Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately 2-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers.

The majority of mild cognitive impairment (MCI) studies use baseline and one follow-up measurement to determine the clinical course of the disorder. This report of MCI clinical course is based on the a statistical evaluation of multiple neurocognitive tests over a 60 month period in elderly normal and MCI cohorts. The data includes serial informant-based measures (Clinical Dementia Rating [CDR]) and a comprehensive battery of neuropsychological tests analyzed by two different regression methods. Twenty-nine elderly participants entered the study as neurocognitively normal; 26 remained normal, 2 progressed to MCI, and 1 progressed to dementia. Eighty-three participants entered the study as multiple domain MCI cases; 10 became normal, 46 remained MCI, and 27 progressed to dementia. Three of the 27 demented died with full necropsies performed (one case was progressive supranuclear palsy and two confirmed Alzheimer's disease with severe cerebral amyloid angiopathy (CAA)). Without serial measures, 1 in 8 MCI could be misclassified as "stable MCI" despite reverting to normal. The stable MCI cohorts did not benefit from practice effects though the normal subjects did. Applying Classification and Regression Tree (CART) analysis enabled prediction of the endpoint status of participants from baseline values with 78.6% accuracy. The fluctuating cognitive status of the multiple domain MCI cases implies a remitting pathologic process with elements of recovery consistent with a progressive microvasculopathy such as CAA.

One of the remaining challenges in Alzheimer's disease (AD) research is the establishment of biomarkers for early disease detection. As part of a prospective study spanning a period of five years, we have collected serial serum samples from cognitively normal, mild cognitively impaired (MCI), and mild AD participants, including same patient samples before and after cognitive decline. Using mass spectrometry we identified several promising leads for biomarker development, such as prosaposin, phospholipase D1, biliverdin reductase B, and S100 calcium binding protein A7. Selected candidate markers were verified using reverse phase protein microarray assays. Of 15 protein/protein abundance ratios that were significantly altered in sera from subjects with mild AD compared to Normal or MCI subjects, 14 were composed of ratios containing heme oxygenase-1, biliverdin reductase A, or biliverdin reductase B. Moreover, an increase in the protein abundance ratio of matrix metallopeptidase 9/biliverdin reductase differentiated stable MCI subjects from MCI subjects progressing into mild AD before the onset of cognitive decline. These findings strongly implicate the heme degradation pathway as a promising source of protein biomarkers for the early detection of AD.

We have found with serial iron sensitive MRI (susceptibility weighted imaging - SWI) that a progressive increase in small brain microbleeds (BMB) correlates with a dementing course in elderly subjects. Our prospective study of sporadic late-onset dementia extended over a five and a half year period enabled the simultaneous detection of both BMB and regional iron levels. BMB are radiologic biomarkers for microvascular fragility. A community-based cohort of 103 participants; 75 mildly cognitively impaired (MCI) and 28 cognitively normal subjects have been studied for a five and a half year period with serial head SWI and serum proteomic tests. SWI is a high resolution, 3D gradient echo sequence that takes advantage of phase information present in every MR image to produce images of BMB. SWI is far more sensitive for BMB detection than the commonly used clinical sequence - gradient echo T2* (GE-T2*). There is a six-fold increase in number of BMB detectable by SWI at 1.5 T than GE-T2*. Changes in cognitive status monitored by comprehensive psychometric evaluations have been correlated with BMB, regional iron levels, serum proteomics and post-mortem brain studies. Counts of BMB when increasing and clustered are correlated with cognitive loss. BMB clustering, as well as radiologic-neuropathological studies of cadaver brains show progressive fragility of proximate small blood vessels. Serial proteomic studies have identified complementary serum biomarkers related to hemoglobin degradation. BMB once initiated activate a cascade of cellular and genetic events that by a positive feedback mechanism further weaken the vasculature, promote additional BMB, with neuronal death. Once BMB appearance has been observed, lesions then avalanche in a domino-effect with clustering and increasing cognitive loss. These observations lead to a conclusion that brain microvascular fragility is a major cause for Alzheimer's disease. This research was funded by NIH Grant #AG20948.

A new iron sensitive MR sequence (susceptibility weighted imaging - SWI) enabling the simultaneous quantitation of regional brain iron levels and brain microbleeds (BMB) has been acquired serially to study dementia. Cohorts of mildly cognitively impaired (MCI) elderly (n = 73) and cognitively normal participants (n = 33) have been serially evaluated for up to 50 months. SWI phase values (putative iron levels) in 14 brain regions were measured and the number of BMB were counted for each SWI study. SWI phase values showed a left putaminal mean increase of iron (decrease of phase values) over the study duration in 27 participants who progressed to dementia compared to Normals (p = 0.035) and stable MCI (p = 0.01). BMB were detected in 9 out of 26 (38%) MCI participants who progressed to dementia and are a significant risk factor for cognitive failure in MCI participants [risk ratio = 2.06 (95% confidence interval 1.37-3.12)]. SWI is useful to measure regional iron changes and presence of BMB, both of which may be important MR-based biomarkers for neurodegenerative diseases.

