Successful surgical cricothyroidotomy following an obstetric "can't oxygenate" scenario: a narrative of enabling factors.

The airway management of a patient requiring emergency caesarean delivery for fetal distress and pre-eclampsia with severe features is described. A difficult obstetric airway was anticipated prior to induction and managed with the use of decision-support guidelines and cognitive aids. Failed tracheal intubation later progressed to a "can't oxygenate" scenario necessitating front-of-neck-access via surgical cricothyroidotomy. We discuss the factors which facilitated the preparation and implementation of interventions required to successfully execute this high-acuity task.

Detection of a microbial metabolite by STING regulates inflammasome activation in response to Chlamydia trachomatis infection.

The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1ß processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1ß processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand-cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.

Observational study comparing non-invasive blood pressure measurement at the arm and ankle during caesarean section.

Upper-arm non-invasive blood pressure measurement during caesarean section can be uncomfortable and unreliable because of movement artefact in the conscious parturient. We aimed to determine whether ankle blood pressure measurement could be used instead in this patient group by comparing concurrent arm and ankle blood pressure measured throughout elective caesarean section under regional anaesthesia in 64 term parturients. Bland-Altman analysis of mean difference (95% limits of agreement [range]) between the ankle and arm was 11.2 (-20.3 to +42.7 [-67 to +102]) mmHg for systolic arterial pressure, -0.5 (-21.0 to +19.9 [-44 to +91]) mmHg for mean arterial pressure and -3.8 (-25.3 to +17.8 [-41 to +94]) mmHg for diastolic arterial pressure. Although ankle blood pressure measurement is well tolerated and allows greater mobility of the arms than measurement from the arm, the degree of discrepancy between the two sites is unacceptable to allow routine use of ankle blood pressure measurement, especially for systolic arterial pressure. However, ankle blood pressure measurement may be a useful alternative in situations where arm blood pressure measurement is difficult or impossible.

Point-of-care testing of coagulation and fibrinolytic status during postpartum haemorrhage: developing a thrombelastography®-guided transfusion algorithm.

Thrombelastography® is a monitor of coagulation and fibrinolytic status, with point-of-care applications in managing haemorrhaging patients. Advocates have suggested a possible role in managing obstetric haemorrhage. This study aims to develop a pregnancy-specific thrombelastography-guided transfusion algorithm, which could be integrated into the management of postpartum haemorrhage. In this prospective observational study, 57 healthy, term-parturients provided pre-caesarean whole blood specimens for thrombelastography analyses. Specimens were processed according to a standardised protocol involving simultaneous analyses using three assays: native (non-activated); kaolin-activated; and kaolin and tissue factor-activated (RapidTEG®). For each assay, the following thrombelastography parameters were measured: reaction time (minutes); clot formation kinetics time (minutes); maximum amplitude (mm); and a angle (degree). Subsequent reference values were used to establish assay-specific reference intervals. For all thrombelastography parameters studied, reference values obtained using a non-activated assay differed from the corresponding values obtained using activated assays, and also demonstrated greater inter-sample variability. From the assay-specific reference intervals obtained, it was possible to establish a pregnancy-specific thrombelastography-guided transfusion algorithm. Specific features of this transfusion algorithm included the preferential use of activated assays, the need for duplicates and a recommendation that an initial baseline thrombelastography measurement is established for subsequent serial comparisons. This transfusion algorithm has been developed to assist with assessment of coagulation and fibrinolytic status during postpartum haemorrhage.

Chloride intracellular channels modulate acute ethanol behaviors in Drosophila, Caenorhabditis elegans and mice.

Identifying genes that influence behavioral responses to alcohol is critical for understanding the molecular basis of alcoholism and ultimately developing therapeutic interventions for the disease. Using an integrated approach that combined the power of the Drosophila, Caenorhabditis elegans and mouse model systems with bioinformatics analyses, we established a novel, conserved role for chloride intracellular channels (CLICs) in alcohol-related behavior. CLIC proteins might have several biochemical functions including intracellular chloride channel activity, modulation of transforming growth factor (TGF)-ß signaling, and regulation of ryanodine receptors and A-kinase anchoring proteins. We initially identified vertebrate Clic4 as a candidate ethanol-responsive gene via bioinformatic analysis of data from published microarray studies of mouse and human ethanol-related genes. We confirmed that Clic4 expression was increased by ethanol treatment in mouse prefrontal cortex and also uncovered a correlation between basal expression of Clic4 in prefrontal cortex and the locomotor activating and sedating properties of ethanol across the BXD mouse genetic reference panel. Furthermore, we found that disruption of the sole Clic Drosophila orthologue significantly blunted sensitivity to alcohol in flies, that mutations in two C. elegans Clic orthologues, exc-4 and exl-1, altered behavioral responses to acute ethanol in worms and that viral-mediated overexpression of Clic4 in mouse brain decreased the sedating properties of ethanol. Together, our studies demonstrate key roles for Clic genes in behavioral responses to acute alcohol in Drosophila, C. elegans and mice.

