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.

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.

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.

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.

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.

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.

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.

Hyaluronan fragments synergize with interferon-gamma to induce the C-X-C chemokines mig and interferon-inducible protein-10 in mouse macrophages.

Hallmarks of chronic inflammation and tissue fibrosis are increased influx of activated inflammatory cells, mediator release, and increased turnover and production of the extracellular matrix (ECM). Recent evidence has suggested that fragments of the ECM component hyaluronan play a role in chronic inflammation by inducing macrophage expression of chemokines. Interferon-gamma (IFN-gamma), an important regulator of macrophage functions, has been shown to induce the C-X-C chemokines Mig and IP-10. These chemokines affect T-cell recruitment and inhibit angiogenesis. The purpose of this investigation was to determine the effect of hyaluronan (HA) on IFN-gamma-induced Mig and IP-10 expression in mouse macrophages. We found a marked synergy between HA and IFN-gamma on Mig and IP-10 mRNA and protein expression in mouse macrophages. This was most significant with Mig, which was not induced by HA alone. The synergy was specific for HA, was not dependent on new protein synthesis, was not mediated by tumor necrosis factor-alpha, was selective for Mig and IP-10, and occurred at the level of gene transcription. These data suggest that the ECM component HA may influence chronic inflammatory states by working in concert with IFN-gamma to alter macrophage chemokine expression.

Inhibition of interferon gamma induced interleukin 12 production: a potential mechanism for the anti-inflammatory activities of tumor necrosis factor.

Inflammation is associated with production of cytokines and chemokines that recruit and activate inflammatory cells. Interleukin (IL) 12 produced by macrophages in response to various stimuli is a potent inducer of interferon (IFN) gamma production. IFN-gamma, in turn, markedly enhances IL-12 production. Although the immune response is typically self-limiting, the mechanisms involved are unclear. We demonstrate that IFN-gamma inhibits production of chemokines (macrophage inflammatory proteins MIP-1alpha and MIP-1beta). Furthermore, pre-exposure to tumor necrosis factor (TNF) inhibited IFN-gamma priming for production of high levels of IL-12 by macrophages in vitro. Inhibition of IL-12 by TNF can be mediated by both IL-10-dependent and IL-10-independent mechanisms. To determine whether TNF inhibition of IFN-gamma-induced IL-12 production contributed to the resolution of an inflammatory response in vivo, the response of TNF+/+ and TNF-/- mice injected with Corynebacterium parvum were compared. TNF-/- mice developed a delayed, but vigorous, inflammatory response leading to death, whereas TNF+/+ mice exhibited a prompt response that resolved. Serum IL-12 levels were elevated 3-fold in C. parvum-treated TNF-/- mice compared with TNF+/+ mice. Treatment with a neutralizing anti-IL-12 antibody led to resolution of the response to C. parvum in TNF-/- mice. We conclude that the role of TNF in limiting the extent and duration of inflammatory responses in vivo involves its capacity to regulate macrophage IL-12 production. IFN-gamma inhibition of chemokine production and inhibition of IFN-gamma-induced IL-12 production by TNF provide potential mechanisms by which these cytokines can exert anti-inflammatory/repair function(s).

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.

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.

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.

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.