Preparation and activity of conjugate of monoclonal antibody HAb18 against hepatoma F( ab′ )_2 fragment and staphylococcal enterotoxin A

AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.

Results of Combining Phosphorylase Kinase Inhibition with Removal of Precipitating Factors in Large Cohort of Psoriatic Patients: A Proof of Concept Study

Background: Phosphorylase kinase (PhK) activity is induced by injurious stimuli, which is known to precipitate psoriasis. We had previously reported that elevated PhK activity in psoriatic epidermis correlated with increased psoriatic activity, and that suppression of PhK activity by its inhibitor, curcumin gel, correlated with disease resolution. Objective: We evaluated the efficacy of a strategy of combining PhK inhibition by topical curcumin with elimination of PhK-generating precipitating factors from various injurious stimuli in producing improvement of psoriatic activity, aiming at complete resolution. Patients and Methods: We studied a cohort of 647 consecutive patients with mild to severe psoriasis in a single center. Our therapeutic regimen consisted of curcumin gel, topical steroids, strict avoidance of contact allergens, avoidance of dairy products in lactose-intolerant patients, and treatment of infections to eliminate bacterial superantigens. Results: PASI scores at 0 wk was 24.7 +/– 17.1 (SD), n = 647. PASI scores improved significantly at 4 weeks to 11.5 +/– 8.1 (n = 638;p < 0.0001), at 8 weeks to 4.5 +/– 4.2 (n = 636, p < 0.0001), and at 16 weeks to 0.9 +/– 2.5 (n = 641, p < 0.0001). At 16 weeks, 72.2% of patients were completely clear of psoriatic activity (PASI = 0). Conclusion: Our results indicate that a regimen of PhK inhibition by topical curcumin with elimination of PhK-generating factors is effective in producing significant reduction of psoriatic activity at 16 weeks, with complete clearance of psoriasis in 72.2% of patients.

Disulfide-stabilized single-chain antibody-targeted superantigen: Construction of a prokaryotic expression system and its functional analysis

AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.RESULTS: The expression vector B3 (scdsFv)-SEA-PETwas constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 ℃.CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.

Cyclosporin A protects Balb/c mice from liver damage induced by superantigen SEB and D-GaIN

AIM To investigate the pathogenic effect ofSEB and D-GalN on liver and the protection ofcyclosporin A, the relationship between hepaticapoptosis and necrosis and the possiblemechanism of acute hepatic necrosis.METHODS After staphylococcal enterotoxin B(SEB ) mixed with D--galactosamine (D-GaiN )were injected intraperitoneally into Balb/c miceand those previously treated with cyclosporin A,blood samples were collected and livers wereisolated at 2, 6, 12 and 24 h. Patterns othepatocellular death were studiedmorphologically and biochemically, circulatingcytokines (TNF-a, IFN--y ) and mice mortalitywithin 24h was assessed.RESU’LTS The SEB could induce the typicalapoptotic changes of hepatocytes, the D-GaiNcould induce hepatocytes apoptosis anddegeneration at the same time, and the micehaving received the SEB + D-GaiN injectionsdeveloped apoptosis at 2 and 6 h, but after 12 hhepatocytes were characterized by severein jury, whereas all the examinations in thecyclosporin A treated mice were normal.CONCLUSION Hepatic cell apoptosis might berelated to necrosis, and massive hepatocyteapoptosis is likely the initiating step of acutehepatic necrosis in mice. The effects induced bySEB and D--GaiN on hepatocytes might bemediated by T cells, and could be prevented bycyclosporin A.

Update on molecular diversity and multipathogenicity of staphylococcal superantigen toxins

Staphylococcal superantigen(SAg)toxins are the most notable virulence factors associated with Staphylococcus aureus,which is a pathogen associated with serious community and hospital acquired infections in humans and various diseases in animals.Recently,SAg toxins have become a superfamily with 29 types,including staphylococcal enterotoxins(SEs)with emetic activity,SE-like toxins(SEIs)that do not induce emesis in primate models or have yet not been tested,and toxic shock syndrome toxin-1(TSST-1).SEs and SEIs can be subdivided into classical types(SEA to SEE)and novel types(SEG to SEIY,SE01,SE02,SEI26 and SEI27).The genes of SAg toxins are located in diverse accessory genetic elements and share certain structural and biological properties.SAg toxins are heat-stable proteins that exhibit pyrogenicity,superantigenicity and capacity to induce lethal hypersensitivity to endotoxin in humans and animals.They have multiple pathogenicities that can interfere with normal immune function of host,increase the chances of survival and transmission of pathogenic baaeria in host,consequently contribute to the occurrence and development of various infeaions,persistent infeaions or food poisoning.This review focuses on the following aspeas of SAg toxins:(1)superfamily members of classic and novelty discovered staphylococcal SAgs;⑵diversity of gene locations and molecular structural characteristics;(3)biological characteristics and activities;(4)multi-pathogenicity of SAgs in animal and human diseases,including bovine mastitis,swine sepsis,abscesses and skin edema in pig,arthritis and septicemia in poultry,and nosocomial infections and food-borne diseases in humans.

