Using Virtual Reality to teach ultrasound-guided needling skills for regional anaesthesia: A randomised controlled trial.

STUDY OBJECTIVE: We previously designed and validated a virtual reality-based simulator to help train novices in ultrasound-guided needling skills necessary for safe and competent ultrasound-guided regional anaesthesia. This study was designed to compare the performance and error rates of novices trained by a human faculty aided with the assistance of this virtual reality simulator (virtual reality-assisted training), versus novices trained wholly by humans (conventional training). DESIGN, SETTING, AND PARTICIPANTS: In this single centre, randomised controlled study, we used a standardised teaching protocol, rigorous blinding, iterative training of assessors, and validated global rating scale and composite error score checklists to assess skills learning of novice participants. MAIN RESULTS: We recruited 45 novices and scored 270 assessments of performance and error rates. Inter-rater correlation coefficient of reliability of scoring between assessors for the global rating scale was 0.84 (95%CI 0.68-0.92) and for the composite error score checklist was 0.87 (95%CI 0.73-0.93). After adjustment for age, sex, Depression, Anxiety and Stress-21, and baseline score, there was no statistical difference for virtual reality-assisted training compared to conventional training in final global rating score (average treatment effect -3.30 (95%CI-13.07-6.48), p = 0.51) or in the final composite error score (average treatment effect 1.14 (95%CI -0.60-2.88), p = 0.20). Realism in the virtual reality simulator was similar to real-life when measured by the Presence Questionnaire, all components p > 0.79; and task workload assessed by the NASA-Task Load Index was not statistically different between groups, average treatment effect 5.02 (95%CI -3.51-13.54), p = 0.25. Results were achieved in the virtual reality-assisted group with half the human faculty involvement. CONCLUSION: Novices trained using a hybrid, virtual reality-assisted teaching program showed no superiority to novices trained using a conventional teaching program, but with less burden on teaching resources.

Disseminated Tuberculosis Involving Lung, Peritoneum, and Endometrium in an Immunocompetent 17-Year-Old Patient.

A 17-year-old Guatemalan female with a recent history of spontaneous abortion requiring dilation and curettage at 16 weeks' gestation presented two weeks post-procedure to a pediatric hospital for three days of worsening generalized abdominal pain, diarrhea, fevers, and cough. The patient's vital signs showed hypoxia, tachypnea, tachycardia, and hypotension; she was alert and oriented with a thin body habitus and suprapubic abdominal tenderness without rebound, guarding, or hepatosplenomegaly. She had no crackles, rales, or wheezing on lung examination. Labs revealed neutrophilic leukocytosis, acute kidney injury, transaminitis, and coagulopathy. Pelvic ultrasound demonstrated a septated pelvic fluid collection with an endometrial thickening. CT abdomen and pelvis showed significant nodular omental thickening and ascites. CT angiogram of the chest demonstrated an apical lung cavity and bilateral micro-nodularity without lymphadenopathy. Due to concern for septic shock secondary to endometritis, the patient was started on broad-spectrum antibiotics and intubated for acute hypoxic respiratory failure. Repeat dilation and evacuation revealed degenerative first trimester products of conception and necrotizing granulomatous endometritis with Mycobacterium tuberculosis (M. tuberculosis) bacteria. Paracentesis indicated tuberculosis (TB) in ascites fluid, and bronchoalveolar lavage (BAL) showed pulmonary TB. Human immunodeficiency virus (HIV) screen and serum QuantiFERON®-TB Gold testing were negative. Rifampin, isoniazid, pyrazinamide, and ethambutol (RIPE) therapy was initiated alongside piperacillin-tazobactam for the treatment of both disseminated TB and septic abortion. She was extubated with hemodynamic stability, but fevers persisted. Repeat fallopian tube fluid sampling after five weeks of RIPE indicated numerous acid-fast bacilli. The patient's septic clinical picture clouded her TB diagnosis as it appeared unusual that a healthy 17-year-old would concurrently have a septic abortion and disseminated TB; the lack of lymphadenopathy on CT scan also contributed to diagnostic uncertainty. Among patients from endemic regions, TB is a cause of spontaneous abortion. Conversely, during pregnancy, progesterone suppresses the T-helper 1 (Th1) proinflammatory response and increases susceptibility to TB. Peripartum women are at higher risk for disseminated TB, and postpartum women are twice as likely to experience reactivation of latent TB than nonpregnant women. Disseminated TB must be considered in pregnant adolescents presenting with appropriate clinical characteristics and imaging findings.

Rapid and profound potentiation of Apo2L/TRAIL-mediated cytotoxicity and apoptosis in thoracic cancer cells by the histone deacetylase inhibitor Trichostatin A: the essential role of the mitochondria-mediated caspase activation cascade.