A Staphylococcus aureus paraspinal abscess associated with epidural analgesia in labour.

A case is described in which a parturient developed a Staphylococcus aureus paraspinal abscess following epidural analgesia in labour. We compared this case with other reported cases of paraspinal abscesses in obstetric patients. The presentation, diagnosis and management of these cases were reviewed. Anaesthetists need to be aware that non-spinal-epidural abscesses can occur in patients with an associated labour epidural.

Probing the 121-136 domain of lecithin:cholesterol acyltransferase using antibodies.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of plasma lipoprotein cholesterol in mammals as part of the reverse cholesterol transport pathway. Studies of the natural mutations of LCAT revealed a region that is highly sensitive to mutations (residues 121-136) and it is highly conserved in six animal species. The purpose of these studies was to investigate the reactivity of wild type and several mutated forms of LCAT, with a series polyclonal antibodies to further characterize this specific domain (residues 121-136). Two polyclonal antibodies directed against the whole enzyme, one against human plasma LCAT and the other against purified recombinant LCAT, and one site specific polyclonal antibody, directed against the 121-136 region of LCAT, were employed. All three antibodies reacted with a recombinant form of purified LCAT; however, only the polyclonal antibodies directed against the whole enzyme were able to recognize the LCAT when it was adsorbed to a hydrophobic surface in a solid phase immunoassay, or when bound to HDL in a sink immunoassay. These findings indicate that the epitope(s) of the 121-136 region are not accessible to antibodies under these conditions. Three mutant forms of LCAT, representing alterations in the 121-136 region, were also examined for their immunoreactivity with the same panel of antibodies and compared to the wild-type enzyme. These studies demonstrate that in its native configuration the 121-136 region of LCAT is likely to reside on a surface of LCAT. Furthermore, mutations within this region appear to markedly impact the exposure of epitopes at additional sites. These findings suggest that the 121-136 region could play an important role in enzyme interaction with its hydrophobic lipoprotein substrates as mutations within this region appear to alter enzyme conformation, catalytic activity, and the specificity of LCAT.

Phase I and pharmacokinetic study of photodynamic therapy for high-grade gliomas using a novel boronated porphyrin.

PURPOSE: To determine the recommended dose, toxicity profile, and pharmacokinetics of a novel boronated porphyrin (BOPP) for photodynamic therapy (PDT) of intracranial tumors. PATIENTS AND METHODS: BOPP was administered alone in increasing doses (0.25, 0.5, 1.0, 2.0, 4.0, or 8.0 mg/kg) preoperatively in patients with intracranial tumors undergoing postresection PDT until dose-limiting toxicity (DLT) was observed. RESULTS: Twenty-nine assessable patients with intracranial tumors received BOPP intravenously 24 hours before surgery. The recommended dose was 4 mg/kg. Dose escalation was limited by thrombocytopenia. The most common nonhematologic toxicity was skin photosensitivity. Pharmacokinetic parameters showed increased area under the plasma concentration-time curve and maximum concentration with increased dose. Tumor BOPP concentrations also increased with increased dose. CONCLUSION: BOPP at a dose of 4 mg/kg was well tolerated. DLT was thrombocytopenia, and photosensitivity was the only other toxicity of note. The efficacy of PDT using BOPP requires further exploration.

Irritable bowel syndrome.

Within the literature, it appears evident that neither a univariate etiologic model nor a single-agent treatment approach is sufficient to address the many diagnostic, assessment, and therapeutic challenges posed by irritable bowel syndrome (IBS). Various scientific advances have been made over the past 5 years, particularly in the areas of nonpharmacologic management of IBS. However, further collaboration between scientists and clinicians from multiple disciplines is strongly encouraged.

Chemical precipitation of apolipoprotein B-containing lipoproteins facilitates determination of LDL particle size.

