bluuebunny/crossref_metadata_2025 huggingface dataset:
Restitution of superoxide generation in autosomal cytochrome-negative chronic granulomatous disease (A22(0) CGD)-derived B lymphocyte cell lines by transfection with p22phax cDNA.
Authors: Maly F E , Schuerer-Maly C C , Quilliam L , Cochrane C G , Newburger P E , Curnutte J T , Gifford M , Dinauer M C
Published: June 2004

The respiratory burst oxidase of phagocytes and B lymphocytes is a multicomponent enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2-production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or physiologic stimuli, such as phagocytosis in phagocytes or cross-linking of surface immunoglobulin in B lymphocytes. The activity of this enzyme is greatly diminished or absent in patients with chronic granulomatous disease (CGD), an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every CGD patient studied so far, an abnormality has been found in a gene encoding one of the four components of the respiratory burst oxidase: the membrane proteins p22phox or gp91phox which together form the cytochrome b558 protein, or the cytosolic proteins p47phox or p67phox. Autosomal recessive cytochrome-negative CGD (A22(0) CGD) is associated with mutations in the gene coding for p22phox. We report here that the capacity for O2- production and cytochrome b558 protein expression were restored to Epstein-Barr virus-transformed B lymphocytes from two A22(0) CGD patients by transfection with an expression plasmid containing a p22phox cDNA. No detectable O2- was generated by untransfected p22phox-deficient lymphocytes. The genetic reconstitution of the respiratory burst in A22(0) CGD B lymphocytes by transfer of the wild-type p22phox cDNA represents a further step towards somatic gene therapy for this subgroup of A22(0) CGD. This system will also be useful for expression of genetically engineered mutant p22phox proteins in intact cells, facilitating the structure-function analysis of cytochrome b558.
Follicular dendritic cells help resting B cells to become effective antigen-presenting cells: induction of B7/BB1 and upregulation of major histocompatibility complex class II molecules.
Authors: Kosco-Vilbois M H , Gray D , Scheidegger D , Julius M
Published: June 2004

This study was designed to investigate whether follicular dendritic cells (FDC) can activate B cells to a state in which they can function as effective antigen-presenting cells (APC). High buoyant density (i.e., resting) B cells specific for 2,4-dinitro-fluorobenzene (DNP) were incubated with DNP-ovalbumin (OVA) bearing FDC, after which their capacity to process and present to an OVA-specific T cell clone was assessed. The efficacies of alternative sources of antigen and activation signals in the induction of B cell APC function were compared with those provided by FDC. Only FDC and Sepharose beads coated with anti-immunoglobulin (Ig)kappa monoclonal antibody provided the necessary stimulus. FDC carrying inappropriate antigens also induced B cell APC function in the presence of exogenous DNP-OVA. However, in circumstances where soluble DNP-OVA was limiting, FDC bearing complexes containing DNP, which could crosslink B cell Ig receptors, induced the most potent APC function. Analysis by flow cytometry revealed that within 24 h of coculture with FDC, a significant percentage of B cells increased in size and expressed higher levels of major histocompatibility complex class II. By 48 h, an upregulation of the costimulatory molecule, B7/BB1, occurred, but only when exposed to the FDC bearing DNP. Taken together, the results demonstrate that FDC have the capacity to activate resting B cells to a state in which they can function as APC for T cells. The stimuli that FDC provide may include: (a) an antigen-dependent signal that influences the upregulation of B7/BB1; and (b) possibly a signal independent of crosslinking mIg that results in Ig internalization. The relevance of these findings to the formation of germinal centers and maintenance of the humoral response is discussed.
Major histocompatibility complex susceptibility genes for dermatitis herpetiformis compared with those for gluten-sensitive enteropathy.
Authors: Ahmed A R , Yunis J J , Marcus-Bagley D , Yunis E J , Salazar M , Katz A J , Awdeh Z , Alper C A
Published: June 2004

Dermatitis herpetiformis (DH) shares some clinical features and major histocompatibility complex (MHC) markers with gluten-sensitive enteropathy (GSE). We compared MHC haplotypes in 27 patients with DH, 35 patients with GSE, and normal controls. As in GSE, the frequencies of two extended haplotypes, [HLA-B8, SC01, DR3] and [HLA-B44, FC31, DR7], were increased in patients with DH. Distributions of fragments of extended haplotypes, consisting of some but not all of the elements of complete extended haplotypes, were analyzed to attempt to localize a susceptibility gene. Besides complete extended susceptibility haplotypes, (DR3, DQ2) and (DR7, DQ2) fragments were most common in GSE. In contrast, DH showed only a few such fragments but many instances of the fragment (SC01). The differences in distribution of these fragments in the two diseases were highly significant (P < 0.002). HLA-DQ2 and DR3 had the highest odds ratios for GSE, but the highest odds ratio for DH was for the complotype SC01. These findings suggest that the MHC susceptibility gene for DH is between class II and complotype regions, closest to the complotype, whereas that for GSE is in the class II region.
High efficiency retroviral mediated gene transduction into single isolated immature and replatable CD34(3+) hematopoietic stem/progenitor cells from human umbilical cord blood.
Authors: Lu L , Xiao M , Clapp D W , Li Z H , Broxmeyer H E
Published: June 2004

