Long-Term Effects of Sirolimus on Human Skin TSC2-Null Fibroblast‒Like Cells.

Tuberous sclerosis complex (TSC) is an autosomal-dominant disorder characterized by hamartomatous tumors of the skin, kidneys, brain, and lungs. TSC is caused by mutations in the TSC1 and TSC2 genes, which result in hyperactivation of the mTOR, leading to dysregulated cell growth and autophagy. Rapamycin (sirolimus) shrinks TSC tumors, but the clinical benefits of sirolimus are not sustained after its withdrawal.

Phosphodiesterase 3B (PDE3B) regulates NLRP3 inflammasome in adipose tissue.

Activation of inflammation in white adipose tissue (WAT), includes infiltration/expansion of WAT macrophages, contributes pathogenesis of obesity, insulin resistance, and metabolic syndrome. The inflammasome comprises an intracellular sensor (NLR), caspase-1 and the adaptor ASC. Inflammasome activation leads to maturation of caspase-1 and processing of IL1β, contributing to many metabolic disorders and directing adipocytes to a more insulin-resistant phenotype.

Antibody αPEP13h reacts with lymphangioleiomyomatosis cells in lung nodules.

Background: Lymphangioleiomyomatosis (LAM) is characterized by the proliferation in the lung, axial lymphatics (eg, lymphangioleiomyomas), and kidney (eg, angiomyolipomas) of abnormal smooth muscle-like LAM cells, which express melanoma antigens such as Pmel17/gp100 and have dysfunctional tumor suppressor tuberous sclerosis complex (TSC) genes TSC2 or TSC1. Histopathologic diagnosis of LAM in lung specimens is based on identification of the Pmel17 protein with the monoclonal antibody HMB-45.

Methods: We compared the sensitivity of HMB-45 to that of antipeptide antibody αPEP13h, which reacts with a C-terminal peptide of Pmel17.

Syndecan-2 exerts antifibrotic effects by promoting caveolin-1-mediated transforming growth factor-β receptor I internalization and inhibiting transforming growth factor-β1 signaling.

Rationale: Alveolar transforming growth factor (TGF)-β1 signaling and expression of TGF-β1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-β receptor TβRI inhibits TGF-β signaling and could attenuate development of experimental lung fibrosis.

Objectives: To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-β1 signaling in alveolar epithelial cells.

Optical spectroscopy in turbid media using an integrating sphere: mitochondrial chromophore analysis during metabolic transitions.

Recent evidence suggests that the activity of mitochondrial oxidative phosphorylation complexes (MOPCs) is modulated at multiple sites. Here, a method of optically monitoring electron distribution within and between MOPCs is described using a center-mounted sample in an integrating sphere (to minimize scattering effects) with a rapid-scanning spectrometer. The redox-sensitive MOPC absorbances (∼465-630 nm) were modeled using linear least squares analysis with individual chromophore spectra.

Brefeldin A-inhibited ADP-ribosylation factor activator BIG2 regulates cell migration via integrin β1 cycling and actin remodeling.

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)2 activates ADP-ribosylation factors, ∼20-kDa GTPase proteins critical for continuity of intracellular vesicular trafficking by accelerating the replacement of ADP-ribosylation factor-bound GDP with GTP. Mechanisms of additional BIG2 function(s) are less clear. Here, the participation of BIG2 in integrin β1 cycling through actin dynamics during cell migration was identified using small interfering RNA (siRNA) and difference gel electrophoresis analyses.

Automatic Spot Identification for High Throughput Microarray Analysis.

High throughput microarray analysis has great potential in scientific research, disease diagnosis, and drug discovery. A major hurdle toward high throughput microarray analysis is the time and effort needed to accurately locate gene spots in microarray images. An automatic microarray image processor will allow accurate and efficient determination of spot locations and sizes so that gene expression information can be reliably extracted in a high throughput manner.

Time course analysis of gene expression identifies multiple genes with differential expression in patients with in-stent restenosis.

Background: The vascular disease in-stent restenosis (ISR) is characterized by formation of neointima and adverse inward remodeling of the artery after injury by coronary stent implantation. We hypothesized that the analysis of gene expression in peripheral blood mononuclear cells (PBMCs) would demonstrate differences in transcript expression between individuals who develop ISR and those who do not.

Methods And Results: We determined and investigated PBMC gene expression of 358 patients undergoing an index procedure to treat in de novo coronary artery lesions with bare metallic stents, using a novel time-varying intercept model to optimally assess the time course of gene expression across a time course of blood samples.

Chemokine-enhanced chemotaxis of lymphangioleiomyomatosis cells with mutations in the tumor suppressor TSC2 gene.

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction caused by LAM cells (smooth-muscle-like cells) that have mutations in the tumor suppressor genes tuberous sclerosis complex (TSC) 1 or 2 and have the capacity to metastasize. Since chemokines and their receptors function in chemotaxis of metastatic cells, we hypothesized that LAM cells may be recruited by chemokine(s) in the lung. Quantification of 25 chemokines in bronchoalveolar lavage fluid from LAM patients and healthy volunteers revealed that concentrations of CCL2, CXCL1, and CXCL5 were significantly higher in samples from LAM patients than those from healthy volunteers.

