bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombin functions as an inflammatory mediator through activation of its receptor.
Authors: Cirino G , Cicala C , Bucci M R , Sorrentino L , Maraganore J M , Stone S R
Published: June 2004

A rat model of inflammation was used to investigate the biological effects of thrombin. The thrombin-specific inhibitor Hirulog markedly attentuated the carrageenin-induced edema of the paw of the rat. Injection of thrombin into the paw also produced edema. The effect of thrombin was due to activation of its receptor; a thrombin receptor activating peptide (TRAP) reproduced the effects of thrombin in causing edema. TRAP also increased vascular permeability as demonstrated by extravasation of Evans blue and 125I-labeled serum albumin. The release of bioactive amines played an important role in mediating the TRAP-induced edema; the serotonin/histamine antagonist cryproheptadine and the histamine H2 receptor antagonist cimetidine reduced significantly the edema caused by TRAP. Treatment of rats with the mast cell degranulator 48/80 to deplete these cells of their stores of histamine and serotonin abolished completely the ability of TRAP to produce edema. Histochemical examination confirmed that TRAP treatment led to mast cell degranulation. Thus, it has been possible to demonstrate that thrombin acts as an inflammatory mediator in vivo by activating its receptor, which in turn leads to release of vasoactive amines from mast cells.
Reversibility of T helper 1 and 2 populations is lost after long-term stimulation.
Authors: Murphy E , Shibuya K , Hosken N , Openshaw P , Maino V , Davis K , Murphy K , O'Garra A
Published: June 2004

Commitment of T helper 1 (Th1) or Th2 populations developing during an immune response to a pathogen, or an inappropriate immune response to an allergen or autoantigen, may determine the difference between health and chronic disease. We show that strongly polarized Th1 and Th2 populations assessed by immunoassay are heterogeneous using flow cytometry to detect single cells producing interferon gamma (IFN-gamma) and interleukin 4 (IL-4). Th1 populations arising after 1 wk of stimulation in IL-12 plus anti-IL-4 antibodies could convert to Th2 cells when restimulated in IL-4. Th2 populations resulting from stimulation for 1 wk in IL-4 could give rise to Th1 cells upon restimulation in IL-12 plus anti-IL-4. In contrast, the cytokine profiles of long-term Th1 and Th2 populations arising originally from repeated stimulation in IL-12 or IL-4 appeared more homogeneous and were not reversible, although IL-4 dramatically reduced the number of IFN-gamma-producing Th1 cells. This may explain previous reports that Th1 cells can be converted to Th2 cells.
Tumor necrosis factor alpha and interleukin 1beta are responsible for in vitro myocardial cell depression induced by human septic shock serum.
Authors: Kumar A , Thota V , Dee L , Olson J , Uretz E , Parrillo J E
Published: June 2004

Previous studies have demonstrated the presence of myocardial depression in clinical and experimental septic shock. This depression is associated with the presence of a circulating myocardial depressant substance with physical characteristics consistent with cytokines. The present study utilized an in vitro myocardial cell assay to examine the role of various human recombinant cytokines, including tumor necrosis factor (TNF)alpha and interleukin (IL)1beta, in depression of cardiac myocyte contractile function induced by serum from humans with septic shock. The extent and velocity of electrically paced rat cardiac myocytes in tissue culture was quantified by a closed loop video tracking system. Individually, TNF-alpha and IL-1beta each caused significant concentration-dependent depression of maximum extent and peak velocity of myocyte shortening in vitro. In combination, TNF-alpha and IL-1beta induced depression of myocardial cell contractility at substantially lower concentrations consistent with a synergistic effect. Using immunoabsorption, removal of both TNF-alpha and IL-1beta (but not either alone) from the serum of five patients with acute septic shock and marked reversible myocardial depression resulted in elimination of serum myocardial depressant activity. IL-2, -4, -6, -8, -10, and interferon gamma failed to cause significant cardiac myocyte depression over a wide range of concentrations. These data demonstrate that TNF-alpha and IL-1beta cause depression of myocardial cell contraction in vitro and suggest that these two cytokines act synergistically to cause sepsis-associated myocardial depression in humans.
The T cell-B cell interaction via OX40-OX40L is necessary for the T cell-dependent humoral immune response.
Authors: Stüber E , Strober W
Published: June 2004

Recent in vitro studies have established that activated B cells express OX40 ligand (L), a member of the tumor necrosis factor/nerve growth factor family of cytokines, and become stimulated to proliferate and secrete immunoglobulin (Ig) after cross-linking of OX40L by its counterreceptor OX40, which is expressed on activated T cells. In the present study we investigated the in vivo role of this receptor-ligand pair for the interaction of T and B cells in the course of the T-dependent B cell response against 2,4,6 trinitro-phenyl-keyhole limpet hemocyanin. First, we showed that OX40 is maximally expressed by T cells in the periarteriolar lymphoid sheath (PALS) 3 d after primary immunization. These OX40+ cells are located in close proximity to antigen-specific, activated B cells. Second, we demonstrated that blocking of OX40-OX40L interaction with polyclonal anti-OX40 antibody or with antibodies against certain peptide sequences within its extracellular domain resulted in a profound decrease of the anti-hapten IgG response, whereas the antihapten IgM response was grossly unchanged. Third, we showed that this antibody treatment leads to an inhibition of the development of PALS-associated B cell foci, whereas the formation of germinal centers remained intact. Finally, our data suggest that, whereas B cell memory development was not impaired by anti-OX40 administration, OX40-OX40L interaction seems to be crucial in the secondary immune response. We conclude from these data that the OX40-OX40L interaction in vivo is necessary for the differentiation of activated B cells into highly Ig-producing cells, but is not involved in other pathways of antigen-driven B cell differentiation such as memory cell development in the germinal centers.
Central role of immunoglobulin (Ig) E in the induction of lung eosinophil infiltration and T helper 2 cell cytokine production: inhibition by a non-anaphylactogenic anti-IgE antibody.
Authors: Coyle A J , Wagner K , Bertrand C , Tsuyuki S , Bews J , Heusser C
Published: June 2004

