bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytochrome C Maintains Mitochondrial Transmembrane Potential and Atp Generation after Outer Mitochondrial Membrane Permeabilization during the Apoptotic Process
Authors: Waterhouse Nigel J. , Goldstein Joshua C. , von Ahsen Oliver , Schuler Martin , Newmeyer Donald D. , Green Douglas R.
Published: July 2002

During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30–60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.
Regulation and Localization of the Bloom Syndrome Protein in Response to DNA Damage
Authors: Bischof Oliver , Kim Sahn-Ho , Irving John , Beresten Sergey , Ellis Nathan A. , Campisi Judith
Published: July 2002

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RecQ-like helicase, presumed to function in DNA replication, recombination, or repair. BLM localizes to promyelocytic leukemia protein (PML) nuclear bodies and is expressed during late S and G2. We show, in normal human cells, that the recombination/repair proteins hRAD51 and replication protein (RP)-A assembled with BLM into a fraction of PML bodies during late S/G2. Biochemical experiments suggested that BLM resides in a nuclear matrix–bound complex in which association with hRAD51 may be direct. DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism. This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed. It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci. After radiation, foci containing BLM and PML formed at sites of single-stranded DNA and presumptive repair in normal cells, but not in cells with defective PML. Our findings suggest that BLM is part of a dynamic nuclear matrix–based complex that requires PML and functions during G2 in undamaged cells and recombinational repair after DNA damage.
Localization of Calmodulin and Dynein Light Chain Lc8 in Flagellar Radial Spokes
Authors: Yang Pinfen , Diener Dennis R. , Rosenbaum Joel L. , Sale Winfield S.
Published: July 2002

Genetic and in vitro analyses have revealed that radial spokes play a crucial role in regulation of ciliary and flagellar motility, including control of waveform. However, the mechanisms of regulation are not understood. Here, we developed a novel procedure to isolate intact radial spokes as a step toward understanding the mechanism by which these complexes regulate dynein activity. The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes. Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14. Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8. Analyses of flagellar mutants and chemical cross-linking studies demonstrated calmodulin and LC8 form a complex located in the radial spoke stalk. We postulate that calmodulin, located in the radial spoke stalk, plays a role in calcium control of flagellar bending.
v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication
Authors: Lin Rui , Warn-Cramer Bonnie J. , Kurata Wendy E. , Lau Alan F.
Published: July 2002

The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap junctional communication (GJC) is not clear. In this study, we determined that Tyr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We established an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in the absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. Most importantly, GJC established by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src. We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.
Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation
Authors: Gilbert Stéphane , Loranger Anne , Daigle Nathalie , Marceau Normand
Published: July 2002

Keratins 8 and 18 belong to the keratin family of intermediate filament (IF) proteins and constitute a hallmark for all simple epithelia, including the liver. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18). In these cells, the loss of one partner via a targeted null mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins. Here, we report that K8-null mouse hepatocytes in primary culture and in vivo are three- to fourfold more sensitive than wild-type (WT) mouse hepatocytes to Fas-mediated apoptosis after stimulation with Jo2, an agonistic antibody of Fas ligand. This increased sensitivity is associated with a higher and more rapid caspase-3 activation and DNA fragmentation. In contrast, no difference in apoptosis is observed between cultured K8-null and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) α or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8-null and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8-null hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8-null versus WT hepatocytes. Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.
A protein interaction map for cell polarity development
Authors: Drees Becky L. , Sundin Bryan , Brazeau Elizabeth , Caviston Juliane P. , Chen Guang-Chao , Guo Wei , Kozminski Keith G. , Lau Michelle W. , Moskow John J. , Tong Amy , Schenkman Laura R. , McKenzie Amos , Brennwald Patrick , Longtine Mark , Bi Erfei , Chan Clarence , Novick Peter , Boone Charles , Pringle John R. , Davis Trisha N. , Fields Stanley , Drubin David G.
Published: July 2002

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.
Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation
Authors: Vieira Otilia V. , Botelho Roberto J. , Rameh Lucia , Brachmann Saskia M. , Matsuo Tsuyoshi , Davidson Howard W. , Schreiber Alan , Backer Jonathan M. , Cantley Lewis C. , Grinstein Sergio
Published: July 2002

