Selection of bacterial strains efficient in decolorization of remazol black-B.

Azo dyes are released into wastewater streams without any pretreatment and polluted water and soil environments. To prevent contamination of our vulnerable resources, removal of these dye pollutants is of great importance. For this purpose, wastewater samples were collected from dye-contaminated sites of Ankleshwar, Gujarat, India. About 50 bacterial isolates were isolated through enrichment and then tested for their potential to remove Remazol Black-B azo dye in liquid medium. Three bacterial isolates capable of degrading Remazol Black-B azo dye efficiently were screened through experimentation on modified mineral salt medium. Isolate ETL-1 was able to completely remove the Remazol Black-B dye from the liquid medium in 18 h. Further, the isolate showed the best performance at the dye concentration of 100 mg L-1 medium (pH 7) and at temperature 35 degrees C. Similarly, yeast extract proved to be the best carbon source for decolorization purpose. The results imply that the isolate ETL-1 could be used for the removal of the reactive dyes from textile effluents.

Establishment of cell line with NK/NKT phenotype from myeloid NK cell acute leukemia.

Acute Myeloid Leukemia (AML) is the most common malignancy in adults with a 5-year survival rate of 27% of the total affected population. For effective treatment and new drug discovery, cell lines are considered as a very important tool. Here we report an establishment of a continuous human cell line AML-004 with a hypo-diploid chromosome 44 and presence of both NK/NKT phenotypes. The cell line was isolated from the blood sample of myeloid NK cell acute leukemia patients and extensively characterized by flow cytometery, morphology, and cytogentic analysis. Cytotoxicity by standard chemotherapeutic drugs was also examined. As characterized by Giemsa staining, the predominant cell type in the culture had high nuclear/cytoplasmic ratio. Cytogenetic analysis revealed high chromosome instability and structural abnormalities confirming the source of cell line from a patient with AML. The karyotype of the isolated cells did not alter up to around 40 passages. These AML-004 cells lacked specific markers for B and T lymphoid cells, but expressed surface receptors for lymphoid/NK cells. Cells also lacked the presence of early progenitors. The proliferation of the isolated cells was inversely proportional to the IL-2 concentration confirming presence of NK phenotype. AML-004 was resistant against standard chemotherapeutic drugs excluding cisplatin. Thus, AML-004 cells provide a continuous source of human cells for designing novel therapies for patients with T-lymphoblastic leukemia/lymphoma.

Internalins from the human pathogen Listeria monocytogenes combine three distinct folds into a contiguous internalin domain.

Listeria monocytogenes is an opportunistic, food-borne human and animal pathogen. Host cell invasion requires the action of the internalins A (InlA) and B (InlB), which are members of a family of listerial cell-surface proteins. Common to these proteins are three distinctive N-terminal domains that have been shown to direct host cell-specific invasion for InlA and InlB. Here, we present the high-resolution crystal structures of these domains present in InlB and InlH, and show that they constitute a single "internalin domain". In this internalin domain, a central LRR region is flanked contiguously by a truncated EF-hand-like cap and an immunoglobulin (Ig)-like fold. The extended beta-sheet, resulting from the distinctive fusion of the LRR and the Ig-like folds, constitutes an adaptable concave interaction surface, which we propose is responsible for the specific recognition of the host cellular binding partners during infection.

The conserved undecapeptide shared by thiol-activated cytolysins is involved in membrane binding.

Thiol-activated cytolysins share a conserved hydrophobic, Trp-rich undecapeptide that is suggested to be involved in membrane binding and intercalation. The neutralizing monoclonal antibody PLY-5 recognizes all members of this toxin family and peptide mapping assigned its epitope to the undecapeptide motif. This antibody inhibited binding of the toxins to host cell membranes and the epitope was no longer available for binding when a preformed toxin/membrane complex was tested. These results confirm the model of cytolysin binding suggested by structural data.

T-cell anergy induced by antigen presenting cells treated with the hemolysin of Listeria monocytogenes.

Antigen presenting cells (APC) that are infected with listeriolysin (LLO) secreting Listeria lack the ability to stimulate MHC class II restricted T-cells by conventional antigens. Similarly, T-cell activation by native proteins but not by peptides was inhibited upon pretreatment of APC with purified listeriolysin. The inhibition is due to an irreversible inactivation of T-cells that recognize antigen on infected or LLO treated APC. Inhibition was found to dominate over stimulation by peptides. This condition is reminiscent of T-cells inactivation by antagonistic peptides and represents a novel type of immune escape.

Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier.

