Revised contraindications for the use of non-medical WB-electromyostimulation. Evidence-based German consensus recommendations.

Whole-body electromyostimulation has proven to be a highly effective alternative to conventional resistance-type exercise training. However, due to adverse effects in the past, very extensive contraindications have been put in place for the commercial, non-medical WB-EMS market. Considering recent positive innovations e.g., federal regulation, mandatory trainer education, revised guidelines, and new scientific studies on WB-EMS application, we believe that a careful revision of the very restrictive contraindications on WB-EMS is needed. This applies all the more because many cohorts with limited options for conventional exercise have so far been excluded. During a first meeting of an evidence-based consensus process, stakeholders from various backgrounds (e.g., research, education, application) set the priorities for revising the contraindications. We decided to focus on four categories of absolute contraindications: "Arteriosclerosis, arterial circulation disorders", "Diabetes mellitus" (DM), "Tumor and cancer" (TC), "Neurologic diseases, neuronal disorders, epilepsy". Based on scientific studies, quality criteria, safety aspects and benefit/risk assessment of the category, DM and TC were moved to the relative contraindication catalogue, while arteriosclerosis/arterial circulation disorders and neurologic diseases/neuronal disorders/epilepsy were still considered as absolute contraindications. While missing evidence suggests maintaining the status of neurologic diseases/neuronal disorders as an absolute contraindication, the risk/benefit-ratio does not support the application of WB-EMS in people with arteriosclerosis/arterial circulation diseases. Despite these very cautious modifications, countries with less restrictive structures for non-medical WB-EMS should consider our approach critically before implementing the present revisions. Considering further the largely increased amount of WB-EMS trials we advice regular updates of the present contraindication list.

Operationalising targeted next-generation sequencing for routine diagnosis of drug-resistant TB.

BACKGROUND: Phenotypic drug susceptibility testing (pDST) for Mycobacterium tuberculosis can take up to 8 weeks, while conventional molecular tests identify a limited set of resistance mutations. Targeted next-generation sequencing (tNGS) offers rapid results for predicting comprehensive drug resistance, and this study sought to explore its operational feasibility within a public health laboratory in Mumbai, India. METHODS: Pulmonary samples from consenting patients testing Xpert MTB-positive were tested for drug resistance by conventional methods and using tNGS. Laboratory operational and logistical implementation experiences from study team members are shared below. RESULTS: Of the total number of patients tested, 70% (113/161) had no history of previous TB or treatment; however, 88.2% (n = 142) had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB). There was a high concordance between resistance predictions of tNGS and pDST for most drugs, with tNGS more accurately identifying resistance overall. tNGS was integrated and adapted into the laboratory workflow; however, batching samples caused significantly longer result turnaround time, fastest at 24 days. Manual DNA extraction caused inefficiencies; thus protocol optimisations were performed. Technical expertise was required for analysis of uncharacterised mutations and interpretation of report templates. tNGS cost per sample was US$230, while for pDST this was US$119. CONCLUSIONS: Implementation of tNGS is feasible in reference laboratories. It can rapidly identify drug resistance and should be considered as a potential alternative to pDST.