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3′-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.
CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis
Authors: Zeitlin Samantha G. , Shelby Richard D. , Sullivan Kevin F.
Published: July 2002

Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1γ1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1γ1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.
The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost
Authors: Roh Michael H. , Makarova Olga , Liu Chia-Jen , Shin  , Lee Seonok , Laurinec Stephanie , Goyal Meera , Wiggins Roger , Margolis Ben
Published: July 2002

Membrane-associated guanylate kinase (Maguk) proteins are scaffold proteins that contain PSD-95–Discs Large–zona occludens-1 (PDZ), Src homology 3, and guanylate kinase domains. A subset of Maguk proteins, such as mLin-2 and protein associated with Lin-7 (Pals)1, also contain two L27 domains: an L27C domain that binds mLin-7 and an L27N domain of unknown function. Here, we demonstrate that the L27N domain targets Pals1 to tight junctions by binding to a PDZ domain protein, Pals1-associated tight junction (PATJ) protein, via a unique Maguk recruitment domain. PATJ is a homologue of Drosophila Discs Lost, a protein that is crucial for epithelial polarity and that exists in a complex with the apical polarity determinant, Crumbs. PATJ and a human Crumbs homologue, CRB1, colocalize with Pals1 to tight junctions, and CRB1 interacts with PATJ albeit indirectly via binding the Pals1 PDZ domain. In agreement, we find that a Drosophila homologue of Pals1 participates in identical interactions with Drosophila Crumbs and Discs Lost. This Drosophila Pals1 homologue has been demonstrated recently to represent Stardust, a crucial polarity gene in Drosophila. Thus, our data identifies a new multiprotein complex that appears to be evolutionarily conserved and likely plays an important role in protein targeting and cell polarity.
Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis
Authors: Lane Jon D. , Lucocq John , Pryde James , Barr Francis A. , Woodman Philip G. , Allan Victoria J. , Lowe Martin
Published: July 2002

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.
Perturbation of the T Lymphocyte Lineage In Transgenic Mice Expressing a Constitutive Repressor of Nuclear Factor (NF)-κB
Authors: Boothby Mark R. , Mora Ana L. , Scherer David C. , Brockman Jeffrey A. , Ballard Dean W.
Published: July 2002

Members of the nuclear factor (NF)-κB/Rel family transcription factors are induced during thymic selection and in mature T lymphocytes after ligation of the T cell antigen receptor (TCR). Despite these findings, disruption of individual NF-κB/Rel genes has revealed no intrinsic defect in the development of mature T cells, perhaps reflecting functional redundancy. To circumvent this possibility, the T cell lineage was targeted to express a trans-dominant form of IκBα that constitutively represses the activity of multiple NF-κB/Rel proteins. Transgenic cells expressing this inhibitor exhibit a significant proliferative defect, which is not reversed by the addition of exogenous interleukin-2. Moreover, mitogenic stimulation of splenocytes leads to increased apoptosis of transgenic T cells as compared with controls. In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes. This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4+ T cells. Taken together, these in vivo findings indicate that the NF-κB/Rel signaling pathway contains compensatory components that are essential for the establishment of normal T cell subsets.
In Vivo Detection of Dendritic Cell Antigen Presentation to CD4+ T Cells
Authors: Ingulli Elizabeth , Mondino Anna , Khoruts Alexander , Jenkins Marc K.
Published: July 2002

Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4+ T cells specific for an OVA peptide–I-Ad complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell–rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.
Syk Tyrosine Kinase Is Required for the Positive Selection of Immature B Cells into the Recirculating B Cell Pool
Authors: Turner Martin , Gulbranson-Judge Adam , Quinn Marian E. , Walters Alice E. , MacLennan Ian C.M. , Tybulewicz Victor L.J.
Published: July 2002

The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.
Proteolytic Cleavage of CD25, the α Subunit of the Human T Cell Interleukin 2 Receptor, by Der p 1, a Major Mite Allergen with Cysteine Protease Activity
Authors: Schulz Oliver , Sewell Herb F. , Shakib Farouk
Published: July 2002