Although several therapies for Alzheimer's disease (AD) are currently in clinical trials, there are no biomarkers available to estimate the effectiveness of treatment. Moreover, AD must be diagnosed early to therapeutically prevent neurodegeneration, which is largely irreversible. Using peripheral blood serum as a diagnostic fluid has several advantages - it is much more accessible and can be collected from patients at different stages of the disease. We have studied serum samples from a community-based cohort of cognitively normal (control) and mildly cognitively impaired (MCI) subjects, of which several have progressed into mild AD. Using reverse phase protein arrays we have measured the abundance of selected protein targets in a low molecular weight (LMW) protein fraction of the serum (n = 10 each for control, MCI and mild AD). Furthermore, serum samples from the same person before and after significant cognitive decline (n = 6) have been investigated. In addition, selected whole serum and LMW serum samples have been analyzed using liquid chromatography coupled with tandem mass spectrometry to identify further candidate biomarker proteins for future studies. Protein ratios of oxidative stress related proteins, quantitatively measured by reverse phase protein microarray technology, showed a significant difference in mild AD participants or subjects after cognitive decline. Of the ratios observed to be significantly altered, 93% (14 of 15) involved either heme oxygenase 1, biliverdin reductase A or biliverdin reductase B. Using mass spectrometry we have identified several other functional protein groups that may be potential biomarkers for AD, such as metal/transition metal binding proteins, proteins with associated receptor activity/binding and proteins with lipid transporter activity. Several proteins have been identified in serum that could potentially serve as biomarkers for the early diagnosis of Alzheimer's disease. Proteins involved in heme degradation and oxidative stress, such as heme oxygenase 1 and biliverdin reductase A and B, were shown to be promising targets for future biomarker validation using a large and blinded study set. We conclude, that although the brain is the center of pathology for AD, peripheral blood is a promising source for AD biomarker development.

Although brain iron metabolism perturbations have been correlated with neurodegeneration its specific role in the pathogenesis of Alzheimer's disease (AD) remains to be investigated. Susceptibility weighted imaging (SWI) with its greater sensitivity and precision for brain iron estimation may help to establish a correlation of disease states with actual iron content in tissue. To validate this technique we have compared SWI signals to conventional T2 imaging, brain iron histochemistry and quantitative measurements of different iron pools extracted from discrete regions of the mouse brain after focused microwave irradiation (MWR). We have developed a dissection protocol based on MWR mouse brain that allowed us to isolate 12 distinct regions in each hemisphere (including hippocampus, nucleus accumbens and frontal, parietal and entorhinal cortex) for subsequent analysis of loosely–bound, non–heme bound, total iron and tissue weight. Measurement of these extracts was performed with graphite furnace atomic absorption spectrometry (SpectrAA 220Z, Varian). Nine iron regulatory protein 2 (IRP–2) knockout mice, known to accumulate higher iron levels in certain areas of the brain, have been compared to nine age and gender matched controls. SWI and T2 imaging was performed in a 11.7T MRI (Bruker Biospin, MA) followed by cardiac perfusion for iron histochemistry or MWR for brain iron extraction. DAB–enhanced Perl's stain was used for iron histochemistry and analyzed semi quantitatively by NIH–SCION. This is the first attempt to correlate quantitative iron measurements of different iron pools (loosely–bound, non–heme and total iron) with brain SWI and T2 signals. Our results have confirmed differences in iron content for distinct brain regions and will allow us to evaluate SWI as an investigative tool for the assessment of steady state iron in the brain non–invasively. Validation of SWI for brain iron measurement will have significant implications for the early radiologic diagnosis of neurodegeneration and AD associated with brain iron metabolism perturbations. This research was funded by NIH Grant #AG20948.

