Volume 115, Issue 12, Pages 3301-3640
45 total articles
Sydney Ringer would be overwhelmed today by the implications of his simple experiment performed over 120 years ago showing that the heart would not beat in the absence of Ca2+. Fascination with the role of Ca2+ has proliferated into all aspects of our understanding of normal cardiac function and the progression of heart disease, including induction of cardiac hypertrophy, heart failure, and sudden death. This review examines the role of Ca2+ and the L-type voltage-dependent Ca2+ channels in cardiac disease.
Ilona Bodi, Gabor Mikala, Sheryl E. Koch, Shahab A. Akhter, Arnold Schwartz
Osteoporosis is a disorder in which loss of bone strength leads to fragility fractures. This review examines the fundamental pathogenetic mechanisms underlying this disorder, which include: (a) failure to achieve a skeleton of optimal strength during growth and development; (b) excessive bone resorption resulting in loss of bone mass and disruption of architecture; and (c) failure to replace lost bone due to defects in bone formation. Estrogen deficiency is known to play a critical role in the development of osteoporosis, while calcium and vitamin D deficiencies and secondary hyperparathyroidism also contribute. There are multiple mechanisms underlying the regulation of bone remodeling, and these involve not only the osteoblastic and osteoclastic cell lineages but also other marrow cells, in addition to the interaction of systemic hormones, local cytokines, growth factors, and transcription factors. Polymorphisms of a large number of genes have been associated with differences in bone mass and fragility. It is now possible to diagnose osteoporosis, assess fracture risk, and reduce that risk with antiresorptive or other available therapies. However, new and more effective approaches are likely to emerge from a better understanding of the regulators of bone cell function.
Lawrence G. Raisz
Biological evolution has struggled to produce mechanisms that can limit blood loss following injury. In humans and other mammals, control of blood loss (hemostasis) is achieved through a combination of plasma proteins, most of which are made in the liver, and platelets, anucleate blood cells that are produced in the bone marrow by megakaryocytes. Much has been learned about the underlying mechanisms, but much remains to be determined. The articles in this series review current ideas about the production of megakaryocytes from undifferentiated hematopoietic precursors, the steps by which megakaryocytes produce platelets, and the molecular mechanisms within platelets that make hemostasis possible. The underlying theme that connects the articles is the intense investigation of a complex system that keeps humans from bleeding to death, but at the same time exposes us to increased risk of thrombosis and vascular disease.
Lawrence F. Brass
Platelets, derived from megakaryocytes, have an essential role in thrombosis and hemostasis. Over the past 10 years, a great deal of new information has been obtained concerning the various aspects of hematopoiesis necessary to maintain a steady-state platelet level to support physiologic hemostasis. Here we discuss the differentiation of HSCs into megakaryocytes, with emphasis on the key cytokine signaling pathways and hematopoietic transcription factors. Recent insight into these processes elucidates the molecular bases of numerous acquired and inherited hematologic disorders. It is anticipated that the growing knowledge in these areas may be exploited for new therapeutic strategies to modulate both platelet numbers and their thrombogenicity.
Liyan Pang, Mitchell J. Weiss, Mortimer Poncz
Our understanding of thrombopoiesis — the formation of blood platelets — has improved greatly in the last decade, with the cloning and characterization of thrombopoietin, the primary regulator of this process. Thrombopoietin affects nearly all aspects of platelet production, from self-renewal and expansion of HSCs, through stimulation of the proliferation of megakaryocyte progenitor cells, to support of the maturation of these cells into platelet-producing cells. The molecular and cellular mechanisms through which thrombopoietin affects platelet production provide new insights into the interplay between intrinsic and extrinsic influences on hematopoiesis and highlight new opportunities to translate basic biology into clinical advances.
Platelets are formed and released into the bloodstream by precursor cells called megakaryocytes that reside within the bone marrow. The production of platelets by megakaryocytes requires an intricate series of remodeling events that result in the release of thousands of platelets from a single megakaryocyte. Abnormalities in this process can result in clinically significant disorders. Thrombocytopenia (platelet counts less than 150,000/μl) can lead to inadequate clot formation and increased risk of bleeding, while thrombocythemia (platelet counts greater than 600,000/μl) can heighten the risk for thrombotic events, including stroke, peripheral ischemia, and myocardial infarction. This Review will describe the process of platelet assembly in detail and discuss several disorders that affect platelet production.
Sunita R. Patel, John H. Hartwig, Joseph E. Italiano Jr.
To examine thrombus formation in a living mouse, new technologies involving intravital videomicroscopy have been applied to the analysis of vascular windows to directly visualize arterioles and venules. After vessel wall injury in the microcirculation, thrombus development can be imaged in real time. These systems have been used to explore the role of platelets, blood coagulation proteins, endothelium, and the vessel wall during thrombus formation. The study of biochemistry and cell biology in a living animal offers new understanding of physiology and pathology in complex biologic systems.
Bruce Furie, Barbara C. Furie
The platelet integrin αIIbβ3 is required for platelet aggregation. Like other integrins, αIIbβ3 resides on cell surfaces in an equilibrium between inactive and active conformations. Recent experiments suggest that the shift between these conformations involves a global reorganization of the αIIbβ3 molecule and disruption of constraints imposed by the heteromeric association of the αIIb and β3 transmembrane and cytoplasmic domains. The biochemical, biophysical, and ultrastructural results that support this conclusion are discussed in this Review.