Recent reports have indicated that the cysteine protease activity of Der p 1 may play a significant role in its ability to elicit IgE antibody responses, mainly through cleavage of membrane CD23 on B cells and interleukin (IL)-4 synthesis and secretion from mast cells and basophils. Here we demonstrate for the first time that Der p 1 also cleaves the α subunit of the IL-2 receptor (IL-2R or CD25) from the surface of human peripheral blood T cells and, as a result, these cells show markedly diminished proliferation and interferon γ secretion in response to potent stimulation by anti-CD3 antibody. Given that the IL-2R is pivotal for the propagation of Th1 cells, its cleavage by Der p 1 may consequently bias the immune response towards Th2 cells, thereby creating an allergic microenvironment.
Expression of the Elm1 Gene, a Novel Gene of the CCN (Connective Tissue Growth Factor, Cyr61/Cef10, and Neuroblastoma Overexpressed Gene) Family, Suppresses In Vivo Tumor Growth and Metastasis of K-1735 Murine Melanoma Cells
Authors: Hashimoto Yasunobu , Shindo-Okada Nobuko , Tani Masachika , Nagamachi Yasuhiro , Takeuchi Kaori , Shiroishi Toshihiko , Toma Hiroshi , Yokota Jun
Published: July 2002

We previously isolated a partial cDNA fragment of a novel gene, Elm1 (expressed in low-metastatic cells), that is expressed in low-metastatic but not in high-metastatic K-1735 mouse melanoma cells. Here we determined the full-length cDNA structure of Elm1 and investigated the effect of Elm1 expression on growth and metastatic potential of K-1735 cells. The Elm1 gene encodes a predicted protein of 367 amino acids showing ∼40% amino acid identity with the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, neuroblastoma overexpressed gene [Nov]) family proteins, which consist of secreted cysteine-rich proteins with growth regulatory functions. Elm1 is also a cysteine-rich protein and contains a signal peptide and four domains conserved in the CCN family proteins. Elm1 was highly conserved, expressed ubiquitously in diverse organs, and mapped to mouse chromosome 15. High-metastatic K-1735 M-2 cells, which did not express Elm1, were transfected with an Elm1 expression vector, and several stable clones with Elm1 expression were established. The in vivo growth rates of cells expressing a high level of Elm1 were remarkably slower than those of cells expressing a low level of Elm1. Metastatic potential of transfectants was reduced in proportion to the level of Elm1 expression. Thus, Elm1 is a novel gene of CCN family that can suppress the in vivo growth and metastatic potential of K-1735 mouse melanoma cells.
Interferon γ Regulates Acute and Latent Murine Cytomegalovirus Infection and Chronic Disease of the Great Vessels
Authors: Presti Rachel M. , Pollock Jessica L. , Dal Canto Albert J. , O'Guin Andrew K. , Virgin Herbert W.
Published: July 2002

To define immune mechanisms that regulate chronic and latent herpesvirus infection, we analyzed the role of interferon γ (IFN-γ) during murine cytomegalovirus (MCMV) infection. Lethality studies demonstrated a net protective role for IFN-γ, independent of IFN-α/β, during acute MCMV infection. Mice lacking the IFN-γ receptor (IFN-γR−/−) developed and maintained striking chronic aortic inflammation. Arteritis was associated with inclusion bodies and MCMV antigen in the aortic media. To understand how lack of IFN-γ responses could lead to chronic vascular disease, we evaluated the role of IFN-γ in MCMV latency. MCMV-infected IFN-γR−/− mice shed preformed infectious MCMV in spleen, peritoneal exudate cells, and salivary gland for up to 6 mo after infection, whereas the majority of congenic control animals cleared chronic productive infection. However, the IFN-γR was not required for establishment of latency. Using an in vitro explant reactivation model, we showed that IFN-γ reversibly inhibited MCMV reactivation from latency. This was at least partly explained by IFN-γ– mediated blockade of growth of low levels of MCMV in tissue explants. These in vivo and in vitro data suggest that IFN-γ regulation of reactivation from latency contributes to control of chronic vascular disease caused by MCMV. These studies are the first to demonstrate that a component of the immune system (IFN-γ) is necessary to regulate MCMV-associated elastic arteritis and latency in vivo and reactivation of a herpesvirus from latency in vitro. This provides a new model for analysis of the interrelationships among herpesvirus latency, the immune system, and chronic disease of the great vessels.
Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Secreting Merozoite Surface Protein 1 (MSP1) Induces Protection against Rodent Malaria Parasite Infection Depending on MSP1-stimulated Interferon γ and Parasite-specific Antibodies
Authors: Matsumoto Sohkichi , Yukitake Hideharu , Kanbara Hiroji , Yamada Takeshi
Published: July 2002

