicular transport. Serine and cysteine protease inhibitors also reduced Alternaria ‐induced ATP release. This result was consistent with our previous data showing that Alternaria ‐evoked increases in [Ca 2+ ] i depend on purinoceptor signaling.

Alternaria aeroallergens have been previously shown to stimulate allergic inflammation and exacerbation of respiratory distress in patients with asthma. An early event following exposure to Alternaria extract is the sustained release of ATP and a persistent increase in intracellular [Ca 2+ ] ([Ca 2+ ] i ) resulting from uptake of Ca 2+ ions from the extracellular solution. In the present study we tested the hyopothesis that the release of ATP and the subsequent increase in [Ca 2+ ] i was due to oxidative stress induced by exposure to Alternaria . ATP release following apical stimulation with Alternaria was measured in human primary bronchial epithelial cells using a luciferase/luciferin ATP measurement kit in combination with a modified luminometer that allows for tracking ATP release in real time. Pretreatment with glutathione (GSH) produced a concentration‐dependent inhibition of ATP release with an EC 50 = 500 μM. Furthermore, GSH blocked the increase in [Ca 2+ ] i typically observed following apical exposure to Alternaria. Moreover an increase in the expression of specific antioxidant protein mRNAs was also observed after 12 hours of treatment with Alternaria extract. These proteins included peroxiredoxins 1‐6, keratin 1, cyclooxygenase 2 (COX2) and oxidative stress responsive 1 protein with COX2 having the highest level of expression (~20 fold). These results implicate oxidative stress as an important initiating event in Alternaria evoked airway inflammation. Grant Funding Source : Supported by NIH: HL110539

Objective: To evaluate the incidence of and characterize the presentation of neuropsychiatric symptoms and/or Parkinsonism as a presentation of central nervous system lymphoma (CNSL) in either its primary CNSL form or when it spreads to the brain in systemic diffuse large B-cell lymphoma (secondary CNSL). Patients and Methods: With Institutional Review Board approval we identified patients who had been treated at Mayo Clinic from 1998 to 2018 and were recorded to have a combination of ICD 9/10 codes for CNSL and various psychiatric diagnoses. Results: A total of 20 of the 232 patients (9%) were noted to have neuropsychiatric symptoms preceding diagnosis. The average age at diagnosis was 62, with even split for sex. The majority (85%) of patients had primary CNSL. The average duration of symptoms before the diagnosis was 4.8 months. Confusion (80%), depression (40%), apathy (30%), anxiety (30%), and agitation (30%) were the most common symptoms identified. The majority (65%) of patients had subcortical lesions followed by the frontal lobe (50%). Parkinsonism was identified in 5 of the 20 patients with 4 demonstrating resolution of symptoms with treatment of the lymphoma. Conclusions: Neuropsychiatric symptoms are a rare but notable symptom before the presentation of CNSL. There is an increasing awareness of neurological illness presenting as pure psychiatric disturbance, prompting the need to exclude organic and treatable diseases, particularly in elderly patients. Acknowledgment and diagnosis are important for an appropriate management as there is a significant impact on patient and caregiver quality of life.

Abstract Background Accurate assessments of patient response to therapy are a critical component of personalized medicine. In glioblastoma (GBM), the most aggressive form of brain cancer, tumor growth dynamics are heterogenous across patients, complicating assessment of treatment response. This study aimed to analyze days gained (DG), a burgeoning model-based dynamic metric, for response assessment in patients with recurrent GBM who received bevacizumab-based therapies. Methods DG response scores were calculated using volumetric tumor segmentations for patients receiving bevacizumab with and without concurrent cytotoxic therapy (N = 62). Kaplan–Meier and Cox proportional hazards analyses were implemented to examine DG prognostic relationship to overall (OS) and progression-free survival (PFS) from the onset of treatment for recurrent GBM. Results In patients receiving concurrent bevacizumab and cytotoxic therapy, Kaplan–Meier analysis showed significant differences in OS and PFS at DG cutoffs consistent with previously identified values from newly diagnosed GBM using T1-weighted gadolinium-enhanced magnetic resonance imaging (T1Gd). DG scores for bevacizumab monotherapy patients only approached significance for PFS. Cox regression showed that increases of 25 DG on T1Gd imaging were significantly associated with a 12.5% reduction in OS hazard for concurrent therapy patients and a 4.4% reduction in PFS hazard for bevacizumab monotherapy patients. Conclusion DG has significant meaning in recurrent therapy as a metric of treatment response, even in the context of anti-angiogenic therapies. This provides further evidence supporting the use of DG as an adjunct response metric that quantitatively connects treatment response and clinical outcomes.