Elevated levels of immunoglobulin (Ig) E are associated with bronchial asthma, a disease characterized by eosinophilic inflammation of the airways. Activation of antigen-specific T helper (Th) 2 cells in the lung with the subsequent release of interleukin (IL) 4 and IL-5 is believed to play an important role in the pathogenesis of this disease. In this study, we have used a non-anaphylactogenic anti-mouse-IgE antibody to investigate the relationship between IgE, airway eosinophil infiltration, and the production of Th2 cytokines. Immunization of mice with house dust mite antigen increased serum levels of IgE and IgG. Antigen challenge of immunized but not control mice induced an infiltration of eosinophils in the bronchoalveolar lavage associated with the production of IL-4 and IL-5 from lung purified Thy1.2+ cells activated through the CD3-T cell receptor complex. Administration of the anti-IgE monoclonal antibody (mAb) 6h before antigen challenge neutralized serum IgE but not IgG and inhibited the recruitment of eosinophils into the lungs and the production of IL-4 and IL-5 but not interferon gamma. Studies performed using an anti-CD23 mAb, CD23 deficient and mast cell deficient mice suggest that anti-IgE mAb suppresses eosinophil infiltration and Th2 cytokine production by inhibiting IgE-CD23-facilitated antigen presentation to T cells. Our results demonstrate that IgE-dependent mechanisms are important in the induction of a Th2 immune response and the subsequent infiltration of eosinophils into the airways. Neutralization of IgE, for example, non-anaphylactogenic anti-IgE mAbs may provide a novel therapeutic approach to the treatment of allergic airway disease.
Visualization, characterization, and turnover of CD8+ memory T cells in virus-infected hosts.
Authors: Zimmerman C , Brduscha-Riem K , Blaser C , Zinkernagel R M , Pircher H
Published: June 2004

The cellular basis of T cell memory is a controversial issue and progress has been hampered by the inability to induce and to trace long-term memory T cells specific for a defined antigen in vivo. By using the murine model of lymphocytic choriomeningitis virus (LCMV) infection and an adoptive transfer system with CD8+ T cells from transgenic mice expressing an LCMV-specific T cell receptor, a population of authentic memory T cells specific for LCMV was generated and analyzed in vivo. The transgenic T cells that have expanded (1,000-fold) and then decreased (10-fold) in LCMV-infected C57BL/6 recipient mice exhibited the following characteristics: they were (a) of larger average cell size than their naive counterparts but smaller than day 8 effector cells; (b) heterogeneous with respect to expression of cell surface "memory" markers; and (c) directly cytolytic when isolated from recipient spleens. The time-dependent proliferative activity of these LCMV-specific memory T cells was analyzed in the recipients by bromodeoxyuridine labeling experiments in vivo. The experiments revealed that LCMV-specific CD8+ memory T cells can persist in LCMV-immune mice for extended periods of time (>2 mo) in the absence of cell division; the memory population as a whole survived beyond 11 mo.
Mice deficient in IL-1beta manifest impaired contact hypersensitivity to trinitrochlorobenzone.
Authors: Shornick L P , De Togni P , Mariathasan S , Goellner J , Strauss-Schoenberger J , Karr R W , Ferguson T A , Chaplin D D
Published: June 2004

Mice rendered deficient in IL-1 beta by gene targeting in embryonic stem cells develop and grow normally in a protected laboratory environment. Endotoxin-stimulated peritoneal macrophages from IL-1beta-deficient mice showed normal synthesis and cellular release of IL-1alpha after treatment with 5 mM ATP demonstrating that IL-1beta is not necessary for expression and release of the IL-1alpha isoform. Mice deficient in IL-1beta showed unaltered sensitivity to endotoxic shock, with or without pretreatment with D-galactosamine. In contrast, IL-1beta-deficient mice showed defective contact hypersensitivity responses to topically applied trinitrochlorobenzene (TNCB). This defect could be overcome either by application of very high doses of sensitizing antigen, or by local intradermal injection of recombinant IL-1beta immediately before antigen application. These data demonstrate an essential role for IL-1beta in contact hypersensitivity and suggest that IL-1beta acts early during the sensitization phase of response. They suggest an important role for IL-1beta in initiation of the host of response at the epidermal barrier.
Distinct regulatory roles of lymphocyte costimulatory pathways on T helper type-2 mediated autoimmune disease.
Authors: Biancone L , Andres G , Ahn H , Lim A , Dai C , Noelle R , Yagita H , De Martino C , Stamenkovic I
Published: June 2004

