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PubMed1985

Transcapillary diffusion of Na-fluorescein measured by a 'large window technique' in skin areas of the forefoot.

A method is introduced for the study of transcapillary diffusion of 20% Na-fluorescein (0.3 ml/l of blood given intravenously) in skin areas by a fluorescence video-microscopy system which has been used earlier for measurements at the single capillary level or on axes crossing capillary groups. Two groups of 17 and 14 healthy volunteers respectively were included in a test-retest experiment (first measurement at day one, second measurement at day three). The appearance of the dye was visualized and the intensity of fluorescent light measured by a quadratic video densitometer window at the forefoot in an area of 2 mm2 containing 78 capillaries on the average. In both groups with and without control of the skin temperature, there were no statistically significant differences of the mean values measured at the first and second day of study. However, the mean intraindividual differences and the coefficients of variance were significantly (p less than 0.005) higher in the subjects without temperature control. It is concluded that the method is suited to detect transcapillary diffusion of Nafluorescein in skin areas by an almost atraumatic procedure with acceptable reproducibility. Potentially, it may be used to follow the natural history of microvascular diseases and to test the effect of various treatment modalities.

Adult↗
PubMed1985

Pharmacokinetics and distribution of a fluoresceinated glycosaminoglycan, sulodexide, in rats. Part II: Organ distribution in rats.

Fluorescein-labelled sulodexide tissue distribution was studied in the rat after intravenous administration at the dosage of 15 mg/kg. A globular fluorescence was observed mainly in the extracellular matrix and a diffuse one was present in liver and kidney parenchyma lasting for a long time, up to 4 h after administration. In particular in the kidney cortex the fluorescent material was present at the higher time intervals. Fluorescence was also observed in the spleen and in the perivascular areas and in the media of the aortic wall.

Animals↗
PubMed1985

Intestinal capillary blood flow studied with fluorescein flowmetry in hemorrhagically shocked rats.

The intestinal capillary blood flow in hemorrhagic shock was studied in 32 rats by fluorescein flowmetry (FF). FF implies the measurement of relative intestinal capillary blood flow, expressed as an index. Exsanguination to 35 mmHg for 180 min resulted in a heterogeneous fluorescence pattern with 51% difference in blood flow index between the areas with most and those with least fluorescence, compared with 14% in the control group. Following retransfusion of the shed blood, the difference in blood flow index between areas with most and least fluorescence was 54%, suggesting that the heterogeneous blood flow was not improved by retransfusion. Heterogeneity of the capillary blood flow in the intestines seems to be a phenomenon of hemorrhagic shock and FF to be a suitable method for studying this condition.

Animals↗
PubMed1983

Catalytic and regulatory ATP-binding sites of the red cell Ca2+ pump studied by irreversible modification with fluorescein isothiocyanate.

Catalytic and regulatory binding sites for ATP on the red cell Ca2+ pump have been investigated using fluorescein isothiocyanate (FITC). Both (Ca2+ + Mg2+)-ATPase activity and ATP-dependent Ca2+ flux are selectively and irreversibly inactivated by FITC and the pump is protected from FITC by the presence of ATP. The time course of inactivation by FITC is characteristically biphasic. Analysis of the kinetics of inactivation by FITC and protection by ATP reveals the participation of both high and low affinity binding sites for ATP and FITC. The sites binding ATP or reacting with FITC do not, however, appear to co-exist on the same enzyme molecules. Thus, "flip-flop" mechanisms for (Ca2+ + Mg2+)-ATPase, involving negative interactions between high and low affinity ATP sites, are considered unlikely. The two affinities for ATP are most simply explained by assuming that the Ca2+ pump protein exists in alternative conformational forms, E1 having a high affinity for ATP and E2 having a low affinity for ATP. Ca2+ pumping and (Ca2+ + Mg2+)-ATPase involve interconversion between these forms. It is suggested that regulation of Ca2+ pump activity by Mg-ATP reflects acceleration of the conformational transition between the E1 and E2 forms, as well as a previously described acceleration of phosphoenzyme hydrolysis (Muallem, S., and Karlish, S. J. D. (1981) Biochim. Biophys. Acta 647, 73-86; Garrahan, P. J., and Rega, A. F. (1978) Biochim. Biophys. Acta 513, 59-65).

Adenosine Triphosphate↗
PubMed1982

Direct staining of mouse T lymphoblasts with fluoresceinated Vicia villosa lectin.

