Host Cell Activation Research Articles

Identification of migratory graft and host cell populations after allogeneic rat small bowel transplantation.

This study used flow cytometry to identify graft cells in the recipient peripheral blood and spleen and host cells infiltrating the graft mesenteric lymph node and Peyer's patches after heterotopic rat small bowel transplantation. Transplantation had little effect on the overall cell subset composition of these compartments and no changes appeared characteristic or indicative of developing rejection, suggesting that physiological control of cell migration remained unaltered. A small and transient population of graft cells was detected in the peripheral blood and spleen of the recipient which disappeared after 5 and 3 days respectively. Graft-derived cells in the peripheral blood comprised predominantly CD4+ cells on day 1 with B cells predominating on day 5. Graft cells infiltrating the spleen were predominantly B cells. Host cells infiltrated the graft mesenteric lymph nodes and Peyer's patches to a lesser extent than previously reported using immunohistochemical analysis. For both tissues, infiltrating host-derived cells initially comprised mainly CD4+ cells. On day 4 approximately equal proportions of CD4+ and B cells were present in the mesenteric lymph node, whereas B cells were predominant in the host cell infiltrate of the graft Peyer's patches. In summary, these findings indicate that the cell subset composition of recipient and graft lymphoid compartments does not change after small bowel transplantation, even in the presence of a substantial recipient cell infiltration. The reasons for the apparent discrepancies in the degree of host cell infiltration when assessed using immunohistochemical and flow cytometric techniques are currently uncertain, but may result from the localised release of soluble MHC class I in graft tissues as a consequence of infiltrating host cell activation or localised cell destruction.

Rapid identification and isolation of transcriptionally active regions from mouse genomes

We report here the design, construction and testing of a self- inactivating (Sin) retrovirus promoter-trap vector suitable for identifying and isolating transcriptionally active regions from the mouse genome. When this vector, which contains the bacterial aph gene as its reporter, is integrated into a site downstream from an active host cell promoter, it expresses aph, whose product, aminoglycoside phosphotransferase, produces resistance to the antibiotic G418 in mammalian cells. The construct also contains a native aph promoter which functions in bacteria, but not in mouse cells, to express kanamycin (Km) resistance, plus an adjacent pBR322-derived replication origin. Thus, mammalian DNA segments containing actively transcribed regions flanking aph can be quickly isolated by restriction endonuclease treatment of total DNA from provirus-containing mouse cells, followed by self-ligation, transformation and Km selection of plasmids carried by bacteria transformed with this DNA. We tested this Sin retrovirus promoter-trap system by isolating eight DNA segments upstream to the provirus integration sites in the genome of virus-infected mouse F9 cells. We found that the Sin retrovirus vector produces a high yield of infectious virus particles carrying aph, and that the isolated genomic DNA fragments of F9 cells are transcriptionally active.

Infection of cultured rat enterocytes by Ileal symbiont intracellularis depends on host cell function and actin polymerisation

The mechanisms of entry of Ileal symbiont intracellularis into IEC-18 rat enterocyte cells and subsequent bacterial proliferation were examined in centrifuge-assisted and static infections. Live, oxygen or neomycin damaged, and formalin killed bacteria, each rapidly entered viable cells. Live or damaged bacteria did not enter cells nor proliferate within cells after static infection of cells cooled to 5°C. Infection of cells was greatly reduced at 20° or 32° compared to infection at 37°C. Centrifuge-assisted infection was also reduced by chilling the cells. Cytochalasin D but not B inhibited the entry process indicating an actin-dependent infection, although other pathways may also be involved in centrifuge-assisted infections. Drugs capable of modifying cell membrane charge, heparin receptors or trypsin-labile proteins were all inactive in preventing or enhancing infection. We therefore conclude that infection of enterocytes by IS intracellularis is dependent on host cell activity and actin polymerization, but is independent of bacterial viability.

