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PubMedDate not supplied

Fluorescein flowmetry in human nasal mucosa.

In sixteen patients a relative blood flow, expressed as an index, in the nasal mucosa was measured by fluorescein flowmetry (FF) using an endoscope technique. Duplicate determination showed slightly lower values in the second measurement performed 5 min after the first one. This difference might be due to a too high background fluorescence in the tissue. The blood flow index was reduced to 1/3 of the original blood flow index by the administration of nose drops. These changes in man due to decongestion are comparable to the changes in blood flow index observed in a previous work (2) in rabbit sinus mucosa, using the corresponding technique.

Adolescent↗
PubMed1989

Increased gut permeability to fluorescein isothiocyanate-dextran after total parenteral nutrition in the rat.

Sepsis with subsequent multiple organ failure is the commonest complication seen in the surgical intensive care unit today. A gut mucosal barrier dysfunction is assuming an increasingly important role as one possible explanation for the initiation of the septic process. It is known that the gut bacteria and endotoxins can, in the presence of a seemingly intact epithelium, translocate to extraintestinal sites, but the exact mechanism behind this process is not understood. In the present study we have approached this problem by testing the gut permeability to two macromolecules, bovine serum albumin (BSA) and fluorescein isothiocyanate (FITC)-dextran, after 7 days of enteral or parenteral nutrition in the rat. The plasma values of FITC-dextran after 4 h of marker feeding showed a significant increase in gut permeability after parenteral but not after enteral nutrition as compared with the controls. The plasma values of BSA, however, did not show any significant change in any of the groups. Thus, parenteral nutrition, with the changes occurring in the gut mucosa, may be one of the etiologic co-factors behind a gut mucosal barrier dysfunction, eventually leading to absorption of noxious agents into the systemic circulation with subsequent multiple organ failure.

Animals↗
PubMed1989

Intraphagosomal pH in alveolar macrophages studied with fluorescein-labeled amorphous silica particles.

Intraphagosomal pH in rabbit alveolar macrophages was studied using amorphous silica particles (FSP) and yeast particles (FYP) labeled with fluorescein. The pH was estimated from the quotient between the fluorescence intensity at wavelength 519 nm with excitation at wavelengths 495 and 450 nm. Within a factor of 10, pH was independent of the number of FSP added to macrophages in vitro. In macrophages cultured for 24 h, the pH obtained with FSP and FYP was about 5. Three hours after lavage, pH was the same as after 24 h for the FSP but significantly higher for the FYP, 5.8. Both 3 and 24 h after lavage, more lysosomes were in contact with the FYP-containing phagosomes than with the FSP-containing ones. Most FSP were in tight contact with the phagosomal membrane, while there was a clear zone between most FYP and the phagosomal membrane. The differences in pH and morphology between cells containing FSP and FYP might be explained by the assumptions that the macrophages disintegrate the FYP, which results in higher pH, and that the disintegration of FYP is more efficient at 3 than at 24 h after the lavage. The intraphagosomal pH was lower when the macrophages were allowed to phagocytize the FSP in vivo. The pH values were 5.1 in vitro, 4.9 at 24 h, and 4.5 at 1 week after the FSP had been instilled via trachea. The FSP should be a useful tool for estimation of intraphagosomal pH at basic conditions, i.e., the milieu to which many inhaled inorganic particles will be exposed.

Animals↗
PubMed1984

The permeability of the corneal endothelium to fluorescein in the normal human eye.

Endothelial permeability to fluorescein was measured as part of fluorophotometric studies of aqueous humor flow in 112 normal subjects whose ages span six decades. The permeability was 2.4 +/- 0.2 X 10(-4) cm/min (mean +/- SD). Females had a slightly higher permeability than males. No correlation between age and permeability was noted. The precision of this method of measurement was estimated to be +/- 22%.

Adult↗
PubMed1996

Effect of food intake on the delivery of fluorescein as a model drug in colon delivery capsule after oral administration to beagle dogs.

