bluuebunny/crossref_metadata_2025 huggingface dataset:
Endotoxin "priming" potentiates lung vascular abnormalities in response to Escherichia coli hemolysin: an example of synergism between endo- and exotoxin.
Authors: Walmrath D , Ghofrani H A , Rosseau S , Schütte H , Cramer A , Kaddus W , Grimminger F , Bhakdi S , Seeger W
Published: June 2004

The pore-forming hemolysin of Escherichia coli (HlyA), an important virulence factor in extraintestinal E. coli infections, causes thromboxane generation and related vasoconstriction in perfused rabbit lungs (Seeger, W., H. Walter, N. Suttorp, M. Muhly, and S. Bhakdi. 1989. J. Clin. Invest. 84:220). We investigated the influence of pulmonary vascular "priming" with endotoxin on the responsiveness of the lung to a low-dose HlyA challenge. Rabbit lungs were perfused with Krebs Henseleit buffer containing 0.1-100 ng/ml Salmonella abortus equii lipopolysaccharide (LPS) for 60-180 min. This treatment caused protracted release of tumor necrosis factor into the recirculating medium, but did not induce significant alterations of pulmonary hemodynamics and fluid balance. At a dose of 1 ng/ml, HlyA elicited only moderate thromboxane release (< 200 pg/ml) and pulmonary artery pressure increase (< or = 6 mmHg) in control lungs. Acceleration and potentiation of both the metabolic and vasoconstrictor response occurred in lungs primed with LPS. This priming effect displayed dose (threshold integral of 0.1-1 ng/ml LPS) and time dependencies (threshold integral of 60-90 min LPS incubation). Maximum thromboxane release and pulmonary artery pressure increase surpassed the responses to HlyA in nonprimed lungs by more than 15-fold. Cyclooxygenase inhibition and thromboxane-receptor antagonism blocked these effects. These data demonstrate that LPS priming synergizes with HlyA challenge to provoke vascular abnormalities that are possibly relevant to the pathogenesis of organ failure in severe local and systemic infections.
A family of serine proteases expressed exclusively in myelo-monocytic cells specifically processes the nuclear factor-kappa B subunit p65 in vitro and may impair human immunodeficiency virus replication in these cells.
Authors: Franzoso G , Biswas P , Poli G , Carlson L M , Brown K D , Tomita-Yamaguchi M , Fauci A S , Siebenlist U K
Published: June 2004

Two groups of U937 promonocytic cells were obtained by limiting dilution cloning which differed strikingly in their ability to support human immunodeficiency virus 1 (HIV-1) replication. "Plus" clones replicated the virus efficiently, whereas "minus" clones did not. We examined these clones for differences in nuclear factor (NF)-kappa B activity which might account for the observed phenomenon. Stimulation of plus clones liberated the classical p50-p65 complex from cytoplasmic pools, whereas minus clones produced an apparently novel, faster-migrating complex, as judged by electrophoretic mobility shift assays. It is surprising that the faster-migrating complex was composed also of p50 and p65. However, the p65 subunit was COOH-terminally truncated, as shown by immunoprecipitation. The truncation resulted from limited proteolysis of p65 during cellular extraction which released particular lysosomal serine proteases, such as elastase, cathepsin G, and proteinase 3. These specific proteases are coordinately expressed and were present exclusively in the minus U937 clones, but not in the plus clones, as demonstrated in the case of cathepsin G. In addition, these proteases were detected in certain subclones of THP-1 and HL-60 cells and in primary monocytes, in each case correlating with the truncated from of p65. We demonstrate in vitro cleavage of p65 by purified elastase and cathepsin G. It is possible that particular serine proteases may have inhibiting effects on the replication of HIV-1 in myelo-monocytic cells. The data also demonstrate that special precautions must be taken when making extracts from myelo-monocytic cells.
Retroviral infection of neonatal Peyer's patch lymphocytes: the mouse mammary tumor virus model.
Authors: Karapetian O , Shakhov A N , Kraehenbuhl J P , Acha-Orbea H
Published: June 2004

Mouse mammary tumor virus is known to infect newborn mice via mother's milk. A proposed key step for viral spread to the mammary gland is by the infection of lymphocytes. We show here that although in suckling mice retroviral proteins are found in all epithelial cells of the gut, viral DNA is exclusively detectable in the Peyer's patches. As early as 5 d after birth the infection leads to a superantigen response in the Peyer's patches but not in other lymphoid organs draining the intestine. Viral DNA can be detected before the superantigen response and becomes first evident in the Peyer's patches followed by mesenteric lymph nodes and finally all lymphoid organs.
Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase.
Authors: Cifone M G , De Maria R , Roncaioli P , Rippo M R , Azuma M , Lanier L L , Santoni A , Testi R
Published: June 2004

Intracellular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed cell surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monoclonal antibody was produced by immunizing mice with human CD95-transfected L cells. Crosslinking of CD95 with DX2 resulted in the activation of a sphingomyelinase (SMase) in promyelocytic U937 cells, as well as in other human tumor cell lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomyelin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labeled SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hydrolysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to cell-permeant C2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be relevant in defining the biochemical nature of intracellular messengers leading to apoptotic cell death.
Induction of interleukin 2 receptor beta chain expression by self-recognition in the thymus.
Authors: Hanke T , Mitnacht R , Boyd R , Hünig T
Published: June 2004

