DNA and a CpG oligonucleotide derived from Babesia bovis are mitogenic for bovine B cells

DNAs from bacteria and variety of nonvertebrate organisms, including nematodes, mollusks, yeasts, and insects, cause polyclonal activation of murine B lymphocytes. Similar studies have not been reported for bovine B cells, and to date no studies have reported mitogenic properties of protozoal DNA for any species. However, we and others have observed that protozoal parasite antigens can induce the proliferation of lymphocytes from nonexposed donors. Extending these studies, we now show that the mitogenic property of protozoal antigen preparations is in part attributable to parasite DNA and that Babesia bovis DNA is directly mitogenic for bovine B cells. DNase treatment of B. bovis extracts abrogated B. bovis-induced proliferation of peripheral blood mononuclear cells from nonexposed cattle. Like DNAs from other organisms that were mitogenic for murine B cells, B. bovis DNA is largely nonmethylated and induced a dose-dependent proliferation of bovine B cells, which was reduced upon methylation. Furthermore, B. bovis and E. coli DNAs enhanced immunoglobulin secretion by cultured B cells, inducing moderate increases in immunoglobulin G1 and stronger increases in immunoglobulin G2. Because certain nonmethylated CpG motifs present in bacterial DNA are known to stimulate proliferation of murine and human B cells, an 11-kb fragment of B. bovis DNA was analyzed for CG dinucleotide content and for the presence of known immunostimulatory sequences (ISS) centered on a CG motif. The frequency of CG dinucleotides was approximately one-half of the expected frequency, and several CpG hexameric sequences with known activity for murine B cells were identified. An oligodeoxynucleotide containing one of these ISS (AACGTT), which is present within the rhoptry-associated protein-1 (rap-1) open reading frame, was shown to stimulate B-cell proliferation. These ISS may be involved in host immune modulation during protozoal infection and may be useful as vaccine adjuvants.     

Renal tolerability profile of novel, potent bisphosphonates in two short-term rat models

Bisphosphonates are used clinically to inhibit bone resorption but they may also cause renal damage. For the profiling of new potent bisphosphonates, their adverse renal effects were investigated in 2 rat models. In the first model, bisphosphonate was repeatedly injected (1 mg/kg, subcutaneously) over 2 weeks and the urinary excretion of malate dehydrogenase was monitored to assess nephrotoxic potential. Of the 6 new compounds tested, 3 markedly elevated malate dehydrogenase whereas 3 others caused only minor changes similar to those observed with 6 reference bisphosphonates that are already used clinically. On the basis of a therapeutic index (inhibition of bone resorption versus renal effects) 7-180 fold greater than that of other analogues, the compound CGP 42446 was further profiled. In the second model, CGP 42446 or pamidronate was infused (1.5-50 mg/kg, intravenously) into anaesthetized rats and the serum urea concentration was monitored as an indicator of renal dysfunction. Both compounds elevated serum urea in a time- and dose-dependent manner, but the ED100 value for CGP 42446 was 3.8-fold higher than that of pamidronate. It is concluded that CGP 42446 (zoledronate) has a low nephrotoxic potential and can be further developed as a new potent inhibitor of bone resorption.     

V beta-specific immunotoxin selectively kills acetylcholine receptor-reactive T lymphocytes from mice with experimental autoimmune myasthenia gravis

Immunization of C57BL/6 mice with purified acetylcholine receptor (AChR) is known to induce a T cell-dependent antibody response that results in experimental autoimmune myasthenia gravis (EAMG). Since past observations link V beta 6+ T cells with a prominent AChR epitope specificity, a V beta 6-specific immunotoxin (VIT6) was tested in vitro for its ability to selectively kill monoclonal and polyclonal T cells that demonstrate reactivity against AChR. Results described below clearly demonstrate the ability to selectively kill AChR-reactive T cells based on their expression of a particular V beta-associated antigen receptor.     

Validation of [13C]urea breath test for Helicobacter pylori using a simple gas chromatograph-mass selective detector

OBJECTIVE: Isotope ratio mass spectrometry (IRMS) is the accepted method for accurately measuring the 13CO2:12CO2 ratio in the non-invasive and non-radioactive [13C]urea breath test (13C-UBT) for Helicobactor pylori. The IRMS instrument, an expensive and highly specialized analyser, is rarely available. The objective of this project was to modify and validate the use of a simple bench-top gas chromatograph-mass selective detector (GC-MSD) for 13C-UBT. METHODS: Breath samples from 71 patients were taken at baseline and 30 min after ingestion of 100 mg [13C]urea. The breath samples were analysed using GC-MSD in the selected ion monitoring mode. The reference 13CO2:12CO2 ratio was from NBS19 obtained from the US National Institute of Standards and Technology. 13CO2:12CO2 ratios of the breath samples were determined. Excess delta per thousand (per mil, delta/thousand) of the 30 min sample over the baseline (deltadelta/thousand) of > or = 6deltadelta/thousand was considered H. pylori positive. Results from 13C-UBT and histology determined blind to each other were compared. RESULTS: The coefficient of variation of the reference 13CO2:12CO2 ratio was 0.06%. Using histology as the 'gold standard', the sensitivity (97.9%) and specificity (95.8%) of the GC-MSD 13C-UBT were comparable to those of other methods of H. pylori diagnosis. CONCLUSION: A gas chromatograph coupled to a mass selective detector that is available in many analytical and biomedical laboratories can be used for the 13C-UBT. This method will increase the availability and reduce the cost of this non-invasive, non-radioactive diagnostic test.     

Multiple risk factors in the development of externalizing behavior problems: group and individual differences

The aim of this study was to test whether individual risk factors as well as the number of risk factors (cumulative risk) predicted children's externalizing behaviors over middle childhood. A sample of 466 European American and 100 African American boys and girls from a broad range of socioeconomic levels was followed from age 5 to 10 years. Twenty risk variables from four domains (child, sociocultural, parenting, and peer-related) were measured using in-home interviews at the beginning of the study, and annual assessments of externalizing behaviors were conducted. Consistent with past research, individual differences in externalizing behavior problems were stable over time and were related to individual risk factors as well as the number of risk factors present. Particular risks accounted for 36% to 45% of the variance, and the number of risks present (cumulative risk status) accounted for 19% to 32% of the variance, in externalizing outcomes. Cumulative risk was related to subsequent externalizing even after initial levels of externalizing had been statistically controlled. All four domains of risk variables made significant unique contributions to this statistical prediction, and there were multiple clusters of risks that led to similar outcomes. There was also evidence that this prediction was moderated by ethnic group status, most of the prediction of externalizing being found for European American children. However, this moderation effect varied depending on the predictor and outcome variables included in the model.