Fluorescein conjugated Vicia villosa (Vv) lectin was used for direct staining of the surface of viable cells of various mouse lymphocyte populations. A varying proportion of polyclonally-activated T cells expressed Vv receptor although less strongly than cells of an influenza virus-specific cytotoxic T lymphocyte (CTL) clone and blasts generated in the mixed lymphocyte reaction. Cyclosporin A (CyA) treatment markedly reduced the expression of Vv receptor following concanavalin A (Con A) stimulation by between 66% and 93% with a concurrent inhibition of blastogenesis and complete abrogation of cytolytic function. Resting mouse lymphocytes and B-cell blasts were always Vv-negative. However, non-specific suppressor factor producing non-cytotoxic T-cell lines also expressed Vv receptor as shown by the weak, but specific, surface fluorescence of EL4 and BW5147 cells stained with Vv. Vv-positive cells were not restricted to a particular Ly phenotype, Vv-positive cells being found among both Lyt 1+ and Lyt2+ MLC stimulated lymphocytes. Our data suggest that the receptor for Vv lectin cannot be regarded as an exclusive differential marker for CTL.

Animals↗
PubMed1983

Granules of blood eosinophils are stained directly by anti-immunoglobulin fluorescein isothiocyanate conjugates.

Direct staining of the granules of blood eosinophils by anti-immunoglobulin fluorescein isothiocyanate (FITC) conjugates was observed when feline blood smears were tested for presence of feline leukemia virus (FeLV) antigen by immunofluorescent antibody. When blood smears of other species including swine, horses, cattle, dogs, sheep, birds, and human beings were examined, direct staining of eosinophils by FITC conjugates was also detected. This FITC staining was restricted to eosinophils and was not observed in neutrophils, lymphocytes, and platelets. Direct FITC staining of eosinophils does not represent a problem in immunofluorescent test for the detection of FeLV infection in cats, as long as the eosinophils, which can easily be recognized as such, are excluded from the spectrum of interpreted cells.

Animals↗
PubMed1983

[Double labeling of antibodies with fluorescein isothiocyanate (FITC) and horseradish peroxidase (HPOD)].

This paper presents a method for labeling antibodies with fluorescein isothiocyanate (FITC) and additional with horseradish peroxidase (HPOD). With this double labelled antisera the fluorescence-serologic antibody technique (FAT) as well as the enzyme-serologic antibody technique (EAT) was done bit by bit for the same object. How the presented pictures demonstrate the result of both techniques, FAT and EAT, are the same with the described method. So, it is possible after fluorescence-microscopical exploitation through a following histochemical proof of the HPOD to get permanent preparations with the same biological result.

Animals↗
PubMed1984

The use of radiolabeled and fluorescein-labeled antiglobulins in assays to predict platelet transfusion outcome.

We used both radiolabeled and fluorescein-labeled antiglobulins in assays to detect antibodies against platelets in multiply transfused patients to determine the value of these tests in predicting the outcome of platelet transfusion in such patients. In 15 allosensitized patients, we studied 68 single-donor platelet transfusions, 43 (63%) of which had a poor outcome, defined as a corrected count increment (CCI), less than 10,000. The results obtained with either test were significantly correlated with the CCI following transfusion (p less than 0.001), but the assay using the radiolabeled antiglobulin had slightly better sensitivity, specificity, and predictive value. When the assays were used in combination, there was again significant correlation with the CCI of the transfusion, p less than 0.001. When both assays predicted failure of the transfusions, 31/31 (100%) such transfusions resulted in a CCI of less than 10,000, and when both assays predicted success of the transfusions, 14/15 (93%) such transfusions resulted in a CCI of greater than 10,000. Both assays are useful in predicting the outcome of the platelet transfusions; when the assay results were concordant, almost total predictive accuracy was obtained.

Adolescent↗
PubMed1984

The appearance of fluorescein-labelled lymphocytes in lymph following in vitro or in vivo labelling: the route of lymphocyte recirculation through mesenteric lymph nodes.

Fluorescein isothiocyanate (FITC) has been used to study lymphocyte migration in sheep. After being labelled in vitro with FITC, lymphocytes migrated from blood into lymph at the same rate and with the same recovery as lymphocytes labelled with with the radioisotope 51chromium. The in vivo labelling of mesenteric lymph nodes (MLN) with FITC resulted in high numbers of labelled lymphocytes appearing in prescapular lymph. However, the appearance of the FITC-labelled lymphocytes in the prescapular lymph could be prevented by cannulating the main intestinal lymph duct prior to the in vivo labelling procedure. It was concluded that lymphocytes labelled in vivo within the MLN required an intact lymphatic system to reach the blood circulation and did not enter the venous circulation directly from the MLN.

Animals↗
PubMed1983

Observations on organ distribution of fluorescein-labelled tumour cells released intravascularly.