Synthesis and anti-HIV-1 activity of bis-ketol AZT monophosphates

The synthesis of five bis-ketol AZT monophosphates and their anti-HIV-1 activity in CEM-SS host cells are reported.

Studies on the antiviral activity of protein kinase inhibitors against the replication of vesicular stomatitis virus.

Several derivatives of K-252a, a protein kinase inhibitor isolated from Nocardiopsis sp., were investigated for their effects on the replication of vesicular stomatitis virus (VSV) in BHK-21 cell cultures. Among those we tested, KT5926, which preferentially inhibits the myosin light chain kinase (MLCK), suppressed the viral replication by 95-99% at 15 microM. K-252a, which inhibits a broad spectrum of cellular protein kinase, similarly affected the viral replication. Other derivatives, KT5720 and KT5823, that are known to inhibit the cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, did not suppress VSV replication even at a high concentration as 15 microM. None of these inhibitors affected the Sindbis virus replication in BHK-21 cells under similar assay conditions as used for VSV. KT5926 and K-252a seemed to affect the VSV replication at the step(s) after the viral invasion, resulting in decreased viral RNA synthesis. Neither substance inhibited cellular casein kinase (CK) II which is known to be involved in phosphorylation of the nonstructural (NS) protein, a non-catalytic subunit of the viral RNA polymerase. These results suggest that the inhibition of VSV replication by KT5926 and K-252a is not a secondary effect due to generalized suppression of host cell activities, and that the VSV replication requires the KT5926-sensitive function(s) in the cell which would be performed by an enzyme(s) other than CK II.

The role of leukocyte adhesion molecules in acute transplant rejection.

The specific adhesion of cells to other cells or to particular tissues or tissue components is a basic function of cell migration and recognition and underlies many biologic processes including embryogenesis, repair, and immunity. Adhesion molecules are involved in and mediate most cell to cell interactions, implement migration of leukocytes to inflammatory or alloantigenic sites, and costimulate well activation and transformation. Because of these functions, the subject of adhesion molecules is gaining broader interest in the field of transplantation, particularly in the conceptualization and development of future treatment strategies. These molecules influence not only the first interaction between host leukocytes and vascular endothelial cells of the graft but also their migration through the grafted tissues. These contacts seem to be based initially on antigen-independent events of adhesion, which then progress to the antigen-dependent events of antigen recognition and host cell activation. This review evaluates current studies concerning the role of adhesion molecules in the context of the multifaceted processes of allograft rejection.

Ultrastructural analysis of the invasion of tick cells by Lyme disease spirochetes (Borrelia burgdorferi) in vitro

The association of Lyme disease spirochetes, Borrelia burgdorferi, with tick cell cultures was characterized by electron microscopy. These cells were active in endocytosis and intracellular digestion, containing coated vesicles, pits, and phagosomes. Borrelia burgdorferi in tick cell cultures resembled those described in tick tissues. In RAE25 cultures, isolated from Rhipicephalus appendiculatus embryos, invasion of cells was mediated by coated pits, indicating active host cell participation. The invading, tapered end of B. burgdorferi contained an electron-dense body that persisted throughout invasion. A host-derived membrane surrounded invading and intracellular borreliae as observed by transmission electron microscopy of cross and longitudinal sections, whereas degenerating ones lay inside secondary lysosomes. Borrelia burgdorferi cocultivated with cell lines from Ixodes scapularis were mainly found at the cell surface or within lysosomes. The differences seen in invasion of these cell lines are interpreted to reflect differences in cell types rather than species. Gemmae, indicative of degenerative changes in the spirochetes, were observed extra- and intra-cellularly. Membrane blebs were liberated by the spirochetes into the medium and onto the cells, and were avidly endocytosed. Tick cell cultures are a useful tool to elucidate spirochete – vector cell interactions that may be obscured in vivo.

The redox status of malaria-infected erythrocytes: an overview with an emphasis on unresolved problems.

