Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.

Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway

Background Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. Objective Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. Methods We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. Results We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. Conclusions and Clinical Relevance Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen-and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.

Disposable polyester-toner electrophoresis microchips for DNA analysis

Microchip electrophoresis has become a powerful tool for DNA separation, offering all of the advantages typically associated with miniaturized techniques: high speed, high resolution, ease of automation, and great versatility for both routine and research applications. Various substrate materials have been used to produce microchips for DNA separations, including conventional (glass, silicon, and quartz) and alternative (polymers) platforms. In this study, we perform DNA separation in a simple and low-cost polyester-toner (PeT)-based electrophoresis microchip. PeT devices were fabricated by a direct-printing process using a 600 dpi-resolution laser printer. DNA separations were performed on PeT chip with channels filled with polymer solutions (0.5% m/v hydroxyethylcellulose or hydroxypropylcellulose) at electric fields ranging from 100 to 300Vcm(-1). Separation of DNA fragments between 100 and 1000 bp, with good correlation of the size of DNA fragments and mobility, was achieved in this system. Although the mobility increased with increasing electric field, separations showed the same profile regardless of the electric field. The system provided good separation efficiency (215 000 plates per m for the 500 bp fragment) and the separation was completed in 4 min for 1000 bp fragment ladder. The cost of a given chip is approximately $0.15 and it takes less than 10 minutes to prepare a single device.

DNA repair gene excision repair cross complementing-group 1 (ERCC1) in head and neck squamous cell carcinoma: analysis of methylation and polymorphism (G19007A), protein expression and association with epidemiological and clinicopathological factors

Aims: To evaluate the associations of excision repair cross complementing-group 1 (ERCC1) (DNA repair protein) (G19007A) polymorphism, methylation and immunohistochemical expression with epidemiological and clinicopathological factors and with overall survival in head and neck squamous cell carcinoma (HNSCC) patients. Methods and results: The study group comprised 84 patients with HNSCC who underwent surgery and adjuvant radiotherapy without chemotherapy. Bivariate and multivariate analyses were used. The allele A genotype variant was observed in 79.8% of the samples, GG in 20.2%, GA in 28.6% and AA in 51.2%. Individuals aged more than 45 years had a higher prevalence of the allelic A variant and a high (83.3%) immunohistochemical expression of ERCC1 protein [odds ratio (OR) = 4.86, 95% confidence interval (CI): 1.2-19.7, P = 0.027], which was also high in patients with advanced stage (OR= 5.04, 95% CI: 1.07-23.7, P = 0.041). Methylated status was found in 51.2% of the samples, and was higher in patients who did not present distant metastasis (OR = 6.67, 95% CI: 1.40-33.33, P = 0.019) and in patients with advanced stage (OR = 5.04, 95% CI: 1.07-23.7, P = 0.041). At 2 and 5 years, overall survival was 55% and 36%, respectively (median = 30 months). Conclusion: Our findings may reflect a high rate of DNA repair due to frequent tissue injury during the lifetime of these individuals, and also more advanced disease presentation in this population with worse prognosis.

Bixin and norbixin protect against DNA-damage and alterations of redox status induced by methylmercury exposure in vivo

Populations in the Amazon are exposed to organic mercury via consumption of contaminated foods. These ethnic groups consume a specific plant seed annatto which contains certain carotenoids. The aim of this study was to find out if these compounds (bixin, BIX and norbixin, NOR), protect against DNA-damage caused by the metal. Therefore, rats were treated orally with methylmercury (MeHg) and with the carotenoids under conditions that are relevant to humans. The animals were treated either with MeHg (30 mu g/kg/bw/day), BIX (0.110 mg/kg/bw/day), NOR (0.011.0 mg/kg/bw/day) or combinations of the metal compound and the carotenoids consecutively for 45 days. Subsequently, the glutathione levels (GSH) and the activity of catalase were determined, and DNA-damage was measured in hepatocytes and leukocytes using single cell gel electrophoresis assays. Treatment with the metal alone caused a decrease in the GSH levels (35%) and induced DNA damage, which resulted in increased DNA migration after electrophoresis in liver and blood cells, whereas no effects were seen with the carotenoids alone. When BIX or NOR were given in combination with organic mercury, the intermediate and the highest concentrations of the carotenoids (1.0 and 10.0 mg/kg/bw/day BIX and 0.1 and 1.0 mg/kg/bw/day NOR) protected against DNA-damage. Furthermore, we found with both carotenoids, a moderate increase in the GSH levels in both metal-treated and untreated animals, while the activities of catalase remained unchanged. Our results indicate that consumption of BIX and NOR may protect humans against the adverse health effects caused by exposure to organic mercury. Environ. Mol. Mutagen., 2012. (c) 2012 Wiley Periodicals, Inc.

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily

Abstract Background The CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism. Results We describe a novel S. mansoni transposon named SmTRC1, for S. mansoni Transposon Related to CACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30–300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence. Conclusion The DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.

