Controlled nanometric phase transitions of phospholipid membranes by plasmonic heating of single gold nanoparticles.

The development of remotely controlled nanoscopic sources of heat is essential for investigating and manipulating temperature sensitive processes at the nanoscale. Here, we use single gold nanoparticles to rapidly deposit controlled amounts of heat in nanoscopic regions of defined size. This allows us to induce and control nanoscale reversible gel-fluid phase transitions in phospholipid membranes. We exploit the optical control over the phase transition to determine the velocity of the fluid phase front into the gel phase membrane and to guide the nanoparticles to specific locations. These results illustrate how single gold nanoparticles enable local thermodynamic investigation and manipulation on nanoscale (bio-) systems.

mTORC2 signalling regulates M2 macrophage differentiation in response to helminth infection and adaptive thermogenesis.

Alternatively activated macrophages (M2) have an important function in innate immune responses to parasitic helminths, and emerging evidence also indicates these cells are regulators of systemic metabolism. Here we show a critical role for mTORC2 signalling in the generation of M2 macrophages. Abrogation of mTORC2 signalling in macrophages by selective conditional deletion of the adaptor molecule Rictor inhibits the generation of M2 macrophages while leaving the generation of classically activated macrophages (M1) intact. Selective deletion of Rictor in macrophages prevents M2 differentiation and clearance of a parasitic helminth infection in mice, and also abrogates the ability of mice to regulate brown fat and maintain core body temperature. Our findings define a role for mTORC2 in macrophages in integrating signals from the immune microenvironment to promote innate type 2 immunity, and also to integrate systemic metabolic and thermogenic responses.

Regional ventilatory clearance by xenon scintigraphy: a critical evaluation of two estimation procedures.

Estimates of ventilatory clearance are usually made by inspecting xenon washout images. Quantitative computer procedures have been described that produce regional clearance rates, yet their accuracy is not well established. We define a mathematical model for scintigraphic ventilation data based on 96 clinical studies, and with this model we test the accuracy of two procedures used to estimate ventilatory clearance. The least-squares curve-fitting technique for both washin and washout data has the same accuracy as a modified Stewart-Hamilton method (A/H) that uses washout data alone. Both procedures demonstrate relative errors of less than 5% and coefficients of variation of 10-20% when regions with equilibrium count rates of 3 cps and clearance times between 10 and 90 sec are examined. Because the A/H procedure is preferred for its simplicity and speed, we analyze two of its main sources of error: early washin/washout termination and background activity. To measure regional ventilation by the A/H procedure, we recommend: (a) washin and washout periods at least three times the largest clearance time of clinical interest; b) a regional equilibrium count rate of at least 3cps; and c) a 25- to 50-sec average of the equilibrium count rate.

Assessment of patients' perceptions and beliefs regarding herbal therapies.

We evaluated the demographics and beliefs regarding safety and efficacy of herbal therapy among individuals in Iowa and assessed the willingness to discuss the use of these products with health care providers. We distributed 1300 surveys to two random samples: patients attending eight clinics, and residents of the state (mailing). Data were categorized according to herb use and compared between users and nonusers. The response rate was 61% (794 people), with 41.6% of respondents reporting herb use. They were predominately white women and were likely to have had education beyond high school (p<0.05). Their use of prescription drugs was high (p<0.05). Although users rated safety and efficacy of herbs higher than nonusers (p<0.05), both groups believed that health care providers should be aware of use and would provide this information.

Revisiting thalidomide: fighting with caution against idiopathic pulmonary fibrosis.

