Recipient iNOS but Not eNOS Deficiency Reduces Luminal Narrowing in Tracheal Allografts

Chronic airway rejection is characterized by prolonged inflammation, epithelial damage, and eventual luminal obliterative bronchiolitis (OB). In cardiac allografts, the inducible nitric oxide synthase (iNOS) promotes acute rejection but paradoxically reduces neointimal formation, the hallmark of chronic rejection. The specific roles of NOS isoforms in modulating lymphocyte traffic and airway rejection are not known. Using a double lumen mouse tracheal transplant model, tracheal grafts from B10.A (allo) or C57BL/6J (iso) mice were transplanted into cyclosporine-treated wild-type (WT) iNOS−/− or endothelial NOS (eNOS)−/− recipients. OB was observed in WT tracheal allografts at 3 weeks (53 ± 2% luminal occlusion vs. 17 ± 1% for isografts, P < 0.05) with sites of obstructive lesion formation coinciding with areas of CD3+ CD8+ T cell–rich lymphocytic bronchitis. In contrast, allografts in iNOS−/− recipients exhibited reductions in local expression of proinflammatory chemokines and cytokines, graft T cell recruitment and apoptosis, and luminal obliteration (29 ± 2%, P < 0.05 vs. WT allografts). Recipient eNOS deficiency, however, suppressed neither chemokine expression, lymphocyte infiltration, nor airway occlusion (54 ± 2%). These data demonstrate that iNOS exacerbates luminal obliteration of airway allografts in contrast with the known suppression by iNOS of cardiac allograft vasculopathy. Because iNOS−/− airways transplanted into WT allograft hosts are not protected from rejection, these data suggest that iNOS expressed by graft-infiltrating leukocytes exerts the dominant influence on airway rejection

The Model Cities Program

The period from 1961 through 1965 saw a dramatic increase in the number of federal grant-in-aid programs and the total federal funding levels directed at curing the ills of the urban community. There was a persistent anxiety, however, that, despite the proliferation of new drugs administered to the patient for his array of symptoms, the progress was not satisfactory, and that time was running out. In October, 1965, a Task Force on Urban Problems was appointed by President Johnson to study urban problems and recommend action. The Task Force looked at the prior efforts and decided a new approach was necessary-a treatment to be commenced in selected cities as a demonstration. They recommended that the federal, state and local medicine men consult with each other in order to develop a program of drug therapy which would be comprehensive, and coordinated. They also recommended that massive new types and higher dosage levels of drugs were necessary if the patient was to be revitalized. The basic Task Force recommendation was accepted by the President and presented to the Congress in his message of January 26, 1966. A proposed Demonstration Cities Act of 1966 was introduced into Congress, which was later consolidated with other provisions into an omnibus bill, and finally was enacted as Title I of the Demonstration Cities and Metropolitan Development Act of 1966. As HUD commenced to implement the new program, it was given its popular name, the Model Cities Program, a designation not appearing in the Act. This article presents this, program in its historical perspective and its posture as developed administratively by the Johnson Administration. Its full potential and direction will unfold during the Nixon Administration. Secretary of Housing and Urban Development, George W. Romney, has endorsed the concept underlying the Program and announced Presidential approval of certain revisions in the administration of the Program.\u2

Molecular regulation of the PAI‐1 gene by hypoxia: contributions of Egr‐1, HIF‐1 α, and C/EBPα

