No difference in survival after HLA mismatched versus HLA matched allogeneic stem cell transplantation in Ewing sarcoma patients with advanced disease.

Patients with advanced Ewing sarcoma (AES) carry a poor prognosis. Retrospectively, we analyzed 66 AES patients treated with allogeneic stem cell transplantation (allo-SCT) receiving HLA-mismatched (group A, n = 39) versus HLA-matched grafts (group B, n = 27). Median age at diagnosis was 13 years, and 15 years (range 3-49 years) at allo-SCT. The two groups did not differ statistically in distribution of gender, age, remission status/number of relapses at allo-SCT, or risk stratum. 9/39 (23%) group A versus 2/27 (7%) group B patients developed severe acute graft versus host disease (GvHD). Of patients alive at day 100, 7/34 (21%) group A versus 9/19 (47%) group B patients had developed chronic GvHD. In group A, 33/39 (85%) versus 20/27 (74%) group B patients died of disease and 1/39 (3%) versus 1/27 (4%) patients died of complications, respectively. Altogether 12/66 (18%) patients survived in CR. Median EFS 24 months after allo-SCT was 20% in both groups, median OS was 27% (group A) versus 17% (group B), respectively. There was no difference in EFS and OS in AES patients transplanted with HLA-mismatched versus HLA-matched graft in univariate and multivariate analyses. In this analysis, CR at allo-SCT is a condition for survival (p < 0.02).

Perspectives for personalized therapy for patients with multidrug-resistant tuberculosis.

According to the World Health Organization (WHO), tuberculosis is the leading cause of death attributed to a single microbial pathogen worldwide. In addition to the large number of patients affected by tuberculosis, the emergence of Mycobacterium tuberculosis drug-resistance is complicating tuberculosis control in many high-burden countries. During the past 5 years, the global number of patients identified with multidrug-resistant tuberculosis (MDR-TB), defined as bacillary resistance at least against rifampicin and isoniazid, the two most active drugs in a treatment regimen, has increased by more than 20% annually. Today we experience a historical peak in the number of patients affected by MDR-TB. The management of MDR-TB is characterized by delayed diagnosis, uncertainty of the extent of bacillary drug-resistance, imprecise standardized drug regimens and dosages, very long duration of therapy and high frequency of adverse events which all translate into a poor prognosis for many of the affected patients. Major scientific and technological advances in recent years provide new perspectives through treatment regimens tailor-made to individual needs. Where available, such personalized treatment has major implications on the treatment outcomes of patients with MDR-TB. The challenge now is to bring these adances to those patients that need them most.

Whole genome sequencing for M/XDR tuberculosis surveillance and for resistance testing.

Whole genome sequencing (WGS) can help to relate Mycobacterium tuberculosis genomes to one another to assess genetic relatedness and infer the likelihood of transmission between cases. The same sequence data are now increasingly being used to predict drug resistance and susceptibility. Controlling the spread of tuberculosis and providing patients with the correct treatment are central to the World Health Organization's target to 'End TB' by 2035, for which the global prevalence of drug-resistant tuberculosis remains one of the main obstacles to success. So far, WGS has been applied largely to drug-susceptible strains for the purposes of understanding transmission, leaving a number of analytical considerations before transferring what has been learnt from drug-susceptible disease to drug-resistant tuberculosis. We discuss these potential problems here, alongside some of the challenges to characterizing the Mycobacterium tuberculosis 'resistome'-the optimal knowledge-base required for WGS-based assays to successfully direct individualized treatment regimens through the prediction of drug resistance and susceptibility in the future.

Treatment of relapse after allogeneic stem cell transplantation in children and adolescents with ALL: the Frankfurt experience.

Therapy for post-transplant relapse of paediatric ALL is limited. Standardised curative approaches are not available. We hereby describe our local procedure in this life-threatening situation. A total of 101 ALL patients received their first allogeneic stem cell transplantation (SCT) in our institution. After relapse, our primary therapeutic goal was to cure the patient with high-dose chemotherapy or specific immunotherapy (HDCHT/SIT) followed by a second SCT from a haploidentical donor (transplant approach). If this was not feasible, low-dose chemotherapy and donor lymphocyte infusions (LDCHT+DLI) were offered (non-transplant approach). A total of 23 patients suffered a post-transplant relapse. Eight patients received HDCHT/SIT, followed by haploidentical SCT in 7/8. Ten received LDCHT+DLI. The eight patients treated with a second transplant and the ten treated with the non-transplant approach had a 4-year overall survival of 56% and 40%, respectively (P=0.232). Prerequisites for successful treatment of post-transplant relapse by either a second transplant or experimental non-transplant approaches are good clinical condition and the capacity to achieve haematological remission by the induction treatment element.

