Human and murine cytomegalovirus evasion of cytotoxic T lymphocyte and natural killer cell-mediated immune responses

Herpesviruses, such as human and murine cytomegalovirus, possess an impressive array of genes believed to assist in virus survival against the host immune response. In this review, we cover the rapidly growing area of cytomegalovirus evasion of cellular immunity, specifically cytotoxic T lymphocytes and natural killer cells. The proposed mechanisms of action of viral proteins involved in blocking peptide presentation to CD8(+) T cells, namely, interference with peptide generation, inhibition of peptide assembly with class I MHC and retention/destabilization of class I MHC complexes, are described. In addition, recent evidence implicating the viral class I MHC-like proteins as inhibitors of natural killer cell-mediated clearance is reviewed, (C) 1998 Academic Press.

m144, a murine cytomegalovirus (MCMV)-encoded major histocompatibility complex class I homologue, confers tumor resistance to natural killer cell-mediated rejection

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

Isolation and characterization of a Clostridium sp with cinnamoyl esterase activity and unusual cell envelope ultrastructure

Microorganisms that hydrolyse the ester linkages between phenolic acids and polysaccharides in plant cell walls are potential sources of enzymes for the degradation of lignocellulosic waste. An anaerobic, mesophilic, spore-forming, xylanolytic bacterium with high hydroxy cinnamic acid esterase activity was isolated from the gut of the grass-eating termite Tumilitermes pastinator. The bacterium was motile and rod-shaped, stained gram-positive, had an eight-layered cell envelope, and.formed endospores. Phylogenetic analysis based on 16S rRNA indicated that the bacterium is closely related to Clostridium xylanolyticum and is grouped with polysaccharolytic strains of clostridia. A wide range of carbohydrates were fermented, and growth was stimulated by either xylan or cellobiose as substrates. The bacterium hydrolysed and then hydrogenated the hydroxy cinnamic acids (ferulic and p-coumaric acids), which are esterified to arabinoxylan in plant cell walls. Three cytoplasmic enzymes with hydroxy cinnamic acid esterase activity were identified using non-denaturing gel electrophoresis. This bacterium possesses an unusual multilayered cell envelope in which both leaflets of the cytoplasmic membrane, the peptidoglycan layer and the S layer are clearly discernible. The fate of all these components was easily followed throughout the endospore formation process. The peptidoglycan component persisted during the entire morphogenesis. It was seen to enter the septum and to pass with the engulfing membranes to surround the prespore. It eventually expanded to form the cortex, verification for the peptidoglycan origin of the cortex. Sporogenic vesicles, which are derived from the cell wall peptidoglycan, were associated with the engulfment process. Spore coat fragments appeared early, in stage II, though spore coat formation was not complete until after cortex formation.

Signaling of neuronal cell death by the p75NTR neurotrophin receptor

The neurotrophin receptor (p75NTR) is best known for mediating tropic support by participating in the formation of high-affinity nerve growth factor (NGF) receptor complexes with trkA, however, p75NTR more recently has been shown to act as a bona fide death-signaling receptor, which can signal independently of trkA. This article discusses the evidence for an active role of p75NTR in neuronal cell death and the mechanisms controlling this process, including roles for Bcl-2 family members, the c-jun stress kinase JNK, the transcription factor nuclear factor kappa B (NF kappa B), and caspases.

Putative beta(4)-adrenoceptors in rat ventricle mediate increases in contractile force and cell Ca2+: comparison with atrial receptors and relationship to (-)-[H-3]-CGP 12177 binding

