Cindy B. Uhl

Mitochondrial injury and caspase activation by the local anesthetic lidocaine

Background:Lidocaine, a local anesthetic, can be neurotoxic. However, the cellular mechanisms of its neurotoxicity at concentrations encountered during spinal anesthesia remain unclear. Methods:The authors examined the mechanisms of lidocaine neurotoxicity in the ND7 cell line derived from rat dorsal root ganglion. Individual neurons were assayed by flow cytometry or microscopy using fluorescent probes of plasma membrane integrity, mitochondrial membrane potential, caspase activity, phospholipid membrane asymmetry, and mitochondrial cytochrome c release. Results:In the ND7 cell line, lidocaine at 185 mm × 10 min to 2.3 mm × 24 h caused necrosis or late apoptosis. Equimolar Tris buffer and equipotent tetrodotoxin controls were not toxic, indicating that neither osmotic nor Na+-blocking effects explain lidocaine neurotoxicity. The earliest manifestation of lidocaine neurotoxicity was complete loss of mitochondrial membrane potential within 5 min after exposure to lidocaine at a concentration of 19 mm or greater. Consistent with these data, 37 mm lidocaine (1%) induced release of mitochondrial cytochrome c into the cytoplasm, as well as plasma membrane blebbing, loss of phosphatidylserine membrane asymmetry, and caspase activation, with release of mitochondrial cytochrome c to the cytoplasm within 2 h. Treatment with z-VAD-fmk, a specific inhibitor of caspases, prevented caspase activation and delayed but did not prevent neuronal death, but did not inhibit the other indicators of apoptosis. Conclusions:Collectively, these data indicate that lidocaine neurotoxicity involves mitochondrial dysfunction with activation of apoptotic pathways.

Effect of local anesthetic on neuronal cytoplasmic calcium and plasma membrane lysis (necrosis) in a cell culture model

Background To investigate the mechanism by which rare cases of spinal local anesthetic (LA) neurotoxicity occur, we have tested the hypotheses that LAs elevate cytoplasmic calcium (Ca2+cyt), that this is associated with a neurotoxic effect, and that lidocaine and bupivacaine differ in their neurotoxicity. Methods Neurons of the ND7 cell culture line, derived from dorsal root ganglion, were loaded with fura-2 and analyzed by digitized video fluorescence microscopy during 60 min LA exposure, allowing determination of Ca2+cyt and time of necrotic cell death (plasma membrane lysis) at the single neuron level. Results Lidocaine 0.1% and bupivacaine 0.025% caused minimal changes in Ca2+cyt. Lidocaine 0.5–5% and bupivacaine 0.125–0.625% caused an early, small (less than threefold), concentration-dependent increase in Ca2+cyt that was transient and returned to near baseline within 10 min. Lidocaine 2.5% and 5% then caused a sustained, greater than ten-fold increase in Ca2+cyt and death in some neurons during the 60 min exposure period. Pretreatment with thapsigargin eliminated the initial transient increase in Ca2+cyt, consistent with endoplasmic reticulum (ER) as its source, and increased neuronal death with 5% lidocaine, suggesting that lidocaine neurotoxicity can be increased by failure of ER to take up elevated Ca2+cyt. The later sustained increase in Ca2+cyt seen with 2.5 and 5% lidocaine was prevented in Ca2+-free medium, and restored when Ca2+ was added back to the buffer in the presence of lidocaine, suggesting that higher concentrations of lidocaine increase influx of Ca2+ through the plasma membrane. Conclusions In this model, lidocaine greater than 2.5% elevates Ca2+cyt to toxic levels. Bupivacaine and lower concentrations of lidocaine transiently alter Ca2+cyt homeostasis for several minutes, but without an immediate neurotoxic effect within 60 min.

A phase II study of ABT-510 (thrombospondin-1 analog) for the treatment of metastatic melanoma.

