Impacts of swine manure pits on groundwater quality.

Manure deep-pits are commonly used to store manure at confined animal feeding operations. However, previous to this study little information had been collected on the impacts of deep-pits on groundwater quality to provide science-based guidance in formulating regulations and waste management strategies that address risks to human health and the environment. Groundwater quality has been monitored since January 1999 at two hog finishing facilities in Illinois that use deep-pit systems for manure storage. Groundwater samples were collected on a monthly basis and analyzed for inorganic and bacteriological constituent concentrations. The two sites are located in areas with geologic environments representing different vulnerabilities for local groundwater contamination. One site is underlain by more than 6 m of clayey silt, and 7-36 m of shale. Concentrations of chloride, ammonium, phosphate, and potassium indicated that local groundwater quality had not been significantly impacted by pit leakage from this facility. Nitrate concentrations were elevated near the pit, often exceeding the 10 mg N/l drinking water standard. Isotopic nitrate signatures suggested that the nitrate was likely derived from soil organic matter and fertilizer applied to adjacent crop fields. At the other site, sandstone is located 4.6-6.1 m below land surface. Chloride concentrations and delta15N and delta15O values of dissolved nitrate indicated that this facility may have limited and localized impacts on groundwater. Other constituents, including ammonia, potassium, phosphate, and sodium were generally at or less than background concentrations. Trace- and heavy-metal concentrations in groundwater samples collected from both facilities were at concentrations less than drinking water standards. The concentration of inorganic constituents in the groundwater would not likely impact human health. Fecal streptococcus bacteria were detected at least once in groundwater from all monitoring wells at both sites. Fecal streptococcus was more common and at greater concentrations than fecal coliform. The microbiological data suggest that filtration of bacteria by soils may not be as effective as commonly assumed. The presence of fecal bacteria in the shallow groundwater may pose a significant threat to human health if the ground water is used for drinking. Both facilities are less than 4 years old and the short-term impacts of these manure storage facilities on groundwater quality have been limited. Continued monitoring of these facilities will determine if they have a long-term impact on groundwater resources.

Phenotypic and functional analysis of T-cell recovery after anti-CD3 immunotoxin treatment for tolerance induction in rhesus macaques.

T-cell reduction utilizing specific antibody has been widely used in human transplantation, and is a cornerstone of several tolerance induction strategies in nonhuman primates. We have established a population of long-term tolerant rhesus macaques induced with an anti-CD3epsilon immunotoxin (IT). This treatment effects transient, specific and profound ablation of T cells in blood and lymphoid tissues. In most instances the IT was used in combination with the NF-kappaB inhibitor, 15-Deoxyspergualin. This 2-week long protocol produces a "window of opportunity" for tolerization in which the animal exhibits an enduring quiescent state of unresponsiveness to the allograft, all accomplished without maintenance immunosuppressive drugs. During this induction period, the treated immune system bears some resemblance to that of the neonate, in that T cell numbers are abnormally low and antigen presentation by dendritic cells is precluded by an arrest in their NF-kappaB dependant maturation. In addition, IL-4 production is prominent during and after the tolerance induction interval. For this study we focused on measuring the monkey's ability to repopulate T cells with particular emphasis on the memory T-cell phenotype. Three "memory" phenotypes were utilized; CD3(+)CD45RO(+), CD3(+)CRTH2(+), and CD3(+)CD4(+)CD8(+). All three phenotypes exhibited different patterns of recovery, all of which included transient bursts in their numbers during repopulation. We also estimated thymic activity after T-cell ablation with the use of a newly-described RTE or recent thymic émigré phenotype (a naïve CD8(+)CD103(+) T cell). This marker revealed production of RTE cells including supranormal levels at approximately 6 months post-transplant, implicating thymic function in the repopulation of T-cells. Finally, we measured antibody responses to a panel of antigens (vaccines, environmental antigen, and foreign proteins) that indicated there was no apparent loss of immunologic function during or after the tolerance induction period. Results of studies of T-cell receptor repertoire expression suggest preservation of the pretreatment repertoire, which is consistent with rapid recovery of immune competence to the test antigens. Taken together, these results suggest that while aggressive, this tolerance induction protocol does not appear to incur a prolonged immunologically-compromised state, if at all.

Cytoprotection of pancreatic islets before and soon after transplantation by gene transfer of the anti-apoptotic Bcl-2 gene.

Isolated pancreatic islet transplantation is a promising alternative to conventional insulin-dependent diabetes treatment but is not yet a practical clinical therapy. In the first few days after pancreatic islet transplantation, substantial donor pancreatic islet dysfunction and apoptosis commonly occur. Islet apoptosis has been documented after extracellular matrix disruption and exposure to proinflammatory cytokines, and during hypoxia before islet revascularization and rejection. These studies show that targeting the apoptosis pathway by adenoviral-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective effect on isolated macaque pancreatic islets. Bcl-2 transfection ex vivo protects these islets from apoptosis induced by disruption of the islet extracellular matrix during pancreatic digestion. Additionally, overexpression of Bcl-2 confers long-term, stable protection and maintenance of functional islet mass after transplantation of macaque islets into diabetic severe combined immunodeficency mice. Notably, genetic modification of pancreatic islets also reduced the islet mass required to achieve stable euglycemia. Ex vivo gene transfer of anti-apoptotic genes has potential as a therapeutic approach to both minimize loss of functional islet mass after transplant and reduce the high donor islet requirement currently needed for successful stable reversal of insulin-dependent diabetes.