OBJECTIVES: To simplify the determination of low-density lipoprotein (LDL) particle size by eliminating the need for ultracentrifugation. DESIGN AND METHODS: We compared LDL particle size determination by gradient gel electrophoresis using two different methods for separation of LDL: (a) by ultracentrifugation with a density between 1.019 and 1.063 g/mL, and (b) by precipitation of the apolipoprotein B-containing lipoproteins from plasma. LDL particle size was determined for 41 individuals using both methods. RESULTS: The correlation between these two methods was r = 0.98; peak particle diameter (nm) was reproducible with a coefficient of variation of 1. 3% for LDL separated by ultracentrifugation and 1.4% for LDL prepared by precipitation. The intra-assay variation within a single gel was 0.2%. CONCLUSION: Elimination of the need for ultracentrifugation or lipid staining significantly reduces the cost and simplifies the procedure of LDL particle sizing. As a result, larger patient populations can be more readily screened for the determination of LDL particle size.

An investigation of the cytotoxicity of the morpholino anthracycline MX2 against glioma cells in vitro.

MX2 is a novel morpholino anthracycline reported to have lower systemic toxicity than other anthracyclines. It has similar antitumour activity to adriamycin and is cytotoxic towards multi-drug resistant cells and anthracycline sensitive sublines of human and murine tumour cells. In this study MX2 showed a marked cytocidal effect compared to M2, the most cytotoxically active metabolite, and the nitrosourea, BCNU, when 30 ng/ml of each drug was added to separate flasks of C6 glioma cells grown in monolayer. The colony formation of C6 glioma cells was markedly inhibited by MX2 in a dose dependent manner. The LD50 values for MX2, M2 and BCNU were 10.5 ng/ml, 15.8 ng/ml and 465 ng/ml respectively. MX2 is likely to be bound to the main plasma protein, albumin, and can also interact with the plasma lipoproteins, particularly high density lipoprotein. The results in this study strongly support the further investigation of MX2 as a potential chemotherapeutic agent against brain tumours.

KRN8602 (MX2-hydrochloride): an active new agent for the treatment of recurrent high-grade glioma.

PURPOSE: To assess the efficacy and toxicity of KRN8602 when administered as an intravenous bolus to patients with recurrent high-grade malignant glioma. PATIENTS AND METHODS: Patients with recurrent or persistent anaplastic astrocytoma or glioblastoma multiforme who had not received recent chemotherapy or radiotherapy and were of good performance status (Eastern Cooperative Oncology Group score < or = 2) were treated with an intravenous bolus of 40 mg/m(2) KRN8602 every 28 days. Tumor responses were assessed radiologically and clinically after every second cycle of therapy. Treatment was continued until documented progression or a total of six cycles. RESULTS: A median of three cycles (range, one to six cycles) of KRN8602 was administered to 55 patients, 49 of whom received at least two cycles and were, therefore, assessable for response. The overall response rate (disease stabilization or better) was 43% (95% confidence interval, 29% to 58%). There were three complete responses, one partial response, seven minor responses, and 10 patients with stable disease. The median time to progression was 2 months (range, 1.5 to 37 months) and overall survival was 11 months (range, 1.5 to 40 months). Neutropenia was the most common toxicity, although it was generally of brief duration, and there were only seven episodes of febrile neutropenia in 176 cycles delivered. Nonhematologic toxicity was mostly gastrointestinal (nausea and vomiting, diarrhea) and events were grade 2 or lower except for a single episode of grade 3 vomiting. CONCLUSION: KRN8602 is an active new agent with minimal toxicity in the treatment of relapsed or refractory high-grade glioma. Further studies with KRN8602 in combination with other cytotoxics and in adjuvant treatment of gliomas are warranted.

Chloromethyl-X-rosamine (MitoTracker Red) photosensitises mitochondria and induces apoptosis in intact human cells.

