Recombinant Mycobacterium bovis BCG for immunotherapy in nonmuscle invasive bladder cancer.

In the past three decades, intravesical instillation of Mycobacterium bovis bacille Calmette-Guérin (BCG) has been used for treating bladder cancer and it still remains at the forefront of immunotherapy for cancer patients. Although BCG-based therapy is the most effective intravesical therapy for this kind of tumor and represents the only agent known to reduce progression into muscle invasive bladder cancer, BCG is ineffective in approximately 30-40 % of cases and disease recurs in up to 50 % of patients. Since that BCG is considered an effective vehicle for delivery of antigens due to its unique characteristics, the genetic manipulation of these mycobacteria has been appealing in the search for less toxic and more potent therapeutic agents for bladder cancer immunotherapy. Herein, we discuss current advances in recombinant BCG construction, research, concerns, and future directions to promote the development of this promising immunotherapeutic approach for bladder cancer.

Genistein induces apoptosis and autophagy in human breast MCF-7 cells by modulating the expression of proapoptotic factors and oxidative stress enzymes.

Breast cancer is one of the common tumors occurring in woman and despite treatment, the prognostic is poor. Genistein, a soy isoflavone, has been reported to have chemopreventive\chemotherapeutic potential in multiple tumor types. Here, we investigated the genistein antiproliferative effect in MCF-7 breast cancer, underlying the molecular mechanisms involved in this effect. MCF-7 cancer and CCD1059sK fibroblast cells were treated with estradiol (10 nM) or genistein (0.01-100 µM) for 24, 48, and 72 h and the cell proliferation was investigated by MTT; membrane cell permeability was evaluated by LDH and PI incorporation; apoptosis was investigated by externalization of phosphatidylserine by FACS; and presence of autophagy was detected by LC3A/B immunostaining. The expression of apoptotic proteins and antioxidant enzymes was evaluated by qPCR. The results demonstrate that genistein (100 µM) for 72 h of treatment selectively reduced MCF-7 cell proliferation independent of estrogen receptor activation, while no cytotoxicity was observed in fibroblast cells. Further experiments showed that genistein induced phosphatidylserine externalization and LC3A/B immunopositivity in MCF-7 cells, indicating apoptosis and autophagy cell death. Genistein increased in three times proapoptotic BAX/Bcl-2 ratio and promoted a parallel downregulation of 20 times of antiapoptotic survivin. In addition, genistein promoted a decrease of 5.5, 9.3, and 3.6 times of MnSOD, CuZnSOD, and TrxR mRNA expression, respectively, while the GPx expression was increased by 6.5 times. These results suggest that the antitumor effect of genistein involved the modulation of antioxidant enzyme and apoptotic signaling expression, which resulted in apoptosis and progression of autophagy.

Ocorrência de Campylobacter em carne de frango, fezes de frango e humanas e pesquisa dos genes cdt / Occurrence of Campylobacter in poultry, meat chicken and human feces, and cdt GENES research

Foram coletadas 100 amostras de conteúdo fecal de aves de corte, 100 de produtos de frango (coxa, sobrecoxa, asa, dorso, carne moída e fígado) e 100 de fezes de humanos, e analisadas para pesquisa de Campylobacter. Realizou-se a determinação da espécie e da presença dos genes cdt, responsáveis pela codificação da toxina citoletal distensiva (CDT), através da técnica da PCR. A bactéria foi isolada de 61% das amostras de fezes de frango, 20% de produtos de frango e 3% de fezes de humanos. A maioria dos isolados foi determinada como C. jejuni . Destes, 93,5% apresentaram os genes para a toxina CDT. Apesar de a ocorrência de Campylobacter em fezes de humanos ter sido baixa, a prevalência em frangos de corte e produtos de frango foi elevada, fato que, aliado à presença dos genes cdt na maioria dos isolados, representa risco potencial para os consumidores. Esses resultados são indicativos da necessidade de medidas preventivas no sistema de produção e de boas práticas de fabricação na indústria, de forma a minimizar a contaminação dos produtos e diminuir o risco para os consumidores.

