Superantigen Activity Research Articles

Staphylococcal enterotoxins: Molecular aspects and detection methods

Members of the Staphylococcus genus, especially Staphylococcus aureus, are the most common pathogens found in hospitals and in community-acquired infections. Some of their pathogenicity is associated with enzyme and toxin production. Until recently, S. aureus was the most studied species in the genus; however, in last few years, the rise of infections caused by coagulase-negative staphylococci has pointed out the need for further studies on virulence factors that have not yet been completely elucidated so as to better characterize the pathogenic potential of this group of microorganisms. Several staphylococcal species produce enterotoxins, a family of related proteins responsible for many diseases, such as the toxic-shock syndrome, septicemia and food poisoning. To this date, 23 different enterotoxin types have been identified besides toxic-shock syndrome toxin-1 (TSST-1), and they can be divided into five phylogenetic groups. The mechanism of action of these toxins includes superantigen activity and emetic properties, which can lead to biological effects of infection. Various methods can detect genes that encode enterotoxins and their production. Molecular methods are the most frequently used at present. This review article has the objective to describe aspects related to the classification, structure and regulation of enterotoxins and toxic-shock syndrome toxin-1 detection methods. Key words: Staphylococcus aureus, coagulase-negative staphylococci, enterotoxins, TSST-1, PCR, molecular detection.

Oral Vaccine Formulations Stimulate Mucosal and Systemic Antibody Responses against Staphylococcal Enterotoxin B in a Piglet Model

Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.

Staphylococcal Superantigen-Like Protein 5 Inhibits Matrix Metalloproteinase 9 from Human Neutrophils

Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass fingerprinting analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [K(D)] = 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (K(i) = 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking.

Neurodermitis

Atopic eczema (AE) is a multifaktoriell skin disease caused by a variety of factors such as genetic conditions, alterated skin structure, immunologic deviations, psychological and environmental factors, among others. There are two main subtypes of AE, i.e. the IgE-associated ("extrinsic atopic eczema") and the non-IgE-associated type ("intrinsic atopic eczema") with different prognosis concerning the development of respiratory diseases ("atopy march"). The role of allergens varies: while in early childhood food allergens may play a role, mites and microbial antigens may become more relevant in adolescence and adulthood. Recently, it was demonstrated that Filaggrin is a major gene for atopic eczema. The altered skin structure and a deficiency in antimicrobial peptides favour colonization with Staphylococcus aureus; their enterotoxins with superantigenic activity stimulate activation of T cells and macrophages. Also sensitization to the yeast Malassezia spp. occurs almost exclusively in AE patients. So far, AE skin lesions are orchestrated by the local tissue expression of proinflammatory cytokines and chemokines with activation of T lymphocytes, dendritic cells, macrophages, keratinocytes, mast cells, and eosinophils which lead to the skin inflammatory responses. From the therapeutic point of view, a step wise approach using emollients as a basic treatment is recommended. Modern topical corticosteroids of moderate to medium potency applied once per day and only on several days per week offer an efficient anti-inflammatory treatment with moderate side effects. Topic immunomodulatory drugs (tacrolimus and pimecrolimus) have in addition substantially improved the treatment of AE. Proactive treatment approaches also in disease-free intervals may reduce exacerbations and total drug use. Phototherapy and wet dressings are both efficient and safe additional tools in more severe forms. For generalized severe forms systemic drugs such as Cyclosporin A are very helpful. Various Biologicals and antipruriginous substances are under clinical investigation and may add to an improved therapy in the future.

Neurodermatitis : Atopic dermatitis - current insights into path physiology and management