Objectives:Thrombospondins are natural inhibitors of angiogenesis, tumor metastases, and tumor growth (melanoma). ABT-510 is a synthetic analog of thrombospondin-1, well tolerated in phase I studies. We conducted a phase II trial evaluating the clinical efficacy of ABT-510 and its effects on biomarkers of angiogenesis and immunity in patients with metastatic melanoma (MM). Patients and Methods:A 2-stage phase II clinical trial was conducted to assess the clinical efficacy, safety, and pharmacodynamic effects (angiogenesis and immunity) of ABT-510 in patients with stage IV melanoma. The primary endpoint was 18-week treatment failure rate. Patients self-administered 100 mg of ABT-510 subcutaneously twice daily. Blood samples were collected at baseline and every 3 weeks while on therapy. Eligible patients demonstrated measurable disease, good performance status and no evidence of intracranial metastases. Correlative laboratory studies evaluated biomarkers of angiogenesis and immunity. Results:Twenty-one patients were enrolled. Most patients were stage M1c (71%) and all had prior therapy for MM. Only 3 of the first 20 patients enrolled were progression free and on treatment at 18 weeks resulting in early termination of the study. Decreases in peripheral blood VEGF-A levels and VEGF-C levels, and CD146 and CD34/133 counts relative to pretreatment were detected. Limited changes in antitumor T cell immunity were observed. Conclusions:ABT-510 therapy administered at 100 mg twice/day in patients with MM did not demonstrate definite clinical efficacy. Further dose escalation or combination with cytotoxic therapy may be more effective therapeutically.

Improved detection of drug cytotoxicity in the soft agar colony formation assay through use of a metabolizable tetrazolium salt

Use of a metabolizable tetrazolium salt was observed to facilitate assessments of tumor cell drug sensitivity in the soft-agar colony formation assay. Enzyme-mediated staining permits discrimination between viable and non-viable groups of cells so that drug-induced cytotoxicity is clearly identifiable by visual inspection as well as by computerized image analysis. The technique appears to be especially useful in the evaluation of primary tumor cell cultures which often contain substantial numbers of non-viable cellular aggregates.

Reactive oxygen species and human platelet GP IIb/IIIa receptor activation

This study concerned reactive oxygen species for their potential to activate human platelet GP IIb/IIIa receptors. All cells produce reactive oxygen species  — radicals that can abstract electrons and hydrogen atoms from biological molecules to alter cell function. In many cells, radicals contribute to cellular signaling. In platelets, the predominant oxidant effect is platelet activation. Less is known concerning oxidants and GP IIb/IIIa receptor activation. The first aim of the current study was to confirm that although both H2O2 and tert butyl hydroperoxide both predispose platelets to aggregation; neither directly activates GP IIb/IIIa receptors. The second aim was to demonstrate that even in the presence of extracellular redox iron; H2O2 does not activate GP IIb/IIIa receptors. The third aim was to determine if extracellular superoxide anions evoke GP IIb/IIIa activation. Finally, a role for intra-platelet iron in GP IIb/IIIa activation was examined. Intracellular superoxide anions are produced in excess during platelet activation and curiously, they are uniquely able to increase intracellular free iron. This iron can, in a redox manner, generate radicals and these iron dependent species modulate signaling systems, including systems associated with adhesion receptor activation. In the current studies, platelets in suspension were exposed to H2O2 and to tert butyl hydroperoxide, to H2O2 plus ferrous or ferric chloride (± ascorbate to enhance iron redox cycling) and to xanthine plus xanthine oxidase to generate extra-platelet superoxide anions. Intra-platelet iron was increased with iron ionophore 8-hydroxyquinoline. During flow cytometry, intra-platelet oxidant state was assessed with the redox sensitive fluorescent indicator H2DCF, while GP IIb/IIIa activation was assessed using fluorescent antibody PAC-1. Results showed that although all the oxidizing systems examined increased intra-platelet oxidant state, GP IIb/IIIa receptors were not activated by H2O2, by tert butyl hydroperoxide, by H2O2 plus iron (± ascorbate) or by xanthine plus xanthine oxidase. In contrast, iron plus ionophore 8-hydroxyquinoline evoked GP IIb/IIIa activation. Platelet positivity for PAC-1 increased from 2 ± 0.2 to 28 ± 7% (P < 0.005). However this response, although vigorous, was less than 56 ± 8% (P < 0.001) evoked by thrombin 0.1 milliunit/ml. In conclusion, the results indicated that oxidant systems external to platelets did not activate GP IIb/IIIa receptors while increased intra-platelet iron was associated with appearance of cytosolic oxidizing species and with GP IIb/IIIa receptor activation.

