Mycolactone cytotoxicity in Schwann cells could explain nerve damage in Buruli ulcer

Buruli ulcer is a chronic painless skin disease caused by Mycobacterium ulcerans. The local nerve damage induced by M. ulcerans invasion is similar to the nerve damage evoked by the injection of mycolactone in a Buruli ulcer mouse model. In order to elucidate the mechanism of this nerve damage, we tested and compared the cytotoxic effect of synthetic mycolactone A/B on cultured Schwann cells, fibroblasts and macrophages. Mycolactone induced much higher cell death and apoptosis in Schwann cell line SW10 than in fibroblast line L929. These results suggest that mycolactone is a key substance in the production of nerve damage of Buruli ulcer

Mycolactone Is Responsible for the Painlessness of Mycobacterium ulcerans Infection (Buruli Ulcer) in a Murine Study▿

Buruli ulcer is a chronic skin disease caused by Mycobacterium ulcerans, which produces a toxic lipid mycolactone. Despite the extensive necrosis and tissue damage, the lesions are painless. This absence of pain prevents patients from seeking early treatment and, as a result, many patients experience severe sequelae, including limb amputation. We have reported that mice inoculated with M. ulcerans show loss of pain sensation and nerve degeneration. However, the molecules responsible for the nerve damage have not been identified. In order to clarify whether mycolactone alone can induce nerve damage, mycolactone A/B was injected to footpads of BALB/c mice. A total of 100 μg of mycolactone induced footpad swelling, redness, and erosion. The von Frey sensory test showed hyperesthesia on day 7, recovery on day 21, and hypoesthesia on day 28. Histologically, the footpads showed epidermal erosion, moderate stromal edema, and moderate neutrophilic infiltration up to day 14, which gradually resolved. Nerve bundles showed intraneural hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Ultrastructurally, vacuolar change of myelin started on day 14 and gradually subsided by day 42, but the density of myelinated fibers remained low. This study demonstrated that initial hyperesthesia is followed by sensory recovery and final hypoesthesia. Our present study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. Furthermore, mice injected with 200 μg of mycolactone showed pulmonary hemorrhage. This is the first study to demonstrate the systemic effects of mycolactone

Detection of apoptosis by fluorescence microscopy.

<p>Fibroblasts and Schwann cells were cultured in chamber slides for 24 hrs. Synthetic mycolactone A/B with a final concentration of 3 ng/ml, 30 ng/ml, or 300ng/ml was added and further cultured for 12 and 24 hrs. Fixed cells were stained with fluorescent reagents. Red: cleaved caspase-3 (Rabbit anti-Cleaved Caspase-3 (1:1000)/Alexa Fluor 594 Goat Anti-Rabbit IgG); Blue: nuclear DNA (Hoechst 33342); and Green: intracellular actin (Alexa Fluor 488 Phalloidin). Cells were examined under a confocal laser scanning microscope (Olympus: FV10i-DOC Laser Scanning Microscope). As a positive control, actinomycin-D was added to the culture. Expression of cleaved caspase-3 was compared at 12 and 24 hrs after administration of mycolactone. Positive cell rate (number of cleaved caspase 3 positive cells/number of Hoechst 33342 positive cells, %) was calculated. (A and B) In the four conditions (12 and 24 hrs, 30 and 300 ng/ml mycolactone), the expression of cleaved caspase 3 was observed in the cytoplasm of SW10 Schwann cells (10–21%) and in some of L929 fibroblasts (2–3%). (C) Actinomycin-D showed the expression of cleaved caspase 3 to SW10 and L929 cells.</p

Comparison of synthetic mycolactone A/B and its remote diastereomer.

<p>L929 fibroblasts and SW10 Schwann cells were cultured and treated with the same concentration of mycolactone A/B or mycolactone A/B remote diastereomer. Trypan blue staining and the TUNEL assay were performed. Synthetic mycolactone A/B (A, B) and its remote diastereomer (C, D) exerted identical cytotoxicity in both fibroblasts and Schwann cells at the same concentration.</p

Effect of mycolactone on cellular morphology.