Hypoxia, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. Plasminogen activator inhibitor‐1 (PAI‐1) is an important factor suppressing fibrinolysis under conditions of low oxygen tension. We previously reported that hypoxia induced PAI‐1 mRNA and antigen expression in murine macrophages secondary to increased de novo transcription as well as increased mRNA stability. We now show in RAW264.7 murine macrophages that the transcription factors early growth response gene‐1 (Egr‐1), hypoxia‐inducible factor‐1α (HIF‐1α), and CCAAT/enhancer binding protein α (C/EBPα) are quickly activated in hypoxia and are responsible for transcription and expression of PAI‐1. Murine PAI‐1 promoter constructs, including Egr, HIF‐1α, and/or C/EBPα binding sites, were transfected into RAW 264.7 murine macrophages. To identify the relative importance of each of these putative hypoxia‐responsive elements, cells were exposed to normobaric hypoxia, and transcriptional activity was recorded. At 16 h of hypoxic exposure, murine PAI‐1 promoter deletion constructs that included Egr, HIF‐1α, and/or C/EBPα binding sites demonstrated increased tran‐scriptional activity. Mutation of each of these three murine PAI‐1 promoter sites (or a combination of them) resulted in a marked reduction in hypoxia sensitivity as detected by transcriptional analysis. Functional data obtained using 32P‐labeled Egr, HIF‐1 α response element (HRE), and C/EBPα oligonucleotides revealed induction of DNA binding activity in nuclear extracts from hypoxic RAW cells, with supershift analysis confirming activation of Egr‐1, HIF‐1 α, or C/EBPα. ChIP analysis confirmed the authenticity of these interactions as each of these transcription factors binds to chromatin under hypoxic conditions. Further, the induction of PAI‐1 by Egr‐1, HIF‐1 α, or C/EBPα was replicated in primary peritoneal macrophages. These data suggest that although HIF‐1 α appears to dominate the PAI‐1 transcriptional response in hyp‐oxia, Egr‐1 and C/EBPα greatly augment this response and can do so independent of HIF‐1α or each other. These studies are relevant to ischemic up‐regulation of the PAI‐1 gene and consequent accrual of micro‐vascular thrombus under ischemic conditions.—Liao, H., Hyman, M. C., Lawrence, D. A., Pinsky, D. J. Molecular regulation of the PAI‐1 gene by hypoxia: contributions of Egr‐1, HIF‐1α, and C/EBPα. FASEB J. 21, 935–949 (2007)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154298/1/fsb2fj066285com.pd

Regulation of ecto‐apyrase CD39 (ENTPD1) expression by phosphodiesterase III (PDE3)

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154432/1/fsb2027011011.pd

Vacuum Structure of Two-Dimensional Gauge Theories on the Light Front

We discuss the problem of vacuum structure in light-front field theory in the context of (1+1)-dimensional gauge theories. We begin by reviewing the known light-front solution of the Schwinger model, highlighting the issues that are relevant for reproducing the $\theta$-structure of the vacuum. The most important of these are the need to introduce degrees of freedom initialized on two different null planes, the proper incorporation of gauge field zero modes when periodicity conditions are used to regulate the infrared, and the importance of carefully regulating singular operator products in a gauge-invariant way. We then consider SU(2) Yang-Mills theory in 1+1 dimensions coupled to massless adjoint fermions. With all fields in the adjoint representation the gauge group is actually SU(2)$/Z_2$, which possesses nontrivial topology. In particular, there are two topological sectors and the physical vacuum state has a structure analogous to a $\theta$ vacuum. We formulate the model using periodicity conditions in $x^\pm$ for infrared regulation, and consider a solution in which the gauge field zero mode is treated as a constrained operator. We obtain the expected $Z_2$ vacuum structure, and verify that the discrete vacuum angle which enters has no effect on the spectrum of the theory. We then calculate the chiral condensate, which is sensitive to the vacuum structure. The result is nonzero, but inversely proportional to the periodicity length, a situation which is familiar from the Schwinger model. The origin of this behavior is discussed.Comment: 29 pages, uses RevTeX. Improved discussion of the physical subspace generally and the vacuum states in particular. Basic conclusions are unchanged, but some specific results are modifie

Reciprocal regulation of airway rejection by the inducible gas-forming enzymes heme oxygenase and nitric oxide synthase

