Intratumoral cytogenetic heterogeneity detected by comparative genomic hybridization and laser scanning cytometry in human gliomas

Although it is well-known that cancers show intratumoral phenotypic heterogeneity, genotypic studies have been scarce. Using comparative genomic hybridization and laser scanning cytometric analyses, we investigated intratumoral cytogenetic heterogeneity in 21 surgically removed gliomas including 11 glioblastomas (GBMs), 8 anaplastic astrocytomas (AAs) and 2 low-grade astrocytomas. Comparative genomic hybridization analysis revealed gain or amplification of 7p in 63%, gain of 7q in 73%, loss of 9p in 53%, loss of 10p in 47%, loss of 10q in 47%, loss of 13q in 53%, and loss of 22q in 37% of high-grade astrocytomas. Because these aberrations were region-independent within the same tumor, they did not contribute to intratumoral cytogenetic heterogeneity. Such heterogeneity was due to cytogenetic changes other than the above region-independent aberrations. Intratumoral cytogenetic heterogeneity was detected in 8 of 11 GBMs, 4 of 8 AAs, and none of the 2 low-grade astrocytomas. These observations suggest that cytogenetic changes at chromosomes 7, 9p, 10, 13, and 22 are primary events in high-grade astrocytomas and that subsequent cytogenetic changes involving increases in copy number provide intratumoral heterogeneity. DNA aneuploidy was detected by laser scanning cytometry in 5 of 11 GBMs and 1 of 8 AAs. All tumors with DNA aneuploidy exhibited intratumoral cytogenetic heterogeneity, and there was a significant correlation between DNA aneuploidy and intratumoral cytogenetic heterogeneity. These results support the notion that cytogenetic heterogeneity results from genetic instability within a tumor.     

Detection of human papillomavirus DNA in CaSki and HeLa cells by fluorescent in situ hybridization. Analysis by flow cytometry and confocal laser scanning microscopy

CaSki and HeLa cell lines, isolated from human uterine carcinomas and containing integrated human papillomavirus (HPV) DNA type 16 and 18, respectively were used to evaluate the sensitivity of HPV-DNA detection on suspended cells by fluorescent in situ hybridization using flow cytometry and on corresponding cell deposits using confocal laser scanning microscopy (CLSM). HPV DNAs were detected in cell suspensions with biotinylated DNA probes and revealed with a three-step technique: a rabbit antibiotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. By flow cytometry, HPV DNA was detectable only in CaSki cells which contained about 600 copies of HPV DNA per cell. In HeLa cells, with only 20-50 copies of HPV DNA, flow cytometry could not detect HPV DNA, whereas CLSM permitted visualization of fluorescent labelling of HPV DNA hybrids. Furthermore, CLSM showed good preservation of cellular morphology and the nucleus was clearly recognizable after fluorescent in situ hybridization and counterstaining with propidium iodide. Moreover, this examination confirmed that the fluorescent foci were specifically confined to the cell nuclei.     

Two-dimensional blood flow determinations in allergic reactions using laser Doppler scanning

The new technique of laser Doppler scanning (LDS) provides a 2-dimensional pattern of cutaneous microcirculation, which offers a visual image and can quantify the intensity and expansion of perfusion. With the help of this technique, we examined the microcirculatory pattern of Type IV reactions to recall antigens, which were applied using a test stamp (Multitest Merieux). The measurements were performed before application of the test stamp as well as 10 min, 24, 48 and 72 h afterwards. The inflammatory hyperemia was evaluated using LDS and unidimensional laser Doppler fluxmetry. The diameter of the inflammatory infiltrate was quantified by means of palpation, the thickness by means of high-resolution 20 MHz sonography. The clinically visible erythema was measured planimetrically. An unspecific hyperemia resulting from the trauma of the stamp revealed no evident infiltrate under sonography 10 min after the test application. Depending of the individual reaction, the mean flux and the expansion of the hyperemia were at their peak after 48 h. The flux values were at a maximum in the center of the inflammatory reaction and dropped continuously toward the periphery. The area of the hyperemia seen in the LDS image was significantly larger than the expansion of the erythema measured planimetrically, but there was a significant correlation. The perfusion correlated significantly with the infiltration diameter (24 h, 48 h, 72 h) and the infiltration thickness 48 h after testing. All in all, it was possible to measure directly and without touching the skin and to quantify a subclinical pattern of skin perfusion as a response to and inflammatory reaction on a 2-dimensional display.     

