Nikolaos Kavantzas

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.

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.

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.

A study of the pathogenesis of rosacea: how angiogenesis and mast cells may participate in a complex multifactorial process.

In the present study we evaluated, in involved and clinically uninvolved skin of Rosacea, microvessels density (MVD) and total vascular area (TVA) in addition to multiple morphologic characteristics of microvessels and also mast cells (MCs) number. We examined also the relationship between angiogenesis, MCs number and disease clinicopathological data. The study included 69 patients with Rosacea. A skin biopsy with a 4-mm punch was performed from clinically involved skin in each case. In nine randomly selected patients, facial biopsy specimens were obtained from both involved and clinically uninvolved skin. Histological sections, immunostained for factor VIII, were evaluated by image analysis for the quantification of MVD, TVA and several morphometric parameters related to the vessel size or shape. MCs detection in the dermis was carried out using the chloracetate esterase method (Fast Blue RR) in parafin sections. Serum antibodies against H.pylori were detected. Statistically important differences concerning the factors of angiogenesis between lesional and clinically non-lesional skin were demonstrated. A statistical important correlation was found also between high vascular density, PPR clinical type and the presence of ocular manifestations. MVD or TVA showed no correlation with the degree of solar elastosis or inflammation and with the Demodex density as well. However, high MVD values were found to correlate with granuloma formation in the dermis. MCs number were significantly greater in lesional compared to clinically non-lesional skin. Statistical significance was shown between MCs density and disease duration. However, no correlation between MCs number and blood vessel density was found. Angiogenesis seems to play an important role in the pathogenesis especially of the more severe clinical form of Rosacea. MCs seem to participate in evolution to disease chronicity by contributing to inflammation, angiogenesis and tissue fibrosis.

Prognostic implications of microvessel morphometry in diffuse astrocytic neoplasms

Astrocytic brain tumours, particularly malignant astrocytomas, are recognized to be highly vascular neoplasms with potent angiogenic activity. Recent research has shown that quantification of microvessel density (MVD), as a measure of the degree of angiogenesis, constitutes a strong prognostic indicator in patients with astrocytomas. However, the significance of other morphometric aspects of microvessel network has not been tested so far. In this report, histological sections from 70 astrocytomas (grades II to IV), immunostained for CD34, were evaluated by image analysis for the quantification of MVD, total vascular area (TVA), and microvascular branching, as well as several morphometric parameters related to vessel size or shape. Minor axis length increased with grade (P = 0.045) but MVD and TVA presented a peak in grade III (P = 0.033 and P < 0.001, respectively). Size and shape related parameters affected survival in univariate analysis of grade IV and grades II/III, respectively. In multivariate analysis, only branching counts, along with age and grade, were the independent predictors of survival. Although MVD, TVA and branching counts were adversely related to disease‐free survival in grades II and III (univariate analysis), only TVA remained statistically significant in multivariate analysis. It is concluded that TVA and branching counts are prognostically more informative than MVD for patients with diffuse astrocytic tumours.

Autologous transplantation of adipose-derived stem cells enhances skin graft survival and wound healing in diabetic rats.

BackgroundDiabetes can lead to impaired wound healing and skin grafts used surgically for diabetic wounds are often complicated with necrosis, although different therapies have been proposed. Adipose-derived stem cells (ASCs) participate in tissue repair processes and may have a role during impaired wound healing. In this study, autologous transplantation of ASCs was used to determine if it increases angiogenesis and skin graft survival and enhances wound healing in diabetic rats. MethodsAdipose-derived stem cells were successfully isolated and cultured. A full-thickness skin graft model was used to determine the effects of locally administered ASCs in 10 rats rendered diabetic (group 1), whereas 10 others served as controls (group 2). Histological examination of skin grafts followed after 1 week. Additionally, immunohistochemical staining intensity of vascular endothelial growth factor (VEGF) and transforming growth factor &bgr;3 (TGF-&bgr;3) was assessed in all grafts. ResultsThe gross and histological results showed significantly increased survival, angiogenesis, and epithelialization. Mean area of graft necrosis was significantly less in group 1 than in group 2 (7.49% vs 39.67%, P < 0.001). Statistically significant increase of capillary density, collagen intensity, VEGF, and TGF-&bgr;3 expression was noted in group 1 compared with group 2. ConclusionsThese findings suggest that autologous ASC transplantation can enhance skin graft survival in diabetic rats through differentiation, vasculogenesis, and secretion of growth factors such as VEGF and TGF-&bgr;3. This might represent a novel therapeutic approach in skin graft surgery for diabetic wounds.

