Developing a control framework for self-adjusting prosthetic sockets incorporating tissue injury risk estimation and generalized predictive control.

To perform activities of daily living (ADL), people with lower limb amputation depend on the prosthetic socket for stability and proprioceptive feedback. Poorly fitting sockets can cause discomfort, pain, limb tissue injuries, limited device usage, and potential rejection. Semi-passively controlled adjustable socket technologies exist, but these depend upon the user's perception to determine safe interfacial pressure levels. This paper presents a framework for automatic control of an adjustable transtibial prosthetic socket that enables active adaptation of residuum-socket interfacial loading through localized actuators, based on soft tissue injury risk estimation. Using finite element analysis, local interfacial pressure vs. compressive tissue strain relationships were estimated for three discrete anatomical actuator locations, for tissue injury risk assessment within a control structure. Generalized Predictive Control of multiple actuators was implemented to maintain interfacial pressure within estimated safe and functional limits. Controller simulation predicted satisfactory dynamic performance in several scenarios. Actuation rates of 0.06-1.51 kPa/s with 0.67% maximum overshoot, and 0.75-1.58 kPa/s were estimated for continuous walking, and for a demonstrative loading sequence of ADL, respectively. The developed platform could be useful for extending recent efforts in adjustable lower limb prosthetic socket design, particularly for individuals with residuum sensory impairment.

Coding of COPD Exacerbations and the Implications on Clinical Practice, Audit and Research.

International Classification of Disease 10 (ICD-10) codes record hospital admissions. We aimed to measure the accuracy of COPD exacerbation (ECOPD) codes and examine coding practices for COPD exacerbation.Prospective screening and ICD-10 codes were used to identify potential ECOPD within the DECAF internal validation cohort. Two coding searches were performed. The first search identified patients with an ECOPD discharge code, and a second, broad search was developed to identify all clinically confirmed ECOPD.717 of 1,122 (64%) patients with an ECOPD code had confirmed ECOPD. Common reasons for misclassification in the 405 patients who did not have an ECOPD included: lack of obstructive spirometry to diagnose COPD; and hospital admission due to progressive malignancy, asthma or cardiovascular disease. The broad search identified an additional 297 patients with ECOPD missed by the ECOPD codes. The vast majority of this group had pneumonia complicating ECOPD.ECOPD codes are insufficiently reliable to identify patients with clinically confirmed ECOPD for the purposes of audit or research. Search strategies should include pneumonia codes, specialist review of medical notes and spirometry confirmation of COPD.

Dry heat and microwave-generated steam protocols for the rapid decontamination of respiratory personal protective equipment in response to COVID-19-related shortages.

BACKGROUND: In the wake of the SARS-CoV-2 pandemic and unprecedented global demand, clinicians are struggling to source adequate access to personal protective equipment. Respirators can be in short supply, though are necessary to protect workers from SARS-CoV-2 exposure. Rapid decontamination and reuse of respirators may provide relief for the strained procurement situation. METHOD: In this study, we investigated the suitability of 70°C dry heat and microwave-generated steam (MGS) for reprocessing of FFP2/N95-type respirators, and Type-II surgical face masks. Staphylococcus aureus was used as a surrogate as it is less susceptible than enveloped viruses to chemical and physical processes. RESULTS: We observed >4 log10 reductions in the viability of dry S. aureus treated by dry heat for 90 min at 70°C and >6 log10 reductions by MGS for 90 s. After 3 reprocessing cycles, neither process was found to negatively impact the bacterial or NaCl filtration efficiency of the respirators that were tested. However, MGS was incompatible with Type-II surgical masks tested, as we confirmed that bacterial filtration capacity was completely lost following reprocessing. MGS was observed to be incompatible with some respirator types due to arcing observed around some types of metal nose clips and by loss of adhesion of clips to the mask. CONCLUSION: Considering the advantages and disadvantages of each approach, we propose a reprocessing personal protective equipment/face mask workflow for use in medical areas.

Impact of rapid microbial identification on clinical outcomes in bloodstream infection: the RAPIDO randomized trial.

OBJECTIVES: Bloodstream infection has a high mortality rate. It is not clear whether laboratory-based rapid identification of the organisms involved would improve outcome. METHODS: The RAPIDO trial was an open parallel-group multicentre randomized controlled trial. We tested all positive blood cultures from hospitalized adults by conventional methods of microbial identification and those from patients randomized (1:1) to rapid diagnosis in addition to matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) performed directly on positive blood cultures. The only primary outcome was 28-day mortality. Clinical advice on patient management was provided to members of both groups by infection specialists. RESULTS: First positive blood culture samples from 8628 patients were randomized, 4312 into rapid diagnosis and 4136 into conventional diagnosis. After prespecified postrandomization exclusions, 2740 in the rapid diagnosis arm and 2810 in the conventional arm were included in the mortality analysis. There was no significant difference in 28-day survival (81.5% 2233/2740 rapid vs. 82.3% 2313/2810 conventional; hazard ratio 1.05, 95% confidence interval 0.93-1.19, p 0.42). Microbial identification was quicker in the rapid diagnosis group (median (interquartile range) 38.5 (26.7-50.3) hours after blood sampling vs. 50.3 (47.1-72.9) hours after blood sampling, p < 0.01), but times to effective antimicrobial therapy were no shorter (respectively median (interquartile range) 24 (2-78) hours vs. 13 (2-69) hours). There were no significant differences in 7-day mortality or total antibiotic consumption; times to resolution of fever, discharge from hospital or de-escalation of broad-spectrum therapy or 28-day Clostridioides difficile incidence. CONCLUSIONS: Rapid identification of bloodstream pathogens by MALDI-TOF MS in this trial did not reduce patient mortality despite delivering laboratory data to clinicians sooner.

Predictive prosthetic socket design: part 1-population-based evaluation of transtibial prosthetic sockets by FEA-driven surrogate modelling.

It has been proposed that finite element analysis can complement clinical decision making for the appropriate design and manufacture of prosthetic sockets for amputees. However, clinical translation has not been achieved, in part due to lengthy solver times and the complexity involved in model development. In this study, a parametric model was created, informed by variation in (i) population-driven residuum shape morphology, (ii) soft tissue compliance and (iii) prosthetic socket design. A Kriging surrogate model was fitted to the response of the analyses across the design space enabling prediction for new residual limb morphologies and socket designs. It was predicted that morphological variability and prosthetic socket design had a substantial effect on socket-limb interfacial pressure and shear conditions as well as sub-dermal soft tissue strains. These relationships were investigated with a higher resolution of anatomical, surgical and design variability than previously reported, with a reduction in computational expense of six orders of magnitude. This enabled real-time predictions (1.6 ms) with error vs the analytical solutions of < 4 kPa in pressure at residuum tip, and < 3% in soft tissue strain. As such, this framework represents a substantial step towards implementation of finite element analysis in the prosthetics clinic.

Predictive prosthetic socket design: part 2-generating person-specific candidate designs using multi-objective genetic algorithms.

