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PubMedDate not supplied

Nerve growth factor (NGF) promotes angiogenesis in the quail chorioallantoic membrane.

Angiogenesis, the formation of new blood vessels, is tightly regulated by growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). The authors hypothesize that nerve growth factor (NGF), a well known neurotrophin, may play a direct angiogenic role. To test this hypothesis, the authors measured the effects of NGF on the natural vascularization of the quail chorioallantoic membrane (CAM). The angiogenic effect of NGF was compared to that of human recombinant VEGF165 (rhVEGF) and basic FGF (rhbFGF). In comparison to phosphate-buffered saline-treated controls, NGFs from different biological sources (mouse, viper, and cobra) increased the rate of angiogenesis in a dose-dependent fashion from 0.5 to 5 microg. For quantitative morphometry, grayscale images of the blood vessels end points of the CAM arteries were binarized for visualization and skeletonized for quantization by fractal analysis. In mouse NGF-treated embryos the fractal dimension (Df), indicative of arterial vessel length and density, increased to 1.266 +/- 0.021 compared to 1.131 +/- 0.018 (p < .001) for control embryos. This effect was similar to that of 0.5 microg rhVEGF (1.290 +/- 0.021, p < .001) and 1.5 microg rhbFGF (1.264 +/- 0.028, p < .001). The mouse NGF-induced angiogenic effect was blocked by 1 microM K252a (1.149 +/- 0.018, p < .001), an antagonist of the NGF/trkA receptor, but not by 1 microM SU-5416 (1.263 +/- 0.029, p < .001), the VEGF/Flk1 receptor antagonist, indicating a direct, selective angiogenic effect of NGF via quail embryo trkA receptor activation. These results confirm previous observations that NGF has angiogenic activity and suggest that this neurotrophin may also play an important role in the cardiovascular system, besides its well-known effects in the nervous system. The angiogenic properties of NGF may be beneficial in engineering new blood vessels and for developing novel antiangiogenesis therapies for cancer.

Angiogenesis Inducing Agents↗
PubMed2004

Detection of isolated tumor cells in peripheral blood and in BM: evaluation of a new enrichment method.

Cell enrichment methods that deal with larger volumes of peripheral blood and BM are needed for increased sensitivity of detection, characterization and quantification of isolated tumor cells (ITC). This study was designed to evaluate a new procedure, the RosetteSep-Applied Imaging Rare Event (RARE) detection method, which depletes the majority of the erythrocytes and leucocytes in a peripheral blood (PB) sample, thereby negatively enriching tumor cells if present. This enrichment procedure allows for increased sensitivity, by analyzing a 5-10 fold larger volume of blood, compared with a direct immunocytochemical (ICC) technique, with minimal impact on laboratory workload. Model experiments showed comparable tumor cell recoveries between the two tested methods, both in PB and BM. Clinical samples were evaluated using paired PB and BM samples from 95 carcinoma patients. Analysis of PB results showed that 25.3% had > or = 1 tumor cell detected by the RARE procedure, compared with 5.2% after direct ICC analysis, analyzing a 10-fold larger volume by the RARE procedure. The direct ICC analysis of BM from the same patients revealed 16.8% positive. The ITC detection differed both quantitatively and qualitatively between BM and PB, as samples with high numbers of ITC in BM were still negative in PB. The clinical significance of ITC in blood still needs to be established. However, the easy access of peripheral blood, and the increased sensitivity obtained by increasing the sample volume with the RARE procedure, suggests that the value of peripheral blood analysis should be tested in parallel in studies where ITC detection in BM is performed.

Antibodies, Monoclonal↗
PubMed2004

Immunomodulation of chickens with the nonionic block polymer T150R1.

Chickens injected subcutaneously at one day of age with oil-in-water emulsions of the nonioinic block polymer T150R1 developed higher titres to sheep erythrocytes and killed Brucella abortus than did untreated chickens when they were immunised with the antigens one and two weeks later. The increase was IgM only. Cell-mediated immunity was not affected as measured by blastogenic responses. Morphometric analysis of tissue sections of bursae of Fabricius suggested the polymers were stimulating more rapid migration of B cells to peripheral organs.