The efficacious delivery of eukaryotic expression plasmids to inductive cells of the immune system constitutes a key prerequisite for the generation of effective DNA vaccines. Here, we have explored the use of bacteria as vehicles to orally deliver expression plasmids. Attenuated Salmonella typhimurium aroA harbouring eukaryotic expression plasmids that encoded virulence factors of Listeria monocytogenes were administered orally to BALB/c mice. Strong cytotoxic and helper T cell responses as well as antibody production were elicited even after a single administration. Mice immunised four times with Salmonella that carried a eukaryotic expression plasmid encoding the secretory listerial protein listeriolysin were protected against a subsequent lethal challenge with this pathogen. A single dose was already partially protective. The efficiency of this vaccination procedure was due to transfer of the expression plasmid from the bacterial carrier to the mammalian host. Evidence for such an event could be obtained in vivo and in vitro. Expression of the desired antigen in various lymphoid tissues was already detectable 1 day after administration of the DNA vaccine and persisted for at least 1 month in spleen and mesenteric lymph nodes. Induction of cytotoxic and helper T cell responses was observed in all mouse strains tested including outbred strains whereas antibodies were mainly detected in BALB/c. Furthermore, we could show that immunogenicity could be improved by increasing the invasiveness of the bacterial carrier.

Hyperexpression of listeriolysin in the nonpathogenic species Listeria innocua and high yield purification.

Listeriolysin, the hemolysin of the pathogenic species Listeria monocytogenes, was expressed in the non-pathogenic species Listeria innocua. Coexpression of the positive regulatory factor prfA in the plasmid vector in conjunction with the structural gene hly increased the expression over 500-fold. Purification from supernatant fluids was achieved by two steps of ion exchange chromatography. The procedure resulted in over 60% yield of a hemolytically active, homogeneous 58 kDa protein which was used to produce monospecific antibodies. As shown by immunoblot the purified listeriolysin was free of p60, a highly immunogenic protein of similar size also produced by Listeria spp., which otherwise would interfere with immunoassays. Listeriolysin retained full activity for more than 6 months at -70 degrees C.

Cytotoxicity of Mycobacterium indicus pranii on Mia-Pa-Ca2 cells

Background: MIP is a cultivable, non-pathogenic organism, which shares several antigens with Mycobacterium tuberculosis and Mycobacterium leprae. It has several proposed clinical applications. However, its cytotoxic effect on pancreatic cancer has not been documented. Hence, the study was conducted to investigate MIP induced cytotoxicity on Mia-Pa-Ca2 cells. To determine the cytotoxic potential of heat killed Mycobacterium indicus pranii (MIP) on pancreatic cancer cells in vitro along with gemcitabine & 5-fluorouracil (5-FU). Mitogen-activated protein kinase (MAPK) level was also studied post MIP treatment. Methods: Cytotoxic effect of MIP, gemcitabine and 5-FU on Mia-Pa-Ca2 cells was determined. We have analyzed extent of apoptosis using flow cytometry and changes in p38 levels, c-Jun N-terminal kinases (JNK) and extracellular signal­regulated kinase (ERK) using ELISA. Results: MIP not only exhibits cell cytotoxicity in dose dependent manner, but also enhances efficacy of gemcitabine and 5-FU when used in combination. Flow cytometry analyses reveals apoptosis of Mia-Pa-Ca2 cells post MIP treatment compared to untreated cells. MAPK pathway study using ELISA shows that p38 and JNK levels are suppressed while there is no change in ERK level. Conclusion: With these results we conclude that MIP is a cytotoxic agent. Cytotoxicity is exhibited by apoptosis. Combining MIP with gemcitabine and 5-FU shows synergistic effect (AU)

Salmonella-mediated oral DNA vaccination using stabilized eukaryotic expression plasmids.

The use of Salmonella for the delivery of plasmid-encoded heterologous antigens to eukaryotic host cells has proven successful in experimental systems, but its general applicability is still hampered by a severe instability of transformants carrying these expression plasmids. To overcome the problem of plasmid instability, new low copy number expression plasmids were constructed using different replicons. Comparative studies between transformants of the high copy number plasmid pCMVbeta and the different low copy number plasmids that contain the pMB1, p15A or pSC101 replicons on the pCMVbeta backbone, revealed a dramatic increase in plasmid stability both in vitro and in vivo. Analysis of the resulting immune responses against antigens encoded by these vectors indicated that the increased stability resulted in a strong and reproducible induction of both antigen-specific CD4(+) and CD8(+) T-cell and antibody responses even after a single application. In addition, protective immunity was induced against Listeria monocytogenes using listeriolysin as antigen, regardless of the copy number of the delivery plasmid employed. Finally, Salmonella expressing two independent antigens on compatible low copy number plasmids elicited robust responses to either antigen that is as effective as Salmonella transformed with each plasmid singly adding further versatility to this delivery system.