A hundred chicken fecal samples, a hundred samples of retail poultry products and a hundred samples of human feces were collected and tested for the presence of Campylobacter. The species identification and the analysis for the presence of cdt genes, responsible for encoding the cytolethal distending toxin, were performed by PCR. Campylobacter was found in 61% of the chicken fecal samples, in 20% of the poultry products and in 3% of the human feces. Most isolates were identified as C. jejuni. In 93.5% of these isolates, the cdt genes have been detected. Despite the occurrence of Campylobacter in feces of humans has been low, the prevalence in broilers and poultry products was high, which, combined with the presence of cdt genes in most isolates, represents a potential risk to consumers. These results suggest there is a need for preventive measures in the production system and good manufacturing practices in the industry so as to minimize contamination of products and reduce the risk to consumers.

Oncogenic somatic events in tissue-specific stem cells: a role in cancer recurrence?

Tissue-specific stem cells (TSSCs) are a very unique cell type, with critical and well-defined roles for the homeostasis of high turnover tissues (such as the blood and the skin). Emerging evidence suggests that TSSCs are implicated in malignancies, with several theories being proposed and tested, including many attempts to identify the cells of origin and studies deigned to understand how TSSCs participate in age-related increase in cancer risk. A currently unexplored possibility in this respect is the plausible theory that an oncogenic event that arises at a TSSC would promote tissue replenishment by cells containing these mutations, with progressive propagation of such mutated TSSCs in the niche. Therefore, the effect of a somatic oncogenic event in a single TSSC may have more important implications than previously anticipated, resulting in sustained and progressively higher cancer risk. This model could have important implications for tumor recurrence, since in some cases the underlying cause might be the development of a new tumor originated from daughter cells of the TSSC that suffered the first oncogenic hit, rather than proliferation of residual cancer cells. In this review, we present and discuss approaches for testing the proposed theory of tumorigenesis and cancer risk, as well as practical implications for biomedical research and clinical practice.

Developments in the use of nanocapsules in oncology

The application of nanotechnology to medicine can provide important benefits, especially in oncology, a fact that has resulted in the emergence of a new field called Nanooncology. Nanoparticles can be engineered to incorporate a wide variety of chemotherapeutic or diagnostic agents. A nanocapsule is a vesicular system that exhibits a typical core-shell structure in which active molecules are confined to a reservoir or within a cavity that is surrounded by a polymer membrane or coating. Delivery systems based on nanocapsules are usually transported to a targeted tumor site and then release their contents upon change in environmental conditions. An effective delivery of the therapeutic agent to the tumor site and to the infiltrating tumor cells is difficult to achieve in many cancer treatments. Therefore, new devices are being developed to facilitate intratumoral distribution, to protect the active agent from premature degradation and to allow its sustained and controlled release. This review focuses on recent studies on the use of nanocapsules for cancer therapy and diagnosis.

Developments in the use of nanocapsules in oncology.

The application of nanotechnology to medicine can provide important benefits, especially in oncology, a fact that has resulted in the emergence of a new field called Nanooncology. Nanoparticles can be engineered to incorporate a wide variety of chemotherapeutic or diagnostic agents. A nanocapsule is a vesicular system that exhibits a typical core-shell structure in which active molecules are confined to a reservoir or within a cavity that is surrounded by a polymer membrane or coating. Delivery systems based on nanocapsules are usually transported to a targeted tumor site and then release their contents upon change in environmental conditions. An effective delivery of the therapeutic agent to the tumor site and to the infiltrating tumor cells is difficult to achieve in many cancer treatments. Therefore, new devices are being developed to facilitate intratumoral distribution, to protect the active agent from premature degradation and to allow its sustained and controlled release. This review focuses on recent studies on the use of nanocapsules for cancer therapy and diagnosis.

Associação entre proteínas do plasma seminal, motilidade e viabilidade espermática em coelhos submetidos a doping genético / Association among seminal plasma proteins, sperm motility and sperm viability in rabbits submitted to gene doping

Neste trabalho foi estudada a correlação entre o perfil proteico do plasma seminal e a motilidade e viabilidade espermática em coelhos submetidos ao tratamento com vetores de expressão contendo o gene da eritropoetina (EPO) e com EPO recombinante humana. Foram identificadas, em coelhos submetidos ao tratamento com vetor de DNA contendo o gene da EPO, duas bandas proteicas associadas a alterações na motilidade espermática - 48kDa à baixa motilidade (P<0,05) e 18kDa à alta motilidade (P<0,05) - e esse fator foi associado a maior viabilidade espermática (P<0,05). Em coelhos submetidos ao tratamento com EPO recombinante, um fator proteico, 63kDa, associou-se à alta motilidade espermática (P<0,05), enquanto dois, 26 e 40kDa, foram associados à alta viabilidade espermática (P<0,05). Esses resultados sugerem que o doping genético pode ocasionar mudanças no perfil proteico do plasma seminal, provocando alterações na motilidade e viabilidade espermática.

