Use of 'rainy day' autologous haemopoietic stem cells: a single-institution experience over 10 years.

BACKGROUND: High-dose chemotherapy and autologous haematopoietic stem cell transplantation is an important therapeutic modality in the treatment of many haematological malignancies. Generally, stem cells are collected close to the time of the transplant, but an alternative is to collect and cryopreserve cells at an early stage of the illness so they are available for later use ('rainy day harvesting'). Although this practice has been commonplace in Australia, there is little evidence to document eventual use of cells collected in this manner. METHODS: We conducted an audit of indications for and eventual transplantation of 'rainy day' harvests performed at our institution over a 10-year period. RESULTS: Although there was some variation across different disease groups, we found that only 14% of cells were transplanted. The median delay to transplantation was 19 months. CONCLUSION: Together with recent advances in stem cell mobilisation techniques, results from this audit suggest that the practice may not be an effective use of limited health resources.

Penetrating paediatric thoracic injury.

Paediatric penetrating chest trauma is common in conflict, but rarely seen in peacetime. We describe the successful hospital management of a five year old female civilian casualty with life threatening penetrating thoracic trauma caused by a fragment from an explosive device.

Use of nasogastric tubes in trauma patients--a review.

Traditionally, suspected basal skull fractures have been considered a relative contra-indication to inserting a nasogastric tube (NGT). In patients with basal skull fractures the Advanced Trauma Life Support protocol recommends that an orogastric tube (OGT) be inserted rather than a nasogastric tube (NGT). This article reviews the available literature. We conclude that in patients with suspected or confirmed basal skull fractures numerous case reports confirm that nasogastric tube insertion may lead to intracranial placement which, although the causal relationship is unclear, is associated with patient death.

Enhanced case management can be delivered for patients with EVD in Africa: Experience from a UK military Ebola treatment centre in Sierra Leone.

BACKGROUND: Limited data exist describing supportive care management, laboratory abnormalities and outcomes in patients with Ebola virus disease (EVD) in West Africa. We report data which constitute the first description of the provision of enhanced EVD case management protocols in a West African setting. METHODS: Demographic, clinical and laboratory data were collected by retrospective review of clinical and laboratory records of patients with confirmed EVD admitted between 5 November 2014 and 30 June 2015. RESULTS: A total of 44 EVD patients were admitted (median age 37 years (range 17-63), 32/44 healthcare workers), and excluding those evacuated, the case fatality rate was 49% (95% CI 33%-65%). No pregnant women were admitted. At admission 9/44 had stage 1 disease (fever and constitutional symptoms only), 12/44 had stage 2 disease (presence of diarrhoea and/or vomiting) and 23/44 had stage 3 disease (presence of diarrhoea and/or vomiting with organ failure), with case fatality rates of 11% (95% CI 1%-58%), 27% (95% CI 6%-61%), and 70% (95% CI 47%-87%) respectively (p = 0.009). Haemorrhage occurred in 17/41 (41%) patients. The majority (21/40) of patients had hypokalaemia with hyperkalaemia occurring in 12/40 patients. Acute kidney injury (AKI) occurred in 20/40 patients, with 14/20 (70%, 95% CI 46%-88%) dying, compared to 5/20 (25%, 95% CI 9%-49%) dying who did not have AKI (p = 0.01). Ebola virus (EBOV) PCR cycle threshold value at baseline was mean 20.3 (SD 4.3) in fatal cases and 24.8 (SD 5.5) in survivors (p = 0.007). Mean national early warning score (NEWS) at admission was 5.5 (SD 4.4) in fatal cases and 3.0 (SD 1.9) in survivors (p = 0.02). Central venous catheters were placed in 37/41 patients and intravenous fluid administered to 40/41 patients (median duration of 5 days). Faecal management systems were inserted in 21/41 patients, urinary catheters placed in 27/41 and blood component therapy administered to 20/41 patients. CONCLUSIONS: EVD is commonly associated life-threatening electrolyte imbalance and organ dysfunction. We believe that the enhanced levels of protocolized care, scale and range of medical interventions we report, offer a blueprint for the future management of EVD in resource-limited settings.

Use of a functional assay to diagnose protein S deficiency; inappropriate testing yields equivocal results.

Inherited deficiency of protein S (PS) is a rare but accepted risk factor for venous thromboembolism. There is accumulating evidence that inherited PS deficiency may be associated with a variety of adverse obstetric events. Acquired PS deficiency may be caused by a variety of clinical states including normal pregnancy. We conducted a retrospective audit of the results of screening for PS deficiency through our reference laboratory. The majority of patients in this audit with significantly reduced (<50%) free functional PS levels had a major confounding factor likely to cause acquired PS deficiency, most frequently pregnancy. Recommendations for PS testing for the diagnosis of hereditary PS deficiency include deferring testing until at least 40 days post-partum. It appears that these recommendations are not being adhered to leading to difficulty in the interpretation of results.

