Th2 lymphoproliferative disorder of LatY136F mutant mice unfolds independently of TCR-MHC engagement and is insensitive to the action of Foxp3+ regulatory T cells.

Mutant mice where tyrosine 136 of linker for activation of T cells (LAT) was replaced with a phenylalanine (Lat(Y136F) mice) develop a fast-onset lymphoproliferative disorder involving polyclonal CD4 T cells that produce massive amounts of Th2 cytokines and trigger severe inflammation and autoantibodies. We analyzed whether the Lat(Y136F) pathology constitutes a bona fide autoimmune disorder dependent on TCR specificity. Using adoptive transfer experiments, we demonstrated that the expansion and uncontrolled Th2-effector function of Lat(Y136F) CD4 cells are not triggered by an MHC class II-driven, autoreactive process. Using Foxp3EGFP reporter mice, we further showed that nonfunctional Foxp3(+) regulatory T cells are present in Lat(Y136F) mice and that pathogenic Lat(Y136F) CD4 T cells were capable of escaping the control of infused wild-type Foxp3(+) regulatory T cells. These results argue against a scenario where the Lat(Y136F) pathology is primarily due to a lack of functional Foxp3(+) regulatory T cells and suggest that a defect intrinsic to Lat(Y136F) CD4 T cells leads to a state of TCR-independent hyperactivity. This abnormal status confers Lat(Y136F) CD4 T cells with the ability to trigger the production of Abs and of autoantibodies in a TCR-independent, quasi-mitogenic fashion. Therefore, despite the presence of autoantibodies causative of severe systemic disease, the pathological conditions observed in Lat(Y136F) mice unfold in an Ag-independent manner and thus do not qualify as a genuine autoimmune disorder.

Microsomal triglyceride transfer protein promotes the secretion of Xenopus laevis vitellogenin A1.

Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.

The inflammasomes: guardians of the body.

The innate immune system relies on its capacity to rapidly detect invading pathogenic microbes as foreign and to eliminate them. The discovery of Toll-like receptors (TLRs) provided a class of membrane receptors that sense extracellular microbes and trigger antipathogen signaling cascades. More recently, intracellular microbial sensors have been identified, including NOD-like receptors (NLRs). Some of the NLRs also sense nonmicrobial danger signals and form large cytoplasmic complexes called inflammasomes that link the sensing of microbial products and metabolic stress to the proteolytic activation of the proinflammatory cytokines IL-1beta and IL-18. The NALP3 inflammasome has been associated with several autoinflammatory conditions including gout. Likewise, the NALP3 inflammasome is a crucial element in the adjuvant effect of aluminum and can direct a humoral adaptive immune response. In this review, we discuss the role of NLRs, and in particular the inflammasomes, in the recognition of microbial and danger components and the role they play in health and disease.

Theileria parva-transformed T cells show enhanced resistance to Fas/Fas ligand-induced apoptosis.

Lymphocyte homeostasis is regulated by mechanisms that control lymphocyte proliferation and apoptosis. Activation-induced cell death is mediated by the expression of death ligands and receptors, which, when triggered, activate an apoptotic cascade. Bovine T cells transformed by the intracellular parasite Theileria parva proliferate in an uncontrolled manner and undergo clonal expansion. They constitutively express the death receptor Fas and its ligand, FasL but do not undergo apoptosis. Upon elimination of the parasite from the host cell by treatment with a theilericidal drug, cells become increasingly sensitive to Fas/FasL-induced apoptosis. In normal T cells, the sensitivity to death receptor killing is regulated by specific inhibitor proteins. We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of caspase-8, and X-chromosome-linked inhibitor of apoptosis protein (IAP) as well as c-IAP, which can block downstream executioner caspases, are constitutively expressed in T. parva-transformed T cells. Expression of these proteins is rapidly down-regulated upon parasite elimination. Antiapoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-x(L) are also expressed but, in contrast to c-FLIP, c-IAP, and X-chromosome-linked IAP, do not appear to be tightly regulated by the presence of the parasite. Finally, we show that, in contrast to the situation in tumor cells, the phosphoinositide 3-kinase/Akt pathway is not essential for c-FLIP expression. Our findings indicate that by inducing the expression of antiapoptotic proteins, T. parva allows the host cell to escape destruction by homeostatic mechanisms that would normally be activated to limit the continuous expansion of a T cell population.

p27 and Skp2 immunoreactivity and its clinical significance with endocrine and chemo-endocrine treatments in node-negative early breast cancer.