Joel S. Bennett
Proteomic and genomic technologies provide powerful tools for characterizing the multitude of events that occur in the anucleate platelet. These technologies are beginning to define the complete platelet transcriptome and proteome as well as the protein-protein interactions critical for platelet function. The integration of these results provides the opportunity to identify those proteins involved in discrete facets of platelet function. Here we summarize the findings of platelet proteome and transcriptome studies and their application to diseases of platelet function.
Iain C. Macaulay ... Des Fitzgerald, Nicholas A. Watkins
Platelets represent an important linkage between inflammation, thrombosis, and atherogenesis. Inflammation is characterized by interactions among platelets, leukocytes, and ECs. These interactions trigger autocrine and paracrine activation processes that lead to leukocyte recruitment into the vascular wall. Platelet-induced chronic inflammatory processes at the vascular wall result in development of atherosclerotic lesions and atherothrombosis. This Review highlights the molecular machinery and inflammatory pathways used by platelets to initiate and accelerate atherothrombosis.
Meinrad Gawaz, Harald Langer, Andreas E. May
Efforts to understand the role of platelets in hemostasis and thrombosis have largely focused on the earliest events of platelet activation, those that lead to aggregation. Although much remains to be learned about those early events, this Review examines a later series of events: the interactions between platelets that can only occur once aggregation has begun, bringing platelets into close contact with each other, creating a protected environment in the gaps between aggregated platelets, and fostering the continued growth and stability of the hemostatic plug.
Lawrence F. Brass, Li Zhu, Timothy J. Stalker
Ghrelin, produced in the stomach, acts on growth hormone secretagogue receptors (GHSRs) in hypothalamic neurons to potently increase food intake. However, male mice with deletions of ghrelin (Ghrl–/– mice) or GHSR (Ghsr–/– mice) display normal growth and regulation of food intake. Furthermore, adult Ghrl–/– mice display a normal sensitivity to high-fat diet–induced obesity. These findings from early studies raised the question as to whether the ghrelin system is an essential component for the regulation of food intake and body weight homeostasis. However, recent studies by Wortley et al. and Zigman et al. demonstrate that Ghrl–/– and Ghsr–/– mice are resistant to diet-induced obesity when fed a high-fat diet during the early post-weaning period. This commentary highlights 3 key issues raised by these 2 reports: (a) the impact of ghrelin on the development of metabolic systems; (b) the constitutive activity of GHSR; and (c) gender differences in the sensitivity to deletion of the ghrelin signaling system.
Kevin L. Grove, Michael A. Cowley
Minor histocompatibility antigens (mHAgs) selectively expressed by cells or cell subsets of the hematopoietic system are targets of the T cell–mediated graft-versus-leukemia response that develops following allogeneic hematopoietic stem cell transplantation (HSCT) for the treatment of hematological malignancies. This observation has served as the rationale for utilizing mHAg-specific immunotherapy for the treatment of particular patients. However, at present, only a select and small number of patients could potentially benefit from mHAg-based immunotherapy. A report from de Rijke et al. in this issue of the JCI describes a new hematopoietic lineage–specific HLA-B7–restricted mHAg associated with remission of chronic myeloid leukemia. This result represents another example of an mHAg-mediated graft-versus-leukemia response, thereby expanding the number of patients eligible for mHAg-based immunotherapy in the setting of HSCT.
Eric Spierings, Els Goulmy
The recent discovery of the Gi protein–coupled receptor GPR109A (HM74A in humans; PUMA-G in mice) as a receptor for nicotinic acid has provided the opportunity to gain greater understanding of the underlying biology contributing to the clinical efficacy (increases in HDL, decreases in VLDL, LDL, and triglycerides) and the characteristic side-effect profile of nicotinic acid. GPR109A has been proven to be the molecular target for the actions of nicotinic acid on adipose tissue, and in this issue of the JCI, Benyó et al. have confirmed the involvement of GPR109A in the nicotinic acid–induced flushing response, a common side effect. The involvement of GPR109A in both the desirable and undesirable clinical actions of nicotinic acid raises interesting questions regarding the function of this receptor.
Nicholas B. Pike
A life-saving response to hypoglycemia requires rapid sensing of decreases in glycemia and consequent brisk glucagon secretion. Preceding studies have shown that mice lacking glucose transporter type 2 (GLUT2) lose this response. In this issue of the JCI, Marty et al. report that glucose sensing and consequent pancreatic glucagon secretion are restored by re-expression of GLUT2 in glial but not neuronal cells. A new, glucose-sensing role is ascribed to GLUT2-expressing glial cells.
Amira Klip, Meredith Hawkins
Glucagon-like peptide–1 (GLP-1) has a diverse set of peripheral actions which all serve to promote enhanced glucose tolerance, and for this reason it has become the basis for new treatments for type 2 diabetes. In this issue of the JCI, Knauf et al. provide clear evidence that GLP-1 signaling in the CNS is also linked to the control of peripheral glucose homeostasis by inhibiting non–insulin-mediated glucose uptake by muscle and increasing insulin secretion from the pancreas. The authors’ work points to an important need to integrate diverse GLP-1 signaling actions and peripheral GLP-1 function in order to better understand both normal and abnormal glucose homeostasis.