Effect of Volatile Anesthetics on Hydrogen Peroxide-induced Injury in Aortic and Pulmonary Arterial Endothelial Cells

Background Oxidant damage to endothelial cells occurs during inflammation and reperfusion after ischemia, mediated in part by endogenously produced hydrogen peroxide (H2 O2). Previous studies have established a role for increased cytosolic calcium in the mechanism of endothelial oxidant injury, and have suggested that volatile anesthetics may exacerbate oxidant injury in pulmonary endothelium. However, the effect of volatile anesthetics on oxidant injury to systemic arterial endothelial cells, and their effect on oxidant‐related changes in cytosolic calcium homeostasis, have not been reported previously. Methods Primary cultures of human aortic and pulmonary arterial endothelial cells were studied. The rate of cell death after H2 O2 exposure was determined in cell suspension by propidium iodide fluorimetry and lactate dehydrogenase release. The final extent of cell death 24 h after H2 O2 exposure was determined in monolayer cultures by methyl thiazolyl tetrazolium reduction. Cytosolic calcium and cell death were determined in single cells using fura‐2 and propidium iodide imaging with digitized, multiparameter, fluorescent video microscopy. Results In aortic endothelial cells, clinical concentrations of halothane (1.0%) and isoflurane (1.5%) decreased both the rate of cell death and the final extent of cell death after H2 O2 exposure, with halothane being more protective. Supraclinical concentrations of halothane (2.7%) and isoflurane (4.0%) were less protective. In pulmonary arterial endothelial cells, halothane and isoflurane had essentially no effect on H2 O2 ‐mediated cell death. The protective effect of anesthetic in aortic endothelial cells was not due to an enhanced removal of H2 O2 by endogenous enzymes. Hydrogen peroxide exposure caused a large increase in cytosolic calcium well before cell death, and this was moderated by anesthetic treatment. Conclusions The effect of volatile anesthetics on oxidant injury to endothelial cells may differ between cells derived from systemic and pulmonary vascular beds. Halothane, and to a lesser extent, isoflurane, protects against oxidant injury in aortic endothelial cells. The mechanism of protection may involve modulation of the interaction of H2 O2 with vital cellular constituents, and/or amelioration of the toxic increase in cytosolic calcium that follows such interaction.

A reproducible method for the enumeration of functional (cytokine producing) versus non-functional peptide-specific cytotoxic T lymphocytes in human peripheral blood

One of the most difficult laboratory challenges in the field of therapeutic cancer vaccines has been the development of uncomplicated/reproducible methods for the quantification of vaccine immunization efficacy in peripheral blood of cancer patients. Existing methods are limited by lack of functional information (tetramers), difficulties with standardization/reproducibility [enzyme‐linked immunosorbent spot (ELISPOT)] and reliance on endogenous (sample‐specific) antigen presentation (cytokine flow cytometry). Herein we present a reproducible method utilizing an artificial antigen‐presenting cell platform for flow cytometry‐based quantification of the frequency and activation status of peptide‐specific cytotoxic T lymphocytes. The methodology [currently presented for cytomegalovirus human leucocyte antigen (HLA)‐A2 cognant peptide antigens] allows simultaneous ex vivo quantification of activated (cytokine‐producing) and inactive tetramer‐positive T cells following HLA class I/peptide/CD28 stimulation independent of endogenous antigen presentation. The simplicity and reliability of the assay provide for high‐throughput applications and automation. The utility and application of this method are discussed.

Diagnostic laboratory standardization and validation of platelet transmission electron microscopy

Abstract Platelet transmission electron microscopy (PTEM) is considered the gold standard test for assessing distinct ultrastructural abnormalities in inherited platelet disorders (IPDs). Nevertheless, PTEM remains mainly a research tool due to the lack of standardized procedures, a validated dense granule (DG) count reference range, and standardized image interpretation criteria. The aim of this study was to standardize and validate PTEM as a clinical laboratory test. Based on previously established methods, we optimized and standardized preanalytical, analytical, and postanalytical procedures for both whole mount (WM) and thin section (TS) PTEM. Mean number of DG/platelet (plt), percentage of plts without DG, platelet count (PC), mean platelet volume (MPV), immature platelet fraction (IPF), and plt light transmission aggregometry analyses were measured on blood samples from 113 healthy donors. Quantile regression was used to estimate the reference range for DG/plt, and linear regression was used to assess the association of DG/plt with other plt measurements. All PTEM procedures were standardized using commercially available materials and reagents. DG interpretation criteria were established based on previous publications and expert consensus, and resulted in improved operator agreement. Mean DG/plt was stable for 2 days after blood sample collection. The median within patient coefficient of variation for mean DG/plt was 22.2%; the mean DG/plt reference range (mid-95th %) was 1.2–4.0. Mean DG/plt was associated with IPF (p = .01, R2 = 0.06) but not age, sex, PC, MPV, or plt maximum aggregation or primary slope of aggregation (p > .17, R2 < 0.02). Baseline ultrastructural features were established for TS-PTEM. PTEM was validated using samples from patients with previously established diagnoses of IPDs. Standardization and validation of PTEM procedures and interpretation, and establishment of the normal mean DG/plt reference range and PTEM baseline ultrastructural features, will facilitate implementation of PTEM as a valid clinical laboratory test for evaluating ultrastructural abnormalities in IPDs.