By labelling cells with fluorescein isothiocyanate (FITC) and other fluorescent dyes and examining frozen sections of several organs of the recipients with an ultraviolet microscope, it has been demonstrated that viable tumour cells arrive in all organs examined within 15 min of inoculation either intravenously or arterially and are still detectable in various organs 30 days later. Although the method is not quantitatively accurate, its advantages are that the cells can be directly visualised and it can be confirmed that the label is attached to whole viable cells and not to cellular fragments. The findings effectively dispose of the possibility that the consistent absence of deposits in certain sites in animals inoculated with cells from spontaneous murine mammary tumours is due to failure of the cells to reach them. It has also been confirmed that FITC-labelled cells are still capable of forming deposits and occasional fluorescent cells are detected in these secondary neoplasms.

Animals↗
PubMed1984

Trypanosoma (Nannomonas) congolense: analysis by fluorescein-conjugated plant lectins of surface saccharides of cloned variant antigen types differing in infectivity for mice.

Surface saccharides of 4 cloned VATs (variant antigen types) of Trypanosoma (Nannomonas) congolense, AmNats (Amherst Nannomonas antigen types) 1.1, 1.2, 2.1, and 3.1, derived from 3 different stocks, were compared by fluorescein-conjugated, plant lectins using a quantitative fluorescence method. It was ascertained by the ID63 assay that the 4 AmNats differed in their infectivity for mice. The lectins employed for AmNats 1.1, 2.1, and 3.1 were concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), garden pea agglutinin (GPA), and gorse seed (Ulex europaeus) agglutinin (UEA). In view of the results obtained with these 3 AmNats, only Con A, WGA, and GPA were used with AmNat 1.2, which was isolated after the lectin analyses of the other cloned VATs were completed. On the basis of experimental results, we concluded that the amounts of saccharide residues binding the several lectins differed among the 4 AmNats. In each instance, the reaction specificity was controlled by inclusion of an appropriate sugar in the incubation mixture. Although the actual numbers of various specific lectin-binding sites differed among the AmNats 1.1, 2.1, and 3.1, all of them were found to have the following sugars on their surfaces: alpha-D-mannose, N-acetyl-D-glucosamine, D-galactose, alpha-D-glucose, and alpha-L-fucose. AmNat 1.2 treated with Con A, WGA, and GPA only had the first 2 sugars named above and alpha-D-glucose residues. The results of the ID63 assay indicated AmNats 1.1 and 2.1 to be significantly more infective for mice than AmNats 1.2 and 3.1. The lectin analysis revealed that the 2, more infective, cloned VATs incubated with Con A or WGA emitted significantly (approximately 39% to approximately 62%) more fluorescence than the less infective ones. Thus there were significantly more numerous Con A and WGA binding sites on the more infective AmNats. The situation was reversed with regard to GPA. Upon treatment with this lectin, fluorescence emitted by AmNats 1.1 and 2.1 was significantly (approximately 56% to approximately 81%) lower than that recorded for the less infective AmNats 1.2 and 3.1. In light of our results, infectivity of T. congolense cloned VATs was correlated with the presence of higher numbers of alpha-D-mannose and N-acetyl-D-glucosamine residues and of lower numbers of alpha-D-glucose residues on the surface of the bloodstream trypanosomes. There appeared to be no correlation between infectivity and the numbers of D-galactose and alpha-L-fucose residues present on these parasites.

Animals↗
PubMed1983

[Photodocumentation of intra-arterial infusions using fluorescein].

Intra-arterial chemotherapy was given to 12 patients with Head and Neck tumours with the aim of selective perfusion of the tumour area, usually located in the area of supply of the lingual and/or facial artery. A catheter was inserted retrograde into the external carotid artery. The skin or mucous membrane area thus selectively infused by the antineoplastic drug and fluorescein solution could be controlled and documented using an excitation- and barrier filter. Serial photographs were used to demonstrate the higher fluorescence in the perfused area during the intra-arterial infusion lasting 60 or 90 minutes. The use of a balloon catheter increased the selectivity of the fluorescent region. At the end of the infusion the fluorescence disappeared rapidly. From our findings we conclude that it is possible to produce a selective higher concentration of a substance in the tumour during the intra-arterial infusion.

Catheterization↗
PubMed1983

[Transcapillary diffusion of Na-fluorescein in skin areas of the dorsum of the foot in juvenile diabetics].

Fluorescence videomicroscopy after intravenous injection of Na-fluorescein serves to quantitate dye diffusion out of single capillaries or capillary groups in an almost atraumatic manner [2, 4, 6, 8, 9]. In 14 young patients with a mean disease duration of 10 years and in 16 healthy controls, fluorescent light intensity was measured continuously during one hour at the dorsum of the foot by a videodensitometer covering an average of 68 capillaries. 1 second and 5 minutes after first appearance of the dye in the foot skin, the amount of the tracer detected in the area of measurement was significantly increased in the diabetics (p less than 0.05-0.001). At 1 minute, for example, mean fluorescent light intensity reached 47.2 +/- 18.1% of the maximal individual intensity and in the normals only 29.3 +/- 10.8% (p less than 0.01). In the patients the capillaroscopic image was characterized by early blurring, whereas the pericapillary halo of the controls contained more dye than the more remote interstitial space and remained well delineated for a much longer time. The new technique offers the possibility of studying transcapillary diffusion as an expression of microangiopathy in different groups of diabetic patients, and of following the effect of therapeutic regimens.

