Pseudomonas exotoxin antisense RNA selectively kills hepatitis B virus infected cells

AIM: To present an approach for selectively killing retrovirus-infected cells that combines the toxicity of Pseudomonas exotoxin (PE) and the presence of reverse transcriptase (RT) in infected cells. METHODS: PE antisense toxin RNA has palindromic stem loops at its 5' and 3' ends enabling self-primed generation of cDNA in the presence of RT. The RT activity expressed in retrovirus-infected cells converts "antisense-toxin-RNA" into a lethal toxin gene exclusively in these cells. RESULTS: Using cotransfection studies with PE-expressing RNAs and β-gal expressing reporter plasmids, we show that, in HepG2 and HepG2.2.15 hepatoma cells as well as in duck hepatitis B virus (DHBV) infected cells, HBV or DHBV-polymerase reverse transcribe a lethal cDNA copy of an antisense toxin RNA, which is composed of sequences complementary to a PE gene and eukaryotic transcription and translation signals. CONCLUSION: This finding may have important implications as a novel therapeutic strategy aimed at the elimination of HBV infection.

Research Advances of Exotoxin Gene and Related Vaccines of Clostridium perfringens

Clostridium peringens, also called clostridium westergren, mainly causes diseases in ruminants, such as gas gangrene, enterotoxaemia, hemorrhagic enteritis of cattle and sheep. The bacteria are divided into five subtypes including A, B, C, D and E subtype, which can secrete more than 10 kinds of exotoxin,In these toxins, a, β, ε and ζ play a key pathogenic role. At present, the disease is prevented mainly by univalent vaccine, combined vaccine and muhivalent inactivated vaccine. And the genetic engineering subunit vaccine and nucleic acid vaccine has wide develop- ment prospects. In order to provide some reference for comprehensive prevention and deep research of C. pe^ringens, the etiological characteristics of C. perfrin- germ, pathogenic mechanism of exotoxin and research progress of related vaccines were reviewed, and the genetic engineering subunit vaccine and nucleic acid vac- cine development prospects were also discussed in this paper.

Streptococcal Pyrogenic Exotoxin Genes SpeA and SpeB in Isolates of Streptococcus pyogenes from Children with Pharyngitis, Gezira State, Sudan

Background: Streptococcus pyogenes (group A streptococcus, GAS) is an important human bacterial pathogen. This organism possesses many virulence factors, Streptococcal pyrogenic exotoxinone of these. Aim: Detection of Streptococcal pyrogenic exotoxin SpeA and SpeB in isolated Streptococcus pyogenes. Methods: Tow hundred throat swab samples were collected from children with pharyngitis referred to Pediatric Teaching hospital and ENT hospital Wad medani, Sudan, from January to November 2021. The questionnaire was filled out to collect clinical and demographic data. Throat swabs were collected and processed with the standard microbiological procedure to isolate Streptococcus pyogenes. Antimicrobial susceptibility testing was done on all GAS isolates using the Kirby Bauer disk diffusion method according to clinical laboratory standard institute (CLSI) guidelines. Detection of Spy 1258 gene and Streptococcal pyrogenic exotoxins SpeA and SpeB were done by using Multiplex PCR. Results: Amongst the Tow hundred collected samples fifty-one isolates (25.5%) were identified as S. pyogenes. Antibiotic susceptibility testing showed that all the GAS isolates were sensitive to Azithromycin and Penicillin. Sensitivity to Erythromycin, Gentamicin, Clarithromycin, Amoxicillin and Cephalexin were 88.2%, 86.3%, 45.1%, 41.2%, 13.7%, respectively. SpeA was detected in 17 (33.3%) and SpeB in 48 (94.1%). Conclusion: Streptococcal pyrogenic exotoxin genes SpeA and SpeB were detected in 17 (33.3%) and 48 (94.1%) respectively of Streptococcus pyogenes isolates.

Intraperitoneal administration of gonadotropin-releasing hormone-PE40 induces castration in male rats

AIM： To evaluate the long-term effects of gonadotropinreleasing hormone （GnRH）-based vaccine on levels of GnRH antibody and testosterone, and vaccine-induced immunocastration on sexual behavior of male rats. METHODS： The rats were treated with GnRH-PE40 intraperitoneally every other day for 12 wk. GnRH antibody and testosterone level in rat blood were determined by EUSA and radioimmunoassay, respectively. Morphological changes in testes and sexual behavior of rats were evaluated. RESULTS： GnRH-PE40 induced a high production in GnRH antibody, decreased the serum testosterone level, testis atrophy and sexual function in rats. CONCLUSION： Intraperitoneal administration of GnRH- PE40 produces structural and functional castration of male rat reproductive system by inducing anti-GnRH antibody.

APOPTOSIS INDUCTION BY THE RECOMBINANT FUSION APOPTOSIS INDUCING FACTOR ON HELA CELLS

Objective: To obtain the recombinant fusion AIF genes inserted into the eukaryotic expression vectorpIRES2-EGFP, to observe the expression and location ofthe fusion AIF genes (3NE: PE(280-358)-AIF1-120, and4NE: PE(280-364)-AIF1-120), and to detect and compare their apoptosis inducing effects on the transfected HeLacells. Methods: Full-length human AIF gene was clonedby RT-PCR, and its N-terminal mitochondrial localization sequence (MLS) was replaced by part sequence ofPsuedomonas exotoxin A (PE) translocation domain(PEII(280-358/364)), then the recombinant fusion geneswere inserted into the pIRES2-EGFP eukaryotic expression vector. After these genes were transiently transfected into HeLa cells with LipofectAmine, the expression of therecombinant fusion AIF genes and their effects on HeLacells were detected by fluorescent microscopy, laser confocal microscopy and electron microscopy. Results: Theeukaryotic expression vectors containing the recombinant fusion AIF genes (pIRES2-EGFP-PEII(280-358/364)-AIF1- 120) were constructed successfully. It wasdemonstrated that the fusion AIF protein genes wereexpressed effectively in the transfected cells, with the GFP co-expressed in cells by indirect immunofluorescence staining analysis. After transfection, expression of the genes could induce HeLa cells to exhibit the typical apoptosis features: such as plasma membrane blebbing and peripheral chromatin condensation. As compared with control groups, the untreated cells and the void vector transfected cells, the living cell number of the AIF gene transfected cells reduced distinctly. Conclusion: Our data prove that the expression of the recombinant human AIF fusion genes could induce apoptosis in transfected HeLa cells, which provides new strategy for cancer killing.

Gene therapy for human colorectal carcinoma using human CEA promoter controlled bacterial ADP-ribosylating toxin genes:PEA and DTA gene transfer

AIM To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes.METHODS Pseudomonas exotoxin A domain Ⅱ+Ⅲ (PEA) was cloned from genomic DNA of Pseudomonas aeruginosa. PEA and diphtheria toxin A chain gene (DTA) were modified to express eukaryotically. After sequencing, the toxin genes under the control of human carcinoembryonic antigen (CEA) promoter were cloned into retroviral vectors to construct CEAPEA and CEADTA respectively. In vitro cotransfection of the constructs with luciferase vectors and in vivo gene transfer in nude mice were subsequently carried out.RESULTS Both CEAPEA and CEADTA specifically inhibited the reporter gene expression in the CEA positive human colorectal carcinoma (CRC) cells in vitro. Direct injection of CEAPEA and CEADTA constructs into the established human tumors in BALB/c nude mice led to significant and selective reductions in CRC tumor size as compared with that in control groups.CONCLUSION The toxin genes, working as therapeutic genes, are suitable for the tissue-specific gene therapy for colorectal carcinoma.

Inhibitory effect of TGF_α-PE40 on neointimal proliferation follow injury to the rabbit common carotid artery

To investigate inhibitory effect of recombinant transforming growth factora-pseudomonas exotoxin fusion protein (TGFInhibitory effect of TGF_α-PE40; TP40) on neointimal proliferation following arterial injury.Methods: Forty-eight male rabbits fed with rich cholesterol diet were randomized into the treatment group(n= 24)and the control group (n=24). The rabbits in the treatment group were treated by local adiministration of TP40 (30 μg) 24 h after arterial injury, and the control group were treated by saline. LM and computer image analysis were used to study the rabbit arterial segments 2, 4 and & weeks after treatment. Results: Irregular thickening of the arterial intima. large amounts of smooth muscle cells within the neointima, and stenosis of the arterial cavity in the control group, and significant inhibition of intimal proliferation and no stenosis of the arterial cavity in the treatment group were observed microscopically. Computer image analysis showed that the neointimal area and the ratio of neointimal/medial area of the treatment group at 2, 4 and 8weeks after treatment were signifantly smaller than those of the control groups (P<0. 01 ). COnclusion: The results suggest that TP40 can significantly inhibit neointimal proliferation following carotid arterial injury.

Creation and anti-cancer potency in HeLa cells of a novel chimeric toxin, HMGNCIDIN, composed of HMGN2 α- helical domain and PE38 KDEL domain Ⅲ

Using targeted toxins is a promising approach for the therapy of cancer and autoimmune diseases, as well as other disorders/The high mobility group chromosomal protein N2 （HMGN2） is one of the most abundant and well-characterized classes of nonhistone nuclear proteins, which seems to function as architectural elements in chromatin.2 Recently our group isolated an antimicrobial polypeptide from human LAK cells and cervical mucus,

Gram-negative bacteria and their lipopolysaccharides in Alzheimer’s disease:pathologic roles and therapeutic implications

Alzheimer's disease(AD)is the most serious age-related neurodegenerative disease and causes destructive and irreversible cognitive decline.Failures in the development of therapeutics targeting amyloid-β(Aβ)and tau;principal proteins inducing pathology in AD,suggest a paradigm shift towards the development of new therapeutic targets.The gram-negative bacteria and lipopolysaccharides(LPS)are attractive new targets for AD treatment.Surprisingly,an altered distribution of gram-negative bacteria and their LPS has been reported in AD patients.Moreover,gram-negative bacteria and their LPS have been shown to affect a variety of AD-related pathologies,such as Aβ homeostasis,tau pathology,neuroinflammation,and neurodegeneration.Moreover,therapeutic approaches targeting gram-negative bacteria or gram-negative bacterial molecules have significantly alleviated AD-related pathology and cognitive dysfunction.Despite multiple evidence showing that the gram-negative bacteria and their LPS play a crucial role in AD pathogenesis,the pathogenic mechanisms of gram-negative bacteria and their LPS have not been clarified.Here,we summarize the roles and pathomechanisms of gram-negative bacteria and LPS in AD.Furthermore,we discuss the possibility of using gram-negative bacteria and gram-negative bacterial molecules as novel therapeutic targets and new pathological characteristics for AD.

Immunotoxins and Cancer Therapy

In the past decade,an increased amount of clinicallyloriented research involving immunotoxins has been published. Immunotoxins are a group of artificially-made cytotoxic molecules targeting cancer cells.These molecules composed of a targeting moiety,such as a ligand or an antibody,linked to toxin moiety,which is a toxin with either truncated or deleted cell-binding domain that prevents it from binding to normal cells.Immunotoxins can be divided into two categories:chemically conjugated immunotoxins and recombinant ones.The immunotoxins of the first category have shown limited efficacy in clinical trials in patients with hematologic malignancies and solid tumors.Within the last few years,single-chain immunotoxins provide enhanced therapeutic efficacy over conjugated forms and result in improved antitumor activity.In this review,we briefly illustrate the design of the immunotoxins and their applications in clinical trials.Cellular & Molecular Immunology.2005;2(2):106-112.