The various mechanisms involved in the redox defence of normal erythrocytes are adequately known. They are herein briefly reviewed, outlining the principal enzymes and metabolic pathways, such as superoxide dismutase, catalase, glutathione peroxidase and reductase, the hexose monophosphate shunt (HMS) and glutathione synthesis and turnover. The intraerythrocytic malaria parasite is imposing an oxidative stress on its host cell. Malaria infected cells produce O2-, H2O2, enhance lipide peroxidation and activate host cell HMS. This stress is produced during the digestion of host cell hemoglobin by the parasite. Hence, both parasite and host cell must be able to confront this stress. The antioxidant defence systems of the parasite and the response of those systems in the infected host cell are reviewed, underscoring unresolved problems. Nothing is virtually known on the parasite's glutathione metabolism, and on possible interactions between host cell and parasite antioxidant defence systems. The postulate that 1. host cell activated HMS in conjunction with purine salvage can provide purine nucleotides to the parasite, and 2. that glutathione transferase can participate in parasite resistance to antimalarial drugs, are also discussed.

Hyperimmediate entry of human cytomegalovirus virions and dense bodies into human fibroblasts

Previous ultrastructural and cytochemical examination of the HCMV inoculum as used in the laboratory enabled the distinction of 7 morphologically different types of structures including complete virions, other enveloped and non-enveloped particles and dense bodies (Topilko and Michelson, 1994). In the present study, electron microscopy was used to investigate the kinetics and modalities of the earliest interactions between these components of inoculum and human foreskin fibroblasts (FSF). Particles did not attach to cells during incubation at 4 degrees C. However, when FSF were adsorbed with virus for 30 seconds at 37 degrees C, HCMV particles attached to the cell surface. Particle attachment was mediated by fine virus envelope-cell membrane bridges. Within 60 seconds, numerous virions had fused with cell membranes, and nucleocapsids had entered the cytoplasm. Enveloped particles with translucent cores, designated non-infectious enveloped particles (NIEP), were also seen to enter cells in the same way and with the same kinetics as complete virions. Uptake of dense bodies followed the same kinetics and mode of penetration as complete virus particles. These findings reveal that in vitro, enveloped particles (virions and NIEP) and dense bodies enter the cytoplasm of the host cell simultaneously, immediately (< 60 seconds) after contact with the cell membrane. Our results suggest that activation of immediate early cellular responses may not simply be due to transmembrane signal transduction, but that hyperimmediate entry of these elements into cells may participate directly in host cell activation.

Graft-versus-host reaction and GvH disease.

Chronic GvHR was induced by inoculating parental lymphoid cells into F1 hybrid mouse. Combination of ATL and ATH, which were congenic recombinant strains differing only in H-2I and S region from each other, was chosen to induce class II-GvHR. Selective activation against partner's alloantigen of graft CD4+ T cells was the primary event of the GvHR and then led to concomitant activation of both graft and host cells. Immune dysregulation among these cells made the GvHR-mouse express various chronic diseases including immune complex glomerulonephritis, autoimmune-like lesions of the liver or the salivary gland, tumor-like proliferations of T cells and abnormal extramedullary hematopoiesis. Chronic GvHR was also induced by a preferential but not a selective activation of graft CD4+ T cells. A combination of DBA/2 and C57BL/6, which differ in whole MHC antigens, was an example. When D2 cells, but not B6 cells, were incoulated into the BDF1 mouse, predominant activation of CD4+ cells over CD8+ cells were observed. Contributing factors to this phenomenon were low responsiveness of graft CD8+ T cells to allogeneic class I MHC antigens and anti-parent activity of host CD8+ cells. Thus both graft and host cells participate either actively or passively in the reaction induced in the parent --> F1 experimental system of GvHR/D.

