Disruption of Akt kinase activation is important for immunosuppression induced by measles virus.

Surface-contact-mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2-dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

The role of NF-AT transcription factors in T cell activation and differentiation.

The family of genuine NF-AT transcription factors consists of four members (NF-AT1 [or NF-ATp], NF-AT2 [or NF-ATc], NF-AT3 and NF-AT4 [or NF-ATx]) which are characterized by a highly conserved DNA binding domain (is designated as Rel similarity domain) and a calcineurin binding domain. The binding of the Ca(2+)-dependent phosphatase calcineurin to this region controls the nuclear import and exit of NF-ATs. This review deals (1) with the structure of NF-AT proteins, (2) the DNA binding of NF-AT factors and their interaction with AP-1, (3) NF-AT target genes, (4) signalling pathways leading to NF-AT activation: the role of protein kinases and calcineurin, (5) the nuclear entry and exit of NF-AT factors, (6) transcriptional transactivation by NF-AT factors, (7) the structure and expression of the chromosomal NF-AT2 gene, and (8) NF-AT factors in Th cell differentiation. The experimental data presented and discussed in the review show that NF-AT factors are major players in the control of T cell activation and differentiation and, in all likelihood, also of the cell cycle and apoptosis of T lymphocytes.

Transactivation of naturally occurring HIV-1 long terminal repeats by the JNK signaling pathway. The most frequent naturally occurring length polymorphism sequence introduces a novel binding site for AP-1 factors.

To study the role of MAPK cascades in the regulation of naturally occurring human immunodeficiency virus type 1 long terminal repeats (HIV-1 LTRs), we analyzed several HIV-1 LTRs from patients at different stages of disease progression. One of these naturally occurring HIV-1 LTRs contains an insertion termed the most frequent naturally occurring length polymorphism (MFNLP) and exhibited high inducibility upon T cell activation. We found that the protein kinase mixed lineage kinase 3/src-homology 3 domain-containing proline-rich kinase, a specific activator of the stress-activated protein kinase (SAPK)/JNK signaling pathway in T lymphocytes, induces high transcriptional activation of this promoter. Promoter inducibility is inhibited by the SAPK/JNK inhibitor, the JNK binding domain of the JNK interacting protein 1, and Tam-67 (N-terminal deletion mutant of c-Jun). In electrophoretic mobility shift assay, several protein complexes were found to bind to the MFNLP sequence in T cells. We identified AP-1 factors c-Fos and JunB as MFNLP-binding proteins, whose binding is abolished by introducing point mutations in the 3'-half of the MFNLP sequence. Introduction of these point mutations into the MFNLP containing HIV-1 LTR reduced src-homology 3 domain-containing proline-rich kinase -mediated transactivation. These data indicate that the AP-1-like binding site in the MFNLP sequence gives rise to a higher inducibility of natural HIV-LTRs by the SAPK/JNK signaling pathway.

CD4-mediated signals induce T cell dysfunction in vivo.

Triggering of CD4 coreceptors on both human and murine T cells can suppress TCR/CD3-induced secretion of IL-2. We show here that pretreatment of murine CD4+ T cells with the CD4-specific mAb YTS177 inhibits the CD3-mediated activation of the IL-2 promoter factors NF-AT and AP-1. Ligation of CD4 molecules on T cells leads to a transient stimulation of extracellular signal-regulated kinase (Erk) 2, but not c-Jun N-terminal kinase (JNK) activity. Pretreatment with anti-CD4 mAb impaired anti-CD3-induced Erk2 activation. Costimulation with anti-CD28 overcame the inhibitory effect of anti-CD4 Abs, by induction of JNK activation. The in vivo relevance of these studies was demonstrated by the observation that CD4+ T cells from BALB/c mice injected with nondepleting anti-CD4 mAb were inhibited in their ability to respond to OVA Ag-induced proliferation and IL-2 secretion. Interestingly, in vivo stimulation with anti-CD28 mAb restored IL-2 secretion. Furthermore, animals pretreated with anti-CD4 elicited enhanced IL-4 secretion induced by OVA and CD28. These observations suggest that CD4-specific Abs can inhibit T cell activation by interfering with signal 1 transduced through the TCR, but potentiate those delivered through the costimulatory molecule CD28. These studies have relevance to understanding the mechanism of tolerance induced by nondepleting anti-CD4 mAb used in animal models for allograft studies, autoimmune pathologies, and for immunosuppressive therapies in humans.

