A CRISPR interference screen reveals a role for cell wall teichoic acids in conjugation in Bacillus subtilis.

Conjugative elements are widespread in bacteria and include plasmids and integrative and conjugative elements (ICEs). They transfer from donor to recipient cells via an element-encoded type IV secretion system. These elements interact with and utilize host functions for their lifecycles. We sought to identify essential host genes involved in the lifecycle of the integrative and conjugative element ICEBs1 of Bacillus subtilis. We constructed a library of strains for inducible knockdown of essential B. subtilis genes using CRISPR interference. Each strain expressed one guide RNA in ICEBs1. We induced partial interference of essential genes and identified those that caused an acute defect in acquisition of ICEBs1 by recipient cells. This screen revealed that reducing expression of genes needed for synthesis of cell wall teichoic acids caused a decrease in conjugation. Using three different ways to reduce their synthesis, we found that wall teichoic acids were necessary in both donors and recipients for efficient conjugative transfer of ICEBs1. Further, we found that depletion of wall teichoic acids caused cells involved in ICEBs1 conjugation to die, most likely from damage to the cell envelope. Our results indicate that wall teichoic acids help protect against envelope stress caused by active conjugation machines.

Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage.

Most bacterial genomes contain horizontally acquired and transmissible mobile genetic elements, including temperate bacteriophages and integrative and conjugative elements. Little is known about how these elements interact and co-evolved as parts of their host genomes. In many cases, it is not known what advantages, if any, these elements provide to their bacterial hosts. Most strains of Bacillus subtilis contain the temperate phage SPß and the integrative and conjugative element ICEBs1. Here we show that the presence of ICEBs1 in cells protects populations of B. subtilis from predation by SPß, likely providing selective pressure for the maintenance of ICEBs1 in B. subtilis. A single gene in ICEBs1 (yddK, now called spbK for SPß killing) was both necessary and sufficient for this protection. spbK inhibited production of SPß, during both activation of a lysogen and following de novo infection. We found that expression spbK, together with the SPß gene yonE constitutes an abortive infection system that leads to cell death. spbK encodes a TIR (Toll-interleukin-1 receptor)-domain protein with similarity to some plant antiviral proteins and animal innate immune signaling proteins. We postulate that many uncharacterized cargo genes in ICEs may confer selective advantage to cells by protecting against other mobile elements.

Exploring what is important to patients with regards to quality of life after experiencing a lower limb reconstructive procedure: a qualitative evidence synthesis.

BACKGROUND: Patient reported outcome measures (PROMs) are used to understand the impact of lower limb reconstruction surgery on patients' quality of life (QOL). Existing measures have not been developed to specifically capture patient experiences amongst adults with lower limb conditions that require reconstruction surgery. This review aimed to synthesise qualitative evidence to identify what is important to patients requiring, undergoing, or following reconstructive surgery for lower limb conditions. METHODS: MEDLINE, Embase, PsychINFO and Cinahl were searched from inception until November 2020. Studies were included if they employed qualitative research methods, involved patients requiring, undergoing or following lower limb reconstruction and explored patients' experiences of care, treatment, recovery and QOL. Mixed methods studies that did not separately report qualitative findings, mixed population studies that were not separately reported and studies in languages other than English were excluded. Included studies were analysed using thematic synthesis. The Critical Appraisal Skills Programme qualitative studies checklist was used to undertake quality assessment. RESULTS: Nine studies met the inclusion criteria. The thematic synthesis identified two overarching themes: (1) areas of living key to QOL for lower limb reconstruction patients and (2) moving towards a new normal. The way in which lower limb reconstruction affects an individual's QOL and their recovery is complex and is influenced by a range of inter-related factors, which will affect patients to varying degrees depending on their individual circumstances. We identified these factors as: pain, daily functioning and lifestyle, identity, income, emotional wellbeing, support, the ability to adapt and adjust and the ability to move forwards. CONCLUSIONS: The way patients' QOL is affected after a lower limb reconstruction is complex, may change over time and is strongly linked to their recovery. These findings will aid us in developing a conceptual framework which identifies the outcomes important to patients and those that should be included in a PROM. Further research is then required to establish whether the range of factors we identified are captured by existing PROMs. Depending on the outcome of this work, a new PROM for patients following lower limb reconstruction may be required.

Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans1-6. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.