Umbilical cord blood is rich in hematopoietic stem and progenitor cells and has recently been used successfully in the clinic as an alternative source of engrafting and marrow repopulating cells. With the likelihood that cord blood stem/progenitor cells will be used for gene therapy to correct genetic disorders, we evaluated if a TK-neo gene could be directly transduced in a stable manner into single isolated subsets of purified immature hematopoietic cells that demonstrate self-renewed ability as estimated by colony replating capacity. Sorted CD34(3+) cells from cord blood were prestimulated with erythropoietin (Epo), steel factor (SLF), interleukin (IL)-3, and granulocyte-macrophage colony stimulating factor (GM-CSF) and transduced with the gene in two ways. CD34(3+) cells were incubated with retroviral-containing supernatant from TK-neo vector-producing cells, washed, and plated directly or resorted as CD34(3+) cells into single wells containing a single cell or 10 cells. Alternatively, CD34(3+) cells were sorted as a single cell/well and then incubated with viral supernatant. These cells were cultured with Epo, SLF, IL-3, and GM-CSF +/- G418. The TK-neo gene was introduced at very high efficiency into low numbers of or isolated single purified CD34(3+) immature hematopoietic cells without stromal cells as a source of virus or accessory cells. Proviral integration was detected in primary G418-resistant(R) colonies derived from single immature hematopoietic cells, and in cells from replated colonies derived from G418R-colony forming unit-granulocyte erythroid macrophage megakaryocyte (CFU-GEMM) and -high proliferative potential colony forming cells (HPP-CFC). This demonstrates stable expression of the transduced gene into single purified stem/progenitor cells with replating capacity, results that should be applicable for future clinical studies that may utilize selected subsets of stem/progenitor cells for gene therapy.
T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).
Authors: da Silva A J , Janssen O , Rudd C E
Published: June 2004

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.
Short consensus repeat-3 domain of recombinant decay-accelerating factor is recognized by Escherichia coli recombinant Dr adhesin in a model of a cell-cell interaction.
Authors: Nowicki B , Hart A , Coyne K E , Lublin D M , Nowicki S
Published: June 2004

A bacterial pathogen that is important in both urinary tract and intestinal infections is Escherichia coli which expresses Dr or related adhesins. In this report, we present a model for testing cell-cell interaction, using both molecularly characterized laboratory cells that express recombinant molecules of human decay-accelerating factor (DAF), and recombinant bacterial Dr colonization factors. Dr adhesin ligand was identified as DAF (CD55), a membrane protein that protects autologous tissues from damage due to the complement system. Structure-function studies mapped the adhesin-binding site on the DAF molecule. A single-point substitution in the third short consensus repeat domain, Ser165 to Leu, corresponding to the Dra to Drb allelic polymorphism, caused complete abolition of adhesin binding to DAF.
Allergen immunotherapy decreases interleukin 4 production in CD4+ T cells from allergic individuals.
Authors: Secrist H , Chelen C J , Wen Y , Marshall J D , Umetsu D T
Published: June 2004

Allergen specific CD4+ T cell clones generated from allergic individuals have been shown to produce increased levels of the cytokine interleukin 4 (IL-4), compared to allergen specific clones generated from nonallergic individuals. This difference between CD4+ T cells from allergic and nonallergic individuals with regard to cytokine production in response to allergen is thought to be responsible for the development of allergic disease with increased IgE synthesis in atopic individuals. We examined the production of IL-4 in subjects with allergic rhinitis and in allergic individuals treated with allergen immunotherapy, a treatment which involves the subcutaneous administration of increasing doses of allergen and which is highly effective and beneficial for individuals with severe allergic rhinitis. We demonstrated that the quantity of IL-4 produced by allergen specific memory CD4+ T cells from allergic individuals could be considerably reduced by in vivo treatment with allergen (allergen immunotherapy). Immunotherapy reduced IL-4 production by allergen specific CD4+ T cells to levels observed with T cells from nonallergic subjects, or to levels induced with nonallergic antigens such as tetanus toxoid. In most cases the levels of IL-4 produced were inversely related to the length of time on immunotherapy. These observations indicate that immunotherapy accomplishes its clinical effects by reducing IL-4 synthesis in allergen specific CD4+ T cells. In addition, these observations indicate that the cytokine profiles of memory CD4+ T cells can indeed be altered by in vivo therapies. Thus, the cytokine profiles of memory CD4+ T cells are mutable, and are not fixed as had been suggested by studies of murine CD4+ memory T cells. Finally, treatment of allergic diseases with allergen immunotherapy may be a model for other diseases which may require therapies that alter inappropriate cytokine profiles of memory CD4+ T cells.
The alpha 3 domain of the Qa-2 molecule is defective for CD8 binding and cytotoxic T lymphocyte activation.
Authors: Teitell M , Holcombe H , Cheroutre H , Aldrich C J , Stroynowski I , Forman J , Kronenberg M
Published: June 2004