The merozoite surface protein 1 (MSP1) has emerged as a leading malaria vaccine candidate at the erythrocytic stage. Recombinant bacillus Calmette-Guérin (rBCG), which expressed a COOH-terminal 15-kD fragment of MSP1 of Plasmodium yoelii (MSP1-15) as a fusion protein with a secretory protein of Mycobacterium kansasii, was constructed. Immunization of mice with this rBCG induced a higher degree of protection against blood-stage parasite infection than with recombinant MSP1-15 in the RIBI adjuvant (RIBI ImmunoChem Research, Inc., Hamilton, MT) or incomplete Freund's adjuvant systems. We studied the mechanism of protection induced by MSP1-15, and found that interferon (IFN)-γ had a major role in protection in all adjuvant systems we examined. Mice that produced low amounts of MSP1-15 stimulated IFN-γ and could not control parasite infection. The antibody against MSP1-15 did not play a major role in protection in this system. After parasite infection, immunoglobulin G2a antibodies, which had been produced by IFN-γ stimulation, were induced and subsequently played an important role in eradicating parasites. Thus, both cellular and humoral immune responses were essential for protection from malaria disease. These data revealed that BCG is a powerful adjuvant to induce such a protective immune response against malaria parasites.
Predominant Role for Directly Transfected Dendritic Cells in Antigen Presentation to CD8+ T Cells after Gene Gun Immunization
Authors: Porgador Angel , Irvine Kari R. , Iwasaki Akiko , Barber Brian H. , Restifo Nicholas P. , Germain Ronald N.
Published: July 2002

Cutaneous gene (DNA) bombardment results in substantial expression of the encoded antigen in the epidermal layer as well as detectable expression in dendritic cells (DC) in draining lymph nodes (LNs). Under these conditions, two possible modes of DC antigen presentation to naive CD8+ T cells might exist: (a) presentation directly by gene-transfected DC trafficking to local lymph nodes, and (b) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells. The relative contributions of these distinct modes of antigen presentation to priming for cytotoxic T cell (CTL) responses have not been clearly established. Here we show that LN cells directly expressing the DNA-encoded antigen are rare; 24 h after five abdominal skin bombardments, the number of these cells does not exceed 50–100 cells in an individual draining LN. However, over this same time period, the total number of CD11c+ DC increases more than twofold, by an average of 20,000–30,000 DC per major draining node. This augmentation is due to gold bombardment and is independent of the presence of plasmid DNA. Most antigen-bearing cells in the LNs draining the site of DNA delivery appear to be DC and can be depleted by antibodies to an intact surface protein encoded by cotransfected DNA. This finding of predominant antigen presentation by directly transfected cells is also consistent with data from studies on cotransfection with antigen and CD86-encoding DNA, showing that priming of anti-mutant influenza nucleoprotein CTLs with a single immunization is dependent upon coexpression of the DNAs encoding nucleoprotein and B7.2 in the same cells. These observations provide insight into the relative roles of direct gene expression and cross-presentation in CD8+ T cell priming using gene gun immunization, and indicate that augmentation of direct DC gene expression may enhance such priming.
Protective Heterologous Antiviral Immunity and Enhanced Immunopathogenesis Mediated by Memory T Cell Populations
Authors: Selin Liisa K. , Varga Steven M. , Wong Iris C. , Welsh Raymond M.
Published: July 2002