Thalidomide is an infamous drug whose use by pregnant women in the middle of last century tragically resulted in serious birth defects. However, as a result of its potent immunomodulatory, anti-inflammatory and antiangiogenic properties, thalidomide may be a potential therapy in many diseases. In recent years, thalidomide has been used effectively to treat various malignancies, including multiple myeloma, myelodysplastic syndromes, renal cell cancer, glioblastoma multiforme and prostate cancer. In addition, thalidomide has also proven effective against other immune-related diseases, including erythema nodosum leprosum and sarcoidosis. Idiopathic pulmonary fibrosis (IPF) is a deadly fibrotic disease with no effective treatment options. However, there is data to suggest that thalidomide may be useful in treating the chronic, disabling cough that accompanies IPF. It remains to be seen whether the immunomodulatory and antiangiogenic properties of thalidomide will also make it a potential therapy against the clinical progression of IPF.

Primary pulmonary arterial hypertension presenting as diffuse micronodules on CT.

Primary pulmonary arterial hypertension is a rare lethal disease that typically presents radiographically with enlarged central pulmonary arteries, pruning of the peripheral vasculature, and cardiomegaly but clear lung fields. Although it is a disease of unknown etiology, primary PAH has been associated with anorexigen use. We present a case of pulmonary arterial hypertension in a woman with a history of fenfluramine and phentermine use who presented with diffuse micronodules on computed tomography scan. Lung biopsy confirmed the micronodules were radiographic manifestations of extensive diffuse plexogenic arterial lesions. This report represents an unusual radiographic presentation of anorexigen related pulmonary arterial hypertension, and to our knowledge, the first case reported as presenting with diffuse micronodules on high resolution computed tomography scan.

A review of algorithms for molecular sequence comparison.

Computers have recently become an essential component of research in molecular biology. Most computer analyses of nucleic acid and protein sequences depend on comparisons between sequences. These comparisons, depending on their purpose, may differ not only in the kinds of comparisons that are done, but also in the way the results of the comparison are used by molecular biologists or by other computer programs. This paper reviews algorithms currently in use to solve comparison problems in molecular biology. Each algorithm is explained in detail and discussed in terms of the molecular biology problems it is most suited to solve.

Suppression of baseline wander in the ECG using a bilinearly transformed, null-phase filter.

The purpose of this study was to design and test a bilinearly transformed, null-phase (BLT/NP) filter for removing baseline wander and to compare it with the cubic spline for performance. For this purpose, the ECG data were filtered to remove high-frequency noise and low-frequency baseline wander to form a set of "clean" ECGs. Artificial low-frequency noise mimicking typical baseline wander was constructed from sine and cosine waves at 0.20 and 0.45 Hz and with amplitudes of 400 and 300 microV, respectively, and added to the "clean" ECGs to form the "test" ECGs. The BLT/NP filter and the cubic spline method each were applied to a "test" ECG to form a "restored" ECG. The measure of performance was the root mean square difference (RMSD) between the "restored" ECG and the initial "clean" ECG. RMSD values showed that on the average the BLT/NP filter performed as well as the cubic spline method and has the advantage that accurate determination of the QRS onset is not required.

Nonparametric comparison of entire ROC curves for computerized ECG left ventricular hypertrophy algorithms using data from the Framingham Heart Study.

A computer program may be capable of several different statements for left ventricular hypertrophy (eg, possible LVH, probable LVH, consistent with LVH), but such statements resulting from discretized levels of sensitivity/specificity would represent only isolated points on a receiver-operating characteristic (ROC) curve, which is a plot of all levels of sensitivity versus specificity. Even if two algorithms use the same discrete scales, their performances may not readily be compared. The authors present a comparison methodology for ROC curves using ROC area as a nonparametric measure of the ability of the algorithm to separate the two populations; the ROC area ranges from 0.5 (no ability) to 1.0 (perfect separation) and is unbiased if the normal versus abnormal populations have no common values for the measurement. The methodology compares the performance of ECG algorithms on the same population of cases by testing for significant differences of ROC areas and incorporating correlation of the algorithms in a nonparametric way. To illustrate this methodology, they use ECG and echocardiographic data from the Framingham Heart Study.

Regulation of hyaluronan-induced chemokine gene expression by IL-10 and IFN-gamma in mouse macrophages.

