Efstratios Patsouris

Staging of Neurofibrillary Pathology in Alzheimer's Disease: A Study of the BrainNet Europe Consortium

It has been recognized that molecular classifications will form the basis for neuropathological diagnostic work in the future. Consequently, in order to reach a diagnosis of Alzheimers disease (AD), the presence of hyperphosphorylated tau (HP‐tau) and β‐amyloid protein in brain tissue must be unequivocal. In addition, the stepwise progression of pathology needs to be assessed. This paper deals exclusively with the regional assessment of AD‐related HP‐tau pathology. The objective was to provide straightforward instructions to aid in the assessment of AD‐related immunohistochemically (IHC) detected HP‐tau pathology and to test the concordance of assessments made by 25 independent evaluators. The assessment of progression in 7‐µm‐thick sections was based on assessment of IHC labeled HP‐tau immunoreactive neuropil threads (NTs). Our results indicate that good agreement can be reached when the lesions are substantial, i.e., the lesions have reached isocortical structures (stage V–VI absolute agreement 91%), whereas when only mild subtle lesions were present the agreement was poorer (I–II absolute agreement 50%). Thus, in a research setting when the extent of lesions is mild, it is strongly recommended that the assessment of lesions should be carried out by at least two independent observers.

Staging/typing of Lewy body related α-synuclein pathology: a study of the BrainNet Europe Consortium

When 22 members of the BrainNet Europe (BNE) consortium assessed 31 cases with α-synuclein (αS) immunoreactive (IR) pathology applying the consensus protocol described by McKeith and colleagues in 2005, the inter-observer agreement was 80%, being lowest in the limbic category (73%). When applying the staging protocol described by Braak and colleagues in 2003, agreement was only 65%, and in some cases as low as 36%. When modifications of these strategies, i.e., McKeith’s protocol by Leverenz and colleagues from 2009, Braak’s staging by Müller and colleagues from 2005 were applied then the agreement increased to 78 and 82%, respectively. In both of these modifications, a reduced number of anatomical regions/blocks are assessed and still in a substantial number of cases, the inter-observer agreement differed significantly. Over 80% agreement in both typing and staging of αS pathology could be achieved when applying a new protocol, jointly designed by the BNE consortium. The BNE-protocol assessing αS-IR lesions in nine blocks offered advantages over the previous modified protocols because the agreement between the 22 observers was over 80% in most cases. Furthermore, in the BNE-protocol, the αS pathology is assessed as being present or absent and thus the quality of staining and the assessment of the severity of αS-IR pathology do not alter the inter-observer agreement, contrary to other assessment strategies. To reach these high agreement rates an entity of amygdala-predominant category was incorporated. In conclusion, here we report a protocol for assessing αS pathology that can achieve a high inter-observer agreement for both the assignment to brainstem, limbic, neocortical and amygdala-predominant categories of synucleinopathy and the Braak stages.

The Influence of Platelet-Rich Plasma on Angiogenesis During the Early Phase of Tendon Healing

Background: The poor vascularity of tendons is a major factor in their limited healing capacity. The aim of this study was to assess the effect of Platelet Rich Plasma (PRP) on angiogenesis during tendon healing. Materials and Methods: Forty-eight skeletally mature New Zealand White rabbits were used. The Achilles tendon was transected transversely and 0.5 ml of PRP was injected into the tendon mass on each side of the incision on both limbs. The injection in the control group consisted of saline. Six animals from each group (12 tendons each) were sacrificed after 1, 2, 3, and 4 weeks following treatment. Three sections from each Achilles were stained with hematoxylinosin for microscopic examination. Further three sections were immunostained with a monoclonal antibody against CD31 (Daco Co), followed by image analysis to count new vessel numbers and statistical analysis was performed. Results: There was significantly more angiogenesis in the PRP group compared to the control group during the first two weeks of the healing process, i.e., inflammatory and proliferative phase (p < 0.0001). The orientation of collagen fibers in the PRP group was better organized. The number of the newly formed vessels in the PRP group were significantly reduced at 4 weeks compared to the controls (p < 0.0001) suggesting the healing process was shortened. Conclusion: PRP seems to enhance neovascularization which may accelerate the healing process and promote scar tissue of better histological quality. Clinical Relevance: Although these results need replication and further biomechanical research, PRP may promote tendon healing acceleration.