1 We identified putative beta(4)-adrenoceptors by radioligand binding, measured increases in ventricular contractile force by (-)-CGP 12177 and (+/-)-cyanopindolol and demonstrated increased Ca2+ transients by (-)-CGP 12177 in rat cardiomyocytes. 2 (-)-[H-3]-CGP 12177 labelled 13-22 fmol mg(-1) protein ventricular beta(1), beta(2)-adrenoceptors (pK(D) similar to 9.0) and 50-90 fmol mg(-1) protein putative beta(4)-adrenoceptors (pK(D) similar to 7.3). The affinity values (PKi) for (beta(1),beta(2)-) and putative beta(4)-adrenoceptors, estimated from binding inhibition, were (-)-propranolol 8.4, 5.7; (-)-bupranolol 9.7, 5.8; (+/-)-cyanopindolol 10.0,7.4. 3 In left ventricular papillary muscle, in the presence of 30 mu M 3-isobutyl-1-methylxanthine, (-)CGP 12177 and (+/-)-cyanopindolol caused positive inotropic effects, (pEC(50) (-)-CGP 12177, 7.6; (+/-)-cyanopindolol, 7.0) which were antagonized by (-)-bupranolol (pK(B) 6.7-7.0) and (-)-CGP 20712A (pK(B) 6.3-6.6). The cardiostimulant effects of(-)-CGP 12177 in papillary muscle, left and right atrium were antagonized by (+/-)-cyanopindolol (pK(i), 7.0-7.4). 4 (-)-CGP 12177 (1 mu M) in the presence of 200 nM (-)-propranolol increased Ca2+ transient amplitude by 56% in atrial myocytes, but only caused a marginal increase in ventricular myocytes. In the presence of 1 mu M 3-isobutyl-1-methylxanthine and 200 nM (-)-propranolol, 1 mu M (-)-CGP 12177 caused a 73% increase in Ca2+ transient amplitude in ventricular myocytes. (-)-CGP 12177 elicited arrhythmic transients in some atrial and ventricular myocytes. 5 Probably by preventing cyclic AMP hydrolysis, 3-isobutyl-1-methylxanthine facilitates the inotropic function of ventricular putative beta(4)-adrenoceptors. suggesting coupling to G(s) protein-adenylyl cyclase. The receptor-mediated increases in contractile force are related to increases of Ca2+ in atrial and ventricular myocytes. The agreement of binding affinities of agonists with cardiostimulant potencies is consistent with mediation through putative beta(4)-adrenoceptors labelled with (-)-[H-3]-CGP 12177.

Novel murine myeloid cell lines that exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the CM-CSF receptor beta subunit

Several activating mutations have recently been described in the common beta subunit for the human interleukin(IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (h beta c), Two of these, FI Delta and 1374N, result, respectively, in a 37-amino acid duplication and an isoleucine-to-asparagine substitution in the extracellular domain. A third, V449E, leads to valine-to-glutamic acid substitution in the transmembrane domain. Previous studies have shown that when expressed in murine hemopoietic cells in vitro, the extracellular mutants can confer factor independence on only the granulocyte-macrophage lineage while the transmembrane mutant can do so to all cell types of the myeloid and erythroid compartments. To further study the signaling properties of the constitutively active hpc mutants, we have used novel murine hemopoietic cell lines, which we describe in this report. These lines, FDB1 and FDB2, proliferate in murine IL-3 and undergo granulocyte-macrophage differentiation in response to murine GM-CSF, We find that while the transmembrane mutant, V449E, confers factor-independent proliferation on these cell lines, the extracellular hpc mutants promote differentiation. Hence, in addition to their ability to confer factor independence on distinct cell types, transmembrane and extracellular activated h beta c mutants deliver distinct signals to the same cell type. Thus, the FDB cell lines, in combination with activated h beta c mutants, constitute a powerful new system to distinguish between signals that determine hemopoietic proliferation or differentiation. (C) 2000 by The American Society of Hematology.

Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates

Objective. Differentiated dendritic cells (DC) and other antigen-presenting cells are characterized by the nuclear location of RelB, a member of the nuclear factor kappa B/Rel family. To characterize and enumerate differentiated DC in rheumatoid arthritis (RA) peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST), the expression and location of RelB were examined. Methods. RelB protein expression and cellular location were determined in RA PB, SF, and ST by flow cytometry and immunohistochemical analysis of purified cells or formalin-fixed tissue. DNA-binding activity of RelB was determined by electrophoretic: mobility shift-Western immunoblotting assays. Results. Circulating RA PBDC resembled normal immature PBDC in that they did not express intracellular RelB protein. In RA ST serial sections, cells containing nuclear RelB (nRelB) were enriched in perivascular regions. A mean +/- SD of 84 +/- 10% of these cells were DC. The remaining nRelB+,HLA-DR+ cells comprised B cells and macrophages. Only 3% of sorted SFDC contained nRelB, However, RelB present in the nucleus of these SFDC was capable of binding DNA, and therefore capable of transcriptional activity. Conclusion. Circulating DC precursors differentiate and express RelB after entry into rheumatoid ST. Differentiated DC can thus be identified by immunohistochemistry in formalin-fixed ST. Signals for DC maturation may differ between RA ST and SF, resulting in nuclear location of RelB predominantly in ST. This is likely to have functional consequences for the DC in these sites.

FHA domain boundaries of the Dun1p and Rad53p cell cycle checkpoint kinases

Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues), In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks, This indicates that the non-catalytic FI-IA domain plays an important role in the transcriptional function of the Dun1p protein kinase. (C) 2000 Federation of European Biochemical Societies.

Role of neurotrophin receptor p75NTR in mediating neuronal cell death following injury

1. The neurotrophin receptor p75NTR has been shown to mediate neuronal cell death after nerve injury. 2. Down-regulation of p75NTR by antisense oligonucleotides is able to inhibit both sensory and motor neuron death and this treatment is more effective than treatment with growth factors. 3. p75NTR induces cell death by a unique death signalling pathway involving transcription factors (nuclear factor kappa B and c-jun), Bcl-2 family members and caspases.

Cell death and immunohistochemistry of p53, c-Fos and c-Jun after spermine infection into the rat striatum

Administration of polyamines into the central nervous system results in tissue damage, possibly through the excitotoxic actions of the NMDA receptor. Direct injection of 100 nmol of spermine into the rat striatum produced a lesion equivalent to approximately 50% of the striatum. Analysis of the DNA in this region revealed the distinct ladder-like pattern of degradation often associated with apoptosis. This DNA fragmentation was confirmed in vivo using terminal deoxynucleotidyl-transferase-mediated biotinylated deoxyuridine triphosphate nick end labelling (TUNEL). The morphology of the TUNEL-positive cells showed marked differences at the needle tract when compared with cells in damaged areas away from the needle tract, suggesting a differential mechanism of cell death in these two regions. The patterns of p53, c-Fos and c-Jun protein expression were determined using immunohistochemistry. The number of p53-immunoreactive cells increased up to 14 h and returned to basal levels by 24 h. c-Fos protein expression transiently increased, peaking at 8 h after injection, c-Jun exhibited a protracted pattern of expression, remaining elevated up to 24 h. p53 protein expression was colocalised with TUNEL staining in areas away from the needle tract, but not in cells at the needle tract, suggesting once again a differential mechanism of cell death. At 14 h, c-Fos and c-Jun were not colocalised with TUNEL staining, suggesting that they are either not involved with the cell death process or that the time course of protein expression and the onset of DNA fragmentation do not overlap. This work represents the first characterisation of processes associated with cell death induced by spermine in vivo.

Comparative analysis of dendritic cell density and total number in commonly transplanted organs: morphometric estimation in normal mice

