Douglas E. Burger

X-ray fluorescence measurements of lead burden in subjects with low-level community lead exposure.

A k-x-ray fluorescence (K-XRF) instrument that can measure in vivo bone lead at low levels was used on a population of 34 adults with no known history of excessive lead exposure. A questionnaire that gathered information relevant to occupational and environmental lead exposure was administered prior to the measurement. A 30-min measurement that produced an average estimated uncertainty of 6 mcg lead/g bone mineral was taken at the mid-tibial diaphysis for each subject. Eighteen subjects had bone lead levels below the measurement uncertainty. The remainder had bone lead levels ranging up to 21 mcg lead/g bone mineral. Bone lead levels were greater among older subjects. Among young adult subjects, bone lead levels greater than the measurement uncertainty were confined entirely to subjects who had grown up in housing that was estimated to have been build prior to 1955. Such a childhood environment is at high risk of fostering exposure to biologically absorbable lead through ingestion of lead paint-contaminated dust and lead pipe-contaminated water. We conclude that the K-XRF technique has the potential to distinguish between low levels of lead burden in epidemiologic studies.

X-ray fluorescence: Issues surrounding the application of a new tool for measuring burden of lead

Studies of lead toxicity would greatly benefit from a convenient, noninvasive measure of body lead burden. X-ray fluorescence (XRF) promises to provide such a measure by rapidly estimating lead content in bone, the repository of greater than 95% of an adults lead stores (73% in children). Two separate XRF techniques exist, L-XRF and K-XRF. They differ in terms of calibration method and type of bone sampled. They also involve different radiation energies; however, radiation doses and concomitant risks are similarly low. Since interpretation of an XRF measurement depends to an extent on the distribution of lead in the skeleton, this topic is reviewed. Available data suggest that trabecular and cortical bone comprise two distinct compartments with regard to lead kinetics. Within each compartment, however, there also appears to be a significant degree of variability. Furthermore, there is evidence to suggest that the subperiosteal surface layer of cortical bone has a pattern of lead absorption and release that is different from the rest of cortical bone. This may have implications for (1) the choice of XRF technique, since the L-XRF technique measures lead in surface bone, whereas the K-XRF technique derives an estimate of lead from the full thickness of bone, and (2) the selection of bone sites for taking XRF measurements. More research is necessary to fully optimize the applicability of the XRF instrument.

Proof-of-Concept, Randomized, Controlled Clinical Trial of Bacillus-Calmette-Guerin for Treatment of Long-Term Type 1 Diabetes

Background No targeted immunotherapies reverse type 1 diabetes in humans. However, in a rodent model of type 1 diabetes, Bacillus Calmette-Guerin (BCG) reverses disease by restoring insulin secretion. Specifically, it stimulates innate immunity by inducing the host to produce tumor necrosis factor (TNF), which, in turn, kills disease-causing autoimmune cells and restores pancreatic beta-cell function through regeneration. Methodology/Principal Findings Translating these findings to humans, we administered BCG, a generic vaccine, in a proof-of-principle, double-blind, placebo-controlled trial of adults with long-term type 1 diabetes (mean: 15.3 years) at one clinical center in North America. Six subjects were randomly assigned to BCG or placebo and compared to self, healthy paired controls (n = 6) or reference subjects with (n = 57) or without (n = 16) type 1 diabetes, depending upon the outcome measure. We monitored weekly blood samples for 20 weeks for insulin-autoreactive T cells, regulatory T cells (Tregs), glutamic acid decarboxylase (GAD) and other autoantibodies, and C-peptide, a marker of insulin secretion. BCG-treated patients and one placebo-treated patient who, after enrollment, unexpectedly developed acute Epstein-Barr virus infection, a known TNF inducer, exclusively showed increases in dead insulin-autoreactive T cells and induction of Tregs. C-peptide levels (pmol/L) significantly rose transiently in two BCG-treated subjects (means: 3.49 pmol/L [95% CI 2.95–3.8], 2.57 [95% CI 1.65–3.49]) and the EBV-infected subject (3.16 [95% CI 2.54–3.69]) vs.1.65 [95% CI 1.55–3.2] in reference diabetic subjects. BCG-treated subjects each had more than 50% of their C-peptide values above the 95th percentile of the reference subjects. The EBV-infected subject had 18% of C-peptide values above this level. Conclusions/Significance We conclude that BCG treatment or EBV infection transiently modified the autoimmunity that underlies type 1 diabetes by stimulating the host innate immune response. This suggests that BCG or other stimulators of host innate immunity may have value in the treatment of long-term diabetes. Trial Registration ClinicalTrials.gov NCT00607230

