Identification of Leishmania Proteins Preferentially Released in Infected Cells Using Change Mediated Antigen Technology (CMAT)

Although Leishmania parasites have been shown to modulate their host cell''s responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus.     

Leishmania parasitophorous vacuoles interact continuously with the host cell's endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments

Macrophages that express representative endoplasmic reticulum (ER) molecules tagged with green fluorescence protein were generated to assess the recruitment of ER molecules to Leishmania parasitophorous vacuoles (PVs). More than 90% of PVs harbouring Leishmania pifanoi or Leishmania donovani parasites recruited calnexin, to their PV membrane. An equivalent proportion of PVs also recruited the membrane‐associated soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), Sec22b. Both ER molecules appeared to be recruited very early in the formation of nascent PVs. Electron microscopy analysis of infected Sec22b/YFP expressing cells confirmed that Sec22b was recruited to Leishmania PVs. In contrast to PVs, it was found that no more than 20% of phagosomes that harboured Zymosan particles recruited calnexin or Sec22b to their limiting phagosomal membrane. The retrograde pathway that ricin employs to access the cell cytosol was exploited to gain further insight into ER–PV interactions. Ricin was delivered to PVs in infected cells incubated with ricin. Incubation of cells with brefeldin A blocked the transfer of ricin to PVs. This implied that molecules that traffic to the ER are transferred to PVs. Moreover the results show that PVs are hybrid compartments that are composed of both host ER and endocytic pathway components.     

Functional Activity of Monocytes and Macrophages in HTLV-1 Infected Subjects

The Human T lymphotropic virus type-1 (HTLV-1) infects predominantly T cells, inducing proliferation and lymphocyte activation. Additionally, HTLV-1 infected subjects are more susceptible to other infections caused by other intracellular agents. Monocytes/macrophages are important cells in the defense against intracellular pathogens. Our aims were to determine the frequency of monocytes subsets, expression of co-stimulatory molecules in these cells and to evaluate microbicidal ability and cytokine and chemokine production by macrophages from HTLV-1 infected subjects. Participants were 23 HTLV-1 carriers (HC), 22 HAM/TSP patients and 22 healthy subjects (HS) not infected with HTLV-1. The frequencies of monocyte subsets and expression of co-stimulatory molecules were determined by flow cytometry. Macrophages were infected with L. braziliensis or stimulated with LPS. Microbicidal activity of macrophages was determined by optic microscopy. Cytokines/chemokines from macrophage supernatants were measured by ELISA. HAM/TSP patients showed an increase frequency of intermediate monocytes, but expression of co-stimulatory molecules was similar between the groups. Macrophages from HTLV-1 infected individuals were infected with L. braziliensis at the same ratio than macrophages from HS, and all the groups had the same ability to kill Leishmania parasites. However, macrophages from HTLV-1 infected subjects produced more CXCL9 and CCL5, and less IL-10 than cells from HS. While there was no correlation between IFN-γ and cytokine/chemokine production by macrophages, there was a correlation between proviral load and TNF and CXCL10. These data showed a dissociation between the inflammatory response and microbicidal ability of macrophages from HTLV-1 infected subjects. While macrophages ability to kill an intracellular pathogen did not differ among HTLV-1 infected subjects, these cells secreted high amount of chemokines even in unstimulated cultures. Moreover the increasing inflammatory activity of macrophages was similar in HAM/TSP patients and HC and it was related to HTLV-1 proviral load rather than the high IFN-γ production observed in these subjects.     

Mycobacterium bovis BCG attenuates surface expression of mature class II molecules through IL-10-dependent inhibition of cathepsin S

We have previously shown that macrophage infection with Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) partially inhibits MHC class II surface expression in response to IFN-gamma. The present study examined the nature of class II molecules that do in fact reach the surface of infected cells. Immunostaining with specific Abs that discriminate between mature and immature class II populations showed a predominance of invariant chain (Ii)-associated class II molecules at the surface of BCG-infected cells suggesting that mycobacteria specifically block the surface export of peptide-loaded class II molecules. This phenotype was due to inhibition of IFN-gamma-induced cathepsin S (Cat S) expression in infected cells and the subsequent intracellular accumulation of alphabeta class II dimers associated with the Cat S substrate Ii p10 fragment. In contrast, infection with BCG was shown to induce secretion of IL-10, and addition of blocking anti-IL-10 Abs to cell cultures restored both expression of active Cat S and export of mature class II molecules to the surface of infected cells. Consistent with these findings, expression of mature class II molecules was also restored in cells infected with BCG and transfected with active recombinant Cat S. Thus, M. bovis BCG exploits IL-10 induction to inhibit Cat S-dependent processing of Ii in human macrophages. This effect results in inhibition of peptide loading of class II molecules and in reduced presentation of mycobacterial peptides to CD4(+) T cells. This ability may represent an effective mycobacterial strategy for eluding immune surveillance and persisting in the host.     