Qa-2 is a nonclassical class I molecule encoded by the Q7 gene within the mouse major histocompatibility complex (MHC). Results from previous experiments on Qa-2, and on a chimeric Ld molecule (LQ3) in which the alpha 3 domain is encoded by Q7b, suggested that the alpha 3 domain of Qa-2 does not carry out the functions typical of the alpha 3 domains in other classical and nonclassical class I antigens. Class I molecules that contain the Qa-2 alpha 3 domain are poorly recognized by primary cytotoxic T lymphocytes (CTLs), and do not function normally in either positive or negative selection in vivo. By employing a cell-cell adhesion assay we demonstrate directly that the Qa-2 alpha 3 domain in the context of the LQ3 hybrid molecule cannot bind to human CD8, although other mouse class I alpha 3 domains bind efficiently. In addition, CD8-dependent CTL-mediated lysis of target cells, in a system which requires mouse CD8-class I alpha 3 domain interactions, is deficient in cells that express the Qa-2 alpha 3 domain. When combined with our earlier work on LQ3 transgenic mice, these results provide additional molecular support for the hypothesis that interaction with CD8 is required for both positive and negative selection of class I restricted T cells in the thymus. As the Qa-2 alpha 3 domain sequence does not differ from the previously defined minimal CD8 binding sequence of other class I molecules, these results also suggest that additional amino acids in the alpha 3 domain must be critical for CD8 binding and CTL activation.
Expression and catalytic activity of the tyrosine phosphatase PTP1C is severely impaired in motheaten and viable motheaten mice.
Authors: Kozlowski M , Mlinaric-Rascan I , Feng G S , Shen R , Pawson T , Siminovitch K A
Published: June 2004

Mutations in the gene encoding the phosphotyrosine phosphatase PTP1C, a cytoplasmic protein containing a COOH-terminal catalytic and two NH2-terminal Src homology 2 (SH2) domains, have been identified in motheaten (me) and viable motheaten (mev) mice and are associated with severe hemopoietic dysregulation. The me mutation is predicted to result in termination of the PTP1C polypeptide within the first SH2 domain, whereas the mev mutation creates an insertion or deletion in the phosphatase domain. No PTP1C RNA or protein could be detected in the hemopoietic tissues of me mice, nor could PTP1C phosphotyrosine phosphatase activity be isolated from cells homozygous for the me mutation. In contrast, mice homozygous for the less severe mev mutation expressed levels of full-length PTP1C protein comparable to those detected in wild type mice and the SH2 domains of mev PTP1C bound normally to phosphotyrosine-containing ligands in vitro. Nevertheless, the mev mutation induced a marked reduction in PTP1C activity. These observations provide strong evidence that the motheaten phenotypic results from loss-of-function mutations in the PTP1C gene and imply a critical role for PTP1C in the regulation of hemopoietic differentiation and immune function.
Advanced glycosylation endproduct-specific receptors on human and rat T-lymphocytes mediate synthesis of interferon gamma: role in tissue remodeling.
Authors: Imani F , Horii Y , Suthanthiran M , Skolnik E Y , Makita Z , Sharma V , Sehajpal P , Vlassara H
Published: June 2004

During normal aging and in chronic diabetes the excessive accumulation of reactive glucose-protein or glucose-lipid adducts known as advanced glycosylation endproducts (AGEs) has been shown to induce tissue dysfunction, in part through interaction with AGE-specific receptors on monocyte/macrophages and other cells. Recognizing that circulating lymphocytes trafficking through tissues interact with tissue AGEs, we searched for the expression of AGE-binding sites on peripheral blood T lymphocytes. Resting rat and human T cells bound 125I-AGE-albumin with an affinity of 7.8 x 10(7) M-1, whereas, after stimulation with phytohemagglutinin (PHA) for 48 h, binding affinity increased to 5.8 x 10(8) M-1. Flow cytometric analysis of resting rat T cells using polyclonal antibodies raised against rat liver AGE-binding proteins (p60 and p90) revealed the constitutive expression of both immunoreactivities. The number of resting CD4+ and CD8+ T cells positive for anti-p60 antibody binding (34.2 and 58.5%, respectively) increased to 92 and 90% of cells after 48-h stimulation with PHA. Exposure of PHA-activated T lymphocytes to AGE-albumin enhanced expression of interferon gamma (IFN-gamma) mRNA 10-fold and induced greater elaboration of the mature protein than did exposure to unmodified protein or PHA treatment alone. These data indicate that T cells contain an inducible system of surface receptors for AGE-modified proteins, and that receptor occupancy is linked to lymphokine production. This T cell AGE-receptor system might serve to target lymphocytes to AGE-rich tissues and involve them in the regulation of tissue homeostasis either by assisting in macrophage-dependent clearance of AGE-proteins, or by exerting direct antiproliferative action on mesenchymal cells. Under conditions of excessive AGE-protein and AGE lipid accumulation (e.g., aging and diabetes), enhanced production of AGE-induced IFN-gamma may accelerate immune responses that contribute to tissue injury.
Murine B7-2, an alternative CTLA4 counter-receptor that costimulates T cell proliferation and interleukin 2 production.
Authors: Freeman G J , Borriello F , Hodes R J , Reiser H , Gribben J G , Ng J W , Kim J , Goldberg J M , Hathcock K , Laszlo G
Published: June 2004