David A. D’Alessio, Darleen A. Sandoval, Randy J. Seeley
During intravascular hemolysis in human disease, vasomotor tone and organ perfusion may be impaired by the increased reactivity of cell-free plasma hemoglobin (Hb) with NO. We experimentally produced acute intravascular hemolysis in a canine model in order to test the hypothesis that low levels of decompartmentalized or cell-free plasma Hb will severely reduce NO bioavailability and produce vasomotor instability. Importantly, in this model the total intravascular Hb level is unchanged; only the compartmentalization of Hb within the erythrocyte membrane is disrupted. Using a full-factorial design, we demonstrate that free water–induced intravascular hemolysis produces dose-dependent systemic vasoconstriction and impairs renal function. We find that these physiologic changes are secondary to the stoichiometric oxidation of endogenous NO by cell-free plasma oxyhemoglobin. In this model, 80 ppm of inhaled NO gas oxidized 85–90% of plasma oxyhemoglobin to methemoglobin, thereby inhibiting endogenous NO scavenging by cell-free Hb. As a result, the vasoconstriction caused by acute hemolysis was attenuated and the responsiveness to systemically infused NO donors was restored. These observations confirm that the acute toxicity of intravascular hemolysis occurs secondarily to the accelerated dioxygenation reaction of plasma oxyhemoglobin with endothelium-derived NO to form bioinactive nitrate. These biochemical and physiological studies demonstrate a major role for the intact erythrocyte in NO homeostasis and provide mechanistic support for the existence of a human syndrome of hemolysis-associated NO dysregulation, which may contribute to the vasculopathy of hereditary, acquired, and iatrogenic hemolytic states.
Peter C. Minneci ... Mark T. Gladwin, Steven B. Solomon
TNF-α is the dominant cytokine in inflammatory osteolysis. Using mice whose BM stromal cells and osteoclast precursors are chimeric for the presence of TNF receptors, we found that both cell types mediated the cytokine’s osteoclastogenic properties. The greater contribution was made, however, by stromal cells that express the osteoclastogenic cytokine M-CSF. TNF-α stimulated M-CSF gene expression, in vivo, only in the presence of TNF-responsive stromal cells. M-CSF, in turn, induced the key osteoclastogenic cytokine receptor, receptor activator of NF-κB (RANK), in osteoclast precursors. In keeping with the proproliferative and survival properties of M-CSF, TNF-α enhanced osteoclast precursor number only in the presence of stromal cells bearing TNF receptors. To determine the clinical relevance of these observations, we induced inflammatory arthritis in wild-type mice and treated them with a mAb directed against the M-CSF receptor, c-Fms. Anti–c-Fms mAb selectively and completely arrested the profound pathological osteoclastogenesis attending this condition, the significance of which is reflected by similar blunting of the in vivo bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b). Confirming that inhibition of the M-CSF signaling pathway targets TNF-α, anti–c-Fms also completely arrested osteolysis in TNF-injected mice with nominal effect on macrophage number. M-CSF and its receptor, c-Fms, therefore present as candidate therapeutic targets in states of inflammatory bone erosion.
Hideki Kitaura, Ping Zhou, Hyun-Ju Kim, Deborah V. Novack, F. Patrick Ross, Steven L. Teitelbaum
Nephrophilic autoantibodies dominate the seroprofile in lupus, but their fine specificities remain ill defined. We constructed a multiplexed proteome microarray bearing about 30 antigens known to be expressed in the glomerular milieu and used it to study serum autoantibodies in lupus. Compared with normal serum, serum from B6.Sle1.lpr lupus mice (C57BL/6 mice homozygous for the NZM2410/NZW allele of Sle1 as well as the FASlpr defect) exhibited high levels of IgG and IgM antiglomerular as well as anti–double-stranded DNA/chromatin Abs and variable levels of Abs to α-actinin, aggrecan, collagen, entactin, fibrinogen, hemocyanin, heparan sulphate, laminin, myosin, proteoglycans, and histones. The use of these glomerular proteome arrays also revealed 5 distinct clusters of IgG autoreactivity in the sera of lupus patients. Whereas 2 of these IgG reactivity clusters (DNA/chromatin/glomeruli and laminin/myosin/Matrigel/vimentin/heparan sulphate) showed association with disease activity, the other 3 reactivity clusters (histones, vitronectin/collagen/chondroitin sulphate, and entactin/fibrinogen/hyaluronic acid) did not. Human lupus sera also displayed 2 distinct IgM autoantibody clusters, one reactive to DNA and the other apparently polyreactive. Interestingly, the presence of IgM polyreactivity in patient sera was associated with reduced disease severity. Hence, the glomerular proteome array promises to be a powerful analytical tool for uncovering novel autoantibody disease associations and for distinguishing patients at high risk for end-organ disease.