Characteristics of commercial pig farms in Great Britain and their use of antimicrobials.

A mail survey of 1889 pig producers in Great Britain with more than 100 sows or more than 1000 pigs was carried out to investigate their use of antimicrobials and other health-related management practices. The response rate was 25.5 per cent. Antimicrobials were most commonly administered in feed. Depending on the production system, between 60 and 75 per cent of the farms had used antimicrobials in their weaner rations and 20 to 62 per cent had used them in their grower rations. Antimicrobial injections had been used on 59 per cent of the farms. The use of antimicrobials in pigs of different age groups was variously associated with the scale of production, membership of a quality assurance scheme, the use of vaccination and whether the farmer thought the farm environment could be improved. Half the respondents (49 per cent) indicated that they used growth promoters in their weaners, but fewer used these in their growers (45 per cent) and finishers (34 per cent). Although 63 per cent of the farmers felt that the continued use of antimicrobials for disease prevention was justified, only 21 per cent felt that their continued use as growth promoters was justified.

SPAK, a STE20/SPS1-related kinase that activates the p38 pathway.

We have cloned a member of the STE20/SPS1 protein kinase family from a transformed rat pancreatic beta cell line. SPAK (STE20/SPS1-related, proline alanine-rich kinase) belongs to the SPS1 subfamily of STE20 kinases and is highly conserved between species. SPAK is expressed ubiquitously, although preferentially in brain and pancreas. Biochemical characterization of SPAK catalytic activity demonstrates that is a serine/threonine kinase that can phosphorylate itself and an exogenous substrate in vitro. SPAK is immunoprecipitated from transfected mammalian cells as a complex with another, as yet uncharacterized, serine/threonine kinase which is capable of phosphorylating catalytically-inactive SPAK and myelin basic protein in an in vitro kinase assay. SPAK specifically activates the p38 pathway in cotransfection assays. Like MST1 and MST2, SPAK contains a putative caspase cleavage site at the junction of the catalytic domain and the C-terminal region. Full-length SPAK is expressed in the cytoplasm in transfected cells, while a mutant corresponding to caspase-cleaved SPAK is expressed predominantly in the nucleus. The similarity of SPAK to other SPS1 family members, its ability to activate the p38 pathway, in addition to its putative caspase cleavage site, provide evidence that SPAK may act as a novel mediator of stress-activated signals. Oncogene (2000) 19, 4290 - 4297

Changes in fatty acid distribution and thermotropic properties of phospholipids following phosphatidylcholine depletion in a choline-requiring mutant of Neurospora crassa.

Growth of a choline requiring auxotroph of Neurospora crassa on medium lacking exogenous choline produces large changes in the levels of phosphatidylethanolamine and phosphatidylcholine. Whole cell fatty acid distributions were found to vary widely between different phospholipid species of normally growing, choline-supplemented cultures with phosphatidylcholine showing the highest levels of unsaturation and anionic phospholipids and cardiolipin having the lowest. In these lipids, choline deprivation produced little change in fatty acid profiles of phosphatidylethanolamine, whereas changes in fatty acids of phosphatidylcholine and acidic phospholipids resulted in increased levels of unsaturation at both growth temperatures. Microsomal phospholipids also showed fatty acid variability with sharp decreases in phosphatidylcholine unsaturates and increases in acidic phospholipid unsaturated fatty acids at low growth temperatures. Fluorescence polarization of 1,6-diphenylhexatriene in vesicles formed from total cellular and microsomal lipids showed that choline deprivation produces changes in thermotropic properties in the lipids in deprived cultures at either growth temperature. The effective differences in fluorescence polarization between choline-deprived and supplemented cultures grown at a given temperature were found to be comparable to those produced by temperature were found to be comparable to those produced by temperature acclimation in normally growing cultures over a temperature range of 22 K.

The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa.

