Optimization of biosensor cell lines for the detection of tau seeding competency in human CSF

AbstractTau protein forms self-replicating assemblies (seeds) that may underlie progression of pathology in Alzheimer’s disease (AD) and related tauopathies. Seeding in recombinant protein preparations and brain homogenates has been quantified with “biosensor” cell lines that express tau with a disease-associated mutation (P301S) fused to complementary fluorescent proteins. Quantification of induced aggregation in cells that score positive by fluorescence resonance energy transfer (FRET) is accomplished by cell imaging or flow cytometry. Several groups have reported seeding activity in antemortem cerebrospinal fluid (CSF) using various methods, but these findings are not yet widely replicated. To address this question, we created two improved FRET-based biosensor cell lines based on tau expression, termed version 2 low (v2L) and version 2 high (v2H). We determined that v2H cells are ~ 100-fold more sensitive to AD-derived tau seeds than our original lines, and coupled with immunoprecipitation reliably detect seeding from samples containing as little as 100 attomoles of recombinant tau fibrils or ~ 32 pg of total protein from AD brain homogenate. We tested antemortem CSF from 11 subjects with a clinical diagnosis of AD, 9 confirmed by validated CSF biomarkers. We used immunoprecipitation coupled with seed detection in v2H cells and detected no tau seeding in any sample. Thus we cannot confirm prior reports of tau seeding activity in the CSF of AD patients. This next generation of ultra-sensitive tau biosensors may nonetheless be useful to the research community to quantify tau pathology as sensitively and specifically as possible.

Download Full-text

(EB) Virus: Latency and Expression in Transplanted and Cultured Human Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100065882 ◽

1971 ◽

Vol 29 ◽

pp. 368-369

Author(s):

B. G. Uzman ◽

M. M. Kasac ◽

H. Saito ◽

A. Krishan

Keyword(s):

Cell Lines ◽

Primary Cultures ◽

Virus Particles ◽

Barr Virus ◽

Virus Latency ◽

Virus Surveillance ◽

Cultured Human Cells ◽

Epstein Barr ◽

Serial Transplantation ◽

Tubular Arrays

In conjunction with the cultivation and transplantation of cells from human tumors by the Programs of Microbiology and Immunogenetics, virus surveillance by electron microscopy has been routinely employed. Of particular interest in this regard have been 3 cell lines cultured from lymph nodes or spleen of 2 patients with Hodgkin's disease and 1 patient with Letterer-Siwe's disease. Each of these cell lines when transplanted in Syrian hamster neonates conditioned with anti-lymphocyte serum grew as serially transplantable tumors; from such transplants of the 3 cell lines cell cultures were retrieved.Herpes type virus particles (Figs. 1, 2, 3) were found in the primary cultures of all three lines, in frozen thawed aliquots of same, and in cultures retrieved from their tumors growing by serial transplantation in hamsters. No virus was detected in sections of 25 of the serially transplanted tumors. However, in 10 such tumors there were repeated instances of tubular arrays in the cisternae of the endoplasmic reticulum (Fig. 4). On serologic examination the herpes virus was shown to be the Epstein-Barr virus.

Download Full-text

Comparison of Choriocarcinoma and Normal Placenta

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100065663 ◽

1971 ◽

Vol 29 ◽

pp. 324-325

Author(s):

John C. Garancis ◽

Roland A. Pattillo ◽

Robert O. Hussa ◽

Jon V. Straumfjord

Keyword(s):

Cell Lines ◽

Small Cells ◽

Tissue Cultures ◽

Glycogen Depletion ◽

Cell Surfaces ◽

Intercellular Spaces ◽

Concomitant Increase ◽

Gestational Choriocarcinoma ◽

Cytoplasmic Glycogen ◽

Normal Placenta

Two different cell lines (Be-Wo and Jar) of human gestational choriocarcinoma have been maintained in continuous tissue culture for a period of four and two years respectively without losing the ability to elaborate human chorionic gonadotropin (HCG). Tissue cultures, as revealed by electron microscopy, consisted of small cells with single nuclei. In some instances cell surfaces were provided with microvilli but more often the intercellular spaces were narrow and bridged by desmosomes. However, syncytium was not formed. Endoplasmic reticulum (ER) was poorly developed in both cell lines, except in some Be-Wo cells it was prominent. Golgi complex, lysosomes and numerous free ribosomes, as well as excessive cytoplasmic glycogen, were present in all cells (Fig. 1). Glycogen depletion and concomitant increase of ER were observed in many cells following a single dose of 10 ugm/ml of adrenalin added to medium (Fig. 2).