Immunologic properties of mast cells from rats infected with Nippostrongylus brasiliensis.

The concentration of IgE in the serum of Sprague-Dawley rats increased after infection with Nippostrongylus brasiliensis (NB). The IgE concentration in normal rats was less than 1 mug/ml. After re-infection with NB, the concentration increased in 100 to 300 mug/ml. Mast cells were purified from peritoneal cells of both normal and NB-infected animals. Purified mast cells from the infected animals released histamine upon exposure to NB antigen. The antibody specific for IgE released histamine from purified mast cells of both normal and infected animals. Dose-reponse curves of histamine release suggested that mast cells from NB-infected animals bear more IgE molecules than normal mast cells. Binding of 125I-labeled rat E myeloma protein with normal mast cells was demonstrated by autoradiography. Under the same experimental conditions, mast cells of infected animals were not labeled with 125I-IgE. Mast cells from both normal and infected animals failed to combine 125I-labeled IgG. The number of IgE molecules bound per mast cell was determined by incubating 125I-labeled IgE with purified mast cells. When mast cells were incubated incubated in 0.6 to 2 mug/ml of IgE, the number of IgE molecules combined with the mast cells from infected animals was about 10% of that bound with normal mast cells. The results indicated that a large proportion of IgE receptors on mast cells of infected animals was occupied by their own IgE. No significant difference was observed between normal mast cells and those of infected animals with respect to histamine content and intracellular levels of cyclic nucleotides.     

Complex of bacteriophage M13 single-stranded DNA and gene 5 protein

Lysates of bacteriophage M13-infected cells contain numerous unbranched filamentous structures approximately 1·1 μm long × 160 Å wide, that is, slightly longer and considerably wider than M13 virions. These structures are complexes of viral single-stranded DNA molecules with M13 gene 5 protein, a non-capsid protein required for single-stranded DNA production. All, or nearly all, of the single-stranded DNA from the infected cells and at least half to two-thirds of the gene 5 protein molecules are found as complex in the lysates. The complex contains about 1300 gene 5 protein molecules per DNA molecule but little if any of the two known capsid proteins. The complex is much less stable than virions in the presence of salt or ionic detergent solutions and in electron micrographs it appears to have a much looser and more open structure. If an excess of M13 single-stranded DNA is added to complex in a lysate, the gene 5 protein molecules from the complex redistribute onto all of the added as well as the original DNA, again suggesting a rather loose association of protein and DNA.By electron microscopy, the complex from infected cells appears to differ structurally from complex formed in vitro between purified single-stranded DNA and purified gene 5 protein. Because of this apparent structural difference and because previous experiments suggested the presence of complex in vivo, we presume that the complex which we have found in lysates of infected cells previously did exist as such inside the cells, but we have been unable to exclude that it formed during or after lysis. If it is assumed that complex does occur in vivo, the results of pulse-chase radioactive labeling experiments on infected cells can be interpreted as showing that with time the single-stranded DNA leaves complex, presumably to be matured into virions, while the gene 5 protein molecules are re-used to form more complex.     

Relationship Between Replication of Simian Virus 40 DNA and Specific Events of the Host Cell Cycle

The relationship between replication of simian virus 40 (SV40) DNA and the various periods of the host-cell cycle was investigated in synchronized CV(1) cells. Cells synchronized through a double excess thymidine procedure were infected with SV40 at the beginning or the middle of S, or in G(2). The first viral progeny DNA molecules were in all instances detected approximately 20 h after release from the thymidine block, independent of the time of infection. The length of the early, prereplicative phase of the virus growth cycle therefore depended upon the period of the cell cycle at which the cells were infected. Infection with SV40 was also performed on cells obtained in early G(1) through selective detachment of cells in metaphase. As long as the cells were in G(1) at the time of infection, the first viral progeny DNA molecules were detected during the S period immediately following, whereas if infection took place once the cells had entered S, no progeny DNA molecule could be detected until the S period of the next cell cycle. These results suggest that the infected cell has to pass through a critical stage situated in late G(1) or early S before SV40 DNA replication can eventually be initiated.     