Adjuvants, Immunologic↗
PubMed2004

The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles.

Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This "poleward flux" of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose-responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.

Animals↗
PubMed2006

From imaging to understanding: Frontiers in Live Cell Imaging, Bethesda, MD, April 19-21, 2006.

A recent meeting entitled Frontiers in Live Cell Imaging was attended by more than 400 cell biologists, physicists, chemists, mathematicians, and engineers. Unlike typical special topics meetings, which bring together investigators in a defined field primarily to review recent progress, the purpose of this meeting was to promote cross-disciplinary interactions by introducing emerging methods on the one hand and important biological applications on the other. The goal was to turn live cell imaging from a "technique" used in cell biology into a new exploratory science that combines a number of research fields.

Animals↗
PubMed2004

CD152 (CTLA-4) determines the unequal resistance of Th1 and Th2 cells against activation-induced cell death by a mechanism requiring PI3 kinase function.

Survival of antigen-experienced T cells is essential for the generation of adaptive immune responses. Here, we show that the genetic and antibody-mediated inactivation of CD152 (cytotoxic T lymphocyte antigen 4) in T helper (Th) effector cells reduced the frequency of nonapoptotic cells in a completely Fas/Fas ligand (FasL)-dependent manner. CD152 cross-linking together with stimulation of CD3 and CD28 on activated Th2 cells prevented activation-induced cell death (AICD) as a result of reduced Fas and FasL expression. Apoptosis protection conferred by CD152 correlated with the up-regulation of Bcl-2 and was mediated by phosphatidylinositol 3 kinase, which prevented FasL expression through the inhibitory phosphorylation of Forkhead transcription factor FKHRL1. We show that signals induced by CD152 act directly on activated T lymphocytes and, due to its differential surface expression on activated Th1 and Th2 cells, induce resistance to AICD mainly in Th2 cells.

Animals↗
PubMed2003

Ratiometric fluorescence polarization as a cytometric functional parameter: theory and practice.

The use of ratiometric fluorescence polarization (RFP) as a functional parameter in monitoring cellular activation is suggested, based on the physical phenomenon of fluorescence polarization dependency on emission wavelengths in multiple (at least binary) solutions. The theoretical basis of this dependency is thoroughly discussed and examined via simulation. For simulation, aimed to imitate a fluorophore-stained cell, real values of the fluorescence spectrum and polarization of different single fluorophore solutions were used. The simulation as well as the experimentally obtained values of RFP indicated the high sensitivity of this measure. Finally, the RFP parameter was utilized as a cytometric measure in three exemplary cellular bioassays. In the first, the apoptotic effect of oxLDL in a human Jurkat FDA-stained T cell line was monitored by RFP. In the second, the interaction between cell surface membrane receptors of human T lymphocyte cells was monitored by RFP measurements as a complementary means to the fluorescence resonance energy transfer (FRET) technique. In the third bioassay, cellular thiol level of FDA- and CMFDA-labelled Jurkat T cells was monitored via RFP.

Computer Simulation↗
PubMed2002

Histomorphometric analysis of a cell-based model of cartilage repair.

This study examined the morphology of chondrocytes in an established model of articular cartilage repair. Articular cartilage was harvested from young sheep and seeded onto pieces of devitalized sheep cartilage. The seeded pieces were stacked in pairs and wrapped in fibrin glue, and then implanted subcutaneously in the dorsum of athymic mice. Samples were harvested after 6 weeks and examined by transmission electron microscopy (TEM) or by light microscopy. TEM revealed that the cells in direct apposition to the devitalized cartilage were elongated, with an enlarged cytoplasm, and a ruffled border. TEM of cells far from the interface with scaffold tissue revealed rounded cells with large nuclei that appeared similar to normal chondrocytes. Quantitative morphometry of histologic specimens revealed that cell area, relative amount of cytoplasm, cell aspect ratio, and relative nuclear displacement were all higher in cells near the interface with the scaffold tissue, and decreased with distance from the interface. These indices of cell morphology are all consistent with an active remodeling of the scaffold at the cell-scaffold interface.