CONTEXTE: Les tests phénotypiques de sensibilité aux médicaments (pDST) pour Mycobacterium tuberculosis peuvent prendre jusqu'à 8 semaines, tandis que les tests moléculaires conventionnels identifient un ensemble limité de mutations de résistance. Le séquençage ciblé de la prochaine génération (tNGS) offre des résultats rapides pour prédire la résistance globale aux médicaments, et cette étude avait pour objectif d'explorer sa faisabilité opérationnelle au sein d'un laboratoire de santé publique à Mumbai, en Inde. MÉTHODES: Des échantillons pulmonaires de patients consentants testés positifs au Xpert MTB ont été testés pour la résistance aux médicaments par des méthodes conventionnelles et en utilisant le tNGS. Les expériences des membres de l'équipe de l'étude en matière de fonctionnement du laboratoire et de mise en œuvre logistique sont présentées ci-dessous. RÉSULTATS: Sur le nombre total de patients testés, 70% (113/161) n'avaient pas d'antécédents de TB ou de traitement ; cependant, 88,2% (n = 142) présentaient une TB résistante à la rifampicine/multirésistante aux médicaments (RR/MDR-TB). La concordance entre les prédictions de résistance de la tNGS et de la pDST était élevée pour la plupart des médicaments, la tNGS identifiant globalement la résistance avec plus de précision. La tNGS a été intégrée et adaptée au flux de travail du laboratoire ; toutefois, la mise en lots des échantillons a entraîné un délai d'obtention des résultats beaucoup plus long, le plus rapide étant de 24 jours. L'extraction manuelle de l'ADN a été source d'inefficacité ; le protocole a donc été optimisé. L'analyse des mutations non caractérisées et l'interprétation des modèles de rapport ont nécessité une expertise technique. Le coût du tNGS par échantillon s'élevait à US$230, contre US$119 pour le pDST. CONCLUSIONS: La mise en œuvre de la tNGS est possible dans les laboratoires de référence. Elle permet d'identifier rapidement la résistance aux médicaments et devrait être considérée comme une alternative potentielle à la pDST.

Clinical utility of target-based next-generation sequencing for drug-resistant TB.

BACKGROUND: In high TB burden countries, access to drug susceptibility testing is a major bottleneck. Targeted next-generation sequencing (tNGS) is a promising technology for rapid resistance detection. This study assessed the role of tNGS for the diagnosis of drug-resistant TB (DR-TB).METHODS: A total of 161 samples from bacteriologically confirmed TB cases were subjected to tNGS using the Deeplex® Myc-TB kit and sequenced using the MiSeq platform. These samples were also processed for conventional phenotypic DST (pDST) using 13 drugs on Mycobacteria Growth Indicator Tube and line-probe assays (MTBDRplus and MTBDRsl).RESULTS: There were 146 DR-TB and 15 drug-susceptible TB (DS-TB) samples. About 70% of patients with DR-TB had no previous TB treatment history. Overall, 88.2% had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB), 58.5% pre-extensively drug-resistant TB (pre-XDR-TB) and 9.2% had XDR-TB as defined by the WHO (2020). Around 8% (n = 13) of samples were non-culturable; however, identified 8 were resistant to first and second-line drugs using tNGS. Resistance frequency was similar across methods, with discordance in drugs less reliable using pDST or with limited mutational representation within databases. Sensitivities were aligned with literature reports for most drugs. We observed 10% heteroresistance, while 75% of strains were of Lineages 2 and 3.CONCLUSIONS: Programme data supported tNGS in the diagnosis of DR-TB for early treatment using individualised regimens.

Rethink Vape: Development and evaluation of a risk communication campaign to prevent youth E-cigarette use.

INTRODUCTION: E-cigarettes are now the most common form of tobacco use among adolescents, and use is associated with increased risk of initiation of cigarette smoking. This project used a community-engaged research process to develop and pilot a risk communication campaign to prevent youth vaping. METHOD: The research team worked with a 36-member Teen Advisory Council and a 19-member Expert Panel. Together, the team employed survey (N = 674) and focus group (N = 82) methodologies, and hired a marketing company to partner on development of the campaign. Campaign concepts were developed, eliminated, and/or modified through an iterative process of feedback and refinement. The final campaign included video ads (https://bit.ly/2QMR8gH) a microsite (rethinkvape.org), and social media sites (@rethinkvape). The campaign communicated three messages to teens: what's in the vapor, health risks, and connections to big tobacco. Prior to launch of the campaign, a randomized controlled 2 (time) × 2 (group) online experiment was conducted to evaluate the campaign (N = 268). RESULTS: Repeated measures mixed analyses of variance indicated that vaping knowledge, perceptions of risk, and anti-vape intentions significantly increased among teens viewing the Rethink Vape Materials compared to their own baseline, while control participants did not change. Following evaluation, the team launched a 6-week online media campaign with a teen-targeted geo-fence radius to deliver 3,838,465 impressions, 770,443 completed video views, and 18,316 clicks in mobile app, Snapchat, YouTube, and Spotify platforms. The majority of placements exceeded industry standards, with mobile pre-roll and Snapchat as top performers. CONCLUSIONS: The e-cigarette campaign showed promising signs of effectiveness and scalability.