Systematic review of the limited evidence base for treatments of Eustachian tube dysfunction: a health technology assessment.

BACKGROUND: The Health Technology Assessment programme commissioned a wide-ranging review of treatments for adult Eustachian tube dysfunction. Treatments range from advice and observation and pharmacological treatments to surgical options. OBJECTIVE: (i) To assess the evidence for interventions for adults with a clinical diagnosis of Eustachian tube dysfunction and (ii) to identify priorities for future research. TYPE OF REVIEW: Systematic review (PROSPERO registration CRD42012003035) adhering to PRISMA guidance. SEARCH: An extensive search of 15 databases including MEDLINE, EMBASE and CENTRAL (up to October 2012). EVALUATION METHOD: Controlled and uncontrolled studies of interventions for adult Eustachian tube dysfunction were included. Because of insufficient data, the protocol was amended to also include controlled studies with mixed adult/child populations. Risk of bias was assessed. Narrative synthesis was employed due to high clinical heterogeneity. RESULTS: Interventions assessed were pharmacological treatments [two randomised controlled trials (RCTs), one controlled non-randomised trial (CCT), 159 patients]; mechanical pressure equalisation devices (one randomised controlled trial, one CCT, 48 patients); and surgery, including laser tuboplasty (seven case series, 192 patients), balloon dilatation (three case series, 103 patients), myringotomy without grommet insertion (two case series, 121 patients), transtubal steroids (one case series, 11 patients) and laser coagulation (one retrospective controlled study, 40 patients). All studies had high risk of bias except two pharmacological trials; one had low risk and one unclear risk. No evidence was found for many treatments. The single low risk of bias RCT (n = 91; 67% adults) showed no effect of nasal steroids and favoured placebo for improved middle ear function (RR 1.20, 95% CI 0.91-1.58) and symptoms (P = 0.07). Other studies showed improvements in middle ear function for mechanical devices, antihistamine/ephedrine and nasal decongestant, but they had significant methodological weaknesses including insufficient length of follow-up. None of the surgical studies were adequately controlled, and many reported high levels of co-intervention. Therefore, observed benefits for tuboplasty and balloon dilatation in symptoms, middle ear function or hearing could not be reliably attributed to the interventions assessed. There was variability in definitions of the condition. CONCLUSION: Eustachian tube dysfunction is a poorly defined condition. Due to the limited and poor-quality evidence, it is inappropriate to make conclusions on the effectiveness of any intervention; the evidence base is insufficient to guide recommendations for a trial of any particular intervention. Consensus on diagnostic criteria for Eustachian tube dysfunction is required to inform inclusion criteria of future trials.

Acupuncture and other physical treatments for the relief of pain due to osteoarthritis of the knee: network meta-analysis.

OBJECTIVE: To compare the effectiveness of acupuncture with other relevant physical treatments for alleviating pain due to knee osteoarthritis. DESIGN: Systematic review with network meta-analysis, to allow comparison of treatments within a coherent framework. Comprehensive searches were undertaken up to January 2013 to identify randomised controlled trials in patients with osteoarthritis of the knee, which reported pain. RESULTS: Of 156 eligible studies, 114 trials (covering 22 treatments and 9,709 patients) provided data suitable for analysis. Most trials studied short-term effects and many were classed as being of poor quality with high risk of bias, commonly associated with lack of blinding (which was sometimes impossible to achieve). End of treatment results showed that eight interventions: interferential therapy, acupuncture, TENS, pulsed electrical stimulation, balneotherapy, aerobic exercise, sham acupuncture, and muscle-strengthening exercise produced a statistically significant reduction in pain when compared with standard care. In a sensitivity analysis of satisfactory and good quality studies, most studies were of acupuncture (11 trials) or muscle-strengthening exercise (9 trials); both interventions were statistically significantly better than standard care, with acupuncture being statistically significantly better than muscle-strengthening exercise (standardised mean difference: 0.49, 95% credible interval 0.00-0.98). CONCLUSIONS: As a summary of the current available research, the network meta-analysis results indicate that acupuncture can be considered as one of the more effective physical treatments for alleviating osteoarthritis knee pain in the short-term. However, much of the evidence in this area of research is of poor quality, meaning there is uncertainty about the efficacy of many physical treatments.

The clinical effectiveness and cost-effectiveness of low-intensity psychological interventions for the secondary prevention of relapse after depression: a systematic review.