TAP-dependent major histocompatibility complex class I presentation of soluble proteins using listeriolysin.

Immunization of mice with mixtures of listeriolysin, a pore-forming hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, together with soluble ovalbumin, nucleoprotein of influenza virus, or beta-galactosidase of Escherichia coli, resulted in strong cytotoxic CD8 T cell responses to each of the respective passenger proteins in vivo. Also, the concomitant addition of either protein with listeriolysin to target cells elicited efficient sensitization of these cells which could be attributed to the pore-forming activity of listeriolysin. This response was dependent upon a functional TAP transporter and was inhibitable by brefeldin A, indicating the transfer of the soluble proteins into the cytosol and the classical major histocompatibility (MHC) class I presentation pathway. The treatment of target cells with listeriolysin under our experimental conditions did not affect cell viability and the pores generated by listeriolysin treatment were repaired within 60 min. Introduction of soluble proteins into the MHC class I presentation pathway by listeriolysin provides a powerful system to study the cytotoxic response towards intracellular pathogens and would allow for rapid screening of potential antigens in vaccine formulations.

Listeriolysin generates a route for the presentation of exogenous antigens by major histocompatibility complex class I.

We have exploited the pore forming activity of listeriolysin, the hemolysin of Listeria monocytogenes, to activate CD8+ T cells with soluble proteins in vivo and in vitro. Immunization with soluble, hemolytically active listeriolysin induces both cytotoxic CD8+ T cells and CD4+ T cells, and the CD8+ T cells can be propagated with soluble listeriolysin in vitro. Moreover, conventional antigens like ovalbumin mixed together with listeriolysin are also efficiently introduced into the MHC class I pathway in vitro and in vivo. Hence, listeriolysin effectively directs itself and passenger molecules into the intracellular compartment that leads to the cytotoxic T cell response. In this way, we circumvent the bias of CD8+ T cells to recognize intracellular antigens presented by major histocompatibility complex class I molecules. As cytotoxic CD8+ T cells are of pivotal importance in eliminating viral and microbial pathogens, the findings reported here could prove to be useful in vaccine development.

In vitro simulation of immunosuppression caused by Trypanosoma brucei.

Macrophage populations derived from Trypanosoma brucei-infected mice suppress both interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) expression. A prostaglandin-independent mechanism accounts for the suppression of IL-2R expression, while the suppression of IL-2 is prostaglandin-dependent. A macrophage hybridoma cell line (i.e. 2C11-12) was used to mimic the parasite-induced immunosuppression in vitro. It was found that 2C11-12 cells acquired a suppressive potential in vitro following interaction with opsonized living parasites. T. brucei-pulsed 2C11-12 cells failed to block IL-2 secretion in the presence of indomethacin but still suppressed the IL-2R expression. In contrast, addition of living T. brucei parasites to the cultures caused a complete suppression of IL-2 secretion and IL-2R expression, even in the presence of indomethacin. Hence, the addition of excess living parasites to the cultures could cause a depression which was different from the suppression associated with infection, whereas the addition of parasite-pulsed 2C11-12 cells mimicked the situation occurring during infection. This model system can be adopted as a suitable tool to unravel the mechanisms underlying the suppression of IL-2R expression during T. brucei infections.

Modulation of IL-1 production and IL-1 release during experimental trypanosome infections.

Macrophage populations derived from Trypanosoma brucei-infected mice suppress both interleukin-2 (IL-2) production and IL-2 receptor expression. To try to identify the regulatory level by which the T-cell activation is switched off, we have analysed the potential of the suppressive macrophage-like cells to block the secretion of the accessory cell-derived T cell co-stimulator interleukin-1 (IL-1). The IL-1 secretion, however, was found to be greatly increased rather than decreased. The increased secretion was in part caused by an increased release rather than by an increased synthesis. In the presence of an in vitro trigger (lipopolysaccharide), the IL-1 secretion was increased 20-30-fold by the infection whereas the total IL-1 production increased only 1.5-2-fold. Macrophages from infected mice thus manifested a marginally increased IL-1 synthesis but released a markedly larger proportion of the synthesized monokine than normal macrophages. In the absence of an in vitro trigger, the infection caused a 10-15-fold increase in IL-1 synthesis as a consequence of an in vivo preactivation. This increase was only observed when the synthesis of prostaglandins was blocked by addition of indomethacin.

