Cows as Bioreactors for the Production of Nutritionally and Biomedically Significant Proteins.

Dairy and beef cattle make a vital contribution to global nutrition, and since their domestication, they have been continuously exposed to natural and artificial selection to improve production characteristics. The technologies of transgenesis and gene editing used in cattle are responsible for generating news characteristics in bovine breeding, such as alteration of nutritional components of milk and meat enhancing human health benefits, disease resistance decreasing production costs and offering safe products for human food, as well as the recombinant protein production of biomedical significance. Different methodologies have been used to generate transgenic cattle as bioreactors. These methods include the microinjection of vectors in pronuclear, oocyte or zygote, sperm-mediate transgenesis, and somatic cell nuclear transfer. Gene editing has been applied to eliminate unwanted genes related to human and animal health, such as allergy, infection, or disease, and to insert transgenes into specific sites in the host genome. Methodologies for the generation of genetically modified cattle are laborious and not very efficient. However, in the last 30 years, transgenic animals were produced using many biotechnological tools. The result of these modifications includes (1) the change of nutritional components, including proteins, amino acids and lipids for human nutrition; (2) the removal allergic proteins milk; (3) the production of cows resistant to disease; or (4) the production of essential proteins used in biomedicine (biomedical proteins) in milk and blood plasma. The genetic modification of cattle is a powerful tool for biotechnology. It allows for the generation of new or modified products and functionality that are not currently available in this species.

Australian action to reduce health risks from radon.

In Australia, worker exposure to radon in underground uranium mines has been a focus of policy makers and regulators, and has been well controlled in the industry sector. That cannot be said for public exposure to radon. Radon exposure studies in the late 1980s and early 1990s demonstrated that the levels of radon in Australian homes were some of the lowest in the world. The International Basic Safety Standards, published by the International Atomic Energy Agency, requires the government to establish and implement an action plan for controlling public exposure due to radon indoors. When considering different policy options, it is important to develop radon prevention and mitigation programmes reflecting elements that are unique to the region or country. The Australian Radon Action Plan is being considered at a national level, and presents a long-range strategy designed to reduce radon-induced lung cancer in Australia, as well as the individual risk for people living with high concentrations of radon. In Australia, workers who are not currently designated as occupationally exposed are also considered as members of the public. In the Australian context, there are only a limited set of scenarios that might give rise to sufficiently high radon concentrations that warrant mitigation. These include highly energy efficient buildings in areas of high radon potential, underground workplaces, workplaces with elevated radon concentrations (e.g. spas using natural spring waters), and enclosed workspaces with limited ventilation. The key elements for a successful plan will rely on collaboration between government sectors and other health promotion programmes, cooperative efforts involving technical and communication experts, and partnering with building professionals and other stakeholders involved in the implementation of radon prevention and mitigation.

Invasion of Wolbachia at the residential block level is associated with local abundance of Stegomyia aegypti, yellow fever mosquito, populations and property attributes.

Wolbachia can suppress dengue and control mosquito populations and this depends on the successful invasion of Wolbachia-infected mosquitoes into local populations. Ovitrap data collected during the recent invasion of wMel-infected Stegomyia aegypti (Diptera: Culicidae) (Linnaeus) into Gordonvale near Cairns, Australia, were used to identify variables that help predict the success of localized invasion. Based on the variance in Wolbachia frequencies across Gordonvale as well as at another release site at Yorkeys Knob in comparison to simulations, it was estimated that on average 2-4 females contributed eggs to an ovitrap. By collating ovitrap data from two collection periods at the start of the release from residential blocks, it was found that uninfected mosquitoes had a patchy distribution across the release site. Residential blocks with relatively high uninfected mosquito numbers were less easily invaded by Wolbachia than blocks with low numbers. The numbers of uninfected mosquitoes in ovitraps were negatively correlated with the proportion of brick houses in a residential block, whereas local Wolbachia frequencies were correlated positively with this variable as well as negatively with the amount of shading in a yard and availability of breeding sites. These findings point to proxy measures for predicting the ease of localized invasion of Wolbachia.

Evaluation of in vivo T cell kinetics: use of heavy isotope labelling in type 1 diabetes.