Anti-tumor Effect and Mechanism of SEA-Fab’ Coupled Protein on Gastric Tumor

Summary： The anti-tumor effect and mechanism of SEA-Fab＇ coupled protein on gastric tumor was studied. The target cell Walke-256 was treated with SEA-Fab＇ synthesized chemically or SEA respectively for 24 h, 36 h or 72 h. PBMC＋Walke-256 cells served as controls. The apoptotic index of SEA-Fab＇ against effector cells was detected. In the mouse gastric cancer models （n=60）, SEA-Fab＇, SEA and normal saline was injected in experimental group, SEA group and control group respectively. The occurrence and weight of tumor was observed. The results showed that the apoptotic index was significantly higher in the SEA-Fab＇ （34.6 -68.9%） and SEA group （15.5-31.9%） than in PBMC＋Walker-256 group （5.5%-12.8%） with the difference being significant （P〈0.01）. And there was significant difference between SEA-Fab＇ group and SEA group （P〈0.01）. The tumor weight in SEA-Fab＇, SEA and control groups was 3.6±0.53 g, 0. 78±0. 26 g and 0.49±0. 17 g respectively with the difference being statistically significant between the SEA-Fab＇ group, SEA group and the control group （P〈0.01）. In the SEA-Fab＇s and SEA groups, there were CD4^＋ T and CD8^＋ T cell infiltrates, but in the cotnrol group, no or few T lymphocytes were seen in the mouse tumor tissue. It was concluded that SEA-Fab＇ was more effective to activate T lymphocytes to kill the tumor cells than SEA used alone. It was feasibility by using the monoclonal antibody as carrier to perform the targeted Immunotherapy of gatric tumor.

A Possible Association of Staphylococcus Enterotoxin B-induced Asthma and Sinusitis

In order to gain insight into a possible association between chronic sinusitis and asthma, 85 patients with sinusitis and asthma underwent functional endoscopic sinus surgical treatment and serum antibodies and cytokines were measured. The results showed that 51 out of 85 patients with high serum anti-Staphylococcus enterotoxin B （SEB） antibody before treatment obtained satisfactory results for both sinusitis and asthma. The high level of Th2 cytokine IL-4 was down regulated to the levels of normal controls after sinus surgery. Thirty-four out of 85 patients did not show high serum anti-SEB antibody before sinus surgery and did not show much improvement in their asthmatic symptoms although sinusitis symptoms were resolved by sinus surgery. It was concluded that bacterial superantigen SEB （in the sinuses） might play a crucial role in the pathogenesis of lower airway hypersensitivity.

Effect and Mechanism of Superantigen Staphylococcal Enterotoxin Therapy for Mouse Gastric Tumor

The anti-tumor effect and mechanism of the staphylococcal enterotoxin A (SEA) were studied. The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumor ceils (MGC80-3). The experimental group was treated with SEA, and the control group was treated with normal saline. The percentage of tumor generation and tumor mass was measured. The results showed that the percentage of the tumor generation in the SEA-treated mice was lower than in the control group, but there was no significant difference (P>0. 05). However, the tumor mass in the experimental group was significantly lighter than in the control group, with the difference being very significant (P<0. 001). There were more CD4+ T cells and CD8+ T cells in the tumor of the mice treated with SEA than those of the control group. SEA has an obvious anti-tumor effect on mice gastric tumor. The mechanism might be that SEA induces the effect of superantigen-dependent cell mediated cytotoxicity to the tumor cells.

