Oral vaccination with Trichinella spiralis DNase II DNA vaccine delivered by attenuated Salmonella induces a protective immunity in BALB/c mice.

Trichinellosis is one of the most serious foodborne parasitic zoonosis with worldwide distribution, and it is necessary to develop a vaccine to interrupt transmission from animals to humans. Trichinella spiralis adult-specific DNase II-1 (TsDNase II) were identified by immunoproteomics in surface or excretory/secretory proteins of adult worms (AW) and intestinal infective larvae (IIL). The aim of this study was to investigate the systemic, mucosal responses and immune protection elicited by oral vaccination with TsDNase II DNA vaccine delivered by attenuated Salmonella typhimurium strain⊿cyaSL1344. Oral vaccination with TsDNase II DNA vaccine triggered an obvious mucosal sIgA response and a systemic IgG response in mice, and IgG1 was predominant. Th1 (IFN-γ) and Th2 (IL-4, 10) cytokines were distinctly increased in the spleen and mesenteric lymph node (MLN) cells of vaccinated mice. An indirect immunofluorescent test revealed that native TsDNase II is present at the cuticle of this nematode after the 2nd molting, further confirming that TsDNase II is adult-specific and expressed at AW and pre-adult stages. Oral immunization of mice with TsDNase II exhibited a 53.85% reduction in AW and a 59.26% reduction in ML after larval challenge. The in vitro NBL production of adult females from TsDNase II-vaccinated mice was also reduced in comparison with pcDNA3.1 or the PBS control group (P < 0.01). Our results show that oral immunization of mice with TsDNase II produced an intestinal and systematic concurrent Th1/Th2 immune response, and a significant immune protection against challenge.

Therapeutic genome editing: prospects and challenges.

Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease-based therapies as well as future prospects and challenges.

Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure.

Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to the controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than the controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of oxidative DNA damage in aniline-induced splenic toxicity.

Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation.

The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.

Effect of xenogenic repair enzymes on photoimmunology and photocarcinogenesis.

Exposure to ultraviolet B (UVB) radiation leads to an increased generation of UVB-induced skin damage in humans. The most important UVB-induced side effects are UVB-induced immunosuppression and photocarcinogenesis and there is a large body of evidence that cyclobutane pyrimidine dimers (CPD) induced by UVB radiation play a pivotal role in both processes. The topical application of DNA repair enzymes is a new innovative strategy to reduce the amount of CPDs in human skin. Two different methods have recently been established. The use of T4 endonuclease V was of clinical efficacy in protecting patients with a nucleotide excision repair defect from premalignant and malignant skin lesions. Application of photolyase, a xenogenic enzyme which has been found in different organisms is also capable of removing UVB-induced CPD from normal human skin cells in vivo and appears to be more effective than T4 endonuclease V in damage removal. Photolyase encapsulated in liposomes may have in the near future a broad use as an active ingredient in modern skin care products.

Photoprotection by topical DNA repair enzymes: molecular correlates of clinical studies.

A new approach to photoprotection is to repair DNA damage after UV exposure. This can be accomplished by delivery of a DNA repair enzyme with specificity to UV-induced cyclobutane pyrimidine dimers into skin by means of specially engineered liposomes. Treatment of DNA-repair-deficient xeroderma pigmentosum patients or skin cancer patients with T4N5 liposome lotion containing such DNA repair liposomes increases the removal of DNA damage in the first few hours after treatment. In these studies, a DNA repair effect was observed in some patients treated with heat-inactivated enzyme. Unexpectedly, it was discovered that the heat-inactivated T4 endonuclease V enzyme refolds and recovers enzymatic activity. These studies demonstrate that measurements of molecular changes induced by biological drugs are useful adjuvants to clinical studies.

Pyrimidine dimer removal enhanced by DNA repair liposomes reduces the incidence of UV skin cancer in mice.

UV exposure has been linked to skin cancer in humans by epidemiology and the rare genetic disease xeroderma pigmentosum. However, UV produces multiple photoproducts in DNA, and their relative contribution is uncertain. An enzyme which specifically repairs cyclobutane pyrimidine dimers in DNA, T4 endonuclease V, was encapsulated in liposomes for topical delivery into mouse and human skin. In both species, liposomes applied after UV exposure localized in the epidermis and stimulated the removal of cyclobutane pyrimidine dimers. UV-irradiated mice treated with these liposomes had a dose-dependent decrease in the incidence of squamous cell carcinoma compared to controls. The results demonstrate that unrepaired cyclobutane pyrimidine dimers in DNA are a direct cause of cancer in mammalian skin.