The B7-1 molecule, expressed on antigen presenting cells (APC), provides a crucial costimulatory signal for T cell activation. Recent studies demonstrate the existence of alternative, non-B7-1 CTLA4 counter-receptors in mice and humans. Here, we describe the molecular cloning and demonstrate costimulatory function of the murine B7-2 (mB7-2) gene. Murine B7-2 cDNA encodes a member of the Ig supergene family that binds CTLA4-Ig and stains with the GL1 but not anti-mB7-1 mAb. Murine B7-2 costimulates the proliferation and interleukin 2 production of CD4+ T cells and this costimulation can be inhibited by either CTLA4-Ig or GL1 mAb. Identification of the B7-2 molecule will permit further manipulation of the B7:CD28/CTLA4 costimulatory pathway which has been shown to be involved in the prevention of tolerance, induction of tumor immunity, and most recently, in the pathogenesis of autoimmunity.
Interleukin 10 (IL-10) inhibits the release of proinflammatory cytokines from human polymorphonuclear leukocytes. Evidence for an autocrine role of tumor necrosis factor and IL-1 beta in mediating the production of IL-8 triggered by lipopolysaccharide.
Authors: Cassatella M A , Meda L , Bonora S , Ceska M , Constantin G
Published: June 2004

In this study we have examined the effects of interleukin 10 (IL-10) on polymorphonuclear leukocytes (PMN), and found that it is a potent inhibitor of tumor necrosis factor (TNF), IL-1 beta, and IL-8 secretion triggered by lipopolysaccharide (LPS). Cytokine production by phagocytosing PMN was also inhibited by IL-10, but to a lesser extent than the LPS-induced production. As shown by Northern blot analysis, IL-10 diminished the levels of TNF, IL-1 beta, and IL-8 mRNAs late after the onset of stimulation of PMN with LPS. In addition, we provide evidence that the kinetics of LPS-induced IL-8 production by PMN is composed of two distinct phases. Specifically, our experiments demonstrated that in the first phase, the production of IL-8 is a process directly induced by LPS that lasts for some hours. After this early wave, a second phase begins that is sustained and leads to an elevated production of IL-8 that appears to be due to the endogenous release of TNF and IL-1 beta. This second wave can in fact be blocked by anti-TNF and anti-IL-1 beta neutralizing antibodies, and by IL-10 as the consequence of its downregulatory effects on TNF and IL-1 beta release. Taken together, these findings identify novel biological actions of IL-10 as a suppressor of the inflammatory response.
The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of Stiff-Man syndrome with breast cancer.
Authors: De Camilli P , Thomas A , Cofiell R , Folli F , Lichte B , Piccolo G , Meinck H M , Austoni M , Fassetta G , Bottazzo G
Published: June 2004

Stiff-Man syndrome (SMS) is a rare disease of the central nervous system (CNS) characterized by progressive rigidity of the body musculature with superimposed painful spasms. An autoimmune origin of the disease has been proposed. In a caseload of more than 100 SMS patients, 60% were found positive for autoantibodies directed against the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Few patients, all women affected by breast cancer, were negative for GAD autoantibodies but positive for autoantibodies directed against a 128-kD synaptic protein. We report here that this antigen is amphiphysin. GAD and amphiphysin are nonintrinsic membrane proteins that are concentrated in nerve terminals, where a pool of both proteins is associated with the cytoplasmic surface of synaptic vesicles. GAD and amphiphysin are the only two known targets of CNS autoimmunity with this distribution. This finding suggests a possible link between autoimmunity directed against cytoplasmic proteins associated with synaptic vesicles and SMS.
Double-negative T cells from MRL-lpr/lpr mice mediate cytolytic activity when triggered through adhesion molecules and constitutively express perforin gene.
Authors: Hammond D M , Nagarkatti P S , Goté L R , Seth A , Hassuneh M R , Nagarkatti M
Published: June 2004

The lpr gene induces in mice, accumulation of large numbers of CD4-CD8- (double negative [DN]) T lymphocytes which bear adhesion molecules not characteristic of normal resting T cells. These cells fail to acquire interleukin 2 (IL-2) receptors, produce IL-2, and proliferate when activated with mitogens or monoclonal antibodies (mAbs) against the T cell receptor (TCR). Because of these poor functions in vitro, the nature and significance of DN T cells in the autoimmune disease process is not clear. In the current study, we describe a surprising finding that mAbs against CD3-TCR-alpha/beta complex can strongly trigger the lytic activity of the DN T cells to induce redirected lysis of Fc receptor-positive targets. Similar redirected lysis was also inducible using mAbs against CD44 and gp90MEL-14, molecules involved in the binding of lymphocytes to endothelial cells. The spontaneous cytotoxic potential of the DN T cells was further corroborated by demonstrating that the lpr DN T cells constitutively transcribed perforin gene but failed to express granzyme A. The current study suggests that DN T cells are capable of mediating lysis of autologous cells bearing the specific ligands for adhesion molecules involved in the signaling of cytotoxicity. These findings provide a novel insight into the functional significance of DN T cells in lpr mice and their potential role in the pathogenesis of autoimmune disease.
Fas transduces activation signals in normal human T lymphocytes.
Authors: Alderson M R , Armitage R J , Maraskovsky E , Tough T W , Roux E , Schooley K , Ramsdell F , Lynch D H
Published: June 2004

The Fas gene encodes a cell surface molecule that is a member of the the nerve growth factor/tumor necrosis factor receptor family of proteins and can mediate programmed cell death (apoptosis) in certain transformed cell lines. To characterize further the biological function of Fas, particularly with regard to its function in normal cells, a panel of monoclonal antibodies (mAbs) was generated against the extracellular portion of human Fas. Some of these mAbs induced apoptosis in transformed cell lines expressing Fas, but only when immobilized on the culture vessel. One of the new Fas mAbs (M38) was used for studies on normal lymphoid cells and found to stimulate the proliferation of purified human T cells and thymocytes when immobilized on culture wells along with CD3 antibody. T cell proliferation induced by Fas mAb was largely interleukin 2 independent and was demonstrated to be due to a direct effect on the precursor T cell. Thus, the data demonstrate that in addition to a role in the induction of apoptosis in certain transformed cell lines, the Fas protein may also play an important role in the activation and proliferation of normal T cells.
An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection.
Authors: Flynn J L , Chan J , Triebold K J , Dalton D K , Stewart T A , Bloom B R
Published: June 2004