1-2% of adult mouse thymocytes express the T cell receptor alpha/beta (TCR-alpha/beta) together with the interleukin (IL) 2R beta (p70), but not the alpha (p 55) chain. We show that the previously described alpha/beta-TCR +CD4-8- and the partially overlapping Ly6C+ thymocytes are contained within this subset. Most IL-2R beta+ alpha/beta-TCR+ cells have a mature and activated (heat stable antigen [HSA]-, thymic shared antigen 1 [TSA-1]-, CD44high, CD69+) phenotype. Overrepresentation of V beta 8.2 in both CD4-8- and CD4 and/or CD8+ IL-2R beta+ thymocytes suggests that IL-2R beta expression is induced by a TCR-mediated activation event. In mice transgenic for an H-2Kb-specific TCR, IL-2R beta+ cells were abundant under conditions of mainstream negative selection, i.e., in the presence of Kb, but absent under conditions of mainstream positive selection or in a nonselecting environment. Together, these results show that in addition to clonal deletion, self-recognition by immature thymocytes leads to phenotypic maturation of a small subset of thymocytes expressing IL-2R beta. IL-2-deficient mice contain normal numbers of IL-2R beta+ alpha/beta-TCR+ thymocytes, indicating that like mainstream T cell development, this minor pathway of positive selection does not depend on IL-2. However, in the absence of IL-2, the CD4/CD8 subset composition of IL-2R beta+ thymocytes is skewed towards CD4-8+, mostly at the expense of CD4-8-. A possible relevance of this finding for the development of the immune pathology of IL-2-deficient mice is discussed.
T cell clones from an X-linked hyper-immunoglobulin (IgM) patient induce IgE synthesis in vitro despite expression of nonfunctional CD40 ligand.
Authors: Life P , Gauchat J F , Schnuriger V , Estoppey S , Mazzei G , Durandy A , Fischer A , Bonnefoy J Y
Published: June 2004

The induction of immunoglobulin E (IgE) switching in B cells requires at least two signals. The first is given by either of the soluble lymphokines interleukin 4 (IL-4) or IL-13, whereas the second is contact dependent. It has been widely reported that a second signal can be provided by the CD40 ligand (CD40L) expressed on the surface of T cells, mast cells, and basophils. A defect in the CD40L has been shown recently to be responsible for the lack of IgE, IgA, and IgG, characteristic of the childhood X-linked immunodeficiency, hyper IgM syndrome (HIGM1). IgE can however be detected in the serum of some HIGM1 patients. In this study, we isolated T cell clones and lines using phytohemagglutinin (PHA) and allergen, respectively, from the peripheral blood of one such patient who expressed a truncated form of CD40L, and investigated their ability to induce IgE switching in highly purified, normal tonsillar B cells in vitro. Unexpectedly, 4 of 12 PHA clones tested induced contact-dependent IgE synthesis in the presence of exogenous IL-4. These clones were also shown to strongly upregulated IL-4-induced germline epsilon RNA and formed dense aggregates with B cells. Of the four helper clones, three were CD8+, of which two were characteristic of the T helper cell 2 (Th2) subtype. Two allergen-specific HIGM1 T cell lines, both of the Th0 subtype, could also drive IgE synthesis when prestimulated using specific allergen. All clones and lines were negative for surface expression of CD40L, and the mutated form of CD40L was confirmed for a representative clone by RNase protection assay and sequencing. The IgE helper activity could not be attributed to membrane tumor necrosis factor alpha (TNF-alpha) although it was strongly expressed on activated clones, and the addition of neutralizing anti-TNF-alpha antibody did not abrogate IgE synthesis. These results therefore suggest the involvement of T cell surface molecules other than CD40L in the induction of IgE synthesis, and that these molecules may also be implicated in other aspects of T-B cell interactions.
Thymic selection and adaptability of cytotoxic T lymphocyte responses in transgenic mice expressing a viral protein in the thymus.
Authors: von Herrath M G , Dockter J , Nerenberg M , Gairin J E , Oldstone M B
Published: June 2004

Upon primary challenge with lymphocytic choriomeningitis virus (LCMV), H-2d (BALB/cByJ) mice mount a cytotoxic T lymphocyte (CTL) response to a single immunodominant domain of the viral nucleoprotein (NP) but no detectable response to the viral glycoprotein (GP). To manipulate this CTL response, the viral NP gene was expressed in the thymus and peripheral T lymphocytes using the murine Thy1.2 promoter. As a result, such Thy1.2-NP (H-2d) transgenic (tg) mice deleted their high-affinity anti-LCMV-NP CTL, but generated equal numbers of lower-affinity NP CTL. Further, they made an alternative anti-LCMV-GP CTL response that is not normally found in non-tg mice indicating a hierarchial control of the CTL response. Unlike the H-2d mice, H-2b (C57Bl/6J) mice normally mount a CTL response to both LCMV-GP and -NP. When the LCMV-NP was expressed using the Thy1.2 promoter in these H-2b mice, the LCMV-NP-specific CTL response was completely aborted and no CTL to new, alternative viral epitopes were generated. Dilutions of H-2b or H-2d NP peptides indicated that 3-4 logs less H-2b NP peptide was required to sensitize syngeneic target cells for CTL-specific lysis, suggesting that the differing affinities of H-2b and H-2d major histocompatibility complex molecules for their peptides likely account for the total removal of NP CTL in the H-2b mice but only partial removal in H-2d mice made to express thymic NP. Thymic grafting experiments done with thymi from newborn Thy1.2-NP tg mice show that selection processes studied in this model are of central (thymic) origin and are not caused by Thy1.2-positive LCMV-NP-expressing T lymphocytes in the periphery.
Thromboxane A2 receptor blocking abrogates donor-specific unresponsiveness to renal allografts induced by thymic recognition of major histocompatibility allopeptides.
Authors: Remuzzi G , Noris M , Benigni A , Imberti O , Sayegh M H , Perico N
Published: June 2004