Apo2L/TRAIL is actively investigated as a novel targeted agent to directly induce apoptosis of susceptible cancer cells. Apo2L/TRAIL-refractory cells can be sensitized to the cytotoxic effect of this ligand by cytotoxic chemotherapeutics. The aim of this study was to evaluate the in vitro tumoricidal activity of the Apo2L/TRAIL + Trichostatin A in cultured thoracic cancer cells and to elucidate the molecular basis of the synergistic cytotoxicity of this combination. Concurrent exposure of cultured cancer cells to sublethal concentrations of Apo2L/TRAIL and Trichostatin A resulted in profound enhancement of Apo2L/TRAIL-mediated cytotoxicity in all cell lines regardless of their intrinsic susceptibility to this ligand. This combination was not toxic to primary normal cells. While Apo2L/TRAIL alone or Trichostatin A alone mediated < 20% cell death, 60 to 90% of cancer cells were apoptotic following treatment with TSA + Apo2L/TRAIL combinations. Complete translocation of Bax from the cytosol to the mitochondria compartment was mainly observed in combination-treated cells and this was correlated with robust elevation of caspase 9 proteolytic activity indicative of activation of the mitochondria apoptogenic effect. Profound TSA + Apo2L/TRAIL-mediated cytotoxicity and apoptosis were completely abrogated by either Bcl2 over-expression or by the selective caspase 9 inhibitor, highlighting the essential role of mitochondria-dependent apoptosis signaling cascade in this process. Moreover, increased caspase 8 activity observed in cells treated with the TSA + Apo2L/TRAIL combination was completely suppressed by Bcl-2 over-expression or by the selective caspase 9 inhibitor indicating that the elevated caspase 8 activity in combination-treated cells was secondary to a mitochondria-mediated amplification feedback loop of caspase activation. These finding form the basis for further development of HDAC inhibitors + Apo2L/TRAIL combination as novel targeted therapy for thoracic malignancies.

Gossypol, a phytochemical with BH3-mimetic property, sensitizes cultured thoracic cancer cells to Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand.

OBJECTIVES: Chemotherapeutic agents sensitize cancer cells to Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) via recruitment of the mitochondria-dependent activation of caspase and induction of apoptosis. This study was designed to evaluate whether gossypol, a phytochemical compound with BH3-mimetic property that functions as an inhibitor of Bcl2/BclXL, would sensitize cultured thoracic cancer cells to this death-inducing ligand. METHODS: Cancer cell lines from the lung (H460, H322), the esophagus (TE2, TE12), and the pleura (H290, H211) or primary normal cells were treated with gossypol+Apo2L/TRAIL combinations. Cell viability and apoptosis were evaluated by (4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assays, respectively. Caspase 9 and 3 specific proteolytic activity in combination-treated cells was determined by fluorometric enzymatic assay. RESULTS: Gossypol, selectively cytotoxic to cancer cells and not primary normal cells, significantly sensitized thoracic cancer cells to Apo2L/TRAIL as indicated by 1.5- to more than 10-fold reduction of Apo2L/TRAIL 50% inhibitory concentration values in cells treated with gossypol+Apo2L/TRAIL combinations. Whereas less than 20% of cancer cells exposed to either gossypol (5 micromol/L) or Apo2L/TRAIL (20 ng/mL) were dead, more than 90% of cells treated with the drug combinations were apoptotic. Combination-induced cytotoxicity and apoptosis was completely abrogated either by overexpression of Bcl2 or by the selective caspase 9 inhibitor. This combination was not toxic to normal cells. CONCLUSION: Gossypol profoundly sensitizes thoracic cancer cells to the cytotoxic effect of Apo2L/TRAIL via activation of the mitochondria-dependent death signaling pathway. This study provides evidence for the profound anticancer activity of this drug combination and should be further evaluated as a novel targeted molecular therapeutic for thoracic cancers.

Enhancement of Apo2L/TRAIL-mediated cytotoxicity in esophageal cancer cells by cisplatin.

Although expressing adequate levels of functional tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4/DR5, significant proportion of cancer cells exhibit resistance to the cytotoxic effect of this ligand. Exposure of Apo2L/TRAIL-refractory cancer cells to cytotoxic chemotherapeutic agents enhances their sensitivity to Apo2L/TRAIL cytotoxicity. This study aims to elucidate the molecular mechanism responsible for the cisplatin-mediated enhancement of Apo2L/TRAIL sensitivity in cultured esophageal cancer cells. Exposure of cancer cells to sublethal concentrations of cisplatin resulted in profound potentiation of their susceptibility to Apo2L/TRAIL cytotoxicity as indicated by 2- to >20-fold reduction in Apo2L/TRAIL IC50 values. Significant activation of caspase-8, caspase-9, and caspase-3 was observed only in cells treated with cisplatin/Apo2L/TRAIL combination and not in those exposed to either agent alone. More importantly, activation of these key caspases was significantly abrogated by overexpression of Bcl2 or by the selective caspase-9 inhibitor. This observation strongly suggested that caspase-8 activation in cells treated with the cisplatin/Apo2L/TRAIL combination was secondary to the mitochondria-mediated amplification feedback loop and activation of the executioner caspase-3 was dependent on the recruitment of the intrinsic pathway characteristic of the type II cell. Profound combination-mediated cytotoxicity and induction of apoptosis was completely suppressed either by Bcl2 overexpression or by inhibition of caspase-9 activity, which conclusively pointed to the essential role of the mitochondria-dependent death signaling cascade in this process. Cisplatin sensitizes esophageal cancer cells to Apo2L/TRAIL cytotoxicity by potentiation of the mitochondria-dependent death signaling pathway that leads to amplification of caspase activation, particularly caspase-8, by the feedback loop to efficiently induce apoptosis.

