In early human gestation, maternal arterial blood flow into the intervillous space of the developing placenta is obstructed by invaded trophoblasts, which form cellular plugs in uterine spiral arteries. These trophoblast plugs have recently been described to be loosely cohesive with clear capillary-sized channels into the intervillous space by 7 weeks of gestation. Here, we analysed localisation of maternal platelets at the maternal-foetal interface of human first trimester pregnancy, and tested the hypothesis whether HLA-G, which is primarily expressed by extravillous trophoblasts, affects aggregation and adhesion of isolated platelets. Immunohistochemistry of first trimester placental sections localised maternal platelets in vessel-like channels and adjacent intercellular gaps of extravillous trophoblasts in distal parts of columns. Furthermore, this localisation was confirmed by transmission electron microscopy. Neither co-incubation of HLA-G overexpressing JAR cells with isolated platelets, nor incubation with cell-derived soluble HLA-G or recombinant HLA-G affected platelet adhesion and aggregation. Our study suggests that maternal platelets flow through vessel-like channels of distal trophoblast columns and spread into adjacent lateral intercellular gaps, where platelet-derived factors could contribute to trophoblast differentiation into the invasive phenotype.

Major pathological hallmarks of Alzheimer's disease (AD) are amyloid plaques, neurofibrillary tangles (NFT) and neuritic plaques (NP). Both NFTs and NPs contain a broad variety of hyperphosphorylated species of Tau proteins. Although manifold hypotheses suggest distinct phospho-sites as so-called triggers for AD progression, evidence is still fragmentary. This study aimed to quantitatively determine the distribution pattern of various forms of hyperphosphorylated Tau in human AD brains. In detail, quantitative immunohistochemistry of ThioflavinS positive NFTs and total Tau as well as its phosphorylated forms (pTau) Thr231, Tyr18, Ser262, Ser202/Thr205, Ser396 and Ser422 are evaluated in four different cortical regions and hippocampi of five AD subjects originating from three different Braak stages and age-matched healthy controls. Tau phosphorylated at site Tyr18 is suggested as early AD marker. However, we found an enormous accumulation of this specific phospho form in end stage cortices and hippocampus. This suggests that even late in the disease “new” hyperphosphorylated Tau is produced. In line with our observations that the amount of regional Tau increases with increasing topographical progression, e.g. more tau in the entorhinal cortex of a Braak IV case as compared to a Braak II case. Beside that we also detected an increased presence of ThioflavinS positive NFTs and pThr231 Tau in late stage somata and neurites. In hippocampi however, elevated hyperphosphorylation of pTyr18 and pThr231 Tau occurs already at Braak stages I/II, while NFT formation follows afterwards. Our results suggest especially Tau hyperphosphorylation at residues Tyr18 and Thr231 are early events in hippocampal degeneration and highlight the possible link to early memory loss in AD subjects. Whereas, NFTs, pSer202/Thr205 and pSer262 IR dystrophic neurites present as later stage features. Further studies are warranted to clarify the role of C terminal phosphorylation at Ser396 and Ser422 for the progression of AD.

Human AD cases were reported to show vascular pathology, also different mouse models of Alzheimer's disease (AD) show vascular alterations. The evident cerebral amyloid angiopathy (CAA) in AD model mice triggers increased vascular diameter and increased IR area of vascular markers. The latter was interpreted as vascular sprouting and/or increase of vascular diameter. Yet, it is not clear, if these mechanisms to increase cerebral blood flow and sustenance can be found in both mice and men, if they are common within mouse models at all, or if there are probably misinterpretations of published data. Real comparative quantitative data between human AD cases and AD model mice are not available today. Thus we created a setup comparing CAA, vascular diameter and number in APP SL model mice at different ages and human AD cases at different Braak stages of disease. CAA was immunohistochemically evaluated using a co-labeling of an amyloid plaque marker (6E10) with Annexin-IV and smooth muscle actin to be able to directly quantify vascular amyloid load on arteries and venules. CAA is related to arteries and does hardly appear at venules in both mice and men. APP SL mice react with an increased vascular diameter on amyloid load rather than with angiogenesis. Also in humans, no increase of the vessel number but rather an increase in vascular diameter of amyloid loaded vessels seems present. Furthermore, and as in mice over age, CAA increases with Braak stage in most cases. Obtained data support the thesis that, CAA is growing with age in APP SL mice and Braak stage in humans, and that the typical reaction to CAA is an increase of blood volume rather than an increase of arterial network. Differences and parallels between mice and men are presented and methodological topics discussed.