Atopic eczema (AE) is a multifaktoriell skin disease caused by a variety of factors such as genetic conditions, alterated skin structure, immunologic deviations, psychological and environmental factors, among others. There are two main subtypes of AE, i.e. the IgE-associated (extrinsic atopic eczema) and the non-IgE-associated type (intrinsic atopic eczema) with different prognosis concerning the development of respiratory diseases (atopy march). The role of allergens varies: while in early childhood food allergens may play a role, mites and microbial antigens may become more relevant in adolescence and adulthood. Recently, it was demonstrated that Filaggrin is a major gene for atopic eczema. The altered skin structure and a deficiency in antimicrobial peptides favour colonization with Staphylococcus aureus; their enterotoxins with superantigenic activity stimulate activation of T cells and macrophages. Also sensitization to the yeast Malassezia spp. occurs almost exclusively in AE patients. So far, AE skin lesions are orchestrated by the local tissue expression of proinflammatory cytokines and chemokines with activation of T lymphocytes, dendritic cells, macrophages, keratinocytes, mast cells, and eosinophils which lead to the skin inflammatory responses. From the therapeutic point of view, a step wise approach using emollients as a basic treatment is recommended. Modern topical corticosteroids of moderate to medium potency applied once per day and only on several days per week offer an efficient anti-inflammatory treatment with moderate side effects. Topic immunomodulatory drugs (tacrolimus and pimecrolimus) have in addition substantially improved the treatment of AE. Proactive treatment approaches also in disease-free intervals may reduce exacerbations and total drug use. Phototherapy and wet dressings are both efficient and safe additional tools in more severe forms. For generalized severe forms systemic drugs such as Cyclosporin A are very helpful. Various Biologicals and antipruriginous substances are under clinical investigation and may add to an improved therapy in the future.

Expression and bioactivity of staphylococcal enterotoxin O

Objective To clone the mature peptide gene of seo, construct two kinds of prokaryotic fusion expression vector, obtain recombinant staphylococcal enterotoxin O(SEO) and evaluate its biological activity. Methods The seo gene was amplified from ZNZ2-3 strain of Staphylococcus xylosus by designed primers and it was cloned into fusion vector pGEX-6P-1 and pET28a. The soluble recombinant protein(GST-SEO) was expressed in E. coli BL21 host cells, the purified GST-SEO was obtained by a single step of affini-ty chromatography using Glutathinione Sepharose 4B colume. Another fusion protein(P28-SEO) with his-tag was expressed in E. coli BL21 ( DE3), the purified P28-SEO protein was harvested by nickel chelate affinity chromatography method. The antigenicity of recombinant SEO was tested by Western blot. The stimulating effects of recombinant SEO on mouse lymphocytes was tested by MTT. The activity of Caspase 3 and the ap-optosis of DNA ladder were tested. Results The nucleotide sequence of the cloned seo gene was the same as that of reported in GenBank. The soluble recombinant protein GST-SEO was expressed at 25% expression level, and P28-SEO was 22%. The Western blot by GST-SEO-positive sera demonstrated that the recombi-nant SEO had good immunogenicity. The MTY assay of the enterotoxin activity showed that low dose of re-combinant SEO stimulated the proliferation of mice spleen lymphocytes, higher doses will lead to apoptosis. Conclusion Two of the recombinant SEO still have superantigen activity, and can lead to apoptosis of mice splenocytes. Key words: Staphylococcus; Enterotoxin O; Bioactivity

Staphylococcal superantigen-like protein 10 (SSL10) binds to human immunoglobulin G (IgG) and inhibits complement activation via the classical pathway

Staphylococcal superantigen-like (SSL) proteins are a family of exoproteins that share structural similarity with staphylococcal superantigens but exhibit no superantigenic activity. It was previously reported that two members (SSL5 and SSL7) bound to serum components and cell adhesion molecules involved in host immune response; however, the other family members have not been functionally characterized. In this study, we attempted to isolate SSL10-binding proteins from human serum and found that recombinant His-tagged SSL10 bound two major polypeptides of ∼50 and ∼25 kDa after affinity purification and SDS-polyacrylamide gel electrophoresis. These polypeptides were identified as heavy and light chains of human IgG by peptide mass fingerprinting analysis. The specific interaction between recombinant SSL10 and human IgG was confirmed by far Western blot analysis using immobilized SSL10 and pull-down analysis using SSL10-conjugated Sepharose. Surface plasmon resonance analysis revealed that the dissociation equilibrium constant for the interaction between human IgG and recombinant SSL10 was estimated to be 220 nM. We also found that recombinant SSL10 inhibited the binding of complement component C1q to IgG-Sepharose and hemolysis of IgG-sensitized sheep erythrocytes via the classical complement activation pathway. These results suggest that SSL10 may play a role in the evasion of Staphylococcus aureus from the host immune system via interfering complement activation.