Turnover of the extracellular matrix (ECM), activation of macrophages, and accumulation of chemokines/cytokines are all hallmarks of chronic inflammation. Extracellular matrix components, such as hyaluronan (HA), have recently been shown to influence macrophage effector functions, such as the release of inflammatory chemokines and cytokines. Although low m.w. fragments of the glycosaminoglycan HA induce macrophages to secrete numerous inflammatory mediators, the mechanisms regulating ECM-induced macrophage activation are poorly understood. We have examined the effects of IL-10 and IFN-gamma on HA-induced chemokine gene expression in primary mouse macrophages. We found that IL-10 and IFN-gamma independently inhibit HA-induced expression of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and KC at both the mRNA and protein levels. Whereas IL-10 inhibited most of the HA-induced chemokines tested, IFN-gamma selectively inhibited only MIP-1alpha, MIP-1beta, and KC. This inhibition did not require prestimulation and occurred even when the cytokines were added up to 3 h after stimulation with HA. For MIP-1alpha, the inhibition by IFN-gamma occurred at the level of transcription, whereas IL-10 predominantly decreased the stability of MIP-1alpha mRNA. IFN-gamma and IL-10 equally inhibited macrophage expression of MIP-1beta mRNA at the level of transcription, but MIP-1beta mRNA stability was decreased to a greater extent by IL-10. These data identify a previously unrecognized role for IL-10 and IFN-gamma as regulators of ECM-induced macrophage expression of inflammatory chemokines.

Distinct requirements for C-C chemokine and IL-2 production by naive, previously activated, and anergic T cells.

Ag presented by activated APCs promote immunogenic responses whereas Ag presented by resting APCs leads to tolerance. In such a model, the regulation of cytokine release by the presence or absence of costimulation might potentially play a critical role in dictating the ultimate outcome of Ag recognition. C-C chemokines are a structurally defined family of chemoattractants that have diverse effects on inflammation. We were interested in determining the activation requirements for chemokine production by CD4+ T cells. Our data demonstrate for T cell clones and previously activated T cells from TCR-transgenic mice that stimulation with anti-TCR alone results in the production of copious amounts of macrophage-inflammatory protein-1alpha (MIP-1alpha) and other C-C chemokines, and that addition of anti-CD28 gives very little augmentation. Furthermore, MIP-1alpha production is nearly equivalent from both anergic and nonanergic cells. For naive T cells, anti-CD3 stimulation alone led to as much MIP-1alpha production as Ag + APC stimulation. The addition of costimulation gave a 3-10-fold enhancement, but this was 70-fold less than the effect of costimulation on IL-2 production. Thus, although C-C chemokines play a broad role in influencing inflammation, their production by signal 1 alone makes them unlikely to play a critical role in the decision between a tolerogenic and an immunogenic response. Furthermore, the production of MIP-1alpha by anergic T cells, as well as following signal 1 alone, raises the possibility that in vivo this chemokine serves to recruit activated T cells to become tolerant.

Induction and regulation of macrophage metalloelastase by hyaluronan fragments in mouse macrophages.

Although the metalloproteinase murine metalloelastase (MME) has been implicated in lung disorders such as emphysema and pulmonary fibrosis, the mechanisms regulating MME expression are unclear. Low m.w. fragments of the extracellular matrix component hyaluronan (HA) that accumulate at sites of lung inflammation are capable of inducing inflammatory gene expression in macrophages (Mphi). The purpose of this study was to examine the effect of HA fragments on the expression of MME in alveolar Mphi. The mouse alveolar Mphi cell line MH-S was stimulated with HA fragments over time, total RNA was isolated, and Northern blot analysis was performed. HA fragments induced MME mRNA in a time-dependent fashion, with maximal levels at 6 h. HA fragments also induced MME protein expression as well as enzyme activity. The induction of MME gene expression was specific for low m.w. HA fragments and dependent upon new protein synthesis; it occurred at the level of gene transcription. We also examined the effect of HA fragments on MME expression in inflammatory alveolar Mphi from bleomycin-injured rat lungs. Although normal rat alveolar Mphi did not express MME mRNA in response to HA fragments, alveolar Mphi from the bleomycin-treated rats responded to HA fragment stimulation by increasing MME mRNA levels. Furthermore, baseline and HA fragment-induced MME gene expression in alveolar Mphi from bleomycin-treated rats was inhibited by IFN-gamma. These data suggest that HA fragments may be an important mechanism for the expression of MME by Mphi in inflammatory lung disorders.

