The pattern and the sequence of tumour necrosis factor‐α (TNFα) induced cell death in the acute T‐lymphoblastic leukaemic cell line CCRF‐CEM and its vinblastine‐resistant subline CEM/VLB100 have been ...studied. Previously, we found that the CEM/VLB100 cell line was more sensitive to TNFα‐induced killing than its parental CCRF‐CEM cell line. TNFα‐induced cell death showed an apoptotic pattern, as detected by agarose electrophoresis, flow cytometry and transmission electron microscopy (TEM). TEM images revealed that autophagy and condensed mitochondria occurred earlier than nuclear fragmentation. The specific inhibitor of autophagy, 3‐methyladenine (3MA), inhibited the formation of autophagosomes. TNFα‐induced DNA fragmentation and cytolysis were completely inhibited by 10 mM 3MA. Inhibition of the fusion of lysosomes with autophagosomes by asparagine did not block TNFα‐induced apoptosis. In addition, amino acid and protein deprivation enhanced TNFα‐induced autophagy but not apoptosis. We propose that the early stages of autophagy are required for, but do not necessarily result in, TNFα‐induced apoptosis.
The effects of priming monocytes from septic patients with granulocyte-macrophage colony-stimulating factor (GM-CSF) ex vivo were investigated. Monocytes from septic patients had depressed plasma ...GM-CSF and dysregulated levels of other cytokines compared with normal subjects. Membrane expression of CD71 and HLA-DR were depressed, and monocytes were anergic to lipopolysaccharide (LPS) stimulation in vitro, which was associated with spontaneous and accelerated activation-induced apoptosis by LPS. Priming monocytes with GMCSF ex vivo augmented membrane cytokine expression, CD71, and HLA-DR. GM-CSF priming augmented cytokine secretion in response to LPS stimulation, restored cytokine secretion in monocytes from septic patients, and reversed their predilection to undergo apoptosis. Thus, monocyte dysfunction in septic shock is associated with depressed plasma levels of GM-CSF and enhanced apoptosis; however, GM-CSF stimulation ex vivo restored normal monocyte function and cytokine secretion by a mechanism that may depend on abrogating apoptosis.
Liposomal amphotericin (AmBisome) 2 mg/kg three times weekly was compared with placebo as prophylaxis against fungal infection in patients undergoing chemotherapy or bone marrow transplantation (BMT) ...for haematological malignancies. Prophylaxis began on day 1 of chemotherapy and continued until neutrophils regenerated or infection was suspected. Of 161 evaluable patients, 74 received AmBisome and 87 received placebo. Proven fungal infections developed in no patients on AmBisome and in three on placebo (3.4%) (P = NS). Suspected fungal infections requiring intervention with systemic antifungal therapy (usually amphotericin B) occurred in 31 patients on AmBisome (42%) and in 40 on placebo (46%) (P = NS). Suspected deep-seated infections developed in 21 (28.3%) and 31 (35.6%) patients, respectively (P = NS). Time to develop a suspected or proven deep-seated infection showed a trend in favour of AmBisome (P = 0.11). Fifty patients had fungal colonisation (48 with Candida spp, two with Aspergillus spp) of at least one body site during prophylaxis; 15 patients while receiving AmBisome (20%) and 35 while on placebo (40%) (P < 0.01). Time to colonisation was significantly delayed in the group receiving AmBisome (P < 0.05). Treatment-related toxicity was modest and no additional toxicity was observed in patients receiving AmBisome. AmBisome 2 mg/kg three times weekly is safe and reduces fungal colonisation in patients receiving intensive chemotherapy or BMT. However, despite encouraging trends, prophylactic AmBisome did not lead to a significant reduction in fungal infection or in requirement for systemic antifungal therapy.
Platelets show proinflammatory as well as prothrombotic properties. Patients with inflammatory bowel disease are at increased risk of systemic thromboembolism, and multifocal microvascular infarction ...has been proposed as a pathogenetic mechanism in Crohn's disease. The aim of this study was to determine if inflammatory bowel disease is associated with abnormal platelet behavior.
Platelet activation and aggregability were assessed using flow cytometry, Born aggregometry, and the modified method of Wu and Hoak. Serum beta-thromboglobulin was measured in patients with Crohn's disease and ulcerative colitis and, as controls, in healthy volunteers and patients with active rheumatoid arthritis.
Platelet surface expression of P-selectin and GP53 (markers of activation) were increased in Crohn's disease (13 of 30 patients abnormal for P-selectin; 9 of 28 abnormal for GP53) (P < 0.01) and ulcerative colitis (9 of 21 for P-selectin; 10 of 21 for GP53) (P < 0.01) compared with healthy controls. Increased circulating platelet aggregates (15 of 24 patients with Crohn's disease and 8 of 16 with ulcerative colitis) (P < 0.01), platelet aggregability in vitro, and serum beta-thromboglobulin were detected in active inflammatory bowel disease compared with healthy controls. Platelet behavior in active rheumatoid arthritis resembled that in healthy controls.