Animals↗
PubMed2005

Lack of a gender difference in post-traumatic neurodegeneration in the mouse controlled cortical impact injury model.

Recent studies using a mouse model of weight-drop-induced "diffuse" traumatic brain injury (TBI) have demonstrated a substantial gender difference in the time course and magnitude of post-traumatic neurodegeneration following a severe level of injury. The time of maximal damage, as assessed by the de Olmos aminocupric silver staining method, occurred at 72 h in male mice, whereas the peak of neurodegeneration was not observed until 14 days in females and was less in magnitude compared to males. This difference, favoring females, has been postulated to relate to the neuroprotective actions of estrogen and progesterone. In the presently reported experiments, we compared the time course and peak of neurodegeneration in male and female mice after a severe level of "focal" controlled cortical impact (CCI; 1 mm, 3.5 m/sec) TBI using the same strain (CF-1) and weight (29-31 g) as employed in the "diffuse" TBI study. The volume of silver staining was measured using image analysis methods at 24, 48, and 72 h, and 1, 2 and 4 weeks. In male and female mice, a significant increase in neurodegeneration was observed at 24 h, and the volume was not significantly different between the two genders. In both gender groups, the maximal neurodegeneration was seen at 48 h after injury. Although the female mice exhibited a trend toward higher mean volumes of silver staining, this difference was not significantly different compared to males. Furthermore, the rate of resolution of staining between 48 h and 4 weeks was similar. However, injured females still exhibited a significantly higher volume of staining compared to sham, non-injured females at 4 weeks, whereas the difference in staining volume between sham and injured males was no longer significant at that time point. These results show that, following a "focal" CCI, there is no gender difference that favors females, in contrast to that seen with the "diffuse" injury paradigm. The disparity between the effects of gender in the two models may be due to the fact that, in the "focal" CCI model, the timing of post-traumatic neurodegeneration is significantly more rapid than that seen in the "diffuse" model, which may overwhelm the neuroprotective effects of estrogen and progesterone and obscure the appearance of a gender difference.

Animals↗
PubMed2002

Protein kinase Cepsilon actin-binding site is important for neurite outgrowth during neuronal differentiation.

We have previously shown that protein kinase Cepsilon (PKCepsilon) induces neurite outgrowth via its regulatory domain and independently of its kinase activity. This study aimed at identifying mechanisms regulating PKCepsilon-mediated neurite induction. We show an increased association of PKCepsilon to the cytoskeleton during neuronal differentiation. Furthermore, neurite induction by overexpression of full-length PKCepsilon is suppressed if serum is removed from the cultures or if an actin-binding site is deleted from the protein. A peptide corresponding to the PKCepsilon actin-binding site suppresses neurite outgrowth during neuronal differentiation and outgrowth elicited by PKCepsilon overexpression. Neither serum removal, deletion of the actin-binding site, nor introduction of the peptide affects neurite induction by the isolated regulatory domain. Membrane targeting by myristoylation renders full-length PKCepsilon independent of both serum and the actin-binding site, and PKCepsilon colocalized with F-actin at the cortical cytoskeleton during neurite outgrowth. These results demonstrate that the actin-binding site is of importance for signals acting on PKCepsilon in a pathway leading to neurite outgrowth. Localization of PKCepsilon to the plasma membrane and/or the cortical cytoskeleton is conceivably important for its effect on neurite outgrowth.

Actins↗
PubMed1996

Quality control of proliferation marker (MIB-1) in image analysis systems utilizing cell culture-based control materials.