<p>SW10 mouse Schwann cells and L929 mouse fibroblast cells were cultured for 24 hrs. Synthetic mycolactone A/B diluted to a final concentration of 3 ng/ml, 30 ng/ml, or 300 ng/ml was added to a cell culture and incubated for 12, 24, 48 and 72 hrs. Photomicrographs were taken by a phase-contrast microscope. Cells treated with 3 ng/ml of mycolactone showed no floating cells at 24, 48, or 72 hrs (A, B). Fibroblasts showed no changes until 48 hrs, but partial detachment began at 72 hrs with 30 ng/ml of mycolactone A/B (C). Schwann cells showed round shrinkage and floating at 24 hrs with 30 ng/ml of mycolactone A/B. Some of the cells remained adherent at 48 hrs, but all cells were detached at 72 hrs (D). Bar = 100 μm.</p

TUNEL assay in SW10 and L929 cells.

<p>L929 fibroblasts and SW10 Schwann cells were cultured on BD Falcon 2 well culture slides for 24 hrs (2.0x10⁴ cells/well) before synthetic mycolactone A/B was added. The cells were fixed with 4% paraformaldehyde-PBS and washed with PBS at 24 hrs and 48 hrs. The TUNEL assay and mild hematoxylin nuclear staining were performed. Total cell number and number of TUNEL-positive cells were counted using photomicrographs. At 24 hrs, 30 ng/ml of mycolactone induced less apoptosis (brown nuclear staining) in fibroblasts (A) than in Schwann cells (B). Three ng/ml of mycolactone did not show significant TUNEL reaction, while 300 ng/ml produced a strong TUNEL reaction in both fibroblasts and Schwann cells. In the quantitative analysis (C), fibroblasts in 3 ng/ml of mycolactone showed no apoptosis at 24 and 48 hrs, but 30 and 300 ng/ml of mycolactone induced apoptosis in a concentration-dependent and time-dependent manner. Schwann cells also showed no apoptosis at 24 and 48 hrs with 3 ng/ml of mycolactone; however, 30 and 300 ng/ml of mycolactone induced more apoptosis in Schwann cells (91% at 48 hrs, 300 ng/mg) than in fibroblasts (48% at 48 hrs, 300 ng/ml). Bar = 100 μm.</p

Risk for Major Bleeding in Patients Receiving Ticagrelor Compared With Aspirin After Transient Ischemic Attack or Acute Ischemic Stroke in the SOCRATES Study (Acute Stroke or Transient Ischemic Attack Treated With Aspirin or Ticagrelor and Patient Outcomes)