Increased platelet activation and aggregation are features of inflammatory bowel disease and may contribute to the risk of systemic thromboembolism and the pathogenesis of mucosal inflammation. Therefore, antiplatelet agents may be valuable in the management of inflammatory bowel disease.
A prospective and retrospective case analysis study of all perioperative cardiac arrests occurring during a 10-yr period from 1989 to 1999 was done to determine the incidence, cause, and outcome of ...cardiac arrests attributable to anesthesia. One hundred forty-four cases of cardiac arrest within 24 h of surgery were identified over a 10-yr period from an anesthesia database of 72,959 anesthetics. Case abstracts were reviewed by a Study Commission composed of external and internal members in order to judge which cardiac arrests were anesthesia-attributable and which were anesthesia-contributory. The rates of anesthesia-attributable and anesthesia-contributory cardiac arrest were estimated. Fifteen cardiac arrests out of a total number of 144 were judged to be related to anesthesia. Five cardiac arrests were anesthesia-attributable, resulting in an anesthesia-attributable cardiac arrest rate of 0.69 per 10,000 anesthetics (95% confidence interval, 0.085-1.29). Ten cardiac arrests were found to be anesthesia-contributory, resulting in an anesthesia-contributory rate of 1.37 per 10,000 anesthetics (95% confidence interval, 0.52-2.22). Causes of the cardiac arrests included medication-related events (40%), complications associated with central venous access (20%), problems in airway management (20%), unknown or possible vagal reaction in (13%), and one perioperative myocardial infarction. The risk of death related to anesthesia-attributable perioperative cardiac arrest was 0.55 per 10,000 anesthetics (95% confidence interval, 0.011-1.09). Most perioperative cardiac arrests were related to medication administration, airway management, and technical problems of central venous access. Improvements focused on these three areas may result in better outcomes.
Smac (or DIABLO) is a recently identified, novel proapoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac functions by eliminating the caspase-inhibitory ...properties of the inhibitors of apoptosis proteins (IAP), particularly XIAP. In this study, we stably transfected both full-length (FL) and mature (MT) Smac genes into the K562 and CEM leukaemic cell lines. Both FL and MT Smac transfectants increased the sensitivity of leukaemic cells to UV light-induced apoptosis and the activation of caspase-9 and caspase-3. Purified cytosol from the mature Smac transfectants, or the addition of human recombinant Smac protein or N-7 peptide into nontransfected cytosol, showed an increased sensitivity to cytochrome c-induced activation of caspase-3. The mature Smac enhanced the susceptibility of both K562 and CEM cells to TRAIL-induced apoptosis. Overexpression of the mature Smac protein also inhibited proliferation, as detected by reduced colony formation and Ki-67 expression in leukaemic cells. Cell cycle analysis revealed that Smac transfectants displayed significant G0/G1 arrest and reduction in 5-bromo-2'-deoxyuridine (BrdU) incorporation. Smac sensitized human acute myeloid leukaemia blasts to cytochrome c-induced activation of caspase-3. However, Smac failed to overcome Apaf-1-deficiency-mediated resistance to cytochrome c in primary leukaemic blasts. In summary, this study reveals that Smac/DIABLO exhibits a potential role in increasing apoptosis and suppressing proliferation in human leukaemic cells. Importantly, it also indicates that it is crucial to evaluate the levels of Apaf-1 and XIAP proteins in patient samples before using Smac peptide therapy in the treatment of human leukaemia.
There are many factors contributing to the resistance to TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. However, it is not clear whether the mechanism of ...resistance to TRAIL is constitutive or inductive. Therefore, the purpose of this study was to investigate the resistant mechanisms to TRAIL at different levels in the apoptotic pathway. The human T-lymphoblastic leukemic CEM cell line showed more resistant to TRAIL-induced apoptosis compared with the human chronic myeloid leukemic K562 cell line. Lower level of constitutive caspase-8 expression in the CEM cell line led to a poor response to both TRAIL-induced activation of caspase-3 and reduction in the mitochondrial membrane potential (DeltaPsim). There was no significant difference in the constitutive levels of NF-kappaB in CEM and K562 cell lines. However, CEM cells showed a faster response to TRAIL-induced NF-kappaB activation than K562 cells. TRAIL-induced regulation of Bcl-2 family of proteins included an up-regulation in Bcl-2/Bcl-XL and a down-regulation in Bax. IAPs, such as XIAP, cIAP-1, cIAP-2 and Survivin were all up-regulated during the treatment with TRAIL. In summary, our data suggest that the leukemic cells resistance to TRAIL-induced apoptosis might be due to the deficiency in the constitutive caspase-8 expression. Development of potential resistance to apoptosis by TRAIL can occur in both TRAIL-resistant and TRAIL-sensitive leukemic cells.