THE EFFECT OF SUPERANTIGEN STAPHYLOCOCCALENTEROTOXIN B AND D-GALACTOSAMINE ON BALB/CMOUSE HEPATOCYTES

Objective To observe the role of superantigen staphylococcal enterotoxin B(SEB) andD - galactosamine (D - GalN) on Balb/c mouse hepatocytes and its mechanism. Methods After Balb/c mice wereinjected intraperitoneally with SEB, D- GalN or both, blood samples were collected and livers were removed at 2,6, 12, 24h. Patterns of hepatocellular death were studied morphologically and biochemically, circulating cytokines(TNF, IFN-γ) were determined, and mice mortality within 24h was assessed. Results SEB could induce thetypical apoptotic changes of hepatocytes morphologically and biochemically. The mechanism is probably associatedwith the production and release of Cytokines (such as TNF, IFN- γ, etc).D - GalN could induce hepatocytesapoptosis and degeneration at the same time. Besides this, we confirmed hepatocytes of the mice which wereadministered SEB and D - GalN developing apoptosis at 2, 6h, but after 12h hepatocytes were characterized bysevere injury, the mice mortality within 24h is 50%. Conclusion SEB or D - GalN alone could induce the typicalapoptotic changes of hepatocytes. SEB+D-GalN developed hepatocytes apoptosis in the early stage and necrosisin the later. It suggests that there is some relationship between hepatic cell apoptosis and necrosis, and massivehepatocyte apoptosis is the probably initiating step of acute hepatic necrosis in mice.

Stimulation of staphylococcal enterotoxin A combined with PML-RARα peptide on the specifical T-cells against NB4 cell line

Objective： To investigate the effects of staphyococcal enterotoxin A （SEA） on the cytotoxicity of T cells stimulated by PML-RARa peptide in vitro. Methods： Peripheral blood mononuclear cells （MNC） from healthy donor were obtained by density gradient centrifugation on Ficoll-Hypaque, MNC were cultured with PML-RARa peptide and SEA for 20 days. After induction, the cytotoxicity of T cells induced against NB4 and K562 cell lines were examined by Cell Counting Kit-8 （CCK-8）. The CD4 and CD8 surface markers on the harvested CD3^＋ T cells were detected by flow cytometry （FCM）. Results： The cytotoxicity of T cells induced by PML-RARa peptide with SEA was higher than that of T cells induced only by PML-RARa peptide against NB4 cells. The FCM assay showed that the ratio of CD4^＋/CD8^＋ T cells were gradually decreased in both groups of PML-RARα peptide whether with SEA or not at the intervals of day 5,10 and 20 day after induction, but the most significantly decreased by PML-RARe peptide with SEA. Conclusion： The specific cytotoxicity of T cells induced by PML-RARa peptide against NB4 cells could be enhanced with superantigen SEA.

Investigation of <i>Streptococcus pyogenes</i>Carriage in Population Vulnerable to Scarlet Fever during 2015-2017 in Shanghai, China

This study aimed to investigate the carriage of Streptococcus pyogenes in population vulnerable to scarlet fever and to compare their genotypic characterization between different age groups. Pharyngeal swabs were collected from 120 - 150 students in each of the three districts in Shanghai in May and December during 2015 to 2017, while emm typing and detection of 12 superantigen genes were performed to characterize the isolates. During 2015-2017, the average carriage rate in students was 5.7% (135/2,371), without significant difference between different years or districts. The carriage rate was significantly different between children from the three age groups, with 2.4% in 3 - 4 years, 5.4% in 5 - 9 years, and 9.1% in 10 - 14 years. Eight emm types were found, including emm 1, emm 4, emm 12, emm 22, emm 75, emm 89, emm 70 and emm 241, among which emm 12 accounted for 60%, and emm 1 27.5%. The predominance of emm 12 was found in each year, but the proportion of emm 12 was lower in 10 - 14 years (43.3%) than in 3 - 4 years (86.7%) and in 5 - 9 years (73.3%) (P = 0.002 and 0.003). Superantigen genes of speB, speC, speG, ssa and smeZ were found in almost all the isolates. The average carriage of S. pyogenes in population vulnerable to scarlet fever was 5.7% in Shanghai, highest in 10 - 14 years (9.1%), while emm 12 was the predominant type.

Colonization by Superantigen Producing Staphylococcus aureus in Mice Enhances the Capacity to Develop Oral Tolerance

Microbial stimulation in early childhood may be necessary for proper maturation of the immune system. Infants colonized with Staphylococcus aureus have low risk of developing food allergy. Neonatal exposure to staphylococcal superantigen improves oral tolerance and enhances protection in experimental allergy models. Here, we used three wild-type strains of S. aureus, naturally harboring genes for different superantigens (SElM/SElO alone, or in combination with SEA or TSST-1). We first investigated their in vitro stimulatory capacity of splenocytes from germ-free mice. Secondly, germ-free mice were colonized with the strains and their capacity to develop oral tolerance was tested in a food allergy model. In vitro, S. aureus with only SElM/SElO genes promoted the strongest B-cell stimulation. S. aureus carrying gene for SEA induced the highest proportion of CD4+FoxP3+ T cells. The proportion of regulatory T cells was inversely correlated to B-cell proliferation, indicating suppressive ability of these cells. All strains were equally able to colonize the germ-free gut, initially achieving 1010CFU/g faeces, which decreased to 105 over a period of six weeks. Mice colonized with S. aureus carrying genes for SEA or TSST-1 had improved capacity to develop tolerance compared to germ-free mice. These results suggest that colonization by S. aureus producing superantigens may improve active tolerance to gut allergens.