Standard controls for quality control of cell growth (proliferation) assays in image analysis systems are not currently available. The authors have developed a system of controls, based on cultured and harvested human cell lines, that can mimic tissue sections. These controls help ensure quality control for the entire cell proliferation analysis process, from the initial cutting of the paraffin block through fixation, immunohistochemical staining, and interactive image analysis. The use of cell line controls is advantageous because of the greater cell population homogeneity and the volume of uniform slides that are obtainable, as opposed to the use of a heterogeneous tissue sample. This system provides an excellent means of evaluating the day-to-day performance of cell proliferation analysis and may also be adapted for use as a method of multi-institutional proficiency testing.

Biomarkers, Tumor↗
PubMed1996

Quantitative immunocytochemical assays on frozen sections of p53: correlation to the follow-up of patients with breast carcinomas.

A series of 222 tumor samples stored at -80 degrees C in the authors' tumor library were investigated with anti-p53 (PA 1801) and streptavidin-biotin-peroxidase complex. The p53 immunoprecipitates were quantified by densitometry assessed by image analysis of digitized microscopic images. Two parameters, percentage of positive surface and mean optical densities, were compared with the patient's outcome (follow-up = 96.8 months) (life table method, Mantel Cox test, BMDP statistical software). The p53 expression significantly correlated with a poor overall survival (P = .0063), metastasis-free survival (P = .024), and recurrence-free survival (P = .022) at a 20% cutoff point of positive immunoreactive tumor surface. A strong prognostic significance was observed in the node-positive subset of patients but not in the node-negative subset, except for recurrence-free survival (P = .047). The results indicate the clinical relevance p53 evaluated by quantitative immunocytochemistry.

Adult↗
PubMedDate not supplied

Cytoarchitectonic definition of prefrontal areas in the normal human cortex: I. Remapping of areas 9 and 46 using quantitative criteria.

The classical cytoarchitectonic maps of human prefrontal areas produced by various cartographers in the early part of this century, though similar in gross topography, differ from one another in their descriptions of the size, shape, and precise location of specific regions within the frontal promontory. The current advances in human neurobiology stimulated us to reinvestigate the cytoarchitecture of the human prefrontal cortex, beginning with areas 9 and 46, to establish a set of objective cytometric criteria for identification of these areas. Nisslstained and Gallyas-stained celloidin-embedded sections were prepared from the left hemispheres of 17 human subjects 23-73 years old, without history of neurological disease. In eight cases, light microscopic observations were supplemented by morphometric data collected on a research microscope equipped with differential interference contrast optics and interfaced to a TV monitor with video mixing equipment and a microcomputer. We used the three-dimensional counting method of Williams and Rakic (1988) to measure (1) total cortical and relative laminar thickness, (2) neuronal packing density per 0.001 mm3 in individual cortical layers, and (3) sizes of neuronal somata in selected cortical layers. Light microscopic analysis confirmed that the cortical layers are more differentiated in area 46 than in area 9, particularly at the borders of layer IV. Layers III and V exhibit clearer sublamination in area 9, while layer IV is also somewhat wider in area 46 than in area 9 (9.3% vs 6.4% of cortical thickness); the overall thickness of the cortex is the same in both areas. Cytometric analysis revealed that layer IV neurons of area 46 are more densely packed than those in area 9 (55.38 +/- 7.26 vs 45.80 +/- 4.45 neurons/0.001 mm3), as are neurons in the supragranular layers II and III combined (53.51 +/ 6.33 vs 45.69 +/ 3.81 neurons/0.001 mm3). Finally, neurons in area 46 are more homogeneous in size than those in area 9. Differences in myeloarchitecture are also evident: each area contains numerous, well-stained radial striae and two pronounced bands of horizontal fibers, but in general, area 46 is less myelinated than area 9. Objective cytometric methods can clearly distinguish two adjacent areas within the human prefrontal lobe. These findings may prove useful in the areal parcellation of the human cerebral cortex as well as provide a baseline for analysis of pathological changes in neurological and psychiatric disorders such as a schizophrenia, Huntington's or Alzheimer's diseases.

Adult↗
PubMed1998

Cytomorphometric analysis of vaginal cells during normal cycle, under oral contraceptive therapy or in-vitro fertilization stimulation protocol.