Experiments were conducted to examine the effects of temperature acclimation on sterol and phospholipid biosynthesis in Neurospora crassa. Cultures grown at high (37 degrees C) and low (15 degrees C) temperatures show significant differences in free and total sterol content, sterol/phospholipid ratios and distribution of major phospholipid species in total lipids and two functionally distinct membrane fractions. The ratio of free sterols to phospholipids in total cellular lipids from 15 degrees C cultures was found to be about one-half that found at 37 degrees C, whereas sterol/phospholipid ratios of mitochondrial and microsomal membranes were found to be higher at the low growth temperature. Total sterol and phospholipid biosynthetic rates showed parallel reductions in cultures acclimating to a shift from 37 to 15 degrees C growth conditions. Distribution of [14C]acetate label into free sterols was significantly lower under these conditions, however; indicating an increase in the conversion rate of sterols to sterol esters at the lower temperature. Mitochondrial and microsomal membrane fractions showed distinct phospholipid distributions which also differed from total lipid distributions at the two growth temperatures. In each case there was a consistent decrease in phosphatidylcholine and a corresponding increase in phosphatidylethanolamine as growth temperatures were lowered.

The effects of altered levels of phosphatidylcholine and phosphatidylethanolamine on fatty acid desaturase activity and sterol metabolism during temperature acclimation in a choline auxotroph of Neurospora crassa.

Experiments were conducted to determine the effects of choline deprivation on levels of phospholipid fatty acids in a choline auxotroph (chol-1; chol-2) of Neurospora crassa with respect to high (37 degrees C) and low (15 degrees C) growth temperatures and during acclimation following a shift from high to low temperature conditions. Although grossly altered levels of phosphatidylethanolamine and phosphatidylcholine were observed at both temperatures, phospholipid fatty acid levels remained virtually identical to those found in a phenotypically wild-type and maximally supplemented chol-1; chol-2 strains grown under the same conditions. Deprivation of choline from supplemented cultures of the mutant followed by a shift from high to low growth temperatures did not significantly affect the level of fatty acid desaturation with respect to control cultures. Free sterols did not significantly affect the level of fatty acid desaturation with respect to control cultures. Free sterols were reduced, however, and sterol ester levels were elevated in choline-deprived cultures, suggesting that sterol interconversions may be closely tied to aspects of phospholipid biosynthesis. These experiments suggest that although major modifications in membrane fluidity may be brought about by thermally induced changes in fatty acid desaturase activity, it seems probable that additional cellular mechanisms may be involved if fluidity is under precise control.

Use of an ultraviolet tracer in simulation training for the clinical management of Ebola virus disease.

In October 2014 the UK military deployed to Sierra Leone to provide care for healthcare workers affected by Ebola virus disease. A training package designed by the Army Medical Services Training Centre prepared the deploying personnel in the required infection prevention and control measures. The training used ultraviolet tracer to provide validation of the skills required when treating patients with Ebola and to confirm subsequent decontamination. This training construct provided useful feedback to clinicians on their infection control measures and would be useful in the context of any infection spread by droplets and fomites.

Inorganic carbon acquisition by aquatic photolithoatrophs of the Dighty Burn, Angus, U.K.: uses and limitations of natural abundance measurements of carbon isotopes.

The 13 C/12 C ratio (expressed as δ13 C) of benrhic photolithotrophs. in the Dighn Water (= Burn) were measured fur comparison with that of the potential inorganic carhun sources. CO2 and HCO3 - , in the Burn. The Burn water contains an average of 65.7 mmol m-3 CO2 with δ13 C of -14.7% and 1600 mmol m-3 HCO3 - with δ13 C of -4.%. δ13 C values of riparian vegetation were also measured as contributors, after respiration in the soil or the Burn, to the δ13 C of inorganic carbon in the Burn. The potential range of differences in 13 C/12C between dissolved CO2 and plant organic C is set by the intrinsic 13 c/12 C discrimination (α value) in CO2 fixation by Rubisco. Main results and conclusions are. as follows, (i) A literature survey suggests that there is no convincing evidence that the α, Values (rate constant for 12 CO2 fixation relative to that for 13 CO2 fixation by Rubisco in the absence of CO2 transport limitation) for the'lower plants'in the Burn (diatoms, green and red algae, mosses) are significantly different from the well-established αp values for the flowering plum enzyme. (ii) In confirmation of earlier work, the semi-erect 'streamer'gametophytes of the red alga Lemanea mamillosa and the moss Fontinalis antipyetica have δ13 C values which can only be interpreted in terms of diffusive CO2 entry with minimal limitation of photosynthesis by CO- diffusion, (iii) The serui-erect grren alga Cladophora glomerata and the flowering plant Ranunculus penicillatus ssp. pseudofluitons (formerly var. calcareus) are- both able to use HCO3 - . Their δ13 C values indicate that, if the HCO3 - -use system does not (as is likely) discriminate significantly between 13 C and 12 C, then a substantial fraction of the inorganic C made available to Rubisco must return to the medium, carrying 13 C-inorganic C not fixed by Rubisco. (iv) Two sets of δ13 C data from different hydrodynamic regimes distance from leading edge of a flat stone; different size of thalli) show that the attainable differences in situ in thickness of the diffusion boundary layer do not alter the fractional limitation of photosynthesis of Cladophora by external diffusion of inorganic C, considered with HCO3 use. (vi) The entrusting red alga Hildenbrandia rivularis has a δ13 C value suggestive of CO2 as the inorganic C source, but not entirely ruling nut HCO3 - . Marine species of both Hildenbrundia and Cladophora have δ13 C values which, even when corrected for source inorganic C δ13 C values, are 10%, more positive than the freshwater species. (vii) Mats of pennate diatoms were shown by pH-drift to by able to use HCO3 - ; the relatively high (i.e. not very negative) δ12 C value of these mats could relate to a relatively'non-leaky'HCO3 - aequisition mechanism and/or to limitation by external diffusion (e.g. through the mat).

