Dementia screening, biomarkers and protein misfolding: Implications for public health and diagnosis

Misfolded proteins are at the core of many neurodegenerative diseases, nearly all of them associated with cognitive impairment. For example, Creutzfeldt-Jacob disease is associated with aggregation of prion protein,^1,2 Lewy body dementia and Parkinson disease with α-synuclein^3,4 and forms of frontotemporal dementia with tau, TDP43 and a host of other proteins.^5,6 Alzheimer disease (AD), the most common cause of dementia,⁷ and its prodromal syndrome mild cognitive impairment (MCI)⁸ are an increasing public health problem and a diagnostic challenge to many clinicians. AD is characterized pathologically by the accumulation of amyloid β-protein (Aβ)^9,10 as senile plaques and in the walls of blood vessels as amyloid angiopathy.^11,12 Additionally, there are accumulations of tau-protein as neurofibrillary tangles and dystrophic neurites.^11,12 Biological markers of AD and MCI can serve as in vivo diagnostic indicators of underlying pathology, particularly when clinical symptoms are mild^13–15 and are likely present years before the onset of clinical symptoms.^16–19 Research to discover and refine fluid and imaging biomarkers of protein aggregation has undergone a rapid evolution^20–22 and combined analysis of different modalities may further increase diagnostic sensitivity and specificity.^23–26 Multi-center trials are now investigating whether imaging and/or cerebrospinal fluid (CSF) biomarker candidates can be used as outcome measures for use in phase III clinical trials for AD.^27–29Key words: dementia, screening, biomarkers, amyloid, tau, Alzheimer disease, preclinical, presymptomaticCurrently, the diagnosis of AD is based on exclusion of other forms of impairment with definitive diagnosis requiring autopsy confirmation.³⁰ Thus, there is a strong need to find easily measurable in vivo AD biomarkers that could facilitate early and accurate diagnosis³¹ as well as prognostic data to assist in monitoring therapeutic efficacy.³² Although biological markers such as MRI, PET scans and CSF increase the diagnostic likelihood that AD is present,^{9,18–20,33,34} biomarkers are invasive, uncomfortable, expensive and may not be readily available to rural areas, underserved communities, underinsured individuals or developing countries, making them impractical for broad use. However, the lessons learned from biomarkers can be applied to increase the likelihood that clinicians will be able to detect disease at earlier stages in the form of dementia screening.Public health may be best defined as the organized efforts of society to improve health, often framed in terms of primary, secondary and tertiary prevention. Prevention encompasses an understanding of causation, alteration of natural history of disease and understanding of pathophysiological mechanisms.³⁵ The clearest application of this from a public health perspective is in the setting of secondary prevention (i.e., screening)—early detection as a core element, coupled with treatments or preventative actions to reduce the burden of disease.³⁵ In this instance we seek to identify individuals in whom a disease has already begun and who may be experiencing very mild clinical symptoms but have not yet sought out medical care. The objective of effective screening is to detect the disease earlier than it would have been detected with usual care. Recent healthcare reform (Accountable Care Act)³⁶ proposes a Personalized Prevention Plan including screening for cognitive disorders, reimbursable through Medicare. Thus tying knowledge about dementia screening with underlying biology of protein misfolding associated with neurodegenerative disease can have enormous implications.A review of the natural history of dementia illustrates this point (Fig. 1). The timeline of disease from presumptive start to the patient demise is plotted. Stage I marks the biologic onset of disease; however this point often cannot be identified and may begin years to decades before any evidence is apparent (represented by dashed lines). As this stage is subclinical, it is difficult to study in humans but lends itself nicely to animal models. At some point in the progression of the biology, stage II begins heralding the first pathologic evidence of disease could be obtained—in the case of AD this could include CSF measurements of amyloid and tau^22,26,27 or PET imaging with amyloid ligands.