We assessed the role of CD40-CD40L, cytotoxic T lymphocyte (CTL)A4/CD28-B7s, and CD2-CD48/CD58 lymphocyte costimulatory pathways in the development of mercury chloride (HgCl2)-induced autoimmune disease in mice, which is believed to be mediated by T helper (Th) subset Th2. Inhibition of CD40-CD40-L and CTLA4/CD28-B7s interactions by anti-CD40-L antibody and soluble CTLA4-immunoglobulin (Ig) fusion protein, respectively, abrogated the autoimmune disease without affecting interleukin 4 (IL-4) production, showing the importance of physical contact between T and B lymphocytes in the Th2-mediated process. In contrast, two anti-CD2 antibodies that have been shown to induce immunosuppression of Th1-mediated events exacerbated the autoantibody response and augmented IgG1, IgE, and IL-4 production, transforming a mild mesangial glomerulopathy into a severe systemic immune complex disease. These observations demonstrate that manipulation of lymphocyte accessory counterreceptor interactions may affect the course of Th2-associated autoimmune disease and suggest that signals resulting from CD2 engagement play an essential role in the regulation of the Th1-Th2 effector equilibrium.
Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase.
Authors: Stenger S , Donhauser N , Thüring H , Röllinghoff M , Bogdan C
Published: June 2004

Nitric oxide (NO) synthase (iNOS) is required for the resolution of acute cutaneous leishmaniasis in resistant C57BL/6 mice. As is the case in several other infections, the clinically cured host organism still harbors small amounts of live Leishmania major parasites. Here, we demonstrate lifelong expression of iNOS at the site of the original skin lesion and in the draining lymph node of long-term-infected C57BL/6 mice. iNOS activity in the lymph node was dependent on CD4+, but not on the CD8+ T cells. By double labeling techniques, iNOS and L. major were each found in macrophages (F4/80+, BM-8+, and/or MOMA-2+) and dendritic cells (NLDC-145+), but not in granulocytes or endothelial cells. In situ triple labeling of lymph node sections revealed that approximately 30-40% of the L. major foci were associated with iNOS-positive macrophages or dendritic cells. The majority of the L. major foci (60-70%), however, was located in areas that were negative for both iNOS and the macrophage and dendritic cell markers. In L. major-infected C57BL/6 mice, which had cured their cutaneous lesions, administration of L-N6-iminoethyl-lysine (L-NIL), a potent inhibitor of iNOS, led to a 10(4)-10(5)-fold increase of the parasite burden in the cutaneous and lymphoid tissue and caused clinical recrudescence of the disease. Persistent expression of iNOS and resumption of parasite replication after application of L-NIL was also observed in resistant C3H/HeN and CBA/J mice. We conclude that iNOS activity is crucial for the control of Leishmania persisting in immunocompetent hosts after resolution of the primary infection. Failure to maintain iNOS activity might be the mechanism underlying endogenous reactivation of latent infections with NO-sensitive microbes during phases of immunosuppression.
Recognition of class I major histocompatibility complex molecules by Ly-49: specificities and domain interactions.
Authors: Brennan J , Mahon G , Mager D L , Jefferies W A , Takei F
Published: June 2004

Ly-49 is a family type II transmembrane proteins encoded by a gene cluster on murine chromosome 6. One member of this family, Ly-49A, is expressed by a natural killer (NK) cell subset, binds to class I major histocompatibility complex (MHC) molecules, and blocks the killing of target cells bearing the appropriate H-2 antigens. Here we show that another member of this family which is expressed by an NK cell subset, Ly-49C, recognizes H-2b and H-2d structures which are distinct from and overlapping with those recognized by Ly-49A. Interactions between Ly-49A and C and their class I ligands are entirely blocked by the antibodies 5E6, YE1/48, YE1/32, and A1, all of which were found to recognize epitopes contained within the carbohydrate recognition domain (CRD). However, cell-cell binding assays revealed that class I binding specificity is conferred by a combination of sequences within both the CRD and a 19-amino acid adjacent region. We also investigated the question of whether Ly-49A and C form dimers on cells which express both receptors. When coexpressed on COS cells, sequential immunoprecipitation demonstrated that these receptors pair exclusively as homodimers, with no evidence for heterodimeric structures. These observations provide insight into both the biochemical nature of the Ly-49 family as well as the receptor functions of Ly-49C on NK cells.
The protease inhibitor, N-acetyl-L-leucyl-L-leucyl-leucyl-L-norleucinal, decreases the pool of major histocompatibility complex class I-binding peptides and inhibits peptide trimming in the endoplasmic reticulum.
Authors: Hughes E A , Ortmann B , Surman M , Cresswell P
Published: June 2004

N-acetyl-L-leucyl-L-leucyl-L-norleucinal, (LLnL), which inhibits proteasomes in addition to other proteases, was found to prolong the association of major histocompatibility complex class I molecules with the transporters associated with antigen processing (TAP), and to slow their transport out of the endoplasmic reticulum (ER). LLnL induced a reversible accumulation of ubiquitinated proteins and changed the spectrum of peptides bound by class I molecules. These effects can probably be attributed to proteasome inhibition. Unexpectedly, in the TAP-deficient cell line .174, the rate of intracellular transport of human histocompatibility leukocyte antigen (HLA) A2 was also reduced by LLnL, and the generation of most HLA-A2-associated signal sequence peptides was inhibited. The inhibition of HLA-A2 transport in .174 cells was found to be less sensitive to LLnL than in wild-type cells, and a similar difference was found for a second protease inhibitor, benzyloxycarbonyl-L-leucyl-L-leucyl-L-phenylalanilal. These data suggest that under some conditions such inhibitors can block trimming of peptides by an ER peptidase in addition to inhibiting cytosolic peptide generation.
Metabolism of Tac (IL2Ralpha): physiology of cell surface shedding and renal catabolism, and suppression of catabolism by antibody binding.
Authors: Junghans R P , Waldmann T A
Published: June 2004

