SHOC1 is a ERCC4-(HhH)2-like protein, integral to the formation of crossover recombination intermediates during mammalian meiosis.

Chromosome segregation errors during meiosis result in the formation of aneuploid gametes and are the leading cause of pregnancy loss and birth defects in humans. Proper chromosome segregation requires pairwise associations of maternal and paternal homologous chromosomes. Chiasmata, which are the cytological manifestations of crossovers (COs), provide a physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Although CO-promoting activities ensure a balanced number and position of COs, their identity and mechanism of action in mammals remain understudied. Previous work in yeast and Arabidopsis has shown that Zip2 and Shoc1 are ortholog proteins with an important role in promoting the formation of COs. Our work is the first study in mammals showing the in vivo and in vitro function of mouse and human SHOC1. We show that purified recombinant human SHOC1, an XPF/MUS81 family member, preferentially binds branched DNA molecules but apparently lacks in vitro endonuclease activity, despite its conserved ERCC4-(HhH)2 core structure. Cytological observations suggest that initial steps of recombination are normal in a majority of spermatocytes from SHOC1 hypomorphic mice. However, late stages of recombination appear abnormal, as chromosomal localization of MLH1 is reduced. In agreement, chiasma formation is reduced, and cells arrest at metaphase I with a few lagging chromosomes and subsequent apoptosis. This analysis of SHOC1-deficient mice and the selective localization of SHOC1 to a subset of recombination sites show that SHOC1 acts at key mid-stage steps of the CO formation process. The formation of chromosome axial elements and homologous pairing are apparently normal, but synapsis is altered with SYCP1 frequently failing to extend the full length of the chromosome axes. Finally, we describe that SHOC1 interacts with TEX11, another protein important for the formation of COs, connecting SHOC1 to chromosome axis and structure.

Low-dose plasmid DNA treatment increases plasma vasopressin and regulates blood pressure in experimental endotoxemia.

BACKGROUND: Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert) as therapeutic agent requires further investigation. RESULTS: Here, we showed that plasmid DNA (pcDNA3) at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS)-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 µg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP) drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP), a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO) production. CONCLUSION: Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.

Endonucleases: tools to correct the dystrophin gene.

BACKGROUND: Various endonucleases can be engineered to induce double-strand breaks (DSBs) in chosen DNA sequences. These DSBs are spontaneously repaired by nonhomologous-end-joining, resulting in micro-insertions or micro-deletions (INDELs). We detected, characterized and quantified the frequency of INDELs produced by one meganuclease (MGN) targeting the RAG1 gene, six MGNs targeting three introns of the human dystrophin gene and one pair of zinc finger nucleases (ZFNs) targeting exon 50 of the human dystrophin gene. The experiments were performed in human cells (i.e. 293 T cells, myoblasts and myotubes). METHODS: To analyse the INDELs produced by the endonucleases the targeted region was polymerase chain reaction amplified and the amplicons were digested with the Surveyor enzyme, cloned in bacteria or deep sequenced. RESULTS: Endonucleases targeting the dystrophin gene produced INDELs of different sizes but there were clear peaks in the size distributions. The positions of these peaks were similar for MGNs but not for ZFNs in 293 T cells and in myoblasts. The size of the INDELs produced by these endonucleases in the dystrophin gene would have permitted a change in the reading frame. In a subsequent experiment, we observed that the frequency of INDELs was increased by re-exposition of the cells to the same endonuclease. CONCLUSIONS: Endonucleases are able to: (i) restore the normal reading of a gene with a frame shift mutation; (ii) delete a nonsense codon; and (iii) knockout a gene. Endonucleases could thus be used to treat Duchenne muscular dystrophy and other hereditary diseases that are the result of a nonsense codon or a frame shift mutation.

B cells Can Modulate the CD8 Memory T Cell after DNA Vaccination Against Experimental Tuberculosis.

BACKGROUND: Although B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown. METHODS: In this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge. RESULTS: In vitro and in vivo studies showed that B cells efficiently present antigens after naked plasmid pcDNA3 encoding M. leprae 65-kDa heat shock protein (pcDNA3-Hsp65) internalization and protect B knock-out (BKO) mice against Mycobacterium tuberculosis infection. pcDNA3-Hsp65-transfected B cells adoptively transferred into BKO mice rescued the memory phenotypes and reduced the number of CFU compared to wild-type mice. CONCLUSIONS: These data not only suggest that B cells play an important role in the induction of CD8 T cells but also that they improve bacterial clearance in DNA vaccine model.

Intranasal vaccination with messenger RNA as a new approach in gene therapy: use against tuberculosis.

BACKGROUND: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. RESULTS: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 µg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). CONCLUSIONS: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells.

This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co-glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice.

Influence of storage time and temperature on pleural fluid adenosine deaminase determination.

OBJECTIVES AND BACKGROUND: The determination of adenosine deaminase (ADA) activity in pleural fluid is important for differentiation of pleural effusions and diagnosing pleural tuberculosis. Although measurement of ADA is simple and inexpensive, controversies exist regarding potential errors caused by time elapsed between sample collection and analysis, storage temperature and the use of anticoagulants. The objective of this study was to evaluate the influence of storage time (1, 3, 7, 10 and 28 days) and temperature (4 degrees C and -20 degrees C) on the determination of ADA in pleural fluid samples collected in EDTA and sent at ambient temperature to the laboratory for initial processing within 1 h of collection. METHODOLOGY: Twenty-seven pleural exudates obtained from 20 patients with neoplastic disease and seven with tuberculosis were analysed. The ADA activity obtained within 1 h of collection was considered the gold standard and was compared with the other measurements. RESULTS: The correlation between the initial measurement and all others was >or=0.90 for both temperatures up to the 10th day after thoracocentesis and tended to decrease by 28 days after collection, but this difference was not significant. CONCLUSIONS: Pleural fluid samples collected in EDTA and sent for analysis within 1 h after collection can be used to determine ADA up to 28 days after collection if stored at 4 degrees C or -20 degrees C, with no evidence of significant increases or decrease in enzyme activity that might distort the results.