In this study the correlation between seminal plasma protein profile and the sperm motility and sperm viability in rabbits submitted to treatment with an expression vector containing EPO gene and with human recombinant EPO was evaluated. In rabbits submitted to treatment with EPO expression vector, two protein bands were associated to sperm motility - 48kDa associated to low motility (P<0.05) and 18kDa to high motility (P<0.05) - and this protein band was also associated to high sperm viability (P<0.05). In rabbits submitted to treatment with human recombinant EPO, a protein factor, 63kDa, was associated to high sperm motility (P<0.05) while two protein factors, 26 and 40kDa, were associated to high sperm viability (P<0.05). These results suggest that gene doping leads to changes in rabbit seminal plasma protein, altering sperm motility and sperm viability.

Erythropoietin non-viral gene therapy does not affect motility, viability, morphology or concentration of rabbit sperm.

Erythropoietin (EPO) gene therapy can be used for several purposes; however, its effects on reproductive performance are unknown. The aim of this study was to evaluate the toxicological effects of non-viral (EPO) gene transfer on sperm motility, viability, morphology and concentration. Rabbit EPO cDNA was cloned into a pTarget mammalian expression vector. Rabbits were administered with: (1) pTarget/EPO vector, (2) recombinant human EPO (rHuEpo) and (3) saline (control). Both pTarget/EPO and rHuEpo significantly increased (P < 0.05) hematocrit levels 1 week after injection and they remained significantly higher than the control for up to 5 weeks (P < 0.05), showing that both EPO treatments were effective in stimulating the production of red blood cells in rabbits. The EPO gene transfer or rHuEPO administration had no significant effect (P > 0.05) on sperm motility, vigor, viability, concentration or morphology in the testis.

Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

Cryopreservation of immature equine oocytes, comparing a solid surface vitrification process with open pulled straws and the use of a synthetic ice blocker.

The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N=269), the OPS (N=159) and the SSV (N=202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P<0.001) and MIn (76.6 vs. 31.1 and 33.7%; P<0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in the OPS vitrification process increased the rates of both IVM (30.5%; P<0.01) and MIn (45.8%; P<0.05) of the oocytes (N=59). Including 0.1% ice blocker in the SSV process improved the IVM rate (20.9%; P<0.05), whereas MIn remained compromised in this group (N=67). However, increasing the concentration of the ice blocker (to 1.0%) in the cryopreservation methods did not significantly improve rates of IVM. In conclusion, the addition of a synthetic ice blocker (0.1%) to both cryopreservation processes significantly increased rates of both IVM and MIn of immature equine oocytes cryopreserved by OPS.

The use of genes for performance enhancement: doping or therapy?

Recent biotechnological advances have permitted the manipulation of genetic sequences to treat several diseases in a process called gene therapy. However, the advance of gene therapy has opened the door to the possibility of using genetic manipulation (GM) to enhance athletic performance. In such ‘gene doping’, exogenous genetic sequences are inserted into a specific tissue, altering cellular gene activity or leading to the expression of a protein product. The exogenous genes most likely to be utilized for gene doping include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor type 1 (IGF-1), myostatin antagonists, and endorphin. However, many other genes could also be used, such as those involved in glucose metabolic pathways. Because gene doping would be very difficult to detect, it is inherently very attractive for those involved in sports who are prepared to cheat. Moreover, the field of gene therapy is constantly and rapidly progressing, and this is likely to generate many new possibilities for gene doping. Thus, as part of the general fight against all forms of doping, it will be necessary to develop and continually improve means of detecting exogenous gene sequences (or their products) in athletes. Nevertheless, some bioethicists have argued for a liberal approach to gene doping.

The use of genes for performance enhancement: doping or therapy?