Potentiation of in vivo Antitumor Effects of Recombinant Interleukin‐1α by Gelatin Conjugation

Chemical conjugation of a recombinant human interleukin‐1α (IL‐1) with gelatin was conducted using a water‐soluble carbodiimide in an attempt to augment the indirect effect of IL‐1 on in vivo tumor cell growth in mice. Chromatographic studies of the IL‐1‐gelatin conjugate demonstrated that the apparent molecular weight of IL‐1 was increased by the gelatin conjugation and about 60% of IL‐1 activity was retained in the prepared conjugate. Intraperitoneal (i.p.) injection of the conjugate significantly suppressed the intraperitoneal growth of a subline of Meth A fibrosarcoma cells (RR1 cells), compared with the effect of free IL‐1 at the same dose, although the cells per se were resistant not only to free IL‐1 but also to gelatin‐conjugated IL‐1. Simple mixing of gelatin with free IL‐1 did not augment the in vivo antitumor effect as compared with that of free IL‐1. Gelatin conjugation improved the in vivo stability of IL‐1. Prolonged retention of IL‐1 activity in the peritoneal cavity as well as the circulation of mice was observed after i.p. injection of the IL‐1‐gelatin conjugate in comparison with free IL‐1 injection, irrespective of the presence of tumor cells. Gelatin conjugation was effective in augmenting the in vivo antitumor effects of IL‐1 to activate host cells, e.g. macrophages (Mø). The i.p. injection of the conjugate enhanced Mø infiltration into the peritoneal cavity of tumor‐bearing mice and peritoneal Mø were strongly activated to inhibit the in vitro growth of RR1 cells. Thus, gelatin conjugation was effective in augmenting the indirect effect of IL‐1 via host cells, leading to a high suppressive effect on in vivo growth of tumor cells.

Human immunodeficiency virus type 1 Nef protein inhibits NF-kappa B induction in human T cells.

Human immunodeficiency virus type 1 (HIV-1) can establish a persistent and latent infection in CD4+ T lymphocytes (W. C. Greene, N. Engl. J. Med. 324:308-317, 1991; S. M. Schnittman, M. C. Psallidopoulos, H. C. Lane, L. Thompson, M. Baseler, F. Massari, C. H. Fox, N. P. Salzman, and A. S. Fauci, Science 245:305-308, 1989). Production of HIV-1 from latently infected cells requires host cell activation by T-cell mitogens (T. Folks, D. M. Powell, M. M. Lightfoote, S. Benn, M. A. Martin, and A. S. Fauci, Science 231:600-602, 1986; D. Zagury, J. Bernard, R. Leonard, R. Cheynier, M. Feldman, P. S. Sarin, and R. C. Gallo, Science 231:850-853, 1986). This activation is mediated by the host transcription factor NF-kappa B [G. Nabel and D. Baltimore, Nature (London) 326:711-717, 1987]. We report here that the HIV-1-encoded Nef protein inhibits the induction of NF-kappa B DNA-binding activity by T-cell mitogens. However, Nef does not affect the DNA-binding activity of other transcription factors implicated in HIV-1 regulation, including SP-1, USF, URS, and NF-AT. Additionally, Nef inhibits the induction of HIV-1- and interleukin 2-directed gene expression, and the effect on HIV-1 transcription depends on an intact NF-kappa B-binding site. These results indicate that defective recruitment of NF-kappa B may underlie Nef's negative transcriptional effects on the HIV-1 and interleukin 2 promoters. Further evidence suggests that Nef inhibits NF-kappa B induction by interfering with a signal derived from the T-cell receptor complex.

Salmonella typhimurium induces an inositol phosphate flux in infected epithelial cells

Salmonella typhimurium, like many other intracellular pathogens, is capable of inducing its own uptake into non-phagocytic cells by a process termed invasion, and residing within a membrane-bound inclusion. During invasion it causes significant rearrangment of the host cytoskeleton, indicating that signals are transduced between the bacterium and the host cell cytoplasm, across the eukaryotic cell membrane. We found that intracellular inositol phosphate concentrations in HeLa cells increased during S. typhimurium entry and returned to normal levels after bacterial internalization. A chelator of intracellular calcium (BAPTA / AM) blocked S. typhimurium uptake into HeLa epithelial cells, but extracellular calcium chelators (BAPTA, EGTA, EDTA) had no effect on bacterial invasion. These results indicate that S. typhimurium may activate host cell phospholipase C activity to form inositol phosphates which in turn stimulate release of intracellular calcium stores to facilitate bacterial uptake.