^18,37 Subsequently, the first signs and symptoms of disease develop (stage III). Till this point, the disease process has been entirely presymptomatic. Beginning with the onset of symptoms, the patient may seek medical care (stage IV) and eventually be diagnosed (stage V). From stage III onwards, the patient enters the symptomatic phase of disease. From this point, the patient is typically treated with various pharmacologic and nonpharmacologic approaches towards some outcome. Another way to envision the disease spectrum is from the biological onset to the seeking of medical attention as the preclinical phase of disease with the clinical phase beginning with the initial clinical investigations into the cause of the patients'' symptoms.Open in a separate windowFigure 1Model of the natural history of AD. Timeline from presumptive start of AD through patient diagnosis is plotted. The initiation of biological changes (stage I) marks the onset of disease and begins years to decades before any evidence is apparent (represented by dashed lines). At some point the first pathologic evidence of disease (stage II) begins and in theory can be detected with biomarkers such as CSF measurements of amyloid and tau or PET imaging with amyloid ligands. Subsequently, the first signs and symptoms of disease develop (stage III) followed by the patient seeking medical attention (stage IV) and finally a diagnosis is established (stage V). This timeline can be clustered into a presymptomatic phase (stages I–III) and a symptomatic phase (stages III–V). An alternative way to envision the disease spectrum is from the biological onset to the seeking of medical attention (stages I–IV) as the preclinical phase of disease with the clinical phase beginning with the initial clinical investigations into the cause of the patients'' symptoms (stages IV and V). Stage III is the ideal time for dementia screening.What is the value of thinking about disease in this fashion? Such models allow researchers and clinicians to model the approach to finding and applying new diagnostics and offering new interventions. From stage I to stage III, the patient is the presymptomatic, preclinical phase of disease. The only means of detection would be with a biological marker that reflected protein misfolding or some proxy marker of these events. Although longitudinal evidence of cognitive change exist from 1–3 years before clinical diagnosis, raw scores on neuropsychological testing during this time remains in the normal range.³⁸ After stage IV, the patient is in the symptomatic, clinical phase of disease. Testing here is centered on confirming the suspected diagnosis, correctly staging the disease and initiating the appropriate therapies. Basic scientific approaches focusing on the presymptomatic, preclinical phase and clinical care approaches focusing on the symptomatic, clinical phase are well established and will continue to benefit from additional research.However, if we focus only on these two phases, an opportunity will be missed to make a decidedly important impact in the patient''s well-being. From stage III to stage IV, the patient enters symptomatic, preclinical phase of disease; symptomatic because the patient or family is beginning to detect some aspect of change, but preclinical because these signs and symptoms have not yet been brought to medical attention. In the case of AD (and the other forms of dementia) this period may go for an extended length of time as patients, families and clinicians dismiss early cognitive symptoms as part of the normal aging process. Thus, the rationale for screening is that if we can identify disease earlier in its natural history than would ordinarily occur, intervention measures (those currently available and those that are being developed) would be more effective. Dementia screening therefore would be best suited to detect cognitive impairment at the beginning of disease signs (stage III), particularly if these screening measures reflect what is known about the symptomatic, clinical phase of disease and correlate with the pathologic changes occurring in the brain during the pre-symptomatic, preclinical phase of disease.In a recent paper, we evaluated the relationship between several dementia screening tests and biomarkers of AD.