Enhancement of HIV-specific CD8 T cell responses by dual costimulation with CD80 and CD137L

HIV-specific CD8 T cell responses are defective in chronic HIV infection. In this study, we report that costimulation with either CD137L (4-1BBL) or CD80 (B7.1) enhanced the Ag-specific expansion and acquisition of effector function by HIV-specific memory CD8 T cells. Ag-specific T cells from recently infected donors showed maximal expansion with single costimulatory molecules. Dual costimulation of T cells from recently infected donors or from healthy donors responding to influenza epitopes led to enhanced responses when the accumulation of cytokines was measured. However, accumulation of regulatory cytokines, particularly IFN-gamma, led to inhibition of further Ag-specific CD8 T cell expansion in the cultures. This inhibition was relieved by neutralization of IFN-gamma or of IFN-gamma, TNF, and IL-10. Thus, strong costimulation of T cells in vitro can lead to induction of regulatory cytokines at levels that limit further T cell expansion. In marked contrast, T cells from long-term (>4 years) infected HIV+ donors exhibited reduced Ag-specific CD8 T cell expansion, reduced CD4 T cell responses, and minimal cytokine accumulation. Dual costimulation with both 4-1BBL and B7.1 enhanced responses of T cells from long-term infected subjects to a level similar to that obtained with T cells from early in HIV infection. Experiments with purified CD8 T cells showed that B7.1 and 4-1BBL could act directly and synergistically on CD8 T cells. Taken together, these data suggest that 4-1BBL and B7.1 have additive or synergistic effects on HIV-specific CD8 T cell responses and represent a promising combination for therapeutic vaccination for HIV.     

Molecular analysis of enhanced replication of UV-damaged simian virus 40 DNA in UV-treated mammalian cells.

Irradiation of simian virus 40 (SV40)-infected cells with low fluences of UV light (20 to 60 J/m2, inducing one to three pyrimidine dimers per SV40 genome) causes a dramatic inhibition of viral DNA replication. However, treatment of cells with UV radiation (20 J/m2) before infection with SV40 virus enhances the replication of UV-damaged viral DNA. To investigate the mechanism of this enhancement of replication, we analyzed the kinetics of synthesis and interconversion of viral replicative intermediates synthesized after UV irradiation of SV40-infected cells that had been pretreated with UV radiation. This enhancement did not appear to be due to an expansion of the size of the pool of replicative intermediates after irradiation of pretreated infected cells; the kinetics of incorporation of labeled thymidine into replicative intermediates were very similar after irradiation of infected control and pretreated cells. The major products of replication of SV40 DNA after UV irradiation at the low UV fluences used here were form II molecules with single-stranded gaps (relaxed circular intermediates). There did not appear to be a change in the proportion of these molecules synthesized when cells were pretreated with UV radiation. Thus, it is unlikely that a substantial amount of DNA synthesis occurs past pyrimidine dimers without leaving gaps. This conclusion is supported by the observation that the proportion of newly synthesized SV40 form I molecules that contain pyrimidine dimers was not increased in pretreated cells. Pulse-chase experiments suggested that there is a more efficient conversion of replicative intermediates into form I molecules in pretreated cells. This could be due to more efficient gap filling in relaxed circular intermediate molecules or to the release of blocked replication forks. Alternatively, the enhanced replication observed here may be due to an increase in the excision repair capacity of the pretreated cells.     

Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cell interactions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediated interactions. These physiological interactions are crucial for normal thymocyte differentiation, but can be disrupted in pathological situations. Indeed, there is severe thymic atrophy in animals acutely infected with Trypanosoma cruzi due to CD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together with changes in ECM deposition and thymocyte migration. We studied an in vitro model of TEC infection by T. cruzi and found that infected TEC cultures show a reduced number of cells, which was likely associated with decreased proliferative capacity, but not with increased cell death, as demonstrated by bromodeoxyuridine and annexin-V labelling. The infected TEC cultures exhibited increased expression of fibronectin (FN), laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relative number of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted in lower infection rates. Consistent with these data, we observed increased thymocyte adhesion to infected TEC cultures. Overall, these results suggest that ECM molecules, particularly FN, facilitate infection of the thymic epithelium and that the consequent enhancement of ECM expression might be associated with changes in TEC-thymocyte interactions.     