Quan Li Zhen, Chun Xie, Tianfu Wu, Meggan Mackay, Cynthia Aranow, Chaim Putterman, Chandra Mohan
Numerous mechanisms of action have been proposed for intravenous Ig (IVIG). In this study, we used IgG passive transfer murine models of bullous pemphigoid (BP), pemphigus foliaceus (PF), and pemphigus vulgaris (PV) to test the hypothesis that the effect of IVIG in autoantibody-mediated cutaneous bullous diseases is to accelerate the degradation of pathogenic IgG by saturation of the MHC-like Fc receptor neonatal Fc receptor (FcRn). BP, PF, and PV are organ-specific antibody-mediated diseases in which autoantibodies target the hemidesmosomal antigen BP180 and desmosomal antigens Dsg1 and Dsg3, respectively. Antibodies against BP180, Dsg1, and Dsg3, when injected into neonatal mice, induce the BP, PF, and PV disease phenotypes, respectively. We found that FcRn-deficient mice were resistant to experimental BP, PF, and PV. Circulating levels of pathogenic IgG in FcRn-deficient mice were significantly reduced compared with those in WT mice. Administration of high-dose human IgG (HDIG) to WT mice also drastically reduced circulating pathogenic IgG levels and prevented blistering. In FcRn-deficient mice, no additional protective effect with HDIG was realized. These data demonstrate that the therapeutic efficacy of HDIG treatment in the pemphigus and pemphigoid models is dependent on FcRn. Thus, FcRn is a promising therapeutic target for treating such IgG-mediated autoimmune diseases.
Ning Li ... Derry C. Roopenian, Zhi Liu
Thrombospondin 1 (TSP-1) is a matricellular protein that inhibits angiogenesis and causes apoptosis in vivo and in vitro in several cancerous cells and tissues. Here we identify TSP-1 as the molecule with the highest induction level at 3 hours of IR injury in rat and mouse kidneys subjected to ischemia/reperfusion (IR) injury using the DNA microarray approach. Northern hybridizations demonstrated that TSP-1 expression was undetectable at baseline, induced at 3 and 12 hours, and returned to baseline levels at 48 hours of reperfusion. Immunocytochemical staining identified the injured proximal tubules as the predominant sites of expression of TSP-1 in IR injury and showed colocalization of TSP-1 with activated caspase-3. Addition of purified TSP-1 to normal kidney proximal tubule cells or cells subjected to ATP depletion in vitro induced injury as demonstrated by cytochrome c immunocytochemical staining and caspase-3 activity. The deleterious role of TSP-1 in ischemic kidney injury was demonstrated directly in TSP-1 null mice, which showed significant protection against IR injury–induced renal failure and tubular damage. We propose that TSP-1 is a novel regulator of ischemic damage in the kidney and may play an important role in the pathophysiology of ischemic kidney failure.
Charuhas V. Thakar ... Hamid Rabb, Manoocher Soleimani
Th1 inflammation and remodeling characterized by tissue destruction frequently coexist in human diseases. To further understand the mechanisms of these responses, we defined the role(s) of CCR5 in the pathogenesis of IFN-γ–induced inflammation and remodeling in a murine emphysema model. IFN-γ was a potent stimulator of the CCR5 ligands macrophage inflammatory protein–1α/CCL-3 (MIP-1α/CCL-3), MIP-1β/CCL-4, and RANTES/CCL-5, among others. Antibody neutralization or null mutation of CCR5 decreased IFN-γ–induced inflammation, DNA injury, apoptosis, and alveolar remodeling. These interventions decreased the expression of select chemokines, including CCR5 ligands and MMP-9, and increased levels of secretory leukocyte protease inhibitor. They also decreased the expression and/or activation of Fas, FasL, TNF, caspase-3, -8, and -9, Bid, and Bax. In accordance with these findings, cigarette smoke induced pulmonary inflammation, DNA injury, apoptosis, and emphysema via an IFN-γ–dependent pathway(s), and a null mutation of CCR5 decreased these responses. These studies demonstrate that IFN-γ is a potent stimulator of CC and CXC chemokines and highlight the importance of CCR5 in the pathogenesis of IFN-γ–induced and cigarette smoke–induced inflammation, tissue remodeling, and emphysema. They also demonstrate that CCR5 is required for optimal IFN-γ stimulation of its own ligands, other chemokines, MMPs, caspases, and cell death regulators and the inhibition of antiproteases.
Bing Ma ... Robert J. Homer, Jack A. Elias
The respiratory tract is a primary site of infection and exposure to environmental antigens and an important site of memory T cell localization. We analyzed the migration and retention of naive and activated CD8+ T cells within the noninflamed lungs and quantitated the partitioning of adoptively transferred T cells between the pulmonary vascular and interstitial compartments. Activated but not naive T cells were retained within the lungs for a prolonged period. Effector CD8+ T cells preferentially egressed from the pulmonary vascular compartment into the noninflamed pulmonary interstitium. T cell retention within the lung vasculature was leukocyte function antigen-1 dependent, while the egress of effector T cells from the vascular to the interstitium functions through a pertussis toxin–sensitive (PTX-sensitive) mechanism driven in part by constitutive CC chemokine ligand 5 expression in the lungs. These results document a novel mechanism of adhesion receptor– and pulmonary chemokine–dependent regulation of the migration of activated CD8+ T cells into an important nonlymphoid peripheral site (i.e., the normal/noninflamed lung).
Elena Galkina, Jayant Thatte, Vrushali Dabak, Mark B. Williams, Klaus Ley, Thomas J. Braciale
We have generated mice that carry a neuron-specific leptin receptor (LEPR) transgene whose expression is driven by the rat synapsin I promoter synapsin–LEPR B (SYN-LEPR-B). We have also generated mice that are compound hemizygotes for the transgenes SYN-LEPR-B and neuron-specific enolase–LEPR B (NSE-LEPR-B). We observed a degree of correction in db/db mice that are hemizygous (Syn db/db) and homozygous (Syn/Syn db/db) for the SYN-LEPR-B transgene similar to that previously reported for the NSE-LEPR-B transgene. We also show complete correction of the obesity and related phenotypes of db/db mice that are hemizygous for both NSE-LEPR-B and SYN-LEPR-B transgenes (Nse+Syn db/db). Body composition, insulin sensitivity, and cold tolerance were completely normalized in Nse+Syn db/db mice at 12 weeks of age compared with lean controls. In situ hybridization for LEPR B isoform expression in Nse+Syn db/db mice showed robust expression in the energy homeostasis–relevant regions of the hypothalamus. Expression of 3 neuropeptide genes, agouti-related peptide (Agrp), neuropeptide Y (Npy), and proopiomelanocortin (Pomc), was fully normalized in dual transgenic db/db mice. The 2 transgenes in concert conferred normal fertility to male and female db/db mice. Male mice with partial peripheral deletion of Lepr, induced in the periweaning phase, did not show alterations in body composition or mass. In summary, we show that brain-specific leptin signaling is sufficient to reverse the obesity, diabetes, and infertility of db/db mice.
Carl de Luca ... Shun-Mei Liu, Streamson C. Chua Jr.
Activation of inflammatory pathways may contribute to the beginning and the progression of both atherosclerosis and type 2 diabetes. Here we report a novel interaction between insulin action and control of inflammation, resulting in glucose intolerance and vascular inflammation and amenable to therapeutic modulation. In insulin receptor heterozygous (Insr+/–) mice, we identified the deficiency of tissue inhibitor of metalloproteinase 3 (Timp3, an inhibitor of both TNF-α–converting enzyme [TACE] and MMPs) as a common bond between glucose intolerance and vascular inflammation. Among Insr+/– mice, those that develop diabetes have reduced Timp3 and increased TACE activity. Unchecked TACE activity causes an increase in levels of soluble TNF-α, which subsequently promotes diabetes and vascular inflammation. Double heterozygous Insr+/–Timp3+/– mice develop mild hyperglycemia and hyperinsulinemia at 3 months and overt glucose intolerance and hyperinsulinemia at 6 months. A therapeutic role for Timp3/TACE modulation is supported by the observation that pharmacological inhibition of TACE led to marked reduction of hyperglycemia and vascular inflammation in Insr+/– diabetic mice, as well as by the observation of increased insulin sensitivity in Tace+/– mice compared with WT mice. Our results suggest that an interplay between reduced insulin action and unchecked TACE activity promotes diabetes and vascular inflammation.
Massimo Federici ... Domenico Accili, Renato Lauro
Minor histocompatibility antigens (mHAgs) constitute the targets of the graft-versus-leukemia response after HLA-identical allogeneic stem cell transplantation. Here, we have used genetic linkage analysis to identify a novel mHAg, designated lymphoid-restricted histocompatibility antigen–1 (LRH-1), which is encoded by the P2X5 gene and elicited an allogeneic CTL response in a patient with chronic myeloid leukemia after donor lymphocyte infusion. We demonstrate that immunogenicity for LRH-1 is due to differential protein expression in recipient and donor cells as a consequence of a homozygous frameshift polymorphism in the donor. Tetramer analysis showed that emergence of LRH-1–specific CD8+ cytotoxic T cells in peripheral blood and bone marrow correlated with complete remission of chronic myeloid leukemia. Furthermore, the restricted expression of LRH-1 in hematopoietic cells including leukemic CD34+ progenitor cells provides evidence of a role for LRH-1–specific CD8+ cytotoxic T cells in selective graft-versus-leukemia reactivity in the absence of severe graft-versus-host disease. These findings illustrate that the P2X5-encoded mHAg LRH-1 could be an attractive target for specific immunotherapy to treat hematological malignancies recurring after allogeneic stem cell transplantation.
Björn de Rijke ... Elly van de Wiel-van Kemenade, Harry Dolstra
Homeostatic proliferation of T cells leads to the generation of effector/memory cells, which have the potential to cause harm to the host. The role of Tregs in the control of homeostatic proliferation is unclear. In this study we utilized mice that either harbor or lack Tregs as recipients of monoclonal or polyclonal T cells. We observed that while Tregs completely prevented cell division of T cells displaying low affinity for self ligands, they had a less marked, albeit significant, effect on cell cycle entry of T cells displaying higher affinity. The presence of Tregs resulted in a lower accumulation of T cells, enhanced apoptosis, and impaired differentiation to a cytokine-producing state. We conclude that Tregs play a major role in the control of homeostatic proliferation.