Hyperphosphorylation of Tau is one of the major hallmarks of Alzheimer’s Disease pathology. Neurofibrillary tangles (NFTs) are used in clinical staging (Braak & Braak 1997). Yet, the tangle reflects an end stage of paired helical filament (PHF) aggregations, which appear much earlier and are typical found in neuropil threads and not neuronal somata. Furthermore, those consist of a large array of differently hyperphosphorylated Tau (pTau), whereas several phospho-sites were found to be especially related to AD. However, today’s knowledge about appearance, quantity and location of PHFs with different phosphorylation and in different brain regions during disease progression is rather poor. To elucidate the quantitative distribution of different pTau sites in the AD brain, we analyzed total human Tau, pThr231, pSer202, pSer262, pTyr18 Tau as well as ThioS positive tangles in four different cortical brain regions. Systematic random sets of brain sections deriving from human AD cases* of three different Braak stages (Braak stages I/II; III/IV; V/VI) were immunohistochemically labeled for detection of different Tau species, imaged and quantitatively evaluated in terms of IR surface area using image analysis software (Axio.Imager Z1 microscope, ImageProPlus). Sections of age-matched non AD human subjects served as controls. Samples of humans were evaluated by rater independent automated counting in mosaic image stripes of a mean of 3 mm² imaged at 20x through white and grey matter per region and subject. Samples of human cingulate, temporal, frontal and occipital cortex were averaged and are shown in graphs as “cortex”, the hippocampus is shown as separate region and average of two large sized mosaic images. Data of the single cortices, however, are available as well but not shown not to overload the presentation. Table 1 shows the primary anti Tau antibodies that were used and indirectly visualized using highly cross-adsorbed DyLight TM secondary antibodies. ThioflavinS (Sigma T1892-25G) was used at 0.5% for 7 minutes. Stained surface or IR area was measured and normalized to the individual AOI size. To account for a significant amount of autofluorescent bias objects (mainly Lipofuscin), an unlabeled channel was imaged, a bilevel mask created and subtracted from the labeled channels ( Fig. 1). *… provided and staged by the Brain Bank of Newcastle; samples were formalin fixed Paraffin embedded.

The aim of the present study was to evaluate the potential of intraoral harvested alveolar bone as an alternative source of multipotent mesenchymal stromal cells for future applications in oral and maxillofacial tissue engineering. Explant cultures were established from 20 alveolar bone samples harvested from the oblique line immediately before wisdom tooth removal. Morphology and proliferation characteristics of the in vitro expanded cells, referred to as human alveolar bone-derived cells (hABDCs), were studied using phase-contrast microscopy. Immunocytochemical analysis of their surface marker expression was conducted using monoclonal antibodies defining mesenchymal stromal cells. To evaluate their multilineage differentiation potential, hABDCs were induced to differentiate along the osteogenic, adipogenic, and chondrogenic lineage and compared to bone marrow mesenchymal stromal cells (hBMSCs) on mRNA and protein levels applying RT-PCR and cytochemical staining methods. hABDCs showed typical morphological characteristics comparable to those of hBMSCs such as being mononuclear, fibroblast-like, spindle-shaped, and plastic adherent. Immunophenotypically, cells were positive for CD105, CD90, and CD73 while negative for CD45, CD34, CD14, CD79α, and HLA-DR surface molecules, indicating an antigen expression pattern considered typical for multipotent mesenchymal stromal cells. As evidenced by RT-PCR and cytochemistry, hABDCs showed multilineage differentiation and similar chondrogenic and osteogenic differentiation potentials when compared to hBMSCs. Our findings demonstrate that human alveolar bone contains mesenchymal progenitor cells that can be isolated and expanded in vitro and are capable of trilineage differentiation, providing a reservoir of multipotent mesenchymal cells from an easily accessible tissue source.