Herpes B Virus,Macacine Herpesvirus 1, Breaks Simplex Virus Tradition via Major Histocompatibility Complex Class I Expression in Cells from Human and Macaque Hosts

B virus of the family Herpesviridae is endemic to rhesus macaques but results in 80% fatality in untreated humans who are zoonotically infected. Downregulation of major histocompatibility complex (MHC) class I in order to evade CD8⁺ T-cell activation is characteristic of most herpesviruses. Here we examined the cell surface presence and total protein expression of MHC class I molecules in B virus-infected human foreskin fibroblast cells and macaque kidney epithelial cells in culture, which are representative of foreign and natural host initial target cells of B virus. Our results show <20% downregulation of surface MHC class I molecules in either type of host cells infected with B virus, which is statistically insignificantly different from that observed in uninfected cells. We also examined the surface expression of MHC class Ib molecules, HLA-E and HLA-G, involved in NK cell inhibition. Our results showed significant upregulation of HLA-E and HLA-G in host cells infected with B virus relative to the amounts observed in other herpesvirus-infected cells. These results suggest that B virus-infected cell surfaces maintain normal levels of MHC class Ia molecules, a finding unique among simplex viruses. This is a unique divergence in immune evasion for B virus, which, unlike human simplex viruses, does not inhibit the transport of peptides for loading onto MHC class Ia molecules because B virus ICP47 lacks a transporter-associated protein binding domain. The fact that MHC class Ib molecules were significantly upregulated has additional implications for host-pathogen interactions.     

Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands.

The molecular mechanisms of in vivo inhibition of mammalian DNA replication by exposure to UV light (at 254 nm) was studied in monkey and human cells infected with simian virus 40. Analysis of viral DNA by electron microscopy and sucrose gradients confirmed that the presence of UV-induced lesions severely blocks DNA synthesis, and thus the conversion of replicative intermediates (RIs) into fully replicated form I DNA is inhibited by UV irradiation. These blocked RI molecules present several special features when visualized by electron microscopy. (i) In excision repair-proficient monkey and human cells they are composed of a double-stranded circular DNA with a double-stranded tail whose size corresponds to the average interpyrimidine dimer distance, as determined by the dimer-specific T4 endonuclease V. (ii) In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Cairns-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. (iii) Single-stranded gaps are visualized in the replicated portions of UV-irradiated RI molecules; such regions are detected and clearly distinguishable from double-stranded DNA when probed by a specific single-stranded DNA-binding protein such as the bacteriophage T4 gene 32 product. Consistent with the presence of gaps in UV-irradiated RI molecules, single-strand-specific S1 nuclease digestion causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules. These results are in agreement with and reinforce the model in which UV lesions are a barrier to the replication fork movement when present in the template for the leading strand; when lesions are in the template for the lagging strand they inhibit synthesis or completion of Okazaki fragments, leaving gaps opposite the lesion. Moreover, cellular DNA repair-linked endonucleolytic activity may induce double-stranded breaks in the blocked region of the replication forks, resulting in the tailed structures observed in viral DNA molecules obtained from excision repair-proficient cell lines.     

Elimination of hepatitis C virus from hepatocytes by a selective activation of therapeutic molecules

To eliminate hepatitis C virus (HCV) from infected hepatocytes, we generated two therapeutic molecules specifically activated in cells infected with HCV. A dominant active mutant of interferon (IFN) regulatory factor 7 (IRF7) and a negative regulator of HCV replication, VAP-C (Vesicle-associated membrane protein-associated protein subtype C), were fused with the C-terminal region of IPS-1 (IFNβ promoter stimulator-1), which includes an HCV protease cleavage site that was modified to be localized on the ER membrane, and designated cIRF7 and cVAP-C, respectively. In cells expressing the HCV protease, cIRF7 was cleaved and the processed fragment was migrated into the nucleus, where it activated various IFN promoters, including promoters of IFNα6, IFNβ, and IFN stimulated response element. Activation of the IFN promoters and suppression of viral RNA replication were observed in the HCV replicon cells and in cells infected with the JFH1 strain of HCV (HCVcc) by expression of cIRF7. Suppression of viral RNA replication was observed even in the IFN-resistant replicon cells by the expression of cIRF7. Expression of the cVAP-C also resulted in suppression of HCV replication in both the replicon and HCVcc infected cells. These results suggest that delivery of the therapeutic molecules into the liver of hepatitis C patients, followed by selective activation of the molecules in HCV-infected hepatocytes, is a feasible method for eliminating HCV.     

Quantitative analysis of the bacteriophage Qbeta infection cycle

In this study, the infection cycle of bacteriophage Qbeta was investigated. Adsorption of bacteriophage Qbeta to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4x10(-10) ml/cells/min. In infected cells, approximately 130 molecules of beta-subunit and 2x10(5) molecules of coat protein were translated in 15 min. Replication of Qbeta RNA proceeded in 2 steps-an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5x10(5) molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.     