Immune reconstitution in human immunodeficiency virus-infected children receiving highly active antiretroviral therapy: a cohort study.

BACKGROUND: Highly active antiretroviral therapy (HAART) has brought about rapid declines in HIV-1 RNA concentrations and an increase in CD4+ counts in HIV-1-infected children. These changes are often accompanied by clinical improvement; however, the extent to which immune reconstitution occurs is not known. DESIGN: We compared two cohorts (n = 35) of HIV-1-infected children to evaluate the effects of HAART on immune recovery. Cohort 1 (C1) included clinically well children receiving HAART with a CD4 >22% at study initiation. Before HAART all children had moderately to severely suppressed immune function by CDC criteria (CD4 <25%) or CDC Category B or C disease. Cohort 2 (C2) included children with no current or past evidence of immunosuppression based on CDC criteria (CD4 >25%) and no evidence of clinical disease. Children in C2 were receiving a non-HAART regimen. METHODS: Immunophenotyping was performed to characterize CD4+ and CD8+ subsets with regard to maturation and activation. T cell rearrangement excision circles (TRECs) were measured to quantify recent thymic emigrants. RESULTS: No difference was found in percent CD4+ or percent CD8+ T cells or maturation markers between C1 and C2. There was significantly less expression of activation markers in both CD4+ and CD8+ cells in C1. There was no difference in TREC production between C1 and C2. CONCLUSION: Moderately to severely suppressed HIV-1-infected children receiving HAART are able to reconstitute their immune systems to a degree that is indistinguishable from that of stable, CDC Class A1 HIV-1-infected children with regard to CD4+ and CD8+ T cell subsets, expression of cellular maturation markers and TREC production.

Predominant transgene expression in exocrine pancreas directed by the CMV promoter.

The enhancer/promoter of the human cytomegalovirus gene encoding the major immediate-early protein (CMVp) is reputed to be one of the strongest and most promiscuous regulatory elements for directing transcription of heterologous genes in vitro. However, transgene expression under the promoter in adult transgenic mice is often more restricted. We selected a CMVp segment from position -350 to +59 to control expression of transgenes for two secretory fusion proteins. Expression was analyzed by immunohistology staining and quantified by Northern blot, Western blot, and ELISA of secretions from explanted tissues. In all six lines of transgenic mice, the highest expression of transgenes at the mRNA and protein level was observed in the exocrine tissue of the pancreas, although the levels of expression varied among the lines. The results indicate not only that CMVp is not a universal promoter in vivo but indeed that it can be relatively specific for the exocrine pancreas, where expression of the gene it controlled was consistently very high.

Animals and antibiotics.

The routine use of antibiotics as growth promoters is no longer acceptable, but there is a continuing requirement to treat infection occurring in herds or flocks and often it is necessary to treat all animals regardless of whether or not they are infected. To minimise the risk of the development of resistant strains the mode of treatment should be carefully considered in order to prevent the transfer of low levels of antibiotics to uninfected animals. Suitable veterinary input into the course of treatment should reduce the chance of transfer of resistant strains or resistance mechanisms to human strains of microorganisms.

Pharmacokinetics, absorption, distribution and disposition of [125I]-defibrotide following intravenous or oral administration in the rat.