Placental trophoblast cells of the semi-allogenic human conceptus invade deeply into maternal uterine tissue. From a classical immunoiogic point of view this invasion and the following growth and development of the fetus in the uterus have to be tolerated by a pregnant woman's immune system. Among the various possible protective mechanisms that may be involved, the unique expression pattern of HLA class I molecules seems to be relevant. Besides many other differences between placentation and organ transplantation, this extraordinary HLA class I expression on trophoblast explains why pregnancy should not be considered an immunologic paradox but rather a fascinating example of a very special challenge for the female immune system.

The role of hyperphosphorylation of the microtubule associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, the development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. We therefore generated and validated a stably transfected SH-SY5Y cell line as well as a transgenic mouse line, both expressing tau441 with two mutations (V337M/R406W). The SH-SY5Y-TMHT441 cell line was characterized for its tau expression levels, tau phosphorylation status as well as the effect of different kinase inhibitors. The TMHT transgenic mice were analyzed for total and phosphorylated tau in different fractions of the brain as well as in different brain areas by biochemical and immunofluorescent labeling methods, respectively. Furthermore mice were analyzed for memory deficits in the Morris water maze as well as in some other tests. Our results show that the SH-SY5Y-TMHT441 cells as well as the transgenic mice express high levels of total and phosphorylated tau at residues Thr181, Thr231/Ser235, and Ser396/Ser404 measured, using Mass Spectrometry (i.e. Phospho-Tau SRM assay). Treatment of SH-SY5Y-TMHT441 cells with different tauopathy related kinase inhibitors shows a distinct inhibition pattern of these phosphorylation sites. Analysis of TMHT transgenic mice over age reveals increased total and ptau levels in the soluble but to a much lesser extent in the insoluble fraction of the hippocampus as early as 3 months of age by MSD-ISA and increasing total and ptau levels in the amygdala by immunofluorescent quantification. Behavioral tests show that TMHT transgenic mice present with distinct memory deficits as early as 5 months of age. Since we did not observe an increase in insoluble total or ptau in these mice, we conclude that hyperphosphorylated soluble tau is efficient to cause severe memory deficits. In summary, we present two new model systems to analyze the efficiency of tau modulating compounds. These models complement each other entirely, since they are generated with the same gene construct and show activation of the same tau phosphorylation sites.

Purpose: The aim of the present study was to compare the influence of 2 different bone scrapers with respect to graft quality.Materials and Methods: The study was conducted as a prospective, controlled experimental study of patients selected from the outpatient unit of the Department of Oral Surgery and Radiology (Dental Clinic, Medical University, Graz, Austria). Bone samples were obtained during routine lower third molar removal. Both a manual bone scraper (MS) and a piezoelectric device (PD) were used in directly adjacent regions in each case. As variables, the chip morphology, cell viability, and osteogenic differentiation were investigated. For statistical analysis, the Student t test and Fisher's exact test (P < .05) were applied.Results: A total of 20 patients (12 women and 8 men, mean age 28.15 +/- 5.8 years) were included in the study. A series of 40 bone samples was obtained during lower third molar removal. MS and PD enabled similar intraoral harvest of bone chips. In vitro outgrowth of adherent cells was found in 90% of the MS and 80% of the PD samples after 7 to 18 days, without statistical significance (P = .67). Similar cell viability of outgrowing cells in both groups was observed (94.7% +/- 2.2% in the MS group and 94.1% +/- 1.6% in the PD group). Reverse transcriptase-polymerase chain reaction analysis and the staining pattern verified osteopotent cells in both groups.Conclusions: Both manual and piezoelectric techniques are adequate harvesting technologies for limited intraoral augmentations. Our results did not show an advantage for the piezoelectric device. (C) 2012 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 70:154-162, 2012