Abstract: Background: Patients with minor acute ischemic stroke or transient ischemic attack are at high risk for subsequent stroke, and more potent antiplatelet therapy in the acute setting is needed. However, the potential benefit of more intense antiplatelet therapy must be assessed in relation to the risk for major bleeding. The SOCRATES trial (Acute Stroke or Transient Ischemic Attack Treated With Aspirin or Ticagrelor and Patient Outcomes) was the first trial with ticagrelor in patients with acute ischemic stroke or transient ischemic attack in which the efficacy and safety of ticagrelor were compared with those of aspirin. The main safety objective was assessment of PLATO (Platelet Inhibition and Patient Outcomes)\u2013defined major bleeds on treatment, with special focus on intracranial hemorrhage (ICrH). Methods: An independent adjudication committee blinded to study treatment classified bleeds according to the PLATO, TIMI (Thrombolysis in Myocardial Infarction), and GUSTO (Global Use of Strategies to Open Occluded Coronary Arteries) definitions. The definitions of ICrH and major bleeding excluded cerebral microbleeds and asymptomatic hemorrhagic transformations of cerebral infarctions so that the definitions better discriminated important events in the acute stroke population. Results: A total of 13 130 of 13 199 randomized patients received at least 1 dose of study drug and were included in the safety analysis set. PLATO major bleeds occurred in 31 patients (0.5%) on ticagrelor and 38 patients (0.6%) on aspirin (hazard ratio, 0.83; 95% confidence interval, 0.52\u20131.34). The most common locations of major bleeds were intracranial and gastrointestinal. ICrH was reported in 12 patients (0.2%) on ticagrelor and 18 patients (0.3%) on aspirin. Thirteen of all 30 ICrHs (4 on ticagrelor and 9 on aspirin) were hemorrhagic strokes, and 4 (2 in each group) were symptomatic hemorrhagic transformations of brain infarctions. The ICrHs were spontaneous in 6 and 13, traumatic in 3 and 3, and procedural in 3 and 2 patients on ticagrelor and aspirin, respectively. In total, 9 fatal bleeds occurred on ticagrelor and 4 on aspirin. The composite of ICrH or fatal bleeding included 15 patients on ticagrelor and 18 on aspirin. Independently of bleeding classification, PLATO, TIMI, or GUSTO, the relative difference between treatments for major/severe bleeds was similar. Nonmajor bleeds were more common on ticagrelor. Conclusions: Antiplatelet therapy with ticagrelor in patients with acute ischemic stroke or transient ischemic attack showed a bleeding profile similar to that of aspirin for major bleeds. There were few ICrHs. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01994720.Abstract: BACKGROUND: Patients with minor acute ischemic stroke or transient ischemic attack are at high risk for subsequent stroke, and more potent antiplatelet therapy in the acute setting is needed. However, the potential benefit of more intense antiplatelet therapy must be assessed in relation to the risk for major bleeding. The SOCRATES trial (Acute Stroke or Transient Ischemic Attack Treated With Aspirin or Ticagrelor and Patient Outcomes) was the first trial with ticagrelor in patients with acute ischemic stroke or transient ischemic attack in which the efficacy and safety of ticagrelor were compared with those of aspirin. The main safety objective was assessment of PLATO (Platelet Inhibition and Patient Outcomes)-defined major bleeds on treatment, with special focus on intracranial hemorrhage (ICrH). METHODS: An independent adjudication committee blinded to study treatment classified bleeds according to the PLATO, TIMI (Thrombolysis in Myocardial Infarction), and GUSTO (Global Use of Strategies to Open Occluded Coronary Arteries) definitions. The definitions of ICrH and major bleeding excluded cerebral microbleeds and asymptomatic hemorrhagic transformations of cerebral infarctions so that the definitions better discriminated important events in the acute stroke population. RESULTS: A total of 13 130 of 13 199 randomized patients received at least 1 dose of study drug and were included in the safety analysis set. PLATO major bleeds occurred in 31 patients (0.5%) on ticagrelor and 38 patients (0.6%) on aspirin (hazard ratio, 0.83; 95% confidence interval, 0.52-1.34). The most common locations of major bleeds were intracranial and gastrointestinal. ICrH was reported in 12 patients (0.2%) on ticagrelor and 18 patients (0.3%) on aspirin. Thirteen of all 30 ICrHs (4 on ticagrelor and 9 on aspirin) were hemorrhagic strokes, and 4 (2 in each group) were symptomatic hemorrhagic transformations of brain infarctions. The ICrHs were spontaneous in 6 and 13, traumatic in 3 and 3, and procedural in 3 and 2 patients on ticagrelor and aspirin, respectively. In total, 9 fatal bleeds occurred on ticagrelor and 4 on aspirin. The composite of ICrH or fatal bleeding included 15 patients on ticagrelor and 18 on aspirin. Independently of bleeding classification, PLATO, TIMI, or GUSTO, the relative difference between treatments for major/severe bleeds was similar. Nonmajor bleeds were more common on ticagrelor. CONCLUSIONS: Antiplatelet therapy with ticagrelor in patients with acute ischemic stroke or transient ischemic attack showed a bleeding profile similar to that of aspirin for major bleeds. There were few ICrHs

Rationale, design, and baseline characteristics in Evaluation of LIXisenatide in Acute Coronary Syndrome, a long-term cardiovascular end point trial of lixisenatide versus placebo

BACKGROUND: Cardiovascular (CV) disease is the leading cause of morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). Furthermore, patients with T2DM and acute coronary syndrome (ACS) have a particularly high risk of CV events. The glucagon-like peptide 1 receptor agonist, lixisenatide, improves glycemia, but its effects on CV events have not been thoroughly evaluated. METHODS: ELIXA (www.clinicaltrials.gov no. NCT01147250) is a randomized, double-blind, placebo-controlled, parallel-group, multicenter study of lixisenatide in patients with T2DM and a recent ACS event. The primary aim is to evaluate the effects of lixisenatide on CV morbidity and mortality in a population at high CV risk. The primary efficacy end point is a composite of time to CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. Data are systematically collected for safety outcomes, including hypoglycemia, pancreatitis, and malignancy. RESULTS: Enrollment began in July 2010 and ended in August 2013; 6,068 patients from 49 countries were randomized. Of these, 69% are men and 75% are white; at baseline, the mean ± SD age was 60.3 ± 9.7 years, body mass index was 30.2 ± 5.7 kg/m(2), and duration of T2DM was 9.3 ± 8.2 years. The qualifying ACS was a myocardial infarction in 83% and unstable angina in 17%. The study will continue until the positive adjudication of the protocol-specified number of primary CV events. CONCLUSION: ELIXA will be the first trial to report the safety and efficacy of a glucagon-like peptide 1 receptor agonist in people with T2DM and high CV event risk