Defibrotide (D) was labelled with 125I. The radiolabelled compound ([125I]-Defibrotide ([125I]-D)) retained the same profibrinolytic activity as the parent drug. Following single intravenous administration of [125I]-D the half lives of radioactivity associated with D components in plasma were 9.45 min and 11.27 h for alpha and beta phases respectively. Following single oral administration of [125I]-D the half life of radioactivity associated with D components in plasma was 12.83 h for the elimination phase. Bioavailability was apparently 58%. The areas under plasma total radioactivity versus time curves were dose-dependent following both intravenous and oral administration. No significant accumulation of total radioactivity in plasma was observed following multiple oral administration of [125I]-D. Following single intravenous administration of [125I]-D a larger proportion of administered radioactivity was excreted via urine than faeces while following single oral administration excretion via urine and faeces accounted for similar proportions of administered radioactivity. Following both single and oral administration the levels of total radioactivity in tissues and organs examined were generally highest in highly perfused organs and were very high in the thyroid despite pretreatment with non-radiolabelled potassium iodide. Radioactivity was also found to be associated with the aorta wall.

The acquisition of inorganic carbon by four red macroalgae.

Photosynthesis was studied in four species of red marine macroalgae: Palmaria palmata, Laurencia pinnatifida, Lomentaria articulata and Delesseria sanguinea. The rate of O2 evolution for submersed photosynthesis was measured as a function of incident photon flux density at normal pH and inorganic carbon concentration (pH 8.0, 2 mol m-3), and as a function of inorganic carbon concentration at pH 8.0 at saturating and at limiting photon flux density. The rate of CO2 uptake was measured for emersed photosynthesis as a function of CO2 partial pressure at saturating photon flux density. Previous pH-drift results suggest that Palmaria and Laurencia are able to use HCO inf3sup- as well as CO2 whereas Lomentaria and Delesseria are restricted to CO2. None of the algae are saturated by 2 mol m-3 inorganic carbon at high light (400 µmol m-2 s-1) but are saturated at low light (35 µmol m-2 s-1). The inorganic C concentration at which half the light-saturated rate of O2 evolution is achieved is higher for Palmaria and Laurencia (1.51 and 1.85 mol m-3) than for Lomentaria and Delesseria (0.772 and 0.841 mol m-3). The lower values for the latter two species could reflect their putative restriction to CO2. If expressed in terms of CO2, the half-saturation values yield 7.2 and 7.8 mmol m-3 respectively, which are very similar to values obtained previously during pH-drift experiments but at lower concentrations of HCO inf3sup- , consistent with restriction to CO2. The photosynthetic conductance (m s-1), calculated from the initial slope for photosynthesis at low concentrations of inorganic carbon, correlates with the suggested ability to extract inorganic carbon based on pH-drift results. Calculations made assuming that CO2 is the only species diffusing across the boundary layer are consistent with boundary layer thicknesses of 20 and 19 µm for Lomentaria and Delesseria respectively, which is feasible given the rapid water movement in the experiments. For Laurencia however, an unreasonably small boundary layer thickness of 6 µm is necessary to explain the flux, which indicates co-diffusion by HCO inf3sup- . In the apparent absence of external carbonic anhydrase, direct uptake of HCO inf3sup- , rather than external conversion to CO2 is indicated in this species. In air, the CO2 concentration at which photosynthesis is half-maximal increases in the same order as the ability to raise pH in drift experiments. At 21 kPa the CO2 compensation partial pressures for Palmaria and Laurencia at 0.56 and 1.3 Pa are low enough to suggest a carbon-concentrating mechanism is operating, while those of Lomentaria at 1.8 Pa and particularly that of Delesseria at 4.5 Pa could be explained without a carbon-concentrating mechanism. The algae tested (all except Delesseria) showed more O2 evolution than could be accounted for with a photosynthetic quotient of 1.0 and uncatalysed conversion of HCO inf3sup- to CO2 outside the cell in high light at pH 8.0 when high algal fresh weight per unit medium was used. These results are concordant with other data suggesting use of HCO inf3sup- by Palmaria and Laurencia, but discordant with the rest of the available information in indicating use of HCO inf3sup- by Lomentaria. The reason for this is unclear. The lightsaturated rate of O2 evolution on an algal area basis and the photon flux density needed to saturate photosynthesis were related partly to the habitat from which the seaweeds were collected, but more strongly to the ability to use HCO inf3sup- . Values for the two users of HCO inf3sup- , Palmaria (population used was intertidal; also occurs subtidally) and Laurencia (intertidal/shaded intertidal), were greater than for Lomentaria (shaded intertidal), which was greater than Delesseria (subtidal), both of which are believed to be restricted to CO2. In accordance with earlier δ13C data and, for Delesseria, estimates of the achieved growth rates in situ, carbon is likely to be saturating and use of HCO inf3sup- is unlikely to occur in the normal low-light habitats of Lomentaria and Delesseria. Analysis of N-use efficiencies show that they are closer to the low-CO2-affinity Laminariales than the high-CO2-affinity Fucaceae.