Claudius Mueller

Correlation of hypointensities in susceptibility-weighted images to tissue histology in dementia patients with cerebral amyloid angiopathy: a postmortem MRI study

Neuroimaging with iron-sensitive MR sequences [gradient echo T2* and susceptibility-weighted imaging (SWI)] identifies small signal voids that are suspected brain microbleeds. Though the clinical significance of these lesions remains uncertain, their distribution and prevalence correlates with cerebral amyloid angiopathy (CAA), hypertension, smoking, and cognitive deficits. Investigation of the pathologies that produce signal voids is necessary to properly interpret these imaging findings. We conducted a systematic correlation of SWI-identified hypointensities to tissue pathology in postmortem brains with Alzheimer’s disease (AD) and varying degrees of CAA. Autopsied brains from eight AD patients, six of which showed advanced CAA, were imaged at 3T; foci corresponding to hypointensities were identified and studied histologically. A variety of lesions was detected; the most common lesions were acute microhemorrhage, hemosiderin residua of old hemorrhages, and small lacunes ringed by hemosiderin. In lesions where the bleeding vessel could be identified, β-amyloid immunohistochemistry confirmed the presence of β-amyloid in the vessel wall. Significant cellular apoptosis was noted in the perifocal region of recent bleeds along with heme oxygenase 1 activity and late complement activation. Acutely extravasated blood and hemosiderin were noted to migrate through enlarged Virchow–Robin spaces propagating an inflammatory reaction along the local microvasculature; a mechanism that may contribute to the formation of lacunar infarcts. Correlation of imaging findings to tissue pathology in our cases indicates that a variety of CAA-related pathologies produce MR-identified signal voids and further supports the use of SWI as a biomarker for this disease.

Iron, zinc and copper in the Alzheimer’s disease brain: a quantitative meta-analysis. Some insight on the influence of citation bias on scientific opinion

Dysfunctional homeostasis of transition metals is believed to play a role in the pathogenesis of Alzheimers disease (AD). Although questioned by some, brain copper, zinc, and particularly iron overload are widely accepted features of AD which have led to the hypothesis that oxidative stress generated from aberrant homeostasis of these transition metals might be a pathogenic mechanism behind AD. This meta-analysis compiled and critically assessed available quantitative data on brain iron, zinc and copper levels in AD patients compared to aged controls. The results were very heterogeneous. A series of heavily cited articles from one laboratory reported a large increase in iron in AD neocortex compared to age-matched controls (p<0.0001) while seven laboratories failed to reproduce these findings reporting no significant difference between the groups (p=0.76). A more than three-fold citation bias was found to favor outlier studies reporting increases in iron and this bias was particularly prominent among narrative review articles. Additionally, while zinc was not significantly changed in the neocortex (p=0.29), copper was significantly depleted in AD (p=0.0003). In light of these findings, it will be important to re-evaluate the hypothesis that transition metal overload accounts for oxidative injury noted in AD.

Reverse phase protein microarrays advance to use in clinical trials

Individualizing cancer therapy for molecular targeted inhibitors requires a new class of molecular profiling technology that can map the functional state of the cancer cell signal pathways containing the drug targets. Reverse phase protein microarrays (RPMA) are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and non‐phosphorylated) forms of cellular proteins from a limited amount of sample. This class of microarray can be used to interrogate tissue samples, cells, serum, or body fluids. RPMA were previously a research tool; now this technology has graduated to use in research clinical trials with clinical grade sensitivity and precision. In this review we describe the application of RPMA for multiplexed signal pathway analysis in therapeutic monitoring, biomarker discovery, and evaluation of pharmaceutical targets, and conclude with a summary of the technical aspects of RPMA construction and analysis.

Serial susceptibility weighted MRI measures brain iron and microbleeds in dementia.

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.

Increased production of inflammatory cytokines in mild cognitive impairment.

Recent studies indicate that chronic inflammation plays a pathogenic role in both the central nervous system (CNS) and periphery in Alzheimers 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-alpha, 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 24 h with phytohemagglutinin (PHA) in MCI subjects compared to healthy elderly controls. However, in PBMCs stimulated for 48 h with lipopolysaccharide (LPS), lower TNF-alpha/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.

Malignant precursor cells pre-exist in human breast DCIS and require autophagy for survival.

Background While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment. Methodology and Principal Findings We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patients breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patients original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation. Conclusions Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.

Tissue is alive: New technologies are needed to address the problems of protein biomarker pre-analytical variability.

Instability of tissue protein biomarkers is a critical issue for molecular profiling. Pre‐analytical variables during tissue procurement, such as time delays during which the tissue remains stored at room temperature, can cause significant variability and bias in downstream molecular analysis. Living tissue, ex vivo, goes through a defined stage of reactive changes that begin with oxidative, hypoxic and metabolic stress, and culminate in apoptosis. Depending on the delay time ex vivo, and reactive stage, protein biomarkers, such as signal pathway phosphoproteins will be elevated or suppressed in a manner which does not represent the biomarker levels at the time of excision. Proteomic data documenting reactive tissue protein changes post collection indicate the need to recognize and address tissue stability, preservation of post‐translational modifications, and preservation of morphologic features for molecular analysis. Based on the analysis of phosphoproteins, one of the most labile tissue protein biomarkers, we set forth tissue procurement guidelines for clinical research. We propose technical solutions for (i) assessing the state of protein analyte preservation and specimen quality via identification of a panel of natural proteins (surrogate stability markers), and (ii) using multi‐purpose fixative solution designed to stabilize, preserve and maintain proteins, nucleic acids, and tissue architecture.

One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens.

Background There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block. Results Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79. Conclusion In a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research.

Iron, copper, and iron regulatory protein 2 in Alzheimer’s disease and related dementias

Accumulating evidence implicates a role for altered iron and copper metabolism in the pathogenesis of neurodegenerative disorders such as Alzheimers disease (AD). However, imbalances in the levels of the various forms of iron at different stages of AD have not been examined. In this pilot study we extracted and measured the levels of loosely bound, non-heme and total iron and copper in the frontal cortex and hippocampus of patients with mild-moderate AD (n=3), severe AD (n=8) and dementia with Lewy bodies (DLB, n=6), using graphite furnace atomic absorption spectrometry (GFAAS). Additionally, the expression of iron regulatory protein 2 (IRP2) was examined in relation to the pathological hallmarks of AD and DLB, amyloid plaques, neurofibrillary tangles (NFT), and Lewy bodies, by immunohistochemistry. We found significantly decreased loosely bound iron in the hippocampal white matter of mild-moderate and severe AD patients and a trend towards increased non-heme iron in the hippocampal gray matter of severe AD patients. Furthermore, decreased levels of total copper were seen in severe AD and DLB frontal cortex compared to controls, suggesting an imbalance in brain metal levels in both AD and DLB. The decrease in loosely bound iron in mild-moderate AD patients may be associated with myelin breakdown seen in the beginning stages of AD and implicates that iron dysregulation is an early event in AD pathogenesis.

The Heme Degradation Pathway is a Promising Serum Biomarker Source for the Early Detection of Alzheimer's Disease

One of the remaining challenges in Alzheimers 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.