A basic principle of immunology is that prior immunity results in complete protection against a homologous agent. In this study, we show that memory T cells specific to unrelated viruses may alter the host's primary immune response to a second virus. Studies with a panel of heterologous viruses, including lymphocytic choriomeningitis (LCMV), Pichinde (PV), vaccinia (VV), and murine cytomegalo (MCMV) viruses showed that prior immunity with one of these viruses in many cases enhanced clearance of a second unrelated virus early in infection. Such protective immunity was common, but it depended on the virus sequence and was not necessarily reciprocal. Cell transfer studies showed that both CD4 and CD8 T cell populations from LCMV-immune mice were required to transfer protective immunity to naive hosts challenged with PV or VV. In the case of LCMV-immune versus naive mice challenged with VV, there was an enhanced early recruitment of memory phenotype interferon (IFN) γ–secreting CD4+ and CD8+ cells into the peritoneal cavity and increased IFN-γ levels in this initial site of virus replication. Studies with IFN-γ receptor knockout mice confirmed a role for IFN-γ in mediating the protective effect by LCMV-immune T cell populations when mice were challenged with VV but not PV. In some virus sequences memory cell populations, although clearing the challenge virus more rapidly, elicited enhanced IFN-γ–dependent immunopathogenesis in the form of acute fatty necrosis. These results indicate that how a host responds to an infectious agent is a function of its history of previous infections and their influence on the memory T cell pool.
DNA Vaccination:  Transfection and Activation of Dendritic Cells as Key Events for Immunity
Authors: Akbari Omid , Panjwani Naveed , Garcia Sylvie , Tascon Ricardo , Lowrie Doug , Stockinger Brigitta
Published: July 2002

The mechanisms underlying initiation and maintenance of CD4 T cell responses after DNA vaccination were studied using a construct coding for nonsecreted fifth component of complement (C5) protein, thus restricting the availability of antigen. The only cell types to express C5 were keratinocytes at the site of DNA application and a small number of dendritic cells present in the draining lymph nodes. Antigen expression persisted for up to 12 wk in keratinocytes, but dendritic cells did not express C5 beyond 2 wk after vaccination. Cross-priming of dendritic cells by C5 expressed in keratinocytes did not occur unless keratinocyte death was induced by irradiation in vitro. CD4 T cells were activated in the draining lymph nodes only and subsequently migrated to the spleen, where memory T cells persisted for longer than 40 wk despite the absence of a source of persistent antigen. While DNA vaccination resulted in transfection of a small proportion of dendritic cells only, it led to general activation of all dendritic cells, thus providing optimal conditions for effective T cell activation and maintenance of memory.
The IKKβ Subunit of IκB Kinase (IKK) is Essential for Nuclear Factor κB Activation and Prevention of Apoptosis
Authors: Li Zhi-Wei , Chu Wenming , Hu Yinling , Delhase Mireille , Deerinck Tom , Ellisman Mark , Johnson Randall , Karin Michael
Published: July 2002

The IκB kinase (IKK) complex is composed of three subunits, IKKα, IKKβ, and IKKγ (NEMO). While IKKα and IKKβ are highly similar catalytic subunits, both capable of IκB phosphorylation in vitro, IKKγ is a regulatory subunit. Previous biochemical and genetic analyses have indicated that despite their similar structures and in vitro kinase activities, IKKα and IKKβ have distinct functions. Surprisingly, disruption of the Ikkα locus did not abolish activation of IKK by proinflammatory stimuli and resulted in only a small decrease in nuclear factor (NF)-κB activation. Now we describe the pathophysiological consequence of disruption of the Ikkβ locus. IKKβ-deficient mice die at mid-gestation from uncontrolled liver apoptosis, a phenotype that is remarkably similar to that of mice deficient in both the RelA (p65) and NF-κB1 (p50/p105) subunits of NF-κB. Accordingly, IKKβ-deficient cells are defective in activation of IKK and NF-κB in response to either tumor necrosis factor α or interleukin 1. Thus IKKβ, but not IKKα, plays the major role in IKK activation and induction of NF-κB activity. In the absence of IKKβ, IKKα is unresponsive to IKK activators.
Natural Killer Cells Determine Development of Allergen-induced Eosinophilic Airway Inflammation in Mice
Authors: Korsgren Magnus , Persson Carl G.A. , Sundler Frank , Bjerke Torbjörn , Hansson Tony , Chambers Benedict J. , Hong Seokmann , Van Kaer Luc , Ljunggren Hans-Gustaf , Korsgren Olle
Published: July 2002