In vitrocell-based assay for activity analysis of staphylococcal enterotoxin A in food

Staphylococcal enterotoxins (SEs) are a leading cause of food poisoning and have two separate biological activities; it causes gastroenteritis and functions as a superantigen that activates large numbers of T cells. In vivo monkey or kitten bioassays were developed for analysis of SEs emetic activity. To overcome the inherent limitations of such bioassays, this study describes an in vitro splenocyte proliferation assay based on SEs superantigen activity as an alternative method for measuring the activity of staphylococcal enterotoxin A (SEA). After incubation of splenocytes with SEA, cell proliferation was measured by labeling the proliferating cells' DNA with bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) and quantifying the incorporated BrdU by immunohistochemistry. BrdU labeling is shown to be highly correlated with SEA concentration (R(2)=0.99) and can detect 20 pg mL(-1) of SEA, which is far more sensitive than most enzyme-linked immunosorbent assays. Our assay can also distinguish between active toxin and inactive forms of the toxin in milk. By applying immunomagnetic beads that capture and concentrate the toxin, our assay was able to overcome matrix interference. These results suggest that our in vitro cell-based assay is an advantageous practical alternative to the in vivo monkey or kitten bioassays for measuring SEA and possibly other SEs activity in food.

Functional analysis of the disulphide loop mutant of staphylococcal enterotoxin C2

The superantigen staphylococcal enterotoxin C2 (SEC2) tremendously activate T lymphocytes bearing certain T-cell receptor Vbeta domains when binding to MHC II molecules, which launches a powerful response of tumour inhibition in vitro as well as in vivo. However, the toxicity of SEC2 performed in clinic limited its broad application for immunotherapy. The previous studies suggested that the disulphide loop may be important for the toxicity of some SEs, which prompted us to investigate the potential roles of the disulphide loop in biological activity of SEC2. Site-directed mutagenesis was used to disturb the formation of the disulphide bond by substituting Ala or Ser for Cys-93 and Cys-110. The expressed mutants in Escherichia coli were used to determine their superantigen activity and toxicity. Results showed that all of the mutated proteins exhibited reduced abilities to induce T-cell proliferation and cytotoxic effects on tumour cells L929 and Hepa1-6, suggesting that the disulphide loop plays functional role in maintaining the maximal superantigen activity of SEC2. Furthermore, the toxicity assays in vivo showed that all of the mutants induced a reduced emetic and pyrogenic responses compared with native SEC2, which might be important for further construction of lowly toxic superantigen agent.

Development of Immunopathobiogenesis on SIRS-Sepsis

Over the past decade, sepsis has been diagnosed according to consensus guidelines established in 1991 as an infection in addition to the symptoms of systemic inflammatory response syndrome (SIRS). In addition to the previous criteria, the 2001 conference added several new diagnostic criteria for sepsis. Of particular interest was the inclusion of the biomarkers procalcitonin (PCT) and C-reactive protein (CRP), despite the overall conclusion that it was premature to use biomarkers for sepsis diagnosis. The primary recommendation of the panel was the implementation of the Predisposition, insult Infection, Response, and Organ dysfunction (PIRO).The immune system has traditionally been devided into innate and adaptive components, each of which has a different role and function in defending the host against infectious agents. Stimulation of different TLRs induces distinct patterns of gene expression, which not only leads to the activation of innate immunity but also increasing evidence supports an additional critical role for TLRs in orchestrating the development of adaptive immune responses.The superantigens are able to induce toxic shock syndrome and can sometimes cause multiple organ failure via adaptive immune system. The superantigenic activity of the bacterial exotoxins can be attributed to their ability to cross-link major histocompatibility complex class II molecules on antigen-presenting cells outside the peptide groove with T-cell receptors to form a trimolecular complex. This trimolecular interaction leads to uncontrolled release of a number of proinflammatory cytokines. Proinflammatory cytokines especially IFN-γ and TNF-α, the key cytokines causing toxic shock syndrome.KEYWORDS: sepsis, innate immunity, adaptive