BACKGROUND: Depression is the most common mental disorder in community settings and a major cause of disability across the world. The objective of treatment is to achieve remission or at least adequate control of depressive symptoms; however, even after successful treatment, the risk of relapse after remission is significant. Although the effectiveness of low-intensity interventions has been extensively evaluated to treat primary symptoms of psychological difficulties, there has been substantially less research examining the use of these interventions as a relapse prevention strategy. OBJECTIVE: To systematically review the clinical effectiveness and cost-effectiveness of low-intensity psychological or psychosocial interventions to prevent relapse or recurrence in patients with depression. As the broader definition of 'low-intensity' psychological intervention is somewhat contested, the review was conducted in two parts: A, a systematic review of all evaluations of 'low-intensity' interventions that were delivered by para-professionals, peer supporters or psychological well-being practitioners as defined by the Improving Access to Psychological Therapies programme; and B, a scoping review of relevant evaluations of interventions involving qualified mental health professionals (e.g. psychiatrists, clinical psychologists, cognitive behavioural therapists) involving < 6 hours of contact per patient. DATA SOURCES: Comprehensive literature searches were developed; electronic databases were searched from inception until September 2010 (including MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations, PsycINFO, EMBASE, The Cochrane Library), internet resources were used to identify guidelines on the treatment of depression, and the bibliographies of relevant reviews, guidelines and included studies were scrutinised. REVIEW METHODS: Two reviewers independently screened titles and abstracts; data were extracted independently by one reviewer using a standardised data extraction form and checked by another. Discrepancies were resolved by consensus, with involvement of a third reviewer when necessary. The inclusion criteria were population - adults or adolescents who had received treatment for depression; intervention - part A, low-intensity interventions, specifically any unsupported psychological/psychosocial interventions or any supported interventions that did not involve highly qualified mental health professionals, and, part B, interventions carried out by qualified mental health professionals that involved < 6 hours of contact per patient; comparator - any, including no treatment, placebo, psychological or pharmacological interventions; outcomes - relapse or recurrence, other outcomes (e.g. social function, quality of life) were recorded where reported; and study design - for clinical effectiveness, randomised, quasi-randomised and non-randomised studies with concurrent control patients. For cost-effectiveness, full economic evaluations that compared two or more treatment options and considered both costs and consequences. No studies met the main part A inclusion criteria. RESULTS: For the clinical effectiveness review, 17 studies (14 completed, three ongoing), reported in 27 publications, met the part B inclusion criteria. These studies were clinically and methodologically diverse, and reported differing degrees of efficacy for the evaluated interventions. One randomised controlled trial (RCT), which evaluated a collaborative care-type programme, was potentially relevant to part A; this study reported no difference between patients receiving the intervention and those receiving usual care in terms of relapse of depression over 12 months. For the cost-effectiveness review, two studies met the criteria for part B. One of these was an economic evaluation of the RCT above, which was potentially relevant to part A. This evaluation found that the intervention may be a cost-effective use of resources when compared with usual care; however, it was unclear how valid these estimates were for the NHS. LIMITATIONS: Although any definition of 'brief' is likely to be somewhat arbitrary, an inclusion threshold of 6 hours contact per patient was used to select brief high-intensity intervention studies. Most excluded studies evaluated clearly resource-intensive interventions, though occasionally, studies were excluded on the basis of having only slightly more than 6 hours contact per patient. CONCLUSIONS: There is inadequate evidence to determine the clinical effectiveness or cost-effectiveness of low-intensity interventions for the prevention of relapse or recurrence of depression. A scoping review of brief high-intensity therapies indicates that some approaches have shown promise in some studies, but findings have not been consistent. Many uncertainties remain and further primary research is required. Careful consideration should be given to the scope of such research; it is important to evaluate the broader patient pathway accounting for the heterogeneous patient groups of interest. Future RCTs conducted in a UK primary care setting should include adult participants in remission or recovery from depression, and evaluate the quality of the intervention and consistency of delivery across practitioners where appropriate. The occurrence of relapse or recurrence should be measured using established methods, and functional outcomes as well as symptoms should be measured; data on quality of life using a generic instrument, such as the European Quality of Life-5 Dimensions (EQ-5D), should be collected. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

The effectiveness of interventions in the management of patients with primary frozen shoulder.