⁴⁰ We tested whether a reliable and validated informant-based dementia screening test (the AD8)^41,42 correlates with changes in AD biomarkers and, if positive, screening with the AD8 clinically supports an AD clinical phenotype, superior to a commonly used performance-based screening tests including the Mini Mental State Exam (MMSE)⁴³ and the Short Blessed Test (SBT).⁴⁴ A total of 257 participants were evaluated, administered a comprehensive clinical and cognitive evaluation with the Clinical Dementia Rating scale (CDR)⁴⁵ used as the gold standard. Participants consented to and completed a variety of biomarker studies including MRI, amyloid imaging using the Pittsburgh Compound B (PiB)^37,46 and CSF studies of Aβ₄₂, tau and phosphorylated tau at Serine 181 (p-tau₁₈₁).^23,24 The sample had a mean age of 75.4 ± 7.3 years with 15.1 ± 3.2 years of education. The sample was 88.7% Caucasian and 45.5% male with a mean MMSE score of 27.2 ± 3.6. The formal diagnoses of the sample was 156 CDR 0 cognitively normal, 23 CDR 0.5 MCI, 53 CDR 0.5 very mild AD and 25 CDR 1 mild AD. Participants with positive AD8 scores (graded as a score of 2 or greater) exhibited the typical AD fluid biomarker phenotype characterized by significantly lower mean levels of CSF Aβ₄₂, greater CSF tau, p-tau₁₈₁ and the tau(s)/Aβ₄₂ ratios.^26,27 They also exhibited smaller temporal lobe volumes and increased mean cortical binding potential (MCBP) for PiB imaging similar to studies of individuals with AD.^18,19 These findings support that informant-based assessments may be superior to performance-based screening measures such as the MMSE or SBT in corresponding to underlying AD pathology, particularly at the earliest stages of decline. The use of a brief test such as the AD8 may improve strategies for detecting dementia in community settings where biomarkers may not be readily available and also may enrich clinical trial recruitment by increasing the likelihood that participants have underlying biomarker abnormalities.⁴⁰To gain a better understanding of changes in biomarkers in the symptomatic, preclinical phase, a post hoc evaluation of the 156 individuals who were rated as CDR 0 no dementia at the time of their Gold Standard assessment was completed. Some of these nondemented individuals have abnormal AD biomarkers, but in the absence of performing lumbar punctures or PET scans, is it possible to detect evidence of change? AD8 scores for 132 individuals were less than 2; thus their screening test suggests no impairment (mean AD8 score = 0.30 ± 0.46). However 25 of these individuals had AD8 scores (≥2) suggesting impairment (mean AD8 score = 2.4 ± 0.91). Applying the model described in Figure 1, some of these individuals are hypothesized to be in the symptomatic, preclinical phase of disease. No difference in age, education, gender or brief performance tests (MMSE or SBT) were detected between groups (45 is increased in the individuals with higher AD8 scores supporting that informants were noticing and reporting changes in the participants cognitive function. A review of the individual AD8 questions that were first reported to change suggest that informants endorsement of subtle changes in memory (repeats questions, forgets appointments) and executive ability (trouble with judgment, appliances, finances) are valuable early signs. This is consistent with previous reports that changes in memory and judgment/problem solving CDR boxscores in nondemented individuals correlate with findings of AD pathology at autopsy.¹⁷ Although biomarkers do not reach significance in this small sample, the direction of change in favor of “Alzheimerization” of this group suggests that some of these individuals may be in the symptomatic, preclinical phase of disease. More research with larger sample sizes and longitudinal follow-up is needed to confirm this hypothesis. It should be also noted that not all individuals with an AD8 score of 2 or greater have AD. The AD8 was designed to detect cognitive impairment from all causes, and as such, these mildly affected individuals may have other causes for their cognitive change such as depression, Lewy body dementia or vascular cognitive impairment.^41,42