Adult↗
PubMed1981

A method for the isolation of homogeneous fluorescein-labeled proteins for studies on the variation in biological and fluorescence properties of the different conjugates.

Different homogeneous fluorescein-labeled conjugates of alpha-chymotrypsin and the pancreas trypsin inhibitor have been analyzed and isolated by isoelectric focusing with and without biospecific sample application. With this procedure the biological activity of the different protein conjugates can be determined from the focusing pattern. The fluorescence quantum yield was found to be decreased with the number of labels per protein. The biological activity, however, was increased in some monolabeled conjugates compared with the activity of the native protein. The quantum yield was found to be markedly (greater than 10%) changed when the monolabeled conjugates formed specific complexes with proteins.

Animals↗
PubMed1982

Pharmacokinetics of fluorescein-labelled glycosaminoglycans and of their lipoprotein lipase-inducing activity in the rat.

Using fluorescein-labelled glycosaminoglycans (F-GAG) an extensive study of their pharmacokinetics and of pharmacokinetics of their lipoprotein lipase-inducing activity was carried out in fed and fasted rats. A two-compartment model was used to describe F-GAG's pharmacokinetics. The parameters of drug disposition were strongly dose-dependent. The dose-response curves for parameters were powers with negative exponent indicating considerable saturation of the F-GAG elimination process. There was also dose dependence for T1/2, Co, AUC and Cl, and the decrease in lipoprotein lipase activity in plasma was strongly dose-dependent. The lipoprotein lipase kinetic parameters also appeared to be dose-dependent.

Animals↗
PubMed1982

Transcapillary and interstitial diffusion of Na-fluorescein in chronic venous insufficiency with white atrophy.

White atrophy (atrophie blanche) in the medial ankle region due to chronic venous insufficiency (stasis syndrome) was studied in 12 patients by fluorescence video microscopy [1,8]. After intravenous bolus injection of 1 ml of 20% Na-fluorescein the dynamic phenomena of transcapillary and interstitial diffusion of the dye were analyzed by a videodensitometer which has moved on single frames of the TV-recordings across white atrophy at different times after dye appearance. White atrophy is characterized by 3 main areas: 1) the avascular field sensu strictu, 2) the border region with enlarged and tortuous capillary loops, and 3) the more remote capillaries showing less altered morphology. The dye reached the ankle skin after 39.2 +/- 13.3 S and leaved rapidly the intravascular compartment. In the region of the border capillaries, the maximal fluorescent light intensity was reached after 5 min, in the centre of the avascular field only after 40 min. Initially, the densitometer curves show a 'valley' which is slowly filled up by the dye. At 40 min and later on the highest light intensities were measured in the central parts of the avascular field ('mountain'), where the clearance was just beginning. The slow diffusion of the small tracer into the avascular field explains that white atrophy is a predilection site for venous ulcer formation.

Adult↗
PubMed1994

Characterization of fluorescein transport in isolated proximal tubular cells of the rat: evidence for mitochondrial accumulation.

The uptake and accumulation of the organic anion fluorescein-sodium (Flu-Na) was investigated in freshly isolated proximal tubular cells (PTC) of the rat kidney. Furthermore, the influence of other organic anions on Flu-Na uptake was studied in order to characterize Flu-Na transport in PTC. Flu-Na showed concentration-dependent, saturable and probenecid-sensitive transport. Comparing the transport parameters with para-aminohippurate (PAH), Flu-Na exhibited a higher affinity, but lower capacity to the organic anion transport system. The apparent Km for Flu-Na transport was 59 +/- 15 microM with a Vmax of 186 +/- 26 pmol/mg of protein/min, and for PAH 207 +/- 11 microM and 740 +/- 46 pmol/mg of protein/min, respectively. Dose-dependent inhibition of Flu-Na uptake with PAH resulted in an apparent inhibition constant Ki of 249 microM. This is in good agreement with the apparent Km of PAH, indicating that Flu-Na uptake is regulated by the PAH transport system. It is suggested that cellular uptake of both organic anions is mediated by a carrier at the basolateral membrane. However, after incubating cells with different concentrations of phenol red the percentage of maximum inhibition was 84%, which was significantly different from the 32% with PAH, suggesting that another transport system may be involved in Flu-Na uptake. Experiments with confocal laser scanning microscopy showed cellular uptake of Flu-Na and accumulation in subcellular structures. After superfusion of PTC with rhodamine 123 these structures were identified as mitochondria.

Animals↗