BACKGROUND: Low p27 and high Skp2 immunoreactivity are associated with a poor prognosis and other poor prognostic features including resistant phenotypes and antiestrogen drug resistance. We investigated these proteins in two International Breast Cancer Study Group trials studying node-negative early breast cancer. PATIENTS AND METHODS: Trial VIII compared chemotherapy followed by goserelin with either modality alone in premenopausal patients. Trial IX compared chemotherapy followed by tamoxifen with tamoxifen alone in postmenopausal patients. Central Pathology Office assessed p27 and Skp2 expression in the primary tumor by immunohistochemistry among 1631 (60%) trial patients. RESULTS: p27 and Skp2 were inversely related; 13% of tumors expressed low p27 and high Skp2. Low p27 and high Skp2 were associated with unfavorable prognostic factors including larger size and higher grade tumors, absence of estrogen receptor and progesterone receptor, human epidermal growth factor receptor 2 overexpression and high Ki-67 (each P < 0.05). Low p27 and high Skp2 were not associated with disease-free survival (P = 0.42 and P = 0.48, respectively). The relative effects of chemo-endocrine versus endocrine therapy were similar regardless of p27 or Skp2. CONCLUSIONS: We confirm the association of low p27 and high Skp2 with other poor prognostic features, but found no predictive or prognostic value, and therefore do not recommend routine determination of p27 and Skp2 for node-negative breast cancer.

Comparative protein profiling identifies elongation factor-1beta and tryparedoxin peroxidase as factors associated with metastasis in Leishmania guyanensis.

Parasites of the Leishmania Viannia subgenus are major causative agents of mucocutaneous leishmaniasis (MCL), a disease characterised by parasite dissemination (metastasis) from the original cutaneous lesion to form debilitating secondary lesions in the nasopharyngeal mucosa. We employed a protein profiling approach to identify potential metastasis factors in laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) through infrequently metastatic (M+/M-) to non-metastatic (M-). Comparison of the soluble proteomes of promastigotes by two-dimensional electrophoresis revealed two abundant protein spots specifically associated with M+ and M+/M- clones (Met2 and Met3) and two others exclusively expressed in M- parasites (Met1 and Met4). The association between clinical disease phenotype and differential expression of Met1-Met4 was less clear in L. Viannia strains from mucosal (M+) or cutaneous (M-) lesions of patients. Identification of Met1-Met4 by biological mass spectrometry (LC-ES-MS/MS) and bioinformatics revealed that M+ and M- clones express distinct acidic and neutral isoforms of both elongation factor-1 subunit beta (EF-1beta) and cytosolic tryparedoxin peroxidase (TXNPx). This interchange of isoforms may relate to the mechanisms by which the activities of EF-1beta and TXNPx are modulated, and/or differential post-translational modification of the gene product(s). The multiple metabolic functions of EF-1 and TXNPx support the plausibility of their participation in parasite survival and persistence and thereby, metastatic disease. Both polypeptides are active in resistance to chemical and oxidant stress, providing a basis for further elucidation of the importance of antioxidant defence in the pathogenesis underlying MCL.

Transgenic expression of Ly49A on T cells impairs a specific antitumor response.

Inhibitory MHC receptors determine the reactivity and specificity of NK cells. These receptors can also regulate T cells by modulating TCR-induced effector functions such as cytotoxicity, cytokine production, and proliferation. Here we have assessed the capacity of mouse T cells expressing the inhibitory MHC class I receptor Ly49A to respond to a well-defined tumor Ag in vivo using Ly49A transgenic mice. We find that the presence of Ly49A on the vast majority of lymphocytes prevents the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor. Despite minor alterations in the TCR repertoire of CD8+ T cells in the transgenic lines, precursors of functional tumor-specific CD8+ T cells exist but could not be activated most likely due to a lack of appropriate CD4+ T cell help. Surprisingly, all of these effects are observed in the absence of a known ligand for the Ly49A receptor as defined by its ability to regulate NK cell function. Indeed, we found that the above effects on T cells may be based on a weak interaction of Ly49A with Kb or Db class I molecules. Thus, our data demonstrate that enforced expression of a Ly49A receptor on conventional T cells prevents a specific immune response in vivo and suggest that the functions of T and NK cells are differentially sensitive to the presence of inhibitory MHC class I receptors.

FLIP prevents apoptosis induced by death receptors but not by perforin/granzyme B, chemotherapeutic drugs, and gamma irradiation.