Table 1

Characteristics of nondemented CDR 0 individuals stratified by AD8 scores

An approach to NMR studies of the metabolism of internal organs using surface coils

This communication describes a surgical preparation of experimental animals to permit NMR spectroscopic studies of the metabolism of internal organs. In the procedure developed, the layer of protective muscle directly above the organ is removed, but the skin is left intact. NMR studies of the metabolism of the organ can then be carried out using surface coils placed externally over the herniated area. Modified probe and stack designs for use with the surgically modified animals in a conventional NMR spectrometer are described. Phosphorus-31 NMR spectra of liver and kidney of the modified animals have been obtained, and data corresponding to the hepatic response to a load of fructose are presented.     

Reconstitution of rabbit thrombomodulin into phospholipid vesicles

The influence of phospholipid on thrombin-thrombomodulin-catalyzed activation of protein C has been studied by incorporating thrombomodulin into vesicles by dialysis from octyl glucoside-phospholipid mixtures. Thrombomodulin was incorporated into vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (30% phosphatidylserine and 70% phosphatidylcholine). Thrombomodulin is randomly oriented in vesicles of different phospholipid composition. Incorporation of thrombomodulin into phosphatidylcholine, with or without phosphatidylserine, alters the Ca2+ concentration dependence of protein C activation. Soluble thrombomodulin showed a half-maximal rate of activation at 580 microM Ca2+, whereas half-maximal rates of activation of liposome-reconstituted thrombomodulin were obtained between 500 microM Ca2+ and 2 mM Ca2+, depending on the composition (protein:phospholipid) of the liposomes. The Ca2+ dependence of protein C activation fits a simple hyperbola for the soluble activator, while the Ca2+ dependence of the membrane-associated complex is distinctly sigmoidal with a Hill coefficient greater than 2.4. In contrast, the Ca2+ dependence of gamma-carboxyglutamic acid (Gla) domainless protein C activation is unchanged by membrane reconstitution (1/2 max = 53 +/- 10 microM) and fits a simple rectangular hyperbola. Incorporation of thrombomodulin into pure phosphatidylcholine vesicles reduces the Km for protein C from 7.6 +/- 2 to 0.7 +/- 0.2 microM. Increasing phosphatidylserine to 20% decreased the Km for protein C further to 0.1 +/- 0.02 microM. Membrane incorporation has no influence on the activation of protein C from which the Gla residues are removed proteolytically (Km = 6.4 +/- 0.5 microM). The Km for protein C observed on endothelial cells is more similar to the Km observed when thrombomodulin (TM) is incorporated into pure phosphatidylcholine vesicles than into negatively charged vesicles, suggesting that the protein C-binding site on endothelial cells does not involve negatively charged phospholipids. In support of this concept, we observed that prothrombin and fragment 1, which bind to negatively charged phospholipids, do not inhibit protein C activation on endothelial cells or TM incorporated into phosphatidylcholine vesicles, but do inhibit when TM is incorporated into phosphatidylcholine:phosphatidylserine vesicles. These studies suggest that neutral phospholipids lead to exposure of a site, probably on thrombomodulin, capable of recognizing the Gla domain of protein C.     

Enterococcus faecium of the vanA genotype in rural drinking water, effluent, and the aqueous environment

Total enterococci and vancomycin-resistant enterococci (VRE) were enumerated in samples of effluent (n = 50) and water (n = 167) from a number of sources. VRE were detected in the outflow of a wastewater treatment plant and in a single rural drinking water supply, suggesting potential for transmission to humans through environmental contamination.     

Gene silencing of TGF-β1 enhances antitumor immunity induced with a dendritic cell vaccine by reducing tumor-associated regulatory T cells

Active immunotherapy and cancer vaccines that promote host antitumor immune responses promise to be effective and less toxic alternatives to current cytotoxic drugs for the treatment of cancer. However, the success of tumor immunotherapeutics and vaccines is dependent on identifying approaches for circumventing the immunosuppressive effects of regulatory T (Treg) cells induced by the growing tumor and by immunotherapeutic molecules, including Toll-like receptor (TLR) agonists. Here, we show that tumors secrete high concentrations of active TGF-β1, a cytokine that can convert naive T cells into Foxp3⁺ Treg cells. Silencing TGF-β1 mRNA using small interfering RNA (siRNA) in tumor cells inhibited active TGF-β1 production in vitro and restrained their growth in vivo. Prophylactic but not therapeutic administration of TGF-β1 siRNA reduced the growth of CT26 tumors in vivo. Furthermore, suppressing TGF-β1 expression at the site of a tumor, using siRNA before, during and after therapeutic administration of a TLR-activated antigen-pulsed dendritic cell vaccine significantly reduced the growth of B16 melanoma in mice. The protective effect of co-administering TGF-β1 siRNA with the DC vaccine was associated with suppression of CD25⁺Foxp3⁺ and CD25⁺IL-10⁺ T cells and enhancement of tumor infiltrating CD4 and CD8 T cells. Our findings suggest that transient suppression of TGF-β1 may be a promising approach for enhancing the efficacy of tumor vaccines in humans.     