Tracing Mycobacterium tuberculosis transmission by whole genome sequencing in a high incidence setting: a retrospective population-based study in East Greenland.

In East Greenland, a dramatic increase of tuberculosis (TB) incidence has been observed in recent years. Classical genotyping suggests a genetically similar Mycobacterium tuberculosis (Mtb) strain population as cause, however, precise transmission patterns are unclear. We performed whole genome sequencing (WGS) of Mtb isolates from 98% of culture-positive TB cases through 21 years (n = 182) which revealed four genomic clusters of the Euro-American lineage (mainly sub-lineage 4.8 (n = 134)). The time to the most recent common ancestor of lineage 4.8 strains was found to be 100 years. This sub-lineage further diversified in the 1970s, and massively expanded in the 1990s, a period of lowered TB awareness in Greenland. Despite the low genetic strain diversity, WGS data revealed several recent short-term transmission events in line with the increasing incidence in the region. Thus, the isolated setting and the uniformity of circulating Mtb strains indicated that the majority of East Greenlandic TB cases originated from one or few strains introduced within the last century. Thereby, the study shows the consequences of even short interruptions in TB control efforts in previously TB high incidence areas and demonstrates the potential role of WGS in detecting ongoing micro epidemics, thus guiding public health efforts in the future.

Nosocomial transmission of multidrug-resistant tuberculosis.

Nosocomial transmission of multidrug-resistant tuberculosis (MDR-TB) was ascertained by 24-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and spoligotyping at four hospitals in the Republic of Moldova, a high MDR-TB burden country. Overall, 5.1% of patients with pan-susceptible TB at baseline were identified with MDR-TB during in-patient treatment. In 75% of cases, the MDR-TB strain was genetically distinct from the non-MDR-TB strain at baseline, suggesting a high rate of nosocomial transmission of MDR-TB. The highest proportion (40.3%) of follow-up MDR-TB isolates was associated with the M. tuberculosis URAL 163-15 strain.

[Undesired side effects of tapentadol in comparison to oxycodone. A meta-analysis of randomized controlled comparative studies]. / Unerwünschte Nebenwirkungen von Tapentadol im Vergleich zu Oxycodon. Eine Metaanalyse randomisierter, kontrollierter Vergleichsstudien.

OBJECTIVE: Tapentadol is a new centrally acting analgesic with a dual mode of action as an agonist of the µ-opioid receptor and as a norepinephrine reuptake inhibitor. The aim of the present study was to evaluate the results from randomized controlled trials investigating the relative amount of adverse effects using tapentadol or oxycodone for the treatment of pain. METHODS: A quantitative systematic review was carried out according to the PRISMA recommendations on randomized controlled trials comparing tapentadol and oxycodone in pain treatment. The incidences of typical adverse side effects of opioid-based analgesic therapy (e.g. nausea, vomiting, obstipation or pruritus) were extracted and the pooled relative risks (RR) with corresponding 95% confidence intervals (CI) were calculated. RESULTS: A total of 9 trials involving 7,948 patients were included and of these 2,810 patients were treated with oxycodone and 5,138 were treated with tapentadol in equivalent analgesic dosages as documented by an equivalent analgesic effect. The risk of typical opioid-based adverse effects, such as nausea (RR 0.61; 95% CI 0.57-0.66), vomiting (RR 0.50, 95% CI: 0.41-0.60), obstipation (RR 0.47, 95%-CI 0.40-0.56), dizziness (RR 0.86, 95% CI 0.78-0.95), somnolence (RR 0.76, 95% CI 0.67-0.86) and pruritus (RR 0.46, 95% CI 0.37-0.58) was reduced when tapentadol was used for analgesic treatment. These adverse effects were investigated in all nine trials. The risk for dryness of the mouth (6 trials, 6,218 patients, RR 1.79, 95% CI 1.40-2.29) and dyspepsia (1 trial, 646 patients, RR 2.75, 95% CI 1.09-6.94) was increased when tapentadol was used instead of oxycodone. There were no significant differences in the relative risk for any other investigated adverse effect such as dysentery, headache or fatigue. CONCLUSION: The results show that using tapentadol significantly reduces the risk of the typical opioid-based adverse effects compared with oxycodone while providing equivalent analgesic treatment.

