Modulation of the phagosome proteome by interferon-gamma.

Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-gamma (IFN-gamma), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-gamma-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-gamma includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-gamma-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-gamma-activated macrophages.

Protein identification and Peptide expression resolver: harmonizing protein identification with protein expression data.

Proteomic discovery platforms generate both peptide expression information and protein identification information. Peptide expression data are used to determine which peptides are differentially expressed between study cohorts, and then these peptides are targeted for protein identification. In this paper, we demonstrate that peptide expression information is also a powerful tool for enhancing confidence in protein identification results. Specifically, we evaluate the following hypothesis: tryptic peptides originating from the same protein have similar expression profiles across samples in the discovery study. Evidence supporting this hypothesis is provided. This hypothesis is integrated into a protein identification tool, PIPER (Protein Identification and Peptide Expression Resolver), that reduces erroneous protein identifications below 5%. PIPER's utility is illustrated by application to a 72-sample biomarker discovery study where it is demonstrated that false positive protein identifications can be reduced below 5%. Consequently, it is recommended that PIPER methodology be incorporated into proteomic studies where both protein expression and identification data are collected.

An evaluation of multidimensional fingerprinting in the context of clinical proteomics.

Multidimensional fingerprinting (MDF) utilizes measurable peptide characteristics to identify proteins. In this study, 3-D fingerprinting, namely, parent protein molecular weight, peptide mass, and peptide retention time on RPLC, is used to identify 331 differentially expressed proteins between normal and human colon cancer plasma membrane samples. A false discovery rate (FDR) procedure is introduced to evaluate the performance of MDF on the colon cancer dataset. This evaluation establishes a false protein identification rate below 15% for this dataset. Western blot analysis is performed to validate the differential expression of the MDF-identified protein VDAC1 on the original tissue samples. The limits of MDF are further assessed by a simulation study where key parameters such as database size, query size, and mass accuracy are varied. The results of this simulation study demonstrate that fingerprinting with three dimensions yields low FDR values even for large queries on the complete human proteome without the need for prior peptide sequencing by tandem mass spectrometry. Specifically, when mass accuracy is 10 ppm or lower, full human proteome searches can achieve FDR values of 10% or less.

High-throughput technology: green fluorescent protein to monitor cell death.

Reliable assessment of cell death is now pivotal to many research programs aiming at generating new antitumor compounds or at screening cDNA libraries to identify genes with pro- or antiapoptotic functions. Such approaches need to rely on reproducible, easy handling, and rapid microplate-based cytotoxicity assays that are amenable to high-throughput screening technologies. We describe here a method for the direct measurement of cell death, based on the detection of a decrease in fluorescence observed following death induction in cells stably expressing enhanced green fluorescent protein (EGFP). Our data clearly show that such a decrease in EGFP fluorescence after cell death induction happens in various cell types, including those routinely used in anticancer drug screening (i.e., murine and human, lymphoid, fibroblastic, or epithelial cell lines). Moreover, the decrease in EGFP fluorescence is observed in cells induced to die by a variety of apoptosis-inducing agents, such as glucocorticoids (dexamethasone), DNA- damaging agents (etoposide, cisplatin), microtubule disorganizers (paclitaxel), protein kinase C inhibitors (staurosporine), or a caspase-independent apoptotic stimulus (CD45 crosslinking). A decrease in fluorescence can be assessed either by flow cytometry or with a fluorescence microplate reader. The kinetics and specificity of this EGFP-based assay were comparable with those of other conventional techniques used to detect cell death. This novel EGFP-based microplate assay combines sensitivity and rapidity and is amenable to high-throughput setups, making it an assay of choice for evaluation of cell cytotoxicity.

Evaluation of risk factors associated with endometriosis.