CD4(+) memory cell development is dependent upon T cell receptor (TCR) signal strength, antigen dose and the cytokine milieu, all of which are altered in type 1 diabetes (T1D). We hypothesized that CD4(+) T cell turnover would be greater in type 1 diabetes subjects compared to controls. In vitro studies of T cell function are unable to evaluate dynamic aspects of immune cell homoeostasis. Therefore, we used deuterium oxide ((2) H(2)O) to assess in vivo turnover of CD4(+) T cell subsets in T1D (n = 10) and control subjects (n = 10). Serial samples of naive, memory and regulatory (T(reg)) CD4(+) T cell subsets were collected and enrichment of deoxyribose was determined by gas chromatography-mass spectrometry (GC-MS). Quantification of T cell turnover was performed using mathematical models to estimate fractional enrichment (f, n = 20), turnover rate (k, n = 20), proliferation (p, n = 10) and disappearance (d*, n = 10). Although turnover of T(regs) was greater than memory and naive cells in both controls and T1D subjects, no differences were seen between T1D and controls in T(reg) or naive kinetics. However, turnover of CD4(+) memory T cells was faster in those with T1D compared to control subjects. Measurement and modelling of incorporated deuterium is useful for evaluating the in vivo kinetics of immune cells in T1D and could be incorporated into studies of the natural history of disease or clinical trials designed to alter the disease course. The enhanced CD4(+) memory T cell turnover in T1D may be important in understanding the pathophysiology and potential treatments of autoimmune diabetes.

An autoimmune-associated variant in PTPN2 reveals an impairment of IL-2R signaling in CD4(+) T cells.

The IL-2/IL-2R signaling pathway has an important role in autoimmunity. Several genes identified in genome-wide association (GWA) studies encode proteins in the IL-2/IL-2R signaling cascade that are associated with autoimmune diseases. One of these, PTPN2, encodes a protein tyrosine phosphatase that is highly expressed in T cells and regulates cytokine signaling. An intronic risk allele in PTPN2, rs1893217(C), correlated with decreased IL-2R signaling in CD4(+) T cells as measured by phosphorylation of STAT5 (phosphorylated STAT5 (pSTAT5)). We modeled an additive single nucleotide polymorphism (SNP) genotype, in which each copy of the risk allele conferred a decrease in IL-2R signaling (P=4.4 × 10(-8)). Decreased pSTAT5 impacted IL-2Rß chain signaling resulting in reduced FOXP3 expression in activated cells. This phenotype was not due to overt differences in expression of the IL-2R, molecules in the IL-2R signaling cascade or defects in STAT5. However, the rs1893217(C) risk variant did correlate with decreased PTPN2 expression in CD4(+)CD45RO T cells (P=0.0002). Thus, the PTPN2rs1893217(C) risk allele associated with reduced pSTAT5 in response to IL-2 and reduced PTPN2 expression. Together, these data suggest that decreased expression of PTPN2 may indirectly modulate IL-2 responsiveness. These findings, identified through genotype/phenotype relationships, may lead to identification of novel mechanisms underlying dysregulation of cytokine signaling in autoimmunity.

A lethal ovitrap-based mass trapping scheme for dengue control in Australia: I. Public acceptability and performance of lethal ovitraps.

We report on the first field evaluation of the public acceptability and performance of two types of lethal ovitrap (LO) in three separate trials in Cairns, Australia. Health workers were able to set standard lethal ovitraps (SLOs) in 75 and 71% of premise yards in the wet and dry season, respectively, and biodegradable lethal ovitraps (BLOs) in 93% of yards. Public acceptance, measured as retention of traps by residents, was high for both trap types, with <9% of traps missing after 4 weeks. Traps retaining water after 4 weeks were 78 and 34% for the two SLO trials and 58% for the BLOs. The 'failure rate' in the 535 BLOs set in the field for 4 weeks was 47%, of which 19% were lost, 51% had holes from probable insect chewing, 23% were knocked over, 7% had dried by evaporation and 1% were split. There was no significant difference in the failure rate of BLOs set on porous (grass, soil and mulch) versus solid (tiles, concrete, wood and stone) substrates. The SLOs and the BLOs were readily acceptable to ovipositing Aedes aegypti L. (Diptera: Culicidae); the mean number of eggs/trap was 6 and 15, for the dry season and wet season SLO trial, respectively, and 15 for the BLO wet season trial. Indeed, 84-94% of premise yards had egg positive SLOs or BLOs. A high percentage of both wet and dry season SLOs (29 and 70%, respectively) and BLOs (62%) that were dry after 4 weeks were egg positive, indicating the traps had functioned. Lethal strips from SLOs and BLOs that had been exposed for 4 weeks killed 83 and 74%, respectively, of gravid Ae. aegypti in laboratory assays. These results indicate that mass trapping schemes using SLOs and BLOs are not rejected by the public and effectively target gravid Ae. aegypti. The impact of the interventions on mosquito populations is described in a companion paper.

