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PubMed1985

Microtechnique for quantitative platelet isolation from blood enabling electronic counting and sizing of animal and human platelets.

A technique for rapidly separating platelets from small quantities of whole blood is described. The isolation method allowed for counting and sizing platelets by electronic means, and counts with this new method were closely correlated with those obtained by phase microscopy. Platelet sizing was possible because of the inert and isotonic nature of the density-gradient medium used in the procedure. The technique was applicable to samples of blood from persons, horses, cattle, sheep, goats, pigs, dogs, mice, rats, guinea pigs, and rabbits. The isolation of platelets from whole blood of various species of animals was necessary for electronic counting because of the great variation in sizes of platelets and erythrocytes and the variable sedimentation properties of animal blood.

Adult↗

PubMed1966

Water-soluble inhibitor(s) of tumor respiration formed from ultraviolet-induced oxidation of linoleic and linolenic acids.

Inhibition of Ehrlich ascites carcinoma respiration by aqueous extracts of oxidized linoleic or linolenic acid (aqueous emulsions UV-irradiated, 90 min) was associated entirely with relatively involatile compounds which were both thiobarbituric acid (TBA)-reactive and peroxidase-reactive. Inhibitory compounds were heat stable and migrated in thin-layer chromatography with aldehydes, "hydroperoxides," and TBA-reactive compounds. Peroxidase-catalyzed reduction of the "hydroperoxide" diminished the inhibition. At 4.7 x 10(-5) M "hydroperoxide" concentration, the residues from both linoleic and linolenic acid inhibited tumor oxygen consumption to a similar degree. However, at this concentration of "hydroperoxide" only the dried extract from linolenic acid was able to produce inhibition (100%) of aerobic glucose utilization by tumor cells. No glycolytic inhibition by the dried residue of oxidized linoleic acid was observed. At least 12 compounds (approximate chain length, 7C-13C) containing alpha,Beta-unsaturated carbonyl groups were isolated by gas-liquid chromatography (GLC) of dried extracts of oxidized linolenic acid. No single fraction inhibited tumor respiration, but the recombined mixture of all compounds caused complete respiratory inhibition of ascites tumor cells. Less material was required to inhibit oxygen consumption before than after GLC presumably because the more highly inhibitory components of the extract (along with "hydroperoxides" and TBA-reactive compounds) were lost during GLC. Extracts from oxidized linolenic acid were found to produce in all tumor cells cytoplasmic evaginations which were readily detected by phase microscopy.

Animals↗

PubMed1984

Platelet cytoskeleton: immunofluorescence studies on ADP and collagen-activated platelets.

Immunofluorescence studies reveal that platelet changes induced by adenosine diphosphate and collagen do not include the reorganization of the cytoskeleton in such a way as to expose actin, alpha-actinin, or vinculin. However, when such platelets were made permeable by saponin, these cytoskeletal proteins were present. In studies with collagen, fluorescence was observed along the fibers at areas of platelet adhesion and where no platelets were seen by phase microscopy. No fluorescence was observed with collagen treated with platelet-poor plasma. Scanning electron microscopy of collagen samples treated with platelet-rich plasma revealed a fibrillar meshwork with single platelets, platelet aggregates, and nodular structures that were smaller in size than individual platelets. These nodular structures may represent remnants of platelets still attached to the collagen after platelet detachment has occurred. These tenacious collagen-platelet membrane-binding sites have associated with them cytoplasmic alpha-actinin and vinculin, proteins that have been proposed by others to anchor actin filaments to the plasma membrane.

Adenosine Diphosphate↗

PubMed1984

Quantitative assessment of platelet morphology by light scattering: a potential method for the evaluation of platelets for transfusion.

Normal fresh platelets are discoid, and platelets that have been stored become spherical. Discoid morphology correlates with normal viability and imparts to a platelet suspension a "streaming" appearance when agitated. We developed a device that quantitates the percent of discoid cells in a suspension by means of laser light scattering at low angles. It performs this assay within 3 minutes on platelets contained in a plastic blood bag. The ability of the device to quantitate discoid platelets was demonstrated in the following ways: (1) comparison of percentage discs calculated from light-scattering data with visually observed optical streaming; (2) correlation of percentage discs from light scattering with the percentage determined by phase microscopy; and (3) observation of changes during room temperature storage. Platelet suspensions containing extreme (dendritic and balloon) forms were examined, and we conclude that these forms do not significantly affect the light-scattering assay. The optical measurements were also used to accurately approximate the total platelet counts of the suspensions. This device may have a role in the evaluation of platelets for transfusion.

Blood Platelets↗

PubMed1984

Platelet concentration and platelet volume distribution in healthy cats.

An electronic particle counter and size analysis system were used to determine platelet count, mean platelet volume (MPV), platelet volume heterogeneity (SD of platelet volumes), and platelet mass values in feline platelet-rich plasma (PRP) prepared by brief centrifugation of blood. Electronic platelet counts correlated well with manual counts done by phase microscopy over a platelet count range of 5,000/microliters to 840,000/microliters (r = 0.93). Variability in manual and electronic counts was not significantly different. However, due to differential stratification of platelet concentration in PRP, electronic counts on aspirated, mixed PRP were significantly less variable (P less than 0.01). Reference values, determined on blood from 25 healthy adult cats, were platelet count: 296,000 to 850,000/microliters; MPV: 11.0 to 18.1 fl; platelet volume heterogeneity: 5.8 to 10.6 fl, and platelet mass: 4.0 to 11.4 microliters/ml of blood. There was a direct relationship between MPV and platelet volume heterogeneity and an inverse relationship between MPV and platelet count. Platelet concentration and size were not constant in different PRP layers. Aliquots of PRP near the top had a significantly lower platelet concentration (P less than 0.001) and lower MPV (P less than 0.001) than did aliquots from near the bottom of the PRP layer. During the 4 hours after sample collection, there was a progressive decrease in platelet count and progressive increase in MPV, but the changes were not significant. When platelets were incubated in counting fluid, there was a slight increase in MPV by 1.5 hours. By 2.5 hours, there was a significant increase in MPV (P less than 0.05).

Animals↗

PubMed1980

[Nonspecific in vitro cytotoxicity of activated human macrophages on human tumor cells (author's transl)].

In experimental animals, it has been established that macrophages activated by various stimulants act as effector cells in the inhibition of tumor growth. The present study aims at determination of in vitro effect of activated human macrophages on the growth of human tumors. In the presence of human macrophages obtained from the peritoneal cavity and activated by bacterial lipopolysaccharide (LPS) in vitro, 3H-thymidine (TdR) uptake by human tumor cells was significantly inhibited but that by normal human diploid cells was not. Neither non-activated macrophages nor culture supernatant of the LPS-activated macrophages inhibited 3H-TdR uptake by the tumor cells. Under a phase microscopy, it was observed that activated macrophages attached to tumor cells and degeneration occurred afterward in the tumor cells attached by macrophages. Electronmicroscopically plasma membranes of activated macrophages fitted in closely with the plasma membranes of tumor cells, and microvilli of both cell types protruded to interdigitate each other. From all these results, it was concluded that the inhibition of tumor growth by the activated macrophages stems from the close attachment of the macrophages to the target tumor cells, but not from any mediators produced by the activated macrophages.

Cells, Cultured↗

PubMed1980

[Innervation of the cutaneous integument of pulmonary mollusks].

Cellular organization of the integumental innervation apparatus was studied in Lymnaea stagnalis and Helix vulgaris by the methods of impregnation with silver nitrate after Golgi, scanning electron microscopy, phase-contrast microscopy and by the technique of semi-thin sections. The integument of the tentacles, mouth lips, head, margins and soles of the foot was subjected to the investigation. In all the areas studied, 6 types of receptors were detected; they varied in number, diameter and form of their peripheral processes, in form of the apical surface, as well as in number, length and position of the receptory cilia. The structure of the subdermal neural plexus was demonstrated. A small number of neurons was present in the distal neural parts and in the subdermal plexus. A possible functional specialization of different receptors and morphological basis for performing peripheral dermal reflexes were discussed.

Animals↗

PubMed1980

Effects of hyperthermia on radiosensitivity of normal and leukaemic lymphocytes.

We measured the effects of X-irradiation plus incubation at elevated temperatures on peripheral blood lymphocytes from normal persons and from patients with chronic lymphocytic leukaemia. Counts of viable lymphocytes were based on morphological characteristics by phase microscopy. Dose-effect curves for X-irradiation were biphasic. The D0S for lymphocytes incubated at 39 and 41 degrees were significantly less than the D0 for 37 degrees. For both normal and leukaemia lymphocytes, X-irradiation was synergistic with hyperthermia at 39 degrees and 41 degrees.

Cell Survival↗

PubMed1977

Stabilization and preservation of the antigenic specificity of Trypanosoma (Trypanozoon) brucei variant specific surface antigens by mild fixation techniques.

Living bloodstream trypansomes fixed by suspension in a 1% formalin solution maintain both their morphology and the immunological specificity of their variant specific surface glycoprotein, so allowing precise identification of the variant types present in a trypanosome population by direct or indirect immunofluorescence combined with phase microscopy. The technique is simple, adaptable to the study of low parasitaemias and should facilitate analysis of the phenomenon of antigenic variation both in the field and the laboratory.

Animals↗

PubMed1994

Nerve growth dynamics. Quantitative models for nerve development and regeneration.

The quantitative analysis of nerve growth dynamics is critical to our understanding of nerve development and regeneration, but only recently has a quantitative framework begun to emerge to help define key objectives and to direct experimental measurements towards achieving this goal. Conceptually, the framework centers on the dynamic processes commonly observed for individual growth cones of growing neurites at the phase microscopy level, namely lamellipodial and filopodial extension and retraction. Because these activities essentially define the position of the axon tip, understanding how they are regulated offers to yield direct insight into factors governing the growth trajectory of the axon. In addition, much biological interest and effort is focused on the lamellipodial and filopodial behavior of the growth cone, which should facilitate experimental quantitation. Characterization of lamellipodial and filopodial activity has not been straightforward, however, because their inherent randomness leads to a requirement for considerable data and for less common mathematical techniques, such as time-series analysis. The work reviewed above has identified key analytical tools and experimental parameters needed to develop an integrated model of growth cone dynamics. Detailed measurement and analysis will be required to carry this development process to the next step. The cellular model of growth cone motility resulting from the characterization of lamellipodial and filopodial dynamics represents an intermediate description that can be extended to encompass both molecular mechanisms of growth cone behavior and axonal growth in multicellular tissue environments. For example, on the molecular level, filopodia contain a central core of actin filaments whose polymerization and depolymerization is thought to correspond to filopodial extension and retraction, with significant regulation possible through receptor-mediated effects on actin dynamics. By rewriting the parameters of filopodia dynamics in the current model in terms of these molecular events, one can begin to investigate their effects on growth cone behavior and to examine hypotheses of molecular mechanisms. Processes underlying lamellipodial behavior can be examined in a similar manner. At the tissue level, the effects of environmental factors on model parameters can be incorporated to yield predictions of the neurite outgrowth response to a particular environment. Such predictions offer a basis for designing microenvironments with optimal characteristics for enhancing nerve regeneration or manipulating the nerve growth response. Although the quantitative framework described here has focused on growth by peripheral nerve cells, it represents concepts known to apply to neurons of the central nervous system, as well.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Characteristics of amphotericin B-induced endothelial cell injury.

The polyene antibiotic amphotericin B has been implicated in vascular injury in human subjects and lung injury in an animal model. Our objective was to determine whether amphotericin B directly injures endothelial cells and to investigate several possible mechanisms of injury. Confluent cultures of bovine endothelial cells were incubated with different concentrations of amphotericin B for varying time periods. Injury was assessed by using a chromium 51 release assay, adherent cell counts, and morphologic changes in the endothelial cell monolayers by phase microscopy. Amphotericin B increased 51Cr release in a dose- and time-dependent fashion. Corresponding to changes in 51Cr release, amphotericin B decreased adherent cell counts and disrupted the monolayers. Incubation with vehicle alone (sodium desoxycholate, 8.2 micrograms/ml) did not alter any of these parameters. Incubation of cells with a dose of antibiotic (1 micrograms/ml), which did not produce overt cell injury, significantly increased membrane permeability to K+ ions and activated the sodium/potassium adenosine triphosphatase (Na/K ATPase). Inhibition of the ATPase at this same antibiotic concentration (1 micrograms/ml) produced endothelial cell injury equivalent to the magnitude of injury observed with high doses of the antibiotic (10 micrograms/ml). In the presence of 10% fetal calf serum, the injury at 24 hours was significantly attenuated. This protective effect could not be attributed to binding of the drug by albumin because varying concentrations of bovine serum albumin in minimal essential medium without other serum constitutents had no effect on the magnitude of injury. Incubation of cells with several exogenous oxygen radical scavengers (dimethylthiourea, catalase, and mannitol) or a decrease in ambient oxygen tension during antibiotic exposure did not alter the magnitude of injury. The results demonstrate that amphotericin B directly injures endothelial cells in a dose- and time-dependent manner and demonstrate the importance of the Na/K ATPase for the maintenance of normal endothelial cell function and viability in response to this form of injury.

Amphotericin B↗

PubMed1993

Development of an in vitro hepatotoxicity assay for assessing the effects of chronic drug exposure.

We have used the human hepatoma cell line HepG2 to compare the hepatotoxic effects of acute (24 hr) and chronic (up to 10 days) exposure to amitriptyline, paracetamol and ondansetron. In acute exposure studies, hepatotoxicity was assessed by the sulforhodamine B protein staining method, where amitriptyline and paracetamol produced 50% hepatotoxicity at concentration of 30 microM and 7 mM, respectively, while ondansetron was non-hepatotoxic at 100 microM, the highest concentration used. In chronic exposure studies, the morphology of HepG2 cells was assessed by phase microscopy every 2 days and the compounds, at concentrations determined from the acute assay, were added fresh every 2 days. Chronic exposure to amitriptyline and paracetamol produced significant morphological changes in HepG2 cells at 3 microM and 1 mM respectively, concentrations which had no significant effect in the acute assay. Ondansetron (100 microM) produced only slight morphological changes in the cells after 10 days of culture. The combination of acute and chronic drug exposure assays with HepG2 cells represents novel in vitro systems for the hepatotoxicological assessment of drugs intended for human use.

Acetaminophen↗

PubMed1998

Kinetics of the structure of neurites in mollusk brain after its dissociation.

Using phase microscopy the behaviour of living neurons directly after their enzymatic isolation from ganglia of Clione limacina and Lymnaea stagnalis was studied. Apart from single-processed unipolar neurons, numerous dipolar and unipolar ones were found in mollusc which does not conform to neuron classification generally adopted. Disrupted neurons were seen to shorten gradually in Ringer solution and -1640 medium at the rate of 0.84-0.27 mm min. Serial photomicrography revealed that the process has two stages. Neurite retraction is interrupted by the expansion stage (extrusion and urgent regeneration). Duration of retraction and extrusion stages made 22.7-3.3 and 6.6-9.1 min respectively. Retraction speed was 24-0.5 mm/min while that of extrusion was 2.8-0.4 mm/min. different processes of the same neuron contract asynchronously. Process invagination leads to its rounding. Thus the cell acquires most thermodynamically stable from which is arranged to prompt its survival. Spherical neurons lacking processes migrate and demonstrate capacity of regeneration. As the conditions somehow resembling the experimental ones (high proteolytic activity of the medium and process loss in part of neurons) arise in cerebral trauma nidus of it is suggested that viable neuron behaviour is alike in this situation.

Animals↗

PubMedDate not supplied

The effects of stains and investigators on assessment of morphology of canine spermatozoa.

Percentage and types of morphological abnormalities found in canine spermatozoa were evaluated by three investigators using three stains (Giemsa-Wright stain [Diff-Quik], eosin Y/nigrosin [Hancock], and eosin B/nigrosin [Society for Theriogenology morphology stain] with conventional light microscopy, compared to phase contrast microscopy on unstained samples. The percentage of spermatozoa with abnormal heads, midpieces, and tails varied by technique and by investigator. Average percentages of morphologically normal spermatozoa were significantly higher in samples stained with Diff-Quik and samples examined by phase contrast microscopy than in samples stained with Hancock or Society for Theriogenology morphology stains. No effect of investigator on the percentage of morphologically normal spermatozoa was assessed. Results suggest that staining or preparation technique may alter the morphology of canine spermatozoa artifactually.

Animals↗

PubMed1984

Subgrouping of human rotavirus strains by complement fixation, indirect double-antibody sandwich enzyme-linked immunosorbent assay and solid-phase immune electron microscopy.

Complement fixation (CF), indirect double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) and solid-phase immune electron microscopy (SPIEM) were compared for their ability to subgroup 73 human rotavirus (HRV) strains from infants and young children with gastroenteritis admitted to one or the other of two different hospitals of Northern Italy. By both indirect DAS ELISA and SPIEM all 73 HRV strains were classified into one or the other of two subgroups. By CF only 67 strains could be subgrouped, as six HRV-positive stools showed anticomplementary activity which could not be eliminated. Indirect DAS ELISA required subgroup-specific, unabsorbed antisera from two different animal species. For SPIEM two antisera from a single animal species were needed, but they had to be absorbed with single-shelled bovine rotavirus for HRV subgrouping to be reliable. Indirect DAS ELISA appeared to be the technique most suitable for extensive application in epidemiological studies of HRV infections by different subgroups. However, SPIEM allowed rapid subgrouping of HRV in stool specimens showing anticomplementary activity in the CF test or non-specific reactions in the ELISA test. In one area of Northern Italy the prevalence of subgroup I HRV infections was 7.8 per cent, while in another it reached 68.1 per cent in the same period.

Antigens, Viral↗

PubMed2006

[Comparison between optical microscopic examination and phase contrast microscopic examination for diagnosing the origin of urinary bleeding].

PURPOSE: Phase contrast microscopy method is useful in the examination of urine sediment to differentiate glomerular(Gl) hematuria from non glomerular(nonGl) hematuria. In this study, we compared the diagnostic value of optical microscopy with that of phase contrast microscopy. METHOD: One hundred and sixty fresh urine samples of microscopic hematuria(5/HPF or more) from 118 patients with renal disease and 42 patients with urologic disease were analyzed. The erythrocytes referred to as Gl, are defined to have acanthocytes, target configuration, or finger ring shape, and a Gl cell/total erythrocyte ratio greater than 3% is defined as reliable marker for Gl bleeding. RESULT: Sensitivity and specificity of this criteria for Gl bleeding were 50.9% and 94.9% in acanthocytes, 69.6% and 89.1% in the target configuration, and 88.4% and 89.1% in the finger ring, respectively. As for the microscopic observation methods, the sensitivity and specificity of this criteria for GI bleeding were 88.1% and 81.0% with phase contrast microscopy, 74.6% and 90.9% with optical microscopy with no dyeing, and 74.6% and 88.6% with optical microscopy with dyeing, respectively. CONCLUSION: GI and nonGl hematuria were correctly diagnosed by counting the urinary Gl cells not only by phase contrast microscopy, but also by optical microscopy. This method seems to have important clinical usefulness by offering information on urinary bleeding.

Adolescent↗

PubMed1990

Sensitive solid-phase immune electron microscopy double-antibody technique with gold-immunoglobulin G complexes for detecting rotavirus in cell culture and feces.

A new solid-phase immune electron microscopy double-antibody colloidal-gold technique (SPIEMDAGT) was developed and compared with direct electron microscopy, direct immune electron microscopy, and enzyme immunoassay for detecting rotavirus. Guinea pig and rabbit antirotavirus antisera were used as capture and detector antibodies, respectively, and goat anti-rabbit immunoglobulin G-gold complexes were employed as a label. Animal rotavirus in cell culture media and human virus in stool specimens were detected by this method. On average, SPIEMDAGT detected 800 times more virus particles than direct electron microscopy and 45 times more particles than direct immune electron microscopy and yielded 20% more positives than enzyme immunoassay. SPIEMDAGT could detect not only viral antigen associated with morphologically recognizable particles but also antigen present when whole virus particles were not visible.

Antibodies, Viral↗

PubMed2001

Imaging the three orientation variants of the DO22 phase by 3D atom probe microscopy.

Three-phase NiAlV alloys were investigated using a three-dimensional atom probe. Ageing at 800 degrees C gives rise to the precipitation of two ordered phases within the supersaturated FCC solid solution, namely Ni3Al (L1(2) structure) and Ni3V (DO22 structure). The DO22 phase has three orientation variants which need to be identified in 3DAP images. It is shown that an appropriate choice of analysis site enables us to image the chemical order within both L1(2) and DO22 ordered phases and to distinguish the three orientation variants of the DO22 phase in reconstructed images. The lateral resolution of 3DAP in these experimental conditions was estimated through simple considerations to be less than 0.3 nm.

Journal Article↗