Allergens derived from the common fungus Alternaria alternata induce oxidative stress in human airway epithelial (HBE) cells that leads to the regulated release of ATP and subsequent activation of P2X 7 receptors that facilitate Ca 2+ uptake into the cell. Sustained increases in [Ca 2+ ] i are required for nuclear processing and secretion of IL‐33, which initiates allergic inflammation by activating ST2 receptors on immune cells to induce Th2 cytokine secretion. Pretreatment of HBE cells with the K2P channel blockers bupivacaine or quinidine produced a significant increase in ATP release following Alternaria stimulation. This response was blocked by inhibiting ATP loading into membrane vesicles by treatment with 200 nM bafilomycin A 1 before allergen exposure. Similarly, pretreatment with the PKC inhibitor Go6976 also blocked approximately 50% of Alternaria ‐induced ATP release and completely abolished the increase in ATP release produced by pretreatment with K2P channel blockers. The effects of bupivacaine and quinidine were comparable to the effect of phorbol 12‐myristate 13‐acetate (PMA, 2 mM), a well‐characterized PKC activating compound prior to Alternaria stimulation. These results support the conclusion that K2P channel inhibition is associated with PKC activation, which functions to potentiate the effects of Alternaria allergens on vesicular ATP release. We propose that enhanced PKC activity is dependent on membrane depolarization associated with inhibition of constitutive, K2P channel mediated K + efflux from these cells. Support or Funding Information This study was supported by grants from the NIH (R01‐AI128729), awarded to HK and SMO and from the Minnesota Agriculture Experiment Station (AES‐0016097) awarded to SMO.

Key points Alternaria aeroallergens induce the release of ATP from human bronchial epithelial (HBE) cells by activating a conductive pathway involving voltage‐dependent anion channel‐1 (VDAC‐1) and by exocytosis of ATP localized within membrane vesicles. Inhibition of VDAC‐1 blocked Alternaria‐evoked Ca2+ uptake across the plasma membrane of HBE cells and interleukin (IL)‐33 release into the extracellular media. Reducing cholesterol content with a cholesterol scavenger (β‐methylcyclodextrin) or statin compound (simvastatin) blocked ATP and IL‐33 release by lowering the expression of VDAC‐1 in the plasma membrane. Pretreatment with simvastatin for 24 h also inhibited the increase in tight junction macromolecule permeability that occurs following Alternaria exposure. These results establish a novel role for VDAC‐1 as a mechanism underlying ATP release induced by fungal allergens and suggests a possible therapeutic use for cholesterol lowering compounds in reducing Alternaria‐stimulated allergic inflammation. AbstractHuman bronchial epithelial (HBE) cells exposed to allergens derived from the common saprophytic fungus, Alternaria alternata release ATP, which in turn stimulates P2X7 receptor‐mediated Ca2+ uptake across the plasma membrane. The subsequent increase in intracellular calcium concentration induces proteolytic processing and secretion of interleukin (IL)‐33, a critical cytokine involved in the initiation of allergic airway inflammation. A major objective of the present study was to identify the mechanism responsible for conductive ATP release. The results show that pretreatment of HBE cells with inhibitors of the voltage‐dependent anion channel‐1 (VDAC‐1) or treatment with a VDAC‐1 selective blocking antibody or silencing mRNA expression of the channel by RNA interference, inhibit Alternaria‐evoked ATP release. Moreover, inhibition of VDAC‐1 channel activity or reducing protein expression blocked the secretion of IL‐33. Similarly, reducing the cholesterol content of HBE cells with simvastatin or the cholesterol scavenger β‐methylcyclodextrin also blocked ATP release and IL‐33 secretion by decreasing the level of VDAC‐1 expression in the plasma membrane. In addition, simvastatin inhibited the increase in tight junction macromolecule permeability that was previously observed after Alternaria exposure. These results demonstrate a novel function for VDAC‐1 as the conductive mechanism responsible for Alternaria‐induced ATP release, an essential early step in the processing, mobilization and secretion of IL‐33 by the airway epithelium. Furthermore, the simvastatin‐evoked reduction of VDAC‐1 expression in the plasma membrane, suggests the possibility that cholesterol lowering compounds may be beneficial in alleviating allergic airway inflammation induced by fungal allergens.