ABSTRACT
The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Method Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). Results The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. Conclusion HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.
Subject(s)
Animals , Antivenins/biosynthesis , Neurotoxins/classification , Neurotoxins/genetics , SnakesABSTRACT
ObjectiveThis study aimed to compare the clinical characteristics and prognoses of primary Waldeyer's ring diffuse large B-cell lymphoma (DLBCL) and extranodal nasal-type NK/T-cell lymphoma ( ENKTCL).MethodsFrom 2000 to 2008,122 patients with primary Waldeyer's ring DLBCL and 44 patients with primary Waldeyer' s ring ENKTCL consecutively diagnosed were retrospectively compared.Patients with DLBCL usually received 4-6 cycles of CHOP-based chemotherapy followed by involved-field radiotherapy.Patients with early stage ENKTCL usually received extended-field radiotherapy with or without subsequent chemotherapy,or short courses ( 1 - 3 cycles ) of chemotherapy followed by radiotherapy.Kaplan-Meier method was used for survival analysis.Logrank method was used for univariate analysis.ResultsThe follow-up rate was 82％.The number of patients followed 5 years were 32 and 15 in DLBCL and ENKTCL.DLBCL mainly presented with stage Ⅱ tonsillar disease with regional lymph node involvement.ENKTCL occurred predominately in young males,as nasopharyngeal stage I disease with B symptoms and involving adjacent structures.The 5-year overall survival (OS) and progression-free survival (PFS) rates were 74％ and 67％ in DLBCL,and 68％ and 59％ in ENKTCL (x2=0.53,1.06,P=0.468,0.303),respectively.In stage Ⅰ and Ⅱ diseases,the 5-year OS and PFS rates were 79％ and 76％ for DLBCL compared to 72％ and 62％ for ENKTCL (x2 =1.20,2.46,P=0.273,0.117).On univariate analysis,age ＞ 60 years,elevated lactate dehydrogenase,eastern cooperative oncology group performance status ＞ 1,international prognosis index ( IPI ) score ≥ 1,stage Ⅲ/Ⅳ diseases and bulky disease were associated with unfavorable survival for DLBCL (x2=9.40,12.72,6.15,10.36,12.48,5.53,P=0.002,0.000,0.013,0.001,0.000,0.019),and only age＞60 years and IPI score ≥ 1 were associated with poor survival for ENKTCL (x2 =3.98,8.41,P =0.046,0.004).ConclusionsThese results indicate that remarkable clinical disparities exist between DLBCL and ENKTCL in Waldeyer's ring. Different treatment strategies for each can result in similarly favorable prognoses.