A procedure for successful nonsurgical embryo transfer in swine

The current study was undertaken to develop a successful procedure for the nonsurgical transfer of pig embryos. A total of 663 embryos were surgically collected on Day 4 or 5 from 55 donors, of which 542 embryos of acceptable quality were nonsurgically transferred to 46 recipients. Nonsurgical recipient gilts were sedated 15 min prior to transfer with 20 mg im acepromazine maleate. A disposable insemination spirette with an attached 3-way stopcock was manipulated into the cervix of each gilt. Embryos were expelled from a tomcat catheter into the spirette, and 10 to 12 ml of Whitten's medium were used to flush embryos through the spirette into the reproductive tract. Sixteen (34.8%) recipient gilts did not return to estrus before Day 36, and 10 (21.7%) gilts farrowed with an average litter size of 4.3 +/- 0.7. Embryos were collected from an additional 20 donors and were surgically transferred to an additional 19 recipients. Surgical transfers conducted at the same time as the nonsurgical transfers resulted in 12 (63.2%) gilts farrowing and 7.1 +/- 0.6 pigs were born per litter. In conclusion, a procedure has been developed for nonsurgical transfer of swine embryos which simplifies the process of embryo transfer and which may increase the potential for utilization of embryo transfer technologies by swine producers.     

Microtubule Associated Proteins in Plants and the Processes They Manage

Microtubule associated proteins （MAPs） are proteins that physically bind to microtubules in eukaryotes. MAPs play important roles in regulating the polymerization and organization of microtubules and in using the ensuing microtubule arrays to carry out a variety of cellular functions. In plants, MAPs manage the construction, repositioning, and dismantling of four distinct microtubule arrays throughout the cell cycle. Three of these arrays, the cortical array, the preprophase band, and the phragmoplast, are prominent to plants and are responsible for facilitating cell wall deposition and modification, transducing signals, demarcating the plane of cell division, and forming the new cell plate during cytokinesis. This review highlights important aspects of how MAPs in plants establish and maintain microtubule arrays as well as regulate cell growth, cell division, and cellular responses to the environment.     

Bacterial diversity in human subgingival plaque

The purpose of this study was to determine the bacterial diversity in the human subgingival plaque by using culture-independent molecular methods as part of an ongoing effort to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species of human oral bacteria. Subgingival plaque was analyzed from healthy subjects and subjects with refractory periodontitis, adult periodontitis, human immunodeficiency virus periodontitis, and acute necrotizing ulcerative gingivitis. 16S ribosomal DNA (rDNA) bacterial genes from DNA isolated from subgingival plaque samples were PCR amplified with all-bacterial or selective primers and cloned into Escherichia coli. The sequences of cloned 16S rDNA inserts were used to determine species identity or closest relatives by comparison with sequences of known species. A total of 2,522 clones were analyzed. Nearly complete sequences of approximately 1,500 bases were obtained for putative new species. About 60% of the clones fell into 132 known species, 70 of which were identified from multiple subjects. About 40% of the clones were novel phylotypes. Of the 215 novel phylotypes, 75 were identified from multiple subjects. Known putative periodontal pathogens such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola were identified from multiple subjects, but typically as a minor component of the plaque as seen in cultivable studies. Several phylotypes fell into two recently described phyla previously associated with extreme natural environments, for which there are no cultivable species. A number of species or phylotypes were found only in subjects with disease, and a few were found only in healthy subjects. The organisms identified only from diseased sites deserve further study as potential pathogens. Based on the sequence data in this study, the predominant subgingival microbial community consisted of 347 species or phylotypes that fall into 9 bacterial phyla. Based on the 347 species seen in our sample of 2,522 clones, we estimate that there are 68 additional unseen species, for a total estimate of 415 species in the subgingival plaque. When organisms found on other oral surfaces such as the cheek, tongue, and teeth are added to this number, the best estimate of the total species diversity in the oral cavity is approximately 500 species, as previously proposed.     

The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

Background  

The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues.