PubMed · 11844551
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Simon J Ellis. 2002-02-02. Bad decision NICE.. https://doi.org/10.1016/s0140-6736(02)07582-7
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Macrophages, T cells, and B cells of the immune system are central targets of deoxynivalenol (DON) and other trichothecenes-mycotoxins that can be immunostimulatory or immunosuppressive depending on dose, exposure frequency and timing of functional immune assay. Notably, low dose trichothecene exposure transcriptionally and post-transcriptionally upregulates expression of cytokines, chemokines and inflammatory genes with concurrent immune stimulation, whereas high dose exposure promotes leukocyte apoptosis with concomitant immune suppression. DON and other trichothecenes, via a mechanism known as the ribotoxic stress response, bind to ribosomes and rapidly activate mitogen-activated protein kinases (MAPKs). The latter are important transducers of downstream signaling events related to immune response and apoptosis. Using cloned macrophages, our laboratory has identified two critical upstream transducers of DON-induced MAPK activation. One transducer is double-stranded RNA-(dsRNA)-activated protein kinase (PKR), a widely-expressed serine/theonine protein kinase that can be activated by dsRNA, interferon, and other agents. The second transducer is hematopoetic cell kinase (Hck), a non-receptor associated Src family kinase. Inhibitors and gene silencing studies have revealed that Hck and PKR play roles in DON induced gene expression and apoptosis. Future studies should focus on the molecular linkages between these kinases and trichothecene toxicity.
The development of nonsurgical contraceptives for cats may facilitate population control of the species. The purpose of this study was to investigate the utility of GnRH for immunocontraception of male cats. Male cats (n=12) were divided into groups of three and were immunized once with 0 (sham), 50, 200, or 400 microg synthetic GnRH coupled to keyhole limpet hemocyanin and combined with a mycobacterial adjuvant to enhance immunogenicity. GnRH antibody titer, serum testosterone concentration, and scrotal size were determined monthly. At 6 months, semen was collected by electroejaculation and testes were examined histologically. GnRH antibodies were detected in all cats receiving GnRH vaccine by 1 month post-treatment and persisted throughout the study. No dose effect of GnRH was observed; titers were not different among cats treated with 50, 200, or 400 microg GnRH (P=0.5). Six of nine treated cats were classified as responders based on high GnRH antibody titers (>32,000). By 3 months post-treatment, responder cats had undetectable testosterone concentrations and testicular atrophy. Nonresponder cats had GnRH titers of 4000-32,000 and testosterone concentrations intermediate between responder and sham-treated cats. At 6 months, total sperm counts were similar for sham-treated cats (3.1+/-1.8 x 10(6) sperm) and nonresponder cats (3.4+/-1.6 x 10(6) sperm; P=0.7). Only one of the six responder cats produced sperm, none of which were motile. Combined testicular weights of responder cats (1.3+/-0.1 g) were lower than sham-treated controls (5.3+/-1.3 g; P=0.02) and nonresponder cats (2.9+/-0.3 g; P=0.02). Histologic evaluation of the testes revealed that in responder cats, the interstitial cells that were present were pale and shrunken compared to the plump, polyhedral eosinophilic cells in sham-treated cats. GnRH responder cats had marked tubular atrophy with vacuolated Sertoli cells and a paucity of germ cells. Single-dose GnRH treatment resulted in testosterone concentrations and semen quality consistent with immunocastration in a majority of cats treated.
AIM: To construct ltB-ureB fusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein. METHODS: The ureB gene from a clinical Helicobacter pylori (H pylori) strain Y06 and the ltB gene from Escherichia coli (E. coli) strain 44851 were linked into ltB-ureB fusion gene by PCR. The fusion gene sequence was analyzed after T-A cloning. A prokaryotic recombinant expression vector pET32a inserted with ltB-ureB fusion gene (pET32a-ltB-ureB) was constructed. Expression of the recombinant LTB-UreB protein (rLTB-UreB) in E. coli BL21DE3 induced by isopropylthio-beta-D-galactoside (IPTG) at different concentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTB-UreB by a commercial antibody against whole cell of H pylori and a self-prepared rabbit anti-rUreB serum, respectively, and determine the antigenicity of the recombinant protein on inducing specific antibody in rabbits. GM1-ELISA was used to demonstrate the adjuvanticity of rLTB-UreB. Immunoreaction of rLTB-UreB to the UreB antibody positive sera from 125 gastric patients was determined by using ELISA. RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ltB-ureB fusion gene were 100%. IPTG with different dosages of 0.1-1.0 mmol/L could efficiently induce pET32a-ltB-ureB-E.coli BL21DE3 to express the rLTB-UreB. The output of the target recombinant protein expressed by pET32a-ureB-E.coli BL21DE3 was approximately 35% of the total bacterial proteins. rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori and anti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB binding bovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from the patients (125/125) were positive for rLTB-UreB. CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target fusion gene ltB-ureB was successfully established. The expressed rLTB-UreB showed qualified immunogenicity, antigenicity and adjuvanticity. All the results mentioned above laid a firm foundation for further development of H pylori genetically engineered vaccine.