The earliest contact between antigen and the innate immune system is thought to direct the subsequent antigen-specific T cell response. We hypothesized that cells of the innate immune system, such as natural killer (NK) cells, NK1.1+ T cells (NKT cells), and γ/δ T cells, may regulate the development of allergic airway disease. We demonstrate here that depletion of NK1.1+ cells (NK cells and NKT cells) before immunization inhibits pulmonary eosinophil and CD3+ T cell infiltration as well as increased levels of interleukin (IL)-4, IL-5, and IL-12 in bronchoalveolar lavage fluid in a murine model of allergic asthma. Moreover, systemic allergen-specific immunoglobulin (Ig)E and IgG2a levels and the number of IL-4 and interferon γ–producing splenic cells were diminished in mice depleted of NK1.1+ cells before the priming regime. Depletion of NK1.1+ cells during the challenge period only did not influence pulmonary eosinophilic inflammation. CD1d1 mutant mice, deficient in NKT cells but with normal NK cells, developed lung tissue eosinophilia and allergen-specific IgE levels not different from those observed in wild-type mice. Mice deficient in γ/δ T cells showed a mild attenuation of lung tissue eosinophilia in this model. Taken together, these findings suggest a critical role of NK cells, but not of NKT cells, for the development of allergen-induced airway inflammation, and that this effect of NK cells is exerted during the immunization. If translatable to humans, these data suggest that NK cells may be critically important for deciding whether allergic eosinophilic airway disease will develop. These observations are also compatible with a pathogenic role for the increased NK cell activity observed in human asthma.
Tetrameric Complexes of Human Histocompatibility Leukocyte Antigen (HLA)-G Bind to Peripheral Blood Myelomonocytic Cells
Authors: Allan David S.J. , Colonna Marco , Lanier Lewis L. , Churakova Tatyana D. , Abrams John S. , Ellis Shirley A. , McMichael Andrew J. , Braud Veronique M.
Published: July 2002

The nonclassical MHC class I molecule human histocompatibility leukocyte antigen (HLA)-G is selectively expressed on fetal trophoblast tissue at the maternal–fetal interface in pregnancy. It has long been suggested that HLA-G may inhibit maternal natural killer (NK) cells through interaction with particular NK cell receptors (KIRs). To investigate interactions of HLA-G, we constructed phycoerythrin-labeled tetrameric complexes of HLA-G refolded with a self-peptide. These HLA-G tetramers failed to bind to NK cells and cells transfected with CD94/NKG2 and killer immunoglobulin-like NK receptors. In contrast, HLA-G tetramers did bind to peripheral blood monocytes, staining a CD16+CD14mid subset with greater intensity. On transfectants, HLA-G tetramers bound to inhibitory immunoglobulin-like transcript (ILT)2 and ILT4 receptors. However, staining in the presence of antibodies reactive with ILT receptors revealed that the interaction of HLA-G tetramers with blood monocytes was largely due to binding to ILT4. These results suggest that the primary role of HLA-G may be the modulation of myelomonocytic cell behavior in pregnancy.
A Proinflammatory Cytokine Inhibits P53 Tumor Suppressor Activity
Authors: Hudson James D. , Shoaibi Mahmood A. , Maestro Roberta , Carnero Amancio , Hannon Gregory J. , Beach David H.
Published: July 2002

p53 has a key role in the negative regulation of cell proliferation, in the maintenance of genomic stability, and in the suppression of transformation and tumorigenesis. To identify novel regulators of p53, we undertook two functional screens to isolate genes which bypassed either p53-mediated growth arrest or apoptosis. In both screens, we isolated cDNAs encoding macrophage migration inhibitory factor (MIF), a cytokine that was shown previously to exert both local and systemic proinflammatory activities. Treatment with MIF overcame p53 activity in three different biological assays, and suppressed its activity as a transcriptional activator. The observation that a proinflammatory cytokine, MIF, is capable of functionally inactivating a tumor suppressor, p53, may provide a link between inflammation and tumorigenesis.
Viral Dynamics of Acute HIV-1 Infection
Authors: Little Susan J. , McLean Angela R. , Spina Celsa A. , Richman Douglas D. , Havlir Diane V.
Published: July 2002