OBJECTIVE: To investigate the association between several factors such as demographics, personal habits, reproductive factors, menstrual characteristics, contraception, and clinical profile and the probability of having endometriosis. SETTING: Ten clinical institutions in the Montreal area. PATIENT(S): A total of 2777 subjects who underwent surgery for diagnostic laparoscopy, tubal ligation, or hysterectomy between January 1998 and July 2002. Eight hundred ninety subjects were classified as cases on the basis of the presence of endometriotic lesions found at the time of surgery, whereas 1881 controls had no surgical evidence of endometriosis. MAIN OUTCOME MEASURE(S): The association between potential risk factors and endometriosis was estimated by Pearson chi(2) and by crude and adjusted odds ratios. RESULT(S): Results indicated a positive association between endometriosis and education level for subjects who underwent hysterectomy. An inverse relation between gravidity and endometriosis was also found within a subgroup of subjects who had diagnostic laparoscopy. Furthermore, the proportion of uterine leiomyoma was significantly higher in cases compared with in controls, among subjects who underwent surgery for diagnostic laparoscopy or hysterectomy but not for tubal ligation. CONCLUSION(S): These observations underline the importance of taking into account the clinical profile of subjects when investigating risk factors for endometriosis.

Quantitative assessment of human endometriotic tissue maintenance and regression in a noninvasive mouse model of endometriosis.

Endometriosis is a prevalent disease characterized by the estrogen-dependent ectopic growth of endometrial tissue. Most of the current medical therapies consist in inducing a hypoestrogenic state in patients, but these treatments are associated with severe side effects and high recurrence rates. The development of convenient and reliable endometriosis animal models would be instrumental to accelerate the emergence of new therapeutic alternatives. Recently, we developed an improved experimental model for endometriosis, relying on the infection of human endometrial fragments by an adenovirus carrying the green fluorescent protein. Following injection of fluorescent fragments into nude mice, the implantation and growth of endometriotic-like lesions could be followed noninvasively. In the present work, we demonstrate that this model can be used to quantify the size of fluorescent endometriotic lesions by in vivo imaging. To this end, we repeatedly measured lesion size over a 4-week period in mice supplemented or not with estradiol. The model was adequate to confirm previous results showing that estrogen is dispensable for the implantation phase of endometrial tissue, whereas it is required for lesion maintenance. As a proof of concept for inducing regression of established lesions, ganciclovir was used to treat animals implanted with human fluorescent endometrial fragments expressing thymidine kinase. A significant decrease in lesion size was observed by in vivo imaging in ganciclovir-treated mice. Together, the data indicate that the noninvasive animal model described here provides a tool for drug testing and/or gene target validation in endometriosis.

Concentrations of alpha-fetoprotein, insulin-like growth factor binding protein-3, c-erbB-2, and epidermal growth factor in serum of patients with endometriosis.

OBJECTIVE: Endometriosis, although it is a benign disorder, shares many similarities with cancer. There is increasing levels of evidence suggesting that some circulating factors involved in gynecologic cancers, such as alpha-fetoprotein (AFP), insulin-like growth factor binding protein-3 (IGFBP-3), c-erbB-2 (HER-2/neu), and epidermal growth factor (EGF), could also play a role in endometriosis. Hence, the present study was aimed at evaluating whether the levels of these molecules are modulated in the serum of patients with endometriosis. METHODS: Levels of AFP, IGFBP-3, c-erbB-2, and EGF were determined by enzyme-linked immunosorbent assay in serum from 36 subjects with surgically confirmed endometriosis and 36 controls with no surgical evidence of the disease. In addition, information such as demographic characteristics, personal habits, menstrual characteristics, and clinical profile was collected from each participating subject. RESULTS: No significant difference was found between serum levels of AFP, IGFBP-3, c-erbB-2, and EGF in patients with endometriosis and controls, even when we adjusted for potential confounders and took into account the menstrual cycle. Moreover, no correlation was observed between the serum concentrations of these molecules and the stage of the disease. However, a correlation was detected between soluble levels of IGFBP-3 and presence of uterine leiomyoma. CONCLUSION: Although AFP, IGFBP-3, c-erbB-2, and EGF are not altered in the circulation of patients with endometriosis, their involvement in the development of endometriotic lesions cannot be excluded.

Serum concentrations of insulin-like growth factor-1, soluble tumor necrosis factor receptor-1 and angiogenin in endometriosis patients.