Tuberculosis, a major health problem in developing countries, has reemerged in recent years in many industrialized countries. The increased susceptibility of immunocompromised individuals to tuberculosis, and many experimental studies indicate that T cell-mediated immunity plays an important role in resistance. The lymphokine interferon gamma (IFN-gamma) is thought to be a principal mediator of macrophage activation and resistance to intracellular pathogens. Mice have been developed which fail to produce IFN-gamma (gko), because of a targeted disruption of the gene for IFN-gamma. Upon infection with Mycobacterium tuberculosis, although they develop granulomas, gko mice fail to produce reactive nitrogen intermediates and are unable to restrict the growth of the bacilli. In contrast to control mice, gko mice exhibit heightened tissue necrosis and succumb to a rapid and fatal course of tuberculosis that could be delayed, but not prevented, by treatment with exogenous recombinant IFN-gamma.
Induction and function of vascular adhesion protein-1 at sites of inflammation.
Authors: Salmi M , Kalimo K , Jalkanen S
Published: June 2004

Emigration of leukocytes from the blood into the tissues is critical in controlling lymphocyte patrolling in different lymphatic organs and in leukocyte accumulation at sites of inflammation. During the first stage of the extravasation process, leukocytes bind to the endothelial lining of vessels. At the molecular level, several adhesion molecules on leukocytes and endothelial cells function as receptor-ligand pairs in mediating this dynamic interaction. Recently, we have identified a novel human endothelial cell molecule, vascular adhesion protein 1 (VAP-1), that mediates lymphocyte binding (Salmi, M., and S. Jalkanen. 1992. Science [Wash. DC] 257:1407). VAP-1 was initially characterized by mAb 1B2 which inhibits lymphocyte adhesion to high endothelial venules (HEV) and to purified VAP-1 protein. Here we report the location and function of VAP-1 in normal and inflamed tissues in humans. VAP-1 is abundant in HEV of lymphatic organs belonging to the peripheral lymph node system, but considerably less is expressed in vessels of mucosa-associated lymphatic tissues. A subset of venules in most normal nonlymphatic tissues like skin, brain, kidney, liver, and heart is also VAP-1 positive. In addition to vessels, VAP-1 is distributed on a few other cell types, most notably in dendritic-like cells of germinal centers. At sites of inflammation, such as in inflammatory bowel diseases and chronic dermatoses, expression of VAP-1 is clearly increased. The induced VAP-1 is functional, since mAb 1B2 inhibits lymphocyte binding to inflamed lamina propria venules by approximately 60%. Thus VAP-1 is an endothelial adhesion molecule that under normal conditions is expressed mainly in HEV of lymphatic tissues. However, expression of functional VAP-1 in vivo is upregulated during an inflammatory reaction at other sites as well. Inducibility of VAP-1 suggests that it may play a significant role, not only in recirculation of lymphocytes, but also in controlling entry of leukocytes into sites of inflammation.
Effect of dietary supplementation with n-9 eicosatrienoic acid on leukotriene B4 synthesis in rats: a novel approach to inhibition of eicosanoid synthesis.
Authors: James M J , Gibson R A , Neumann M A , Cleland L G
Published: June 2004

Studies were undertaken to assess the biochemical effects of dietary supplementation with n-9 eicosatrienoic acid (ETrA), an arachidonic acid analogue that is normally present in cell membranes at very low levels but is raised in the presence of essential fatty acid deficiency (EFAD). The incorporation of dietary ETrA into rat neutrophils and its effect on A23187-stimulated 5-lipoxygenase metabolism in these cells was examined; in addition, the effect of ETrA was compared with that of another arachidonic acid analogue, eicosapentaenoic acid (EPA), which is known to accumulate in cell membranes and inhibit synthesis of leukotriene B4 (LTB4) a product of the 5-lipoxygenase metabolic pathway. Rats were fed a defined diet that was sufficient in essential fatty acids and that contained EPA or ETrA (0.014% of energy) or no added fatty acid, for 3 wk. In the cells from ETrA-fed rats, LTB4 synthesis was inhibited relative to control values, but synthesis of the other products of 5-lipoxygenase metabolism, 5-hydroxyeicosatetraenoic acid (5-HETE) and the all-trans isomers of LTB4, were not inhibited. This pattern indicates inhibition of LTA hydrolase in ETrA-fed rats. In EPA-fed rats, there was inhibition of LTB4 and the all-trans isomers of LTB4, but there was no inhibition of 5-HETE. This pattern indicates inhibition of LTA synthase in EPA-fed rats. The results establish that dietary ETrA effectively inhibits synthesis of the inflammatory mediator, LTB4, and suggest that ETrA may confer antiinflammatory benefits similar to those observed with EFAD or dietary fish oil (which contains EPA). Because ETrA is substantially less unsaturated than EPA, it can be expected to have greater chemical stability, which could be an important practical advantage when used as a dietary constituent or supplement.
The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages.
Authors: Miller M D , Warmerdam M T , Gaston I , Greene W C , Feinberg M B
Published: June 2004