Different mechanisms account for the suppression of interleukin 2 production and the suppression of interleukin 2 receptor expression in Trypanosoma brucei-infected mice.

Lymph node cell populations derived from Trypanosoma brucei-infected mice failed to produce interleukin 2 (IL 2) in response to a potent mitogenic trigger and suppress the potential of normal lymph node cells to secrete IL 2 in co-culture assays. This suppression is promptly restored by the addition of indomethacin, which blocks prostaglandin synthesis, but is not markedly affected by the addition of catalase, which degrades H2O2. The suppression of the IL 2 receptor expression, on the other hand, is not restored by the addition of indomethacin, nor by the simultaneous supply of both indomethacin and catalase. This discrepancy is not caused by an extreme susceptibility of the receptor expression to low prostaglandin (PG) concentrations, but rather by the presence of suppressive cells that operate through a PG-independent mechanism. This suppressive mechanism accounts for the loss of the IL 2 receptors on both the Ly-2+ and the L3T4+ T cell compartment. The indomethacin-treated co-cultures, which manifest a normal IL 2 production but lack the IL 2 receptors, manifest an impaired DNA synthesis and contain a decreased number of T cell blasts.

Dual role of macrophages in the suppression of interleukin 2 production and interleukin 2 receptor expression in trypanosome-infected mice.

Lymph node cells derived from T. brucei-infected mice fail to produce interleukin 2-(IL2) subsequent to a potent mitogenic trigger and actively suppress the capacity of normal cells to produce IL2 in co-culture experiments. The depletion of Thy-1+ cells does not decrease but rather increases the suppressive potential of the LNC derived from infected mice. A T cell-enriched nylon wool-nonadherent fraction, on the other hand, is not suppressive. The suppression of IL2 production is promptly restored by the addition of prostaglandin synthesis inhibitors suggesting a key role of the prostaglandin-producing macrophages. Our data indicate that such macrophages do not act indirectly through the induction of suppressor T cells, but rather directly interfere with the normal lymph node cells. In contrast to the essential role of prostaglandins in the impairment of IL2 production, these mediators are not involved in the suppression of IL2 receptor expression. Lymph node cells derived from Trypanosoma brucei-infected mice fail to produce interleukin 2 (IL2) subsequent to a potent mitogenic trigger and actively suppress the capacity of normal cells to produce IL2 in co-culture experiments. The depletion of Thy-1+ cells does not decrease but rather increases the suppressive potential of the LNC derived from infected mice. A T cell-enriched nylon wool-nonadherent fraction, on the other hand, is not suppressive. The suppression of IL2 production is promptly restored by the addition of prostaglandin synthesis inhibitors suggesting a key role of the prostaglandin-producing macrophages. Our data indicate that such macrophages do not act indirectly through the induction of suppressor T cells, but rather interfere directly with the normal lymph node cells.

Antigen-specific T cell receptor antagonism by antigen-presenting cells treated with the hemolysin of Listeria monocytogenes: a novel type of immune escape.

We have examined the influence of listeriolysin O (LLO), the hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, on major histocompatibility complex class II-dependent T cell activation. Stimulation of T cells by native antigens but not by peptides is inhibited upon pretreatment of antigen-presenting cells (APC) with LLO. Experiments presented here reveal that this inhibition is not due to a lack in processing of antigen by APC but is the result of an irreversible inactivation of T cells that recognize antigen on LLO-treated APC. Incubation of mixtures of two different T cells where only one antigen was presented on LLO-treated APC suggested that T cell inactivation is antigen specific. The inactivation was dominant and could be observed even in the presence of amounts of synthetic peptides that normally lead to T cell responses. This condition is reminiscent of the T cell inhibition observed when antagonistic and stimulatory peptides are added to APC at the same time. Our results thus reveal a novel type of interference by pathogens with antigen presentation and T cell stimulation that could give the pathogen a decisive advantage in dissemination and disease.

Murine tumour necrosis factor plays a protective role during the initial phase of the experimental infection with Trypanosoma brucei brucei.