[Prophylaxis of nausea and vomiting in the postoperative phase: relative effectiveness of droperidol and metoclopramide]. / Prophylaxe von Übelkeit und Erbrechen in der postoperativen Phase : Relative Effektivität von Droperidol und Metoclopramid.

OBJECTIVE: The aim of the present study was to conduct a meta-analysis of the results from randomized controlled trials investigating the relative efficacy of droperidol versus metoclopramide for the prevention of postoperative nausea and vomiting (PONV). METHODS: A systematic literature search for randomized controlled trials comparing droperidol and metoclopramide for the prevention of PONV was performed according to the PRISMA recommendations. The incidence of PONV within the early (0-6 h) and cumulative postoperative periods (0-48 h) was collated and the pooled relative risk (RR) with the corresponding 95% confidence interval (CI) was calculated. Results from a subgroup analysis are presented excluding the data of a Japanese group (Fujii et al.) which are given in parentheses. RESULTS: A total of 41 (30) trials with a total number of 3,491 (2,721) patients were included and of these 12 (8) trials with 1,403 (1,083) patients reported data of the early period and 32 (21) studies with 2,656 (1,836) patients comprised data of the cumulative period. A total of 1,797 (1,309) patients were treated with droperidol (0.25-5 mg) and 1,694 (1,412) with metoclopramide (5-50 mg). In the early period the risk for PONV after metoclopramide was 35% (95%-CI: 17-57%) higher than after prophylaxis with droperidol (without Fujii data: 46%; 23-73%). During the cumulative period the risk for PONV after metoclopramide was increased by 20% (95%-CI: 7-37%) compared to droperidol (without Fujii data: 25%; 4-50%). Due to heterogenous dosing of both drugs subgroup analyses with distinct dose intervals were performed with increments of 0.75 mg for droperidol and 7 mg for metoclopramide. Droperidol was superior in 17 (12) out of 19 (14) subgroup analyses. Comparing recommended doses of droperidol (0.75-1.5 mg) with low doses of metoclopramide (7-14 mg) and medium metoclopramide doses (14-21 mg) PONV was increased by 12% (95%-CI: -11% to 42%) and 32% (95%-CI: 4%-66%), respectively when metoclopramide instead of droperidol was used. When higher doses of metoclopramide (>20 mg) were used the superiority of droperidol was less pronounced and did not reach statistical significance due to the limited numbers of trials included in this analysis (3 studies, 662 patients). The risk for PONV after high-dose metoclopramide was increased by 13% (95%-CI: -21% to +61%) for the early period and by 19% (95%-CI: -11% to +57%) for the cumulative observation period. CONCLUSION: For the prevention of postoperative nausea and vomiting droperidol is significantly superior to metoclopramide doses below 20 mg. There was no obvious positive dose response with respect to increasing doses of metoclopramide. There was also a trend towards higher efficacy of droperidol compared to higher doses of metoclopramide (≥20 mg). However, there were not enough comparative studies to show a statistically significant result in this subgroup analysis. These data support the notion that droperidol in low doses may represent the more effective D(2)-antagonist for a pharmacological armamentarium to cope with PONV.

Pulmonary reduction of an intravascular redox polymer.