PROBLEM: Many soluble factors contributing to the pathophysiology of endometriosis are found at abnormal levels in patients suffering from the disease. We postulated that levels of these factors could also be altered in the serum of patients. We compared levels of insulin-like growth factor-1 (IGF-1), soluble form of tumor necrosis factor-alpha (TNF-alpha) receptor-1 (sTNFR-1) and angiogenin in the serum of patients with endometriosis and controls. METHOD OF STUDY: Levels of IGF-1, sTNFR-1 and angiogenin were measured by enzyme-linked immunosorbent assay in samples from 148 patients (77 cases and 71 controls) with diagnostic confirmed by laparoscopy. Correlations with demographic data and stage of the disease were evaluated and potential confounders in the study population were controlled. RESULTS: A significant increase in sTNFR-1 and angiogenin serum levels was observed in cases in comparison with controls, but only for patients in the follicular phase of the cycle. No significant difference was found in serum levels of IGF-1, sTNFR-1 and angiogenin between cases and controls in the luteal phase of the cycle. Correlations between levels of angiogenin and stage of the disease could also be observed. CONCLUSION: sTNFR-1 and angiogenin represent potential blood markers for endometriosis.

Development of a nonsurgical diagnostic tool for endometriosis based on the detection of endometrial leukocyte subsets and serum CA-125 levels.

OBJECTIVE: To determine whether the proportion of several leukocyte subsets is modulated in the endometrium of patients with endometriosis and, if yes, whether it can be used for diagnostic purposes. DESIGN: Case-control study. SETTING: Eight clinical institutions of the Montreal area. PATIENT(S): Women who underwent laparoscopy or laparotomy between 1997 and 2001, who had regular menstrual cycles and were not under hormone treatment for the previous 3 months were selected. This study included 368 women, 173 with surgically confirmed endometriosis and 195 controls with no surgical evidence of endometriosis. MAIN OUTCOME MEASURE(S): Cytometry analysis was used to measure the proportion of several leukocyte subsets among CD45(+) endometrial cells. RESULT(S): The proportion of CD3(+), CD16(+), CD3(-)HLADR(-), CD3(-)CD45RA(-), CD3(+)CD16(-), CD3(+)CD56(-), CD56(-)CD16(+), and CD16b(+) leukocytes was significantly altered in the endometrium of cases compared with controls. A multiple logistic regression model was adjusted with these endometrial leukocytes, serum CA-125 levels, risk factors, and confounders. The diagnostic performance of this predictive model was defined by a specificity of 95% and a sensitivity of 61%. Furthermore, the positive and negative predictive values were 91% and 75%, respectively. CONCLUSION(S): This predictive model represents a novel diagnostic tool to identify women with a high likelihood of suffering from endometriosis.

An improved mouse model for endometriosis allows noninvasive assessment of lesion implantation and development.

OBJECTIVE: To test whether fragments of human endometrium transduced with the green fluorescent protein (GFP) cDNA and transplanted into nude mice can be noninvasively visualized. DESIGN: A murine experimental model for human endometriosis. SETTING: A biotechnology company. ANIMAL(S): Ovariectomized nude mice. INTERVENTION(S): Whole fragments of human endometrium were transduced in vitro by adenoviral infection with the GFP cDNA before transplantation into nude mice. Animals were noninvasively and repeatedly imaged before lesion collection. MAIN OUTCOME MEASURE(S): Fluorescence of GFP-expressing human endometrial fragments was evaluated before transplantation into animals. Development of endometriotic lesions was monitored through direct visualization of fluorescent tissue in the living animal or through conventional dissection. RESULT(S): GFP gene transfer into whole endometrial fragments can be performed, and a high proportion of cells express the reporter gene. Fluorescent endometrial fragments implant in nude mice and form endometriotic-like lesions, which can be directly visualized through the skin of living mice using a simple imaging device. CONCLUSION(S): This improved mouse model allows noninvasive and dynamic studies of lesion implantation and development to be conducted. In addition to helping better understand the pathophysiology of the disease, this model represents a valuable preclinical tool for testing the efficacy of new drugs targeting endometriosis, which should ultimately accelerate their development phase.

Blood leukocyte subsets are modulated in patients with endometriosis.