Frequency of LCT -13910C>T single nucleotide polymorphism associated with adult-type hypolactasia/lactase persistence among Brazilians of different ethnic groups

Abstract Background Adult-type hypolactasia, the physiological decline of lactase some time after weaning, was previously associated with the LCT -13910C>T polymorphism worldwide except in Africa. Lactase non-persistence is the most common phenotype in humans, except in northwestern Europe with its long history of pastoralism and milking. We had previously shown association of LCT -13910C>T polymorphism with adult-type hypolactasia in Brazilians; thus, we assessed its frequency among different Brazilian ethnic groups. Methods We investigated the ethnicity-related frequency of this polymorphism in 567 Brazilians [mean age, 42.1 ± 16.8 years; 157 (27.7%) men]; 399 (70.4%) White, 50 (8.8%) Black, 65 (11.5%) Brown, and 53 (9.3%) Japanese-Brazilian. DNA was extracted from leukocytes; LCT -13910C>T polymorphism was analyzed by PCR-restriction fragment length polymorphism. Results Prevalence of the CC genotype associated with hypolactasia was similar (57%) among White and Brown groups; however, prevalence was higher among Blacks (80%) and those of Japanese descent (100%). Only 2 (4%) Blacks had TT genotype, and 8 (16%) had the CT genotype. Assuming an association between CC genotype and hypolactasia, and CT and TT genotypes with lactase persistence, 356 (62.8%) individuals had hypolactasia and 211 (37.2%) had lactase persistence. The White and Brown groups had the same hypolactasia prevalence (~57%); nevertheless, was 80% among Black individuals and 100% among Japanese-Brazilians (P < 0.01). Conclusion The lactase persistence allele, LCT -13910T, was found in about 43% of both White and Brown and 20% of the Black Brazilians, but was absent among all Japanese Brazilians studied.

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma.

Chemopreventive effect of Copaifera langsdorffii leaves hydroalcoholic extract on 1,2-dimethylhydrazine-induced DNA damage and preneoplastic lesions in rat colon

Background Natural antioxidants present in common foods and beverages have drawn great attention to cancer prevention due to its health benefits, remarkable lack of toxicity and side effects. Copaifera langsdorffii, known as “copaiba”, “capaiva”, or “pau-de-óleo“, belongs to the Leguminosae family and occurs in fields and grasslands in the northern and northeastern parts of Brazil. Biological studies of Copaifera corroborate its widespread use by the population. This paper describes the effects of C. langsdorffii leaves hydroalcoholic extract on the 1,2-dimethylhydrazine (DMH)-induced DNA damage and aberrant crypt foci (ACF) in the colon of male Wistar rats. Methods The hydroalcoholic extract of C. langsdorffii was administered to rats by gavage at daily doses of 20, 40 and 80 mg/kg body weight. To evaluate DNA damage by the comet assay, animals received the C. langsdorffii extract for seven days and a single subcutaneous injection (sc) of 1,2-dimethylhydrazine (DMH) at a dose of 40 mg/kg on day 7. Animals were sacrificed 4 h after injection of DMH, to assess DNA damage. For the ACF assay, animals were acclimatized for one week (week 1) and then treated with the C. langsdorffii extract five times a week for four weeks (weeks 2 to 5). The rats received sc injections of DMH (40 mg/kg) on days 2 and 5 of weeks 2 and 3, to induce ACF. Animals were euthanized at week 5; i.e., four weeks after the first DMH treatment. Results Animals treated with different doses of the C. langsdorffii extract combined with DMH had significantly lower frequency of DNA damage as compared with the positive control (animals treated with DMH only). The percentage of reduction in the frequency of DNA damage ranged from 14.30% to 38.8%. The groups treated with 40 and 80 mg/kg C. langsdorffii extract during and after DMH treatment presented significantly lower numbers of ACF and aberrant crypts compared with the control. Conclusion The C. langsdorffii extract significantly reduced the extent of DNA damage and ACF induced by DMH, suggesting that the extract has a protective effect against colon carcinogenesis.

Use of the checkerboard DNA-DNA hybridization technique for bacteria detection in Aedes aegypti (Diptera:Culicidae) (L.)

Abstract Background Bacteria associated with insects can have a substantial impact on the biology and life cycle of their host. The checkerboard DNA-DNA hybridization technique is a semi-quantitative technique that has been previously employed in odontology to detect and quantify a variety of bacterial species in dental samples. Here we tested the applicability of the checkerboard DNA-DNA hybridization technique to detect the presence of Aedes aegypti-associated bacterial species in larvae, pupae and adults of A. aegypti. Findings Using the checkerboard DNA-DNA hybridization technique we could detect and estimate the number of four bacterial species in total DNA samples extracted from A. aegypti single whole individuals and midguts. A. aegypti associated bacterial species were also detected in the midgut of four other insect species, Lutzomyia longipalpis, Drosophila melanogaster, Bradysia hygida and Apis mellifera. Conclusions Our results demonstrate that the checkerboard DNA-DNA hybridization technique can be employed to study the microbiota composition of mosquitoes. The method has the sensitivity to detect bacteria in single individuals, as well as in a single organ, and therefore can be employed to evaluate the differences in bacterial counts amongst individuals in a given mosquito population. We suggest that the checkerboard DNA-DNA hybridization technique is a straightforward technique that can be widely used for the characterization of the microbiota in mosquito populations.