Comparing multivariate and univariate subject-specific reference regions for blood constituents in healthy persons.

We examined the comparative behavior of subject-specific multivariate and univariate reference regions, using both computer-generated data and serial (semi-annual) measurements of selected analytes in subjects from a large health-maintenance program. Univariate studies under both homeostatic and random-walk time-series models were helpful in defining expected results, but only the homeostatic model was used in multivariate as well as univariate forms. Analysis of the computer-generated data and the real biochemical series produced similar findings, which showed the multivariate subject-specific reference region to be much more conservative than corresponding univariate intervals. That is, a multidimensional point of p correlated observations is quite likely to lie within the individual's multivariate reference region (based on past observation vectors), even when one or more of the observations lie outside their separate reference intervals for that individual. One consequence of this high specificity against univariate false positives in a large surveillance program is a higher than expected proportion of positive multivariate vectors in which none of the values lie outside their univariate ranges. Thus, although the development of multivariate reference regions should be encouraged, they should be used in conjunction with, not instead of, univariate ranges.

Comparative evaluation of the IBM (12-lead) and Royal Infirmary (orthogonal three-lead) ECG computer programs.

A comparison of two computer programs for ECG interpretation was undertaken. Twelve-lead ECGs from 300 patients with various clinical abnormalities were interpreted at the National Institutes of Health using version 1 of the IBM program and corresponding orthogonal three-lead ECGs were analyzed by the Glasgow Royal Infirmary (GRI) program. Interpretations were compared with respect to the clinical documentation, wherever possible, and with each other directly in the case of diagnostic statements for which non-ECG documentation was not available. The two programs had a similar performance in determining abnormalities such as myocardial infarction and ventricular hypertrophy. However, with respect to conduction defects and ST-T-wave statements, certain discrepancies between the two program performances were revealed. There were 222 disagreements between various diagnostic statements. GRI was judged correct in 119 of these disagreements and IBM in 70. In these 189 cases the disagreement could most often be accounted for by different criteria and/or algorithms in the two programs or by the use of different ECG lead sets. The remaining 33 disagreements had to be classified as inconclusive.

Electrocardiographic changes with advancing age. A cross-sectional study of the association of age with QRS axis, duration and voltage.

In order to examine the electrocardiographic (ECG) changes which occur with advancing age we defined an apparently healthy reference population derived from prospectively followed subjects of the Framingham Heart Study. Healthy subjects were clinically free of hypertension, coronary artery disease, congestive heart failure and valve disease and were not taking antihypertensive or other cardiac medications. ECG tracings were analyzed by the IBM Bonner (V2) program. Mean values and correlations with age for PR duration, QRS duration and axis, S wave voltage V1 and R wave voltage V5 are presented. With advancing age in men there is a narrowing of QRS, a leftward QRS axis shift, and a loss of S V1 and R V5 amplitude. In women only a leftward QRS axis shift is associated with advancing age. These changes should be considered in defining normal age- and sex-specific reference values. These findings underscore theoretical limitations of commonly-used criteria for the ECG diagnosis of conditions such as left ventricular hypertrophy.

Regulation of plasminogen activator inhibitor-1 and urokinase by hyaluronan fragments in mouse macrophages.