Prenatal diagnosis of a (X;X) translocation by fluorescence in situ hybridization and laser scanning image cytometry

A de novo structural abnormality of one X chromosome was prenatally detected in a female fetus. This chromosomal abnormality has been analyzed by conventional cytogenetic methods, fluorescence in situ hybridization, and laser scanning image cytometry. The association of these techniques has demonstrated that this anomaly corresponds to a (X;X) translocation. Analysis of hybridization signals by laser scanning image cytometry allowed to localize that the breakpoints were at the X-centromeric region and Xp11.3, respectively. These results show the usefulness of image analysis and fluorescence in situ hybridization for a rapid characterization of de novo structural chromosome anomalies in prenatal diagnosis.     

Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry

To measure the average length of telomere repeats at chromosome ends in individual cells we developed a flow cytometry method using fluorescence in situ hybridization (flow FISH) with labeled peptide nucleic acid (PNA) probes. Results of flow FISH measurements correlated with results of conventional telomere length measurements by Southern blot analysis (R = 0.9). Consistent differences in telomere length in CD8+ T-cell subsets were identified. Naive and memory CD4+ T lymphocytes in normal adults differed by around 2.5 kb in telomere length, in agreement with known replicative shortening of telomeres in lymphocytes in vivo. T-cell clones grown in vitro showed stabilization of telomere length after an initial decline and rare clones capable of growing beyond 100 population doublings showed variable telomere length. These results show that flow FISH can be used to measure specific nucleotide repeat sequences in single cells and indicate that the very large replicative potential of lymphocytes is only indirectly related to telomere length.     

Visual and laser Doppler perfusion scanning assessments of Kathon CG patch test reactions

Metastases are the most common malignant tumors of the skeleton. Several imaging modalities can be engaged for the diagnosis of skeletal metastases. They may be combined, depending on the individual clinical setting. Plain films are used for evaluation of symptomatic regions of the skeleton. Sensitivity is low, but benign skeletal disorders causing clinical symptoms can usually be identified in plain films. Bone scintigraphy is employed for a survey of the entire skeleton with high sensitivity for the presence of metastases, but only poor specificity. Computed tomography (CT) and magnetic resonance imaging (MRI) are advanced diagnostic methods, essential particularly in the diagnosis of metastatic spinal disease. Using CT, additional percutaneous bone biopsies can be performed easily and safely. Of all imaging modalities, MRI has the highest sensitivity for skeletal metastases. Other advantages are the possibility of multidirectional slice positioning and excellent soft tissue contrast.     

Demonstration of an Na+/H+ exchanger in mouse keratinocytes measured by the novel pH-sensitive fluorochrome SNARF-calcein

In many cell types cytoplasmic alkalization is an early marker for cell activation. An amiloride-sensitive Na+/H+ exchanger is an important regulator of this process. However, in keratinocytes the existence of a Na+/H+ exchanger nor a proliferation-associated increase in intracellular pH (pHi) has been demonstrated. The aim of this study was to investigate whether or not keratinocytes, derived from the BALB/MK cell line, contain a Na+/H+ exchanger and whether cytoplasmic alkalization is proliferation-associated in these cells. This mouse keratinocyte cell line can easily be switched between a proliferative and a quiescent state under defined culture conditions. The novel pH-sensitive dye seminaphthorhodafluor (SNARF)-calcein proved to be very suitable for flow cytometric pHi measurements in BALB/MK cells. Initial measurements of the pHi using a cocktail of the established fluorochromes 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and SNARF-1 failed because of the differential uptake and binding kinetics of these pH-sensitive dyes. Using SNARF-calcein we were able to show proliferation to be associated with increased pHi. However, culture conditions were critical for these measurements. Our data indicate that the Na+/H+ exchanger is involved in this process, since acid load and pHi-recovery experiments showed the alkalization to be amiloride-sensitive.     

Estimation of specific glucose uptake rates in cultures of hybridoma cells

The specific glucose uptake rate of a hybridoma cell line was determined in batch and continuous suspension cultures. For high density cultivation a membrane dialysis reactor was used. The aim was to obtain data, which can be used for process design and control. A decrease of the specific glucose uptake rate was observed during the course of batch cultures, even when the specific growth rate remained constant. These results are valid for cell densities between 2 x 10(5) and 2 x 10(6) ml-1. At cell densities between 2 x 10(6) and 1.5 x 10(7) ml-1 during continuous cultivation the specific glucose uptake rate remained constant. A relationship between the specific glucose uptake rate and the cell number was found and formulated in a mathematical equation. Literature data for different hybridoma cell lines fit in this plot very well. Applying this relationship, a correct estimation of the course of the glucose concentration in batch and continuous cultivation is possible.     

Three-dimensional appearance of Langerhans cells in human gingival epithelium as revealed by confocal laser scanning microscopy

Despite the reducing exposure to allogeneic blood in cardiac surgery, most of patients with anemia still require allogeneic blood. In this study, we have attempted to harvest the blood from cardiac patients with baseline hemoglobin levels below 11.0 g/dl using recombinant human erythropoietin (rHuEPO). 29 anemic patients undergoing cardiac surgery at our hospital between January 1994 and March 1997 were divided into two groups: 3 weeks' treatment with recombinant human erythropoietin (rHuEPO) and blood donation (group 1, n = 15) and iron supplementation alone (group 2, n = 14). There were no statistically significant differences among the two groups in patients characteristic and surgical data. No serious adverse events after phlebotomy were apparent in patients donating autologous blood. Patients in group 1 had significantly higher hemoglobin levels than patients in group 2 at 7 days before operation. The number of reticulocytes were increased at just before operation in group 1, whereas group 2 showed no significant increase. The estimated hemoglobin increase in group 1 were higher at 7 days and just before operation. In 75% of group 1, allogeneic blood transfusion could be avoided, while all patients in group 2 received allogeneic blood transfusion. This study suggests that the combination of rHuEPO administration and autologous blood donation would be beneficial for anemic patients in elective cardiac surgery. The use of rHuEPO should not be restricted to anemic patients.     

The incorporation of various porphyrins into blood cells measured via flow cytometry, absorption and emission spectroscopy

The incorporation of the five following porphyrins: meso-tetra(4-phenyl)porphyrin (TPP); meso-tetra(4-sulfonato-phenyl)porphyrin (TPPS4); meso-tetra(4-naphthyl)porphyrin (TNP); tri-sulfo-tetra-phenyl porphyrin (TPPS3) and tetra-sulfonato-naphthyl porphyrin (TNPS4) into human blood cells was investigated using flow cytometry, and absorption and emission spectroscopy. The percentage of stained cells, measured in a fluorescence cytometer, provided information on the efficiency of incorporation of fluorescent dye molecules into different types of cells. The yield of the incorporation of a dye was dependent on the type of dye and the solvent used for cell incubation. The degree of dye aggregation and ionization varied with the incubation medium, but dye molecules incorporated into cells seemed to be restricted to those in the monomeric state, exhibiting similar fluorescence yield. Of the three sulfonated porphyrins investigated only TPPS4 was efficiently incorporated into leukocytes. In the incubation solvent, this dye was in monomeric and neutral form. TPPS3 which was also in monomeric form, practically was not incorporated into cells. TPP and TNP dissolved in 5% aqueous dimethyl sulfoxide were present mostly in aggregated forms but they penetrated the cells with a high efficiency.     

DNA content and kinetic characteristics of non-Hodgkin's lymphoma: determined by flow cytometry and autoradiography

The determination of DNA content and [3H]thymidine labeling index was carried out on malignant lymph nodes from 74 patients with non-Hodgkin's lymphoma. Analysis of cellular DNA content was performed using propidium iodide as DNA-specific fluorescence dye. The ploidy was expressed as the DNA ratio between the relative DNA content of the human lymphoid G0/1 cells to that of chicken red blood cells. Forty-five of the 74 non-Hodgkin's lymphomas (61%) were aneuploid populations and the majority of these (91%) showed a hyperdiploid DNA content. A higher frequency of aneuploidy (72%) was observed in tumors with unfavorable histology than in those with a favorable histology (55%). Moreover, among aneuploid lymphomas heterogeneous populations were observed in 24% of the cases. The evaluation of flow cytometric data using Fried's deconvolution procedure showed no statistically different frequency of G0/1, S and G2 + M cells between the two groups of tumors with favorable and unfavorable histology. on the contrary, a statistically different frequency of G0/1 and S cells was observed between the two groups of tumors with low and high labeling indices (P less than 0.01). A correlation was found between autoradiographic and flow cytometric determination of S phase cells (P less than 0.001).     

Expression of cyclins A, D2 and D3 in individual normal mitogen stimulated lymphocytes and in MOLT-4 leukemic cells analyzed by multiparameter flow cytometry

Cyclins are regulatory subunits of the cyclin dependent kinases (CDKs), the enzymes that drive the cell through the respective phases and check-points of the cell cycle. The expression of cyclins in non-tumor cells, regulated by timely induction of their synthesis and proteolysis, is scheduled, occurring at discrete periods of the cell cycle. Using multiparameter flow cytometry we have recently observed that expression of cyclins B1 and E in individual normal lymphocytes mitogenically stimulated by phytohemagglutinin (PHA) and lymphocytic leukemic MOLT-4 cells was similar, restricted to particular phases of the cycle: cyclin B1 was detected only in G2+M- and cyclin E in late G1 and early S-phase cells. In the present study we have measured the expression of cyclins A, D2 and D3 in these cells. The presence of cyclin A was restricted to late S and G2 phases, both in the case of lymphocytes and of MOLT-4 cells. Over 95% of the non-stimulated lymphocytes were both cyclin D2 and D3 negative. Mitogenic stimulation with PHA-induced expression of cyclins D2 and D3 in over 50% cells, which corresponds to the percentage of cells that respond to this mitogen in cultures. Expression of these proteins peaked between 8 and 24 h after addition of PHA, and then decreased at the time of cell entrance to S. During exponential growth (48-72 h after stimulation with PHA) expression of the D-type cyclins was diminished: only between 5-10% of the lymphocytes had levels of cyclin D3 as high as G1 cells between 8-24 h after PHA stimulation. Populations of proliferating lymphocytes and MOLT-4 cells were very heterogeneous in terms of expression of D-type cyclins by individual cells. While expression of cyclin D2 in exponentially growing MOLT-4 cells was similar to that of proliferating lymphocytes, the percent of cells expressing cyclin D3 as well as the degree of expression, was higher in MOLT-4 cells, regardless of the phase of the cycle. These results, with our earlier observations of the untimely expression of cyclins B1 and E in several other tumor lines, suggest that altered expression of cyclins may be a frequent feature of malignancy.     

Assessment of cornea viability by confocal laser scanning microscopy and MTT assay

PURPOSE: Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness. METHODS: New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature. At various times, corneas were stained with an EH-1/CAM solution and observed, without further treatment, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay. RESULTS: Stromal swelling, shedding of the upper epithelial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was observed, along with loss of endothelial structure. Corneas stored in similar conditions in Optisol were indistinguishable from controls. Storage in Optisol at 4 degrees C affected the superficial layers of the corneal epithelium similarly at both 7 and 14 days. Extensive epithelial shedding and wing-cell death were observed at 25 days, but the basal layer remained approximately 50% healthy. Significant endothelial cell loss was observed at 25 days. MTT results were consistent with CLSM data in the medium-term storage study only. CONCLUSIONS: This CAM/EH-1 CLSM fluorescence assay is a sensitive index of viability in cornea, and thus may prove useful in investigations in which maintenance of vital functions in different cell layers is critical.     

Comparison of bone formation rates measured by radiocalcium kinetics and double-tetracycline labeling in maintenance dialysis patients

We report 23 prospective studies on 18 maintenance dialysis patients in whom we measured skeletal mineralization rate (m) using 47Ca, analyzed by the expanding pool model, and compared it with the histologic bone formation rate (bfr), volume referent, estimated on tetracycline-labeled iliac crest bone. The patients showed a spectrum of bone disease types including adynamic bone, aluminum-related osteomalacia, and various degrees of secondary hyperparathyroidism. The mean width between double labels, on which mineral apposition rate depended, was estimated using a simple formula relating area to perimeter for each feature enclosed by the labels. Values for m ranged from 0 to 155 mmol calcium per day and for bfr from 0 to 124% per year. There was close correlation between m and bfr (r = 0.976), serum alkaline phosphatase (r = 0.968), and serum immunological parathyroid hormone (iPTH) (r = 0.868). When the volumetric bfr was converted to mass units and applied to the whole skeleton, using literature values for mineral density and cortical and trabecular mass, there was close agreement between the histologic and isotopic estimates of m (r = 0.959). The results validate the two methods and suggest they are interchangeable. However, use of a rigorous method to determine bfr appears to be essential.     

Specificity of seven monoclonal antibodies against p53 evaluated with Western blotting, immunohistochemistry, confocal laser scanning microscopy, and flow cytometry

The use of occupation as a therapeutic method is the essence of the profession of occupational therapy. This core of therapeutic occupation is flexible across cultures, times, health care environments, and different philosophies of the nature of the human being. Given this adaptability, the profession espouses diverse models of practice--the multiple frames of reference that guide therapeutic occupation for different populations in different settings. Across the history of the profession, therapeutic occupation has been the common core of otherwise different approaches to intervention. Although each of the many past and current models of practice has a different viewpoint, the common factor is the synthesis of occupational forms designed to elicit meaningful and purposeful occupational performance. Occupational synthesis is the essential act of the occupational therapist. It is necessary that occupational therapists confirm the power of therapeutic occupation through research that examines the profession's central principles. Occupational therapists are also urged to use the term occupation consistently and proudly in their interactions with recipients of therapy, fellow health care professionals, and each other. The profession of occupational therapy will flourish because occupation, its core, is so basic to human health yet so flexible, depending on the needs of the individual human being.     

18F]fluorodeoxyglucose uptake in neonatal acute lung injury measured by positron emission tomography

PURPOSE: Our goal was to investigate the role of serial dynamic contrast-enhanced SPGR MRI in the nonsurgical follow-up of patients with temporomandibular joint (TMJ) pain. METHOD: Ten patients (10 joints) with internal derangement of the TMJ were imaged with T1-weighted SE and serial postgadolinium SPGR MR pulse sequences. RESULTS: On T1-weighted images prior to treatment, the disk position was normal in one joint and anteriorly displaced without reduction in nine joints. After treatment, the disk remained normally positioned in one joint, was anteriorly displaced without reduction in eight joints, and was anteriorly displaced with reduction in one joint. The dynamic study after treatment showed a decrease in contrast enhancement of the posterior disk attachment in 7 of 10 joints. These seven patients had resolution or reduction in joint pain. CONCLUSION: These results suggest an association between a decrease in contrast enhancement of the posterior disk attachment and resolution or reduction in joint pain. This association was much stronger than the association between the clinical findings and the anatomy of the disk.     

Cell contraction caused by microtubule disruption is accompanied by shape changes and an increased elasticity measured by scanning acoustic microscopy

The relationships within the superorder Archonta, which contains the orders Dermoptera (flying lemurs), Scandentia (tree shrews), Chiroptera (bats), and Primates, were examined through the analysis of five newly derived and complete mitochondrial 12S rRNA sequences. The new data is combined with 83 additional known mammalian sequences to provide a full phylogenetic sampling. Phylogenetic hypotheses are generated using PAUP 3.1.1 (Swofford [1993] Illinois Natural History Survey, Champaign, IL) through analyses of all characters equally weighted, transversions only, and the effect of alignment gaps on phylogeny. The Parsimony Jackknifer (Farris et al. [1996] Cladistics 12:99-124) was used to assess the level of ambiguity present in the sequence data, and therefore the strength of the tree topologies. The conclusions of Springer and Douzery (1996, J. Mol. Evol. 43:357-373) which states that 12S rRNA is reliable to a time depth of 100 mya is unsupported by these analyses. The usefulness of 12S rRNA to aid in solving Archonta relationships and others of similar time depth is found to be suspect.