Pulmonary endothelial cells in culture reduce external electron acceptors via transplasma membrane electron transport (TPMET). In studying endothelial TPMET in intact lungs, it is difficult to exclude intracellular reduction and reducing agents released by the lung. Therefore, we evaluated the role of endothelial TPMET in the reduction of a cell-impermeant redox polymer, toluidine blue O polyacrylamide (TBOP(+)), in intact rat lungs. When added to the perfusate recirculating through the lungs, the venous effluent TBOP(+) concentration decreased to an equilibrium level reflecting TBOP(+) reduction and autooxidation of its reduced (TBOPH) form. Adding superoxide dismutase (SOD) to the perfusate increased the equilibrium TBOP(+) concentration. Kinetic analysis indicated that the SOD effect could be attributed to elimination of the superoxide product of TBOPH autooxidation rather than of superoxide released by the lungs, and experiments with lung-conditioned perfusate excluded release of other TBOP(+) reductants in sufficient quantities to cause significant TBOP(+) reduction. Thus the results indicate that TBOP(+) reduction is via TPMET and support the utility of TBOP(+) and the kinetic model for investigating TPMET mechanisms and their adaptations to physiological and pathophysiological stresses in the intact lung.

Lung redox homeostasis: emerging concepts.

This symposium was organized to present some aspects of current research pertaining to lung redox function. Focuses of the symposium were on roles of pulmonary endothelial NADPH oxidase, xanthine oxidase (XO)/xanthine dehydrogenase (XDH), heme oxygenase (HO), transplasma membrane electron transport (TPMET), and the zinc binding protein metallothionein (MT) in the propagation and/or protection of the lung or other organs from oxidative injury. The presentations were chosen to reflect the roles of both intracellular (metallothionein, XO/XDH, and HO) and plasma membrane (NADPH oxidase, XO/XDH, and unidentified TPMET) redox proteins in these processes. Although the lung endothelium was the predominant cell type under consideration, at least some of the proposed mechanisms operate in or affect other cell types and organs as well.

Cyanide increases reduction but decreases sequestration of methylene blue by endothelial cells.

The mechanisms of endothelial cell transplasma membrane electron transport (TMET) have not been completely identified. Redox probes such as methylene blue (MB) can be useful tools, but the complexity of their disposition upon exposure to the cells can hinder interpretation. For example, MB is reduced on the cell surface by TMET, but after entering the cell in reduced form, it is reoxidized and sequestered within the cell. We developed a method to separately quantify the reduction and reoxidation rates such that it can be determined whether a metabolic inhibitor such as cyanide affects the reduction or oxidation process. MB was introduced at the inlet to a column filled with endothelial cell covered beads either as a short 12 s injection (bolus) or a long 45 min infusion (pulse), and its effluent concentration was measured as a function of time. The cells extracted 56% of the MB from the bolus, but only 41% during the pulse steady state. In the presence of cyanide, these extractions increased to 70% and decreased to 4%, respectively. Mathematical model results support the interpretation that these paradoxical effects on bolus and pulse extractions reflect the differential effects of cyanide on extracellular reduction and intracellular oxidation, i.e., cyanide increased the reduction rate from 7.3 to 13.0 cm s-1 X 10(-5) and decreased the oxidation rate from 1.09 to 0.02 cm s-1 X 10(-3). Cyanide also increased intracellular NADH by almost eight times, suggesting that TMET is sensitive to the cell redox status, i.e., NADH is a direct or indirect electron source. The cyanide-induced decrease in sequestration indicates a cyanide-sensitive intracellular oxidation mechanism. The results also demonstrate the potential utility of this approach for further evaluation of these endothelial redox mechanisms.

Transport and reaction at endothelial plasmalemma: distinguishing intra- from extracellular events.

The pulmonary endothelium is a chemical reactor that modifies blood composition in several ways, including reduction of the oxidized forms of certain redox active substances in the blood. The physiological functions of the transplasma membrane electron transport systems involved in the latter are not fully understood, but an argument is made that they are involved in antioxidant defense. In addition, the experimental approaches used to characterize the process, including studies at whole organ, cell culture, and subcellular levels, along with the use of mathematical modeling, may be representative of the physiome concept wherein a goal is the integration of information obtained at all levels of biological organization. In this article, separation of intra- and extracellular events involved in the disposition of redox active probes within the lungs is the particular example.

Proline in vasoactive peptides: consequences for peptide hydrolysis in the lung.