Study of phospho- β -catenin subcellular distribution in invasive breast carcinomas in relation to their phenotype and the clinical outcome

β-Catenin has a crucial role in cell–cell adhesion as well as a signaling role as a member of the Wnt pathway. The aim of this study was to examine the clinicopathological and prognostic value of phosphorylated β-catenin, as well as its relation to the tumors’ phenotype, in breast cancer. Immunohistochemistry was applied on 141 paraffin-embedded breast tissue specimens for the detection of phospho-β-catenin, ER, PR, c-erbB-2, p53, Ki-67, bcl-2, uPAR and TIMP-1. For each case, a phospho-β-catenin index was determined by image analysis. Phospho-β-catenin staining was detected in the cytoplasm and the nucleus of the malignant cells. Cytoplasmic phospho-β-catenin was statistically higher in carcinomas of smaller tumor size (P=0.030), lower stage (P=0.026), decreased Ki-67 and high c-erbB-2 immunoreactivity (P=0.052 and P=0.037, respectively). Nuclear phospho-β-catenin showed a parallel correlation with ER and ERβ (P=0.022 and P=0.043, respectively), bcl-2 (P=0.042), uPAR in cancer cells (P=0.041) and TIMP-1, although the correlation was borderline (P=0.066). Cytoplasmic phospho-β-catenin was found to be independently correlated with prolonged disease-free and overall survival (P=0.046 and P=0.002, respectively), whereas nuclear localization was correlated with a shortened overall survival (P=0.046). In conclusion, phospho-β-catenin may have a different involvement in invasive breast carcinomas, according to its subcellular distribution. Nuclear localization seems to be related to an aggressive tumor phenotype, negatively affecting patients’ overall survival, whereas cytoplasmic localization is associated with a favorable tumor phenotype and a longer disease-free and overall survival.

Differential Expression of bcl-2 Family Proteins in Bladder Carcinomas: Relationship with Apoptotic Rate and Survival

OBJECTIVE To elucidate the role of various bcl-2 family molecules in the regulation of apoptosis and the progression of urothelial cancer, in relation to standard prognosticators. METHODS Paraffin-embedded archival tissue from 103 N0M0 consecutive patients with invasive bladder cancer (28 T1, 57 T2, 13 T3 and 5 T4) was immunostained for bcl-2, bax, bcl-XL, bcl-Xs, p53, Ki-67 and with an anti-single stranded DNA monoclonal antibody recognizing the apoptotic cells. Survival analysis was restricted to T2-T4 tumours. Patients were followed-up until death (n = 27) or for a mean (+/- S.D.) follow-up of 37.6 (+/- 17.4) months. Within this period, 39 patients relapsed after a mean (+/- S.D.) period of 13.6 (+/- 12.3) months. RESULTS Most tumours were immunoreactive for bax (73.1%) and bcl-XL (80.9%) whereas bcl-2 and bcl-XS expression was comparatively less common (44.4 and 28.9%, respectively). The bcl-XL and bcl-XS positivity was related to high grade (P = 0.007) and advanced stage (P = 0.010), respectively. On the contrary, bax and bcl-2 positivity was unrelated to stage or grade. Apoptotic rate was independently influenced only by p53, bcl-2 and proliferation rate. In multivariate analysis of T2-T4 urothelial carcinomas (UC)s, only bax along with T-category and age were the significant predictors of disease-free survival. Increased apoptosis and T-category were also independently related to the overall survival in T2-T4 UCs. CONCLUSIONS The expression of bcl-2 family members appears to be differentially regulated in association with UC evolution. Most importantly, bax immunostaining offers additional information to that provided by traditional prognosticators, with regard to disease-free survival of T2-T4 UCs.