Detecting tuberculosis: rapid tools but slow progress.

The World Health Organization (WHO) currently recommends Xpert® MTB/RIF as the initial test for all people with presumptive tuberculosis (TB). A number of challenges have been reported, however, in using this technology, particularly in low-resource settings. Here we examine these challenges, and provide our perspective of the barriers to Xpert scale-up as assessed through a survey in 16 TB burden countries in which the Médecins Sans Frontières is present. We observed that the key barriers to scale-up include a lack of policy adoption and implementation of WHO recommendations for the use of Xpert, resulting from high costs, poor sensitisation of clinical staff and a high turnover of trained laboratory staff; insufficient service and maintenance provision provided by the manufacturer; and inadequate resources for sustainability and expansion. Funding is a critical issue as countries begin to transition out of support from the Global Fund. While it is clear that there is still an urgent need for research into and development of a rapid, affordable point-of-care test for TB that is truly adapted for use in low-resource settings, countries in the meantime need to develop functional and sustainable Xpert networks in order to close the existing diagnostic gap.

L'Organisation Mondiale de la Santé (OMS) recommande actuellement l'Xpert® MTB/RIF comme test initial pour toutes les personnes suspectes de tuberculose (TB). Le recours à cette technique s'est cependant heurté à un certain nombre de défis, particulièrement dans les contextes de faibles ressources. Nous examinons ici ces défis et proposons notre vision des contraintes à l'expansion comme l'a montré une enquête dans 16 pays très frappés par la TB où Médecins Sans Frontières est présent. Nous affirmons que les entraves majeures à l'expansion incluent un manque d'adoption politique et de mise en œuvre des recommandations de l'OMS relatives à l'utilisation de l'Xpert, résultant des coûts élevés, d'une sensibilisation insuffisante du personnel clinique et d'un taux élevé de renouvellement du personnel de laboratoire formé ; une offre insuffisante de service et de maintenance de la part du fabricant ; et des ressources insuffisantes pour la pérennité et l'expansion. Le financement est un problème crucial à mesure que les pays commencent leur transition vers l'arrêt du soutien du Fonds Mondial. Il est clair qu'il y a toujours un besoin urgent de recherche et de développement d'un test de TB rapide, abordable, réalisable sur place et réellement adapté à une utilisation dans des contextes de faibles ressources. Dans l'intervalle, les pays ont cependant besoin d'élaborer des réseaux d'Xpert fonctionnels et pérennisables de manière à combler le fossé existant en matière de diagnostic.

En la actualidad, la Organización Mundial de la Salud (OMS) recomienda como primer método diagnóstico la prueba Xpert® MTB/RIF en todas las personas con presunción clínica de tuberculosis (TB). Se ha comunicado, no obstante, una serie de dificultades con la utilización de esta técnica sobre todo en entornos con recursos limitados. En el presente estudio se examinan estas dificultades y se ofrece una perspectiva de los obstáculos a la ampliación de escala de la recomendación, a partir de una encuesta realizada en 16 países con carga de morbilidad por TB donde está presente Médicos Sin Fronteras. Los autores afirman que los principales obstáculos a la ampliación de escala son los siguientes: la falta de adopción de políticas en favor de las recomendaciones de la OMS sobre la utilización de la prueba Xpert y el refuerzo de su cumplimiento, debido a los costos altos, la escasa sensibilización del personal médico y una alta rotación del personal de laboratorio capacitado; la insuficiencia de la prestación de servicios y mantenimiento por parte del fabricante; y los recursos insuficientes para la sostenibilidad y la ampliación. El financiamiento constituye un elemento primordial a medida que los países dejan de percibir el apoyo del Fondo Mundial. Es evidente que existe una necesidad urgente de investigación y desarrollo de una prueba en el lugar de la consulta que sea asequible y efectivamente adaptada a los entornos con recursos escasos, pero mientras tanto, los países deben crear redes funcionales y sostenibles de la prueba Xpert a fin de subsanar las deficiencias actuales en el diagnóstico.