A cytomorphometric analysis of superficial vaginal cells in women in three groups of different types of hormonal concentration was made. There were 15 women in each group. Group I was studied during a natural cycle, group II under oral contraceptive therapy and group III during an in-vitro fertilization (IVF) stimulation protocol. Morphometric parameters were measured on an image analyser. The area, perimeter and several form factors were measured separately for nuclei and cytoplasm. The nucleus:cytoplasmic ratio was also determined. The cytoplasmic area was significantly reduced in group II and was associated with a statistically significant reduction of the nuclear area. The nucleus:cytoplasmic ratio appeared significantly increased in group II and reduced in group III. Low oestradiol impregnation obtained with an oral minidosed contraceptive interfered with vaginal cell maturation. High oestradiol concentrations obtained during IVF protocols induced marked nuclear pycnosis but did not induce supra-physiological cell enlargement. Maximal cell size is genetically regulated according to Driesch's law of volume invariance and hormonal over-stimulation has no effect on cell size. The nucleus:cytoplasmic ratio appears to be a powerful parameter reflecting the opposite effects of hormones on cell compartments.

Adult↗
PubMed1999

Morphometric assessment of mature and diminished-maturity human spermatozoa: sperm regions that reflect differences in maturity.

As part of our studies on sperm maturity and function, we examined the head, midpiece and tail of human spermatozoa using computerized morphometry in order to determine which regions reflect the differences between mature spermatozoa and spermatozoa of diminished cellular maturity. We studied 20 men, who were divided into two groups based on their lower (LCKM: 14.6 +/- 7.0%, n = 8) and higher sperm creatine kinase (CK-M) isoform ratios (HCKM: 48.0 +/- 4.3%, n = 12) in the initial semen. Using a sequential centrifugation method which relies on the lower density of immature spermatozoa with retained extra cytoplasm, we prepared three sperm fractions with progressively declining maturity, as confirmed with CK-M isoform ratio measurements. Following the sequential fractionation, we affixed the spermatozoa to glass slides, stained the midpiece and the sperm contour, and photographed 25 spermatozoa in each of the 60 fractions (1509 spermatozoa in all). The spermatozoa were then individually digitized on the Image-1 system, and the dimensions of the head, midpiece, and tail were determined. While the data showed significant differences in the midpiece and tail dimensions between the mature and diminished-maturity sperm fractions, the head dimensions were similar and did not reflect sperm maturity. We postulated that the relationship between the biochemical markers of sperm maturity and sperm morphology is based on common spermiogenic events. The data support this idea. In immature spermatozoa in which cytoplasmic extrusion, CK-M isoform expression, and tail sprouting are all diminished, the retained extra cytoplasm in the midpiece and shorter tail length contribute to the morphological variations that we identified by morphometry and considered in sperm morphology. These morphometric features, in association with fluorochrome-coupled biochemical probes, can facilitate the identification of mature spermatozoa in computer-assisted semen analysis.

Adult↗
PubMed1995

Sperm morphology assessment: historical review in relation to fertility.

Careful analysis of sperm morphology has always been an important part of a routine semen examination. However, the usefulness of sperm morphology assessment as a predictor of a man's fertilizing potential has often been challenged due to different classification systems, various slide preparation techniques and inconsistency of analyses within and between laboratories. Automated sperm morphology analysis instruments may overcome the subjective nature of visual assessments of sperm morphology, but the technical problems are numerous and the validity of these instruments has still to be proven. Having reviewed the literature, it seems clear that there is general agreement concerning the clinical relevance and predictive value of this single semen parameter in vivo and in vitro. Nevertheless, even in cases of severe teratozoospermia, fertilization may be possible. Studies on the acrosome reaction are very promising for patients with severe sperm morphology abnormalities that do not have major effects on the fertilizing potential. Most promising is the development of intracytoplasmic sperm injection (ICSI) as the treatment of first choice in cases of severe teratozoospermia with failed fertilization in vitro. Normal fertilization and pregnancy rates can be obtained with ICSI in the presence of extreme teratozoospermia, suggesting that sperm morphology may be important in spermatozoa-zona binding, penetration and spermatozoon-oocyte fusion but fails to be of any predictive value once the spermatozoon reaches the cytoplasm of the oocyte. To conclude, accurate and strict sperm morphology assessment is very useful in evaluating a patient's fertilizing potential, with the main advantage that the methods used to examine this parameter are easy to learn on equipment found in most laboratories.