The effect of food on the release time of a model drug, fluorescein (FL), has been studied after oral administration to beagle dogs in colon delivery capsule in comparison to conventional gelatin capsule and enteric capsules. The dose of FL was 30 mg for each animal. After oral administration of each test preparation in fasted or postprandial condition (100 grams of commercial solid food was given at 30 min before drug administration), blood samples were collected and plasma FL concentrations were measured spectrofluorometrically. Pharmacokinetic analysis was performed with plasma FL concentration vs. time data and the following parameters were determined; Tmax (the time when plasma FL concentration reaches to its maximum concentration), Cmax (peak plasma FL concentration), Tlag (the time when FL appeared at first into the systemic circulation), AUC (area under the plasma FL concentration vs. Time curve) and MRT (mean residence time). For gelatin capsule, mean Tmax appeared at 0.83 +/- 0.33 (S.E.) h after administration and MRT was 2.67 +/- 0.21 h in fasted condition. By feeding, Tmax and MRT increased to 1.50 +/- 0.76 h and 3.09 +/- 0.49 h. For two enteric HPMCP and Eudragit S capsules, MRT were 2.90 +/- 0.48 h and 5.24 +/- 0.32 h in fasted condition, and 11.30 +/- 1.10 h and 12.83 +/- 0.34 h in postprandial condition, respectively. Tlag also increased by postprandial administration. As colon delivery capsule, time-controlled release capsule (TCC) and two types of intestinal inner pressure-controlled release capsules (PCC) (#1 is a separate type and #2 is a seamless one) were tested. MRT of TCC was 4.76 +/- 0.29 h and 6.43 +/- 0.66 h in fasted and postprandial conditions, respectively. This capsule did not receive the effect of food intake. For #1 PCC, MRTs were 5.32 +/- 0.22 h and 12.28 +/- 0.26 h in fasted and postprandial conditions, respectively. For #2 PCC, MRTs were 5.51 +/- 0.26 h and 13.36 +/- 0.84 h in fasted and postprandial conditions, respectively. In addition, the effect of two times feedings was studied with two PCCs and longer MRTs, 28.44 +/- 1.39 h and 26.32 +/- 1.64 h, were obtained. The release time of FL from PCCs increased by postprandial administration. As compared to the results on two enteric capsules, these PCCs are thought to disintegrate in the colon. However, TCC is thought to disintegrate in the stomach after postprandial administration.

Administration, Oral↗
PubMedDate not supplied

The fluorescein disappearance test (FDT): an evaluation of its use in infants.

We report a prospective evaluation of the fluorescein disappearance test (FDT) in the diagnosis of lacrimal outflow obstruction in infants under 1 year of age. In a preliminary study of 80 lacrimal systems, we showed that the FDT is a sensitive (90%) and specific (100%) test in aiding the diagnosis of nasolacrimal obstruction in infants. In the main study, we examined 288 eyes of 232 children with epiphora. The FDT was abnormal in 237 (82%). Of the remaining 51 eyes, there was a clinical explanation for this in 33 cases (epiblepharon, conjunctivitis, etc). We have found that this simple and reliable test taken in conjunction with clinical examination provides an objective assessment of the lacrimal outflow status of young children.

Evaluation Studies as Topic↗
PubMed1992

Binding of fluorescein-labeled anaphylatoxin C5a to human peripheral blood, spleen, and bone marrow leukocytes.

The expression of C5a receptors (C5aR) on human leukocytes was evaluated by flow cytometry using fluorescein-labeled human C5a (C5a-F). Granulocytes and CD14+ mononuclear cells (MNL) but not CD3+, CD20+, CD16+, CD56+, or CD11b+ lymphocytes in peripheral blood and spleen bound C5a-F. C5a-F binding was saturable and inhibitable by anti-C5a monoclonal antibody (MoAb) C17/5 or unlabeled C5a. During hemodialysis, which led to the generation of C5a, only granulocytes and monocytes increased their expression of the adhesion molecule CD11b (CR3). In vitro, C5a induced an increase of CR3 and p 150/95 (CD11c/CR4) only on myeloid cells. However, treatment of leukocytes with phorbol 12-myristate 13 acetate increased CR3 and CR4 expression on both myeloid cells and a lymphocyte subpopulation. Stimulation of MNL in mixed lymphocyte cultures or by treatment with conditioned medium or with IFN-gamma did not induce binding sites for C5aR on lymphocytes and reduced the binding of C5a-F to monocytes. The expression of C5aR on low-density bone marrow cells was analyzed by setting appropriate gates during flow cytometry. Cells that bound C5a-F were found in all populations that contained granulocyte and monocyte precursors, but not in lymphocyte precursor populations. All C5aR+ bone marrow cells were CD34 and expressed high levels of CR3, which suggests a late appearance of C5aR during myeloid cell maturation. Our results indicate that C5aR is exclusively expressed on myeloid cells within the hematopoetic cell population.

Antibodies, Monoclonal↗
PubMed1992

Construction, characterization, and mutagenesis of an anti-fluorescein single chain antibody idiotype family.