To examine the hypothesis that trans isomers of bradykinin and [Gly6]bradykinin are preferentially hydrolyzed by lung peptidases, we studied the fractional inactivation of these peptides in the perfused rat lung using a bioassay after a single-pass bolus injection and high-performance liquid chromatography after lung recirculation. In the bioassay studies, when the peptides passed through the lung, 25.6-fold more bradykinin or 7-fold more [Gly6]bradykinin was required to elicit a contraction equivalent to that produced when the peptides did not pass through the lung. In the recirculation studies, hydrolysis progress curves with rapid and slow phases were observed, with a higher fraction of bradykinin than [Gly6]bradykinin hydrolyzed in the rapid phase. Cyclophilin increased the hydrolysis rate during the slow phase for both peptides. Kinetic analysis indicated that the slowly hydrolyzed peptide fraction, presumably the cis fraction, was 0.13 for bradykinin and 0.43 for [Gly6]bradykinin with cis-trans isomerization rate constants of 0.074 and 0.049 s-1, respectively, consistent with published nuclear magnetic resonance studies.

Ascorbate-mediated transplasma membrane electron transport in pulmonary arterial endothelial cells.

Pulmonary endothelial cells are capable of reducing certain electron acceptors at the luminal plasma membrane surface. Motivation for studying this phenomenon comes in part from the expectation that it may be important both as an endothelial antioxidant defense mechanism and in redox cycling of toxic free radicals. Pulmonary arterial endothelial cells in culture reduce the oxidized forms of thiazine compounds that have been used as electron acceptor probes for studying the mechanisms of transplasma membrane electron transport. However, they reduce another commonly studied electron acceptor, ferricyanide, only very slowly by comparison. In the present study, we examined the influence of ascorbate [ascorbic acid (AA)] and dehydroascorbate [dehydroascorbic acid (DHAA)] on the ferricyanide and thiazine reductase activities of the bovine pulmonary arterial endothelial cell surface. The endothelial cells were grown on microcarrier beads so that the reduction of ferricyanide and methylene blue could be studied colorimetrically in spectrophotometer cuvettes and in flow-through cell columns. The ferricyanide reductase activity could be increased 80-fold by adding DHAA to the medium, with virtually no effect on methylene blue reduction. The DHAA effect persisted after the DHAA was removed from the medium. AA also stimulated the ferricyanide reductase activity but was less potent, and the relative potencies of AA and DHAA correlated with their relative rates of uptake by the cells. The results are consistent with the hypothesis that AA is an intracellular electron donor for an endothelial plasma membrane ferricyanide reductase and that the stimulatory effect of DHAA is the result of increasing intracellular AA. Adding sufficient DHAA to markedly increase extracellular ferricyanide reduction had little effect on the plasma membrane methylene blue reductase activity, suggesting that pulmonary arterial endothelial cells have at least two separate transplasma membrane electron transport systems.

Kinetics of plasma membrane electron transport in a pulmonary endothelial cell-column.

Thiazine dyes such as toluidine blue O (TBO) are reduced at the luminal endothelial surface. The purpose of this study was to determine the rate of this reaction in endothelial cells in culture. A multiple indicator dilution method was used to measure the reaction kinetics during transient passage of a TBO-containing bolus through a chromatographic column filled with bovine pulmonary arterial endothelial cells grown on microcarrier beads (cell-column). A bolus containing TBO and an inert extracellular reference indicator (FITC-Dextran) was injected upstream of the cell-column, and the indicator concentrations were measured downstream using on-line photodetection. The effects of column flow rate, PO2, and TBO concentration were studied. The fraction of TBO reduced upon passage through the cell-column decreased with increasing flow indicating that the reaction rate rather than TBO delivery controlled TBO reduction. The fraction of TBO reduced did not change with PO2 or dose in the ranges studied. TBO reduction was about 10 times that for steady state TBO sequestration by these cells which, along with the lack of a PO2 effect indicates that the rapid rate of reduction is not the rate-limiting step in steady state sequestration.

Pulmonary endothelial thiazine uptake: separation of cell surface reduction from intracellular reoxidation.