Heterogeneity of human macrophages in culture and in atherosclerotic plaques.

Research suggests that monocytes differentiate into unique lineage-determined macrophage subpopulations in response to the local cytokine environment. The present study evaluated the atherogenic potential of two divergent lineage-determined human monocyte-derived macrophage subpopulations. Monocytes were differentiated for 7 days in the presence of alternative macrophage development cytokines: granulocyte-macrophage colony-stimulating factor to produce granulocyte-macrophage-CSF macrophages (GM-Mac), or macrophage colony-stimulating factor (M-CSF) to produce M-Mac.

Mesenchymal-epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas.

Patients with tuberous sclerosis complex (TSC) develop hamartomas containing biallelic inactivating mutations in either TSC1 or TSC2, resulting in mammalian target of rapamycin (mTOR) activation. Hamartomas overgrow epithelial and mesenchymal cells in TSC skin. The pathogenetic mechanisms for these changes had not been investigated, and the existence or location of cells with biallelic mutations ("two-hit" cells) was unclear.

Detection of venous thromboembolism by proteomic serum biomarkers.

Background: Available blood assays for venous thromboembolism (VTE) suffer from diminished specificity. Compared with single marker tests, such as D-dimer, a multi-marker strategy may improve diagnostic ability. We used direct mass spectrometry (MS) analysis of serum from patients with VTE to determine whether protein expression profiles would predict diagnosis.

Impairment of alveolar macrophage transcription in idiopathic pulmonary fibrosis.

Rationale: Alveolar macrophages are inflammatory cells that may contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF), which is characterized by excessive alveolar aggregation of cells and extracellular matrix proteins.

Objectives: To identify potential molecular mechanisms of IPF.

Methods: To examine large-scale gene expression, messenger RNA isolated from alveolar macrophages and peripheral blood mononuclear cells from subjects with IPF and normal volunteers was hybridized to cDNA filters.

ABCA1 overexpression in the liver of LDLr-KO mice leads to accumulation of pro-atherogenic lipoproteins and enhanced atherosclerosis.

The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg x LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.

Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells.

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors.

Rationale and study design of the CardioGene Study: genomics of in-stent restenosis.

Background And Aims: in-stent restenosis is a major limitation of stent therapy for atherosclerosis coronary artery disease. The CardioGene Study is an ongoing study of restenosis in bare mental stents (BMS) for the treatment of coronary artery disease. The overall goal is to understand the genetic determinants of the responses to vascular injury that result in the development of restenosis in some patients but not in others.

Distinctive gene expression profiles of CD34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities.

Aneuploidy, especially monosomy 7 and trisomy 8, is a frequent cytogenetic abnormality in the myelodysplastic syndromes (MDSs). Patients with monosomy 7 and trisomy 8 have distinctly different clinical courses, responses to therapy, and survival probabilities. To determine disease-specific molecular characteristics, we analyzed the gene expression pattern in purified CD34 hematopoietic progenitor cells obtained from MDS patients with monosomy 7 and trisomy 8 using Affymetrix GeneChips.

3D structural model of the G-protein-coupled cannabinoid CB2 receptor.

The potential for therapeutic specificity in regulating diseases and for reduced side effects has made cannabinoid (CB) receptors one of the most important G-protein-coupled receptor (GPCR) targets for drug discovery. The cannabinoid (CB) receptor subtype CB2 is of particular interest due to its involvement in signal transduction in the immune system and its increased characterization by mutational and other studies. However, our understanding of their mode of action has been limited by the absence of an experimental receptor structure.

Gene expression profiling in CD34 cells to identify differences between aplastic anemia patients and healthy volunteers.

An immune pathophysiology for acquired aplastic anemia (AA) has been inferred from the responsiveness of the patients to immunosuppressive therapies and experimental laboratory data. To address the transcriptome of hematopoietic cells in AA, we undertook GeneChip analysis of the extremely limited numbers of progenitor and stem cells in the marrow of patients with this disease. We pooled total RNA from highly enriched bone marrow CD34 cells of 36 patients with newly diagnosed AA and 12 healthy volunteers for analysis on oligonucleotide chips.

Stimulation of signal transducer and activator of transcription-1 (STAT1)-dependent gene transcription by lipopolysaccharide and interferon-gamma is regulated by mammalian target of rapamycin.

Mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) regulate cell growth, protein synthesis, and apoptosis in response to nutrients and mitogens. As an important source of nitric oxide during inflammation, human inducible nitric oxide synthase also plays a role in the regulation of cytokine-driven cell proliferation and apoptosis. The role of mTOR and PI3K in the activation of human inducible nitric oxide synthase transcription by cytokines and lipopolysaccharide (LPS) was investigated in lung epithelial adenocarcinoma (A549) cells.

F(ab)'2-mediated neutralization of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins.

High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)2-IVIG and irrelevant human monoclonal antibody.