A lethal ovitrap-based mass trapping scheme for dengue control in Australia: II. Impact on populations of the mosquito Aedes aegypti.

In Cairns, Australia, the impacts on Aedes aegypti L. (Diptera: Culicidae) populations of two types of 'lure & kill' (L&K) lethal ovitraps (LOs), the standard lethal ovitrap (SLO) and the biodegradable lethal ovitrap (BLO) were measured during three mass-trapping interventions. To assess the efficacy of the SLO, two interventions (one dry season and one wet season) were conducted in three discrete areas, each lasting 4 weeks, with the following treatments: (i) SLOs (>200 traps, approximately 4/premise), BG-sentinel traps (BGSs; approximately 15, 1/premise) and larval control (container reduction and methoprene treatment) and (ii) larval control alone, and (iii) untreated control. Female Ae. aegypti populations were monitored for 4 weeks pre- and post-treatment in all three areas using BGSs and sticky ovitraps (SOs) or non-lethal regular ovitraps (ROs). In the dry season, 206 SLOs and 15 BGSs set at 54 and 15 houses, respectively, caught and killed an estimated 419 and 73 female Ae. aegypti, respectively. No significant decrease in collection size of female Ae. aegypti could be attributed to the treatments. In the wet season, 243 SLOs and 15 BGSs killed approximately 993 and 119 female Ae. aegypti, respectively. The mean number of female Ae. aegypti collected after 4 weeks with SOs and BGSs was significantly less than the control (LSD post-hoc test). The third mass-trapping intervention was conducted using the BLO during the wet season in Cairns. For this trial, three treatment areas were each provided with BLOs (>500, approximately 4/premise) plus larval control, and an untreated control area was designated. Adult female Ae. aegypti were collected for 4 weeks pre- and post-treatment using 15 BGSs and 20 SOs. During this period, 53.2% of BLOs contained a total of 6654 Ae. aegypti eggs. Over the intervention period, collections of Ae. aegypti in the treatment areas were significantly less than in the control area for BGSs but not SOs. An influx of relatively large numbers of young females may have confounded the measurement of changes in populations of older females in these studies. This is an important issue, with implications for assessing delayed action control measures, such as LOs and parasites/pathogens that aim to change mosquito age structure. Finally, the high public acceptability of SLOs and BLOs, coupled with significant impacts on female Ae. aegypti populations in two of the three interventions reported here, suggest that mass trapping with SLOs and BLOs can be an effective component of a dengue control strategy.

Difficulties in obtaining an HPGe detector for low-level measurements.

All low-level laboratories require HPGe detectors to meet certain technical specifications, some of which are not available from manufacturers prior to purchase. Ensuring an HPGe detector is fit for purpose requires the purchase and installation of a detector in the laboratory, incurring both financial risk and considerable time and effort. We show that the optimal HPGe crystal for low-level laboratories has a diameter matched to the source and a length providing 70% absorption of the gamma-rays of interest.

Optimization of human T-cell expansion ex vivo using magnetic beads conjugated with anti-CD3 and Anti-CD28 antibodies.

T-cell receptor engagement and accompanying costimulatory signals control the level of activation and functional potential of individual T cells. The authors previously developed a novel technology in which human T cells are activated and expanded in culture ex vivo using anti-CD3 and anti-CD28 monoclonal antibodies covalently linked to superparamagnetic beads (Xcyte Dynabeads). In this study the addition of N-acetyl L-cysteine (NAC) to the cultures markedly increased the expansion of T cells from human peripheral blood mononuclear cells without diminishing cell function. NAC increased the rate of T-cell division, reduced apoptosis, and increased the percentage of antigen-specific memory T cells in the cultures. The effect of varying the ratio of beads to T cells (1:10-10:1) at culture initiation was also evaluated. Polyclonal T cells were expanded at all bead-to-T cell ratios tested (range 1:10-10:1). While high bead-to-T cell ratios (5:1 and 10:1) deleted, low ratios (1:10 and 1:5) preserved memory T cells directed against cytomegalovirus, Epstein-Barr virus, and influenza virus antigens. Adding more anti-CD3/anti-CD28 beads during the culture led to further expansion of T cells. Experiments also revealed that reducing the amount of anti-CD3 antibodies relative to the amount of anti-CD28 antibodies on the beads favored the proliferation of antigen-specific T cells. In summary, these data indicate that T cell-stimulating effects of anti-CD3/anti-CD28 beads can be further manipulated to control the expansion of antigen-specific memory T cells and can be used to rapidly expand antigen-specific T cells ex vivo for potential clinical applications.