There are many types of treatment used to manage the frozen shoulder, but there is no consensus on how best to manage patients with this painful and debilitating condition. We conducted a review of the evidence of the effectiveness of interventions used to manage primary frozen shoulder using the Cochrane Database of Systematic Reviews, the Database of Abstracts of Reviews of Effects, the Physiotherapy Evidence Database, MEDLINE and EMBASE without language or date restrictions up to April 2009. Two authors independently applied selection criteria and assessed the quality of systematic reviews using the Assessment of Multiple Systematic Reviews (AMSTAR) tool. Data were synthesised narratively, with emphasis placed on assessing the quality of evidence. In total, 758 titles and abstracts were identified and screened, which resulted in the inclusion of 11 systematic reviews. Although these met most of the AMSTAR quality criteria, there was insufficient evidence to draw firm conclusions about the effectiveness of treatments commonly used to manage a frozen shoulder. This was mostly due to poor methodological quality and small sample size in primary studies included in the reviews. We found no reviews evaluating surgical interventions. More rigorous randomised trials are needed to evaluate the treatments used for frozen shoulder.

A systematic review and economic evaluation of the clinical effectiveness and cost-effectiveness of aldosterone antagonists for postmyocardial infarction heart failure.

BACKGROUND: Two aldosterone inhibitors are currently licensed for heart failure (HF) in the UK: spironolactone and eplerenone. Recent clinical guidelines recommend eplerenone after an acute myocardial infarction (MI) for patients with symptoms and/or signs of HF and left ventricular dysfunction. OBJECTIVES: The primary objective was to evaluate relative clinical effectiveness and cost-effectiveness of spironolactone and eplerenone in patients with postMI HF and explore the possibility of conducting an indirect comparison of spironolactone and eplerenone. A second objective was to undertake value-of-information (VOI) analyses to determine the need for further research to identify research questions critical to decision-making and to help inform the design of future studies. DATA SOURCES: Relevant databases including MEDLINE, EMBASE and CENTRAL were searched between September and December 2008. Randomised controlled trials (RCTs) of spironolactone, eplerenone, canrenone or potassium canrenoate were included if conducted in a postMI HF population. Trials of general HF patients with a subgroup of postMI HF patients were considered if they had at least 100 ischaemic participants per arm and the authors provided subgroup data when contacted. Adverse events summary data were sought from recognised reference sources and RCTs or observational studies in any population that recruited more than 100 participants. REVIEW METHODS: The comparative clinical effectiveness and cost-effectiveness of spironolactone and eplerenone was derived using Bayesian meta-regression drawing on a wider 'network' of aldosterone trials to those considered in the main clinical effectiveness review. An alternative scenario was also considered assuming a 'class effect' for the aldosterone antagonists in terms of major clinical events, but allowing for potential differences in side effect profiles. Cost-effectiveness was assessed using incremental cost-effectiveness ratios (ICERs) where appropriate. Uncertainty in cost-effectiveness results was also presented and used to inform future research priorities using VOI analyses based on expected value of perfect information (EVPI). A probabilistic decision analytic model was developed to estimate cost-effectiveness of spironolactone, eplerenone and standard care for management of postMI HF, provide estimates relevant to the NHS and explore alternative approaches to an indirect comparison between spironolactone and eplerenone. The model incorporated a lifetime horizon to estimate outcomes in terms of quality-adjusted life-years (QALYs) and costs from the NHS persepctive. In the base-case analysis, 2-year treatment duration was assumed, consistent with the follow-up in the main RCTs. Other scenarios were explored to examine the robustness of alternative assumptions including impact of different treatment durations. RESULTS: Searches yielded five RCTs: two spironolactone trials of poor methodological quality and three trials of which only one (of eplerenone) specifically examined postMI HF (Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study, EPHESUS). One trial of spironolactone (Randomised Aldactone Evaluation Study, RALES) and one of canrenone (Antiremodelling Effect of Aldosterone receptors blockade with canrenone In mild Chronic Heart Failure, AREA IN-CHF) comprised general HF, but data were available for an ischaemic subgroup. Structural similarity of spironolactone and eplerenone suggests that they may be interchangeable, but formal indirect comparison between the three trials was severely limited by trial differences. Relative safety data were limited from RCTs and observational sources. Hyperkalaemia rates varied, but were generally higher than for placebo; data were insufficient to assess discontinuation because of hyperkalaemia.Gynaecomastia rates were higher with spironolactone. Adverse event data were sparse. Systematic review of economic evidence identified three main published studies but none used a UK perspective or attempted to compare cost-effectiveness in postMI HF. The new decision model indicated that eplerenone was the most cost-effective strategy for postMI HF (ICER of eplerenone compared with standard care was 4457 pounds per QALY, increasing to 7893 pounds per QALY if treatment continued over the patient's lifetime); in neither scenario did spironolactone appear cost-effective. The ICER of eplerenone was consistently under the 20,000-30,000 pounds per QALY threshold used to establish value for money in the NHS. Uncertainty resulted in EVPI estimates between 820M pounds (base-case) and 1265M pounds (lifetime treatment duration scenario). When class effect for mortality and hospitalisations was assumed spironolactone emerged as the most cost-effective treatment and EVPI estimates were negligible. If class effect is considered more plausible than the results of the evidence synthesis model then there would be limited value in additional research. LIMITATIONS: Exchangeability between trials was poor and there was a lack of robust data in RCTs. CONCLUSIONS: Only two good-quality trials of aldosterone inhibitors in the postMI HF population were found, but lack of exchangeability with respect to study populations, meant that a comparison between these drugs could not be done. It consistently emerged that, compared with usual care, use of an aldosterone antagonist appears to be a highly cost-effective strategy for the management of postMI HF patients in the NHS. An adequately powered, well-conducted RCT that directly compares spironolactone and eplerenone is required to provide more robust evidence on the optimal management of postMI HF patients.