Successful reversal of streptozotocin-induced diabetes with stable allogeneic islet function in a preclinical model of type 1 diabetes.

The recent focus on islet transplantation as primary therapy for type 1 diabetes has heightened interest in the reversal of type 1 diabetes in preclinical models using minimal immunosuppression. Here, we demonstrated in a preclinical rhesus model a consistent reversal of all measured glycemic patterns of streptozotocin-induced type 1 diabetes. The model used single-donor islet transplantation with induction of operational tolerance. The term "operational tolerance" is used to indicate durable survival of single-donor major histocompatibility complex (MHC)-mismatched islet allografts without maintenance immunosuppressive therapy and without rejection or loss of functional islet mass or insulin secretory reserve. In this operational tolerance model, all immunosuppression was discontinued after day 14 posttransplant, and recipients recovered with excellent health. The operational tolerance induction protocol combined peritransplant anti-CD3 immunotoxin to deplete T-cells and 15-deoxyspergualin to arrest proinflammatory cytokine production and maturation of dendritic cells. T-cell deficiency was specific but temporary, in that T-cell-dependent responses in long-term survivors recovered to normal, and there was no evidence of increased susceptibility to infection. Anti-donor mixed lymphocyte reaction responses were positive in the long-term survivors, but all showed clear evidence of systemic T-helper 2 deviation, suggesting that an immunoregulatory rather than a deletional process underlies this operational tolerance model. This study provides the first evidence that operational tolerance can protect MHC nonhuman primate islets from rejection as well as loss of functional islet mass. Such an approach has potential to optimize individual recipient recovery from diabetes as well as permitting more widespread islet transplantation with the limited supply of donor islets.

Specific targeting of activated endothelium in rat adjuvant arthritis with a 99mTc-radiolabeled E-selectin-binding peptide.

OBJECTIVE: To determine the potential of an E-selectin-binding peptide (ESbp) to specifically bind activated endothelium in rheumatoid arthritis (RA) animal models. METHODS: ESbp (KYDGDITWDQLWDLMK; 2,027 daltons) was labeled with biotin and 99mTc. The affinity of ESbp derivatives for E-selectin was measured by enzyme-linked immunosorbent assay. The binding of biotin-ESbp was compared with that of an anti-E-selectin antibody, by immunohistochemical analyses of human synovial sections and sections from the Mycoplasma pulmonis MRL-lpr/lpr mouse arthritis model. 99mTc-ESbp was sequentially imaged in vivo with a gamma camera in the rat adjuvant-induced arthritis model. RESULTS: E-selectin expression was detected in human RA synovium and mouse arthritic synovium using biotin-ESbp. Both biotin-ESbp and 99mTc-labeled ESbp had high affinity for E-selectin (dissociation constant 2-5 nM). In vivo imaging showed specific binding of 99mTc-ESbp to the rat ankle joint prior to clinical manifestations of inflammation. CONCLUSION: These results demonstrate that activated endothelium can be targeted with 99mTc-ESbp. The specificity of targeting can be used to evaluate up-regulation of E-selectin in RA models, and to follow changes in this up-regulation during treatment trials.

Imaging and tissue biodistribution of 99mTc-labeled adenovirus knob (serotype 5).

Hepatic sequestration of systemically administered adenoviral vectors reduces the number of viral particles available for delivery to other tissues. The biological basis of this phenomenon was investigated using a new in vivo technique which permitted imaging in real time. Recombinant adenovirus serotype 5 knob (Ad5K) was radiolabeled with the gamma-emitter 99mTc (half-life = 6 h). Scatchard analysis of the 99mTc-Ad5K showed specific, high-affinity binding to U293 cells (Kd = 1.4 +/- 0.5 nM), demonstrating that the radiolabeling process had no effect on receptor binding. In vivo dynamic imaging with an Anger gamma camera revealed that the liver binding followed an exponential rise to maximum, with a measured 100% extraction efficiency. Initially, the liver binding capacity was 3.1 +/- 0.4 micrograms Ad5K, equivalent to approximately 17,000 Ad5K molecules per liver cell. Liver binding was blocked by preincubation of Ad5K with neutralizing anti-Ad5K antibody; a 50% reduction in liver uptake was demonstrated by imaging. Unlabeled Ad5K was more effective in blocking liver uptake of 99mTc-Ad5K, whereas irrelevant unlabeled Ad3K had no effect. Imaging data for the liver uptake studies were in agreement with biodistribution determined by removing and measuring tissues. These data demonstrated that in vivo imaging is a sensitive tool for measuring changes to liver tropism. Similar imaging techniques can be applied to adenovirus vectors to measure specific targeting for gene therapy.

REALIZED GENE FLOW VIA POLLEN IN ARTIFICIAL POPULATIONS OF MUSK THISTLE, CARDUUS NUTANS L.

Realized gene flow via pollen was measured in four adjacent, artificial populations of musk thistle, Carduus nutans L. by observing the distribution of electrophoretic markers at two allozyme loci. Realized gene movement declined exponentially with distance. Dispersal distances of the marker alleles averaged 5.0 m. Estimates of effective neighborhood sizes based on the movement of these genetic markers ranged from 126 to 378 individuals. When measures of seed movement were included, estimates of effective population sizes range from 1,281 to 3,844 individuals. It is, therefore, unlikely that chance effects play a major role in shaping the genetic structure of well-established musk thistle populations.