Preparation and In Vitro Evaluation of Antitumor Activity of TGFαL3-SEB as a Ligand-Targeted Superantigen.

Tumor-targeted superantigens (TTSs) have been used to treat a variety of tumors in preclinical studies. The TTS utilizes the powerful T-cell activation strategy by means of staphylococcal enterotoxins (SEs) as superantigens (Sags) to target tumor cells. Monoclonal antibodies and tumor-related ligands have been used as targeting molecules of Sag. In this study, we assessed the antitumor potency of tumor-targeted superantigen (TTS) strategy to design and produce fusion protein as a new antitumor candidate. The third loop (L3) of transforming growth factor α (TGF-α) was genetically conjugated to staphylococcal enterotoxin type B (TGFαL3-SEB), and its in vitro antitumor activity against murine breast cancer cells (A431 cell line) was evaluated. We designed and prepared TGFαL3-SEB chimeric protein and evaluated superantigenic activity, binding property to cancer cells, overexpression of epidermal growth factor receptor (EGFR), and in vitro antitumor activities. Cloning of tgfαl3-seb was confirmed by colony-polymerase chain reaction, enzymatic digestion, and sequencing. The recombinant TGFαL3-SEB fusion protein with molecular weight of 31 kDa was expressed and confirmed by anti-His Western-blot analysis. The TGFαL3-SEB fusion protein attached to A431 cell line with proper affinity and induced dose-dependent cytotoxicity against EGFR-expressing cancer cells in vitro. The TGFαL3-SEB chimeric protein exhibited potent in vitro antitumor activity. Our findings indicated that TGFαL3-SEB may be a promising anticancer candidate in cancer immunotherapy, and further studies are required to explore its potential in vivo therapeutic applications.

Novel antimicrobial peptides that inhibit gram positive bacterial exotoxin synthesis.

Gram-positive bacteria, such as Staphylococcus aureus, cause serious human illnesses through combinations of surface virulence factors and secretion of exotoxins. Our prior studies using the protein synthesis inhibitor clindamycin and signal transduction inhibitors glycerol monolaurate and α-globin and ß-globin chains of hemoglobin indicate that their abilities to inhibit exotoxin production by S. aureus are separable from abilities to inhibit growth of the organism. Additionally, our previous studies suggest that inhibition of exotoxin production, in absence of ability to kill S. aureus and normal flora lactobacilli, will prevent colonization by pathogenic S. aureus, while not interfering with lactobacilli colonization. These disparate activities may be important in development of novel anti-infective agents that do not alter normal flora. We initiated studies to explore the exotoxin-synthesis-inhibition activity of hemoglobin peptides further to develop potential agents to prevent S. aureus infections. We tested synthesized α-globin chain peptides, synthetic variants of α-globin chain peptides, and two human defensins for ability to inhibit exotoxin production without significantly inhibiting S. aureus growth. All of these peptides were weakly or not inhibitory to bacterial growth. However, the peptides were inhibitory to exotoxin production with increasing activity dependent on increasing numbers of positively-charged amino acids. Additionally, the peptides could be immobilized on agarose beads or have amino acid sequences scrambled and still retain exotoxin-synthesis-inhibition. The peptides are not toxic to human vaginal epithelial cells and do not inhibit growth of normal flora L. crispatus. These peptides may interfere with plasma membrane signal transduction in S. aureus due to their positive charges.

Target expression of Staphylococcus enterotoxin A from an oncolytic adenovirus suppresses mouse bladder tumor growth and recruits CD3+ T cell.

We recently engineered an oncolytic adenovirus (PPE3-SEA) that expresses the superantigen, Staphylococcus enterotoxin A (SEA), and that has enhanced tumor specificity because the telomerase reverse transcriptase and hypoxia-inducible factor promoters regulate expression of E1A and E1B genes, respectively. Here, we evaluated the PPE3-SEA adenovirus anti-tumor activity against MB49 mouse bladder cancer cell proliferation in vitro and in vivo. PPE3-SEA infection of murine MB49 cells in vitro induced cytopathic effects, and significant expression of SEA mRNA and protein, as measured by RT-PCR and western blot, respectively. Subcutaneous MB29 bladder tumors were established in syngeneic C57BL/6 mice. After 10 days, tumors were injected with either oncolytic virus or PBS. Tumor dimensions were measured on days 1, 3, 5, 7, 9, and 11 post-treatment and tumor volumes were calculated. One of eight PPE3-SEA-treated mice had no tumor by day 9. PPE3-SEA treated group had significantly lower mean tumor volume beginning on day 5 post-treatment (p < 0.01), more fibrous tissue in the tumor, and increased presence of infiltrating CD3+ T cells than those of the control group. Gross appearance and histologic sections from the livers and kidneys of the PPE3-SEA-treated group were similar to those of the control group. In conclusion, oncolytic adenoviruses can provide a novel delivery vehicle for SEA to tumor sites, and PPE3-SEA warrants further study as a potential anti-tumor agent for bladder cancer.

In vivo VL-targeted microbial superantigen induced global shifts in the B cell repertoire.

To subvert host defenses, some microbial pathogens produce proteins that interact with conserved motifs in V regions of B cell Ag receptor shared by large sets of lymphocytes, which define the properties of a superantigen. Because the clonal composition of the lymphocyte pool is a major determinant of immune responsiveness, this study was undertaken to examine the in vivo effect on the host immune system of exposure to a B cell superantigen, protein L (PpL), a product of the common commensal bacterial species, Finegoldia magna, which is one of the most common pathogenic species among Gram-positive anaerobic cocci. Libraries of Vκ L chain transcripts were generated from the spleens of control and PpL-exposed mice, and the expressed Vκ rearrangements were characterized by high-throughput sequencing. A total of 120,855 sequencing reads could be assigned to a germline Vκ gene, with all 20 known Vκ subgroups represented. In control mice, we found a recurrent and consistent hierarchy of Vκ gene usage, as well as patterns of preferential Vκ-Jκ pairing. PpL exposure induced significant targeted global shifts in repertoire with reduction of Vκ that contain the superantigen binding motif in all exposed mice. We found significant targeted reductions in the expression of clonotypes encoded by 14 specific Vκ genes with the predicted PpL binding motif. These rigorous surveys document the capacity of a microbial protein to modulate the composition of the expressed lymphocyte repertoire, which also has broad potential implications for host-microbiome and host-pathogen relationships.

The effects of subinhibitory concentrations of costus oil on virulence factor production in Staphylococcus aureus.

AIM: To determine the antimicrobial activity of costus (Saussurea lappa) oil against Staphylococcus aureus, and to evaluate the influence of subinhibitory concentrations of costus oil on virulence-related exoprotein production in staph. aureus. METHODS AND RESULTS: Minimal inhibitory concentrations (MICs) were determined using a broth microdilution method, and the MICs of costus oil against 32 Staph. aureus strains ranged from 0.15 to 0.6 µl ml(-1) . The MIC(50) and MIC(90) were 0.3 and 0.6 µl ml(-1) , respectively. Western blot, haemolytic, tumour necrosis factor (TNF) release and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of costus oil on virulence-associated exoprotein production in Staph. aureus. The data presented here show that costus oil dose dependently decreased the production of α-toxin, toxic shock syndrome toxin 1 (TSST-1) and enterotoxins A and B in both methicillin-sensitive Staph. aureus (MSSA) and methicillin-resistant Staph. aureus (MRSA). CONCLUSION: Costus oil has potent antimicrobial activity against Staph. aureus, and the production of α-toxin, TSST-1 and enterotoxins A and B in Staph. aureus was decreased by costus oil. SIGNIFICANCE AND IMPACT OF THE STUDY: The data suggest that costus oil may deserve further investigation for its potential therapeutic value in treating Staph. aureus infections. Furthermore, costus oil could be rationally applied in food products as a novel food preservative both to inhibit the growth of Staph. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.

Superantigens.