Considerable controversy and uncertainty have surrounded the biological function of the Human Immunodeficiency Virus (HIV)-1 nef gene product. Initial studies suggested that this early, nonstructural viral protein functioned as a negative regulatory factor; thus, it was proposed to play a role in establishing or maintaining viral latency. In contrast, studies in Simian Immunodeficiency Virus (SIV)mac-infected rhesus monkeys have suggested that Nef is not a negative factor but rather plays a central role in promoting high-level viral replication and is required for viral pathogenesis in vivo. We sought to define a tissue culture system that would approximate the in vivo setting for virus infection in order to assess the role of HIV-1 Nef in viral replication. We show that infection of mitogen-activated peripheral blood mononuclear cells (PBMC) with Nef+ HIV results in enhanced replication as evidenced by earlier gag p24 expression when compared with infections performed with nef mutant viruses. Moreover, when unstimulated freshly isolated PBMC are infected with Nef+ and Nef- viruses and then subsequently activated with mitogen, the Nef-induced difference in viral replication kinetics is even more pronounced, with the Nef- viruses requiring much more time in culture for appreciable growth. A positive effect of Nef on viral replication was also observed in primary macrophages infected with a recombinant of YU-2, a patient-derived molecular clone with macrophage tropism. These positive effects of Nef on viral replication are dependent on the initial multiplicity of infection (MOI), in that infections of unstimulated PBMC at low MOI are most dependent upon intact nef for subsequent viral growth. We now provide evidence that the Nef+ HIV is more infectious than Nef- HIV from both a tissue culture infectious dose analysis, and a single-cell HIV infection assay. In the latter case, we demonstrate that infection with equivalent doses of HIV based on virion-associated gag p24 yields five- to sixfold more infected cells if Nef+ viral stocks were used. Furthermore, we find that the differential infectivity is not dependent on CD4 down-regulation as Nef+ virus produced from transfected COS cells lacking CD4 is also more infectious. However, normalization of PBMC infections to equivalent infectivity between that of the Nef+ and Nef- viruses continues to reveal delayed viral replication in the absence of Nef, suggesting that secondary viral spread in PBMC is also enhanced in Nef+ infections. We demonstrate this directly by showing a 13-15-fold increase in infectivity of PBMC-derived Nef+ HIC.(ABSTRACT TRUNCATED AT 400 WORDS)
The T cell receptor repertoire of CD4-8+ thymocytes is altered by overexpression of the BCL-2 protooncogene in the thymus.
Authors: Tao W , Teh S J , Melhado I , Jirik F , Korsmeyer S J , Teh H S
Published: June 2004

The bcl-2 gene encodes an intracellular, membrane-associated protein that protects immature cortical thymocytes from a wide variety of apoptotic stimuli, including glucocorticoids, radiation, and anti-CD3 treatment. Since cortical thymocytes are the primary target cells for thymic positive and negative selection processes, and since these processes are associated with cell death, we evaluated the role of bcl-2 in T cell development in two ways. In the first approach, transgenic mice expressing high levels of Bcl-2 in cortical thymocytes were mated with H-Y T cell receptor (TCR) transgenic mice, the latter being a well-defined system for the study of positive and negative selection of T cells. We found that the bcl-2 transgene had a dramatic effect on positive selection. This was manifested by a greatly increased production of mature thymocytes that were highly skewed towards the CD4-8+ lineage. The change involving CD4-8+ thymocytes occurred not only in bcl-2 transgenic mice, but was also observed in H-Y TCR/bcl-2 doubly transgenic mice, regardless of whether the H-Y TCR was expressed in the selecting (H-2b) or nonselecting (H-2d) environments. Furthermore, a large proportion of CD4-8+ thymocytes produced in H-2b H-Y TCR/bcl-2 doubly transgenic female mice expressed endogenous TCR alpha chains rather than the transgenic TCR alpha chain. These observations are consistent with the model that high expression of Bcl-2 in cortical thymocytes overrides the normal apoptotic pathway. This then allows the selection of CD4-8+ thymocytes expressing TCRs that are otherwise nonselectable. However, the bcl-2 transgene did not protect CD4+8+ thymocytes expressing the male-specific TCR from deletion in male doubly transgenic mice. In the second approach, we determined the level of bcl-2 mRNA expression in populations of thymocytes defined by their CD4/CD8 phenotypes using quantitative reversed transcriptase PCR techniques. Our results indicate that bcl-2 mRNA was expressed at a high level in immature CD4-8- thymocytes and in mature CD4+8- thymocytes. There is a dramatic downregulation of bcl-2 mRNA in CD4+8+ thymocytes, particularly those expressing a low level of TCR. CD4+8+ thymocytes that upregulated their TCR, likely as a result of receiving positive selection signals, also upregulated bcl-2 mRNA. This observation suggests that rescue of immature thymocytes from the programmed cell death pathway by positive selection signals is accompanied by the upregulation of bcl-2 mRNA.
Coupling between p210bcr-abl and Shc and Grb2 adaptor proteins in hematopoietic cells permits growth factor receptor-independent link to ras activation pathway.
Authors: Tauchi T , Boswell H S , Leibowitz D , Broxmeyer H E
Published: June 2004