Shiqian Shen ... Maria A. Curotto de Lafaille, Juan J. Lafaille
Recovery of the mitochondrial inner membrane potential (ΔΨm) is a key determinant of postischemic functional recovery of the heart. Mitochondrial ROS-induced ROS release causes the collapse of ΔΨm and the destabilization of the action potential (AP) through a mechanism involving a mitochondrial inner membrane anion channel (IMAC) modulated by the mitochondrial benzodiazepine receptor (mBzR). Here, we test the hypothesis that this mechanism contributes to spatiotemporal heterogeneity of ΔΨm during ischemia-reperfusion (IR), thereby promoting abnormal electrical activation and arrhythmias in the whole heart. High-resolution optical AP mapping was performed in perfused guinea pig hearts subjected to 30 minutes of global ischemia followed by reperfusion. Typical electrophysiological responses, including progressive AP shortening followed by membrane inexcitablity in ischemia and ventricular fibrillation upon reperfusion, were observed in control hearts. These responses were reduced or eliminated by treatment with the mBzR antagonist 4′-chlorodiazepam (4′-Cl-DZP), which blocks depolarization of ΔΨm. When applied throughout the IR protocol, 4′-Cl-DZP blunted AP shortening and prevented reperfusion arrhythmias. Inhibition of ventricular fibrillation was also achieved by bolus infusion of 4′-Cl-DZP just before reperfusion. Conversely, treatment with an agonist of the mBzR that promotes ΔΨm depolarization exacerbated IR-induced electrophysiological changes and failed to prevent arrhythmias. The effects of these compounds were consistent with their actions on IMAC and ΔΨm. These findings directly link instability of ΔΨm to the heterogeneous electrophysiological substrate of the postischemic heart and highlight the mitochondrial membrane as a new therapeutic target for arrhythmia prevention in ischemic heart disease.
Fadi G. Akar, Miguel A. Aon, Gordon F. Tomaselli, Brian O’Rourke
Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell–independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modifed DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens.
Mingquan Zheng ... Qiu Zhong, Jay K. Kolls
Ripglut1;glut2–/– mice have no endogenous glucose transporter type 2 (glut2) gene expression but rescue glucose-regulated insulin secretion. Control of glucagon plasma levels is, however, abnormal, with fed hyperglucagonemia and insensitivity to physiological hypo- or hyperglycemia, indicating that GLUT2-dependent sensors control glucagon secretion. Here, we evaluated whether these sensors were located centrally and whether GLUT2 was expressed in glial cells or in neurons. We showed that ripglut1;glut2–/– mice failed to increase plasma glucagon levels following glucoprivation induced either by i.p. or intracerebroventricular 2-deoxy-D-glucose injections. This was accompanied by failure of 2-deoxy-D-glucose injections to activate c-Fos–like immunoreactivity in the nucleus of the tractus solitarius and the dorsal motor nucleus of the vagus. When glut2 was expressed by transgenesis in glial cells but not in neurons of ripglut1;glut2–/– mice, stimulated glucagon secretion was restored as was c-Fos–like immunoreactive labeling in the brainstem. When ripglut1;glut2–/– mice were backcrossed into the C57BL/6 genetic background, fed plasma glucagon levels were also elevated due to abnormal autonomic input to the α cells; glucagon secretion was, however, stimulated by hypoglycemic stimuli to levels similar to those in control mice. These studies identify the existence of central glucose sensors requiring glut2 expression in glial cells and therefore functional coupling between glial cells and neurons. These sensors may be activated at different glycemic levels depending on the genetic background.
Nell Marty ... Friedrich Beermann, Bernard Thorens
Intestinal glucagon-like peptide–1 (GLP-1) is a hormone released into the hepatoportal circulation that stimulates pancreatic insulin secretion. GLP-1 also acts as a neuropeptide to control food intake and cardiovascular functions, but its neural role in glucose homeostasis is unknown. We show that brain GLP-1 controlled whole-body glucose fate during hyperglycemic conditions. In mice undergoing a hyperglycemic hyperinsulinemic clamp, icv administration of the specific GLP-1 receptor antagonist exendin 9–39 (Ex9) increased muscle glucose utilization and glycogen content. This effect did not require muscle insulin action, as it also occurred in muscle insulin receptor KO mice. Conversely, icv infusion of the GLP-1 receptor agonist exendin 4 (Ex4) reduced insulin-stimulated muscle glucose utilization. In hyperglycemia achieved by i.v. infusion of glucose, icv Ex4, but not Ex9, caused a 4-fold increase in insulin secretion and enhanced liver glycogen storage. However, when glucose was infused intragastrically, icv Ex9 infusion lowered insulin secretion and hepatic glycogen levels, whereas no effects of icv Ex4 were observed. In diabetic mice fed a high-fat diet, a 1-month chronic i.p. Ex9 treatment improved glucose tolerance and fasting glycemia. Our data show that during hyperglycemia, brain GLP-1 inhibited muscle glucose utilization and increased insulin secretion to favor hepatic glycogen stores, preparing efficiently for the next fasting state.
Claude Knauf ... Catherine Postic, Rémy Burcelin
Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor (GHSR; ghrelin receptor). Since its discovery, accumulating evidence has suggested that ghrelin may play a role in signaling and reversing states of energy insufficiency. For example, ghrelin levels rise following food deprivation, and ghrelin administration stimulates feeding and increases body weight and adiposity. However, recent loss-of-function studies have raised questions regarding the physiological significance of ghrelin in regulating these processes. Here, we present results of a study using a novel GHSR-null mouse model, in which ghrelin administration fails to acutely stimulate food intake or activate arcuate nucleus neurons. We show that when fed a high-fat diet, both female and male GHSR-null mice eat less food, store less of their consumed calories, preferentially utilize fat as an energy substrate, and accumulate less body weight and adiposity than control mice. Similar effects on body weight and adiposity were also observed in female, but not male, GHSR-null mice fed standard chow. GHSR deletion also affected locomotor activity and levels of glycemia. These findings support the hypothesis that ghrelin-responsive pathways are an important component of coordinated body weight control. Moreover, our data suggest that ghrelin signaling is required for development of the full phenotype of diet-induced obesity.
