Remote ischemic preconditioning (IPC) reduces tissue injury caused by ischemia-reperfusion (IR) in distant organs. We tested the hypothesis that remote IPC (rIPC) modifies inflammatory gene transcription in humans. Using a microarray method, we demonstrated that a simple model of brief forearm ischemia suppresses proinflammatory gene expression in circulating leukocytes. Genes encoding key proteins involved in cytokine synthesis, leukocyte chemotaxis, adhesion and migration, exocytosis, innate immunity signaling pathways, and apoptosis were all suppressed within 15 min (early phase IPC) and more so after 24 h (second window IPC). Changes in leukocyte CD11b expression measured by flow cytometry mirrored this pattern, with there being a significant (P = 0.01) reduction at 24 h. The results of this study show that the rIPC stimulus modifies leukocyte inflammatory gene expression. This effect may contribute to the protective effect of IPC against IR injury and may have broader implications in other inflammatory processes. This is the first study of human gene expression following rIPC stimulus. rIPC stimulus suppressed proinflammatory gene transcription in human leukocytes.

We examined the molecular identity of K+channel genes underlying the delayed rectifier (IK) in differentiated cultured oligodendrocytes (OLGs) and oligodendrocyte progenitor (OP) cells. Using reverse transcription-PCR cloning, we found that OP cells and OLGs expressed multiple Kv transcripts, namely Kv1.2, Kv1.4, Kv.1.5, and Kv1.6. Immunocytochemical and Western blot analyses revealed that Kv1.5 and Kv1.6 as well as Kv1.2 and Kv1.4 channel proteins could be detected in these cells, but definitive evidence for functional K+channel expression was obtained only for the Kv1.5 channel. In addition, mRNA and immunoreactive protein levels of both Kv1.5 and Kv1.6 channels were significantly lower in differentiated OLGs when compared with levels in OP cells. Proliferation of OP cells was inhibited by K+channel blockers, but not by incubation with either Kv1.5 or Kv1.6 antisense oligonucleotides. We conclude that (1)IKin OP cells and OLGs is encoded partly by Kv1.5 subunits, possibly forming heteromultimeric channels with Kv1.6 or other Kv subunits; and (2) inhibition of Kv1.5 or Kv1.6 channel expression alone does not prevent mitogenesis. Concomitant inhibition of other Kv channels underlyingIKmay be necessary for OP cells to exit from cell cycle.

The tyrosine phosphatase PTP-MEG2 is targeted by its amino-terminal Sec14p homology domain to the membrane of secretory vesicles. There it regulates vesicle size by promoting homotypic vesicle fusion by a mechanism that requires its catalytic activity. Here, we identify N-ethylmaleimide-sensitive factor (NSF), a key regulator of vesicle fusion, as a substrate for PTP-MEG2. PTP-MEG2 reduced the phosphotyrosine content of NSF and co-localized with NSF and syntaxin 6 in intact cells. Furthermore, endogenous PTP-MEG2 co-immunoprecipitated with endogenous NSF. Phosphorylation of NSF at Tyr 83, as well as an acidic substitution at the same site, increased its ATPase activity and prevented alphaSNAP binding. Conversely, expression of a Y83F mutant of NSF caused spontaneous fusion events. Our results suggest that the molecular mechanism by which PTP-MEG2 promotes secretory vesicle fusion involves the local release of NSF from a tyrosine-phosphorylated, inactive state. This represents a novel mechanism for localized regulation of NSF and the first demonstrated role for a protein tyrosine phosphatase in the regulated secretory pathway.

The role of a cytosolic phospholipase A 2 -α (cPLA 2 -α) in neutrophil arachidonic acid release, plateletactivating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA 2 -α activity was blocked with the specific cPLA 2 -α inhibitor, Pyrrolidine-1 (human cells), or by cPLA 2 -α gene disruption (mice). cPLA 2 -α inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcγII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA 2 -α inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B 4 . Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA 2 -α (−/−) mice compared with wild type littermates. These studies identify a novel * This work was supported by grants from The Physicians of Ontario through The P.S.I. Foundation (Grant 01-12 (to B. B. R.) and 98-049

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Availability of organs is a limiting factor for lung transplantation, leading to substantial mortality rates on the wait list. Use of organs from donors with transmissible viral infections, such as hepatitis C virus (HCV), would increase organ donation, but these organs are generally not offered for transplantation due to a high risk of transmission. Here, we develop a method for treatment of HCV-infected human donor lungs that prevents HCV transmission. Physical viral clearance in combination with germicidal light-based therapies during normothermic ex-vivo Lung Perfusion (EVLP), a method for assessment and treatment of injured donor lungs, inactivates HCV virus in a short period of time. Such treatment is shown to be safe using a large animal EVLP-to-lung transplantation model. This strategy of treating viral infection in a donor organ during preservation could significantly increase the availability of organs for transplantation and encourages further clinical development.