Enforced expression of p210bcr-abl transforms interleukin 3 (IL-3)-dependent hematopoietic cell lines to growth factor-independent proliferation. It has been demonstrated that nonreceptor tyrosine kinase oncogenes may couple to the p21ras pathway to exert their transforming effect. In particular, p210bcr-abl was recently found to effect p21ras activation in hematopoietic cells. In this context, experiments were performed to evaluate a protein signaling pathway by which p210bcr-abl might regulate p21ras. It was asked whether Shc p46/p52, a protein containing a src-homology region 2 (SH2) domain, and known to function upstream from p21ras, might form specific complexes with p210bcr-abl and thus, possibly alter p21ras activity by coupling to the guanine nucleotide exchange factor (Sos/CDC25) through the Grb2 protein-Sos complex. This latter complex has been previously demonstrated to occur ubiquitously. We found that p210bcr-abl formed a specific complex with Shc and with Grb2 in three different murine cell lines transfected with a p210bcr-abl expression vector. There appeared to be a higher order complex containing Shc, Grb2, and bcr-abl proteins. In contrast to p210bcr-abl transformed cells, in which there was constitutive tight association between Grb2 and Shc, binding between Grb2 and Shc was Steel factor (SLF)-dependent in a SLF-responsive, nontransformed parental cell line. The SLF-dependent association between Grb2 and Shc in nontransformed cells involved formation of a complex of Grb2 with c-kit receptor after SLF treatment. Thus, p210bcr-abl appears to function in a hematopoietic p21ras activation pathway to allow growth factor-independent coupling between Grb2, which exists in a complex with the guanine nucleotide exchange factor (Sos), and p21ras. Shc may not be required for Grb2-c-kit interaction, because it fails to bind strongly to c-kit.
Defective development of thymocytes overexpressing the costimulatory molecule, heat-stable antigen.
Authors: Hough M R , Takei F , Humphries R K , Kay R
Published: June 2004

Heat-stable antigen (HSA) is a small, glycosyl phosphatidylinositol-anchored protein that can act as a costimulatory molecule for antigen-dependent activation of helper T cells. In addition to being expressed on antigen-presenting B cells, HSA is also expressed during the initial stages of T cell development in the thymus. HSA levels are very high on immature CD4-, CD8- double negative thymocytes, but are reduced on CD4+, CD8+ double positive cells undergoing selection in the thymus, and are entirely eliminated when these cells differentiate into immunologically competent CD4+ or CD8+ single positive T cells. To examine the potential roles of this molecule in T cell development and selection, we generated transgenic mice in which HSA was highly expressed on all classes of thymocytes. The consequence of deregulated HSA expression was a pronounced reduction in the numbers of double positive and single positive thymocytes, whereas the numbers of their double negative precursors were largely unaffected. These results demonstrate that downregulation of HSA expression at the double positive stage is a critical event in thymocyte development. The depletion of thymocytes resulting from HSA overexpression begins at the same time as the onset of negative selection, suggesting that HSA may provide signals that contribute to determining the efficiency of this process.
The conditions of primary infection define the load of latent viral genome in organs and the risk of recurrent cytomegalovirus disease.
Authors: Reddehase M J , Balthesen M , Rapp M , Jonjić S , Pavić I , Koszinowski U H
Published: June 2004

Recurrence of cytomegalovirus (CMV) from latency is a frequent cause of disease in immunocompromised patients. To date, there is no explanation for the diversity in the clinical manifestations. Primary infection can occur perinatally or later in life, and inevitably results in latent infection. Seropositivity for antibodies against CMV is indicative of latent infection, but is insufficient as a predictor for the risk of recurrence. As a model for this important medical problem, we compared the risks of murine CMV recurrence from latency established after neonatal primary infection and after infection at adult age. The risk of CMV recurrence was high only after neonatal infection. The copy number of latent viral genome in tissues was identified as the key parameter that determines the overall and organ-specific risks of recurrence. Latent CMV burden and risk of recurrence were related to the extent of virus multiplication during primary infection. The presence of latent CMV in multiple organs provides the molecular basis for stochastic events of recurrence in single organs or in any combination thereof. These findings are discussed as a concept of multifocal CMV latency and recurrence. It provides a rationale for the diversity in the clinical outcome of CMV disease.
Somatic generation of hybrid antibody H chain genes in transgenic mice via interchromosomal gene conversion.
Authors: Giusti A M , Manser T
Published: June 2004

We have constructed lines of mice with transgenes containing an antibody heavy (H) chain variable region (VHDJH) gene and various amounts of natural immunoglobulin (Ig) and plasmid flanking DNA. In these lines, recombination of the transgene and the endogenous Igh locus takes place in B cells, leading to the expression of functional H chains partially encoded by the transgenic VHDJH gene. Here, we demonstrate that the transgenic VHDJH gene, and various amounts of flanking sequence are recombined with Igh locus DNA via interchromosomal gene conversion. The structures of the resulting "hybrid" transgene-Igh H chain loci are consistent with the 3' end of the conversion occurring in regions of sequence identity, and the 5' end taking place between regions of little or no homology. This mode of antibody transgene recombination with the Igh locus is fundamentally different from the previously reported "trans H chain class switching" that results in reciprocal translocations. In contrast, this recombination resembles events previously observed in mammalian tissue culture cells between adjacent homologous chromosomal sequences, or transfected DNA and a homologous chromosomal target. Our data indicate that this recombination takes place at a low frequency, and that the frequency is influenced by both the length and extent of homology between the transgene and the Igh locus, but is not greatly affected by transgene copy number. This recombination pathway provides a novel approach for the subtle alteration of the clonal composition of the mouse B cell compartment in vivo using VH genes with defined structures and functions.
Tolerogenicity of resting and activated B cells.
Authors: Gilbert K M , Weigle W O
Published: June 2004