Recent in vitro studies have documented that thromboxane (Tx)A2 induces thymocyte apoptosis by acting on specific receptors abundantly expressed on the surface of immature T lymphocytes. No information is available on the in vivo relevance of this observation in development of self- or acquired tolerance. We and others have previously documented that injection of donor cells into adult thymus of experimental animals induced specific systemic unresponsiveness to allografts in the rat and mouse models. More recently, we have shown that intrathymic injection of synthetic class II major histocompatibility complex (MHC) allopeptides resulted in donor-specific unresponsiveness to renal allografts. The induction of unresponsiveness was abrogated by recipient thymectomy within the first week. We now report the effect of TxA2 blockade on acquired thymic tolerance to renal allografts induced by intrathymic injection of synthetic class II MHC allopeptides in the Wistar-Furth (WF) to Lewis rat strain combination. Administration of the TxA2 receptor blocker prior to transplantation or 2 wk postengraftment completely abrogated the unresponsive state. In addition, inhibiting the TxA2-forming enzyme by aspirin or dexamethasone also abolished the induction of acquired thymic tolerance. Evidence is also provided for a critical "dose" of peptides to be injected into the thymus to induce systemic unresponsiveness to renal allografts. These data, coupled with observations that activated peripheral T cells can circulate through the thymus, provide evidence that TxA2/TxA2 receptor interaction in the thymic microenvironment, leading to anergy/programmed cell death of activated T cells, may play an important role in the development of acquired unresponsiveness in vivo.
Persistence of human T cell lymphotropic virus type 1 (HTLV-1) sequences in peripheral blood mononuclear cells from patients with mycosis fungoides.
Authors: Manca N , Piacentini E , Gelmi M , Calzavara P , Manganoni M A , Glukhov A , Gargiulo F , De Francesco M , Pirali F , De Panfilis G , Turano A
Published: June 2004

Mycosis fungoides (MF) is a rare form of cutaneous T cell lymphoma suspected of having a viral etiology. As in adult T cell leukemia, the virus involved may be human T lymphotropic virus type 1 (HTLV-1). We cultured the peripheral blood mononuclear cells (PBMC) of 29 patients with MF HTLV-1 seronegative by enzyme-linked immunosorbent assay and Western blot. The presence of reverse transcriptase (RT) and p24 antigen was investigated in the concentrate supernatant of the culture. The DNA of all studied patients was submitted to polymerase chain reaction and Southern blot analysis using primers and probes recognizing the tax region of HTLV-1/2 and the pol region of HTLV-1. 10 of 29 patients were found positive to HTLV-1, whereas they were always negative to RT and p24. The same results were confirmed in double blind after 6 mo. Our findings suggest HTLV-1 may be involved in the etiology of MF, at least in certain cases.
T cell cross-reactivity between coxsackievirus and glutamate decarboxylase is associated with a murine diabetes susceptibility allele.
Authors: Tian J , Lehmann P V , Kaufman D L
Published: June 2004

Limited regions of amino acid sequence similarity frequently occur between microbial antigens and host proteins. It has been widely anticipated that during infection such sequence similarities could induce cross-reactive T cell responses, thereby initiating T cell-mediated autoimmune disease. However, the nature of major histocompatibility complex (MHC)-restricted antigen presentation confers a number of constraints that should make this type of T cell cross-reactivity a rare, MHC allele-dependent event. We tested this prediction using two insulin-dependent diabetes mellitus (IDDM)-associated antigens, coxsackievirus P2-C (Cox P2-C) protein and glutamate decarboxylase (GAD65), which share a prototypic sequence similarity of six consecutive amino acids within otherwise unrelated proteins. We surveyed a panel of 10 murine MHC class II alleles that encompass the spectrum of standard alleles for the ability to cross-reactively present Cox P2-C and GAD65. Out of the 10 restriction elements tested, the sequence similarity regions were both dominant determinants and were cross-reactively displayed after the natural processing of whole antigens, only in the context of I-Anod. These data show that cross-reactive T cell recognition of sequence similarity regions in unrelated proteins is confined to certain MHC alleles, which may explain MHC association with autoimmune disease. It is striking that these two diabetes-associated antigens were cross-reactively recognized only in the context of a diabetes susceptibility allele. Since the human and the murine class II alleles associated with IDDM share conserved features, cross-reactive T cell recognition of GAD65 and Cox P2-C may contribute to the pathogenesis of human IDDM and account for the epidemiological association of coxsackievirus with IDDM.
Protective effect of 55- but not 75-kD soluble tumor necrosis factor receptor-immunoglobulin G fusion proteins in an animal model of gram-negative sepsis.
Authors: Evans T J , Moyes D , Carpenter A , Martin R , Loetscher H , Lesslauer W , Cohen J
Published: June 2004