Salmonella typhimuriuminduces an inositol phosphate flux in infected epithelial cells

Salmonella typhimurium, like many other intracellular pathogens, is capable of inducing its own uptake into non-phagocytic cells by a process termed invasion, and residing within a membrane-bound inclusion. During invasion it causes significant rearrangment of the host cytoskeleton, indicating that signals are transduced between the bacterium and the host cell cytoplasm, across the eukaryotic cell membrane. We found that intracellular inositol phosphate concentrations in HeLa cells increased during S. typhimurium entry and returned to normal levels after bacterial internalization. A chelator of intracellular calcium (BAPTA / AM) blocked S. typhimurium uptake into HeLa epithelial cells, but extracellular calcium chelators (BAPTA, EGTA, EDTA) had no effect on bacterial invasion. These results indicate that S. typhimurium may activate host cell phospholipase C activity to form inositol phosphates which in turn stimulate release of intracellular calcium stores to facilitate bacterial uptake.

Herbicide-induced resistance of cotton to Verticillium wilt disease and activation of host cells to produce the phytoalexin gossypol

Pretreating cotton seeds with selected herbicide markedly increased cotton resistance to vascular wilt caused by Verticillium albo-atrum. The present investigation studied the mechanism of resistance to V. albo-atrum induced by some herbicides in susceptible cotton plants. The three herbicides tested repressed mycelial growth. Prometryn and dalapon caused a marked reduction in the appearance of Verticillium wilt disease and were the most effective in controlling the disease. Phytoalexin (gossypol) production was greatly increased in herbicide-treated inoculated cotton plants. The level of gossypol was higher in the root than stem. Maximum production of gossypol was achieved in cotton plants treated with the herbicides prometryn and dalapon and inoculated with fungal spores but it was not present in uninoculated control plants. Key words: cotton, Verticillium albo-atrum, herbicides, phytoalexin, disease resistance.

SYNERGY BETWEEN CYCLOSPORINE AND ANTI-IL-2 RECEPTOR MONOCLONAL ANTIBODIES IN RATS

Although cyclosporine has improved results of organ transplantation, treatment regimens using multiple agents are being evaluated both experimentally and clinically in attempts to diminish its often profound nephrotoxicity; some therapies act synergistically by differential inhibition of distinct steps of the rejection cascade. The effects on graft function of a full dose or a subclinical dose of CsA, ART-18, a monoclonal antibody (mAb) directed against the IL-2 receptor expressed on activated host cells, and a combination of low-dose CsA and ART-18, have been tested in rat recipients of both heart and kidney allografts. Renal graft function was assessed by several classic techniques; heart function by isolated perfusion methods. Full-dose CsA and combination treatment were most effective in both organ graft systems, with at least one-third of grafts surviving indefinitely. At seven days after transplantation, glomerular filtration rates and renal plasma flow of all grafted recipients were decreased as compared with normal; at 14 days, function in the best treatment groups had improved toward that of isografts. Similarly, cardiac output and stroke work index of best treatment groups were comparable to that of isografts. These functional studies complement previously reported immunological and immunohistological findings stressing that synergy occurs between subclinical doses of CsA and anti-IL-2-R mAb in two rat organ graft systems.