Superantigens (SAgs) are derived from diverse sources, including bacteria, viruses, and human hepatic tissue. SAgs initially cause lymphocyte activation but then result in clonal deletion and anergy, leading to immune tolerance. They can also act as superallergens by stimulating a broad spectrum of mast cells and basophils in patients with allergic conditions. The newly described staphylococcal SAg-like proteins subvert innate immune function by several mechanisms, which are distinct from SAgs' effects on lymphocytes and other acquired immune processes. There is mounting evidence to suggest that SAgs play a role in the pathophysiology of inflammatory airway disease. The pathophysiologic role of SAg-like proteins awaits clarification.

Peripheral B cell tolerance and function in transgenic mice expressing an IgD superantigen.

Transitional B cells turn over rapidly in vivo and are sensitive to apoptosis upon BCR ligation in vitro. However, little direct evidence addresses their tolerance sensitivity in vivo. A key marker used to distinguish these cells is IgD, which, through alternative RNA splicing of H chain transcripts, begins to be coexpressed with IgM at this stage. IgD is also expressed at high levels on naive follicular (B-2) and at lower levels on marginal zone and B-1 B cells. In this study, mice were generated to ubiquitously express a membrane-bound IgD-superantigen. These mice supported virtually no B-2 development, a greatly reduced marginal zone B cell population, but a relatively normal B-1 compartment. B cell development in the spleen abruptly halted at the transitional B cell population 1 to 2 stage, a block that could not be rescued by either Bcl-2 or BAFF overexpression. The developmentally arrested B cells appeared less mature and turned over more rapidly than nontransgenic T2 cells, exhibiting neither conventional features of anergy nor appreciable receptor editing. Paradoxically, type-2 T-independent responses were more robust in the transgenic mice, although T-dependent responses were reduced and had skewed IgL and IgH isotype usages. Nevertheless, an augmented memory response to secondary challenge was evident. The transgenic mice also had increased serum IgM, but diminished IgG, levels mirrored by the increased numbers of IgM(+) plasma cells. This model should facilitate studies of peripheral B cell tolerance, with the advantages of allowing analysis of polyclonal populations, and of B cells naturally lacking IgD.

Staphylococcal infection and toxin production in chronic rhinosinusitis.

BACKGROUND: The association between bacterial colonization and different forms of chronic rhinosinusitis (CRS) has not been well documented. One of the most recent hypotheses is superantigen (SA)-induced inflammation, resulting in up-regulation of lymphocytes to produce cytokines, and other inflammatory mediators that strongly modify the disease. Staphylococcus aureus, frequently encountered in nasal passages, can produce enterotoxins that can act as SAs. METHODS: A prospective case control study was performed. Sixty-four patients diagnosed with CRS (group 1), CRS with nasal polyps (CRSwNP) (group 2), and 15 control subjects were enrolled. Swabs were taken from the middle meatus of all subjects for identification of S. aureus carriers. Positive carriers were analyzed for the presence of toxic shock syndrome toxin (TSST) 1 using reverse passive latex agglutination as well as polymerase chain reaction. RESULTS: The rate of nasal carriage of S. aureus in CRS was 42.8%, that of CRSwNP was 45.4%, and that of the control group was 13.3%. The difference between both groups of CRS and the control group was found to be highly significant (p < 0.001). The detection of TSST-1 was significantly higher (p < 0.001) in both groups of CRS patients than in the control group. Finally, the difference in colonization of TSST-1 was highly significant (p < 0.001) between the CRS group 1 and CRSwNP group 2 patients. CONCLUSION: Identifying SAs and understanding how they elicit the pathogenic condition in CRS will be central in revealing ways to ameliorate their effects and properly treat these conditions.

Expression and bioactivity analysis of Staphylococcal enterotoxin M and N.

Staphylococcal enterotoxins (SEs) are powerful superantigens that stimulate non-specific T-cell proliferation produced by Staphylococcus aureus and draw considerable attention as ideal drugs for cancer therapy. The filtrate of S. aureus culture has been used as ampul named Staphylococcal enterotoxin C injection in clinic for 10 years in China and proved to be effective. The superantigen SEC claimed to be the only active component without certifiable evidences. For further investigations of the active components of this injection and establishment of foundations for the development of novel anti-cancer drugs, in this research we extracted total DNA from S. aureus (FRI 1230), cloned, expressed and purified recombinant proteins of Staphylococcal enterotoxin M and N (rSEM and rSEN). The MTT assay of the purified rSEM and rSEN demonstrated that their abilities of stimulating T cells and inhibiting the proliferation of K562-ADM cells and B16 cells were equivalent to that of purified SEC2 in vitro. These findings suggested that SEC was not the only active component of Staphylococcal enterotoxin C injection and the effective procedure of expression and purification may be useful for mass productions of these therapeutically important proteins.