Immunoreactivity of organic mimeotopes of the E2 component of pyruvate dehydrogenase: connecting xenobiotics with primary biliary cirrhosis.

In primary biliary cirrhosis (PBC), the major autoepitope recognized by both T and B cells is the inner lipoyl domain of the E2 component of pyruvate dehydrogenase. To address the hypothesis that PBC is induced by xenobiotic exposure, we took advantage of ab initio quantum chemistry and synthesized the inner lipoyl domain of E2 component of pyruvate dehydrogenase, replacing the lipoic acid moiety with synthetic structures designed to mimic a xenobiotically modified lipoyl hapten, and we quantitated the reactivity of these structures with sera from PBC patients. Interestingly, antimitochondrial Abs from all seropositive patients with PBC, but no controls, reacted against 3 of the 18 organic modified autoepitopes significantly better than to the native domain. By structural analysis, the features that correlated with autoantibody binding included synthetic domain peptides with a halide or methyl halide in the meta or para position containing no strong hydrogen bond accepting groups on the phenyl ring of the lysine substituents, and synthetic domain peptides with a relatively low rotation barrier about the linkage bond. Many chemicals including pharmaceuticals and household detergents have the potential to form such halogenated derivatives as metabolites. These data reflect the first time that an organic compound has been shown to serve as a mimeotope for an autoantigen and further provide evidence for a potential mechanism by which environmental organic compounds may cause PBC.

Efficacy of an aerosol surface spray against container-breeding Aedes.

The effectiveness of a residual insect surface spray against container-breeding mosquitoes was tested in Cairns, Australia. A formulation containing imiprothrin and cypermethrin (Mortein Plus Cockroach Lure 'n' Kill High Performance Surface Spray) was selected based on the label claim to "kill continuously for up to 6 months." A 1-sec spray was applied to the water and interior surface of partially flooded tires and terra-cotta pots. Treatments were paired with a control and replicated at 2 sites within 3 residential properties for a total of 6 replicates. All mosquito larvae were removed and counted weekly, and a representative sample was identified in the laboratory. Complete control of Aedes species was achieved for 4 and 5 months in all tires and pots, respectively.

Potent and selective nonpeptide inhibitors of caspases 3 and 7.

5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Bovine insulin and porcine or human insulin prime distinct CD4(+) T cell subsets in C57BL/6 mice.

H-2(b) mice produce insulin-specific antibody when injected with bovine but not porcine or human insulin. Nevertheless, CD4(+) T cells have been cloned from C57BL/6 mice primed with porcine, human, and bovine insulin. Here we tested the hypothesis that CD4(+) T cells from C57BL/6 mice primed with porcine or human insulin are functionally distinct from those primed with bovine insulin. Our results show that variants of insulin that stimulate antibody responses induced Th2 clones, whereas variants of insulin that fail to stimulate antibody induced Th0 clones. Th0 clones triggered delayed-type hypersensitivity (DTH) in adoptive recipients, whereas Th2 clones did not. Insulin variants that primed Th0 clones also directly primed for DTH responses, while variants that activated Th2 clones did not. Thus, induction of Th2 clones correlated with the ability of mice to make antibody responses to insulin while development of Th0 clones correlated with DTH responses and the failure to produce antibody.

The use of antioxidant nutrients to prevent and treat cancers of the aerodigestive tract.

Detection and prevention of oral cancer is an integral part of dental hygiene practice. Currently, much is known about the etiology and prevention of cancers of the aerodigestive tract. Poor nutrition is responsible for one-third of all cases. Antioxidant nutrients are assuming more importance in cancer research, including those of the aerodigestive tract. This review article will provide current information on antioxidant nutrients and their possible role as a chemopreventive agent in the development and treatment of oral cancer. In addition, recommendations will be made for incorporating nutrition education into dental hygiene practice.

Incorporating veterinary preventive dental instruction into a traditional dental hygiene program.

A traditional dental hygiene program at the University of Texas Health Science Center, San Antonio, (UTHSCSA) incorporates veterinary preventive dental instruction into its community dentistry course. Students in the Certificate in Dental Hygiene Program attend two hours of didactic instruction on veterinary dentistry during the fall semester of their final year. The following spring semester students may choose a rotation in a veterinary clinic as one of their optional alternative practice sites. In addition, students are given the opportunity to participate in community service activities that are structured around veterinary dental care.