Soluble extracts from salivarian trypanosomes (Trypanosoma brucei brucei, T. evansi and T. congolense) were shown to be capable of inducing murine tumour necrosis factor (mTNF) secretion, both in vivo and in vitro, whereas the soluble extract of an intracellular trypanosome (T. cruzi) failed to do so. Furthermore, the role of mTNF during the initial phase of experimental infections with T. brucei was studied by treating infected mice with mTNF-inducing trypanosoma soluble extract and with neutralizing monoclonal anti-mTNF antibodies. Treatment of the infected animals with different doses of T. brucei soluble extract resulted in a lower first parasitaemia peak (low lysate dose) and in a longer survival time or in a nearly total inhibition of parasite development (high lysate dose). Cotreatment of the infected mice with both anti-mTNF antibodies and a high dose of soluble extract completely restored the parasite development in both trypanosusceptible C3H/He mice and trypanosubtolerant CBA Ca mice, indicating a protective role of mTNF during the parasitaemia. Collectively these results suggest a negative influence of mTNF on T. brucei development in vivo.

In vitro simulation of immunosuppression caused by Trypanosoma brucei: active involvement of gamma interferon and tumor necrosis factor in the pathway of suppression.

Experimental infections of mice with the African trypanosome Trypanosoma brucei lead to a profound state of T-cell unresponsiveness in the lymph node cell (LNC) compartment. This suppression is mediated by macrophage-like cells which inhibit interleukin 2 (IL-2) secretion and down-regulate IL-2 receptor expression (M. Sileghem, A. Darji, R. Hamers, M. Van de Winkel, and P. De Baetselier, Eur. J. Immunol. 19:829-835, 1989). Similar suppressive cells can be generated in vitro by pulsing 2C11-12 macrophage hybridoma cells with opsonized T. brucei parasites (2C11-12P cells). Cocultures of 2C11-12P cells and LNCs secrete higher levels of gamma interferon (IFN-gamma), and the hyperproduction of IFN-gamma was found to be confined to CD8+ lymphoid cells. Elimination of CD8+ cells from cocultures of 2C11-12P cells and LNCs restores the T-cell proliferative response. Furthermore, addition of neutralizing anti-IFN-gamma antibodies to the cocultures reduces the level of suppression and concomitantly restores the level of IL-2 receptor expression. Hence, IFN-gamma plays a cardinal role in this in vitro model for T. brucei-elicited immunosuppression. Cocultures of LNCs and 2C11-12P cells in a two-chamber culture system further demonstrated that cell-cell contact is required for hyperproduction of IFN-gamma and, moreover, that IFN-gamma cooperates with a 2C11-12P-derived diffusible factor to exert its suppressive activity. Finally, tumor necrosis factor alpha (TNF-alpha produced by 2C11-12P cells was found to be implicated in the hyperproduction of IFN-gamma, since addition of neutralizing anti-TNF-alpha antibodies to cocultures reduced the level of suppression and concomitantly abrogated the hyperproduction of IFN-gamma. Collectively, our findings indicate that T. brucei-elicited suppressive 2C11-12 macrophage cells differentially influence T-cell subpopulations: (i) CD8+ cells are signaled via cell-cell contact to produce IFN-gamma, and TNF-alpha is implicated in this process, and (ii) locally produced IFN-gamma and macrophage-released factors act in concert to inhibit CD4+ and CD8+ T-cell proliferative responses.

Neutralizing monoclonal antibodies against listeriolysin: mapping of epitopes involved in pore formation.

Six different mouse monoclonal antibodies (MAbs) and a specific rabbit polygonal antibody were raised against listeriolysin. Four of the MAbs also recognized seeligeriolysin, and five cross-reacted with ivanolysin. The hemolytic activity could be neutralized by the polygonal antibody as well as by five of the MAbs. None of the neutralizing antibodies interfered with the binding of listeriolysin to the cellular membrane. The epitopes recognized by the MAbs were localized by using overlapping synthetic peptides between positions 59 and 279, a region hitherto not implicated in mediating hemolytic activity.

Listeriolysin O: cholesterol inhibits cytolysis but not binding to cellular membranes.

Listeriolysin O (LLO) binds to cholesterol-containing membranes in which it oligomerizes to form pores. Preincubation of the toxin with cholesterol is known to inhibit haemolysis, whereas the oxidized form of cholesterol has no inhibitory effect. Using immunoblot analyses and flow cytometry we demonstrate that preincubation with cholesterol does not influence binding of the listeriolysin-cholesterol complex to red blood cells, eukaryotic cells or artificial membranes. Lytic activity of membrane-bound LLO inactivated by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. To determine the step at which cholesterol inhibits lytic activity, we looked for pore formation using electron microscopy. Pores formed by purified listeriolysin could be directly visualized using erythrocyte ghosts. This property was lost upon incubation of the toxin with cholesterol. Quantitative analysis strongly suggest that inhibition of lysis by cholesterol is not due to decreased binding of listeriolysin to target membranes, but rather to an interference with a subsequent step leading to polymerization of the toxin.