European all-cause excess and influenza-attributable mortality in the 2017/18 season: should the burden of influenza B be reconsidered?

OBJECTIVES: Weekly monitoring of European all-cause excess mortality, the EuroMOMO network, observed high excess mortality during the influenza B/Yamagata dominated 2017/18 winter season, especially among elderly. We describe all-cause excess and influenza-attributable mortality during the season 2017/18 in Europe. METHODS: Based on weekly reporting of mortality from 24 European countries or sub-national regions, representing 60% of the European population excluding the Russian and Turkish parts of Europe, we estimated age stratified all-cause excess morality using the EuroMOMO model. In addition, age stratified all-cause influenza-attributable mortality was estimated using the FluMOMO algorithm, incorporating influenza activity based on clinical and virological surveillance data, and adjusting for extreme temperatures. RESULTS: Excess mortality was mainly attributable to influenza activity from December 2017 to April 2018, but also due to exceptionally low temperatures in February-March 2018. The pattern and extent of mortality excess was similar to the previous A(H3N2) dominated seasons, 2014/15 and 2016/17. The 2017/18 overall all-cause influenza-attributable mortality was estimated to be 25.4 (95%CI 25.0-25.8) per 100,000 population; 118.2 (116.4-119.9) for persons aged 65. Extending to the European population this translates into over-all 152,000 deaths. CONCLUSIONS: The high mortality among elderly was unexpected in an influenza B dominated season, which commonly are considered to cause mild illness, mainly among children. Even though A(H3N2) also circulated in the 2017/18 season and may have contributed to the excess mortality among the elderly, the common perception of influenza B only having a modest impact on excess mortality in the older population may need to be reconsidered.

Interferon-gamma response to the treatment of active pulmonary and extra-pulmonary tuberculosis.

BACKGROUND: Interferon-gamma (IFN-γ) release assays (IGRAs) are used to diagnose tuberculosis (TB) but not to measure treatment response. OBJECTIVE: To measure IFN-γ response to active anti-tuberculosis treatment. DESIGN: Patients from the Henan Provincial Chest Hospital, Henan, China, with TB symptoms and/or signs were enrolled into this prospective, observational cohort study and followed for 6 months of treatment, with blood and sputum samples collected at 0, 2, 4, 6, 8, 16 and 24 weeks. The QuantiFERON® TB-Gold assay was run on collected blood samples. Participants received a follow-up telephone call at 24 months to determine relapse status. RESULTS: Of the 152 TB patients enrolled, 135 were eligible for this analysis: 118 pulmonary (PTB) and 17 extra-pulmonary TB (EPTB) patients. IFN-γ levels declined significantly over time among all patients (P = 0.002), with this decline driven by PTB patients (P = 0.001), largely during the initial 8 weeks of treatment (P = 0.019). IFN-γ levels did not change among EPTB patients over time or against baseline culture or drug resistance status. CONCLUSION: After 6 months of effective anti-tuberculosis treatment, IFN-γ levels decreased significantly in PTB patients, largely over the initial 8 weeks of treatment. IFN-γ concentrations may offer some value for monitoring anti-tuberculosis treatment response among PTB patients.

The impact of mode of acquisition on biological markers of paediatric hepatitis C virus infection.