Jeffrey M. Zigman ... Bradford B. Lowell, Joel K. Elmquist
The gut peptide ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, has been implicated not only in the regulation of pituitary growth hormone (GH) secretion but in a number of endocrine and nonendocrine functions, including appetitive behavior and carbohydrate substrate utilization. Nevertheless, recent genetic studies have failed to show any significant defects in GH levels, food intake, or body weight in adult ghrelin-deficient (Ghrl−/−) mice. Here we demonstrate that male Ghrl−/− mice are protected from the rapid weight gain induced by early exposure to a high-fat diet 3 weeks after weaning (6 weeks of age). This reduced weight gain was associated with decreased adiposity and increased energy expenditure and locomotor activity as the animals aged. Despite the absence of ghrelin, these Ghrl−/− mice showed a paradoxical preservation of the GH/IGF-1 axis, similar to that reported in lean compared with obese humans. These findings suggest an important role for endogenous ghrelin in the metabolic adaptation to nutrient availability.
Katherine E. Wortley ... Michael O. Thorner, Mark W. Sleeman
Maintenance of a reduced body weight is accompanied by decreased energy expenditure that is due largely to increased skeletal muscle work efficiency. In addition, decreased sympathetic nervous system tone and circulating concentrations of leptin, thyroxine, and triiodothyronine act coordinately to favor weight regain. These “weight-reduced” phenotypes are similar to those of leptin-deficient humans and rodents. We examined metabolic, autonomic, and neuroendocrine phenotypes in 10 inpatient subjects (5 males, 5 females [3 never-obese, 7 obese]) under 3 sets of experimental conditions: (a) maintaining usual weight by ingesting a liquid formula diet; (b) maintaining a 10% reduced weight by ingesting a liquid formula diet; and (c) receiving twice-daily subcutaneous doses of leptin sufficient to restore 8 am circulating leptin concentrations to pre–weight-loss levels and remaining on the same liquid formula diet required to maintain a 10% reduced weight. During leptin administration, energy expenditure, skeletal muscle work efficiency, sympathetic nervous system tone, and circulating concentrations of thyroxine and triiodothyronine returned to pre–weight-loss levels. These responses suggest that the weight-reduced state may be regarded as a condition of relative leptin insufficiency. Prevention of weight regain might be achievable by strategies relevant to reversing this leptin-insufficient state.
Michael Rosenbaum ... Ellen Murphy, Rudolph L. Leibel
To further explore the nature of the mitochondrial dysfunction and insulin resistance that occur in the muscle of young, lean, normoglycemic, insulin-resistant offspring of parents with type 2 diabetes (IR offspring), we measured mitochondrial content by electron microscopy and insulin signaling in muscle biopsy samples obtained from these individuals before and during a hyperinsulinemic-euglycemic clamp. The rate of insulin-stimulated muscle glucose uptake was approximately 60% lower in the IR offspring than the control subjects and was associated with an approximately 60% increase in the intramyocellular lipid content as assessed by 1H magnetic resonance spectroscopy. Muscle mitochondrial density was 38% lower in the IR offspring. These changes were associated with a 50% increase in IRS-1 Ser312 and IRS-1 Ser636 phosphorylation and an approximately 60% reduction in insulin-stimulated Akt activation in the IR offspring. These data provide new insights into the earliest defects that may be responsible for the development of type 2 diabetes and support the hypothesis that reductions in mitochondrial content result in decreased mitochondrial function, which predisposes IR offspring to intramyocellular lipid accumulation, which in turn activates a serine kinase cascade that leads to defects in insulin signaling and action in muscle.
Katsutaro Morino ... Marc Pypaert, Gerald I. Shulman
Whooping cough is considered a childhood disease, although there is growing evidence that children are infected by adult carriers. Additionally, increasing numbers of vaccinated adults are being diagnosed with Bordetella pertussis disease. Thus it is critical to understand how B. pertussis remains endemic even in highly vaccinated or immune populations. Here we used the mouse model to examine the nature of sterilizing immunity to B. pertussis. Antibodies were necessary to control infection but did not rapidly clear B. pertussis from the lungs. However, antibodies affected B. pertussis after a delay of at least a week by a mechanism that involved neutrophils and Fc receptors, suggesting that neutrophils phagocytose and clear antibody-opsonized bacteria via Fc receptors. B. pertussis blocked migration of neutrophils and inhibited their recruitment to the lungs during the first week of infection by a pertussis toxin–dependent (PTx-dependent) mechanism; a PTx mutant of B. pertussis induced rapid neutrophil recruitment and was rapidly cleared from the lungs by adoptively transferred antibodies. Depletion of neutrophils abrogated the defects of the PTx mutant. Together these results indicate that PTx inhibits neutrophil recruitment, which consequently allows B. pertussis to avoid rapid antibody-mediated clearance and therefore successfully infect immune hosts.