We report that chloromethyl-X-rosamine (MitoTracker Red), a mitochondrion-selective fluorescent probe, has a strong photosensitising action. Photoirradiation of intact cells loaded with chloromethyl-X-rosamine induces depolarisation of the inner mitochondrial membrane and swelling of mitochondria, subsequently resulting in apoptosis. We have studied human osteosarcoma 143B TK-(rho+) cells and the derived (rho)0 206 cell line devoid of mitochondrial DNA. Colony formation tests revealed that chloromethyl-X-rosamine itself has no toxicity to either cell line in the concentration range 100-250 nM (unless photoirradiated). Chloromethyl-X-rosamine has potent phototoxicity such that almost quantitative cell killing was achieved at light doses of >2 J/cm2. These photodamaged cells initially showed swollen degenerative mitochondria and, later, uptake of propidium iodide in their apoptotic nuclei was observed. When cells were loaded with chloromethyl-X-rosamine (100 nM) and imaged by laser scanning confocal microscopy, photoirradiation by the laser beam under routine scanning conditions was sufficient to induce mitochondrial damage in both cell lines. This was evidenced by a rapid decrease of fluorescence intensity of co-loaded rhodamine 123 (indicative of mitochondrial depolarisation). Globular swelling of mitochondria took place within 15 minutes, imaged by the residual fluorescence of chloromethyl-X-rosamine itself, which also markedly decreased in intensity after imaging. Mitochondrial membrane depolarisation of cells loaded with chloromethyl-X-rosamine after photoirradiation using a measured dose of visible light was independently confirmed in 143B TK- and (rho)0 206 cells, by the significant decrease in uptake into cells of [3H]methyltriphenylphosphonium ions. Photoactivation of chloromethyl-X-rosamine in 143B TK-(rho+) cells, whose mitochondria had previously been loaded with calcein, caused rapid release of the mitochondrially entrapped calcein into the cytosol and nucleus. This major change in permeability of the mitochondrial inner membrane could not be prevented by cyclosporin A. Immunohistochemical study of cytochrome c revealed its diffuse redistribution into the cytoplasm in chloromethyl-X-rosamine-loaded cells after irradiation, as opposed to its specific mitochondrial localisation in non-irradiated cells. As a photosensitiser specifically targeted to mitochondria, and also a reporter of membrane potential and morphology, chloromethyl-X-rosamine may provide versatile new applications in studies of mitochondrial roles in cell death.

Modulation of light delivery in photodynamic therapy of brain tumours.

This study was performed to determine whether modulation of light delivery could improve tumour kill in photodynamic therapy (PDT) of brain tumours, as optimal dosimetry has not been fully established. One hundred and sixty-five adult Wistar rats were treated, of which 70 had an implanted C6 cerebral glioma. Haematoporphyrin derivative (HpD) was injected at doses between 0 and 20 mg/kg, 24 h prior to irradiation with 630 nm laser light. The total energy dose was varied from 0 to 1200 J/cm(2), with fluence rates of 625, 3125 or 9375 mW/cm(2). In some studies, the light delivered at 3125 mW/cm(2) was divided into 10 fractions of approximately 13 s, with refractory intervals of 60 s. The most striking finding was that HpD was much more potent than previously reported. All doses greater than 1.0 mg/kg resulted in normal brain damage with light doses above 50 J/cm(2). However, at 1.0 mg/kg, significant normal injury was not apparent until 1200 J/cm(2). Failure of drug-light dose reciprocity indicated that photobleaching occurred, protecting normal tissue. Selective tumour kill was observed to 2.2. mm depth (SE +/- 0.44 mm). Using lower power or fractionated light did not improve tumour kill and normal tissue injury occured with fluence rates of 9375 mW/cm(2). In conclusion, the doses of HpD currently used in clinical brain tumour trials may be too high to achieve selective tumour kill. Higher light fluence rates allowed shorter intraoperative irradiation times with no loss of efficacy. Photodynamic therapy continues to demonstrate potential as an effective treatment for local control of cerebral lesions.

Subdomain chimeras of hepatic lipase and lipoprotein lipase. Localization of heparin and cofactor binding.

To specify and localize carboxyl-terminal domain functions of human hepatic lipase (HL) and human lipoprotein lipase (LPL), two subdomain chimeras were created in which portions of the carboxyl-terminal domain were exchanged between the two lipases. The first chimera (HL-LPLC1) was composed of residues 1-344 of human HL, residues 331-388 of human LPL, and residues 415-476 of human HL. The second chimera (HL-LPLC2) consisted of just two segments, residues 1-414 of human HL and residues 389-448 of human LPL. These chimeric constructs effectively divided the HL C-terminal domain into halves, with corresponding LPL sequences either in the first or second portion of that domain. Both chimeras were lipolytically active and hydrolyzed triolein emulsions to a similar extent compared with native HL and LPL. Heparin-Sepharose chromatography demonstrated that HL-LPLC1 and HL-LPLC2 eluted at 0.80 and 1.3 M NaCl, respectively, elution positions that corresponded to native HL and LPL. Hence, substitution of LPL sequences into the HL carboxyl-terminal domain resulted in the production of functional lipases, but with distinct heparin binding properties. In addition, HL-LPLC2 trioleinase activity was responsive to apoC-II activation, although the -fold stimulation was less than that observed with native LPL. Moreover, an apoC-II fragment (residues 44-79) was specifically cross-linked to LPL and HL-LPLC2, but not to HL or HL-LPLC1. Finally, both chimeras hydrolyzed phospholipid with a specific activity similar to that of HL, which was unaffected by the presence of apoC-II. These findings indicated that in addition to a region found within the amino-terminal domain of LPL, apoC-II also interacted with the last half of the carboxyl-terminal domain (residues 389-448) to achieve maximal lipolytic activation. In addition, the relative heparin affinity of HL and LPL was determined by the final 60 carboxyl-terminal residues of each enzyme.