The interleukin 2 receptor alpha (IL2Ralpha; CD25; Tac) is the prototypic model for soluble receptor studies. It exists in vivo as a transmembrane complete molecule (TM-Tac) on cell surfaces and as a truncated soluble form (sTac; sIL2R alpha). sTac has been used as a serum marker of T cell activation in immune disorders and of tumor burden in Tac-expressing malignancies. In vivo, serum levels of all soluble proteins depend on the balance between production and catabolism, but little is known about the metabolic features of this class of molecules. We have developed a model for Tac metabolism that incorporates new insights in its production and catabolism. Tac was shed from the surface of malignant and activated human T cells with a model half-life (t1/2) of 2-6h, but which was prolonged under certain circumstances. The rate of shedding is first order overall and nonsaturable over a two order of magnitude range of substrate (TM-Tac) expression. Once shed from cells Tac is subject to catabolic activities in the host. In vivo studies in mice showed that 90% of Tac was catabolized by the kidney with a t1/2 of 1 h and a filtration fraction of 0.11 relative to creatinine. The remaining 10% of catabolism was mediated by other tissues with a t1/2 of 10 h. Approximately 1-3% of sTac is excreted intact as proteinuria with the remaining 97-99% catabolized to amino acids. Antibody to the receptor induced a marked delay in sTac catabolism by preventing filtration of the smaller protein through the renal glomerulus and additionally suppressing other nonrenal catabolic mechanisms. A discrepancy between the catabolic rats for Tac and anti-Tac in the same complex was interpreted as a previously unrecognized differential catabolic mechanism, suggesting features of the Brambell hypothesis and immunoglobulin G transport and catabolism, in which the antigen-in-complex in intracellular vesicles is relatively less protected from catabolism than the associated antibody. In light of the pivotal role played by the kidney in sTac catabolism and the impact of administered antibody, the serum concentration of Tac in the settings of renal dysfunction or antibody therapy is not a suitable surrogate of activated T cells or of the body burden of tumor. These results provide parameters for assessing soluble receptor-ligand interactions generally.
Neonatal peptide exposure can prime T cells and, upon subsequent immunization, induce their immune deviation: implications for antibody vs. T cell-mediated autoimmunity.
Authors: Singh R R , Hahn B H , Sercarz E E
Published: June 2004

Neonatal exposure to antigen is believed to result in T cell clonal inactivation or deletion. Here we report that, contrary to this notion, neonatal injection of BALB/c mice with a hen egg lysozyme peptide 106-116 in putative "tolergenic" doses induced a T cell proliferative and an immunoglobulin G (IgG) antibody (Ab) response of both T helper cell 1 (Th1)- (IgG2a, IgG2b, and IgG 3) and Th2-dependent (IgG1) isotopes. Upon subsequent challenge with the peptide in complete Freund's adjuvant in adult life, although this neonatal regimen suppressed proliferation and the production of Th1 cytokines (interleukin[IL]-2 and interferon gamma), Th2 cytokine (IL-5, IL-4, and IL-10) secretion was increased, and the serum levels of Th1- and Th2-dependent isotypes of peptide-specific Ab remained elevated. The in vitro proliferative unresponsiveness in Th1 cells could be reversed by Abs to Th2 cytokines (IL-4 and IL-10). Thus, neonatal treatment with a peptide antigen induces T cell priming including production of IgG Abs of both Th1- and Th2-dependent isotypes. Upon subsequent peptide exposure, the peptide-specific T cell responses undergo an effective class switch in the direction of Th2, resulting in T cell proliferative unresponsiveness. Accordingly, this shift towards increased Ab production to autoantigen could be deleterious in individuals prone to antibody-mediated diseases. Indeed, neonatal treatment with a self-autoantigenic peptide from an anti-DNA monoclonal Ab (A6H 58-69) significantly increased the IgG anti-double-stranded DNA Ab levels in lupus-prone NZB/NZW F1 mice, despite suppressing peptide-specific T cell proliferation. This adverse clinical response is in sharp contrast to the beneficial outcome of neonatal treatment with autoantigens in Th1-mediated autoimmune diseases, such as autoimmune encephalomyelitis, as reported by others. A Th1 to Th2 immune deviation can explain the discordant biological responses after the presumed induction of neonatal tolerance in autoantibody- vs. Th-1 mediated autoimmune diseases.
5(S),15(S)-dihydroxyeicosatetraenoic acid and lipoxin generation in human polymorphonuclear cells: dual specificity of 5-lipoxygenase towards endogenous and exogenous precursors.
Authors: Chavis C , Vachier I , Chanez P , Bousquet J , Godard P
Published: June 2004