DNA topoisomerase II-alpha immunoreactivity as a marker of tumor aggressiveness in invasive breast cancer.

Objective: The nuclear enzyme DNA topoisomerase (topo) II breaks and rejoins DNA strands; its isoform topo IIα is associated with active cell proliferation of mammalian cells. The aim of this study was to examine the relationship between the expression of topo IIα and biological behavior markers in breast cancer. Methods: Formalin-fixed, paraffin-embedded tissue from 88 samples of infiltrating breast cancer was immunohistochemically stained for topo IIα. For each case, a topo IIα index was determined by image analysis. Similar indexes were available for Ki-67 protein, a known cell proliferation marker, and p53, bcl-2 and c-erbB-2 oncoproteins. Each case had been staged and graded and the patients had been followed up for a mean period of 61.62 months. Results: Elevated topo IIα immunopositivity (in >10% of malignant nuclei) was detected in 22 tumors, and this immunostatus was statistically associated with poor nuclear differentiation, absence of steroid hormone receptors, high Ki-67 immunoexpression, p53 protein accumulation and c-erbB-2 protein overexpression. Topo IIα expression was not linked with disease extent (stage or lymph node status). Neither proliferation marker (topo IIα or Ki-67) had any significant influence on the patients’ recurrence-free survival. Conclusion: From the above results, we conclude that topo IIα overexpression appears to be linked with cellular dedifferentiation and potentially aggressive tumor phenotype in invasive breast cancer.

Oleuropein prevents doxorubicin-induced cardiomyopathy interfering with signaling molecules and cardiomyocyte metabolism

Oleuropein, a natural phenolic compound, prevents acute doxorubicin (DXR)-induced cardiotoxicity but there is no evidence regarding its role in chronic DXR-induced cardiomyopathy (DXR-CM). In the present study, we investigated the role of oleuropein in DXR-CM by addressing cardiac geometry and function (transthoracic echocardiography), cardiac histopathology, nitro-oxidative stress (MDA, PCs, NT), inflammatory cytokines (IL-6, Big ET-1), NO homeostasis (iNOS and eNOS expressions), kinases involved in apoptosis and metabolism (Akt, AMPK) and myocardial metabonomics. Rats were randomly divided into 6 groups: Control, OLEU-1 and OLEU-2 [oleuropein at 1000 and 2000 mg/kg in total, respectively, intraperitoneally (i.p.) for 14 days], DXR (18 mg/kg, i.p. divided into 6 equal doses for 2 weeks), DXR-OLEU-1 and DXR-OLEU-2 (both oleuropein and DXR as previously described). Impaired left ventricular contractility and inflammatory and degenerative pathology lesions were encountered only in the DXR group. The DXR group also had higher MDA, PCs, NT, IL-6 and Big ET-1 levels, higher iNOS and lower eNOS, Akt and AMPK activation compared to controls and the oleuropein-treated groups. Metabonomics depicted significant metabolite alterations in the DXR group suggesting perturbed energy metabolism and protein biosynthesis. The effectiveness of DXR in inhibiting cell proliferation is not compromised when oleuropein is present. We documented an imbalance between iNOS and eNOS expressions and a disturbed protein biosynthesis and metabolism in DXR-CM; these newly recognized pathways in DXR cardiotoxicity may help identifying novel therapeutic targets. Activation of AMPK and suppression of iNOS by oleuropein seem to prevent the structural, functional and histopathological cardiac effects of chronic DXR toxicity.