Phase I and pharmacokinetic study of KRN8602 alone and with filgrastim in patients with advanced cancer.

PURPOSE: To determine the recommended dose, toxicity profile, and pharmacokinetics of KRN8602 (MX2-hydrochloride), a novel morpholino anthracycline with potent cytotoxicity against anthracycline-sensitive and resistant experimental tumors in vitro and in vivo. PATIENTS AND METHODS: KRN8602 was administered alone in increasing doses to patients with advanced cancer or high-grade gliomas until dose-limiting toxicity (DLT) was observed in three or more of five patients treated in a dose level. Because neutropenia was dose limiting, further escalation was investigated with filgrastim support. RESULTS: Fifty-six assessable patients completed at least one cycle of chemotherapy. The recommended dose of KRN8602 alone was 40 mg/m2. Dose escalation was limited by neutropenia. The recommended dose of KRN8602 with filgrastim was 70 mg/m2, and limiting toxicities were neutropenia, diarrhea, and vomiting. The most commonly experienced nonhematologic toxicity was nausea and vomiting. Alopecia and mucositis were infrequent and mild. Pharmacokinetic parameters showed substantial variation, although the area under the plasma concentration-time curve (AUC) and maximum concentration both increased with dose. There was no relationship between pharmacokinetic parameters and toxicity. CONCLUSION: KRN8602 at doses of 40 mg/m2 when administered alone and 70 mg/m2 when administered with filgrastim appeared to be manageable. The major DLTs were neutropenia and, at higher doses, diarrhea and vomiting. The efficacy of this drug is currently being tested in phase II studies.

Upregulation of P-glycoprotein in rat hepatoma rho(o) cells: implications for drug-DNA interactions.

Rat hepatoma cells lacking mitochondrial DNA (rho(o) cells) were used as a model system to examine the possible roles of mitochondrial DNA as a target for the DNA-acting anticancer drug Adriamycin (doxorubicin). The rho(o) cells were 45-fold less sensitive to Adriamycin than the parental rho+ cells containing mitochondrial DNA. Other non-DNA-acting drugs also exhibited similar behaviour, and this was shown to be due to a multidrug resistance (MDR) phenotype in the rho(o) cells. This was indicated by confocal microscopy where rho+ cells exhibited thirteenfold higher cellular levels of Adriamycin than rho(o) cells. Upregulation (tenfold) of P-glycoprotein in rho(o) cells was also confirmed by Northern dot blot analysis. Since the MDR phenotype is present in rho(o) cells and upregulation of P-glycoprotein is maintained in these cells, rho(o) cells are not a good model system for drug-DNA studies (where the drug is susceptible to extrusion by P-glycoprotein), and any such results obtained with this system must be treated with considerable caution.

Effect of filgrastim on the pharmacokinetics of MX2 hydrochloride in patients with advanced malignant disease.

PURPOSE: To investigate the effect of granulocyte colony-stimulating factor (G-CSF) on the pharmacokinetics and pharmacodynamics of the new morpholino anthracycline drug MX2. METHODS: A total of 25 patients with advanced malignant disease participated in a dose-escalation study in the first cycle of treatment given i.v. at doses of 50-80 mg/m2 (74-152 mg) with concomitant filgrastim (G-CSF, 5 microg/kg) given daily beginning at 24 h after the dose of MX2. RESULTS: The mean fast distribution half-life (1.5 +/- 1.0 min) and the mean plasma clearance (2.18 +/- 0.95 l/min) were significantly lower than the respective mean values found in a previous study in which 27 patients had received MX2 (16.8-107.5 mg) alone (3.3 +/- 2.2 min and 2.98 +/- 1.68 l/min, respectively; P < 0.05). There was no correlation between plasma clearance and the delivered dose for the combined MX2-alone and MX2-filgrastim groups, indicating that the lower clearance observed in the G-CSF group was probably not due to the higher dose. Elimination half-lives of the metabolites M1 and M4 were significantly greater in the filgrastim group (19.8 +/- 14.7 and 11.8 +/- 5.0 h for M1 and 14.8 +/- 4.1 and 12.3 +/- 6.3 h for M2, respectively). Unlike the MX2-alone group, there was no relationship in the MX2-filgrastim group between the relative nadir neutrophil count and the dose or between the duration of grade IV neutropenia and the dose of MX2. CONCLUSIONS: This study shows that filgrastim decreased the plasma clearance of MX2 by approximately 25%, possibly by inhibition of metabolism.