Obliterative bronchiolitis (OB) develops insidiously in nearly half of all lung transplant recipients. Although typically preceded by a CD8+ T cell–rich lymphocytic bronchitis, it remains unresponsive to conventional immunosuppression. Using an airflow permissive model to study the role of gases flowing over the transplanted airway, it is shown that prolonged inhalation of sublethal doses of carbon monoxide (CO), but not nitric oxide (NO), obliterate the appearance of the obstructive airway lesion. Induction of the enzyme responsible for the synthesis of CO, heme oxygenase (Hmox) 1, increased carboxyhemoglobin levels and suppressed lymphocytic bronchitis and airway luminal occlusion after transplantation. In contrast, zinc protoporphyrin IX, a competitive inhibitor of Hmox, increased airway luminal occlusion. Compared with wild-type allografts, expression of inducible NO synthase (iNOS), which promotes the influx of cytoeffector leukocytes and airway graft rejection, was strikingly reduced by either enhanced expression of Hmox-1 or exogenous CO. Hmox-1/CO decreased nuclear factor (NF)-κB binding activity to the iNOS promoter region and iNOS expression. Inhibition of soluble guanylate cyclase did not interfere with the ability of CO to suppress OB, implicating a cyclic guanosine 3′,5′-monophosphate–independent mechanism through which CO suppresses NF-κB, iNOS transcription, and OB. Prolonged CO inhalation represents a new immunosuppresive strategy to prevent OB

Cloning of a Putative Vesicle Transport-related Protein, RA410, from Cultured Rat Astrocytes and Its Expression in Ischemic Rat Brain

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was evaluated in cultured rat astrocytes subjected to hypoxia/reoxygenation. Using differential display, a novel putative vesicle transport-related factor (RA410) was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed RA410 to be composed of domains common to vesicle transport-related proteins of the Sec1/Unc18 family, including Sly1p and Sec1p (yeast), Rop (Drosophila), Unc18 (Caenorhabditis elegans), and Munc18 (mammalian), suggesting its possible role in vesicular transport. Northern analysis of normal rat tissues showed the highest expression of RA410 transcripts in testis. When astrocyte cultures were subjected to a period of hypoxia followed by reoxygenation, induction of RA410 mRNA was observed within 15 min of reoxygenation, reaching a maximum by 60 min. At the start of reoxygenation, the addition of diphenyl iodonium, an NADPH oxidase inhibitor, blocked in parallel astrocyte generation of reactive oxygen intermediates and expression of RA410 message. In contrast, cycloheximide did not affect RA410 mRNA levels, indicating that RA410 is an immediate-early gene in the setting of reoxygenation. Using polyclonal antibody raised against an RA410-derived synthetic peptide, Western blotting of lysates from reoxygenated astrocytes displayed an immunoreactive band of ≈70 kDa, the expression of which followed induction of the mRNA. Fractionation of astrocyte lysates on sucrose gradients showed RA410 antigen to be predominantly in the plasma membrane. Immunoelectron microscopic analysis demonstrated RA410 in large vesicles associated with the Golgi, but not in the Golgi apparatus itself, consistent with its participation in post-Golgi transport. Consistent with thesein vitro data, RA410 expression was observed in rat brain astrocytes following transient occlusion of the middle cerebral artery. These data provide insight into a new protein (RA410) that participates in the ischemia-related stress response in astrocytes

Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation.

This is the published version. Copyright 1997 American Society for Clinical Investigation.Clinical conditions associated with local or systemic hypoxemia can lead to prothrombotic diatheses. This study was undertaken to establish a model of whole-animal hypoxia wherein oxygen deprivation by itself would be sufficient to trigger tissue thrombosis. Furthermore, this model was used to test the hypothesis that hypoxia-induced mononuclear phagocyte (MP) recruitment and tissue factor (TF) expression may trigger the local deposition of fibrin which occurs in response to oxygen deprivation. Using an environmental chamber in which inhaled oxygen tension was lowered to 6%, hypoxic induction of thrombosis was demonstrated in murine pulmonary vasculature by 8 h based upon: (a) immunohistologic evidence of fibrin formation in hypoxic lung tissue using an antifibrin antibody, confirmed by 22.5-nm strand periodicity by electron microscopy; (b) immunoblots revealing fibrin gamma-gamma chain dimers in lungs from hypoxic but not normoxic mice or hypoxic mice treated with hirudin; (c) accelerated deposition of 125I-fibrin/fibrinogen and 111In-labeled platelets in the lung tissue of hypoxic compared with normoxic animals; (d) reduction of tissue 125I-fibrin/fibrinogen accumulation in animals which had either been treated with hirudin or depleted of platelets before hypoxic exposure. Because immunohistochemical analysis of hypoxic pulmonary tissue revealed strong MP staining for TF, confirmed by increased TF RNA in hypoxic lungs, and because 111In-labeled murine MPs accumulated in hypoxic pulmonary tissue, we evaluated whether recruited MPs might be responsible for initiation of hypoxia-induced thrombosis. This hypothesis was supported by several lines of evidence: (a) MP depletion before hypoxia reduced thrombosis, as measured by reduced 125I-fibrin/fibrinogen deposition and reduced accumulation of cross-linked fibrin by immunoblot; (b) isolated murine MPs demonstrated increased TF immunostaining when exposed to hypoxia; and (c) administration of an anti-rabbit TF antibody that cross-reacts with murine TF decreased 125I-fibrin/fibrinogen accumulation and cross-linked fibrin accumulation in response to hypoxia in vivo. In summary, these studies using a novel in vivo model suggest that MP accumulation and TF expression may promote hypoxia-induced thrombosis