Despite the introduction of blood donor screening, worldwide, children continue to become infected with hepatitis C virus (HCV) via un-sterile medical injections, receipt of unscreened blood and isolated hospital contamination outbreaks. It is plausible that the natural history and disease progression in these children might differ from that of their vertically infected counterparts. Vertically and parenterally HCV-infected children were prospectively followed within the European Paediatric HCV Network and the UK National HCV Register, respectively. Biological profiles were compared. Vertically and parenterally HCV-infected children differed in terms of some key characteristics including the male to female ratio and the proportion of children receiving therapy. Parenterally infected children were more likely to have at least one hepatomegaly event during follow-up, 20%vs 10%. Parenteral infection did not significantly affect the odds of being consistently viraemic (AOR 1.14, P = 0.703) and there was no significant difference in the odds of having consistently elevated ALT levels and mode of acquisition (AOR 0.83, P = 0.748). The proportion of children with 2 or more markers of HCV infection did not differ significantly by mode of acquisition (χ(2) 1.13, P = 0.288). This analysis does not support substantial differences between vertically and parenterally infected groups, but there are specific mechanisms identified requiring further investigation. Given the continued parenteral infection of children worldwide, it is vital that knowledge of disease progression in this group is accurate and that the differences in comparison with vertically infected children are clarified to inform more accurate and individualized clinical management.

Effects of selected fertilizers on the life history of Bemisia tabaci (Hemiptera: Aleyrodidae) biotype B.

We tested the effects among a purportedly sustainable water-soluble fertilizer, a conventional water-soluble fertilizer, an alternation of these, a controlled-release fertilizer, and a clear water control on the life-history traits of sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae; =Bemisia argentifolii Bellows & Perring) biotype B reared on poinsettia (Euphorbia pulcherrima Willdenow ex Klotzch). Free amino acids in petioles were measured to estimate plant nutrient assimilation and phloem nutritional quality for B. tabaci biotype B. The sustainable fertilizer produced plants with the highest concentration of amino acids. In contrast, fecundity of whiteflies was lowest in plants treated with the sustainable fertilizer and the water control. The relationship between total amino acids in phloem and survival was significantly quadratic, with the highest survival at intermediate levels. Fecundity, however, was negatively correlated with total amino acid content of the maternal host plant. Variation in total amino acid concentration in petioles of plants treated within fertilizer treatments makes it difficult to predict whether a particular fertilizer will produce plants with enough amino acids to deleteriously affect both survivorship and fecundity and yet yield a plant of good quality. Despite this limitation, we can conclude that the use of this sustainable fertilizer will not cause increases in whitefly populations relative to plants fertilized with water-soluble and slow-release fertilizers that deliver the same level of nitrogen to the plant.

Filaggrin loss-of-function mutations are associated with early-onset eczema, eczema severity and transepidermal water loss at 3 months of age.

BACKGROUND: Filaggrin loss-of-function (FLG) mutations are associated with eczema and skin barrier impairment, but it is unclear whether skin barrier impairment precedes phenotypic eczema in FLG mutation carriers. OBJECTIVES: To study the association between FLG mutations, skin barrier impairment and clinical eczema at 3 months of age. METHODS: A total of 88 infants were examined for eczema. Disease severity was determined by the SCORAD eczema severity score. Transepidermal water loss (TEWL) was measured on unaffected forearm skin. Venous blood samples were screened for the four most common FLG mutations found in the U.K. white population (R501X, 2282del4, R2447X and S3247X). Median SCORAD and TEWL measurements in children with and without eczema and FLG mutations were compared. RESULTS: Thirty-three per cent (29/88) of children had clinical eczema. Median SCORAD was 10·6 (range 3·5-31·0). TEWL (g m⁻² h⁻¹) was higher in children with eczema compared with unaffected infants (median TEWL 14·24 vs. 11·24, P < 0·001). Higher TEWL was associated with more severe disease (r = 0·59, P < 0·001, median TEWL, SCORAD < 15, 13·1 vs. 29·6, SCORAD ≥ 15, P = 0·029). Clinically dry skin was associated with higher TEWL, even in the absence of eczema (median TEWL 17·55 vs. 11·08, P = 0·008). Seventeen per cent (15/88) of children carried at least one FLG mutation. FLG mutation carriers were significantly more likely to have clinically dry skin, even in the absence of eczema [odds ratio (OR) 8·50, 95% confidence interval (CI) 1·09-66·58, P = 0·042]. FLG mutation carriers were also more likely to have eczema by 3 months of age (OR 4·26, 95% CI 1·34-13·57, P = 0·014). FLG mutations were significantly associated with higher median TEWL (all children, FLG 'yes' 21·59 vs. FLG 'no' 11·24, P < 0·001), even without clinical eczema (FLG 'yes' 15·99 vs. FLG 'no' 10·82, P = 0·01). CONCLUSIONS: By the age of 3 months, FLG mutations are associated with an eczema phenotype, dry skin and TEWL. The observation that TEWL is elevated in unaffected FLG mutation carriers suggests that skin barrier impairment precedes clinical eczema.