Investigação eletroquímica e calorimétrica da interação de novos agentes antitumorais biscatiônicos com DNA

Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-α,ω-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Ornithodoros peropteryx (Acari: Argasidae) in Bolivia: an argasid tick with a single nymphal stage

By the end of the 1960s, the argasid tick Ornithodoros peropteryx was described from larval specimens collected from the bat Peropteryx macrotis in Colombia. Since its original description, no additional record of O. peropteryx has been reported, and its post-larval stages have remained unknown. During July 2010, 18 larvae were collected from 9 bats (Centronycteris maximiliani), resulting in a mean infestation of 2.0 ± 2.2 ticks per bat (range 1–8). These bats were captured in a farm in northeastern Bolivia close to Guaporé River in the border with Brazil. Morphological examinations of the larvae revealed them to represent the species O. peropteryx. One engorged larva that was kept alive in the laboratory moulted to a nymph after 9 days. Fourteen days after the larval moulting, the nymph moulted to an adult female without taking any blood meal during the nymphal period. This adult female was used for a morphological description of the female stage of O. peropteryx. In addition, the larvae were used for a morphological redescription of this stage. One larva and two legs extirpated from the adult female were submitted to DNA extraction and PCR targeting a fragment of the mitochondrial 16S rDNA gene, which yielded DNA sequences at least 11 % divergent from any available argasid sequence in Genbank. We show that O. peropteryx ontogeny is characterized by a single, non-feeding, nymphal stage. This condition has never been reported for ticks.

Overtraining is associated with DNA damage in blood and skeletal muscle cells of Swiss mice

Abstract: Background: The alkaline version of the single-cell gel (comet) assay is a useful method for quantifying DNA damage. Although some studies on chronic and acute effects of exercise on DNA damage measured by the comet assay have been performed, it is unknown if an aerobic training protocol with intensity, volume, and load clearly defined will improve performance without leading to peripheral blood cell DNA damage. In addition, the effects of overtraining on DNA damage are unknown. Therefore, this study aimed to examine the effects of aerobic training and overtraining on DNA damage in peripheral blood and skeletal muscle cells in Swiss mice. To examine possible changes in these parameters with oxidative stress, we measured reduced glutathione (GSH) levels in total blood, and GSH levels and lipid peroxidation in muscle samples. Results: Performance evaluations (i.e., incremental load and exhaustive tests) showed significant intra and inter-group differences. The overtrained (OTR) group showed a significant increase in the percentage of DNA in the tail compared with the control (C) and trained (TR) groups. GSH levels were significantly lower in the OTR group than in the C and TR groups. The OTR group had significantly higher lipid peroxidation levels compared with the C and TR groups. Conclusions Aerobic and anaerobic performance parameters can be improved in training at maximal lactate steady state during 8 weeks without leading to DNA damage in peripheral blood and skeletal muscle cells or to oxidative stress in skeletal muscle cells. However, overtraining induced by downhill running training sessions is associated with DNA damage in peripheral blood and skeletal muscle cells, and with oxidative stress in skeletal muscle cells and total blood.

Ornithodoros peropteryx (Acari Argasidae) in Bolivia an argasid tick with a single nymphal stage

By the end of the 1960s, the argasid tick Ornithodoros peropteryx was described from larval specimens collected from the bat Peropteryx macrotis in Colombia. Since its original description, no additional record of O. peropteryx has been reported, and its post-larval stages have remained unknown. During July 2010, 18 larvae were collected from 9 bats (Centronycteris maximiliani), resulting in a mean infestation of 2.0 ± 2.2 ticks per bat (range 1–8). These bats were captured in a farm in northeastern Bolivia close to Guapore´ River in the border with Brazil. Morphological examinations of the larvae revealed them to represent the species O. peropteryx. One engorged larva that was kept alive in the laboratory moulted to a nymph after 9 days. Fourteen days after the larval moulting, the nymph moulted to an adult female without taking any blood meal during the nymphal period. This adult female was used for a morphological description of the female stage of O. peropteryx. In addition, the larvae were used for a morphological redescription of this stage. One larva and two legs extirpated from the adult female were submitted to DNA extraction and PCR targeting a fragment of the mitochondrial 16S rDNA gene, which yielded DNA sequences at least 11 % divergent from any available argasid sequence in Genbank. We show that O. peropteryx ontogeny is characterized by a single, non-feeding, nymphal stage. This condition has never been reported for ticks.