The aim of this study was to compare the ability of both a 55- and 75-kD soluble tumor necrosis factor receptor immunoglobulin G fusion protein (sTNFR-IgG) in protecting against death in a murine model of gram-negative sepsis. Pretreatment with 250 micrograms of the p75 construct delayed but did not avert death in this model, reducing peak bioactive TNF-alpha levels after infection from 76.4 ng ml-1 in control mice to 4.7 ng ml-1 in the treated group (p < 0.05, two-sample t test). However, these low levels of bioactive TNF-alpha persisted in the p75 fusion protein-treated animals compared with the controls and were sufficient to mediate delayed death. In contrast, pretreatment with 200 micrograms of the p55 sTNFR-IgG gave excellent protection against death with complete neutralization of circulating TNF. Studies of the binding of TNF-alpha with the soluble TNFR fusion proteins showed that the p75 fusion construct exchanges bound TNF-alpha about 50-100-fold faster than the p55 fusion protein. Thus, although both fusion proteins in equilibrium bind TNF-alpha with high affinity, the TNF-alpha p55 fusion protein complex is kinetically more stable than the p75 fusion construct, which thus acts as a TNF carrier. The persistent release of TNF-alpha from the p75 fusion construct limits its therapeutic effect in this model of sepsis.
Delivery of a viral antigen to the class I processing and presentation pathway by Listeria monocytogenes.
Authors: Ikonomidis G , Paterson Y , Kos F J , Portnoy D A
Published: June 2004

Listeria monocytogenes is a facultative intracellular pathogen that grows in the cytoplasm of infected host cells. We examined the capacity of L. monocytogenes to introduce influenza nucleoprotein (NP) into the class I pathway of antigen presentation both in vitro and in vivo. Recombinant L. monocytogenes secreting a fusion of listeriolysin O and NP (LLO-NP) targeted infected cells for lysis by NP-specific class I-restricted cytotoxic T cells. Antigen presentation occurred in the context of three different class I haplotypes in vitro. A hemolysin-negative L. monocytogenes strain expressing LLO-NP was able to present in a class II-restricted manner. However, it failed to target infected cells for lysis by CD8+ T cells, indicating that hemolysin-dependent bacterial escape from the vacuole is necessary for class I presentation in vitro. Immunization of mice with a recombinant L. monocytogenes strain that stably expressed and secreted LLO-NP induced NP-specific CD8+ cytotoxic T lymphocytes. These studies have implications for the use of L. monocytogenes to deliver potentially any antigen to the class I pathway in vivo.
Reversal of experimental autoimmune encephalomyelitis by a soluble peptide variant of a myelin basic protein epitope: T cell receptor antagonism and reduction of interferon gamma and tumor necrosis factor alpha production.
Authors: Karin N , Mitchell D J , Brocke S , Ling N , Steinman L
Published: June 2004

An immunodominant epitope of myelin basic protein (MBP), VHFFKNIVTPRTP (p87-99), is a major target of T cells in lesions of multiple sclerosis (MS) and in experimental allergic encephalomyelitis (EAE). T cells found in EAE lesions bear the same amino acids in the third complementary determining region of the T cell receptor (TCR) as those found in MS lesions. We analyzed the trimolecular interactions between MBP p87-99, class II major histocompatibility complex (MHC), and TCR, and designed soluble inhibitors for therapy. F, N, I, and V at positions 90, 92, 93, and 94 interact with MHC, whereas K, T, and P at positions 91, 95, and 96 interact with TCR. The peptides, p87-99[95T > A] and p87-99[96P > A] could compete more effectively with p87-99 for binding to MHC and could antagonize the in vitro response to T cells to p87-99 more effectively than p87-99[91K > A]. However, only p87-99[91K > A] prevented and reversed EAE, indicating that the extent of MHC or TCR competition does not predict success in treating EAE. To elucidate the mechanism of inhibition of EAE, draining lymph node cells from rats immunized with the native peptide alone or together with each of the three TCR antagonists were challenged in vitro with p87-99. Administration of p87-99[91K > A], but not p87-99 [95T > A] or p87-99[96P > A], reduced the production of tumor necrosis factor (TNF)- alpha and interferon (IFN) gamma. IFN-gamma and TNF-alpha are two cytokines that are critical in the pathogenesis of EAE and MS.
The germfree state prevents development of gut and joint inflammatory disease in HLA-B27 transgenic rats.
Authors: Taurog J D , Richardson J A , Croft J T , Simmons W A , Zhou M , Fernández-Sueiro J L , Balish E , Hammer R E
Published: June 2004

A number of inflammatory disease states occur with greatly increased frequency in individuals inheriting the human major histocompatibility complex class I allele HLA-B27. In a minority of cases, namely those with B27-associated reactive arthritis, there is good evidence that the disease state is triggered by infection with an enteric or genitourinary bacterial pathogen. For the majority of B27-associated disease, no definite pathogenetic role for bacteria has been established. However, in these latter cases intestinal inflammation can often be demonstrated, and it sometimes occupies a major part of the clinical picture. Rats transgenic for B27 are known to develop a disorder resembling B27-associated human disease, with prominent intestinal, joint, skin, and male genital inflammatory lesions. We report here that B27 transgenic rats raised in a germfree environment do not develop inflammatory intestinal or peripheral joint disease, whereas the skin and genital inflammatory lesions are unaffected by the germfree state. These findings support the concept that gut and joint inflammation are pathogenetically closely related, and they provide direct evidence that the commensal gut flora play an important role in the pathogenesis of B27-associated gut and joint inflammation.
Functional Myc-Max heterodimer is required for activation-induced apoptosis in T cell hybridomas.
Authors: Bissonnette R P , McGahon A , Mahboubi A , Green D R
Published: June 2004