OBJECTIVE: To determine whether blood leukocyte populations could be modulated in endometriosis. DESIGN: Case-control study. SETTING: Eight clinical institutions of the Montreal area. PATIENT(S): Women with regular menstrual cycles who underwent laparoscopy or laparotomy between 1997 and 2001 and who were not under hormonal treatment for at least 3 months were selected. This study includes 175 cases and 131 controls. MAIN OUTCOME MEASURE(S): The proportion of blood leukocytes expressing markers for T, B lymphocytes, monocytes, or natural killer (NK) cells were compared by flow cytometric analysis between cases and controls. RESULT(S): Age and parity were identified as important confounders. Given that smoking, history of acute infection, and previous use of oral contraceptives strongly correlate with the level of some blood leukocyte populations, these parameters were taken into account in addition with age and parity when the level of blood leukocyte subsets were evaluated in cases and controls. Blood monocytes expressing CD14 and CD44 molecules were increased in patients with endometriosis. Alternatively, B lymphocytes were shown to be significantly decreased in cases compared with controls. CONCLUSION(S): Although these results suggest that endometriosis is associated with some systemic manifestations, the exact role of these modulations remains unclear.

Mek2 is dispensable for mouse growth and development.

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.

Weak agonist self-peptides promote selection and tuning of virus-specific T cells.

Recent progress has begun to define the interactions and signaling pathways that are triggered during positive selection. To identify and further examine self-peptides that can mediate positive selection, we searched a protein-database to find peptides that have minimal homology with the viral peptide (p33) that activates a defined P14 transgenic TCR. We identified four peptides that could bind the restriction element H-2D(b) and induce proliferation of P14 transgenic splenocytes at high concentration. Two of the four peptides (DBM and RPP) were able to positively select the virus-specific TCR in fetal thymic organ culture but were unable to induce clonal deletion. Reverse-phase HPLC and mass spectrometry demonstrated that these peptides were presented by H-2D(b) molecules on thymic epithelial cell lines. We also examined whether the selecting ligands altered T cell responsiveness in vitro. DBM-selected T cells lost their ability to respond to the positively selecting ligand DBM, whereas RPP-selected T cells only retained their ability to respond to high concentrations of RPP. These results demonstrate that self-peptides that mediate positive selection can differentially "tune" the activation threshold of T cells and alter the functional repertoire of T cells.

Low activation threshold as a mechanism for ligand-independent signaling in pre-T cells.

Pre-TCR complexes are thought to signal in a ligand-independent manner because they are constitutively targeted to lipid rafts. We report that ligand-independent signaling is not a unique capability of the pre-TCR complex. Indeed, the TCR alpha subunit restores development of pT alpha-deficient thymocytes to the CD4(+)CD8(+) stage even in the absence of conventional MHC class I and class II ligands. Moreover, we found that pre-TCR and alpha beta TCR complexes exhibit no appreciable difference in their association with lipid rafts, suggesting that ligand-independence is a function of the CD4(-)CD8(-) (DN) thymocytes in which pre-TCR signaling occurs. In agreement, we found that only CD44(-)CD25(+) DN thymocytes (DN3) enabled activation of extracellular signal-regulated kinases by the pre-TCR complex. DN thymocytes also exhibited a lower signaling threshold relative to CD4(+)CD8(+) thymocytes, which was associated with both the markedly elevated lipid raft content of their plasma membranes and more robust capacitative Ca(2+) entry. Taken together these data suggest that cell-autonomous, ligand-independent signaling is primarily a property of the thymocytes in which pre-TCR signaling occurs.

Monoglycerides induce apoptosis in human leukemic cells while sparing normal peripheral blood mononuclear cells.

A major drawback of the current antineoplastic treatments is their lack of specificity toward cancer cells, because they are most often cytotoxic to normal cells, thus creating related side effects. Hence, the identification of new apoptosis-inducing agents, specifically targeting malignant cells while sparing their normal counterparts, is of crucial interest. We show here that monoglycerides, a family of lipids consisting of a single fatty acid attached to a glycerol backbone, induce cell death in several human leukemic cell lines. Importantly, treatment of primary leukemic cells, obtained from B-cell chronic lymphocytic leukemia patients, resulted in rapid apoptosis. In striking contrast, resting or activated human peripheral blood mononuclear cells from healthy individuals were resistant to the same treatment. Therefore, these compounds could represent potential antileukemic drugs or could allow for the design of novel therapeutic agents applied to leukemia.