Real-time three-dimensional myocardial contrast echocardiography: is it clinically feasible?

AIMS: Real-time 3D echocardiography (RT3DE) and 2D low mechanical index (LMI), contrast specific, myocardial perfusion imaging are now both accepted techniques. We evaluated the feasibility of an RT3DE LMI implementation in unselected patients. METHODS AND RESULTS: Forty-six patients undergoing contrast enhanced dobutamine stress echo were imaged with novel 3D LMI power modulation software. All patients underwent contrast enhanced 2D and RT3DE acquisitions, in left ventricular opacification (LVO), and LMI perfusion modes. The data sets were evaluated segmentally for wall motion (WM) and myocardial contrast enhancement. Of the 736 evaluated segments, WM could be assessed in 726 (98.6%) of the 2D and 708 (96.2%) 3D segments (P = 0.007). Perfusion could be assessed in 721 (98%) of 2D and 701 (95.2%) of 3D segments (P = 0.006). Six hundred and sixty-one segments had normal WM and thickening in 2D and of these RT3DE demonstrated normal myocardial opacification in 77.2% of basal, 85% of mid, and 91.8% of apical segments. Thirty-four segments were akinetic, with no evidence of perfusion in 2D, and of these RT3DE revealed a perfusion defect in 31 (91%, P = NS). CONCLUSION: LMI RT3DE evaluation of myocardial perfusion is feasible in most segments. It has the potential to accurately locate and possibly quantify perfusion defects.

Understanding membranes.

The fluorescence intensity fluctuations of fluorescent lipid probes seen in giant plasma membrane blebs tell an important story about the physical state of the lipids from animal cell membranes. The fluctuations are associated with lateral liquid-liquid phase separation observed in these same membrane blebs at lower temperatures. Remarkably, the intensity fluctuations in the membrane blebs are found to be the same as those expected for the theoretical 2D Ising ferromagnet!

Non-contingent positive and negative reinforcement schedules of superstitious behaviors.

The role of schedules of reinforcement on the development of superstitious conditioning was investigated in a college age population. Participants were randomly assigned to one of eight operant schedules and instructed to remove (escape), prevent and/or remove (avoidance and escape) or produce (positive) the appearance of a computer generated stimulus using a response pad. Results from the experiment indicate that concomitant (escape and avoidance) schedules of reinforcement are most effective in facilitating acquisition of superstitious behavior as measured by self-reports of participants.

Cholesterol depletion induces solid-like regions in the plasma membrane.