5-Lipoxygenase activation of human blood polymorphonuclear cells (PMN) from asthmatic patients (asthmatics) was studied to investigate whether differences may exist with healthy subjects (controls). The respective cell capacities to produce lipoxins (LXs), leukotrienes, and 5(S), 15(S)-dihydroxyeicosatetraenoic acid [5(S),15(S)-diHETE] were compared under in vitro stimulation by ionophore A23187, with or without exogenous 15(S)-hydroxyeicosatetraenoic acid [15(S)-diHETE]. Eicosanoids were analyzed by elution with an isocratic reverse-phase high performance liquid chromatography system, and their profiles, detected by simultaneous monitoring at 302, 280, and 246 nm, were evaluated on the basis of chromatographic behavior: UV spectral characteristics and coelution with synthetic standards. In the presence of exogenous 15(S)-HETE, human PMN were able to produce LXs and 5(S),15(S)-diHETE, PMN from asthmatics were able to produce 5(S), 5(S),15(S)-diHETE, and LXs from endogenous sources, whereas in the same experimental conditions, no detectable amounts of these compounds were released by PMN from controls. The levels of 5(S),15(S)-diHETE, and LXs biosynthesized from endogenous arachidonic acid were highly correlated. Two different LX patterns were observed involving two possible metabolic pathways: (a) via the intermediate 5,6-epoxytetraene alone for LXs generation from exogenous 15(S)-HETE; and (b) via 5,6- and/or 14,15-epoxytetraenes leading to the formation of an enzyme-bound delocalized carbocation for LXs generation from endogenous arachidonate, respectively. The enhanced 5-lipoxygenase activation of blood PMN from asthmatics and the metabolism of exogenous 15(S)-HETE may reflect a priming induced by various mediators released from environmental cells, and could be considered as a model of transcellular signalization between PMN and endothelial cells.
Infection of human immunodeficiency virus 1 transgenic mice with Toxoplasma gondii stimulates proviral transcription in macrophages in vivo.
Authors: Gazzinelli R T , Sher A , Cheever A , Gerstberger S , Martin M A , Dickie P
Published: June 2004

Human immunodeficiency virus (HIV) 1 transgenic mice expressing low or undetectable levels of viral mRNA in lymphoid tissue were infected with the intracellular protozoan Toxoplasma gondii. Exposure to this parasite resulted in an increase in HIV-1 transcript in lymph nodes, spleens, and lungs during the acute phase of infection and in the central nervous system during the chronic stage of disease. In vivo and ex vivo experiments identified macrophages as a major source of the induced HIV-1 transcripts. In contrast, T. gondii infection failed to stimulate HIV-1 transcription in tissues of two HIV-1 transgenic mouse strains harboring a HIV-1 proviral DNA in which the nuclear factor (NF) kappa B binding motifs from the viral long terminal repeats had been replaced with a duplicated Moloney murine leukemia virus core enhancer. A role for NF-kappaB in the activation of the HIV-1 by T. gondii was also suggested by the simultaneous induction of NF-kappaB binding activity and tumor necrosis factor alpha synthesis in transgenic mouse macrophages stimulated by exposure to parasite extracts. These results demonstrate the potential of an opportunistic pathogen to induce HIV-1 transcription in vivo and suggest a mechanism for the in vivo dissemination of HIV-1 by macrophages.
Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B1 expression.
Authors: Mohapatra S , Yang X , Wright J A , Turley E A , Greenberg A H
Published: June 2004

The hyaluronan (HA) receptor RHAMM is an important regulator of cell growth. Overexpression of RHAMM is transforming and is required for H-ras transformation. The molecular mechanism underlying growth control by RHAMM and other extracellular matrix receptors remains largely unknown. We report that soluble RHAMM induces G2/M arrest by suppressing the expression of Cdc2/Cyclin B1, a protein kinase complex essential for mitosis. Down-regulation of RHAMM by use of dominant negative mutants or antisense of mRNA also decreases Cdc2 protein levels. Suppression of Cdc2 occurs as a result of an increased rate of cdc2 mRNA degradation. Moreover, tumor cells treated with soluble RHAMM are unable to form lung metastases. Thus, we show that mitosis is directly linked to RHAMM through control of Cdc2 and Cyclin B1 expression. Failure to sustain levels of Cdc2 and Cyclin B1 proteins leads to cell cycle arrest.
T cell receptor usage and fine specificity of human immunodeficiency virus 1-specific cytotoxic T lymphocyte clones: analysis of quasispecies recognition reveals a dominant response directed against a minor in vivo variant.
Authors: Kalams S A , Johnson R P , Dynan M J , Hartman K E , Harrer T , Harrer E , Trocha A K , Blattner W A , Buchbinder S P , Walker B D
Published: June 2004

Numerous virus-specific, class I-restricted cytotoxic T lymphocyte (CTL) epitopes have been identified, yet little information is available regarding the specificity of the CTL response in persons of the same human histocompatibility leukocyte antigen (HLA) type. In this study, the human immunodeficiency virus (HIV) 1 envelope-specific CTL response was evaluated in five HLA-B14-positive persons. CTL responses specific for a previously described nine-amino acid epitope in gp41 (aa 584-592, ERYLKDQQL) could be identified in all subjects, and CTL clones specific for this epitope could be isolated from four persons. Despite heterogeneous T cell receptor usage, the fine specificity of the clones was similar, as defined by recognition of alanine-substituted peptides as well as peptides representing natural HIV-1 sequence variants. Correlation with in vivo virus sequences revealed that the dominant species in two of the subjects represented poorly recognized variants, with a K-->Q substitution at amino acid 588, whereas no variants were observed in the other two subjects. Although clonal type-specific responses to these dominant variants could be identified, the magnitude of these responses remained small, and the dominant CTL response was directed at the minor in vivo variant. These studies indicate that despite similar epitope-specific immunologic pressure in persons of the same HLA type, the in vivo quasispecies may differ, and that the major in vivo immune response to a given CTL epitope can be directed at a minor variant.
Human intestinal Vdelta1+ lymphocytes recognize tumor cells of epithelial origin.
Authors: Maeurer M J , Martin D , Walter W , Liu K , Zitvogel L , Halusczcak K , Rabinowich H , Duquesnoy R , Storkus W , Lotze M T
Published: June 2004