Viral dynamics were intensively investigated in eight patients with acute HIV infection to define the earliest rates of change in plasma HIV RNA before and after the start of antiretroviral therapy. We report the first estimates of the basic reproductive number (R0), the number of cells infected by the progeny of an infected cell during its lifetime when target cells are not depleted. The mean initial viral doubling time was 10 h, and the peak of viremia occurred 21 d after reported HIV exposure. The spontaneous rate of decline (α) was highly variable among individuals. The phase 1 viral decay rate (δI = 0.3/day) in subjects initiating potent antiretroviral therapy during acute HIV infection was similar to estimates from treated subjects with chronic HIV infection. The doubling time in two subjects who discontinued antiretroviral therapy was almost five times slower than during acute infection. The mean basic reproductive number (R0) of 19.3 during the logarithmic growth phase of primary HIV infection suggested that a vaccine or postexposure prophylaxis of at least 95% efficacy would be needed to extinguish productive viral infection in the absence of drug resistance or viral latency. These measurements provide a basis for comparison of vaccine and other strategies and support the validity of the simian immunodeficiency virus macaque model of acute HIV infection.
Markedly Different Pathogenicity of Four Immunoglobulin G Isotype-Switch Variants of an Antierythrocyte Autoantibody Is Based on Their Capacity to Interact in Vivo with the Low-Affinity Fcγ Receptor III
Authors: Fossati-Jimack Liliane , Ioan-Facsinay Andreea , Reininger Luc , Chicheportiche Yves , Watanabe Norihiko , Saito Takashi , Hofhuis Frans M. A. , Gessner J. Engelbert , Schiller Carsten , Schmidt Reinhold E. , Honjo Tasuku , Verbeek J. Sjef , Izui Shozo
Published: July 2002

Using three different Fcγ receptor (FcγR)-deficient mouse strains, we examined the induction of autoimmune hemolytic anemia by each of the four immunoglobulin (Ig)G isotype-switch variants of a 4C8 IgM antierythrocyte autoantibody and its relation to the contributions of the two FcγR, FcγRI, and FcγRIII, operative in the phagocytosis of opsonized particles. We found that the four IgG isotypes of this antibody displayed striking differences in pathogenicity, which were related to their respective capacity to interact in vivo with the two phagocytic FcγRs, defined as follows: IgG2a > IgG2b > IgG3/IgG1 for FcγRI, and IgG2a > IgG1 > IgG2b > IgG3 for FcγRIII. Accordingly, the IgG2a autoantibody exhibited the highest pathogenicity, ∼20–100-fold more potent than its IgG1 and IgG2b variants, respectively, while the IgG3 variant, which displays little interaction with these FcγRs, was not pathogenic at all. An unexpected critical role of the low-affinity FcγRIII was revealed by the use of two different IgG2a anti–red blood cell autoantibodies, which displayed a striking preferential utilization of FcγRIII, compared with the high-affinity FcγRI. This demonstration of the respective roles in vivo of four different IgG isotypes, and of two phagocytic FcγRs, in autoimmune hemolytic anemia highlights the major importance of the regulation of IgG isotype responses in autoantibody-mediated pathology and humoral immunity.
Influenza Virus Lung Infection Protects from Respiratory Syncytial Virus–Induced Immunopathology
Authors: Walzl Gerhard , Tafuro Sabrina , Moss Paul , Openshaw Peter J.M. , Hussell Tracy
Published: July 2002

The effect of infection history is ignored in most animal models of infectious disease. The attachment protein of respiratory syncytial virus (RSV) induces T helper cell type 2–driven pulmonary eosinophilia in mice similar to that seen in the failed infant vaccinations in the 1960s. We show that previous influenza virus infection of mice: (a) protects against weight loss, illness, and lung eosinophilia; (b) attenuates recruitment of inflammatory cells; and (c) reduces cytokine secretion caused by RSV attachment protein without affecting RSV clearance. This protective effect can be transferred via influenza-immune splenocytes to naive mice and is long lived. Previous immunity to lung infection clearly plays an important and underestimated role in subsequent vaccination and infection. The data have important implications for the timing of vaccinations in certain patient groups, and may contribute to variability in disease susceptibility observed in humans.
The Relative Roles of Passive Surface Forces and Active Ion Transport in the Modulation of Airway Surface Liquid Volume and Composition
Authors: Tarran Robert , Grubb Barbara R. , Gatzy John T. , Davis C. William , Boucher Richard C.
Published: July 2002