Recent studies indicate that chronic inflammation plays a pathogenic role in both the central nervous system (CNS) and periphery in Alzheimer’s disease (AD). We have screened for cytokines differentially produced by peripheral blood mononuclear cells (PBMCs) isolated from subjects with mild cognitive impairment (MCI) and mild AD subjects who had progressed from MCI using a commercially available cytokine array. Following determination of expressed cytokines, we quantified levels of the proinflammatory cytokines TNF-α, IL-6, and IL-8, and the anti-inflammatory cytokine IL-10 using flow cytometry. We have found a significant increase in the levels of IL-6, IL-8, and IL-10 produced by PBMCs stimulated for 24h with phytohemagglutinin (PHA) in MCI subjects compared to healthy elderly controls. However, in PBMCs stimulated for 48h with lipopolysaccharide (LPS), lower TNF-α/IL-10, IL-6/IL-10, and IL-8/IL-10 ratios were seen in MCI subjects. There were no differences in plasma levels of IL-8 between aged controls, MCI, and mild AD, and the levels of circulating IL-6 and IL-10 were below detection limits. Our data indicate that changes in cytokine production by PBMCs may be detected early in MCI, and an alteration of the immune response may precede clinical AD.

Recent studies indicate an early role for inflammation in the pathogenesis of Alzheimer's disease (AD). The inflammatory process in the AD brain may be expressed in circulating peripheral blood mononuclear cells and thus warrants study. We have screened for cytokines differentially produced by peripheral blood mononuclear cells (PBMCs) isolated from, healthy aged controls (n = 7), patients with mild cognitive impairment (MCI) (n = 4), and patients who had progressed to mild AD from MCI (PMCI) (n = 3) using a commercially available array of 20 cytokines involved in inflammation. Following determination of relevant cytokines, we quantified levels of the proinflammatory cytokines TNF–α, IL–10, IL–6, and IL–8 and the antiinflammatory cytokines IL–12p70 and IL–10 produced by unstimulated and stimulated (with PHA or LPS) PBMCs isolated from 22 healthy aged controls, 32 patients with MCI, and 8 patients with PMCI using flow cytometry. We have found a significant increase in the levels of the proinflammatory cytokines IL–6 and IL–8 and the antiinflammatory cytokine IL–10 produced by PBMCs stimulated for 24 h with PHA in MCI versus controls. This difference disappears after culturing for 48 hours. There were no significant differences in the production of cytokines when PBMCs were stimulated with LPS. Cytokine levels were below the limit of detection in unstimulated PBMCs cultured for 24 or 48 hours except for IL–8, and IL–8 levels did not differ between aged controls, MCI patients, and PMCI patients. Our data indicate that alterations in cytokine production by PBMCs may be detected early in MCI and that a shift toward the proinflammatory phenotype may precede the development of AD. Interestingly, the trend in proinflammatory cytokine production seems to peak in MCI patients and return to control or below control levels once they progress to AD. This is in agreement with findings that intrathecal inflammation precedes the development of AD which gives support to the hypothesis that inflammation is an initiating factor rather than a late consequence of the disease. This research was funded by NIH Grant #AG20948.

Correlations of cognitive test–retest score changes of elderly patients in dementia studies remains controversial. To determine associations between novel test–retest neuropsychological indicators of elderly controls and Mild Cognitive Impairment (MCI) participants (Petersen Mayo criteria). 1) Serial cognitive performance at 6–month to 1–year–intervals using standard neuropsychological and informant–based measures. 2) Practice effects on performance between the two groups. 3) Characterization of participants failing to benefit from practice effects. 4) Determining the effect of education on brain resiliency. 5) Correlations with blood and neuroimaging studies. Screening 1300 volunteers selected 183 individuals for testing with (comprehensive neuropsychological batteries) and videotaped Clinical Dementia Ratings (CDR). Twenty–eight controls and 76 MCI participants have been studied the past 36 months. Sixteen MCI participants have withdrawn; all controls remain. Serial MR (SWI, MP–RAGE, MRS, FLAIR, T1, T2) and blood studies (iron regulatory proteins, cytokines) are correlated with test–retest cognitive status. Control and MCI cohorts test significantly different for MMSE, Logical Memory raw score, CDR raw score, total sum of CDR boxes, Trail Making Test B, Word Fluency: Phonemic and Semantic, Boston Naming Test (short version), Wisconsin Card Sorting Perseverative Errors and Categories Complete, and Draw–A–Clock at baseline. Of the 76 MCI cases at baseline, 68 are multiple domain and 8 are single domain MCI amnestic. Subsequent tests performed at 6–months to 1–year after initial examination for 36 months revealed significant differences between control and MCI performance for the same set of tests. MR and blood studies form baseline values for further comparison. 1) Control participants performed significantly better than MCI participants on both sequential neuropsychological and informant–based measures. 2) Control participants have improved cognitive performance over time due to practice effects in semantic memory. 3) MCI participants did not benefit from practice effects. 4) Progressive MCI participants have low semantic memory scores, those who have not progressed show more variance. The cognitive decline of the progressive MCI participants became rapidly precipitous during the time frame of this study and can be correlated with MR and blood studies. This research was funded by NIH Grant #AG20948.