gammadelta T cells can be grouped into discrete subsets based upon their expression of T cell receptor (TCR) variable (V) region families, their tissue distribution, and their specificity. Vdelta2+ T cells constitute the majority of gammadelta T cells in peripheral blood whereas Vdelta1+T cells reside preferentially in skin epithelium and in the intestine. gammadelta T cells are envisioned as first line host defense mechanisms capable of providing a source of immune effector T cells and immunomodulating cytokines such as interleukin (IL) 4 or interferon (IFN) gamma. We describe here the fine specificity of three distinct gammadelta+ tumor-infiltrating lymphocytes (TIL) obtained from patients with primary or metastatic colorectal cancer, that could be readily expanded in vitro in the presence of IL-1beta and IL-7. Irrespective of donor, these individual gammadelta T cells exhibited a similar pattern of reactivity defined by recognition of autologous and allogeneic colorectal cancer cells, renal cell cancer, pancreatic cancer, and a freshly isolated explant from human intestine as measured by cytolytic T cell responses and by IFN-gamma release. In contrast, tumors of alternate histologies were not lysed, including lung cancer, squamous cell cancer, as well as the natural/lymphocyte-activated killer cell-sensitive hematopoietic cell lines T2, C1R, or Daudi. The cell line K562 was only poorly lysed when compared with colorectal cancer targets. Target cell reactivity mediated by Vdelta1+ T cells was partially blocked with Abs directed against the TCR, the beta2 or beta7 integrin chains, or fibronectin receptor. Marker analysis using flow cytometry revealed that all three gammadelta T cell lines exhibit a similar phenotype. Analysis of the gammadelta TCR junctional suggested exclusive usage of the Vdelta1/Ddelta3/Jdelta1 TCR segments with extensive (< or = 29 bp) N/P region diversity. T cell recognition of target cells did not appear to be a major histocompatibility complex restricted or to be correlated with target cell expression of heat-shock proteins. Based on the ability of some epithelial tumors, including colorectal, pancreatic, and renal cell cancers to effectively cold target inhibit the lysis of colorectal cancer cell lines by these Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cells are capable of recognizing cell surface Ag(s) shared by tumors of epithelial origin.
Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CD8+ cell-, interferon gamma-, and nitric oxide-dependent immunity.
Authors: Doolan D L , Sedegah M , Hedstrom R C , Hobart P , Charoenvit Y , Hoffman S L
Published: June 2004

Despite efforts to develop vaccines that protect against malaria by inducing CD8+ T cells that kill infected hepatocytes, no subunit vaccine has been shown to circumvent the genetic restriction inherent in this approach, and little is known about the interaction of subunit vaccine-induced immune effectors and infected hepatocytes. We now report that immunization with plasmid DNA encoding the plasmodium yoelii circumsporozoite protein protected one of five strains of mice against malaria (H-2d, 75%); a PyHEP17 DNA vaccine protected three of the five strains (H-2a, 71%; H-2k, 54%; H-2d, 26%); and the combination protected 82% of H-2a, 90% of H-2k, and 88% of H-2d mice. Protection was absolutely dependent on CD8+ T cells, INF-gamma, or nitric oxide. These data introduce a new target of protective preerythrocytic immune responses, PyHEP 17 and its P. falciparum homologue, and provide a realistic perspective on the opportunities and challenges inherent in developing malaria vaccines that target the infected hepatocyte.
Inhibition of leukotriene B4-receptor interaction suppresses eosinophil infiltration and disease pathology in a murine model of experimental allergic encephalomyelitis.
Authors: Gladue R P , Carroll L A , Milici A J , Scampoli D N , Stukenbrok H A , Pettipher E R , Salter E D , Contillo L , Showell H J
Published: June 2004

Leukotriene B4 (LTB4) is a chemotactic and cell-activating factor present at inflammatory sites in a variety of autoimmune diseases including multiple sclerosis (MS). In this study, we used a murine model of MS, experimental allergic encephalomyelitis (EAE), to assess the potential role of LTB4 on cell infiltration and paralysis. Injection of encephalogenic T cells into naive animals induced paralysis and weight loss that was completely inhibited by treatment with the selective LTB4 receptor antagonist CP-105,696 (ED50= 8.6 mg/kg orally). Although migration of lymphocytes into the central nervous system was unaffected, the efficacious effects of CP-105,696 correlated with up to a 97% decrease in eosinophil infiltration into the lower spinal cord as determined by light and electron microscopy and quantitated by levels of the specific enzyme marker eosinophil peroxidase. These results demonstrate that eosinophil recruitment in EAE is dependent on LTB4 receptor ligation and further reveal a previously unrecognized role for eosinophils in the pathogenesis of this disease.
The beta subunit of human granulocyte-macrophage colony-stimulating factor receptor forms a homodimer and is activated via association with the alpha subunit.
Authors: Muto A , Watanabe S , Miyajima A , Yokota T , Arai K
Published: June 2004