The objective of this study was to further evaluate the hypothesis that the accumulation of thiazine dyes, such as methylene blue, by cultured bovine pulmonary arterial endothelial cells involves reduction on the cell surface, followed by diffusion of the lipophilic reduced form of the dye into the cells and intracellular reoxidation to the relatively membrane-impermeant hydrophilic form. The specific question addressed was whether inhibition of methylene blue uptake by cyanide and azide is via inhibition of extracellular reduction or inhibition of intracellular reoxidation. We used the cell membrane-impermeant ferricyanide ion as a secondary electron acceptor to measure the extracellular reduction of methylene blue independently from its uptake by the cells. In addition, toluidine blue O, incorporated into an acrylamide polymer so that it could not permeate the cells in either its reduced or oxidized forms, was used to examine the effects of cyanide and azide on the extracellular reduction. Microscopic observations of the effect of the inhibitors on the intracellular accumulation of methylene blue were also made. The results indicate that the reduction and intracellular sequestration are separate processes and that, in doses that inhibited intracellular reoxidation, and therefore uptake and sequestration, neither cyanide nor azide had an inhibitory effect on extracellular reduction. The intracellular distribution of the observable oxidized form of the dye was consistent with oxidation of the reduced dye within subcellular organelles. The demonstration that extracellular reduction and intracellular sequestration are separate events is consistent with the hypothesized sequence of events.

Biotinylation of membrane proteins accessible via the pulmonary circulation in normal and hyperoxic rats.

It is well established that the phenotype of the pulmonary vascular surface can be affected by injurious stimuli, but the few proteins for which the expression and/or activity have been studied make up only a small fraction of the entire spectrum of luminal cell membrane proteins. To expand the capability for studying such proteins, we developed a method for biotinylating cell membrane proteins accessible via the vascular lumen in the isolated-perfused rat lung and examined the impact of hyperoxia on the spectrum of the biotinylated proteins. Labeling was carried out either by single-pass bolus injection of the cell impermeant biotinylation reagent sulfosuccinimidyl 6-biotin-amido hexanoate (NHS-LC-biotin) into the pulmonary artery cannula or by the addition of NHS-LC-biotin to a lung homogenate. Lung membrane fractions were prepared, and the proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by electroblotting. The biotinylated proteins were visualized using a chemiluminescent substrate for streptavidin-linked horseradish peroxidase. The spectrum of proteins biotinylated via the vasculature was distinct from that of the biotinylated lung homogenate. Lectin affinity purification of biotinylated proteins from the lung membrane fractions of normal lungs biotinylated via the vasculature revealed characteristic spectra that were reproducibly different from those from rats exposed to hyperoxia for 48-60 h. These results demonstrate that biotinylation of membrane proteins accessible to an extracellular reagent during a single transit through the pulmonary vascular bed is feasible and that the spectrum of these labeled proteins reveals the effects of hyperoxic lung injury. The affinity of biotin for streptavidin makes this procedure potentially useful as a means of separating the labeled membrane proteins from the much larger population of membrane proteins that are not accessible via the vasculature, e.g., intracellular membrane proteins and plasma membrane proteins of cell types in luminally inaccessible regions of the intact lung. The consistent changes in the spectrum of labeled proteins seen with hyperoxia suggest that in itself the spectrum may be a useful encryption of certain aspects of vascular pathophysiology.

Impact of angiotensin-converting enzyme substrate conformation on fractional hydrolysis in lung.

We examined the hydrolysis kinetics of benzoyl-phenylalanyl-glycyl-proline (BPGP) in the isolated perfused lung and in vitro for evidence of preferential hydrolysis of the trans isomer by angiotensin-converting enzyme (ACE). Nuclear magnetic resonance spectroscopy showed that BPGP exists as cis and trans isomers in a ratio of 44:56. After a single pass through the perfused rabbit lung over a wide range of infused BPGP concentrations, 42% of the BPGP was not hydrolyzed. In single-pass bolus-injection studies, 41% of the injected BPGP was not hydrolyzed, and very little further hydrolysis occurred in a second passage of the bolus through the lungs. In rat lung recirculation and in vitro studies of BPGP hydrolysis by ACE, approximately 60% of the substrate was hydrolyzed rapidly compared with the remaining approximately 40%, and the peptidyl-prolyl cis-trans isomerase cyclophilin increased the rate of the slower phase of the reaction in both kinds of experiments. We conclude that the rapid hydrolysis phase represents primarily the hydrolysis rate of the trans isomer and the slower phase the cis-trans isomerization rate, suggesting that the trans isomer of BPGP is preferentially hydrolyzed by ACE in the perfused lung and in vitro.