In post-amputation rehabilitation, a common goal is to return to ambulation using a prosthetic limb, suspended by a customised socket. Prosthetic socket design aims to optimise load transfer between the residual limb and mechanical limb, by customisation to the user. This is a time-consuming process, and with the increase in people requiring these prosthetics, it is vital that these personalised devices can be produced rapidly while maintaining excellent fit, to maximise function and comfort. Prosthetic sockets are designed by capturing the residual limb's shape and applying a series of geometrical modifications, called rectifications. Expert knowledge is required to achieve a comfortable fit in this iterative process. A variety of rectifications can be made, grouped into established strategies [e.g. in transtibial sockets: patellar tendon bearing (PTB) and total surface bearing (TSB)], creating a complex design space. To date, adoption of advanced engineering solutions to support fitting has been limited. One method is numerical optimisation, which allows the designer a number of likely candidate solutions to start the design process. Numerical optimisation is commonly used in many industries but not prevalent in the design of prosthetic sockets. This paper therefore presents candidate shape optimisation methods which might benefit the prosthetist and the limb user, by blending the state of the art from prosthetic mechanical design, surrogate modelling and evolutionary computation. The result of the analysis is a series of prosthetic socket designs that preferentially load and unload the pressure tolerant and intolerant regions of the residual limb. This spectrum is bounded by the general forms of the PTB and TSB designs, with a series of variations in between that represent a compromise between these accepted approaches. This results in a difference in pressure of up to 31 kPa over the fibula head and 14 kPa over the residuum tip. The presented methods would allow a trained prosthetist to rapidly assess these likely candidates and then to make final detailed modifications and fine-tuning. Importantly, insights gained about the design should be seen as a compliment, not a replacement, for the prosthetist's skill and experience. We propose instead that this method might reduce the time spent on the early stages of socket design and allow prosthetists to focus on the most skilled and creative tasks of fine-tuning the design, in face-to-face consultation with their client.

Natural products show diverse mechanisms of action against Clostridium difficile.

AIMS: To investigate the mechanisms of action of natural products with bactericidal (cinnamon root powder, peppermint oil, trans-cinnamaldehyde, menthol and zingerone) or bacteriostatic (fresh garlic bulb extract, garlic clove powder, Leptospermum honey and allicin) activity against two Clostridium difficile strains. METHODS AND RESULTS: Bactericidal products significantly reduced intracellular ATP after 1 h (P ≤ 0·01), quantified using the BacTiter-Glo reagent, and damaged the cell membrane, shown by the leakage of both 260-nm-absorbing materials and protein, and the uptake of propidium iodide. Bacteriolysis was not observed, determined by measuring optical density of treated cell suspensions at 620-nm. The effect of three bacteriostatic products on protein synthesis was quantified using an Escherichia coli S30 extract system, with Leptospermum honey (16% w/v) showing significant inhibition (P < 0·01). Lastly, no products showed elevated minimum inhibitory concentrations against antimicrobial-resistant C. difficile, determined by broth microdilution. CONCLUSIONS: Cytoplasmic membrane damage was identified as a mechanism of action that may contribute to the activity of several natural products against C. difficile. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the possible mechanisms of action of natural products against C. difficile, yet the efficacy in vivo to be determined.

Finite element analysis of the amputated lower limb: A systematic review and recommendations.

The care and rehabilitation of individuals after lower limb amputation presents a substantial and growing socioeconomic challenge. Clinical outcome is closely linked to successful functional rehabilitation with a prosthetic limb, which depends upon comfortable prosthetic limb - residual limb load transfer. Despite early interest in the 1980s, the amputated limb has received considerably less attention in computational biomechanical analysis than other subjects, such as arthroplasty. This systematic literature review investigates the state of the art in residual limb finite element analysis published since 2000. The identified studies were grouped into the following categories: (1) residuum-prosthesis interface mechanics; (2) residuum soft tissue internal mechanics; (3) identification of residuum tissue characteristics; (4) proposals for incorporating FEA into the prosthesis fitting process; (5) analysis of the influence of prosthetic componentry concepts to improve load transfer to the residuum, such as the monolimb and structural socket compliance; and (6) analysis of osseointegrated (OI) prostheses. The state of the art is critically appraised in order to form recommendations for future modeling studies in terms of geometry, material properties, boundary conditions, interface models, and relevant but un-investigated issues. Finally, the practical implementation of these approaches is discussed.

The PEARL score predicts 90-day readmission or death after hospitalisation for acute exacerbation of COPD.

BACKGROUND: One in three patients hospitalised due to acute exacerbation of COPD (AECOPD) is readmitted within 90 days. No tool has been developed specifically in this population to predict readmission or death. Clinicians are unable to identify patients at particular risk, yet resources to prevent readmission are allocated based on clinical judgement. METHODS: In participating hospitals, consecutive admissions of patients with AECOPD were identified by screening wards and reviewing coding records. A tool to predict 90-day readmission or death without readmission was developed in two hospitals (the derivation cohort) and validated in: (a) the same hospitals at a later timeframe (internal validation cohort) and (b) four further UK hospitals (external validation cohort). Performance was compared with ADO, BODEX, CODEX, DOSE and LACE scores. RESULTS: Of 2417 patients, 936 were readmitted or died within 90 days of discharge. The five independent variables in the final model were: Previous admissions, eMRCD score, Age, Right-sided heart failure and Left-sided heart failure (PEARL). The PEARL score was consistently discriminative and accurate with a c-statistic of 0.73, 0.68 and 0.70 in the derivation, internal validation and external validation cohorts. Higher PEARL scores were associated with a shorter time to readmission. CONCLUSIONS: The PEARL score is a simple tool that can effectively stratify patients' risk of 90-day readmission or death, which could help guide readmission avoidance strategies within the clinical and research setting. It is superior to other scores that have been used in this population. TRIAL REGISTRATION NUMBER: UKCRN ID 14214.

Validation of the DECAF score to predict hospital mortality in acute exacerbations of COPD.

BACKGROUND: Hospitalisation due to acute exacerbations of COPD (AECOPD) is common, and subsequent mortality high. The DECAF score was derived for accurate prediction of mortality and risk stratification to inform patient care. We aimed to validate the DECAF score, internally and externally, and to compare its performance to other predictive tools. METHODS: The study took place in the two hospitals within the derivation study (internal validation) and in four additional hospitals (external validation) between January 2012 and May 2014. Consecutive admissions were identified by screening admissions and searching coding records. Admission clinical data, including DECAF indices, and mortality were recorded. The prognostic value of DECAF and other scores were assessed by the area under the receiver operator characteristic (AUROC) curve. RESULTS: In the internal and external validation cohorts, 880 and 845 patients were recruited. Mean age was 73.1 (SD 10.3) years, 54.3% were female, and mean (SD) FEV1 45.5 (18.3) per cent predicted. Overall mortality was 7.7%. The DECAF AUROC curve for inhospital mortality was 0.83 (95% CI 0.78 to 0.87) in the internal cohort and 0.82 (95% CI 0.77 to 0.87) in the external cohort, and was superior to other prognostic scores for inhospital or 30-day mortality. CONCLUSIONS: DECAF is a robust predictor of mortality, using indices routinely available on admission. Its generalisability is supported by consistent strong performance; it can identify low-risk patients (DECAF 0-1) potentially suitable for Hospital at Home or early supported discharge services, and high-risk patients (DECAF 3-6) for escalation planning or appropriate early palliation. TRIAL REGISTRATION NUMBER: UKCRN ID 14214.

An in vitro study assessing the effect of mesh morphology and suture fixation on bacterial adherence.

PURPOSE: Prosthetic infections, although relatively uncommon in hernia surgery, are a source of considerable morbidity and cost. The aims of this experimental study were to assess the influence of the morphological properties of the mesh on bacterial adherence in vitro. The morphological properties assessed were the polymer type, filament type, filament diameter, mesh weight, mean pore size, and the addition of silver chlorhexidine and titanium coatings. In addition, the study assessed the effect on bacterial adherence of adding a commonly used suture to the mesh and compared adherence rates to self-gripping mesh that does not require suture fixation. METHODS: Eight commercially sourced flat hernia meshes with different material characteristics were included in the study. These were Prolene(®) (Ethicon(®)), DualMesh(®) (Gore(®)), DualMesh(®) Plus (Gore(®)), Parietex™ ProGrip (Covidien™), TiMesh(®) Light (GfE Medical), Bard(®) Soft Mesh (Bard(®)), Vypro(®) (Ethicon(®)), and Omyra(®) (Braun(®)). Individual meshes were inoculated with Staphylococcus epidermidis and Staphylococcus aureus with a bacterial inoculum of 10(2) bacteria. To assess the effect of suture material on bacterial adhesion, a sterile piece of commonly used monofilament suture material (2.0 Prolene(®), ZB370 Ethicon(®)) was sutured to selected meshes (chosen to represent different commonly used polymers and/or the presence of an antibacterial coating). Inoculated meshes were incubated for 18 h in tryptone soy broth and then analysed using scanning electron microscopy. A previously validated method for enumeration of bacteria using automated stage movement electron microscopy was used for direct bacterial counting. The final fraction of the bacteria adherent to the mesh was compared between the meshes and for each morphological variable. One-way analysis of variance (ANOVA) was performed on the bacterial counts. Tukey's test was used to determine the difference between the different biomaterials in the event the ANOVA was significant. RESULTS: Properties that significantly increased the mean bacterial adherence were the expanded polytetrafluoroethylene polymer (P < 0.001); multifilament meshes (P < 0.001); increased filament diameter (P < 0.001); increased mesh weight (P < 0.001); and smaller mean pore size (P < 0.001). In contrast, mesh coating with antibacterial silver chlorhexidine significantly reduced bacterial adhesion (S. epidermidis mean bacterial count 140.7 ± 19.1 SE with DualMesh(®) vs. 2.3 ± 1.2 SE with DualMesh(®) Plus, P < 0.001; S. aureus mean bacterial count 371.7 ± 22.7 SE with DualMesh(®) vs. 19.3 ± 4.7 SE with DualMesh(®) Plus, P = 0.002). The addition of 2.0 Prolene suture material significantly increased the mean number of adherent bacteria independent of the mesh polymer or mesh coating (P = 0.04 to <0.001). CONCLUSION: The present study demonstrates the significant influence of the prosthetic load on bacterial adherence. In patients at increased risk of infection, low prosthetic load materials, i.e., lightweight meshes with large pores, may be beneficial. Furthermore self-fixing meshes, which avoid increasing the prosthetic load and antibacterial impregnated meshes, may have an advantage in this setting.

Predicting outcomes following hospitalization for acute exacerbations of COPD.

Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are a frequent cause of hospital admission and are associated with significant morbidity, mortality, high readmission rates and high resource utilization. More accurate prediction of survival and readmission in patients hospitalized with AECOPD should help to optimize clinical management and allocation of resources, including targeting of palliative care and strategies to reduce readmissions. We have reviewed the published retrospective and prospective studies in this field to identify the factors most likely to be of value in predicting in-hospital and post-discharge mortality, and readmission of patients hospitalized for AECOPD. The prognostic factors which appear most important vary with the particular outcome under consideration. In-hospital mortality is related most clearly to the patient's acute physiological state and to the development of acute comorbidity, while post-discharge mortality particularly reflects the severity of the underlying COPD, as well as specific comorbidities, especially cardiac disease. Important factors influencing the frequency of readmission include functional limitation and poor health-related quality of life. Large prospective studies which incorporate all the potentially relevant variables are required to refine prediction of the important outcomes of AECOPD and thus to inform clinical decision making, for example on escalation of care, facilitated discharge and provision of palliative care.

Panton-Valentine leukocidin Staphylococcus aureus osteomyelitis of the adult tibia--a case report.

Panton-Valentine leukocidin toxin producing Staphylococcus aureus (PVLSA) is known to be responsible for recurrent soft tissue infections and more serious invasive infections including necrotising pneumonia, pyomyositis, and osteomyelitis. Most reported cases involving musculoskeletal infection in adults are associated with methicillin-resistant S. aureus (MRSA) PVL-producing strains. We present the case of an adult male with PVL toxin-producing methicillin-sensitive S. aureus (MSSA) osteomyelitis of the tibia which has not previously been described in adults and highlight issues of recognition, treatment, and surgical management of PVLSA osteomyelitis.

Life after first-line chemotherapy in malignant pleural mesothelioma: a North-East England experience.

AIMS: The benefit of first-line chemotherapy in malignant pleural mesothelioma (MPM) has been established. However, this disease invariably progresses and little is known about how this disease subsequently relapses after initial treatment. Data on second-line treatment are also scarce, especially outside the context of a clinical trial. We conducted a review to observe the presentation of MPM patients when their disease progresses after initial therapy and the use of second-line therapy and its associated outcomes. MATERIALS AND METHODS: Patients were retrospectively identified from the Sunderland Royal Hospital and the Northern Centre for Cancer Care, Newcastle upon Tyne, UK. Data, including demographics, clinical presentation and treatment details at first line and beyond, together with its associated benefits, were collected. Related times to treatment failure (TTF), rates of symptom improvement and survival data were also collated. RESULTS: There were 62 evaluable patients in our series. At the time of data collection, 58 patients (94%) had relapsed. At disease progression, symptoms were usually similar to those at initial presentation, but in patients with prolonged TTF (>9 months) they were more likely to relapse with clinical lymphadenopathy in the neck and axilla compared with patients with TTF < or =9 months (52% vs 13%, respectively, P<0.05). Second-line treatment was given in 45% of patients. Twenty-one patients (36%) received second-line chemotherapy outside the context of a clinical trial and most had retreatment with pemetrexed-based chemotherapy due to a prolonged TTF. In patients treated with second-line therapy outside the remit of a clinical trial, a disease control rate was achieved in nine patients (43%, 95% confidence interval 22-64), whereas improvement in symptoms were noted in 13 patients (62%, 95% confidence interval 41-83). The median TTF in this setting was 6.5 months. CONCLUSION: Patients with a prolonged TTF after first-line treatment are more likely to relapse with neck and axillary lymphadenopathy. The use of second-line chemotherapy, including rechallenge treatment, in this disease is a viable option for a selected group of MPM patients.

Targets of glucocorticoid action on TNF-alpha release by macrophages.

Glucocorticoid drugs affect virtually every cell type involved in inflammatory response, to some degree. Macrophage/monocytes (Mphi) are particularly sensitive, and glucocorticoids suppress release of most known Mphi inflammatory mediators, including TNF-alpha. In the case of TNF-alpha, several levels of regulation are already characterised and ongoing research hints at further glucocorticoid targets. The relative importance of transcriptional and post-transcriptional regulation is lineage-dependent and may also change during the course of Mphi differentiation. In human monocytic cell lines, glucocorticoids primarily suppress transcriptional activation through adjacent promoter binding sites for NF-kappaB transcription factor complexes and for complexes of c-Jun with activating transcription factor-2 (ATF-2). The goal of glucocorticoid research in inflammation is to develop drugs with the anti-inflammatory potential of glucocorticoids, but without the systemic toxicity. Each of the multiple targets for glucocorticoid action presents an opportunity for anti-inflammatory drug development. However, none of the known targets is unique to Mphi, and no single pathway is preeminent in all situations. Research is now directed at characterising targets and regulating them without systemic activation of the glucocorticoid receptor.

NF-kappaB and cell-cycle regulation: the cyclin connection.

The cyclins are a family of proteins that are centrally involved in cell cycle regulation and which are structurally identified by conserved "cyclin box" regions. They are regulatory subunits of holoenzyme cyclin-dependent kinase (CDK) complexes controlling progression through cell cycle checkpoints by phosphorylating and inactivating target substrates. CDK activity is controlled by cyclin abundance and subcellular location and by the activity of two families of inhibitors, the cyclin-dependent kinase inhibitors (CKI). Many hormones and growth factors influence cell growth through signal transduction pathways that modify the activity of the cyclins. Dysregulated cyclin activity in transformed cells contributes to accelerated cell cycle progression and may arise because of dysregulated activity in pathways that control the abundance of a cyclin or because of loss-of-function mutations in inhibitory proteins.Analysis of transformed cells and cells undergoing mitogen-stimulated growth implicate proteins of the NF-kappaB family in cell cycle regulation, through actions on the CDK/CKI system. The mammalian members of this family are Rel-A (p65), NF-kappaB(1) (p50; p105), NF-kappaB(2) (p52; p100), c-Rel and Rel-B. These proteins are structurally identified by an amino-terminal region of about 300 amino acids, known as the Rel-homology domain. They exist in cytoplasmic complexes with inhibitory proteins of the IkappaB family, and translocate to the nucleus to act as transcription factors when activated. NF-kappaB pathway activation occurs during transformation induced by a number of classical oncogenes, including Bcr/Abl, Ras and Rac, and is necessary for full transforming potential. The avian viral oncogene, v-Rel is an NF-kappaB protein. The best explored link between NF-kappaB activation and cell cycle progression involves cyclin D(1), a cyclin which is expressed relatively early in the cell cycle and which is crucial to commitment to DNA synthesis. This review examines the interactions between NF-kappaB signaling and the CDK/CKI system in cell cycle progression in normal and transformed cells. The growth-promoting actions of NF-kappaB factors are accompanied, in some instances, by inhibition of cellular differentiation and by inhibition of programmed cell death, which involve related response pathways and which contribute to the overall increase in mass of undifferentiated tissue.

Glucocorticoids suppress tumor necrosis factor-alpha expression by human monocytic THP-1 cells by suppressing transactivation through adjacent NF-kappa B and c-Jun-activating transcription factor-2 binding sites in the promoter.

Glucocorticoid drugs suppress tumor necrosis factor-alpha (TNF-alpha) synthesis by activated monocyte/macrophages, contributing to an anti-inflammatory action in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells, glucocorticoids acted primarily on the TNF-alpha promoter to suppress a burst of transcriptional activity that occurred between 90 min and 3 h after LPS exposure. LPS increased nuclear c-Jun/ATF-2, NF-kappaB(1)/Rel-A, and Rel-A/C-Rel transcription factor complexes, which bound specifically to oligonucleotide sequences from the -106 to -88 base pair (bp) region of the promoter. The glucocorticoid, dexamethasone, suppressed nuclear binding activity of these complexes prior to and during the critical phase of TNF-alpha transcription. Site-directed mutagenesis in TNF-alpha promoter-luciferase reporter constructs showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-kappaB (-97 to -88 bp) binding sites each contributed to the LPS-stimulated expression. Mutating both sites largely prevented dexamethasone from suppressing TNF-alpha promoter-luciferase reporters. LPS exposure also increased nuclear Egr-1 and PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated expression but not to glucocorticoid response. Dexamethasone suppressed the abundance of the c-Fos/c-Jun complex in THP-1 cell nuclei, but there was no direct evidence for c-Fos/c-Jun transactivation through sites in the -172 to -52 bp region. Small contributions to glucocorticoid response were attributable to promoter sequences outside the -172 to -88 bp region and to sequences in the TNF-alpha 3'-untranslated region. We conclude that glucocorticoids suppress LPS-stimulated secretion of TNF-alpha from human monocytic cells largely through antagonizing transactivation by c-Jun/ATF-2 and NF-kappaB complexes at binding sites in the -106 to -88 bp region of the TNF-alpha promoter.

Effects of stimulus and cell type on the expression of the -308 tumour necrosis factor promoter polymorphism.

The authors have previously demonstrated that the tumour necrosis factor (TNF) -308 G/A polymorphism affects the binding of transcription factors. In transient transfection assays in PMA stimulated U937 monocytes and Jurkat T cells, the A-containing TNF2 promoter has a 2-3-fold greater transcriptional activity than the TNF1 promoter in the presence of the TNF 3'UTR. In this study it was found that a difference in TNF1 and TNF2 promoter activities was only observed in U937 and Jurkat cells, and not in Raji (B cell line), HeLa (epithelial carcinoma cell line), HepG2 (hepatoma cell line) or THP-1 (monocyte), suggesting cell-type specific transcription factors or modifications may be involved in the formation of the -308 protein/DNA complex. Physiological stimulators, TNF and interferon gamma (IFN-gamma) did not cause differential promoter activity between TNF1 and TNF2, but LPS did with only the TNF2 promoter/3'UTR construct being significantly responsive to lipopolysaccharide (LPS) in U937 cells. In U937 cells, the -308 polymorphism affected transcription following differentiation by phorbol myristate acetate (PMA), retinoic acid, PMA plus LPS and PMA plus retinoic acid with an increase in nuclear factor binding to both TNF1 and TNF2 in the -323 to -285 region being observed. The greatest difference between TNF2 and TNF1 promoter activities (5-fold) was observed following PMA plus retinoic acid treatment of transfected U937 cells for 48h. During this time, U937 differentiated into cells with a macrophage-like morphology. An understanding of the cell type and stimuli specific requirements for differential expression of the -308 polymorphism may help elucidate the role the TNF -308 polymorphism plays in diseases where elevated TNF levels are thought to be important.

Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappaB-dependent pathway.

The small GTP-binding protein Rac1, a member of the Ras superfamily, plays a fundamental role in cytoskeleton reorganization, cellular transformation, the induction of DNA synthesis, and superoxide production. Cyclin D1 abundance is rate-limiting in normal G(1) phase progression, and the abundance of cyclin D1 is induced by activating mutations of both Ras and Rac1. Nuclear factor-kappaB (NF-kappaB) proteins consist of cytoplasmic hetero- or homodimeric Rel-related proteins complexed to a member of the IkappaB family of inhibitor proteins. In the current studies, activating mutants of Rac1 (Rac(Leu-61), Rac(Val-12)) induced cyclin D1 expression and the cyclin D1 promoter in NIH 3T3 cells. Induction of cyclin D1 by Rac1 required both an NF-kappaB and an ATF-2 binding site. Inhibiting NF-kappaB by overexpression of an NF-kappaB trans-dominant inhibitor (nonphosphorylatable IkappaBalpha) reduced cyclin D1 promoter activation by the Rac1 mutants, placing NF-kappaB in a pathway of Rac1 activation of cyclin D1. Specific amino acid mutations in the amino-terminal effector domain of Rac(Leu-61) had comparable effects on NF-kappaB transcriptional activity and activation of the cyclin D1 promoter. The NF-kappaB factors Rel A (p65) and NF-kappaB(1) (p50) induced the cyclin D1 promoter, requiring both the NF-kappaB binding site and the ATF-2 site. Stable overexpression of Rac(Leu-61) increased binding of Rel A and NF-kappaB(1) to the cyclin D1 promoter NF-kappaB site. Activation of Rac1 in NIH 3T3 cells induces both NF-kappaB binding and activity and enhances expression of cyclin D1 through an NF-kappaB and ATF-2 site in the proximal promoter, suggesting a critical role for NF-kappaB in cell cycle regulation through cyclin D1 and Rac1.

Effect of teicoplanin on isolation of Staphylococcus aureus from blood culture media.

Intraoperative bacteraemia has been used as an indicator of the efficacy of prophylactic antibiotics. Two clinical isolates of Staphylococcus aureus in nutrient broth, with or without human serum, were exposed to teicoplanin (50 mg/L) and, either immediately or after 30 min, inoculated into blood culture bottles. Bottles with and without resin were used and the experiment was repeated five times with one strain. In the absence of teicoplanin, an inoculum of 10 cfu/mL produced growth in both resin and non-resin bottles. In the presence of teicoplanin, an inoculum of at least 10(5) cfu/mL was required in non-resin bottles to obtain growth, but this was reduced to 10(2)-10(3) cfu/mL for resin bottles. Intraoperative blood cultures overestimate the efficacy of bacterial killing by prophylactic antibiotics during surgery.