Girish S. Kirimanjeswara, Luis M. Agosto, Mary J. Kennett, Ottar N. Bjornstad, Eric T. Harvill
The marked proliferation of activated CD8+ T cells is pathognomonic of EBV-associated infectious mononucleosis (IM), common in young adults. Since the diversity and size of the memory CD8+ T cell population increase with age, we questioned whether IM was mediated by the reactivation of memory CD8+ T cells specific to previously encountered pathogens but cross-reactive with EBV. Of 8 HLA-A2+ IM patients, 5 had activated T cells specific to another common virus, as evidenced by a significantly higher number of peripheral blood influenza A virus M158–66–specific T cells compared with healthy immune donors. Two patients with an augmented M1 response had tetramer-defined cross-reactive cells recognizing influenza M1 and EBV-BMLF1280–288, which accounted for up to one-third of their BMLF1-specific population and likely contributed to a skewed M1-specific T cell receptor repertoire. These epitopes, with only 33% sequence similarity, mediated differential effects on the function of the cross-reactive T cells, which may contribute to alterations in disease outcome. EBV could potentially encode an extensive pool of T cell epitopes that activate other cross-reactive memory T cells. Our results support the concept that cross-reactive memory CD8+ T cells activated by EBV contribute to the characteristic lymphoproliferation of IM.
Shalyn C. Clute ... Raymond M. Welsh, Liisa K. Selin
Previous research on proteins that inhibit kidney stone formation has identified a relatively small number of well-characterized inhibitors. Identification of additional stone inhibitors would increase understanding of the pathogenesis and pathophysiology of nephrolithiasis. We have combined conventional biochemical methods with recent advances in mass spectrometry (MS) to identify a novel calcium oxalate (CaOx) crystal growth inhibitor in normal human urine. Anionic proteins were isolated by DEAE adsorption and separated by HiLoad 16/60 Superdex 75 gel filtration. A fraction with potent inhibitory activity against CaOx crystal growth was isolated and purified by anion exchange chromatography. The protein in 2 subfractions that retained inhibitory activity was identified by matrix-assisted laser desorption/ionization–time-of-flight MS and electrospray ionization–quadrupole–time-of-flight tandem MS as human trefoil factor 1 (TFF1). Western blot analysis confirmed the mass spectrometric protein identification. Functional studies of urinary TFF1 demonstrated that its inhibitory potency was similar to that of nephrocalcin. The inhibitory activity of urinary TFF1 was dose dependent and was inhibited by TFF1 antisera. Anti–C-terminal antibody was particularly effective, consistent with our proposed model in which the 4 C-terminal glutamic residues of TFF1 interact with calcium ions to prevent CaOx crystal growth. Concentrations and relative amounts of TFF1 in the urine of patients with idiopathic CaOx kidney stone were significantly less (2.5-fold for the concentrations and 5- to 22-fold for the relative amounts) than those found in controls. These data indicate that TFF1 is a novel potent CaOx crystal growth inhibitor with a potential pathophysiological role in nephrolithiasis.
Somchai Chutipongtanate ... Prida Malasit, Visith Thongboonkerd
In this study, we investigated whether elimination of CD4+/CD25+ Tregs using the recombinant IL-2 diphtheria toxin conjugate DAB389IL-2 (also known as denileukin diftitox and ONTAK) is capable of enhancing the immunostimulatory efficacy of tumor RNA-transfected DC vaccines. We show that DAB389IL-2 is capable of selectively eliminating CD25-expressing Tregs from the PBMCs of cancer patients without inducing toxicity on other cellular subsets with intermediate or low expression of CD25. DAB389IL-2–mediated Treg depletion resulted in enhanced stimulation of proliferative and cytotoxic T cell responses in vitro but only when DAB389IL-2 was omitted during T cell priming. DAB389IL-2 significantly reduced the number of Tregs present in the peripheral blood of metastatic renal cell carcinoma (RCC) patients and abrogated Treg-mediated immunosuppressive activity in vivo. Moreover, DAB389IL-2–mediated elimination of Tregs followed by vaccination with RNA-transfected DCs significantly improved the stimulation of tumor-specific T cell responses in RCC patients when compared with vaccination alone. Our findings may have implications in the design of immune-based strategies that may incorporate the Treg depletion strategy to achieve potent antitumor immunity with therapeutic impact.
Jens Dannull ... Eli Gilboa, Johannes Vieweg
Nicotinic acid (niacin) has long been used as an antidyslipidemic drug. Its special profile of actions, especially the rise in HDL-cholesterol levels induced by nicotinic acid, is unique among the currently available pharmacological tools to treat lipid disorders. Recently, a G-protein–coupled receptor, termed GPR109A (HM74A in humans, PUMA-G in mice), was described and shown to mediate the nicotinic acid–induced antilipolytic effects in adipocytes. One of the major problems of the pharmacotherapeutical use of nicotinic acid is a strong flushing response. This side effect, although harmless, strongly affects patient compliance. In the present study, we show that mice lacking PUMA-G did not show nicotinic acid–induced flushing. In addition, flushing in response to nicotinic acid was also abrogated in the absence of cyclooxygenase type 1, and mice lacking prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) receptors had reduced flushing responses. The mouse orthologue of GPR109A, PUMA-G, is highly expressed in macrophages and other immune cells, and transplantation of wild-type bone marrow into irradiated PUMA-G–deficient mice restored the nicotinic acid–induced flushing response. Our data clearly indicate that GPR109A mediates nicotinic acid–induced flushing and that this effect involves release of PGE2 and PGD2, most likely from immune cells of the skin.
Zoltán Benyó ... Klaus Pfeffer, Stefan Offermanns
Copyright © 2014 American Society for Clinical Investigation