FLICE-inhibitory protein, FLIP (Casper/I-FLICE/FLAME-1/CASH/CLARP/MRIT), which contains two death effector domains and an inactive caspase domain, binds to FADD and caspase-8, and thereby inhibits death receptor-mediated apoptosis. Here, we characterize the inhibitory effect of FLIP on a variety of apoptotic pathways. Human Jurkat T cells undergoing Fas ligand-mediated apoptosis in response to CD3 activation were completely resistant when transfected with FLIP. In contrast, the presence of FLIP did not affect apoptosis induced by granzyme B in combination with adenovirus or perforin. Moreover, the Fas ligand, but not the perforin/granzyme B-dependent lytic pathway of CTL, was inhibited by FLIP. Apoptosis mediated by chemotherapeutic drugs (i.e., doxorubicin, etoposide, and vincristine) and gamma irradiation was not affected by FLIP or the absence of Fas, indicating that these treatments can induce cell death in a Fas-independent and FLIP-insensitive manner.

p53-induced protein with a death domain (PIDD) isoforms differentially activate nuclear factor-kappaB and caspase-2 in response to genotoxic stress

Cells respond to DNA damage in a complex way and the fate of damaged cells depends on the balance between pro- and antiapoptotic signals. This is of crucial importance in cancer as genotoxic stress is implied both in oncogenesis and in classical tumor therapies. p53-induced protein with a death domain (PIDD), initially described as a p53-inducible gene, is one of the molecular switches able to activate a survival or apoptotic program. Two isoforms of PIDD, PIDD (isoform 1) and LRDD (isoform 2), have already been reported and we describe here a third isoform. These three isoforms are differentially expressed in tissues and cell lines. Genotoxic stress only affects PIDD isoform 3 mRNA levels, whereas isoforms 1 and 2 mRNA levels remain unchanged. All isoforms are capable of activating nuclear factor-kappaB in response to genotoxic stress, but only isoform 1 interacts with RIP-associated ICH-1/CED-3 homologous protein with a death domain and activates caspase-2. Isoform 2 counteracts the pro-apoptotic function of isoform 1, whereas isoform 3 enhances it. Thus, the differential splicing of PIDD mRNA leads to the formation of at least three proteins with antagonizing/agonizing functions, thereby regulating cell fate in response to DNA damage

Rapid death and regeneration of NKT cells in anti-CD3epsilon- or IL-12-treated mice: a major role for bone marrow in NKT cell homeostasis.

Natural killer T (NKT) cells express a T cell receptor (TCR) and markers common to NK cells, including NK1.1. In vivo, NKT cells are triggered by anti-CD3epsilon MAb to rapidly produce large amounts of IL-4 and by IL-12 to reject tumors. We show here that anti-CD3epsilon MAb treatment rapidly depletes the liver (and partially the spleen) of NKT cells and that homeostasis is achieved 1 to 2 days later via NKT cell proliferation that occurs mainly in bone marrow. Similar results were obtained in mice treated with IL-12. Collectively, our data demonstrate that peripheral NKT cells are highly sensitive to activation-induced cell death and that bone marrow plays a major role in restoring NKT cell homeostasis.

Regulation of lymphocyte proliferation and death by FLIP.

Lymphocyte homeostasis is a balance between lymphocyte proliferation and lymphocyte death. Tight control of apoptosis is essential for immune function, because its altered regulation can result in cancer and autoimmunity. Signals from members of the tumour-necrosis-factor receptor (TNF-R) family, such as Fas and TNF-R1, activate the caspase cascade and result in lymphocyte death by apoptosis. Anti-apoptotic proteins, such as FLIP (also known as FLICE/caspase-8 inhibitory protein) have recently been identified. FLIP expression is tightly regulated in T cells and might be involved in the control of both T-cell activation and death. Abnormal expression of FLIP might have a role not only in autoimmune diseases, but also in tumour development and cardiovascular disorders.

Regulation of glucose transporter expression in cardiac myocytes: p38 MAPK is a strong inducer of GLUT4.

OBJECTIVE: In vivo differentiation of cardiac myocytes is associated with downregulation of the glucose transporter isoform GLUT1 and upregulation of the isoform GLUT4. Adult rat cardiomyocytes in primary culture undergo spontaneous dedifferentiation, followed by spreading and partial redifferentiation, which can be influenced by growth factors. We used this model to study the signaling mechanisms modifying the expression of GLUT4 in cardiac myocytes. RESULTS: Adult rat cardiomyocytes in primary culture exhibited spontaneous upregulation of GLUT1 and downregulation of GLUT4, suggesting resumption of a fetal program of GLUT gene expression. Treatment with IGF-1 and, to a minor extent, FGF-2 resulted in restored expression of GLUT4 protein and mRNA. Activation of p38 MAPK mediated the increased expression of GLUT4 in response to IGF-1. Transient transfection experiments in neonatal cardiac myocytes confirmed that p38 MAPK could activate the glut4 promoter. Electrophoretic mobility shift assay in adult rat cardiomyocytes and transient transfection experiments in neonatal cardiac myocytes indicated that MEF2 was the main transcription factor transducing the effect of p38 MAPK activation on the glut4 promoter. CONCLUSION: Spontaneous dedifferentiation of adult rat cardiomyocytes in vitro is associated with downregulation of GLUT4, which can be reversed by treatment with IGF-1. The effect of IGF-1 is mediated by the p38 MAPK/MEF2 axis, which is a strong inducer of GLUT4 expression.