Biological analysis of the deletion mutants of Staphylococcal enterotoxin C2

To investigate the functional domains involved in the biological activity of staphylococcal enterotoxin (SEC2), a series of SEC2 mutants were constructed. Deletion of the last 77 amino acids at the C-terminus of SEC2 did not affect its native superantigen and fever activities, and further removal of the C-terminal residues reduced SEC2 activities significantly. On the other hand, the mutants lacking 18 or more N-terminal residues severely impaired superantigen activity. These data indicated that the functional regions for the biological activities of SEC2 were confined to N-terminal domain, further implied that the proper three-dimensional structure of SEC2 is not needed for its biological activities. Our results deliver valuable information that it is possible to design new SEC2 immunotherapeutic agents which have the superantigen activity and low molecular weight for permeability.

Staphylococcus aureusSuperantigens Elicit Redundant and Extensive Human Vβ Patterns

Staphylococcus aureus can produce a wide variety of exotoxins, including toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxins, and staphylococcal enterotoxin-like toxins. These toxins share superantigenic activity. To investigate the beta chain (Vbeta) specificities of each of these toxins, TSST-1 and all known S. aureus enterotoxins and enterotoxin-like toxins were produced as recombinant proteins and tested for their ability to induce the selective in vitro expansion of human T cells bearing particular Vbeta T-cell receptors (TCR). Although redundancies were observed between the toxins and the Vbeta populations, each toxin induced the expansion of distinct Vbeta subsets, including enterotoxin H and enterotoxin-like toxin J. Surprisingly, the Vbeta signatures were not associated with a specific phylogenic group of toxins. Interestingly, each human Vbeta analyzed in this study was stimulated by at least one staphylococcal superantigen, suggesting that the bacterium derives a selective advantage from targeting the entire human TCR Vbeta panel.

Biological characterization of the zinc site coordinating histidine residues of staphylococcal enterotoxin C2

The bacterial toxin staphylococcal enterotoxin C2 (SEC2) can cause staphylococcal toxic shock syndrome and food poisoning. Although the previously determined crystal structure of SEC2 revealed that some histidine residues (His47, His118 and His122) contribute to the binding of zinc ions, little is known about their biological roles in SEC2. This prompted us to investigate the role of the zinc site coordinating histidine residues in the biological activities of SEC2. The mutants with substitutions at positions 118 and 122 all retained T-cell stimulatory activity, whereas the histidine mutants at position 47 were defective in the ability to stimulate T-cell proliferation. Further toxicity assays in vivo indicated that mutants SEC2-H118A and SEC2-H122A were defective in emetic and febrile activities. However, mutant SEC2-H47A could cause significant emetic and febrile responses in comparison with the other two histidine mutants. These findings suggested that the zinc-coordinating histidine residues play significant roles in superantigen and toxic activities of SEC2 and further implied that superantigen and febrile activities could be separable in staphylococcal enterotoxins. The results also show that it should be possible to design new SEC2 immunotherapeutic agents that have superantigen activity and low toxicity.

Immunization with a Nontoxic Mutant of Staphylococcal Enterotoxin A, SEAD227A, Protects against Enterotoxin‐Induced Emesis in House Musk Shrews

Staphylococcal enterotoxins (SEs) are the most common cause of foodborne diseases and toxic shock throughout the world. However, no vaccine that prevents emesis induced by SEs has been described. A nontoxic mutant of SEA, SEAD227A, was constructed by site-directed mutagenesis and was purified by means of the Escherichia coli expression system. House musk shrews, a small emetic animal model, were immunized with SEAD227A and then challenged with wild-type SEA. SEA-induced emesis was recorded for 3 h. Antibody production was analyzed by gel double-immunodiffusion assay. Neutralizing activities of the antibodies with respect to superantigenic and emetic activities were analyzed in vitro and in vivo. SEAD227A was devoid of both superantigenic and emetic activities but still retained its immunological activity. Immunization with SEAD227A strongly induced specific antibody production and provided significant protection against SEA-induced emesis. Antibodies from immunized shrews markedly inhibited the SEA-induced proliferation of spleen cells and also significantly ablated SEA-induced vomiting in the animals. These results suggest that vaccination with SEAD227A, which is devoid of toxic properties, provides protection against SEA-induced emesis. This nontoxic mutant and its specific antibodies might be useful in the prevention and treatment of staphylococcal food poisoning.