Multiple NF-ATc isoforms with individual transcriptional properties are synthesized in T lymphocytes.

The transcription factor NF-ATc that controls gene expression in T lymphocytes and embryonic cardiac cells is expressed in three prominent isoforms. This is due to alternative splice/polyadenylation events that lead to the predominant synthesis of two long isoforms in naive T cells and a shorter NF-ATc isoform in effector T cells. Whereas the previously described isoform NF-ATc/A contains a relatively short C terminus, the longer isoforms, B and C, span extra C-terminal peptides of 128 and 246 aa, respectively. We show here that in addition to the strong N-terminal trans-activation domain, TAD-A, which is common to all three NF-ATc isoforms, NF-ATc/C contains a second trans-activation domain, TAD-B, in its C-terminal peptide. Various stimuli of T cells that induce the activity of TAD-A also enhance the activity of TAD-B, but, unlike TAD-A, TAD-B remains unphosphorylated by protein from 12-O-tetradecanoyl 12-phorbol 13-acetate-stimulated T cells. The shorter C-terminal peptide of isoform NF-ATc/B exerts a suppressive transcriptional effect. These properties of NF-ATc/B and -C might be of importance for gene regulation in naive T lymphocytes in which NF-ATc/B and -C are predominantly synthesized.

CBP/p300 integrates Raf/Rac-signaling pathways in the transcriptional induction of NF-ATc during T cell activation.

NF-ATc, an inducibly expressed transcription factor, controls gene expression in T lymphocytes and cardiomyocytes. We show here that the transcriptional co-activators CBP/p300 bind to and control the activity of the inducible N-terminal transactivation domain of NF-ATc, TAD-A. Similar to the N terminal transactivation domain of c-Jun, TAD-A is inducibly phosphorylated, but this phosphorylation is dispensable for the interaction with CBP/p300. Constitutive active versions of c-Raf and Rac synergistically enhance the CBP/p300-mediated increase of TAD-A activity, indicating the important role CBP/p300 plays in the integration of T cell activation signals. Since a mutation of CBP abolishing HAT activity is almost as active as wild-type CBP in T cells, functions of CBP/p300 other than histone acetylation appear to control the NF-AT-dependent transcription in T cells.

Alternative polyadenylation events contribute to the induction of NF-ATc in effector T cells.

The transcription factor NF-ATc is synthesized in three prominent isoforms. These differ in the length of their C terminal peptides and mode of synthesis. Due to a switch from the use of a 3' polyA site to a more proximal polyA site, NF-ATc expression switches from the synthesis of the two longer isoforms in naive T cells to that of short isoform A in T effector cells. The relative low binding affinity of cleavage stimulation factor CstF-64 to the proximal polyA site seems to contribute to its neglect in naive T cells. These alternative polyadenylation events ensure the rapid accumulation of high concentrations of NF-ATc necessary to exceed critical threshold levels of NF-ATc for gene induction in effector T cells.

GA-binding protein factors, in concert with the coactivator CREB binding protein/p300, control the induction of the interleukin 16 promoter in T lymphocytes.