Immunohistochemical localization of advanced glycation end-products (AGEs) and their receptor (RAGE) in polycystic and normal ovaries

The aim of the present study was to investigate the localization/immunohistochemical distribution of AGEs and RAGE, as well as their putative signalling mediator NF-κB in ovaries of women with polycystic ovary syndrome (PCOS) compared to normal. Archival ovarian-tissue samples from biopsies of six women with PCOS and from six healthy of similar age women, were examined immunohistochemically with monoclonal anti-AGEs, anti-RAGE and anti-NF-κB(p50/p65) specific antibodies. In healthy women, AGE immunoreactivity was observed in follicular cell layers (granulosa and theca) and luteinized cells, but not in endothelial cells. PCOS specimens displayed AGE immunoexpression in theca interna and granulosa cells as well as in endothelial cells, but staining of granulosa cells was stronger than in that of normal ovaries. RAGE was highly expressed in normal and PCOS tissues. Normal tissue exhibited no staining differences between granulosa cell layer and theca interna. However, in PCOS ovaries, granulosa cells displayed stronger RAGE expression compared to theca interna cells in comparison to controls. NF-κB(p50/p65) was expressed in the cytoplasm of theca interna and granulosa cells of both normal and PCOS ovaries; whereas the NF-κB p65 subunit was only observed in granulosa cells nuclei in PCOS tissue. In conclusion, these findings demonstrate for the first time that RAGE and AGE-modified proteins with activated NF-κB are expressed in human ovarian tissue. Furthermore, a differential qualitative distribution of AGE, RAGE and NF-κB p65 subunit was observed in women with PCOS compared to healthy controls, where a stronger localization of both AGE and RAGE was observed in the granulosa cell layer of PCOS ovaries.

Interlaboratory comparison of assessments of Alzheimer disease-related lesions: a study of the BrainNet Europe Consortium

Abstract This interlaboratory study evaluated the reproducibility of the assessments of neuritic plaques and neurofibrillary tangles (NFTs)-the hallmark lesions of Alzheimer disease-and compared the staining between the BrainNet Europe centers. To reduce the topography-related inconsistencies in assessments, we used a 2-mm tissue microarray (TMA) technique. The TMA block included 42 core samples taken from 21 paraffin blocks. The assessments were done on Bielschowsky and Gallyas silver stains using an immunohistochemical (IHC) method with antibodies directed to beta-amyloid (IHC/A&bgr;) and hyperphosphorylated tau (IHC/HPtau). The staining quality and the assessments differed between the participants, being most diverse with Bielschowsky (good/acceptable stain in 53% of centers) followed by Gallyas (good/acceptable stain in 57%) and IHC/A&bgr; (good/acceptable stain in 71%). The most uniform staining quality and assessment was obtained with the IHC/HPtau method (good/acceptable stain in 94% of centers). The neuropathologic diagnostic protocol (Consortium to Establish a Registry for Alzheimer Disease, Braak and Braak, and the National Institute of Aging and Reagan [NIA-Reagan] Institute) that was used significantly influenced the agreement, being highest with NIA-Reagan (54%) recommendations. This agreement was improved by visualization of NFTs using the IHC/HPtau method. Therefore, the IHC/HPtau methodology to visualize NFTs and neuropil threads should be considered as a method of choice in a future diagnostic protocol for Alzheimer disease.

Proposal for a 10-high-power-fields scoring method for the assessment of tumor budding in colorectal cancer

Although tumor budding is linked to adverse prognosis in colorectal cancer, it remains largely unreported in daily diagnostic work due to the absence of a standardized scoring method. Our aim was to assess the inter-observer agreement of a novel 10-high-power-fields method for assessment of tumor budding at the invasive front and to confirm the prognostic value of tumor budding in our setting of colorectal cancers. Whole tissue sections of 215 colorectal cancers with full clinico-pathological and follow-up information were stained with cytokeratin AE1/AE3 antibody. Presence of buds was scored across 10-high-power fields at the invasive front by two pathologists and two additional observers were asked to score 50 cases of tumor budding randomly selected from the larger cohort. The measurements were correlated to the patient and tumor characteristics. Inter-observer agreement and correlation between observers’ scores were excellent (P<0.0001; intraclass correlation coefficient=0.96). A test subgroup of 65 patients (30%) was used to define a valid cutoff score for high-grade tumor budding and the remaining 70% of the patients were entered into the analysis. High-grade budding was defined as an average of ≥10 buds across 10-high-power fields. High-grade budding was associated with a higher tumor grade (P<0.0001), higher TNM stage (P=0.0003), vascular invasion (P<0.0001), infiltrating tumor border configuration (P<0.0001) and reduced survival (P<0.0001). Multivariate analysis confirmed its independent prognostic effect (P=0.007) when adjusting for TNM stage and adjuvant therapy. Using 10-high-power fields for evaluating tumor budding has independent prognostic value and shows excellent inter-observer agreement. Like the BRE and Gleason scores in breast and prostate cancers, respectively, tumor budding could be a basis for a prognostic score in colorectal cancer.