Platelet activation in the postoperative period after lung transplantation

Objective During lung transplantation, cells in the pulmonary parenchyma are subjected to ischemia, hypothermic storage, and reperfusion injury. Platelets, whose granular contents include adhesion receptors, chemokines, and coactivating substances that activate inflammatory and coagulant cascades, likely play a critical role in the lung allograft response to ischemia and reperfusion. The platelet response to the pulmonary allograft, however, has never been studied. Here we report significant platelet activation immediately after lung transplantation. Methods We performed a prospective cohort study comparing markers of platelet activation in patients undergoing lung transplantation and patients undergoing nontransplant thoracotomy. Plasma levels of soluble P-selectin, soluble CD40 ligand, and platelet–leukocyte conjugates were measured before surgery, after skin closure, and at 6 postoperative hours. Results Both soluble P-selectin and soluble CD40 ligand levels increased significantly after lung transplantation but not after thoracotomy. Additionally, platelet–monocyte conjugate fluorescence was significantly higher after lung transplantation than after thoracotomy alone. Conclusion These findings suggest that platelet activation is significantly increased after lung transplantation beyond that expected from the postoperative state. The increase in circulating platelet–monocyte conjugates suggests an important interaction between platelets and inflammatory cells. Further research should examine whether platelet activation affects early graft function after lung transplantation

Hypoxic induction of interleukin-8 gene expression in human endothelial cells.

This is the published version. Copyright 1994 American Society for Clinical Investigation.Because leukocyte-mediated tissue damage is an important component of the pathologic picture in ischemia/reperfusion, we have sought mechanisms by which PMNs are directed into hypoxic tissue. Incubation of human endothelial cells (ECs) in hypoxia, PO2 approximately 14-18 Torr, led to time-dependent release of IL-8 antigen into the conditioned medium; this was accompanied by increased chemotactic activity for PMNs, blocked by antibody to IL-8. Production of IL-8 by hypoxic ECs occurred concomitantly with both increased levels of IL-8 mRNA, based on polymerase chain reaction analysis, and increased IL-8 transcription, based on nuclear run-on assays. Northern analysis of mRNA from hypoxic ECs also demonstrated increased levels of mRNA for macrophage chemotactic protein-1, another member of the chemokine superfamily of proinflammatory cytokines. IL-8 gene induction was associated with the presence of increased binding activity in nuclear extracts from hypoxic ECs for the NF-kB site. Studies with human umbilical vein segments exposed to hypoxia also demonstrated increased elaboration of IL-8 antigen compared with normoxic controls. In mice exposed to hypoxia (PO2 approximately 30-40 Torr), there was increased pulmonary leukostasis, as evidenced by increased myeloperoxidase activity in tissue homogenates. In parallel, increased levels of transcripts for IP-10, a murine homologue in the chemokine family related to IL-8, were observed in hypoxic lung tissue. Taken together, these data suggest that hypoxia constitutes a stimulus for leukocyte chemotaxis and tissue leukostasis