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor (hGMR) consists of alpha and beta subunits, and the precise stoichiometry of these subunits has remained to be determined. In this work, oligomerization of the beta subunit was studied using a chemical cross-linker. In Ba/F3, a mouse interleukin-3-dependent cell line expressing both subunits of hGMR (Ba/F3-alpha,beta), a protein with a molecular mass corresponding to that of a homodimer of the beta subunit (beta homodimer) was detected only when cells were treated with the cross-linker. Dimerization of the beta subunit was confirmed by coimmunoprecipitation of a tagged beta subunit with the wild type beta subunit COS7 cells. The beta homodimer had already formed in the absence of hGM-CSF, whereas stimulation with the ligand brought both alpha and beta subunits into a complex, the result being tyrosine phosphorylation of the beta homodimer. Tyrosine phosphorylation of the subunit was impaired by deletion of the cytoplasmic domain of the alpha subunit without interfering with the association of both subunits. These results indicate that the beta homodimer, which alone is insufficient for signaling, forms the functional hGMR with the alpha subunit in response to hGM-CSF.
Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production.
Authors: Motto D G , Ross S E , Wu J , Hendricks-Taylor L R , Koretzky G A
Published: June 2004

Recently we described the molecular cloning of SLP-76, a hematopoietic cell-specific 76-kD protein that was first identified through its association with GST/Grb2 fusion proteins. The primary sequence of SLP-76 predicts a protein of 533 amino acids comprising an amino-terminal region with numerous potential tyrosine phosphorylation sites, a central region rich in proline residues, and a single carboxy-terminal SH2 domain. Here we demonstrate formally that Grb2 associates with unphosphorylated SLP-76 and map the Grb2 binding site on SLP-76 undergoes rapid tyrosine phosphorylation and associates with tyrosine phosphoproteins of 36, 62, and 130 kD. In vitro experiments show that the SH2 domain of SLP-76 associates with the 62- and 130-kD proteins and additionally with a serine/threonine kinase. Finally, we demonstrate that transient overexpression of SLP-76 results in dramatically enhanced TCR-mediated induction of nuclear factor of activated T cells (NFAT) and interleukin (IL) 2 promoter activity; and we provide evidence that a functional SLP-76 SH2 domain is required for this effect. Our data document the in vivo associations of SLP-76 with several proteins that potentially participate in T cell activation and implicate SLP-76 itself as an important molecule in TCR-mediated IL-2 production.
Melanoma-specific CD4+ T cells recognize nonmutated HLA-DR-restricted tyrosinase epitopes.
Authors: Topalian S L , Gonzales M I , Parkhurst M , Li Y F , Southwood S , Sette A , Rosenberg S A , Robbins P F
Published: June 2004

Tyrosinase was the first melanoma-associated antigen shown to be recognized by CD4+ T cells. In this study, we have identified two HLA-DRB1*0401-restricted peptides recognized by these T cells: Ty 56-70 and Ty 448-462. As with many of the MHC class I-restricted melanoma epitopes, both are nonmutated self peptides that have intermediate and weak MHC binding affinities, respectively. Mutated and truncated versions of these peptides were used to define their MHC binding anchor residues. Anchor residues were then modified to derive peptides with increased MHC binding affinities and T cell stimulatory properties. Ty 56-70 and Ty 448-462 enhance the list of immunogenic HLA-A2-, A24-, and B44-restricted tyrosinase peptides already described. Thus, tyrosinase provides a model for anti-melanoma vaccines in which a single molecule can generate multivalent immunization incorporating both CD4+ and CD8+ T cell responses.
Hybrid antibody mediated veto of cytotoxic T lymphocyte responses.
Authors: Qi Y , Berg R , Singleton M A , Debrick J E , Staerz U D
Published: June 2004

Strategies are being sought that allow the induction of specific tolerance to allogeneic transplants without affecting other immune functions. The so-called veto effect has been described as one such technology where CD8+ cells suppress responses of class I MHC-restricted T-lymphocyte precursors to antigens expressed by those CD8+ veto cells. Yet, veto inhibition will not be able to provide complete tolerance to allogeneic grafts since it only operates on cell populations that express CD8. Other types of cells prevalent in most organs express different tissue-specific antigens that are recognized by alloreactive T-cells. Therefore, complete tolerance to an allogeneic transplant can only be achieved if all cellular components within the graft acquire the immune-inhibitory function. Here, we studied whether the veto effect could be exploited for this purpose nevertheless. We produced a hybrid antibody (HAb) combining a mAb specific for a class I MHC molecule with a soluble CD8 molecule. We found that this HAb specifically and effectively transferred veto inhibition to different stimulator cell populations. Thus, we have developed a strategy that promises to selectively and completely tolerize graft-specific CTLs without affecting normal immune responses.
Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin.
Authors: Feng D , Nagy J A , Hipp J , Dvorak H F , Dvorak A M
Published: June 2004