[Detection rate of enterotoxigenic Staphylococcus aureus isolated from children with intestinal disbacteriosis].

Detection rate of enterotoxigenic Staphylococcus aureus isolated from faeces of 62 children aged from 3 months old to 7 years old with intestinal dysbacteriosis was studied by indirect hemagglutination assay and enzume immunoassay. It was shown that strains of S.aureus producing staphylococcal enterotoxin A (SEA) are prevailed (40.3%) in children with disturbances of intestinal microflora while staphylococcal enterotoxin B (SEB)-producing strains were detected in 20.9% of children. Amount of produced enterotoxin varied for SEA from 125 ng/ml to 2000 ng/ml and for SEB--from 125 ng/ml to 250 ng/ml. Inverse dependence of detection rate of enterotoxin-producing strains in faeces on age of children was established. The most number of enterotoxigenic strains of S.aureus was detected in infants. These data point to expediency of determination of enterotoxin-producing ability of S. aureus strains isolated from children with dysbacteriosis as measure of danger of this microorganism for children's health and indication for adequate actions for its elimination.

Failure of viridans group streptococci causing bacteremia in pediatric oncology patients to express superantigens.

Group A Streptococcus pyogenes causes a distinctive clinical disorder, streptococcal toxic shock syndrome, mediated by superantigenic bacterial exotoxins. Oncology patients with viridans group streptococcal sepsis frequently present with a streptococcal toxic shocklike syndrome of unclear pathogenesis. Viridans group streptococci isolated from pediatric oncology patients with streptococcal toxic shocklike illnesses do not possess homologs of known superantigen genes. Supernatants from cultures of these bacteria also fail to stimulate T-cell proliferation, suggesting these bacteria do not commonly elaborate superantigens. Adjunctive treatment with intravenous immunoglobulin, which is advantageous in streptococcal toxic shock syndrome, may not benefit these patients.

Cutting edge: Epstein-Barr virus transactivates the HERV-K18 superantigen by docking to the human complement receptor 2 (CD21) on primary B cells.

EBV, a ubiquitous human herpesvirus, is the causative agent of infectious mononucleosis and is associated with many carcinomas. We have previously shown that the EBV latent genes LMP-1 and LMP-2A (for latent membrane proteins 1 and 2A), transactivate a human endogenous retrovirus (HERV), HERV-K18, in infected B lymphocytes. The envelope (Env) protein of HERV-K18 encodes a superantigen that strongly stimulates a large number of T cells. In this study we report that HERV-K18 env is transactivated even earlier in the infection process, before the establishment of latency; namely, we found that EBV, through its interaction with its cellular receptor CD21, induces the HERV-K18 env gene in resting B lymphocytes. This transactivation is direct and immediate, as up-regulation of transcripts can be detected within 30 min after EBV exposure. Thus, EBV binding to human CD21 on resting B cells triggers the expression of an endogenous superantigen. The biological significance of this superantigen expression for the EBV life cycle is discussed.

Agonist ligands expressed by thymic epithelium enhance positive selection of regulatory T lymphocytes from precursors with a normally diverse TCR repertoire.