Interleukin 16 (IL-16) is a chemotactic cytokine that binds to the CD4 receptor and affects the activation of T cells and replication of HIV. It is expressed as a large 67-kDa precursor protein (pro-IL-16) in lymphocytes, macrophages, and mast cells, as well as in airway epithelial cells from asthmatics after challenge with allergen. This pro-IL-16 is subsequently processed to the mature cytokine of 13 kDa. To study the expression of IL-16 at the transcriptional level, we cloned the human chromosomal IL-16 gene and analyzed its promoter. The human IL-16 gene consists of seven exons and six introns. The 5' sequences up to nucleotide -120 of the human and murine IL-16 genes share >84% sequence homology and harbor promoter elements for constitutive and inducible transcription in T cells. Although both promoters lack any TATA box, they contain two CAAT box-like motifs and three binding sites of GA-binding protein (GABP) transcription factors. Two of these motifs are part of a highly conserved and inducible dyad symmetry element shown previously to control a remote IL-2 enhancer and the CD18 promoter. In concert with the coactivator CREB binding protein/p300, which interacts with GABPalpha, the binding of GABPalpha and -beta to the dyad symmetry element controls the induction of IL-16 promoter in T cells. Supplementing the data on the processing of pro-IL-16, our results indicate the complexity of IL-16 expression, which is tightly controlled at the transcriptional and posttranslational levels in T lymphocytes.

The GABP-responsive element of the interleukin-2 enhancer is regulated by JNK/SAPK-activating pathways in T lymphocytes.

T cell activation leads via multiple intracellular signaling pathways to rapid induction of interleukin-2 (IL-2) expression, which can be mimicked by costimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin. We have identified a distal IL-2 enhancer regulated by the Raf-MEK-ERK signaling pathway, which can be induced by TPA/ionomycin treatment. It contains a dyad symmetry element (DSE) controlled by the Ets-like transcription factor GA-binding protein (GABP), a target of activated ERK. TPA/ionomycin treatment of T cells stimulates both mitogen-activated ERK, as well as the stress-activated mitogen-activated protein kinase family members JNK/SAPK and p38. In this study, we investigated the contribution of the stress-activated pathways to the induction of the distal IL-2 enhancer. We show that JNK- but not p38-activating pathways regulate the DSE activity. Furthermore, the JNK/SAPK signaling pathway cooperates with the Raf-MEK-ERK cascade in TPA/ionomycin-induced DSE activity. In T cells, overexpression of SPRK/MLK3, an activator of JNK/SAPK, strongly induces DSE-dependent transcription and dominant negative kinases of SEK and SAPK impair TPA/ionomycin-induced DSE activity. Blocking both ERK and JNK/SAPK pathways abolishes the DSE induction. The inducibility of the DSE is strongly dependent on the Ets-core motifs, which are bound by GABP. Both subunits of GABP are phosphorylated upon JNK activation in vivo and three different isoforms of JNK/SAPK, but not p38, in vitro. Our data suggest that GABP is targeted by signaling events from both ERK and JNK/SAPK pathways. GABP therefore is a candidate for signal integration and regulation of IL-2 transcription in T lymphocytes.

GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction.

Triggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E. Flory, A. Hoffmeyer, U. Smola, U. R. Rapp, and J. T. Bruder, J. Virol. 70:2260-2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation.

Cyclosporin A interferes with the inducible degradation of NF-kappa B inhibitors, but not with the processing of p105/NF-kappa B1 in T cells.

The transcription factor NF-kappa B controls the induction of numerous cytokine promoters during the activation of T lymphocytes. Inhibition of T cell activation by the immunosuppressants cyclosporin A (CsA) and FK506 exerts a suppressive effect on the induction of these NF-kappa B-controlled cytokine promoters. We show for human Jurkat T leukemia cells, as well as human and mouse primary T lymphocytes, that this inhibitory effect is accompanied by an impaired nuclear translocation of the Rel proteins c-Rel, RelA/p65 and NF-kappa B1/p50, whereas the nuclear appearance of RelB remains unaffected. CsA does not interfere with the synthesis of Rel proteins, but prevents the inducible degradation of cytosolic NF-kappa B inhibitors I kappa B alpha and I kappa B beta upon T cell activation. CsA neither inhibits the processing of the NF-kappa B1 precursor p105 to p50, nor does it "stabilize" the C-terminal portion of p105, I kappa B gamma, which is degraded during p105 processing to mature p50. These results indicate that CsA interferes with a specific event in the signal-induced degradation of I kappa B alpha and I kappa B beta, but does not affect the processing of NF-kappa B1/p105 to p50.