Shwachman–Diamond syndrome (SDS) is an inherited multisystem disorder characterized by exocrine pancreatic dysfunction and varying degrees of cytopenia. In addition, various immunological abnormalities have been noted. To clarify the issue of immunological competence or incompetence in SDS, we prospectively studied immune function in 11 patients with SDS. Seven suffered from recurrent bacterial infections and six from recurrent viral infections. Varying degrees of impairment were readily identified. All patients had neutropenia; total lymphocyte counts, however, were normal in all except one patient. Nine patients had B‐cell defects comprising one or more of the following abnormalities: low IgG or IgG subclasses, low percentage of circulating B lymphocytes, decreased in vitro B‐lymphocyte proliferation and a lack of specific antibody production. Seven out of nine patients studied had at least one T‐cell abnormality comprising a low percentage of total circulating T lymphocytes or CD3+/CD4+ cell subpopulations or decreased in vitro T‐lymphocyte proliferation. Five out of six patients studied had decreased percentages of circulating natural killer cells. Moreover, neutrophil chemotaxis was significantly low in all the patients studied. These data point to a major immunodeficiency component in SDS that places patients at heightened risk of infections, even if neutrophil numbers are protective. This finding broadens the definition of the syndrome substantially: it suggests that the SDS marrow defect occurs at the level of an early haematological–lymphocytic stem cell or that a combined marrow and thymic stromal defect accounts for the aberrant function of haematopoietic and lymphopoietic lineages.

Diverse receptors, including Fc␥ receptors and ␤ 2 integrins (complement receptor-3 [CR3], CD11b/CD18), have been implicated in phagocytosis, but their distinct roles and interactions with other receptors in particle engulfment are not well defined. CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, may have additional functions in regulation of inflammation and phagocytosis. We have recently reported that CD44 is a fully competent phagocytic receptor that is able to trigger ingestion of large particles by macrophages. Here, we investigated the role of coreceptors and intracellular signaling pathways in modulation of CD44-mediated phagocytosis. Using biotinylated erythrocytes coated with specific antibodies (anti-CD44-coated erythrocytes [Ebabs]) as the phagocytic prey, we determined that CD44-mediated phagocytosis is reduced by 45% by a blocking CD11b antibody. Further, CD44-mediated phagocytosis was substantially (42%) reduced in CD18-null mice. Immunofluorescence microscopy revealed that CD11b is recruited to the phagocytic cup.The mechanism of integrin activation and mobilization involved activation of the GTPase Rap1. CD44-mediated phagocytosis was also sensitive to the extracellular concentration of the divalent cation Mg 2؉ but not Ca 2؉ . In addition, buffering of intracellular Ca 2؉ did not affect CD44-mediated phagocytosis. Taken together, these data suggest that CD44 stimulation induces inside-out activation of CR3 through the GTPase Rap1. IntroductionComplement receptor-3 (CR3, Mac1, CD11b/CD18) is a member of the ␤ 2 integrin family of adhesion receptors, which includes CR4 (CD11c/CD18) and LFA-1 (CD11a/CD18). These heterodimeric transmembrane receptors, composed of an ␣ chain (CD11a, CD11b, or CD11c) and the common ␤ 2 chain (CD18), are expressed exclusively on cells of hematopoietic origin. CR3 is multifunctional and is known to be involved in leukocyte adhesion, diapedesis, and phagocytosis. 1 Unlike phagocytosis mediated by Fc receptors, CR3-mediated phagocytosis is regulated by the activation state of the receptor. On quiescent (resting) leukocytes and monocytes, CR3 is capable of binding but not ingesting iC3b-coated phagocytic prey. In contrast, prior activation of leukocytes by diverse agents such as phorbol ester 2 and extracellular matrix proteins such as fibrinogen [3][4][5] activates CR3 and renders it capable of particle ingestion (phagocytosis).While many studies of phagocytosis have used artificial particles such as erythrocytes or latex beads opsonized with monospecific ligands as phagocytic prey, microbial pathogens such as bacteria and fungi express multiple ligands on their cell surfaces that can be recognized by phagocytes. 6,7 The simultaneous engagement of multiple receptors on the host cell by the pathogen ensures that diverse cellular signaling pathways are activated, and the resultant cellular response will therefore reflect a composite of the effects of activation of the different receptors. Examples of cross-talk between receptors in...