The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa.

A novel two-component system, CbrA-CbrB, was discovered in Pseudomonas aeruginosa; cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino-terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy-terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot-argR operon was not induced by arginine, indicating an essential role for this two-component system in the expression of the ArgR-dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild-type strain. When succinate or other C4-dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate-dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA-CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB-NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.

Increased connexin43 expression in human saphenous veins in culture is associated with intimal hyperplasia.

OBJECTIVE: Intimal hyperplasia is a vascular remodelling process that occurs after a vascular injury. The mechanisms involved in intimal hyperplasia are proliferation, dedifferentiation, and migration of medial smooth muscle cells towards the subintimal space. We postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, might participate in the development of intimal hyperplasia. Connexin43 (Cx43) expression levels may be altered in intimal hyperplasia, and we therefore evaluated the regulated expression of Cx43 in human saphenous veins in culture in the presence or not of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. METHODS: Segments of harvested human saphenous veins, obtained at the time of bypass graft, were opened longitudinally with the luminal surface uppermost and maintained in culture for 14 days. Vein fragments were then processed for histologic examination, neointimal thickness measurements, immunocytochemistry, RNA, and proteins analysis. RESULTS: Of the four connexins (Cx37, 40, 43, and 45), we focused on Cx43 and Cx40, which we found by real-time polymerase chain reaction to be expressed in the saphenous vein because they are the predominant connexins expressed by smooth muscle cells and endothelial cells. After 14 days of culture, histomorphometric analysis showed a significant increase in the intimal thickness as observed during the process of intimal hyperplasia. A time-course analysis revealed a progressive upregulation of Cx43 to reach a maximal increase of sixfold to eightfold at both transcript and protein levels after 14 days in culture. In contrast, the expression of Cx40, abundantly expressed in the endothelial cells, was not altered. Immunofluorescence showed a large increase in Cx43 within smooth muscle cell membranes of the media layer. The development of intimal hyperplasia in vitro was decreased in presence of fluvastatin and was associated with reduced Cx43 expression. CONCLUSIONS: These data show that Cx43 is increased in vitro during the process of intimal hyperplasia and that fluvastatin could prevent this induction, supporting a critical role for Cx43-mediated gap-junctional communication in the human vein during the development of intimal hyperplasia. CLINICAL RELEVANCE: Stenosis due to intimal hyperplasia is the most common cause of failure of venous bypass grafts. To better understand the development of intimal hyperplasia, we used an ex vivo organ culture model to study saphenous veins harvested from patients undergoing a lower limb bypass surgery. In this model, the morphologic and functional integrity of the vessel wall is maintained and significant intimal hyperplasia development occurs after 14 days in culture. We have postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, may participate in the development of intimal hyperplasia. Indeed, intimal hyperplasia consists of proliferation and migration of smooth muscle cells into the subendothelial space. Intercellular communication is responsible for the direct transfer of ions and small molecules from one cell to the other through gap-junction channels found at cell-cell appositions. No study to date has evaluated whether gap junctional communication is involved in the process of intimal hyperplasia in humans. This assertion was investigated by using the aforementioned organ culture model of intimal hyperplasia in human saphenous veins, and our data support a critical role for Cx43-mediated gap junctional communication in human vein during the development of intimal hyperplasia.

Anterior cingulate cortex pathology in schizophrenia and bipolar disorder.

To explore possible morphological abnormalities in the dorsal and subgenual parts of anterior cingulate cortex in mood disorders and schizophrenia, we performed a quantitative postmortem study of 44 schizophrenic patients, 21 patients with sporadic bipolar disorder, 20 patients with sporadic major depression, and 55 age- and sex-matched control cases. All individuals were drug naïve or had received psychotropic medication for less than 6 months, and had no history of substance abuse. Neuron densities and size were estimated on cresyl violet-stained sections using a stereological counting approach. The distribution and density of microtubule-associated (MAP2, MAP1b) and tau proteins were assessed by immunocytochemistry and quantitative immunodot assay. Mean total and laminar cortical thicknesses as well as mean pyramidal neuron size were significantly decreased in the dorsal and subgenual parts of areas 24 (24sg) in schizophrenic cases. Patients with bipolar disorder showed a substantial decrease in laminar thickness and neuron densities in layers III, V, and VI of the subgenual part of area 24, whereas patients with major depression were comparable to controls. Immunodot assay showed a significant decrease of both MAP2 and MAP1b proteins in bipolar patients but not in patients with schizophrenia and major depression. The neuroanatomical and functional significance of these findings are discussed in the light of current hypotheses regarding the role of areas 24 and 24sg in schizophrenia and bipolar disorder.