Abstract Background Up to 21% of pediatric visits result in an antibiotic prescription, and a large portion of these are unnecessary. Objective To determine if educational sessions would reduce ...inappropriate antibiotic use. Methods Intervention study evaluating antibiotic prescribing following educational sessions for urinary tract infection, skin and soft tissue infection, pharyngitis, upper respiratory tract infection, acute otitis media, and acute bacterial sinusitis. Results A total of 26 out of 43 (60%) nurse practitioners in 4 urgent care centers were enrolled in the study. The rate of inappropriate antibiotic use among all conditions was 10% before and 8% after the intervention ( p = .02). A decrease in inappropriate antibiotic prescribing was seen after the educational session ( p < .01). The most common reasons for inappropriate antibiotic prescribing were too broad (41%), wrong dosage (22%), and not indicated (17%). Conclusions Educational sessions led to improvement in overall inappropriate antibiotic use. Additional stewardship interventions are needed to further reduce unnecessary antibiotic use.
Previous studies have shown that the lymphoblastic leukemia CEM cell line is resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis because of a low expression ...of caspase-8. Bcl-2 inhibitors, BH3I-2' and HA14-1, are small cell-permeable nonpeptide compounds, are able to induce apoptosis by mediating cytochrome c release, and also lead to dissipation of the mitochondrial membrane potential (DeltaPsim). This study aimed to use the Bcl-2 inhibitors to sensitize CEM cells to TRAIL-induced apoptosis by switching on the mitochondrial apoptotic pathway. We found that a low dose of BH3I-2' or HA14-1, which did not induce cytochrome c release, greatly sensitized CEM cells to TRAIL-induced apoptosis. In a similar manner to the classical uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), both BH3I-2' and HA14-1 induced a reduction in DeltaPsim, a generation of reactive oxygen species (ROS), an increased mitochondrial respiration, and a decreased ATP synthesis. This uncoupling function of the Bcl-2 inhibitors was responsible for the synergy with TRAIL-induced apoptosis. CCCP per se did not induce apoptosis but again sensitized CEM cells to TRAIL-induced apoptosis by uncoupling mitochondrial respiration. The uncoupling effect facilitated TRAIL-induced Bax conformational change and cytochrome c release from mitochondria. Inhibition of caspases failed to block TRAIL-mediated cell death when mitochondrial respiration was uncoupled. We observed that BH3I-2', HA14-1, or CCCP can overcome resistance to TRAIL-induced apoptosis in TRAIL-resistant cell lines, such as CEM, HL-60, and U937. Our results suggest that the uncoupling of mitochondrial respiration can sensitize leukemic cells to TRAIL-induced apoptosis. However, caspase activation per se does not represent an irreversible point of commitment to TRAIL-induced cell death when mitochondrial respiration is uncoupled.
Bax translocation from cytosol to mitochondria is believed to be a crucial step for triggering cytochrome c release from mitochondria. However, it is unclear whether Bax translocation is associated ...with Bax induction by DNA damaging agents. The induction of Bax in response to DNA damaging agents has been considered to be linked with p53. In this study, we used the p53 negative human chronic myeloid leukaemia K562 cell line. Bax up-regulation occurred at the whole cell level after DNA damage induced by etoposide. However, after incubation with etoposide, Bax failed to translocate to mitochondria and as a result, the apoptotic process was blocked. A Bax stable transfectant, the K/Bax cell line, expressed more Bax protein in the cytosol, mitochondria and nuclei. This Bax overexpression induced cytochrome c release, a reduction of cytochrome c oxidase activity and mitochondrial membrane potential (Delta(Psi)m). However, Bax-induced apoptosis was blocked downstream of mitochondria in K562 cells. The increased levels of mitochondrial Bax sensitized cells to etoposide-induced activation of caspases-2, -3 and -9 and apoptosis. However, after transient transfection with the Apaf-1 gene, K/Bax cells were sensitized to etoposide-induced caspase activation and apoptosis to a larger extent compared with Bax or Apaf-1 transfection alone. We therefore conclude that two mechanisms contribute to the resistance of K562 cells to etoposide-induced apoptosis; firstly failure of Bax targeting to mitochondria and, secondly, deficiency of Apaf-1. Uncoupling of Bax translocation from Bax induction can occur in response to etoposide-induced DNA damage.
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