The role of increased brain iron in the pathogenesis of Alzheimer's Disease (AD) remains unsettled – is it causing oxidative stress or merely a bystander effort? Two groups of elderly participants – one cognitively intact and the other mildly cognitively impaired (MCI) – have been studied sequentially over the past 36 months by technologies to determine if altered brain iron metabolism constitutes a risk for progressive neurodegeneration and AD. 28 control and 76 MCI participants are studied by new minimally invasive magnetic resonance (MR) and ex vivo technologies to correlate changes in regional brain iron levels with detailed sequential psychometric evaluations. MR imaging includes susceptibility weighted imaging (SWI), magnet prepared rapid acquisition gradient echo (MP–RAGE), fluid attenuated inversion recovery (FLAIR), and MR spectroscopy (MRS). SWI does not currently have FDA approval, but is available to investigators with Institutional Review Board permission for experimental use. SWI has advantages over conventional MR sequences for tissue iron estimation because of greater sensitivity and precision. Regional brain iron levels determined by SWI are expressed in phase units ± standard deviation. Blood RNA, proteomic, and flow cytometric studies assay iron metabolism parameters. The control group has remained cognitively stable over a 36 month period whereas significant co–morbidity and cognitive decline have been noted in the MCI cohort. Fourteen MCI cases have become demented with confirmed sequential hippocampal volume loss. SWI imaging gives quantitative and sensitive phase measures of regional brain iron with apparent enhanced sensitivity for non–invasively identifying amyloid angiography and microhemorrhages (5 of 14 progressively dementing MCI cases). FLAIR enables estimation of white matter hyperdensities, indicative of small vessel disease. This study is the first application of brain SWI for documenting changes in regional brain iron content and microhemorrhages during progressive dementia. MRS–detected changes in cingulate gyrus metabolite levels associated with progressive dementias are consistent with previous reports. We conclude that a uniquely monitored cohort of MCI individuals at risk for progressive dementia yields valid statistical associations for predictor variables never before measured. This research was funded by NIH Grant #AG20948.

ObjectiveThere is an urgent and compelling need to reduce the morbidity and expense of maintaining hemodialysis vascular access patency. This large, long-term, retrospective, multicenter study, which compared access patency of autogenous arteriovenous fistulas (AVF) and synthetic bridge grafts (AVG) created with conventional sutures or nonpenetrating clips, was undertaken to resolve conflicting results from previous smaller studies.DesignPatency data for 1385 vascular access anastomoses (clipped or sutured) was obtained from 17 hospitals and dialysis centers (Appendix). Five hundred eighteen AVF (242 clip, 276 suture) and 827 AVG (440 clip, 384 suture) were analyzed. Statistical comparisons were made with Kaplan-Meier survival analysis, log-rank test, two-sample t test, and X2 test. The Cox proportional hazards model was used to confirm Kaplan-Meier analysis.ResultsAccess patency (primary, secondary, overall, and intention to treat) was significantly improved in access anastomoses constructed with clips. In the intention-to-treat group, primary patency at 24 months was 0.54 for clipped AVF and 0.34 for sutured AVF, and was 0.36 for clipped AVG and 0.17 for sutured AVG. At 24 months, primary patency rate for AVF successfully used for dialysis was 0.67 for clips and 0.48 for sutures, and for AVG was 0.39 for clips and 0.19 for sutured constructs. Interventions necessary to maintain patency were significantly fewer in clipped anastomoses.ConclusionReplacing conventional suture with clips significantly reduces morbidity associated with maintaining permanent hemodialysis vascular access. This beneficial effect may be due to the biologic superiority of interrupted, nonpenetrating vascular anastomoses.

The purpose of this study was to compare the effectiveness of the VCS® vascular clip approximating system for the creation of hemodialysis access fistulas. 173 new vascular accesses were created, 92 prosthetic grafts and 81 autologous fistulas over a 40-month period. 50 sutured and 42 clipped fistulas comprised the prosthetic graft series, and 33 sutured and 48 clipped cases the autologous series. Risk factors associated with access patency were correlated and the primary patencies compared in the suture versus clipped group. A significant improvement in primary patency was noted for the clipped prosthetic group at all time points studied, with a positive trend also noted for clipped autologous fistulae. Conclusions: The vascular clipping system (the VCS system) provides both long-term and immediate advantages for vascular access construction. Clips have been used successfully to treat seven consecutive patients with upper extremity arterial steal syndrome without the technical difficulties associated with traditional methods of repair.