Two hallmarks of AD are amyloid plaques and neurofibrillary tangle pathology. These two were so far not successfully combined in one transgenic mouse model. Progressive amyloid and TAU pathology are described to lead to increased oxidative stress, mitochondrial impairment, inflammatory reactions, cytoskeletal deterioration, apoptosis and finally to loss of synapses and neurons. Crossbreeding of two well characterized transgenic mouse lines of AD, a hAPP over-expression mouse with Swedish and London mutations and a hTAU overexpressor was initiated. The goal was to get further insights whether these two transgenes synergistically induce the occurrence of AD related pathologic alterations and behavioral disturbances. Mice with a C57BL/6 background overexpressing TAU441 with the missense mutations V337M and R406W under the control of the brain specific murine Thy-1 promoter and mice overexpressing human APP carrying the Swedish and London mutations using the same promoter were crossbred. Behavioral changes were investigated at ages of 4, 5 and 6 months followed by evaluation of brain pathology using immunohistochemical staining e.g. with several anti-human PHF-TAU and amyloid antibodies. Furthermore brain inflammatory reaction, number of synapses and neurons were investigated. Evaluation of the cognitive function in the Morris Water Maze (MWM) showed a significant disturbance of the spatial navigation behavior in these mice already at an age of 4 months. Brain TAU pathology revealed numerous densely packed human TAU-positive lesioned neurons. In addition to that the typical amyloid pathology like extracellular beta-amyloid depositions was observed in this age group. The hAPP-SL x hTAU crossbreds were found to effectively simulate AD pathology. The model is expected to be a proper tool to investigate the efficacy of compounds that address the combination of amyloid and TAU pathology as present in humans.

The microtubule-associated CNS protein TAU, is expressed in neurons and at lower level in astrocytes and oligodendrocytes. Its major function is to regulate stability and assembly of microtubules, thus playing an important role in axonal transport and cytoskeletal organization. TAU pathology concerns several familial or sporadic neurodegenerative diseases, e.g. Alzheimer's disease (AD), FTDP-17, Pick's disease, myotonic dystrophy, corticobasal degeneration, and many others. CSF TAU and hyperphosphorylated TAU are the main biochemical marker of neurofibrillary degeneration, well correlated with clinical manifestations of the corresponding disease.Several FTDP-17 missense TAU mutations, including V337M and R406W have been demonstrated to reduce the ability of bacterially expressed recombinant TAU protein to bind to and promote the assembly of microtubules. We have generated transgenic mice that over-express TAU441 bearing the missense mutations V337M and R406W under the control of the brain specific murine Thy-1 promoter. TMHT mice show no motor deficits in the Rota Rod, the beam walking, the wire suspension or in any other motor test. However distinct impairments of reference and working memory in the Morris water maze task (MWM) as well as a decreased recognition index in the object recognition task (ORT) can be detected compared to age matched non transgenic littermates. Brain pathology was investigated by immunohistochemical staining with several anti-human PHF as well as TAU antibodies. Results showed numerous densely packed human TAU-positive lesioned neuronal perikaria and neurites (axons and dendrites) in hippocampus, cortex and amygdala. Besides the specific TAU pathology also the number of neurons and synapses as well as other relevant pathological markers will be determined to further characterize this animal model. The TMHT mouse showing severe behavioural deficits and distinctive brain pathology is a relevant model to study the influence of drugs on TAU phosphorylation, sequestration and deposition in vivo.