Construction and Characterization of a Novel Superantigen Fusion Protein: bFGF/SEB

Background: As one of the bacterial superantigens, Staphylococcal enterotoxins (SEs) are potent activators of T cells, especially for those expressing T cell receptor Vβ chains, and can induce the production of cytokines such as IFN-γ, TNF-α, IL-1, IL-2, IL-6, IL-12, etc. Thus, SEs could be used in tumor-targeting therapy when cooperated with the vectors that can specifically recognize the tumor cells. Materials and methods: The coding sequences of Staphylococcal enterotxin B (SEB) was amplified and fused with human basic fibroblast growth factor (bFGF). Recombinant protein SEB and fusion protein bFGF/SEB were expressed and purified. The biological activity was detected, including splenocytes proliferation, cytokine production, and cytotoxicity in tumor cells in vitro. In addition, the binding of bFGF/SEB with tumor cells and the tumor cell apoptosis were also tested by immunofluorescent technique. Results: The fusion protein bFGF/SEB had similar biological activities compared with natural SEA and recombinant SEB, including tumor-inhibition ratio. Conclusion: The recombinant bFGF/SEB-fusion protein was shown to retain the superantigenic activity of SEB, and might be a novel promising immunotherapeutic agent for the treatment of some carcinomas.

A broad-spectrum inhibitory peptide against staphylococcal enterotoxin superantigen SEA, SEB and SEC

The family of staphylococcal enterotoxin (SE) superantigen has been known to contribute to a broad-spectrum of diseases. In an attempt to screen the peptides with broad-spectrum inhibition against the superantigen activity of SEs, nine low-molecular peptides were designed on the basis of high conservative regions of amino acid sequences and structures of the SEs, the effects of these inhibitory peptides on the biological activity of SEs were detected by cellular and animal models, competition assay was used to detect the ability of the inhibitory peptide binding to MHC class II molecules. The results indicated that one dodecapeptide P72 possessed a broad-spectrum antagonist activity against superantigen SEA, SEB and SEC. The peptide P72 could protect mice from lethal toxic shock in the “two-hit” animal model. Competition experiment demonstrated peptide P72 could not bind to MHC class II molecules, which indicated the inhibitory activity of P72 may not due to binding to MHC class II. The peptide P72 with effective inhibition against the superantigen SEA, SEB and SEC may lead to a novel therapeutic modality in treatment of superantigen-mediated diseases.

Staphylococcal toxin-induced T cell proliferation in atopic eczema correlates with increased use of superantigen-reactive Vbeta-chains in cutaneous lymphocyte-associated antigen (CLA)-positive lymphocytes.

Staphylococcal superantigens have been implicated in the pathogenesis of atopic dermatitis (AD). This may occur through superantigenic activation of T lymphocytes and their subsequent induction of the skin homing receptor CLA on activated cells. We investigated the proliferative responses of peripheral blood mononuclear cells (PBMC) from 10 patients with an infective exacerbation of AD and six normal controls to the staphylococcal superantigens, staphylococcal enterotoxin A and B (SEA, SEB) and toxic shock syndrome toxin-1 (TSST-1), and the mitogens phytohaemagglutinin (PHA) and concanavalin A (Con A). We also assessed CLA and T cell receptor (TCR) Vbeta-chain expression by immunofluorescence and flow cytometry before and after stimulation. PBMC from AD patients showed two-fold increased proliferation to SEA and SEB (P < 0.01) compared with normals, whereas the response to mitogenic stimulation was identical. Analysis of (TCR) Vbeta-chain expression demonstrated increased use of superantigen-reactive Vbeta families in freshly isolated PBMC in AD patients compared with controls. This pattern of Vbeta-chain expression was only observed in the CLA+ but not the total population of T cells. Furthermore, there was a positive correlation between the enhanced PBMC proliferative response and increased expression of superantigen-reactive Vbeta families in atopic patients. These data support the concept that superantigens are important in the pathogenesis of this common condition, and also provide evidence that the increased use of certain Vbeta families in circulating, CLA+, skin homing lymphocytes is of functional significance.