Antigen presentation by resting splenic B cells has been shown previously to induce T helper 1 cell (Th1) anergy. In contrast to expectations, it was found here that B cells treated with F(ab')2 goat anti-mouse immunoglobulin (IgM) for 24 or 48 h also presented antigen (Ag) to Th1 cells in a manner that induced dramatic Ag-specific proliferative inactivation. The tolerogenicity of the anti-Ig-treated B cells was consistent with the observation that these B cells were only slightly more efficient than resting B cells in stimulating human gamma globulin (HGG)-induced proliferation of HGG-specific Th1 cells in primary cultures. The activated B cells were, however, more efficient than resting B cells in stimulating a primary mixed leukocyte reaction, and exhibited increased expression of major histocompatibility complex class II molecules, RL388 Ag and transferrin receptor. In addition, unlike resting B cells, which expressed little detectable B7, anti-Ig-treated B cells expressed high levels of B7. The functional capacity of the B7 expressed on the activated B cells was demonstrated by the fact that the Ag-presenting capacity of these B cells was inhibited by the addition to culture of CTLA4Ig, a soluble receptor for B7. It is unlikely that the tolerogenicity of the activated B cells was due to an inability of the Th1 cells to respond to B7 signals; the Th1 clones used in the experiments, unlike the Th2 clones tested, expressed CD28, the ligand for B7. In addition, anti-CD28 monoclonal antibody inhibited the induction of Th1 cell anergy when added to cultures of Th1 cells and Ag-pulsed fixed antigen-presenting cells. Taken together, the results indicate that B cells, even when activated, do not satisfy the costimulatory requirements of the Th1 cells used here, and therefore can present Ag in a tolerogenic fashion to Th1 cells. The costimulator deficiency of activated B cells may reflect an inadequacy in the level of B7 expressed or a lack of some other molecule.
NADPH-binding component of the respiratory burst oxidase system: studies using neutrophil membranes from patients with chronic granulomatous disease lacking the beta-subunit of cytochrome b558.
Authors: Tsunawaki S , Mizunari H , Namiki H , Kuratsuji T
Published: June 2004

The NADPH-binding site of the respiratory burst oxidase system of neutrophils has been proposed to be either at a cytosolic component or at the beta-subunit of cytochrome b558. In this study, affinity labeling of resting and stimulated membranes, the latter having been assembled by all of the oxidase components from both membrane and cytosol, was carried out using [32P]NADPH dialdehyde (oNADPH). Stimulation of human neutrophils with PMA greatly increased O2(-)-generating activity and caused considerable translocation of the cytosolic components p47phox and p67phox. Nevertheless, PMA stimulation did not produce a labeled band which included positions at 47, 67, and approximately 32 kD. The most intense band reflected a molecular mass of 84 kD regardless of the state of activation, but a labeled band was never found near the beta-subunit (91 kD) of cytochrome b558. This 84-kD protein was further confirmed in neutrophils of 14 patients with gp91phox-deficient X-linked chronic granulomatous disease. These results indicate that the NADPH-binding component is not recruited from the cytosol, and also, that a membranous redox component besides cytochrome b558 must be involved in the NADPH oxidase system.
CD28-mediated costimulation of interleukin 2 (IL-2) production plays a critical role in T cell priming for IL-4 and interferon gamma production.
Authors: Seder R A , Germain R N , Linsley P S , Paul W E
Published: June 2004

Naive T cells require interleukin 4 (IL-4) to develop into IL-4-producing T cells and IL-4 blocks development of such cells into interferon gamma (IFN-gamma) producers. Prior studies in accessory cell-independent priming systems using antireceptor antibodies as agonists have demonstrated that IL-2 is also necessary for the development of IL-4-producing cells under these culture conditions. Here we have examined the role of IL-2 and the CD28 costimulation pathway in priming for IL-4 and IFN-gamma production using a more physiologic model. This involved antigen presentation by accessory cells to naive CD4+ T cells from transgenic mice whose cells express a T cell receptor (TCR) specific for a cytochrome c peptide in association with I-Ek. With splenic antigen-presenting cells (APCs), inhibition of CD28 costimulation by the fusion protein CTLA4-immunoglobulin (Ig) blocked effective priming. Similarly, transfected fibroblasts expressing both MHC class II and the CD28 ligand B7 could prime for IL-4 production and such priming also was blocked by CTLA4-Ig. However, APCs deficient in CD28 ligands also could prime TCR transgenic T cells to become IL-4 producers if an exogenous source of IL-2, as well as IL-4, was provided, and the inhibition of priming seen with splenic or transfected fibroblast APCs in the presence of CTLA4-Ig could be reversed by addition of IL-2. Likewise, priming for IFN-gamma production could be blocked by CTLA4-Ig and reversed by IL-2. Thus, we conclude that IL-2 plays a critical role in priming naive CD4+ T cells to become IL-4 or IFN-gamma producers. Engagement of the CD28 pathway, although normally important in such priming, is unnecessary in the presence of exogenous IL-2.