Practice patterns in the diagnosis of inherited platelet disorders within a single institution.

&NA; The diagnosis of inherited platelet disorders (IPDs) is challenging with variable diagnostic practices existing between institutions. To determine patterns and utility of diagnostic testing practices for IPDs within a single institution, a retrospective cohort study was performed. Records of 50 patients (50% women), median age 32 years (1 day to 81 years) were analyzed. In total, 28 (53%) had a positive International Society of Thrombosis and Hemostasis Bleeding Assessment Tool score. Test-ordering patterns were highly variable. All patients had platelet morphology analysis by light microscopy. In total, 42 (84%) underwent light transmission aggregometry, 43 (86%) platelet function analyzer, 37 (74%) platelet electron microscopy, 25 (50%) flow cytometry, and 15 (30%) genetic testing. Platelet function analyzer and light transmission aggregometry were always used as first-order tests, followed by platelet transmission electron microscopy and flow cytometry (81 and 84%, respectively). Genetic testing was obtained up front in five cases (33% of orders), mostly in patients with syndromic thrombocytopenia or in the setting of a known genetic disorder. Test-ordering practices did not adhere to published algorithms. Even within a single institution, great heterogeneity exists in the testing approach to IPDs. Although, a large proportion of cases were studied with platelet transmission electron microscopy and flow cytometry, standard platelet assays established the diagnosis in a great majority. Standardization of testing practices, first beginning at the institutional level is a much needed step forward.

Myeloid malignancy presenting with a platelet storage pool disorder

Platelet storage pool disorders (SPDs) are collectively characterized by either a congenital or an acquired defi ciency of alpha and/or dense platelet granules, resulting in a qualitative platelet function defect. Storage pool defi ciency in association with myeloid malignancies has been described [1,2], but rarely is the presenting manifestation of the disease. A 56-year-old female with a history of polymyalgia rheumatica, on corticosteroid therapy, was referred from her rheumatologist with a 2-year history of easy bruisability, prolonged bleeding with minor cuts, and recurrent bleeding from a gastric ulcer that was initially attributed to her steroid use. During her initial work-up she was found to have mild normocytic anemia (11 g/dL) and thrombocytopenia (115 000 / μ L). Her leukocyte count was 3800/ μ L with a normal diff erential. A peripheral smear revealed hypogranular platelets. Complete blood counts over the past 2 years had revealed normocytic anemia but a normal platelet and leukocyte count. Th rombocytopenia was fi rst noted 5 months prior to her presentation. Routine coagulation testing revealed a normal prothrombin time (PT) and a normal activated partial thromboplastin time (APTT). Qualitative platelet function tests revealed both epinephrine and adenosine diphosphate (ADP) closure times longer than 300 s. Platelet light transmission aggregation studies demonstrated absent aggregation in response to arachidonate (1.7 mM) and epinephrine (50 μ M) and normal aggregation in response to ADP (5 μ M), collagen (250 μ g/ mL) and ristocetin (0.75 mg/mL). Platelet electron microscopy revealed signifi cant platelet ultrastructural abnormalities (Figure 1), including a decrease in the number of dense bodies (mean dense body count/platelet was 0.5; normal range is 2) and alpha granules, abnormal platelet to platelet attachments, the presence of Golgi complexes and complex tubular networks. Platelet fl ow cytometry studies did not identify glycoprotein IIb, IIIa, Ib, IX, IV or Ia/IIa defi ciency. A bone marrow biopsy was performed. Th is revealed 60% cellularity, moderate megakaryocytic hyperplasia with occasional clusters and variable morphology, including small, monolobated forms, large, hyperlobated forms and hyperchromatic nuclei, grade 1 reticulin fi brosis, and no increase in blasts. JAK2V617F mutation analysis was negative and the karyotype revealed del20q in all metaphases. Th ese fi ndings were thought to be consistent with involvement by a chronic myeloid neoplasm that could not be further classifi ed. Repeat bone marrow biopsy 7 months after her initial diagnosis revealed marked hypercellularity (95%), an increase in the number of megakaryocytes with abnormal morphology and clustering, dysplastic granulocytes and grade 2 – 3 reticulin fi brosis, with no increase in myeloblasts. Collectively these fi ndings were thought to represent an acquired SPD that had developed in the setting of an underlying myelodysplastic/myeloproliferative neoplasm (MDS/MPN) overlap syndrome. Abnormalities of epinephrine, ADP or collagen-induced platelet aggregation have been shown in patients with myeloid malignancies [3 – 5]. Th e patients almost always