CD4+CD25+ regulatory T lymphocytes play a crucial role in inhibition of autoimmune pathology. In accordance with this physiological role, it is now well established that the repertoire of these lymphocytes is strongly enriched in autospecific cells. However, despite extensive investigation, the thymic mechanisms involved in development of regulatory T cells remain incompletely defined. To address the issue of selection of regulatory T cell precursors in mice with a naturally diverse TCR repertoire, we have analyzed development of superantigen-specific regulatory T cells in hemopoietic chimeras in which endogenous super-antigens are exclusively presented by thymic epithelial cells. Our results demonstrate that recognition of agonist ligands expressed by thymic epithelium does not lead to deletion but substantially enhances development of mature regulatory T cells. Interestingly, also development of a small subpopulation of CD25-expressing T cells lacking expression of the transcription factor Foxp3, thought to be autospecific, is enhanced by expression of the agonist ligand on thymic epithelium. Based on quantitative arguments, we propose that commitment to the regulatory T cell lineage is not dictated by the specificity of precursors, but that recognition of the agonist ligand expressed by thymic epithelium substantially enhances their positive selection.

Modulation of expression of superantigens by human transferrin and lactoferrin: a novel mechanism in host-Streptococcus interactions.

The role played by host-pathogen interactions in regulation of expression of streptococcal virulence factors in vivo is beginning to become clear. We have reported that the expression of 2 streptococcal virulence factors, the streptococcal pyrogenic exotoxin (Spe) A and the cysteine protease SpeB, was reciprocally modulated during infection with Streptococcus pyogenes. To identify host signals mediating this reciprocal regulation, we cocultured clonal M1T1 isolates with human peripheral blood mononuclear cells (PBMCs). In accordance with our in vivo findings, when bacteria were in direct contact with human PBMCs or were separated in transwells, expression of speA was induced, whereas expression of speB was down-regulated. This phenomenon was mediated by transferrin and lactoferrin and was influenced by the iron-saturation status of these proteins. Iron chelation from media induced expression of speA, but to a much lesser degree than did that with apotransferrin and lactoferrin, suggesting additional effects of these ferrins on modulation of expression of speA and speB. Thus, ferrins may play an important role in host-pathogen interactions in skin and mucosal tissues.

Ikaros null mice display defects in T cell selection and CD4 versus CD8 lineage decisions.

Previous evidence suggested that the hemopoietic-specific nuclear factor Ikaros regulates TCR signaling thresholds in mature T cells. In this study, we test the hypothesis that Ikaros also sets TCR signaling thresholds to regulate selection events and CD4 vs CD8 lineage determination in developing thymocytes. Ikaros null mice were crossed to three lines of TCR-transgenic mice, and positive selection, negative selection, and CD4 vs CD8 lineage decisions were analyzed. Mice expressing a polyclonal repertoire or a MHC class II-restricted TCR transgene exhibited enhanced positive selection toward the CD4 lineage. Moreover, in the absence of Ikaros, CD4 development can occur with decreased thresholds of TCR signaling. In addition, CD4 single-positive thymocytes were detected in MHC class I-restricted TCR-transgenic Ikaros null mice. To assess the role of Ikaros in negative selection, we analyzed deletion of T cells induced by conventional Ag or by endogenous superantigen. Surprisingly, negative selection was impaired in Ikaros null thymocytes despite evidence of high levels of TCR signal and no intrinsic defect in apoptosis ex vivo. To our knowledge, these data identify Ikaros as the first nuclear factor that plays a critical role in regulating negative selection as well as CD4 vs CD8 lineage decisions during positive selection.

[Preparation and the anti-tumor activity of mutant Staphylococcal enterotoxin B].

AIM: To prepare mutant Staphylococcal enterotoxin B(SEB) and observe its anti-tumor activity. METHODS: The expressed mutant SEB-K172E in inclusion body was denatured and renatured, and then isolated and purified. The anti-tumor activity of the mutant SEB-K172E was compared with wild-type SEB. RESULTS: Renaturation and purification method of the mutant SEB-K172E was developed. The anti-tumor activity of acquired mutant protein was ten times higher than that of wild-type SEB. CONCLUSION: The anti-tumor activity of the mutant SEB-K172E is much higher than that of wild-type SEB, suggesting that the mutant SEB-K172E may become a potential anti-tumor drug.

A role for the B7-1/B7-2:CD28/CTLA-4 pathway during negative selection.