Nuclear NF-ATp is a hallmark of unstimulated B cells from B-CLL patients.

B lymphocytes from the peripheral blood of patients with chronic lymphocytic leukaemia (CLL) were analysed for the nuclear presence and DNA binding of a panel of transcription factors which are involved in the gene control of lymphoid cells. The following transcription factors were studied: the Octamer factors Oct-1 and Oct-2, members of the AP-1 factor family, NF-AT factors, in particular NF-ATp, and members of the Rel/NF-kB family. We show that the constitutive nuclear translocation of NF-ATp, a member of the growing family of NF-AT factors, is a hallmark of nonstimulated B cells from CLL patients that distinguishes B-CLL cells from 'normal' B lymphocytes. Constitutive nuclear appearance was also observed for NF-kB2/p52. Constitutive binding of further factor proteins to DNA, such as JunD, c-Fos and FosB, was detected in several patients whereas the localisation and DNA binding of other factors such as c-Jun, RelA/p65 and c-Rel was unaltered. It is remarkable that in B-CLL cells the nuclear appearance and DNA binding of specific transcription factors is dramatically affected whereas other members of the same factor family remained unaltered in these leukemic cells. It remains to be shown which molecular events lead to the specific 'pre-activation', i.e. constitutive nuclear translocation and DNA binding, of these members of NF-AT, NF-kB and AP-1 factor families.

Combined laparoscopic and endoscopic management of cholelithiasis and choledocholithiasis.

With the advent of laparoscopic cholecystectomy, optimal management of common duct stones remains controversial. Seven hundred six patients underwent laparoscopic cholecystectomy in our institution from January 1990 through January 1992. From this group of patients, 50 were identified as having clinical or radiographic evidence of common duct stones. Thirty-one patients demonstrated preoperative risk factors for common duct stones and underwent preoperative endoscopic retrograde cholangiopancreatography (ERCP). The risk factors included jaundice (19%), pancreatitis (23%), elevated liver function tests (52%), and ultrasound evidence of choledocholithiasis (6%). Preoperative ERCP was performed in 94% of patients. There were two failures due to periampullary diverticula. Common duct stones were identified in 18 patients (62%) and successfully removed by endoscopic sphincterotomy in all of these patients. Nineteen patients were found to have unsuspected common duct stones on intraoperative cholangiography. Eighteen patients (95%) underwent successful ERCP and endoscopic sphincterotomy with stone extraction. Overall, major morbidity was 2% and included one patient who experienced endoscopic sphincteroplasty. The three endoscopic failures were managed by open common duct exploration, laparoscopic duct exploration, and combined laparoscopic and open common duct exploration. We conclude that combined laparoscopic and endoscopic therapy is a viable option for the management of cholelithiasis with choledocholithiasis.

Complete nucleotide sequence of the group I RNA bacteriophage fr.

We report the complete nucleotide sequence of the group I RNA bacteriophage fr. The entire genome consists of 3575 nucleotides, six nucleotides more than the only other sequenced group I representative, MS2. The greatest divergence between these phages occurs in the 5' terminal region of the A gene, while the lysis-replicase gene overlap, the coat gene and the central region of the replicase gene are highly conserved. Overall sequence homology between fr and MS2 is 77%. Here, we present a general comparison between the two phages. In the accompanying paper we use phylogenetic sequence comparison between MS2 and fr to deduce the secondary structure at the 3' untranslated region.

[Primary structure of a fragment of cDNA from phage fr]. / Pervichnaia struktura fragmenta kDNK faga fr.

The nucleotide sequence of a 1392 bp fragment of phage fr cDNA has been determined. The fragment contains 3'-terminal part of the A-protein gene, the complete coat protein gene, and beginning of the replicase gene. A comparison between the sequences of the corresponding genes and regulatory regions from the phage fr and MS2 genomes reveals 320 base changes.