In contrast to normal microvessels, those that supply tumors are strikingly hyperpermeable to circulating macromolecules such as plasma proteins. This leakiness is largely attributable to a tumor-secreted cytokine, vascular permeability factor (VPF). Tracer studies have shown that macromolecules cross tumor vascular endothelium by way of a recently described cytoplasmic organelle, the vesiculo-vacuolar organelle or VVO (VVOs are grapelike clusters of interconnected, uncoated vesicles and vacuoles). However, equivalent VVOs are also present in the cytoplasm of normal venules that do not leak substantial amounts of plasma protein. To explain these findings, we hypothesized that VPF increased the permeability of tumor blood vessels by increasing VVO function and that the VVOs of normal venules were relatively impermeable in the absence of VPF stimulation. To test this hypothesis, VPF was injected intradermally in normal animals after intravenous injection of a soluble macromolecular tracer, ferritin, whose extravasation could be followed by electron microscopy. VPF caused normal venules to leak ferritin, and, as predicted by our hypothesis, ferritin extravasated by way of VVOs, just as in hyperpermeable tumor microvessels. Ultrathin (14-nm) serial electron microscopic sections and computer-aided three-dimensional reconstructions better defined VVO structure. VVOs occupied 16-18% of endothelial cytoplasm in normal venules. Individual VVOs were clusters of numerous (median, 124) interconnected vesicles and vacuoles that formed complex pathways across venular endothelium with multiple openings to both luminal and abluminal surfaces. Like VPF, histamine and serotonin also stimulated ferritin extravasation across venules by way of VVOs. Together, these data establish VVOs as the major pathway by which soluble plasma proteins exit venules in response to several mediators that increase venular hyperpermeability. These same mediators also increased the extravasation of colloidal carbon, but this large particulate nonphysiological tracer exited venules primarily through endothelial gaps.
Induction of passive Heymann nephritis with antibodies specific for a synthetic peptide derived from the receptor-associated protein.
Authors: Kerjaschki D , Ullrich R , Exner M , Orlando R A , Farquhar M G
Published: June 2004

Passive Heymann nephritis (pHN) is an experimental rat model for human membranous glomerulopathy. In pHN, the formation of subepithelial immune deposits (ID) involves as antigenic targets the membrane glycoprotein gp330/megalin and the 44-kD receptor-associated protein (RAP). A single binding site for ID- inducing antibodies (Abs) was previously mapped to the 86 NH2-terminal amino acids of RAP (RAP1-86). To further narrow this epitope, Abs eluted from the glomeruli were immunoblotted on membranes that were loaded with overlapping synthetic peptides representing the amino acid sequence of RAP (SPOTs system). Two adjacent Ab-binding domains with the sequences PVRLAF, (amino acids 39-44) and HSD-LKIQE (amino acids 46-53), which were separated by a single L residue at amino acid 45, were detected. Rabbit Abs raised against synthetic peptides containing these domains individually (P31-44 and P46-53) failed to procedure glomerular IDs. By contrast, Abs raised against a larger composite peptide (P31-53) induced IDs within 3d that were firmly cross linked to the glomerular basement membrane. These data suggest that Ab binding in vivo depends on the conformation of the antigenic target sequence that is preserved in the synthetic peptide P31-53, which covers the entire Ab-binding domain of RAP but not in its subdomains, P31-44 and P46-53. Collectively, these results locate the sole ID-inducing epitope of RAP to amino acids 39-53.
T cell leukemia-associated human Notch/translocation-associated Notch homologue has I kappa B-like activity and physically interacts with nuclear factor-kappa B proteins in T cells.
Authors: Guan E , Wang J , Laborda J , Norcross M , Baeuerle P A , Hoffman T
Published: June 2004

Translocation-associated Notch homologue (TAN-1), a gene originally cloned from the translocation breakpoint of a human T cell leukemia carrying a 9:7(q34.3) translocation, encodes a protein belonging to the Notch/Lin-12/Glp-1 receptor family. These receptors mediate the specification of numerous cell fates during development in invertebrates and vertebrates. The intracellular portion of Notch/TAN-1 contains six ankyrin repeats that are similar to those found in cytoplasmic I kappa B proteins. I kappa B proteins are specific inhibitors of nuclear factor (NF)-kappa B/Rel transcription factors. Here we show that TAN-1 has functional properties of an I kappa B-like regulator with specificity for the NF-kappa B p50 subunit. A recombinant polypeptide corresponding to the cytoplasmic portion of TAN-1 (TAN-1C) specifically inhibited the DNA binding of p50-containing NF-kappa B complexes. When overexpressed in an appropriate cell line, TAN-1C prevented kappa B-dependent transactivation in transient reporter gene assays in a fashion similar to the structurally related protein, Bcl-3. TAN-1C could activate kappa B-dependent gene expression by attenuating the inhibitory effect of an excess of p50 homodimers. Immunoprecipitation experiments showed that the TAN-1 from a T cell line is associated with NF-kappa B containing p50 and p65 subunits. These observations indicate that TAN-1C may directly engage NF-kappa B transcription factors and modulate nuclear gene expression.
Positive selection is not required for thymic maturation of transgenic gamma delta T cells.
Authors: Schweighoffer E , Fowlkes B J
Published: June 2004

Previously published reports describing thymic differentiation in two TCR gamma delta transgenic mouse models have suggested that gamma delta T cells require MHC-mediated positive selection to reach full maturity. Recent studies indicate that recognition of antigen by mature gamma delta T cells is not MHC restricted, raising the issue of why developing gamma delta T cells would even require MHC-driven positive selection. Therefore, we have reinvestigated the requirements for development and selection in G8 gamma delta T cell receptor (TCR) transgenic mice. Analyses of absolute cell numbers, phenotypic subsets, and functional competence of thymic and peripheral G8 gamma delta T cells indicate that these cells can fully mature in class I MHC-deficient mice. Moreover, mixed bone marrow chimeras demonstrate that gamma delta T cells of mutant B2-microglobulin (beta 2m zero) origin are partially deleted in the presence of H-2d-bearing thymocytes (previously believed to be the haplotype mediating positive selection). We conclude that there is no requirement for class I-like molecules for the maturation/development of these transgenic gamma delta T cells and that the differences in thymocyte phenotype and number observed are, instead, attributable to effects of clonal deletion.