T cell hybridomas respond to activation signals by undergoing apoptotic cell death, and this is likely to represent comparable events related to tolerance induction in immature and mature T cells in vivo. Previous studies using antisense oligonucleotides implicated the c-Myc protein in the phenomenon of activation-induced apoptosis. This role for c-Myc in apoptosis is now confirmed in studies using a dominant negative form of its heterodimeric binding partner, Max, which we show here inhibits activation-induced apoptosis. Further, coexpression of a reciprocally mutant Myc protein capable of forming functional heterodimers with the mutant Max can compensate for the dominant negative activity and restore activation-induced apoptosis. These results imply that Myc promotes activation-induced apoptosis by obligatory heterodimerization with Max, and therefore, by regulating gene transcription.
Different roles for the Fc epsilon RI gamma chain as a function of the receptor context.
Authors: Paolini R , Renard V , Vivier E , Ochiai K , Jouvin M H , Malissen B , Kinet J P
Published: June 2004

The high affinity immunoglobulin E receptor (Fc epsilon RI) and the B and T cell antigen receptors (TCR) are multimeric complexes containing subunits with cytoplasmic antigen recognition activation motifs (ARAMs). The presence of multiple motifs may be a way to amplify a single signal or provide independent activation modules. Here we have compared the signaling capacity of the same Fc epsilon RI gamma motif in the context of two different receptors, Fc epsilon RI and TCR/CD3, simultaneously reconstituted on the surface of the same zeta-deficient T cell line. Both reconstituted receptors mediate early (phosphorylation) and late (interleukin [IL]-2 release) signals. Mutation of the two tyrosine residues of ARAM gamma alters early signaling by both receptors, but the set of substrates phosphorylated via ARAM gamma is different for each receptor and is thus dependent on the receptor context. Furthermore, the mutations prevent Fc epsilon RI- but not TCR/CD3-mediated IL-2 release. These data demonstrate that ARAM gamma is necessary for allowing both receptors to phosphorylate the complete set of substrates, and that the CD3 complex, unlike the Fc epsilon RI beta chain, contains activation modules capable of compensating for the absence of a functional ARAM gamma in generating late signals such as IL-2 release.
Expression of the herpes simplex virus type 2 latency-associated transcript enhances spontaneous reactivation of genital herpes in latently infected guinea pigs.
Authors: Krause P R , Stanberry L R , Bourne N , Connelly B , Kurawadwala J F , Patel A , Straus S E
Published: June 2004

The latency-associated transcript (LAT) is the only herpes simplex virus (HSV) gene product detectable in latently infected humans and animals. In this report, we show that a 624-bp deletion in the promoter of the HSV-2 LAT had no discernable effect on viral growth in tissue culture or in acute genital infection of guinea pigs, but impaired LAT accumulation and led to a marked decrease in spontaneous genital recurrences when compared with the behavior of wild-type and rescuant strains. Differences in the ability of the mutant to replicate, or in how readily it established or maintained latency did not account for this finding. Thus, HSV LAT expression facilitates the spontaneous reactivation of latent virus.
CTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells.
Authors: Schneider H , Prasad K V , Shoelson S E , Rudd C E
Published: June 2004

CTLA-4 is a T cell antigen that is structurally related to CD28 and serves as a high affinity ligand for the B cell antigen B7-1/2. Unlike CD28, the function of CTLA-4 is unclear, although reports have implicated the antigen in the costimulation of T cells. Recently, phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated in the costimulatory function of CD28 by virtue of its ability to bind to a pYMNM motif within the cytoplasmic tail of the antigen. In this study, we show that CTLA-4 can also associate with PI 3-kinase as detected by lipid kinase analysis and immunoblotting with anti-p85 antiserum. High pressure liquid chromatographic separation of deacylated lipids showed the presence of a peak corresponding to PI-3-P. Anti-CTLA-4 ligation of the receptor induced a significant increase in the levels of precipitable PI 3-kinase activity. Peptide binding studies revealed that the NH2- and COOH-terminal SH2 domains of p85 bind the CTLA-4 cytoplasmic pYVKM motif with an affinity (ID50: 0.6 and 0.04 microM), that is similar to CD28. CTLA-4 binding to PI 3-kinase provides further evidence that CTLA-4 is not an inert counterreceptor, but rather is coupled to an intracellular signaling molecule with the capacity to regulate cell growth.
Mobilization of long-term reconstituting hematopoietic stem cells in mice by recombinant human interleukin 7.
Authors: Grzegorzewski K J , Komschlies K L , Jacobsen S E , Ruscetti F W , Keller J R , Wiltrout R H
Published: June 2004