Two hypotheses have been proposed recently that offer different views on the role of airway surface liquid (ASL) in lung defense. The “compositional” hypothesis predicts that ASL [NaCl] is kept low (<50 mM) by passive forces to permit antimicrobial factors to act as a chemical defense. The “volume” hypothesis predicts that ASL volume (height) is regulated isotonically by active ion transport to maintain efficient mechanical mucus clearance as the primary form of lung defense. To compare these hypotheses, we searched for roles for: (1) passive forces (surface tension, ciliary tip capillarity, Donnan, and nonionic osmolytes) in the regulation of ASL composition; and (2) active ion transport in ASL volume regulation. In primary human tracheobronchial cultures, we found no evidence that a low [NaCl] ASL could be produced by passive forces, or that nonionic osmolytes contributed substantially to ASL osmolality. Instead, we found that active ion transport regulated ASL volume (height), and that feedback existed between the ASL and airway epithelia to govern the rate of ion transport and volume absorption. The mucus layer acted as a “reservoir” to buffer periciliary liquid layer height (7 μm) at a level optimal for mucus transport by donating or accepting liquid to or from the periciliary liquid layer, respectively. These data favor the active ion transport/volume model hypothesis to describe ASL physiology.
Local differentiability of distance functions
Authors: Poliquin R. , Rockafellar R. , Thibault L.
Published: July 2002

Recently Clarke, Stern and Wolenski characterized, in a Hilbert space, the closed subsets

C
C

for which the distance function

d

C

d_{C}

is continuously differentiable everywhere on an open “tube” of uniform thickness around

C
C

. Here a corresponding local theory is developed for the property of

d

C

d_{C}

being continuously differentiable outside of

C
C

on some neighborhood of a point

x
∈
C

x\in C

. This is shown to be equivalent to the prox-regularity of

C
C

at

x
x

, which is a condition on normal vectors that is commonly fulfilled in variational analysis and has the advantage of being verifiable by calculation. Additional characterizations are provided in terms of

d

C

2

d_{C}^{2}

being locally of class

C

1
+

C^{1+}

or such that

d

C

2

+
σ

|

⋅

|

2

d_{C}^{2}+\sigma |\cdot |^{2}

is convex around

x
x

for some

σ
>
0

\sigma >0

. Prox-regularity of

C
C

at

x
x

corresponds further to the normal cone mapping

N

C

N_{C}

having a hypomonotone truncation around

x
x

, and leads to a formula for

P

C

P_{C}

by way of

N

C

N_{C}

. The local theory also yields new insights on the global level of the Clarke-Stern-Wolenski results, and on a property of sets introduced by Shapiro, as well as on the concept of sets with positive reach considered by Federer in the finite dimensional setting.
Protein Kinase Cε Actin-binding Site Is Important for Neurite Outgrowth during Neuronal Differentiation
Authors: Zeidman Ruth , Trollér Ulrika , Raghunath Arathi , Påhlman Sven , Larsson Christer
Published: July 2002

We have previously shown that protein kinase Cε (PKCε) induces neurite outgrowth via its regulatory domain and independently of its kinase activity. This study aimed at identifying mechanisms regulating PKCε-mediated neurite induction. We show an increased association of PKCε to the cytoskeleton during neuronal differentiation. Furthermore, neurite induction by overexpression of full-length PKCε is suppressed if serum is removed from the cultures or if an actin-binding site is deleted from the protein. A peptide corresponding to the PKCε actin-binding site suppresses neurite outgrowth during neuronal differentiation and outgrowth elicited by PKCε overexpression. Neither serum removal, deletion of the actin-binding site, nor introduction of the peptide affects neurite induction by the isolated regulatory domain. Membrane targeting by myristoylation renders full-length PKCε independent of both serum and the actin-binding site, and PKCε colocalized with F-actin at the cortical cytoskeleton during neurite outgrowth. These results demonstrate that the actin-binding site is of importance for signals acting on PKCε in a pathway leading to neurite outgrowth. Localization of PKCε to the plasma membrane and/or the cortical cytoskeleton is conceivably important for its effect on neurite outgrowth.