Expression and functional detection of recombinant immunotoxins anti-B7-H4-scFv/PE38KDEL / 中国免疫学杂志

Objective:To construct anti-B7-H4-scFv-PE38KDEL,a recombinant toxin based on anti-B7-H4 single chain antibody (scFv),to detect anti-tumor effect of toxin protein.Methods:The anti-B7-H4-scFv gene was ligated with the toxin PE38KDEL gene by overlapping extension PCR(SOE-PCR).The recombinant gene was cloned into prokaryotic expression vector pET28a(+),and the protein was renatured and purified by chromatography (Ni-NTA),and was identified by Western blot.Indirect ELISA and flow analysis technology were used for specific identification.The inhibitory effects of toxins on tumor cells were detected by MTT assay and subcutaneous xenograft model in vitro and in vivo.HE staining and immunohistochemical analysis were performed on tumor tissues.Results:The recombinant expression vector pET28a-anti-B7-H4-scFv-PE38KDEL was obtained by restriction endonuclease di-gestion.The purified toxin protein was inoculated on the tumor cells.The tumor growth was inhibited in the tumor model.Conclusion:The recombinant toxin expression system based on anti-B7-H4 single chain antibody was successfully constructed.The recombinant toxin protein had good biological activity and anti-tumor activity.

Selection of Bacillus thuringiensis strains toxic to cotton boll weevil (Anthonomus grandis, Coleoptera: Curculionidae) larvae / Selección de cepas de Bacillus thuringiensis tóxicas para larvas del picudo del algodonero (Anthonomus grandis, Coleoptera: Curculionidae)

Preliminary bioassays with whole cultures (WC) of 124 Bacillus thuringiensis strains were performed with neonate larvae of Anthonomus grandis, a major cotton pest in Argentina and other regions of the Americas. Three exotic and four native strains were selected for causing more than 50% mortality. All of them were β-exotoxin producers. The native strains shared similar morphology of parasporal crystals, similar protein pattern and identical insecticidal gene profiles. These features resembled Lepidoptera-toxic strains. Furthermore, these strains showed a Rep-PCR pattern identical to lepidoptericidal strain HD-1, suggesting that these strains may belong to serovar kurstaki. However, some differences were observed in the plasmid profiles and in the production of β-exotoxin. To determine the culture fractions where the insecticidal metabolites were present, bioassays including resuspended spore-crystal pellets, filtered supernatants (FS) were compared with those of WC. Both fractions tested showed some level of insecticidal activity. The results may suggest that the main toxic factors can be found in FS and could be directly correlated with the presence of β-exotoxin. Based on the bioassays with FS and autoclaved FS, the participation of thermolabile virulence factors such as Cry1I in toxicity is neither discarded. In the selected strainsβ-exotoxin would be the major associated virulence factor; therefore, their use in biological control of A. grandis should be restricted. Nevertheless, these strains could be the source of genes (e.g., crylla) to produce transgenic cotton plants resistant to this pest.

Se realizaron ensayos preliminares con cultivos completos de 124 cepas de Bacillus thuringiensis utilizando larvas neonatas de Anthonomus grandis, una plaga principal del algodón en Argentina y otras regiones de América. Se seleccionaron 3 cepas exóticas y 4 nativas por producir mortalidad superior al 50%, todas ellas productoras de β-exotoxina. Las cepas nativas presentan la misma morfología de cristales, un perfil de proteínas similar y los mismos genes insecticidas. Estas características hacen que se parezcan a cepas tóxicas para lepidópteros. Además, mostraron un perfil de Rep-PCR idéntico al de la cepa lepidoptericida HD-1, lo que indica que podrían pertenecer al serovar kurstaki. Sin embargo, se observaron diferencias en el perfil plasmídico y en la producción de β-exotoxina. Para determinar en qué fracción del cultivo se encontraban los metabolitos responsables de la toxicidad, se compararon los resultados de bioensayos en los que se utilizó biomasa, sobrenadante filtrado (SF) o cultivos completos. Ambas fracciones mostraron cierto grado de toxicidad. Los resultados indican que los principales factores tóxicos se encuentran en el sobrenadante y estarían directamente relacionados con la presencia de β-exotoxina. De acuerdo con los bioensayos de SF y SF autoclavado, no se descarta también la participación en la toxicidad de factores de virulencia termolábiles, como CrylIa. En las cepas seleccionadas, el principal factor de virulencia es la β-exotoxina, por lo que su uso debería restringirse para el control biológico de A. grandis. No obstante, estas podrían ser fuente de genes (p. ej., crylIa) para la producción de plantas de algodón transgénicas resistentes a dicha plaga.