Administration of recombinant human interleukin 7 (rh)IL-7 to mice has been reported by our group to increase the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakarocyte macrophage) from the bone marrow to peripheral organs (blood, spleen[s], and liver). We now report that IL-7 also stimulates a sixfold increase in the number of more primitive CFU-S day 8 (CFU-S8) and day 12 (CFU-S12) in the peripheral blood leukocytes (PBL) of mice treated with rhIL-7 for 7 d. Moreover, > 90% of lethally irradiated recipient mice that received PBL from rhIL-7-treated donor mice have survived for > 6 mo whereas none of the recipient mice that received an equal number of PBL from diluent-treated donors survived. Flow cytometry analysis at 3 and 6 mo after transplantation revealed complete trilineage (T, B, and myelomonocytic cell) repopulation of bone marrow, thymus, and spleen by blood-borne stem/progenitor cells obtained from rhIL-7-treated donor mice. Thus, IL-7 may prove valuable for mobilizing pluripotent stem cells with long-term repopulating activity from the bone marrow to the peripheral blood for the purpose of gene modification and/or autologous or allogeneic stem cell transplantation.
Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12.
Authors: Leonard J P , Waldburger K E , Goldman S J
Published: June 2004

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that can be transferred to naive mice via CD4+ T cells isolated from appropriately immunized mice. We have evaluated the effects of recombinant murine interleukin 12 (rmIL-12), a potent inducer of interferon gamma (IFN-gamma) and promoter of Th1 T cell development, on the course of adoptively transferred EAE. The transfer of lymph node cells (LNC) isolated from proteolipid protein (PLP)-primed animals and stimulated in vitro with PLP to naive mice resulted in a progressive paralytic disease culminating in complete hind limb paralysis in the majority of the recipients. When mice were injected with LNC that had been stimulated in vitro with PLP in the presence of rmIL-12, the subsequent course of disease was more severe and prolonged. The addition of rmIL-12 during the in vitro stimulation with PLP resulted in a 10-fold increase in IFN-gamma and a 2-fold increase in tumor necrosis factor (TNF) alpha in the supernatants, relative to LNC stimulated with PLP alone. However, neutralization of IFN-gamma or TNF-alpha in vitro with specific antibodies did not abrogate the ability of rmIL-12 to exacerbate the subsequent disease. Similarly, mice treated with rmIL-12 in vivo after the transfer of antigen-stimulated LNC developed a more severe and prolonged course of disease compared with vehicle-treated control animals. In contrast, treatment of mice with an antibody to murine IL-12 after cell transfer completely prevented paralysis, with only 40% of the mice developing mild disease. These results demonstrate that in vitro stimulation of antigen primed LNC with PLP and rmIL-12 enhances their subsequent encephalitogenicity. Furthermore, inhibition of endogenous IL-12 in vivo after LNC transfer prevented paralysis, suggesting that endogenous IL-12 plays a pivotal role in the pathogenesis of this model of autoimmune disease.
Activation of the p21ras pathway couples antigen receptor stimulation to induction of the primary response gene egr-1 in B lymphocytes.
Authors: McMahon S B , Monroe J G
Published: June 2004

The primary response gene egr-1 encodes a sequence-specific transcription factor whose expression is necessary for antigen receptor-stimulated activation of B lymphocytes. The molecular processes involved in linking egr-1 induction to antigen receptor signaling have not been defined. The present study demonstrates that expression of an activated form of p21ras results in egr-1 induction similar to that previously shown after antigen receptor cross-linking. In addition, both antigen receptor cross-linking and p21ras use the same element in the egr-1 promoter to exert their effects. Using dominant-negative mutants of p21ras and raf-1, we demonstrate that induction of egr-1 after antigen receptor cross-linking is mediated by activation of the p21ras/mitogen-activated protein kinase signaling pathway. While regulation of the p21ras pathway during B cell activation has been intensively studied, this report represents the first description of a biologically relevant event associated with its activation.
Fas ligand mediates activation-induced cell death in human T lymphocytes.
Authors: Alderson M R , Tough T W , Davis-Smith T , Braddy S , Falk B , Schooley K A , Goodwin R G , Smith C A , Ramsdell F , Lynch D H
Published: June 2004

A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T cells resulted in the expression of Fas-L mRNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T cells is mediated by Fas/Fas-L interactions.
Intraislet release of interleukin 1 inhibits beta cell function by inducing beta cell expression of inducible nitric oxide synthase.
Authors: Corbett J A , McDaniel M L
Published: June 2004

Cytokines, released in and around pancreatic islets during insulitis, have been proposed to participate in beta-cell destruction associated with autoimmune diabetes. In this study we have evaluated the hypothesis that local release of the cytokine interleukin 1 (IL-1) by nonendocrine cells of the islet induce the expression of inducible nitric oxide synthase (iNOS) by beta cells which results in the inhibition of beta cell function. Treatment of rat islets with a combination of tumor necrosis factor (TNF) and lipopolysaccharide (LPS), conditions known to activate macrophages, stimulate the expression of iNOS and the formation of nitrite. Although TNF+LPS induce iNOS expression and inhibit insulin secretion by intact islets, this combination does not induce the expression of iNOS by beta or alpha cells purified by fluorescence activated cell sorting (Facs). In contrast, IL-1 beta induces the expression of iNOS and also inhibits insulin secretion by both intact islets and Facs-purified beta cells, whereas TNF+LPS have no inhibitory effects on insulin secretion by purified beta cells. Evidence suggests that TNF+LPS inhibit insulin secretion from islets by stimulating the release of IL-1 which subsequently induces the expression of iNOS by beta cells. The IL-1 receptor antagonist protein completely prevents TNF+LPS-induced inhibition of insulin secretion and attenuates nitrite formation from islets, and neutralization of IL-1 with antisera specific for IL-1 alpha and IL-1 beta attenuates TNF+LPS-induced nitrite formation by islets. Immunohistochemical localization of iNOS and insulin confirm that TNF+LPS induce the expression of iNOS by islet beta cells, and that a small percentage of noninsulin-containing cells also express iNOS. Local release of IL-1 within islets appears to be required for TNF+LPS-induced inhibition of insulin secretion because TNF+LPS do not stimulate nitrite formation from islets physically separated into individual cells. These findings provide the first evidence that a limited number of nonendocrine cells can release sufficient quantities of IL-1 in islets to induce iNOS expression and inhibit the function of the beta cell, which is selectively destroyed during the development of autoimmune diabetes.
Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton.
Authors: Valitutti S , Dessing M , Aktories K , Gallati H , Lanzavecchia A
Published: June 2004