Linkage between PTK Signaling Pathway “Crosstalking” and Caspase-3/CPP32-like Proteases Activation in Signaling Transduction of CD4^+ T Lymphocytes Apoptosis Induced by Superantigen SEB

Exposure of naive murine CD4 + T lymphocytes to superantigen such as staphylococcal enterotoxin B (SEB) induces a strong proliferative response. Prolonged exposure or subsequent restimulation of the responding T cell population with SEB leads to the apoptotic events of activation-induced cell death (AICD). The signaling mechanism responsible for the AICD is a target of intensive investigation. However, the precise downstream signaling pathways of SEB-induced AICD remains unclear. Our results here show that the sequential activation of caspase-1/ICE-like and caspase-3/CPP32-like cysteine proteases probably plays a role in the signaling transduction of SEB-induced AICD, but caspase-3/CPP32-like proteases activation does not depend on caspase-1-like proteases activation. Herbimycin A, a specific inhibitor of protein tyrosine kinases, inhibit caspase-3/CPP32-like cysteine proteases activation. However, it does not prevent DNA fragmentation of CD4 + T cells apoptosis induced by SEB. These results indicate that protein tyrosine kinases pathway is probably involved in the signaling transduction of CD4 + T cells apoptosis induced by SEB and “crosstalks” with the pathway of caspase-3/CPP32-like proteases activation.

A non-viral gene therapy for melanoma by staphylococcal enterotoxin A

Staphylococcal enterotoxin A(SEA)derived from Staphylococcus aureus,as a superantigen,shows potential for cancer immunotherapy,but systemic immunotoxicity restricts its clinical application.Targeted delivery of SEA to tumor site provides a promising option for reducing the systemic toxicity.Here,we constructed an iRGD peptide(H-[Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys]-NH_(2))modified nanoparticle(iDPP)to deliver plasmids encoding SEA for melanoma treatment.The iDPP/SEA nanocomplexes efficiently mediated SEA expression in B16-F10 cells in vivo and in vitro and induced the activation of lymphocytes and maturation of murine bone marrow-derived dendritic cells(BMDCs)in vitro.In the subcutaneous B16-F10 melanoma model,the iDPP/SEA nanocomplexes could effectively enhance immune response and T lymphocytes infiltration in tumor site after intravenous administration,thereby considerably decreased melanoma growth.Meanwhile,no obvious adverse effect was observed after intravenous administration of the iDPP/SEA nanocomplexes in vivo.Our findings demonstrated that gene therapy of SEA is a potential candidate for melanoma treatment.

超抗原与鼻息肉

目前，国际上关于慢性鼻及鼻窦炎（chronicrhinosinusitis，CRS）主要的诊疗指南将CRS分为伴鼻息肉（CRSwithnasalpolyps，CRSwNP）和不伴鼻息肉I^CRS（CRSwithoutnasalpolyps，CRSsNP）2种亚型。

Effect of superantigen on ion electrophysiology and permeability in rabbit maxillary sinus epithelia

Obejctive Superantigens are potent inflammatory stimuli which derive from pathogenic microbes such as bacteria, viruses and protozoa The aim of this study was to investigate the role of superantigens on the function of rabbit maxillary sinus epithelium Methods Twenty New Zealand white rabbits were divided into 4 groups Rabbit sinus mucosa was separated under a surgical microscope and mounted in Ussing chambers to record short circuit current, conductance and permeability to horseradish peroxidase (HRP) Group A was used as normal control Group B was stimulated with an injection of superantigen into the sinus for 4 hours The sinus mucosa of Group C was stimulated by the addition of tumor necrosis factor α (TNF α) into Ussing chambers Group D sinus mucosa was stimulated by superantigen after pretreatment with anti TNF α antibody Results Superantigen evoked increases in sinus epithelial cell baseline short circuit current, conductance and permeability to HRP stimulated by the addition of TNF α into Ussing chambers These were similar to results from superantigen stimulation in vivo The effect of superantigen on sinus epithelial cells could be blocked by pretreatment with anti TNF α antibody Conclusions Superantigen affected the function of sinus epithelial cells, including the capability of epithelial defensive barrier, which might be mediated by TNF α