Evaluation of hypoxia‐inducible factor 1α overexpression as a predictor of tumour recurrence and progression in superficial urothelial bladder carcinoma

To investigate the possible role of hypoxia‐inducible factor 1α (HIF‐1α, a transcription factor important in regulating O2 homeostasis and physiological responses to oxygen deprivation) in the recurrence and progression of superficial urothelial bladder cancer, and to examine its expression in relation to proliferation status, apoptotic activity and intratumoral angiogenesis.

Hypoxia-inducible factor 1α/vascular endothelial growth factor axis in astrocytomas. Associations with microvessel morphometry, proliferation and prognosis

Hypoxia‐inducible factor (HIF)‐1α is a transcription factor that promotes ischaemia‐driven angiogenesis. The aim of this study was to determine the relation of HIF‐1α to vascular endothelial growth factor (VEGF; an important angiogenic molecule in brain tumours), p53 expression, angiogenesis, proliferative potential and clinical outcome in a large series of diffuse astrocytomas. Expression of HIF‐1α, VEGF, Ki‐67 (a proliferation‐associated marker) and p53 was determined immunohistochemically in 83 adult patients with supratentorial diffuse astrocytomas. Microvessels, highlighted by means of anti‐CD34 immunohistochemistry, were enumerated with computer‐assisted image analysis. Although HIF‐1α and VEGF were expressed in the majority of cases, their levels increased significantly with increasing grade and proliferative potential. HIF‐1α positively correlated with microvessel counts and VEGF with total vascular area and the presence of rounder vessel sections. There was a positive correlation of VEGF with p53 expression in astrocytomas and anaplastic astrocytomas. In univariate analysis, both VEGF and HIF‐1α were associated with shortened survival in the entire cohort, but lost significance when grades II/III and grade IV were analysed separately. Multivariate analysis revealed that the combination of HIF‐1α with grade was a significant prognostic indicator. HIF‐1α expression may be used to refine the prognostic information provided by grade in patients with diffuse astrocytomas. Its adverse prognostic effect is most likely mediated by hypoxia, the driving force for HIF‐1α accumulation.

Local and systemic toxicity of nanoscale debris particles in total hip arthroplasty

Over the past 30 years joint replacement prostheses have been developed and refined to enhance durability and reproducibility. Total hip joint arthroplasty is being performed in an increasing number of younger patients; therefore orthopaedic surgeons seek implants with a longer life span. With regards to the progress of mechanical behaviour of the biomaterials used in an arthroplasty, little is known about the long‐term biological effects of wear debris. Owing to the composition of the prostheses currently in use, systemic exposure to chromium (Cr), cobalt (Co), nickel (Ni) and aluminium (Al) alloys occurs as a result of the formation of metal wear nano‐particles that are released both from metal‐on‐metal and polyethylene‐on‐metal bearings, resulting in a postoperative increase in metal ion levels at different organ sites. These particles circulate both locally and systemically, penetrate cell plasma membranes, bind to cellular proteins and enzymes and modulate cytokine expression. Their physiologic effects are poorly understood and their potential toxicity, hypersensitivity and carcinogenicity remain a cause for concern. In this article we will address the issue of whether these nanoscale degradation products are associated with adverse, clinically significant local or systemic toxicologic sequelae. Copyright

Inter-laboratory comparison of neuropathological assessments of beta-amyloid protein: a study of the BrainNet Europe consortium.

Amyloid-β-protein (Aβ) is generally assessed by neuropathologists in diagnostics. This BrainNet Europe (http://www.brainnet-europe.org/) (15 centres and 26 participants) study was carried out to investigate the reliability of such an assessment. In the first part of this trial, tissue microarray sections were stained with the antibody of each centre’s choice. Reflecting the reality, seven antibodies and a plethora of pretreatment strategies were used. Ninety-two percent of the stainings were of good/acceptable quality and the estimation of presence of Aβ aggregates yielded good results. However, a poor agreement was reached particularly regarding quantitative (density) and qualitative (diffuse/cored plaques) results. During a joint meeting, the clone 4G8 was determined to label best the fleecy/diffuse plaques, and thus, this clone and the formic acid pretreatment technique were selected for the second part of this study. Subsequently, all stained sections were of good/acceptable quality and again a high level of concordance of the dichotomized (presence/absence) assessment of plaques and CAA was achieved. However, even when only one antibody was used, the type of Aβ-aggregates (diffuse/cored), type of vessel and Vonsattel grade, were not reliably assigned. Furthermore, the quantification of lesions was far from reliable. In line with the first trial, the agreement while assessing density (some, moderate and many) was unimpressive. In conclusion, we can confirm the utility of immunohistochemical detection of Aβ-protein in diagnostics and research. It is noteworthy that to reach reproducible results a dichotomized assessment of Aβ-immunoreactivity rather than quantification and assignment of various types of lesions should be applied, particularly when comparing results obtained by different neuropathologists.