Although costimulation plays an important role in activating naive T cells, its role in negative selection is controversial. By following thymocyte deletion induced by endogenous superantigens in mice lacking B7-1 and/or B7-2, we have identified a role for both B7-1 and B7-2 in negative selection. Studies using CD28-deficient and CD28/CTLA-4-double-deficient mice have revealed that either CD28 or another as yet undefined coreceptor can mediate these B7-dependent signals that promote negative selection. Finally, CTLA-4 delivers signals that inhibit selection, suggesting that CTLA-4 and CD28 have opposing functions in thymic development. Combined, the data demonstrate that B7-1/B7-2-dependent signals help shape the T cell repertoire.

Protein Fv produced during viral hepatitis is an endogenous immunoglobulin superantigen activating human heart mast cells.

Protein Fv, an endogenous protein produced in the liver, is released in biological fluids during viral hepatitis. Acute and chronic viral hepatitis can be associated with cardiovascular derangements. Protein Fv induced the release of histamine, tryptase and the de novo synthesis of prostaglandin D(2) and cysteinyl leukotriene C(4) from mast cells isolated from human heart tissue (HHMC). Protein Fv absorbed with protein A-Sepharose coated with polyclonal IgG did not induce histamine secretion. The maximal percent histamine secretion induced by protein Fv correlated (r(s) = 0.60; p < 0.05) with that induced by anti-IgE, whereas there was no correlation between the release caused by proteins Fv and C5a. Preincubation of HHMC with protein Fv or anti-IgE caused complete cross-desensitization to subsequent challenge with heterologous stimulus. HHMC from which IgE had been dissociated no longer released histamine in response to anti-IgE and protein Fv. A human monoclonal IgE blocked both anti-IgE- and protein Fv-induced release. Three human monoclonal IgM V(H)3(+) inhibited protein-Fv-induced secretion of histamine from HHMC, whereas monoclonal IgM V(H)6(+) did not inhibit the release induced by protein Fv. Protein Fv acts as an endogenous immunoglobulin superantigen by interacting with the V(H)3 domain of IgE to induce the release of mediators from HHMC.

Preferential infection of immature dendritic cells and B cells by mouse mammary tumor virus.

Until now it was thought that the retrovirus mouse mammary tumor virus preferentially infects B cells, which thereafter proliferate and differentiate due to superantigen-mediated T cell help. We describe in this study that dendritic cells are infectable at levels comparable to B cells in the first days after virus injection. Moreover, IgM knockout mice have chronically deleted superantigen-reactive T cells after MMTV injection, indicating that superantigen presentation by dendritic cells is sufficient for T cell deletion. In both subsets initially only few cells were infected, but there was an exponential increase in numbers of infected B cells due to superantigen-mediated T cell help, explaining that at the peak of the response infection is almost exclusively found in B cells. The level of infection in vivo was below 1 in 1000 dendritic cells or B cells. Infection levels in freshly isolated dendritic cells from spleen, Langerhans cells from skin, or bone marrow-derived dendritic cells were compared in an in vitro infection assay. Immature dendritic cells such as Langerhans cells or bone marrow-derived dendritic cells were infected 10- to 30-fold more efficiently than mature splenic dendritic cells. Bone marrow-derived dendritic cells carrying an endogenous mouse mammary tumor virus superantigen were highly efficient at inducing a superantigen response in vivo. These results highlight the importance of professional APC and efficient T cell priming for the establishment of a persistent infection by mouse mammary tumor virus.

Epstein-Barr virus transactivates the human endogenous retrovirus HERV-K18 that encodes a superantigen.

Superantigens (SAgs) are proteins produced by pathogenic microbes to elicit potent, antigen-independent T cell responses that are believed to enhance the microbes' pathogenicity. Here we show that the human lymphotropic herpesvirus Epstein-Barr virus (EBV) transcriptionally activates the env gene of an endogenous retrovirus, HERV-K18, that possesses SAg activity. SAg activity was demonstrated by MHC class II dependent preferential activation of TCRVB13 T cells in response to murine B cells transfected with the HERV-K18 env gene. This is a unique demonstration of a pathogen inducing a host-encoded Sag and accounts for the previously described EBV associated Sag activity. The T cell activation elicited by the Sag could play a central role in EBV infection and associated diseases.