Mode of delivery in HIV-infected pregnant women and prevention of mother-to-child transmission: changing practices in Western Europe.

OBJECTIVES: The aim of the study was to examine temporal and geographical patterns of mode of delivery in the European Collaborative Study (ECS), identify factors associated with elective caesarean section (CS) delivery in the highly active antiretroviral therapy (HAART) era and explore associations between mode of delivery and mother-to-child transmission (MTCT). METHODS: The ECS is a cohort study in which HIV-infected pregnant women are enrolled and their infants prospectively followed. Data on 5238 mother-child pairs (MCPs) enrolled in Western European ECS sites between 1985 and 2007 were analysed. RESULTS: The elective CS rate increased from 16% in 1985-1993 to 67% in 1999-2001, declining to 51% by 2005-2007. In 2002-2004, 10% of infants were delivered vaginally, increasing to 34% by 2005-2007. During the HAART era, women in Belgium, the United Kingdom and the Netherlands were less likely to deliver by elective CS than those in Italy and Spain [adjusted odds ratio (AOR) 0.07; 95% confidence interval (CI) 0.04-0.12]. The MTCT rate in 2005-2007 was 1%. Among MCPs with maternal HIV RNA<400 HIV-1 RNA copies/mL (n=960), elective CS was associated with 80% decreased MTCT risk (AOR 0.20; 95% CI 0.05-0.65) adjusting for HAART and prematurity. Two infants born to 559 women with viral loads <50 copies/mL were infected, one of whom was delivered by elective CS (MTCT rate 0.4%; 95% CI 0.04-1.29). CONCLUSIONS: Our findings suggest that elective CS prevents MTCT even at low maternal viral loads, but the study was insufficiently powered to enable a conclusion to be drawn as to whether this applies for viral loads <50 copies/mL. Diverging mode of delivery patterns in Europe reflect uncertainties regarding the risk-benefit balance of elective CS for women on successful HAART.

Critical care at Role 4.

This descriptive paper focuses on the sequence of events that occur during the admission and ongoing management of the Military Polytrauma patient to Critical Care, Area B, Queen Elizabeth Hospital Birmingham (QEHB). It is intended to inform new clinical staff, the wider DMS, and potentially other NHS intensive care units which may be called upon to manage such patients during a military surge or following a U.K. domestic major incident.

Salmonella typhimurium stimulation combined with tumour-derived heat shock proteins induces potent dendritic cell anti-tumour responses in a murine model.

Appropriate activation of the immune system and effective targeting of tumour cells are the primary hurdles to be overcome for cancer immunotherapy to be successful and applicable to a wide range of tumour types. Our studies have examined the ability of bacterial-stimulated dendritic cells (DCs), loaded with tumour-associated antigens, to inhibit tumour growth in a murine model. Immature murine bone marrow-derived DCs were stimulated in vitro with the cytoplasmic fraction (CM) of Salmonella typhimurium in combination with heat shock proteins (hsps) from 4T1 tumours, isolated using heparin affinity chromatography. Activated DCs were administered subcutaneously. Tumours were generated by orthotopic inoculation of 4T1 cells in Balb/c mice. Primary tumour growth was measured using Vernier calipers, while lung metastases were measured using the clonogenic assay. S. typhimurium CM induced potent tumour necrosis factor (TNF)-alpha responses from DCs accompanied by significant up-regulation of CD80 and CD86 expression. When injected into mice, bacterial-stimulated DCs loaded with 4T1 hsps inhibited the formation of new 4T1 tumours and reduced the growth rate of established tumours. In addition, the number of lung metastatic nodules was reduced significantly in the DC-treated mice (1.6 +/- 0.6 versus 245.9 +/- 55.6, P = 0.0015). DCs stimulated with CM alone, exposed to tumour hsps alone or exposed to tumour hsps from an unrelated tumour cell line did not induce a protective immune response. Dendritic cells primed with a proinflammatory bacterial stimulus and tumour-associated antigens induce a protective anti-tumour immune response in this murine model.

Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis.

The production of ROS is an inevitable consequence of metabolism. However, high levels of ROS within a cell can be lethal and so the cell has a number of defences against oxidative cell stress. Occasionally the cell's antioxidant mechanisms fail and oxidative stress occurs. High levels of ROS within a cell have a number of direct and indirect consequences on cell signalling pathways and may result in apoptosis or necrosis. Although some of the indirect effects of ROS are well known, limitations in technology mean that the direct effects of the cell's redox environment upon proteins are less understood. Recent work by a number of groups has demonstrated that ROS can directly modify signalling proteins through different modifications, for example by nitrosylation, carbonylation, di-sulphide bond formation and glutathionylation. These modifications modulate a protein's activity and several recent papers have demonstrated their importance in cell signalling events, especially those involved in cell death/survival. Redox modification of proteins allows for further regulation of cell signalling pathways in response to the cellular environment. Understanding them may be critical for us to modulate cell pathways for our own means, such as in cytotoxic drug treatments of cancer cells. Protein modifications mediated by oxidative stress can modulate apoptosis, either through specific protein modifications resulting in regulation of signalling pathways, or through a general increase in oxidised proteins resulting in reduced cellular function. This review discusses direct oxidative protein modifications and their effects on apoptosis.

Hypotension and acute pulmonary insufficiency following transfusion of autologous red blood cells during surgery: a case report and review of the literature.

Transfusion of autologous blood is associated with fewer complications, although all untoward events of transfusion may not be negated with this strategy. We report a case of acute pulmonary insufficiency and hypotension following transfusion of autologous packed red blood cells (PRBCs) in a patient, who was undergoing major surgery. Anti-HLA class-I and class-II and anti-granulocyte antibodies were measured in the unit and in the recipient. Neutrophil (PMN)-priming activity was measured as the augmentation of the formyl-Met-Leu-Phe-activated respiratory burst. No immunoglobulins were identified; however, significant lipid-priming activity was present in the implicated, autologous PRBC unit that primed PMNs from both healthy people and the recipient. In addition, lipids, identical to those that accumulate during PRBC storage, caused significant hypotension when infused into rats at similar concentrations found in stored PRBCs. We conclude that the observed transfusion-related acute lung injury reaction with significant hypotension may be the result of two independent events: the first is related to inherent host factors, in this case major surgery, and the second is the infusion of lipids that accumulate during the routine storage of PRBCs.

Identification of carbonylated proteins by MALDI-TOF mass spectroscopy reveals susceptibility of ER.

Reactive oxygen species are produced by metabolism over time, but can also be produced in more acute conditions of cell stress such as treatment with cytotoxic drugs. Treatment of HL-60 cells with peroxide results in cell death and protein carbonylation, a non-enzymatic protein modification that typically results from oxidative stress within cells. It has recently become clear that protein carbonylation during ageing is confined to specific proteins. It is therefore of interest to be able to identify which proteins are susceptible to protein carbonylation. Here we demonstrate immunoprecipitation of carbonylated proteins coupled with 2D-gel electrophoresis to identify carbonylated proteins by MALDI-TOF m/s fingerprinting. The results show that some ER proteins are readily carbonylated in response to peroxide treatment of HL-60 cells. This is likely to have implications for the induction of cell death in such cells.

The anticancer drug mithramycin A sensitises tumour cells to apoptosis induced by tumour necrosis factor (TNF).