Recent biotechnological advances have permitted the manipulation of genetic sequences to treat several diseases in a process called gene therapy. However, the advance of gene therapy has opened the door to the possibility of using genetic manipulation (GM) to enhance athletic performance. In such 'gene doping', exogenous genetic sequences are inserted into a specific tissue, altering cellular gene activity or leading to the expression of a protein product. The exogenous genes most likely to be utilized for gene doping include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor type 1 (IGF-1), myostatin antagonists, and endorphin. However, many other genes could also be used, such as those involved in glucose metabolic pathways. Because gene doping would be very difficult to detect, it is inherently very attractive for those involved in sports who are prepared to cheat. Moreover, the field of gene therapy is constantly and rapidly progressing, and this is likely to generate many new possibilities for gene doping. Thus, as part of the general fight against all forms of doping, it will be necessary to develop and continually improve means of detecting exogenous gene sequences (or their products) in athletes. Nevertheless, some bioethicists have argued for a liberal approach to gene doping.

NanoSMGT: transfection of exogenous DNA on sex-sorted bovine sperm using nanopolymer.

The objective was to introduce exogenous DNA into commercially sex-sorted bovine sperm using nanopolymer for transfection. In the first experiment, the optimal concentration and ratio of linear-to-circular plasmid was determined for NanoSMGT in unsorted sperm. A second experiment was conducted to transfect exogenous DNA into sex-sorted sperm. Exogenous DNA uptake occurred in a dose-dependent manner (P < 0.05). The optimal amount of DNA was 10 µg/10(6) cells. The ratios of linear-to-circular plasmid do not influence the uptake by unsorted sperm cells and none of the tested treatments affected sperm motility and viability. Commercially sex-sorted bovine sperm were able to uptake exogenous DNA using nanopolymer; however, both X- and Y-sorted sperm had decreased DNA uptake in comparison to unsorted sperm (P < 0.05). Neither sperm motility nor viability were affected by nanotransfection. In conclusion, nanopolymer efficiently introduced exogenous DNA into commercially sex-sorted bovine sperm; we inferred that these sperm could be used for production of embryos of the desired sex, a technique named NanoSMGT.

Clonagem e avaliação da expressão gênica do sbGnRH em machos juvenis e adultos de linguado, Paralichthys orbignyanus / Cloning and evaluation of sbGnRH gene expression in juvenile and adult males of Brazilian flounder Paralichthys orbignyanus

Este estudo buscou clonar o cDNA do sbGnRH, identificar sua expressão em diferentes tecidos do linguado, bem como avaliar possíveis diferenças no RNA mensageiro (RNAm) desse gene no cérebro de linguados machos juvenis e adultos. Por meio da RT-PCR, demonstrou-se pela primeira vez, a clonagem da região codificadora do sbGnRH contendo 297 nucleotídeos do cérebro do linguado. A expressão do sbGnRH foi detectada em vários tecidos periféricos. Foram detectados níveis mais elevados de RNAm do sbGnRH no hipotálamo dos animais adultos. Estes resultados sugerem que o sbGnRH está envolvido na puberdade do linguado.

The objectives of this study were to clone sbGnRH cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain sbGnRH expression in juvenile and adult males. Using RT-PCR the cloning of a 297 nucleotides coding region of sbGnRH from Brazilian flounder brain was demonstrated for the first time. Expression of sbGnRH was detected in several peripheral tissues. Brain gene expression in the adult flounder was higher than those found in juvenile. These results suggest that sbGnRH is involved on the Brazilian flounder puberty.

Fator do plasma seminal associado à integridade de membrana de espermatozóides suínos pós-descongelamento / Seminal plasma factor associated to post-thawing swine sperm membrane integrity

Neste estudo, identificaram-se polipeptídeos associados à integridade da membrana plasmática (IMP) de espermatozóides suínos após o processo de congelamento/descongelamento. Por meio do perfil protéico do plasma seminal em SDS-PAGE, observou-se a presença de nove bandas polipeptídicas com pesos moleculares que variaram de 11,97 a 122,52kDa. Detectou-se que uma banda de 26,58kDa esteve associada à baixa IMP (<55 por cento). Não foi verificada associação entre as outras bandas e a IMP. Conclui-se que o fator polipeptídico de 26,58kDa está associado à baixa integridade da membrana plasmática do espermatozóide suíno após o congelamento/descongelamento.

Polypeptides associate to membrane integrity (MI) of swine spermatozoa submitted to freezing and thawing were identified. The protein profile of seminal plasma analyzed by SDS-PAGE allowed the identification of nine polypeptide bands with molecular weight ranging from 11.97 to 122.52kDa. One 26.58kDa band was associated with reduced MI (<55 percent). No associations among other bands and MI were observed. The 26.58kDa factor is associated with reduction of membrane integrity of swine spermatozoa after freezing and thawing.