Enhancement of superantigen activity and antitumor response of staphylococcal enterotoxin C2 by site-directed mutagenesis.

Bacterial superantigen staphylococcal enterotoxins (SEs) tremendously stimulate polyclonal T cells bearing particular TCR Vbeta domains when binding to MHC II molecules, suggesting that they could be a candidate of new antitumor agent. SEC2, an important member of superantigen family, has been used in clinical trial as an immunotherapy agent for cancer treatment in China, and obtained some encouraging effects. However, the presence of immunosuppression and endotoxic activity limits the therapeutic dosage of SEC2, and influences its antitumor effect in clinic. Therefore, the enhancement of superantigen activity and antitumor effect of SEC2 could effectively make compensation for the disadvantages mentioned above. In this study, a superantigen SEC2(T20L/G22E) mutant was generated by site-directed mutagenesis, and efficiently expressed in E. coli BL21(DE3). The results showed that SEC2(T20L/G22E) mutant exhibited a significantly enhanced superantigen activity and antitumor response, compared with native SEC2 in vitro. Further toxicity assay in vivo indicated that SEC2(T20L/G22E) mutant had no significant increase in emetic and pyrogenic activity compared with SEC2, which suggested that the mutant SEC2(T20L/G22E) could be used as a potentially powerful candidate for cancer immunotherapy, and could make compensation for the deficiency of native SEC2 in clinic.

Human herpesvirus 7 detection by quantitative real time polymerase chain reaction in primary cutaneous T-cell lymphomas and healthy subjects: lack of a pathogenic role

Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of lymphomas where the tumour population emerges within a multiple subclone pattern. Mycosis fungoides (MF) and Sézary syndrome (SS) are characterized by the expansion of clonal CD4+/CD45RO+ memory T cells. Lymphomatoid papulosis (LyP) is a chronic, lymphoproliferative disorder included in the CD30+ primary CTCL spectrum. Several studies have suggested a role of viral infection for super-antigenic activation of T lymphocytes; however, evidence of their association with CTCLs is still lacking. Human herpesvirus (HHV) 7 is a CD4+ T-lymphotropic herpesvirus; its restricted cellular tropism and the ability to induce cytokine production in infected cells could make it an important pathogenic cofactor in lymphoproliferative disorders. To investigate the presence of HHV7 DNA on CTCL and healthy skin donors (HD). We used quantitative real time polymerase chain reaction to evaluate the potential pathogenic role of HHV7. Twenty-seven of 84 (32.1%) HD were positive for HHV7 DNA. Twenty-one of 148 (14.2%) patients with CTCLs were positive for HHV7 DNA: nine of 39 (23.1%) SS, six of 14 (42.9%) CD30+ CTCLs and six of 24 (25.0%) LyP, and HHV7 DNA was negative in all 71 patients with MF. These results seem to exclude a pathogenic role of HHV7 in CTCLs, suggesting the possibility of skin as a latency site.

Staphylococcus aureus enterotoxin C2 mutants: biological activity assay in vitro

Staphylococcal enterotoxin C2 (SEC2) is one member of bacterial superantigens produced by Staphylococcus aureus. It can be attributed to its superantigenic activity to cross-link major histocompatibility complex class II molecules with T-cell receptors and activate a large number of resting T cells resulting in release of massive cytokines, which will produce significant tumor inhibition in vivo and in vitro. However, it could be not broadly applied to cure malignant tumors in clinic because of emetic activity of SEC2. The aim of this study was to inactivate emetic activity of SEC2 through site-directed mutagenesis. Cys93, Cys110 and His118 were selected as substitutional sites based on the functional sites responsible for emesis. The mutated proteins were used to determine Peripheral blood mononuclear cell proliferation activity and anti-tumor activity in vitro. Results showed that these mutated proteins efficiently stimulated T cell and exhibited the same tumor-inhibition effect as SEC2. It is possible to inactivate emetic activity of SEC2 through site-directed mutagenesis and provide satisfying agents for tumor treatment in clinic.