In this report we show that mithramycin considerably increases the direct cytotoxic effect of tumour necrosis factor (TNF) on tumour cells in vitro. Sensitisation to TNF-induced apoptosis was prevented by the broad caspase inhibitor zVAD-fmk, whereas overexpression of Bcl-2 had no effect. Mithramycin also potentiated cell death induced by Fas agonistic antibodies. In contrast, mithramycin reduced the percentage of cells undergoing apoptosis due to factor withdrawal. TNF-induced activation of NF-kappaB (NF-kappaB)-dependent gene expression was not modulated by mithramycin treatment. Concomitantly with the increased sensitivity, the protein level of the short-spliced cFLIP variant was downregulated. These results indicate that mithramycin enhances TNF-induced cell death in an NF-kappaB-independent manner, and suggest that the Fas-associated death domain protein plays a crucial role in the TNF-sensitising effect of mithramycin.

Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis.

Recent work has highlighted the importance of protein post-translational modifications such as phosphorylation (enzymatic) and nitrosylation (nonenzymatic) in the early stages of apoptosis. In this study, we have investigated the levels of protein carbonylation, a nonenzymatic protein modification that occurs in conditions of cellular oxidative stress, during etopside-induced apoptosis of HL60 cells. Within 1 h of VP16 treatment, a number of proteins underwent carbonylation due to oxidative stress. This was inhibited by the antioxidant N-acetyl-L-cysteine. Among the proteins found to be carbonylated were glycolytic enzymes. Subsequently, we found that the rate of glycolysis was significantly reduced, probably due to a carbonylation mediated reduction in enzymatic activity of glycolytic enzymes. Our work demonstrates that protein carbonylation can be rapidly induced through cytotoxic drug treatment and may specifically inhibit the glycolytic pathway. Given the importance of glycolysis as a source of cellular ATP, this has severe implications for cell function.

VEGF induces airway epithelial cell proliferation in human fetal lung in vitro.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen involved in normal and abnormal angiogenesis. VEGF mRNA and protein are abundant in distal epithelium of midtrimester human fetal lung. In the present study, we identified immunoreactivity for KDR, a major VEGF-specific receptor, in distal lung epithelial cells of human fetal lung tissue, suggesting a possible autocrine or paracrine regulatory role for VEGF in pulmonary epithelial cell growth and differentiation. Addition of exogenous VEGF to human fetal lung explants resulted in increased epithelium volume density and lumen volume density in the tissues, both morphometric parameters of tissue differentiation. Cellular proliferation demonstrated by bromodeoxyuridine uptake was prominent in distal airway epithelial cells and increased in the VEGF-treated explants. VEGF-treated explants also demonstrated increased surfactant protein (SP) A mRNA, SP-C mRNA, and SP-A protein levels compared with controls. However, SP-B mRNA levels were unaffected by VEGF treatment. [(3)H]choline incorporation into total phosphatidylcholine was increased by VEGF treatment, but incorporation into disaturated phosphatidylcholine was not affected by exogenous VEGF. Based on these observations, we conclude that VEGF may be an important autocrine growth factor for distal airway epithelial cells in the developing human lung.

Signalling pathways regulating the dephosphorylation of Ser729 in the hydrophobic domain of protein kinase Cepsilon upon cell passage.

We have recently demonstrated that in quiescent fibroblasts protein kinase C (PKC) epsilon(95) is phosphorylated at Ser(729), Ser(703), and Thr(566) and that upon passage of quiescent cells phosphorylation at Ser(729) is lost, giving rise to PKCepsilon(87). Ser(729) may be rephosphorylated later, suggesting cycling between PKCepsilon(87) and PKCepsilon(95). Here we show that the dephosphorylation at Ser(729) is insensitive to okadaic acid, calyculin, ascomycin C, and cyclosporin A, suggesting that dephosphorylation at this site is not mediated through protein phosphatases 1, 2A or 2B. We demonstrate that this dephosphorylation at Ser(729) requires serum and cell readhesion and is sensitive to rapamycin, PD98059, chelerythrine, and Ro-31-8220. These results suggest that the phosphorylation status of Ser(729) in the hydrophobic domain at Ser(729) is regulated independently of the phosphorylation status of other sites in PKCepsilon, by a mTOR-sensitive phosphatase. The mitogen-activated protein kinase pathway and PKC are also implicated in regulating the dephosphorylation at Ser(729).