Low levels of C-peptide have clinical significance for established Type 1 diabetes.

To determine whether the low C‐peptide levels (< 50 pmol/l) produced by the pancreas for decades after onset of Type 1 diabetes have clinical significance.

Fetal Hox11 expression patterns predict defective target organs: a novel link between developmental biology and autoimmunity.

Developmental biology has long been ignored in the etiology and diverse manifestations of autoimmune diseases. Yet a role for development is suggested by intriguing overlaps in particular organs targeted in autoimmune diseases, in this case type 1 diabetes and Sjogrens syndrome. Patients with type 1 diabetes have high rates of co‐occurring Sjogrens syndrome, and both conditions are associated with hearing loss and tongue abnormalities. All of these co‐occurrences are found in organs tracing their lineage to the developmental transcription factor Hox11, which is expressed in embryonic cells destined for the pancreas, salivary glands, tongue, cranial nerves and cochlea. To determine whether development contributes to autoimmunity, we compared four target organs in NOD mice (an animal model for type 1 diabetes and Sjogrens syndrome) with NOD‐SCID mice (which lack lymphocytes) and normal controls. We examined the structure and/or function of the cochlea, salivary glands, pancreas and tongue at early time points after birth. Before the usual time of the onset of type 1 diabetes or Sjogrens syndrome, we show that all four Hox11‐derived organs are structurally abnormal in both NOD mice and NOD‐SCID mice versus controls. The most striking functional defect is near complete hearing loss occurring before the normal time of the onset of autoimmunity. The hearing loss is associated with severe structural defects in the cochlea, suggesting that near‐deafness occurs independent of autoimmune attack. The pancreas and salivary glands are also structurally abnormal in NOD and NOD‐SCID mice, but they are functionally normal. This suggests that autoimmune attack of these two organs is required for functional failure. We conclude that a developmental lineage of cells contributes to autoimmunity and predicts which organs may be targeted, either structurally and/or functionally. Taken together, our findings challenge the orthodoxy that autoimmunity is solely caused by a defective immune system.

Novel Automated Blood Separations Validate Whole Cell Biomarkers

Background Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples. Methods and Findings To replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficolls uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes. Conclusions Improving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials.

Possible Transient Benefits of Epstein Barr Virus Infection in Three Subjects with Established Type 1 Diabetes

Tumor necrosis factor (TNF) is a novel immunotherapy for type I diabetes because it selectively kills insulin autoreactive T-cells, which enables recovery of insulin production by pancreatic islet cells. The TNF inducer Bacillus Calmette-Guerin (BCG) also has therapeutic value by activating innate immunity that beneficially modifies the course of type 1 diabetes (T1D) in the same manner as TNF. Epstein Barr Virus (EBV) infection is also an inducer of TNF. This observational study was undertaken to determine whether EBV has the same beneficial effects as BCG in a recent clinical trial. We describe three cases of long-term diabetic subjects with active mononucleosis that were followed for 15 weeks to determine the impact of EBV infection on established T1D. In comparison to non-EBVinfected long-term diabetics, EBV transiently diminished the autoimmune response in two of three cases. EBV infection triggered rapid increase of circulating insulin-B autoreactive T-cells whose striking loss of CD8 marker indicated that the cells were injured or apoptotic. EBV infection also caused a transient surge in the secretion of C-peptide, a marker for pancreatic insulin secretion. EBV acts like BCG in eliciting innate immunity and beneficially modifying the course of TID.