Preparation of immunotoxin BI7D12-PE38KDEL directed to EGFR and determination of its activity in vitro / 中国免疫学杂志

Objective:To prepare nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR and to examine its cytotoxicity against EGFR positive tumor cells.Methods:By using molecular cloning strategy,prokaryotic expression construct of pET28a-BI7D12-PE38KDEL was generated which consisted of nanobody 7D12 targeting EGFR in the form of a divalent fused with PE38KDEL,a truncated form of pseudomonas exotoxin A via a flexible peptide(G4S)4,and then transformed into E.coli BL21(DE3).Protein expression was induced by adding IPTG,purified by Ni-affinity column chromatography,and verified by Western blot.The binding capacity of the resulted immunotoxin to EGFR-positive cells A549,HT29,MCF-7 and EGFR-negative cells CEM,Jurkat were determined by flow cytometry assay,and its cytotoxicity against the target cells was examined.Briefly,tumor cells were treated with different dosage of the immunotoxin,and the killing efficacy of BI7D12-PE38KDEL on these cells were assessed by WST-1 assay after 72 hours.Results:The SDS-PAGE and Western blot results showed the recombinant immunotoxin BI7D12-PE38KDEL was successfully prepared,and majority of them was expressed in soluble form.BI7D12-PE38KDEL could selectively bind to EGFR-positive cells of A549,HT29,and MCF-7.More importantly,the immunotoxin exhibited much more significant killing effect on these EGFR positive cells compared to the negative control group of CEM and Jurkat cells(P<0.01).Conclusion:In the current study,the nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR was successfully prepared and exhibited a superior inhibition effect for the growth of EGFR-positive cells.

Construction and eukaryotic expression of a recombinant plasmid encoding Pseudomonas aeruginosa toxA and typeⅢsecretion system pcrV / 医学研究生学报

Objective Exotoxin A ( encoded by gene toxA ) , one of the most toxic protein secreted by pseudomonas aerugi-nosa(P.a.), and PcrV (encoded by gene pcrV), key component to type Ⅲsecretion system of P.a., both matter significantly to the virulence of P.a.The article was to construct a novel DNA vaccine encoding a mutated toxA gene and the pcrV gene of P .a.and i-dentify gene expressions in eukaryotic cells . Methods The genes of toxA and pcrV were amplified by PCR , and the toxA gene was mutated to reduce the toxicity of Exotoxin A .Then gene fragments toxA m and pcrV were inserted into eukaryotic expression plasmid pIRES simultaneously to construct a recombinant DNA vaccine pIRES-toxAm-pcrV.The novel plasmid was transfected into HEK-293 cells by lipofectamine 2000 .The expressions of toxA m and pcrV were detected by Western blot . Results Gel electrophoresis demon-strated the target gene fragments encoding Exotoxin A and PcrV .Western blot exhibited proteins encoded toxA and pcrV expressed by HEK 293 cells. Conclusion The recombinant plasmid pIRES-toxAm-pcrV was successfully constructed .Western blot analysis indi-cated the expressions of toxA m and pcrV in HEK-293 cells.It may be used as a potential candidate of preventive vaccine of Pseudo-monas aeruginosa .

Systemic administration of low dosage of tetanus toxin decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus / 한국실험동물학회지

In the present study, we investigated the effect of Tetaus toxin (TeT) on cell proliferation and neuroblast differentiation using specific markers: 5-bromo-2-deoxyuridine (BrdU) as an exogenous marker for cell proliferation, Ki-67 as an endogenous marker for cell proliferation and doublecortin (DCX) as a marker for neuroblasts in the mouse hippocampal dentate gyrus (DG) after TeT treatment. Mice were intraperitoneally administered 2.5 and 10 ng/kg TeT and sacrificed 15 days after the treatment. In both the TeT-treated groups, no neuronal death occurred in any layers of the DG using neuronal nuclei (NeuN, a neuron nuclei maker) and Fluoro-Jade B (F-J B, a high-affinity fluorescent marker for the localization of neuronal degeneration). In addition, no significant change in glial activation in both the 2.5 and 10 ng/kg TeT-treated-groups was found by GFAP (a marker for astrocytes) and Iba-1 (a marker for microglia) immunohistochemistry. However, in the 2.5 ng/kg TeT-treated-group, the mean number of BrdU, Ki-67 and DCX immunoreactive cells, respectively, were apparently decreased compared to the control group, and the mean number of each in the 10 ng/kg TeT-treated-group was much more decreased. In addition, processes of DCX-immunoreactive cells, which projected into the molecular layer, were short compared to those in the control group. In brief, our present results show that low dosage (10 ng/kg) TeT treatment apparently decreased cell proliferation and neuroblast differentiation in the mouse hippocampal DG without distinct gliosis as well as any loss of adult neurons.

Response of Peripheral Blood Mononuclear Cells to Staphylococcus Aureus Exotoxin in Nasal Polyposis

BACKGROUND AND OBJECTIVES: Superantigens such as Staphylococcus aureus exotoxin (SE) have been implicated in the pathogenesis of chronic rhinosinusitis with nasal polyposis (NP). The aim of this study was to determine the immunologic response of peripheral blood mononuclear cells (PBMCs) to staphylococcal exotoxin B (SEB) in patients with NP. METHODS: The interleukin (IL)-4, IL-5, and interferon-gamma(IFN-gamma) responses of PBMCs to nonspecific mitogens such as phylohemagglutin (PHA) and SEB were examined in 24 NP patients and 16 control subjects. The presence or absence of atopy and asthma was determined to evaluate the correlation of these conditions with the levels of cytokines. RESULTS: PBMCs from the NP patients were more likely to produce IL-4 and IL-5 in response to SEB than those from controls. There was no difference in the mitogen-induced cytokine responses between NP patients and controls. SEB-induced IL-5 and IL-4 levels were higher in patients with NP with asthma than in patients with NP without asthma. CONCLUSION: Patients with NP show an exaggerated Th2 cytokine response of PBMCs to SEB.

Pseudomonas aeruginosa Exotoxin A Reduces Chemoresistance of Oral Squamous Carcinoma Cell via Inhibition of Heat Shock Proteins 70 (HSP70)

PURPOSE: Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9). MATERIALS AND METHODS: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis. RESULTS: While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint. CONCLUSION: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.

Role of Staphylococcus Aureus Exotoxins in Chronic Rhinosinusitis with Nasal Polyp / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Staphylococcal exotoxins (SEs) have been implicated in the pathogenesis of chronic rhinosinusitis with nasal polyp (CRSwNP). In the current study, we determined the prevalence of specific immunoglobulin E (IgE) antibodies to SEs in serum and polyp tissues of patients with CRSwNP and tried to find out whether there is an association between the presence of SEs-IgE antibody and eosinophilic inflammation. SUBJECTS AND METHOD: Blood, nasal polyp and mucosa samples were obtained from 43 patients undergoing endoscopic sinus surgery for CRSwNP and 11 controls undergoing septoplasty without CRS. Specimens were analyzed for the presence of specific IgE antibody to four SEs [staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC), toxic shock syndrome toxin 1 (TSST-1)] using ImmunoCAP assay. Eosinophil cationic protein (ECP) in serum and nasal polyp tissue were also analyzed using ImmunoCAP. Eosinophil counts were estimated in polyp tissue. RESULTS: SEs-specific IgE antibodies were detected in 13 (30.2%) patients of the CRSwNP group. In contrast, only one (9.1%) control patient had IgE to SEs. Serum ECP level was increased significantly in the CRSwNP group compared with controls. However, there were no significant differences in Lund-MacKay score, the ECP level in the serum and polyp tissue, and eosinophil count in the polyp tissue between the SEs-IgE antibody positive [SEs-IgE Ab (+)] group and the SEs-IgE antibody negative [SEs-IgE Ab (-)] group. CONCLUSION: SEs may play a certain role in the pathogenesis of CRSwNP. However, there is no close correlation between the presence of SEs-IgE antibody and eosinophilic inflammation.

A novel VEGF16S-PE38 fusion gene for anti-angiogenic therapy in malignant glioma of nude mice model / 中华神经医学杂志

Objective To evaluate the anti-angiogenic effect of eukaryotic vector containing human VEGF 165 and truncated pseudomonas exotoxin A (PE38) fusion gene on malignant glioma in nude mice, and explore a novel anti-angiogenic therapy for cancer. Methods The models were established through hypodermic injection of human U251 glioma cells into the nude mice and randomly divided into untreated group, PBS group, pIRES2-EGFP group and pIRES2-VEGF165-PE38-EGFP group on the 9th day. H&E staining was performed to observe the morphological changes of the glioma tissues; SP immunohistochemistry was employed to detect the expression of GFAP, CD31 andPE; quantitative pathologic image analysis system was used to investigate the microvessel density (MVD) in the tumor tissues. Results At day 16, the pIRES2-VEGF165PE38-EGFP group showed significantly lower tumor volume of mice and significantly decreased MVD than the other three groups (P<0.05). Positive expression of PE was shown in the pIRES2-VEGF165PE38-EGFP group, but negative in the other three groups. Conclusion The expression products of VEGFJ65-PE38 fusion gene can obviously inhibit the growth and angiogenesis of U2S1 cells in nude mouse flank tumor models, suggesting that it may be a novel therapeutic approach for anti-angiogenic therapy of cancer.

Detection of Staphylococcus Aureus Exotoxins(SEA, TSST-1) in Nasal Polyp with Chronic Rhinosinusitis Patients / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Staphylococcus aureus (S. aureus) exotoxins have been implicated in the pathogenesis of chronic sinusitis with nasal polyp. The aim of this study was to identify the interplay of S. aureus exotoxins between the nasal mucus and nasal polyp tissue. SUBJECTS AND METHOD: We have selected 30 nasal polyp with chronic sinusitis patients and 10 controls withrhinoplasty without sinusitis. Nasal mucus culture was done by smearing in nasal polyp and middle meatus. PCR analysis of the nasal lavage and immunohistochemical stainingin nasal tissue were done for the presence of S. aureus exotoxins (SEA and TSST-1). RESULTS: Nasal culture results were positive for S. aureus in 27% of the nasal polyp group compared to 10% ofthe control group. PCR analysis for SEA and TSST-1 in the nasal lavage demonstrated remarkable expression in the nasal polyp group (SEA:53%, TSST-1:60%) compared to the control group (SEA:20%, TSST-1:10%). In addition, immunohistochemical staining of nasal tissues reflected significantly higher expression of S. aureus exotoxin in the nasal polyp group (SEA:20%, TSST-1:33%) compared to the control group (SEA:0%, TSST-1:0%). There was a significant correlation between the exotoxins of nasal lavage and nasal polyp. CONCLUSION: Our results demonstrated that the S. aureus exotoxin in the nasal cavity might invade the nasal mucosa and have some role to play in the pathogenesis of nasal polyp.

The Correlation Study between the Specific IgE for Staphylococcus Aureus Exotoxin and Nasal Mucus Culture in Allergic Rhinitis / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: There is a reported association between the increased levels of specific IgE and Staphylococcus aureus exotoxins (SE) and eosinophilic inflammation in nasal polyp tissue. However, the role of IgE to SE in allergic rhinitis has not been known definitely. We sought to find whether the specific IgE to SE has a correlation with the allergic rhinitis. SUBJECTS AND METHOD: Nasal mucosa and serum were obtained from 30 patients undergoing submucous turbinectomy of inferior turbinates for allergic rhinitis and 20 control patients undergoing septoplasty. Nasal culture was performed for each patient. Specific IgE levels for S. aureus exotoxin A (SEA), S. aureus exotoxin B (SEB), and toxic shock syndrome toxin 1 (TSST-1) were measured using ImmunoCAP method in both nasal mucosa and serum. RESULTS: The culture rate for S. aureus was 13.3% for allergic rhinitis and 10% for the control. The specific IgE for S. aureus in serum was significantly expressed in allergic rhinitis (30%) compared to in the control (10%). In nasal mucosa of allergic rhinitis patients, the specific IgE has shown higher expression rate (20%) than the control (0%). CONCLUSION: Our results demonstrated that there is a correlation between allergic rhinitis and specific IgE to S. aureus exotoxin in both nasal mucosa and serum. These results suggest that S. aureus exotoxin can act as a traditional allergen and induce the development of allergic rhinitis.

Expression and activity detection of IL3-PE38KDEL / 重庆医科大学学报

Objective:To prepare a new type of anti-leukemia immunotoxin with killing activity.Methods:The method of cytotoxicity was used to study the activity of the immunotoxin after the induction of IPTG. Results:The expressed fusion proteins were detected mostly as inclusion bodies at high level.The result showed IL3-PE38KDEL had liable activity of toxicity. Conclusion:The fusion protein IL3-PE38KDEL has good biological activity,which paves way for the further study on its treatment of leukemia.

The Correlation Study between the Specific IgE for Staphylococcus Aureus Exotoxin and Nasal Mucus Culture in Chronic Rhinosinusitis with Nasal Polyposis / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Staphylococcus aureus (S. aureus) exotoxins (SE) have been implicated in the pathogenesis of chronic sinusitis with nasal polyposis (CRSwNP). The aim of this study was to identify the contribution of S. aureus exotoxin as allergen in the development of nasal polyp. MATERIALS AND METHOD: Nasal polyp and serum were obtained from 30 patients who underwent endoscopic sinus surgery for CRSwNP and 10 control turbinate mucosae were used. Nasal culture was done for each patients. Specific IgE levels for S. aureus exotoxin A (SEA), S. aureus exotoxin B (SEB), and toxic shock syndrome toxin 1 (TSST-1) were measured using ImmunoCAP method in nasal tissue and serum. The patients were divided into three groups : A, nasal polyp (+)/culture (+) ; B, nasal polyp (+)/culture (-) ; C, nasal polyp (-)/culture (-). RESULTS: The culture for S. aureus was 27% in CRSwNP when compared to 10% in control. The specific IgE for S. aureus in serum was significantly expressed in nasal polyp (30%) compared to control (0%). Also the Group A showed a significant high rate of specific IgE (63%) compared to Group B (18%) and Group C (0%) in serum. In nasal polyp tissue, the specific IgE has showed no specific difference between nasal polyp (7%) and control (10%). However, it was also increased in Group A (25%), compared to Group B (0%) and Group C (11%). CONCLUSION: Our results demonstrated that there was a correlation between nasal polyp and specific IgE levels to S. aureus exotoxin. These results suggest that S. aureus exotoxin can act as a traditional allergen and induce the inflammatory reaction in CRSwNP. Background and Objectives : Staphylococcus aureus (S. aureus) exotoxins (SE) have been implicated in the pathogenesis of chronic sinusitis with nasal polyposis (CRSwNP). The aim of this study was to identify the contribution of S. aureus exotoxin as allergen in the development of nasal polyp.

The Effect of Skin-colonizing Staphylococcus aureus and Its Exotoxins on Childhood Atopic Dermatitis / 소아알레르기및호흡기학회지

PURPOSE: Staphylococcus aureus and its exotoxins have been regarded as having an influence on atopic dermatitis(AD). We aimed to examine the prevalence of S. aureus in the AD lesion, the types of the exotoxins, and the relationship between S. aureus and AD. METHODS: AD patients(n=32) and a normal control group(n=20) were enrolled. The severity of AD was measured by SCORAD index. Through skin culture and PCR, we tried to identify S. aureus and its exotoxins. RESULTS: S. aureus was isolated from 18(56 percent) out of 32 AD patients and its exotoxins were identified from 10(31 percent) out of them. The exotoxin types were as follows; sea in 4, eta in 3, sea+tst-1 in 1, sea+see in 2 patients. On the contrary, S. aureus was isolated from only 1(5 percent) out of 20 subjects of the normal control group, and its exotoxin type was sea. The SCORAD index in the S. aureus(+) group was higher than in the S. aureus(-) group, however it was not significant.(44+/-14.2 vs 38+/-17.1, P= 0.304) The SCORAD index was higher in the exotoxin(+) group than in the exotoxin(-) group(49+/-11.2 vs 38+/-16.2, P<0.05). The prevalence of S. aureus and its exotoxins in the AD group was higher than in the normal control group(P<0.001, P<0.05, respectively). The difference of SCORAD index was significant between the exotoxin(+) group and the exotoxin(-) group, but not between the S. aureus(+) group and S. aureus(-) group.(P<0.05, P= 0.304, respectively) CONCLUSION: The exotoxins of S. aureus were found to influence the severity of AD.

A Comparison Study of the Staphylococcal Exotoxins and Staphylococcal Enterotoxin A-specific IgE Antibody between Childhood and Adulthood Atopic Dermatitis

BACKGROUND: The skin of patients with atopic dermatitis (AD) exhibits a striking susceptibility to colonization with Staphylococcus aureus (S. aureus). Superantigens produced by S. aureus and their specific IgE antibodies are thought to be important precipitating factors of AD, but there are few reports evaluating these 2 factors at the same time, particularly in adult AD patients. OBJECTS: Our purpose was to investigate the differences in the culture degree of S. aureus from the lesion, non-lesion, and control group of child and adult AD patients, to research the correlation between the exotoxin production, total IgE, anti-SEA IgE and the disease severity by SCORAD index, to ascertain the differences between child and adult AD patients. METHODS: The clinical severity of 30 child (2 to 15 years of age) and 30 adult patients (16 to 40 years of age) with AD was evaluated by using SCORAD index. S. aureus was isolated from lesional and non-lesional skin of AD patients, and from healthy controls. Staphylococcal exotoxins were detected by using reversed passive latex agglutination toxin detection kits. Anti-SEA IgE antibody was determined by using AlaSTATt assay RESULTS: S. aureus colonizations were found in 11 (36.7%) of the lesional skin, in 5 (16.7%) of the non-lesional skin of 30 child AD patients, and in 26 (86.7%), in 20 (66.7%) of 30 adult AD patients, respectively. The colonization rates of S. aureus in child patients were much lower than those in adult patients, both form lesional skin and non-lesional skin. Staphylococcal exotoxins were detected in 5 (45.5%) of the 11 colonizations from lesional skin, in 2 (40%) of the 5 colonizations from non-lesional skin of children, and in 10 (38.5%) of the 26 colonizations, in 9 (45%) of the 20 colonizations of adults, respectively. Staphylococcal enterotoxin A (SEA) was most frequently detected in both groups. S. aureus colonization was correlated with the severity of AD in childhood, but not in adulthood. However, there were no statistical significances between severity of AD and others such as exotoxin production, and the level of total IgE and anti-SEA IgE in both groups. CONCLUSION: The colonization of S. aureus was more common in adult AD patients than child AD patients. Anti-SEA IgE level was much higher in adult AD patients than in child AD patients. It is tempting to speculate that the colonization of S. aureus and exotoxin production might be related to the disease. duration rather than clinical severity of AD.

Genotype Analysis of Pyrogenic Exotoxin and emm Genes of Streptococcus pyogenes Clinical Isolates

A variety of proteins produced by Streptococcus pyogenes contribute to the virulence of the pathogen. Among the proteins, the M protein and streptococcal pyrogenic exotoxins (Spe) are considered the major S. pyogenes virulence factors. To better characterize the correlation of M protein type and pyrogenic exotoxins with clinical diseases, we tested 269 S. pyogenes clinical isolates from patients with scarlet fever, pharyngitis, skin infection, otitis media, or other invasive streptococcal infections that provided appropriate clinical data. The strains were genotyped (M type) and assayed for speA, speB, and speC genes. The speB gene was detected in all isolates. Also, speA and speC genes were detected in 54 strains (18.2%) and 140 strains (47.3%), respectively. The strains isolated from invasive disease patients showed the highest frequency of speA gene (40.5%). The correlation among emm genotype, speA gene, and clinical patterns was analyzed. Genotypes emm1 (55.6%) and emm3 (22.2%) were predominant in stains with speA gene. The distribution of emm genotypes did not significantly associate with clinical patterns. These data suggest that SpeA is significantly associated with specific emm genotypes, and the exotoxin serve a dominant virulence factor.

A Study on the Evaluation of the Staphylococcal Exotoxins and Staphylococcal Enterotoxin A-specific IgE Antibody in Childhood Atopic Dermatitis / 대한피부과학회지

BACKGROUND: The skin of patients with atopic dermatitis (AD) exhibits a striking susceptibility to colonization with Staphylococcus aureus (S. aureus). Superantigenic exotoxins produced by S. aureus and their specific IgE antibodies are thought to be important precipitating factors of AD, but there are few reports evaluating these 2 factors at the same time. OBJECT: Our purpose was to examine whether the isolation of S. aureus colonies and the presence of the exotoxins from the skin of childhood AD patients and the level of anti-staphylococcal enterotoxin A(SEA) IgE antibody in their sera correlated with their severity of AD. METHODS: Thirty patients with mild-to-severe AD, 2 to 15 years of age, were evaluated by using SCORAD index. S. aureus was isolated from lesional and non-lesional skin of AD patients, and from healthy controls. By using reversed passive latex agglutination toxin detection kits, we examined whether staphylococcal exotoxins could be detected. Anti-SEA IgE antibody was determined by using AlaSTAT(R)assay. RESULTS: S. aureus colonizations were found in 11(36.7%) of the lesional skin and in 5(16.7%) of the non-lesional skin of 30 AD patients. Staphylococcal exotoxins were detected in 5(45.5%) of the 11 colonizations from lesional skin and in 2(40%) of the 5 colonizations from non-lesional skin. SEA was most frequently detected. S. aureus colonization was correlated with the severity of AD. However, there were no statistical significances between severity of AD and others such as exotoxin production, and the level of total IgE and anti-SEA IgE. Total IgE level was significantly higher in the group of exotoxin production, and correlated with the level of anti-SEA IgE. CONCLUSION: The correlation between S. aureus colonization and severity of AD in our study might support the role of S. aureus in patients with AD. On the other hand, it could be considered that exacerbation of AD trigger more colonization of S. aureus by way of disruption of skin barrier function from scratching or reduced immune responses needed for defense against bacteria. Although there was no correlation between AD severity and exotoxin production and the level of anti-SEA IgE in this study, staphylococcal exotoxins and their specific IgE antibodies might play a role at least in a subset of AD patients.

Analysis of biochemical and physical properties for a new recombinant B7-Pseudomonas exotoxin fusion protein B7-2-PE40KDEL / 中国免疫学杂志

Objective:Identification of some biochemical and physical properties for a new recombinant B7-2-PE40KDEL exotoxin fusion protein.Methods:12%SDS-PAGE separating and gel imaging analyzing,peptide mass fingerprinting,Western blot and MTT assasying were used respectively for identification of the protein.Results:Molecular weight of the recombinant B7-2-PE40KDEL was 72 628,5% of the difference to its theoretical value 69 561.The result of Western blot indicated that the purified recombinant B7-2-PE40KDEL could specifically bind with mAb anti-human B7-2 and the antibody against PEA,while the negative control did not.The recombinant B7-2-PE40KDEL digested with trypsin and then detected by MOLTI-TOF-MS.It was shown that the detected 15 peptides lied in the extracellular part of B7-2 and the truncated Pseudomonas extoxin PE40KDEL.Searching in the peptident data bank of Expasy website,we did not find any known proteins which was accordant with the above terms.The cytotoxic activity of the recombinant toxin with MTT method showed that the B7-2-PE40KDEL selectively killed Jurkat cell line which expressesed CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor.Conclusion:Recombinant B7-2-PE40KDEL exotoxin fusion protein we construct proves to be a new one with targeted killing bioactivity to B7:CD28 system.

Experimental Study on Target Cells of Pseudomonas aeroginosa Exotoxin A / 第三军医大学学报

Pseudomonas aeroginosa exotoxin A(PEA) was purified from the strain PA103 and labelled with Ⅰ~(125).Then autoradiographic tracing,histopathological observations with light and electron microscopes,and antiserum protestion test were carried out on inbred mice BALB/C.The results indicate that the liver is the target organ of PEA,and both the hepatocytes and kupffer cells the target cells,on which the severe damage may be responsible for the acute death of the toxicated animals.

Construction of recombinant plasmid of Pseudomonas aeruginosa exotoxin A and its secreting expression in E. coli / 解放军医学杂志

Objective To clone and to express the full-length Pseudomonas aeruginosa exotoxin A gene and to purify the expressed protein using affinity chromatography. Methods Exotoxin A gene was amplified from the recombinant plasmid pSK/PEA-T vector and subcloned into the pMAL-P2X vector. Then the recombinant plasmid pMAL- PEA was transformed to E.coli BL21. After induction with IPTG, the expressed protein was analyzed by SDS-PAGE and purified with affinity chromatography. Results The recombinants expressed the fusion protein at a level of about 20% of total cell proteins, and 80% of the fusion protein was secreted into the supernatant. Conclusion Successful expression and purification of PEA are of significance for in-depth study of the pathogenic mechanism of Pseudomonas aeruginosa and preparing immunotoxin.