A cell death pathway induced by antibody-mediated cross-linking of CD45 on lymphocytes.

The protein tyrosine phosphatase CD45 is a highly expressed glycoprotein present on all nucleated cells of hematopoietic origin. To date, all the functions attributed to CD45 are inherently coupled to its phosphatase activity. For instance, the regulation of lymphocyte antigen receptor signaling is mediated through the dephosphorylation, and hence activation, of Src-family kinases by CD45. Moreover, signaling via cytokine receptors is negatively modulated by CD45 by dephosphorylation of Janus kinase family members. Recently, another function for CD45, unrelated to regulation of surface receptor signaling, has been unraveled. Specific engagement of CD45 by monoclonal antibodies at the surface of lymphocytes induced their death, through an alternative caspase-independent pathway. In striking contrast to all other previously reported functions for CD45, its phosphatase activity is completely dispensable for the induction of cell death. This article reviews the current knowledge on the death pathway triggered by CD45 ligation on lymphocytes. In an attempt to better elucidate the mechanism of cell death induction through CD45, we also provide original data regarding the susceptibility of various subsets of immature and mature T and B cells to death induced by CD45 engagement. The physiological significance and therapeutic potential of CD45-induced death are also discussed.

Cellular specificity related to monoglyceride-induced cell death.

We have recently observed that monoglycerides (MGs), a family of lipids consisting of a single fatty acid moiety attached to a glycerol backbone, induce rapid dose-dependent apoptosis in murine thymocytes. In this work, we evaluated the sensitivity of various normal and malignant immune and non-immune cells to MGs. We demonstrate that the propensity to MG-induced death displayed by both T and B lymphocytes is clearly modulated according to their differentiation and activation status. For instance, the earliest T and B cell precursors are refractory to MG-mediated cell death. In the T-cell lineage, immature thymocytes are the most susceptible to MG treatment, while B cells from peripheral lymphoid organs appear more sensitive than B-cell subsets from the bone marrow. On the other hand, both activated T and B cells are more resistant to MG exposure than their non-activated counterparts. In addition, other hematopoietic lineages such as natural killer cells, macrophages, and erythroid cells are quite resistant to MG-induced death. Furthermore, using various immortalized cell lines from different tissues, we found that lymphomas and thymomas are the most sensitive among all lineages tested, while epithelial cells and fibroblasts are unaffected by MG treatment. Finally, MG-induced death was shown to be independent of Fas/Fas ligand (FasL) interactions. Altogether, our findings indicate that there is a cellular specificity related to MG-mediated cell death biased towards T and B lymphocytes. This suggests that MGs could potentially be used in the treatment of specific lymphoid disorders by bypassing the requirement for the Fas/FasL system.

Apoptosis mediated through CD45 is independent of its phosphatase activity and association with leukocyte phosphatase-associated phosphoprotein.

Besides the well-recognized role of CD45 as a major player in TCR signaling, we and others have demonstrated that cross-linking of CD45 with mAbs can induce cell death in T lymphocytes. To investigate the role of CD45 phosphatase activity in apoptosis induction, we expressed either wild-type or phosphatase-dead CD45 molecules in a CD45-deficient BW5147 T cell line. We show here that the phosphatase activity of CD45 was not required for apoptosis triggering after cross-linking of the molecule. It is noteworthy that a revertant of the CD45-negative BW5147 cell line, expressing a truncated form of CD45 lacking most of the cytoplasmic domain, was also susceptible to CD45-mediated death. Moreover, we also demonstrate that leukocyte phosphatase-associated phosphoprotein expression is totally dispensable for CD45-mediated apoptosis to occur. Taken together, these results strongly suggest a role for the extracellular and/or the transmembrane portion of CD45 in apoptosis signaling, which contrasts with the previously reported functions for CD45 in T lymphocytes.