Glycosylphosphatidylinositol-linked and transmembrane major histocompatibility complex (MHC) class II I-E(k) proteins, as well as N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Tritc-DHPE), are used as probes to determine the effect of cholesterol concentration on the organization of the plasma membrane at temperatures in the range 22 degrees C-42 degrees C. Cholesterol depletion caused a decrease in the diffusion coefficients for the MHC II proteins and also for a slow fraction of the Tritc-DHPE population. At 37 degrees C, reduction of the total cell cholesterol concentration results in a smaller suppression of the translational diffusion for I-E(k) proteins (twofold) than was observed in earlier work at 22 degrees C (five sevenfold) Vrljic, M., S. Y. Nishimura, W. E. Moerner, and H. M. McConnell. 2005. Biophys. J. 88:334-347. At 37 degrees C, the diffusion of both I-E(k) proteins is Brownian (0.9 < alpha-parameter < 1.1). More than 99% of the protein population diffuses homogeneously when imaged at 65 frames per s. As the temperature is raised from 22 degrees C to 42 degrees C, a change in activation energy is seen at approximately 35 degrees C in the Arrhenius plots. Cytoskeletal effects appear to be minimal. These results are consistent with a previously described model of solid-like domain formation in the plasma membrane.

Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane.

Glycosylphosphatidylinositol (GPI)-linked and native major histocompatibility complex class II I-E(k) were used as probes to determine the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane. These proteins were imaged in Chinese hamster ovary cells using single-molecule fluorescence microscopy. Observed diffusion coefficients of both native and GPI-linked I-E(k) proteins were found to depend on cholesterol concentration. As the cholesterol concentration decreases the diffusion coefficients decrease by up to a factor of 7 for native and 5 for GPI-linked I-E(k). At low cholesterol concentrations, after sphingomyelinase treatment, the diffusion coefficients are reduced by up to a factor of 60 for native and 190 for GPI-linked I-E(k). The effect is reversible on cholesterol reintroduction. Diffusion at all studied cholesterol concentrations, for both proteins, appears to be predominantly Brownian for time lags up to 2.5 s when imaged at 10 Hz. A decrease in diffusion coefficients is observed for other membrane proteins and lipid probes, DiIC12 and DiIC18. Fluorescence recovery after photobleaching measurements shows that the fraction of immobile lipid probe increases from 8 to approximately 40% after cholesterol extraction. These results are consistent with the previous work on cholesterol-phospholipid interactions. That is, cholesterol extraction destroys liquid cholesterol-phospholipid complexes, leaving solid-like high melting phospholipid domains that inhibit the lateral diffusion of membrane components.

Cytokines elicited by T cell epitopes from a synovial autoantigen: altered peptide ligands can reduce interferon-gamma and interleukin-10 production.

OBJECTIVE: To explore the cytokine responses associated with T cell epitopes from human cartilage glycoprotein 39 (HC gp-39) and the potential for modifying cytokine secretion using altered peptide ligands (APLs). METHODS: Draining lymph node cells were harvested from HLA-DR*0401 transgenic mice that had been immunized with HC gp-39. Cytokine responses to 5 previously identified HLA-DR*0401-restricted HC gp-39 T cell epitopes were studied in vitro. The anchor and T cell receptor (TCR) contact residues of peptide 322-337 were identified, and this information was used to design alanine-substituted APLs. T cells were primed in vivo with wild-type peptide 322-337, restimulated with wild-type peptide or APLs, and the cytokine profiles were compared. RESULTS: Restimulation with individual peptides elicited distinct cytokine profiles. HC gp-39 (peptide 322-337) elicited a dominant interferon-gamma (IFNgamma) response. Residues within the core (positions P1-P9) 322-337 peptide sequence were critical for T cell recognition. Surprisingly, the N-terminal flanking region was also important for recognition by 6 of 10 specific T cell hybridomas. Substitutions of charged TCR contact residues in the 322-337 core epitope (E332A and K335A) were associated with a significant reduction in the IFNgamma and interleukin-10 (IL-10) stimulation indices. Restimulation with peptides W325A and V326A was also associated with a trend toward reduced IFNgamma and IL-10 secretion. In contrast, restimulation with peptide D330N elicited cytokine profiles more comparable with those resulting from restimulation with wild-type peptide. CONCLUSION: This study indicates that APLs of a proinflammatory HC gp-39 T cell epitope may be used to alter the cytokine response from a memory T cell population.

Condensed complexes of cholesterol and phospholipids.

There is overwhelming evidence that lipid bilayer regions of animal cell membranes are in a liquid state. Quantitative models of these bilayer regions must then be models of liquids. These liquids are highly non-ideal. For example, it has been known for more than 75 years that mixtures of cholesterol and certain phospholipids undergo an area contraction or condensation in lipid monolayers at the air-water interface. In the past 3 years, a thermodynamic model of "condensed complexes" has been proposed to account for this non-ideal behavior. Here we give an overview of the model, its relation to other models, and to modern views of the properties of animal cell membranes.