Classification↗
PubMed2023

GBMdeconvoluteR accurately infers proportions of neoplastic and immune cell populations from bulk glioblastoma transcriptomics data.

BACKGROUND: Characterizing and quantifying cell types within glioblastoma (GBM) tumors at scale will facilitate a better understanding of the association between the cellular landscape and tumor phenotypes or clinical correlates. We aimed to develop a tool that deconvolutes immune and neoplastic cells within the GBM tumor microenvironment from bulk RNA sequencing data. METHODS: We developed an IDH wild-type (IDHwt) GBM-specific single immune cell reference consisting of B cells, T-cells, NK-cells, microglia, tumor associated macrophages, monocytes, mast and DC cells. We used this alongside an existing neoplastic single cell-type reference for astrocyte-like, oligodendrocyte- and neuronal progenitor-like and mesenchymal GBM cancer cells to create both marker and gene signature matrix-based deconvolution tools. We applied single-cell resolution imaging mass cytometry (IMC) to ten IDHwt GBM samples, five paired primary and recurrent tumors, to determine which deconvolution approach performed best. RESULTS: Marker-based deconvolution using GBM-tissue specific markers was most accurate for both immune cells and cancer cells, so we packaged this approach as GBMdeconvoluteR. We applied GBMdeconvoluteR to bulk GBM RNAseq data from The Cancer Genome Atlas and recapitulated recent findings from multi-omics single cell studies with regards associations between mesenchymal GBM cancer cells and both lymphoid and myeloid cells. Furthermore, we expanded upon this to show that these associations are stronger in patients with worse prognosis. CONCLUSIONS: GBMdeconvoluteR accurately quantifies immune and neoplastic cell proportions in IDHwt GBM bulk RNA sequencing data and is accessible here: https://gbmdeconvoluter.leeds.ac.uk.

Humans↗
PubMed2002

Characterization of calcium oscillations in normal and benzo[a]pyrene-treated clone 9 cells.

Intracellular Ca2+ oscillations induced by oxytocin and vasopressin were analyzed in a rat liver cell line (Clone 9) in order to identify mechanisms by which benzo[a]pyrene (BaP) alters Ca2+ signaling patterns in these cells. Clone 9 cells exhibit an initial Ca2+ spike, followed by Ca2+ oscillations upon oxytocin or vasopressin treatment. The range of frequencies (maximum 110 mHz) was dependent on agonist concentration with a constant amplitude less than or equal to the amount of Ca2+ generated from the inositol trisphosphate (InsP(3))-sensitive pool. This study examined contributions of extracellular and intracellular pools to the frequency of Ca2+ oscillations and the role of membrane channels, second messengers, and different pharmacological reagents on the regulation of oscillation frequency in both control and BaP-treated cells. Results indicated that the Ca2+ oscillations are mainly due to inositol 1,4,5-triphosphate (InsP(3))-sensitive stores and that extracellular Ca2+ contributes to refilling of this intracellular Ca2+ pool. The frequency of Ca2+ oscillations is also sharply affected by protein kinase C activated by phospholipase C. In BaP-treated Clone 9 cells, basal Ca2+ levels were elevated and the frequency of Ca2+ oscillations was suppressed in a dose-dependent fashion. Suppression of Ca2+ oscillations is due, at least in part, to an effect of BaP on enhanced opening of K+ channels. This was confirmed by showing that inhibition of the K+ channel opening by tetraethylammonium chloride can reverse the effect of BaP on oxytocin-induced Ca2+ oscillations, and potentially decrease the toxicity of BaP.

Animals↗