Dendritic cells (DC) are considered to be the major cell type responsible for induction of primary immune responses. While they have been shown to play a critical role in eliciting allosensitization via the direct pathway, there is evidence that maturational and/or activational heterogeneity between DC in different donor organs may be crucial to allograft outcome. Despite such an important perceived role for DC, no accurate estimates of their number in commonly transplanted organs have been reported. Therefore, leukocytes and DC were visualized and enumerated in cryostat sections of normal mouse (C57BL/10, B10.BR, C3H) liver, heart, kidney and pancreas by immunohistochemistry (CD45 and MHC class II staining, respectively). Total immunopositive cell number and MHC class II+ cell density (C57BL/10 mice only) were estimated using established morphometric techniques - the fractionator and disector principles, respectively. Liver contained considerably more leukocytes (similar to 5-20 x 10(6)) and DC (similar to 1-3 x 10(6)) than the other organs examined (pancreas: similar to 0.6 x 10(6) and similar to 0.35 x 10(6): heart: similar to 0.8 x 10(6) and similar to 0.4 x 10(6); kidney similar to 1.2 x 10(6) and 0.65 x 10(6), respectively). In liver, DC comprised a lower proportion of all leukocytes (similar to 15-25%) than in the other parenchymal organs examined (similar to 40-60%). Comparatively, DC density in C57BL/10 mice was heart > kidney > pancreas much greater than liver (similar to 6.6 x 10(6), 5 x 10(6), 4.5 x 10(6) and 1.1 x 10(6) cells/cm(3), respectively). When compared to previously published data on allograft survival, the results indicate that the absolute number of MHC class II+ DC present in a donor organ is a poor predictor of graft outcome. Survival of solid organ allografts is more closely related to the density of the donor DC network within the graft. (C) 2000 Elsevier Science B.V. All rights reserved.

Identification of the glycosaminoglycan keratan sulfate in the prostatic secretory cell

BACKGROUND. Prostate secretory granules (PSG) represent the basic secretory unit of the prostate gland, containing many of its exocrine proteases. Recent analysis of intraluminal corpora amylacea, a proposed by-product of PSG secretion, detected sulfated glycosaminoglycans (GAG) possibly keratan sulfate (KS),indicating a secretory mechanism for GAG in the human prostate surface epithelial cell. METHODS. Immunostains using anti-KS and anti-prostate-specific antigen (PSA) were evaluated on 10 sequential radical prostatectomy specimens, three of which had received neoadjuvant antiandrogen therapy. Extracts of normal secretory tissue as well as a sample composed almost entirely of prostatic stroma were subjected to Western blot analysis, using the same antibody panel. RESULTS. Keratan sulfate secretion from the normal prostate epithelial cell has been confirmed and correlates, as does PSA, with the presence of cytoplasmic PSG. No such correlation exists in most adenocarcinomas or in benign epithelium after androgen ablation. Western blot analyses confirmed tissue immunostains and demonstrated a secretory proteoglycan of 70-95 kDa. CONCLUSIONS. Recognition of PSG heralds a novel secretory mechanism within the human prostate gland that is linked to the secretion of KS. The role of KS in normal prostate secretion remains unknown, although it appears downregulated in neoplastic and androgen-ablated cells. (C) 2000 Wiley-Liss, Inc.

Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes

Chimeric papillomavirus (PV) virus-like particles (VLPs) based on the bovine papillomavirus type 1 (BPV-1) L1 protein were constructed by replacing the 23-carboxyl-terminal amino acids of the BPV1 major protein L1 with an artificial polytope minigene, containing known CTL epitopes of human PV16 E7 protein, HIV IIIB gp120 P18, Nef, and reverse transcriptase (RT) proteins, and an HPV16 E7 linear B epitope. The CTL epitopes were restricted by three different MHC class 1 alleles (H-2(b), H-2(d), HLA-A*0201). The chimeric L1 protein assembled into VLPs when expressed in SF-9 cells by recombinant baculovirus. After immunization of mice with polytope VLPs in the absence of adjuvant, serum antibodies were detected which reacted with both polytope VLPs and wild-type BPV1L1 VLPs, in addition to the HPV16E7 linear B cell epitope. CTL precursors specific for the HPV16 E7, HIV P18, and RT CTL epitopes were also detected in the spleen of immunized mice. Polytope VLPs can thus deliver multiple B and T epitopes as immunogens to the MHC class I and class II pathways, extending the utility of VLPs as self-adjuvanting immunogen delivery systems. (C) 2000 Academic Press.