The essential role of the mitochondria-dependent death-signaling cascade in chemotherapy-induced potentiation of Apo2L/TRAIL cytotoxicity in cultured thoracic cancer cells: amplified caspase 8 is indispensable for combination-mediated massive cell death.

PURPOSE: Despite adequately expressing functional receptors for tumor necrosis factor receptor apoptosis-inducing ligand (TRAIL), many cultured tumor cells are refractory to the cytotoxic effect of this ligand. Cytotoxic chemotherapeutic drugs have been shown to synergize with Apo2L/TRAIL to mediate apoptosis in cancer cells. The main goal of this study was to evaluate the effect of either cisplatin or paclitaxel, two common used chemotherapeutic agents for solid tumors, on enhancing Apo2L/TRAIL cytotoxicity in a panel-cultured thoracic cancer cells and to examine the role of the mitochondria-dependent caspase activation cascade in mediating apoptosis of combination-treated cells. METHODS: Cultured thoracic cancer cells were treated with cisplatin/Apo2L/TRAIL or paclitaxel/Apo2L/TRAIL sequential combinations in vitro. Cell viability and apoptosis were determined by 4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays. Stable transfectants expressing high levels of Bcl-2 were created by retroviral gene transfer. Specific proteolytic activity of caspases 3, 6, 8, and 9 were measured by commercially available kits using fluorescent substrates. RESULTS: All cell lines preferentially expressed high levels of DR4 and/or DR5 and low levels of DcR1/DcR2; all of which were not altered by chemotherapeutic drug treatments. Pretreatment of these cancer cells with sublethal concentrations of either cisplatin or paclitaxel increased their susceptibility to Apo2L/TRAIL by twofold to >20-fold. Profound synergistic induction of apoptosis was observed in combination-treated cells. Viability of primary normal cells was affected by neither Apo2L/TRAIL nor the combinations of chemotherapy and Apo2L/TRAIL. Overexpression of Bcl-2 or inhibition of caspase 9 activity completely abrogated combination-induced cytotoxicity and apoptosis, indicating the essential role of the mitochondria-dependent death signaling cascade in this process. Robust activation of caspase 8 in combination-treated cells was completely suppressed either by Bcl-2 overexpression or by blocking of the activity of the mitochondria-regulated caspase 9, thus identifying the amplification feedback loop as the source of elevated caspase 8 activity. Finally, mitochondria-mediated amplification of caspase 8 activity was indispensable for complete caspase activation and full execution of apoptosis, because suppression of its activity using the selective caspase 6 inhibitor (located downstream of the caspase 3 but upstream of the caspase 8 in the feedback loop) resulted in profound suppression of not only caspase 8 activity but also those of caspases 9 and 3, as well as complete protection of cancer cells from combination-induced cytotoxicity. CONCLUSION: Cisplatin or paclitaxel synergistically interacts with Apo2L/TRAIL to mediate profound induction of apoptosis. The mitochondria-dependent caspase activation cascade and the amplification feedback loop are essential for the complete execution of the cell death program. Furthermore, our data identify mitochondria as the direct target for the development of more refined strategies to enhance the therapeutic effect of Apo2L/TRAIL as an anticancer agent.

Valproic acid, an antiepileptic drug with histone deacetylase inhibitory activity, potentiates the cytotoxic effect of Apo2L/TRAIL on cultured thoracic cancer cells through mitochondria-dependent caspase activation.

Inhibitors of histone deacetylases have been shown to enhance the sensitivity of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand TRAIL-mediated cytotoxicity. Valproic acid (VA), a commonly used antiepileptic agent whose pharmacokinetics and toxicity profiles are well described, is a histone deacetylase inhibitor. This project aims to evaluate if VA can potentiate Apo2L/TRAIL-mediated cytotoxicity in cultured thoracic cancer cells and to elucidate the underlying molecular mechanism responsible for this effect. VA sensitized cultured thoracic cancer cells to Apo2L/TRAIL, as indicated by a 4-fold to a >20-fold reduction of Apo2L/TRAIL IC50 values in combination-treated cells. Although VA (0.5-5 mM) or Apo2L/TRAIL (20 ng/ml) induced less than 20% cell death, VA + Apo2L/TRAIL combinations caused 60% to 90% apoptosis of cancer cells. Moreover, substantial activation of caspases 8, 9, and 3, which was observed only in cells treated with the drug combination, was completely suppressed by Bcl2 overexpression or by the caspase 9 inhibitor. Both the caspase 9 inhibitor and Bcl2 completely abrogated the substantial cytotoxicity and apoptosis induced by this combination, thus highlighting the pivotal role of the type II pathway in this process. These findings provide a rationale for the development of VA and Apo2L/TRAIL combination as a novel molecular therapeutic for thoracic cancers.