Pulmonary inflammation and fibrosis are characterized by increased turnover and production of the extracellular matrix as well as an impairment of lung fibrinolytic activity. Although fragments of the extracellular matrix component hyaluronan induce macrophage production of inflammatory mediators, the effect of hyaluronan on the fibrinolytic mediators plasminogen activator inhibitor (PAI)-1 and urokinase-type plasminogen activator (uPA) is unknown. This study demonstrates that hyaluronan fragments augment steady-state mRNA, protein, and inhibitory activity of PAI-1 as well as diminish the baseline levels of uPA mRNA and inhibit uPA activity in an alveolar macrophage cell line. Hyaluronan fragments alter macrophage expression of PAI-1 and uPA at the level of gene transcription. Similarly, hyaluronan fragments augment PAI-1 and diminish uPA mRNA levels in freshly isolated inflammatory alveolar macrophages from bleomycin-treated rats. These data suggest that hyaluronan fragments influence alveolar macrophage expression of PAI-1 and uPA and may be a mechanism for regulating fibrinolytic activity during lung inflammation.

Hyaluronan fragments induce nitric-oxide synthase in murine macrophages through a nuclear factor kappaB-dependent mechanism.

Activated macrophages play a critical role in controlling chronic tissue inflammation through the release of a variety of mediators including cytokines, chemokines, growth factors, active lipids, reactive oxygen, and nitrogen species. The mechanisms that regulate macrophage activation in chronic inflammation are poorly understood. A hallmark of chronic inflammation is the turnover of extracellular matrix components, and recent work has suggested that interactions with the extracellular matrix can exert important influences on macrophage effector functions. We have examined the effect of low molecular weight fragments of the extracellular matrix glycosaminoglycan hyaluronan (HA) on the induction of nitric-oxide synthase (iNOS) in macrophages. We found that HA fragments induce iNOS mRNA, protein and activity alone, and markedly synergize with interferon-gamma to induce iNOS gene expression in murine macrophages. In addition, we found that resident tissue alveolar macrophages respond minimally, but inflammatory alveolar macrophages exhibit a marked induction in iNOS expression in response to HA fragments. Finally, we demonstrate that the mechanism of HA fragment-induced expression of iNOS requires activation of the transcriptional regulator nuclear factor kappaB. These data support the hypothesis that HA may be an important regulator of macrophage activation at sites of chronic tissue inflammation.

Induction of IL-12 and chemokines by hyaluronan requires adhesion-dependent priming of resident but not elicited macrophages.

Components of the extracellular matrix (ECM) can regulate leukocyte activation and function at inflammatory sites. Low molecular weight fragments of the ECM glycosaminoglycan hyaluronan (LMW-HA) that accumulate in inflammation, but not the ubiquitous high molecular weight form of HA (HMW-HA), have been shown to induce cytokine and/or chemokine production by alveolar and bone-marrow derived macrophages. To determine the cellular requirements for responsiveness to HA, we compared the effects of HMW-HA and LMW-HA on resident and thioglycollate-elicited murine peritoneal macrophages. We demonstrate that treatment of elicited macrophages with LMW-HA, but not with HMW-HA, stimulated production of the chemokines RANTES and macrophage inflammatory protein-1alpha and -1beta. Further, we demonstrate that LMW-HA induced the production of biologically active IL-12, a proinflammatory cytokine not previously known to be regulated by cell-matrix interactions. The LMW-HA-induced production of IL-12 by elicited macrophages was inhibited by an anti-CD44 mAb that blocks HA binding. In contrast to elicited macrophages, freshly explanted resident peritoneal macrophages did not respond to LMW-HA. However, preculture in vitro before stimulation led to adhesion-dependent priming for LMW-HA-induced cytokine and chemokine production by resident macrophages. These results provide further evidence of the potential importance of CD44/LMW-HA interactions in regulating the immune response at sites of inflammation and demonstrate that the state of differentiation of macrophages may determine their sensitivities to matrix components.