Programmed activation of T-lymphocytes. A theoretical basis for short term treatment of AIDS with azidothymidine

When its T-lymphocyte host cell is activated, the latent (DNA) form of human immunodeficiency virus (HIV) is activated to produce RNA copies which are liberated as virus particles from the cell. In this process the cell is destroyed together with the latent virus. If administered at this time, 3′-azidothymidine (AZT) would specifically prevent the liberated RNA copies replicating and establishing latency in new host cells. The RNA copies would then be degraded by viral or host ribonucleases. Thus, one DNA copy of HIV and its RNA progeny would be eliminated from the body. However, many DNA copies of HIV would remain in other cells. The main problem of therapy with AZT is that activation of host cells to become permissive for production of virus is random in time. Activation depends on chance encounters of an infected person with the particular foreign antigens to which individual T-cells bearing latent HIV can specifically respond. It is primarily for this reason that AZT must be administered continuously. If all T-cells could be polyclonally stimulated at one time, all HIV-bearing T-cells would be destroyed and concomitant administration of AZT for a short term would prevent the replication of all liberated viruses. Unlike most renewable ‘end’ cells in the body, the maturation of T cells involves processes of positive and negative selection. To preserve the ‘educated’ T-cell population, T-cell renewal occurs at the end cell, rather than at the stem cell level. It is possible that normal physiological signals concerned with this homeostatic regulation of T-lymphocyte population size could be harnessed to produce synchronous activation of all T-lymphocytes. Tumor necrosis factor-α has some of the properties expected of a postulated polyclonal activator needed for this programmed activation of T-lymphocytes.

Growth of Chlamydia trachomatis in enucleated cells

Chlamydia trachomatis is an obligate intracellular parasite of eucaryotic cells. Little is known about the role of the host in supporting chlamydial replication beyond the facts that host cells provide ATP and that de novo host protein synthesis is not required for bacterial growth. To further explore potential contributions of host nuclear function to chlamydial development, we questioned whether murine C. trachomatis could grow in mouse L cells that had been enucleated with cytochalasin B. Following enucleation, cells were infected with chlamydiae and analyzed morphologically and biochemically. Late in infection, substantial numbers of chlamydiae of all developmental stages were seen within large cytoplasmic inclusions that were indistinguishable from those seen in infected intact cells. Normal numbers of infectious progeny particles were produced from enucleated cultures. We conclude that active host cell nuclear function is not required to support the growth of chlamydiae.

Casein kinase II activities related to hyper-phosphorylation of human papillomavirus type 16-E7 oncoprotein in epidermal keratinocytes

The present study describes hyper-phosphorylation of E7-oncoprotein of human papillomavirus (HPV) type 16 in epidermal keratinocytes. We found that highly phosphorylated E7-oncoprotein was present in epidermal keratinocytes but little in fibroblasts. The E7 oncoprotein contains serine residues (Ser-Ser-Glu-Glu-Glu) capable of being phosphorylated by casein kinase II (CK II). Extracts from various cell lines including human origins transformed by HPV 16 were examined for the casein kinase activity. The results showed that CK II activity was present at significantly high levels in keratinocytes but little or no detectable levels of the activity in human fibroblasts. These differential CK II activities in host cells may play a part in the differential transforming activity by E7-oncoprotein.

Inhibition by Mannose of In Vitro Colonization of Chicken Small Intestine by Salmonella typhimurium

The in vitro adherence of [3 H]thymidine-labeled Salmonella typhimurium isolates to the small intestine of one-day-old chickens was investigated. Bacteria were screened for mannose sensitivity and mannose-resistance binding properties. Type 1 fimbriae positive strains adhered significantly better than Type 2 fimbriae-negative strains. Adherence was significantly (P<.05) inhibited by D-mannose, methyl-α-D-mannoside, arabinose, and galactose. Adherence was both time and temperature dependent. These findings suggest that the small intestine of the chicken has receptors for bacteria with Type 1 fimbriae. The function of the receptors is dependent on a mannose moiety. Bacteria adhered better to fresh intestine cells than to cells held overnight at 4 C. Thus, adherence was dependent upon a metabolically active host cell. The in vitro adherence assay may further be used to study the interaction of bacteria with chicken enterocytes.