Using antigen-specific T cell clones and peptide-pulsed antigen-presenting cells (APCs) we investigated the mechanisms that lead to sustained signaling, known to be required for activation of effector function. Four lines of evidence indicate that the T cell actin cytoskeleton plays a crucial role in T cell activation by antigen-pulsed APCs, but is not required when T cell receptor (TCR) is cross-linked by soluble antibodies. First, addition of antibodies to the major histocompatibility complex molecules recognized by the TCR aborts the ongoing intracellular calcium concentration ([Ca2+]i) increase in performed T-APC conjugates, indicating that the sustained signaling requires the continuous occupancy of TCR. Second, time-lapse image recording shows that T lymphocytes conjugated to peptide-pulsed APCs undergo a sustained [Ca2+]i increase, which is accompanied by the formation of a large and changing area of contact between the two opposing membranes. Third, drugs that disrupt the actin cytoskeleton, Cytochalasin D and and C2 Clostridium botulinum toxin induce a rapid block of [Ca2+]i rise, coincident with a block of the cyclic changes in T cell shape. Finally, the addition of Cytochalasin D or of anti-MHC antibodies to preformed conjugates inhibits interferon gamma production in an 1-antigen dose- and time-dependent fashion. These results identify T cell actin cytoskeleton as a major motor for sustaining signal transduction and possibly for driving TCR cross-linking and offer an explanation for how T cells equipped with low affinity TCR can be triggered by a small number of complexes on APCs.
Monoclonal antibodies specific for murine p55 and p75 tumor necrosis factor receptors: identification of a novel in vivo role for p75.
Authors: Sheehan K C , Pinckard J K , Arthur C D , Dehner L P , Goeddel D V , Schreiber R D
Published: June 2004

Monoclonal antibodies (mAbs) specific for the murine p55 and p75 tumor necrosis factor (TNF) receptors were produced after immunization of Armenian hamsters with the purified soluble extracellular domains of each receptor protein. Four p55- (55R) and five p75 (TR75)-reactive mAbs immunoprecipitated the appropriate receptor from the surface of L929 cells. None of the mAbs cross-reacted with the other TNF receptor form. The mAbs were functionally characterized by their ability to inhibit ligand binding and influence TNF-dependent L cell cytolytic activity or proliferation of the murine cytolytic T cell clone CT6. One p55-specific mAb, 55R-593, displayed agonist activity, while two other p55-specific mAbs (55R-170 and -176) were found to be TNF antagonists. The fourth mAb (55R-286) had no functional effects on cells. Several antibodies specific for the p75 TNF receptor partially inhibited recombinant murine TNF-alpha-dependent cytolytic activity (60%). Blocking mAbs specific for p75 but not anti-p55 inhibited TNF-mediated proliferation of CT6 T cells. When used in vivo, p55- but not p75-specific mAbs protected mice from lethal endotoxin shock and blocked development of a protective response against Listeria monocytogenes infection. In contrast, both p55 and p75 mAbs individually blocked development of skin necrosis in mice treated with murine TNF-alpha. These data thus demonstrate the utility of the two families of murine TNF receptor-specific mAbs and identify a novel function of the p75 TNF receptor in vivo.
Interleukin 12 administration induces T helper type 1 cells and accelerates autoimmune diabetes in NOD mice.
Authors: Trembleau S , Penna G , Bosi E , Mortara A , Gately M K , Adorini L
Published: June 2004

T cells play a major role in the development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. Administration of interleukin 12 (IL-12), a key cytokine which guides the development of T helper type 1 (Th1) CD4+ T cells, induces rapid onset of IDDM in NOD, but not in BALB/c mice. Histologically, IL-12 administration induces massive infiltration of lymphoid cells, mostly T cells, in the pancreatic islets of NOD mice. CD4+ pancreas-infiltrating T cells, after activation by insolubilized anti T cell receptor antibody, secrete high levels of interferon gamma and low levels of IL-4. Therefore, IL-12 administration accelerates IDDM development in genetically susceptible NOD mice, and this correlates with increased Th1 cytokine production by islet-infiltrating cells. These results hold implications for the pathogenesis, and possibly for the therapy of IDDM and of other Th1 cell-mediated autoimmune diseases.
Ligation of CD38 suppresses human B lymphopoiesis.
Authors: Kumagai M , Coustan-Smith E , Murray D J , Silvennoinen O , Murti K G , Evans W E , Malavasi F , Campana D
Published: June 2004

CD38 is a transmembrane glycoprotein expressed in many cell types, including lymphoid progenitors and activated lymphocytes. High levels of CD38 expression on immature lymphoid cells suggest its role in the regulation of cell growth and differentiation, but there is no evidence demonstrating a functional activity of CD38 on these cells. We used stroma-supported cultures of B cell progenitors and anti-CD38 monoclonal antibodies (T16 and IB4) to study CD38 function. In cultures of normal bone marrow CD19+ cells (n = 5), addition of anti-CD38 markedly reduced the number of cells recovered after 7 d. Cell loss was greatest among CD19+ sIg- B cell progenitors (mean cell recovery +/- SD = 7.2 +/- 11.7% of recovery in control cultures) and extended to CD19+CD34+ B cells (the most immature subset; 7.6 +/- 2.2%). In contrast, CD38 ligation did not substantially affect cell numbers in cultures of normal peripheral blood or tonsillar B cells. In stroma-supported cultures of 22 B-lineage acute lymphoblastic leukemia cases, anti-CD38 suppressed recovery of CD19+ sIg- leukemic cells. CD38 ligation also suppressed the growth of immature lymphoid cell lines cultured on stroma and, in some cases, in the presence of stroma-derived cytokines (interleukin [IL] 7, IL-3, and/or stem cell factor), but did not inhibit growth in stroma- or cytokine-free cultures. DNA content and DNA fragmentation studies showed that CD38 ligation of stroma-supported cells resulted in both inhibition of DNA synthesis and induction of apoptosis. It is known that CD38 catalyzes nicotinamide adenine dinucleotide (NAD+) hydrolysis into cyclic ADP-ribose (cADPR) and ADPR. However, no changes in NAD+ hydrolysis or cADPR and ADPR production after CD38 ligation were found by high-performance liquid chromatography; addition of NAD+, ADPR, or cADPR to cultures of lymphoid progenitors did not offset the inhibitory effects of anti-CD38. Thus, anti-CD38 does not suppress B lymphopoiesis by altering the enzymatic function of the molecule. In conclusion, these data show that CD38 ligation inhibits the growth of immature B lymphoid cells in the bone marrow microenvironment, and suggest that CD38 interaction with a putative ligand represents a novel regulatory mechanism of B lymphopoiesis.
Antibody-mediated autoimmune myocarditis depends on genetically determined target organ sensitivity.
Authors: Liao L , Sindhwani R , Rojkind M , Factor S , Leinwand L , Diamond B
Published: June 2004

Injury to cardiac myocytes often leads to the production of anti-myosin antibodies. While these antibodies are a marker of myocardial injury, their contribution to pathogenesis in diseases such as autoimmune myocarditis or rheumatic fever is much less clear. We demonstrate in this report that monoclonal anti-myosin antibodies can mediate myocarditis in a susceptible mouse strain. Additionally, we show disease susceptibility depends on the presence of myosin or a myosin-like molecule in cardiac extracellular matrix. This study demonstrates that susceptibility to autoimmune heart disease depends not only on the activation of self-reactive lymphocytes but also on genetically determined target organ sensitivity to autoantibodies.
Breakdown of B cell tolerance in a mouse model of systemic lupus erythematosus.
Authors: Roark J H , Kuntz C L , Nguyen K A , Caton A J , Erikson J
Published: June 2004

Anti-DNA antibodies, specifically those that stain nuclei in a homogenous nuclear (HN) fashion, are diagnostic of systemic lupus erythematosus (SLE) and the MRL-lpr/lpr SLE murine model. We have used a heavy chain transgene that increases the frequency of anti-HN antibodies to address whether their production in SLE is the consequence of a defect in B cell tolerance. Anti-HN B cells were undetectable in nonautoimmune-prone transgenic mice, but in MRL-lpr/lpr transgenic mice their Ig was evident in the sera and they were readily retrievable as hybridomas. We conclude that nonautoimmune animals actively delete anti-HN-specific B cells, and that MRL-lpr/lpr mice are defective in this process possibly because of the lpr defect in the fas gene.
κ+λ+ dual receptor B cells are present in the human peripheral repertoire.
Authors: Giachino C , Padovan E , Lanzavecchia A
Published: June 2004

It is a common notion that mature B lymphocytes express either kappa or lambda light (L) chains, although the mechanism that leads to such isotypic exclusion is still debated. We have investigated the extent of L chain isotypic exclusion in normal human peripheral blood B lymphocytes. By three-color staining with anti-CD19, anti-kappa, and anti-lambda antibodies we could estimate that 0.2-0.5% of peripheral blood B cells from healthy adults express both kappa and lambda on the cell surface. The kappa+lambda+ cells were sorted, immortalized by Epstein-Barr virus, and five independent clones were characterized in detail. All clones express both kappa and lambda on the cell surface and produce immunoglobulin M that contain both kappa and lambda chains in the same molecule, i.e., hybrid antibodies. Sequencing of the L chains revealed in three out of five clones evidence for somatic mutations. It is interesting to note that among a panel of single receptor B cell clones we identified two lambda+ clones that carried a productively rearranged kappa, which was inactivated by a stop codon generated by somatic mutation. These findings indicate that dual receptor B lymphocytes can be found among mature antigen-selected B cells and suggest that somatic mutation can contribute to increase the degree of isotypic exclusion by inactivating a passenger, nonselected L chain.