Download Full-text

Immunocytochemical localization of β1-4 galactosyltransferase in endometrial carcinoma cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162284 ◽

1990 ◽

Vol 48 (3) ◽

pp. 944-945

Author(s):

Ichiro Yamamoto ◽

Toshiaki Tachibana ◽

Hiroko Maruyama ◽

Noriyuki Komatsu ◽

Hiroyuki Kuramoto ◽

...

Keyword(s):

Endometrial Carcinoma ◽

Cell Lines ◽

Endometrial Adenocarcinoma ◽

Protein A ◽

Rabbit Antiserum ◽

Carcinoma Cells ◽

Affinity Column ◽

Antibody Technique ◽

Galactosyl Transferase ◽

C Terminus

We have paid attention to the alteration of glycosyltransferase in carcinoma cells, because it might be related to the malignancy of the cells. In this connection, localization of β1-4 galactosyl transferase (β1-4 Gal T) in human endometrial carcinoma cells was examined immunocytochemically using two kinds of cell lines, each of which showed different degree of differentiation.An antibody was purified from the rabbit antiserum against the synthetic peptide, IFNRLVFRGMSC (W89) of human β1-4 Gal T coupled with KLH (keyhole limpet hemocyanine) by protein A column and peptide-affinity column chromatography. The anti-W89 serum reacts to the C-terminus of human β 1-4 Gal T and to both membrane-bound and soluble forms of the enzyme. Cell line of well differentiated endometrial adenocarcinoma (I) and that of poorly differentiated endometrial adenocarcinoma (50B) were cultivated respectively in MEM medium containing 15% FCS and 2 mM glutamine for 4 d at 37°C under 5% CO2. The cells were fixed in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M Soerensen’s phosphate buffer (pH 7.4) at 4°C for 30 min, washed with PBS, then freezed and thawed. The indirect method of the peroxidase- labeled antibody technique was used for immunocytochemistry of both LM and TEM on the cell lines. The cells were dehydrated in ethanol and embedded in TAAB 812. Ultrathin sections were observed under a TEM, JEM-100S.

Download Full-text

Absence of temporal ordering of apoptotic features in heat-shock treated leukemia and lymphoma cell lines

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166257 ◽

1996 ◽

Vol 54 ◽

pp. 758-759

Author(s):

D. W. Fairbain ◽

M.D. Standing ◽

K.L. O'Neill

Keyword(s):

Heat Shock ◽

Cell Lines ◽

Chromatin Condensation ◽

Membrane Integrity ◽

Resistant Cell ◽

Membrane Blebbing ◽

Late Loss ◽

Induced Apoptosis ◽

Dying Cells ◽

In Cells

Apoptosis is a genetically defined response to physiological stimuli that results in cellular suicide. Features common to apoptotic cells include chromatin condensation, oligonucleosomal DNA fragmentation, membrane blebbing, nuclear destruction, and late loss of ability to exclude vital dyes. These characteristics contrast markedly from pathological necrosis, in which membrane integrity loss is demonstrated early, and other features of apoptosis, which allow a non-inflammatory removal of dead and dying cells, are absent. Using heat shock-induced apoptosis as a model for examining stress response in cells, we undertook to categorize a variety of human leukemias and lymphomas with regard to their response to heat shock. We were also interested in determining whether a common temporal order was followed in cells dying by apoptosis. In addition, based on our previous results, we investigated whether increasing heat load resulted in increased apoptosis, with particular interest in relatively resistant cell lines, or whether the mode of death changed from apoptosis to necrosis.

Download Full-text