Liquid-liquid immiscibility in membranes.

The observation of liquid-liquid immiscibility in cholesterol-phospholipid mixtures in monolayers and bilayers has opened a broad field of research into their physical chemistry. Some mixtures exhibit multiple immiscibilities. This unusual property has led to a thermodynamic model of "condensed complexes." These complexes are the consequence of an exothermic, reversible reaction between cholesterol and phospholipids. In this quantitative model the complexes are sometimes concentrated in a separate liquid phase. The phase separation into a complex-rich phase depends on membrane composition and intensive variables such as temperature. The properties of defined cholesterol-phospholipid mixtures provide a conceptual foundation for the exploration of a number of aspects of the biophysics and biochemistry of animal cell membranes.

Formation of two peptide/MHC II isomers is catalyzed differentially by HLA-DM.

Major histocompatability class II proteins are transmembrane alphabeta-heterodimers that present peptides to T-cells. MHC II may bind exogenous peptides directly at the cell surface. Alternatively, peptides derived from processing of endosomal protein may bind to MHC II in endosomal compartments. There, HLA-DM catalyzes the formation of peptide/MHC complexes, which are then transported to the cell surface. Here we report evidence that the peptide Ii CLIP 81-104 binds to DR*0404 in two alternate registries, whose dissociation rates, while kinetically indistinguishable at pH 5.3 and 37 degrees C, are kinetically resolved in the presence of HLA-DM. In one registry isomer, CLIP Met 91 is placed in the N-terminal P1 pocket of DR*0404, and peptide dissociation is readily catalyzed by HLA-DM. In a second proposed registry, likely with CLIP Leu 97 in the P1 pocket, the complex is substantially less sensitive to HLA-DM catalysis. Without HLA-DM, or at pH 7, the fraction of each isomer formed in solution is relatively insensitive to the duration of incubation with peptide. However, with HLA-DM, the fraction of the DM-insensitive isomer is dramatically influenced by peptide incubation time. The mechanism of isomer formation appears to be determined by the HLA-DM-modified relative association to the two registries, followed by HLA-DM-catalyzed dissociation of each isomer and rebinding, leading to a final isomer composition determined by these kinetic constants. Intramolecular isomer interconversion does not appear to be involved. The behavior of these complexes may provide a model for peptide editing by DM in endosomes.

Translational diffusion of individual class II MHC membrane proteins in cells.

Single-molecule epifluorescence microscopy was used to observe the translational motion of GPI-linked and native I-E(k) class II MHC membrane proteins in the plasma membrane of CHO cells. The purpose of the study was to look for deviations from Brownian diffusion that might arise from barriers to this motion. Detergent extraction had suggested that these proteins may be confined to lipid microdomains in the plasma membrane. The individual I-E(k) proteins were visualized with a Cy5-labeled peptide that binds to a specific extracytoplasmic site common to both proteins. Single-molecule trajectories were used to compute a radial distribution of displacements, yielding average diffusion coefficients equal to 0.22 (GPI-linked I-E(k)) and 0.18 microm(2)/s (native I-E(k)). The relative diffusion of pairs of proteins was also studied for intermolecular separations in the range 0.3-1.0 microm, to distinguish between free diffusion of a protein molecule and diffusion of proteins restricted to a rapidly diffusing small domain. Both analyses show that motion is predominantly Brownian. This study finds no strong evidence for significant confinement of either GPI-linked or native I-E(k) in the plasma membrane of CHO cells.

Critical points in charged membranes containing cholesterol.

Epifluorescence microscopy is used to determine phase diagrams for lipid monolayers containing binary mixtures of cholesterol or dihydrocholesterol and dimyristoylphosphatidylserine, as well as ternary mixtures that also contain ganglioside G(M1). The phase diagrams are unusual in that they show multiple critical points: two upper miscibility critical points and one lower miscibility critical point. These critical points all are associated with the formation of condensed complexes between these lipids and cholesterol (or dihydrocholesterol). The miscibility critical pressures depend on subphase pH and ionic strength. Changes in critical pressures due to changes in subphase composition are closely related to changes in membrane electrostatic pressure and surface ionization.