Sites of integration of infectious DNA of avian reticuloendotheliosis viruses in different avian cellular DNAs.

The pattern of integration for the infectious DNA of two avian reticuloendotheliosis viruses whose DNA is not inactivated by digestion with the restriction endonuclease, Eco RI was determined. High molecular weight DNA from infected chicken, turkey and pheasant cells was digested with Eco RI, electrophoresed through agarose gels and assayed for infectivity. The same patterns of integration of infectious viral DNA were found for these species of avian cells infected at high or low multiplicities with two reticuloendotheliosis viruses. There were multiple sites of integration in acutely infected cells with concomitant cell death. There was a single site of integration in chronically infected cells with no cell death. There were more integrated infectious viral DNA molecules per cell in acutely infected cells than in chronically infected cells. These results are consistent with the hypotheses that the cell death in the acute phase of infection is a result of the integration of the infectious viral DNA at multiple sites, and that only those cells survive that have the infectious viral DNA integrated exclusively at the single site.     

Cell killing by spleen necrosis virus is correlated with a transient accumulation of spleen necrosis virus DNA.

Spleen necrosis virus productively infects avian and rat cells. The average number of molecules of unintegrated and integrated viral DNA in cells at different times after infection was determined by hybridization and transfection assays. Shortly after infection, there was a transient accumulation of an average of about 150 to 200 molecules of unintegrated linear spleen necrosis virus DNA per chicken, turkey, or pheasant cell. No such accumulation was seen in infected rat cells. Soon after infection there was in chicken cells, but not inturkey, pheasant, or rat cells, also a transient integration of an average of 35 copies of viral DNA per cell. By 10 days after infection, the majority of this integrated viral DNA was lost from the population of infected chicken cells. At the same time, the majority of the unintegrated viral DNA was also lost from infected chicken, turkey, and pheasant cells. The transient cytopathic effect seen in these infected cells also occurred at this time. Late after infection about five copies of apparently nondefective spleen necrosis proviruses were stably integrated at multiple sites in chicken, turkey, pheasant, and rat DNA. These results demonstrate a correlation between the transient accumulation of large numbers of spleen necrosis virus DNA molecules and the transient occurrence of cytopathic effects.     

Staphylococcal exotoxin superantigens induce human immunodeficiency virus type 1 expression in naturally infected CD4+ T cells.

A high proportion of Staphylococcus aureus strains of human origin produce one or more exotoxins. In vivo, these toxins may give rise to a variety of clinical syndromes. In vitro, staphylococcal exotoxins have been shown to bind both to human leukocyte antigen (HLA) class II molecules on antigen-presenting cells and to the T-cell receptors on large fractions of T cells. The result of this interaction may be proliferation of the T cells, T-cell anergy, or apoptosis, depending on several factors, including the state of the responding cells and the presence of accessory molecules. Using naturally infected peripheral blood mononuclear cells depleted of CD8+ T cells, we have shown that staphylococcal exotoxins are powerful inducers of human immunodeficiency virus type 1 expression and that they induce expression at low concentrations and with greater efficiency than other T-cell mitogens. Human immunodeficiency virus type 1 was produced entirely by CD4+ T cells in this model; monocytes were expendable both as a source of virus and as a source of HLA class II molecules as long as other cells expressing HLA class II molecules were present. The results suggest that infection by S. aureus may be a cofactor in the immunopathogenesis of AIDS.     

Characterization of interaction between porcine reproductive and respiratory syndrome virus and porcine dendritic cells

The porcine reproductive and respiratory syndrome Virus (PRRSV) is an infectious disease that causes abortions and respiratory disorders in swine. In this study, the interaction between PRRSV and porcine dendritic cells generated from CD14(+) monocytes in the presence of GM-CSF and IL-4 was examined. As a result, it was shown that immature and mature dendritic cells can be productively infected with PRRSV. When the expression of surface MHC molecules on infected dendritic cells was determined, MHC classes I and II were found to be downregulated when compared with uninfected dendritic cells. With the exception of the IL-4 and IFN-gamma cytokines, the induction of the IL-10, IL-12, and TNF-alpha cytokines all increased in dendritic cells infected with PRRSV. A mixed lymphocyte reaction showed that peripheral blood mononuclear cells cocultured with PRRSVinfected dendritic cells were less stimulated than peripheral blood mononuclear cells cocultured with dendritic cells treated with PBS, LPS, or UV-inactivated PRRSV. Therefore, these results suggest that PRRSV would appear to modulate the immune stimulatory function of porcine dendritic cells.