In addition to crystallographic studies that determined antigen contact residues for monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 2.5 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal antibodies (mAbs) 9-40 (Ka = 4.4 x 10(7) M-1), 12-40 (Ka = 4.0 x 10(8) M-1), and 5-14 (Ka = 2.4 x 10(8) M-1) possessed identical Fl contact residues, with the exception of L34His for L34Arg. Site-specific mutagenesis of single chain antibody (SCA) 4-4-20 in which L34Arg was changed to L34His resulted in approximately 1000- and 3-fold decreases in binding affinity and Qmax (maximum quenching of bound Fl), respectively, which suggested that L34Arg was directly involved in increased binding affinity and fluorescence quenching. Therefore, substitution of Arg for His at residue L34 in mAbs 9-40, 12-40, and 5-14 should result in increased binding affinity and Qmax. To facilitate site-specific mutagenesis studies, single chain derivatives of mAbs 9-40, 12-40, and 5-14 were constructed. Following expression in Escherichia coli, characterization of the SCAs demonstrated that when compared with the respective parental mAb, the SCAs possessed identical binding affinities and similar Qmax and lambda max (absorption profiles of bound Fl) values. These results validated SCA 9-40, 12-40, and 5-14 for use in site-directed mutagenesis studies. Results of mutagenesis studies indicated that substitution of L34Arg into the active sites of 9-40, 12-40, and 5-14 was not enough to produce 4-4-20-like binding characteristics. Therefore, the following single chain mutants were constructed: 9-40L34Arg/L46Val, 12-40L34Arg/L46Val and 5-14L34Arg/L46Val, 9-40L34Arg/L46Val/H101Asp and 4-4-20H101Ala. Results demonstrated that these mutations were not able to render the mutant SCAs with increased binding affinity and fluorescence quenching values. Collectively, these results suggest that the combining sites of mAb 9-40, 12-40, and 5-14 may possess different active site structures than mAb 4-4-20.

Amino Acid Sequence↗
PubMed1990

Improved purification of human lactate dehydrogenase isoenzyme-3 and studies with its fluorescein isothiocyanate and biotin conjugates.

Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) isoenzyme-3 (LD-3) has been isolated in milligram quantities from human erythrocytes. Using an improved procedure--which involves complete hemolysis of the erythrocytes, diethylaminoethyl (DEAE)-Sephacel column chromatography, and 5'-AMP-Sepharose 4B affinity chromatography--we obtained 23,000-fold purified isoenzyme from the crude hemolysate (overall yield about 90%). The final product was homogeneous on polyacrylamide disc gel electrophoresis and had a specific activity of about 435 kU/g. Its amino acid composition is presented. With the eventual aim to make visible and isolate IgA kappa antibody-secreting B lymphocytes, we developed reproducible methods for preparing fluorescein isothiocyanate isomer-1-conjugated LD-3 with a fluorescein/LD-3 molar ratio between 1.3 and 3.3, and biotinylated LD-3 with a biotin/LD-3 molar ratio between 1.3 and 2.5. In evaluating the stability of these two conjugates, we determined that they still can react with IgA kappa to form the IgA kappa (LD-3)2 complex.

Affinity Labels↗
PubMed1991

Nucleotide specificity of cardiac sarcoplasmic reticulum. Inhibition of GTPase activity by ATP analogue in fluorescein isothiocyanate-modified calcium ATPase.

Unlike skeletal muscle sarcoplasmic reticulum, canine cardiac sarcoplasmic reticulum hydrolyzes GTP in ways that are similar and different from ATP hydrolysis. Also, ATP and ATP analogues inhibit GTPase activity noncompetitively with a Ki compatible with the high affinity ATP-binding site (c.f. Tate, C.A., Bick, R.J., Blaylock, S., Youker, K., Scherer, N.M., and Entman, M.L. (1989) J. Biol. Chem. 264, 7809-7813). This suggested that ATP and GTP may enter the reaction pathway at separate nucleotide-binding sites on the CaATPase. To test this hypothesis, cardiac sarcoplasmic reticulum was incorporated with fluorescein isothiocyanate (FITC), which apparently binds at or near the ATP-binding site of the enzyme, preventing ATP binding. After FITC incorporation, calcium-dependent ATPase activity, but not GTPase activity, was completely inhibited. Adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), but not guanyl-5'-yl imidodiphosphate, protected against FITC incorporation and the inhibition of calcium-dependent ATPase activity; at least 100 microM AMP-P(NH)P was required for some protection. Despite FITC incorporation, AMP-P(NH)P still inhibited the GTPase activity with a Ki of 3-7 microM. Direct photo-affinity labeling with either 0.2 microM [alpha-32P]ATP or 0.2 microM [alpha-32P]GTP demonstrated that FITC incorporation did not prevent ATP or GTP binding. The mechanism of FITC inhibition of calcium-dependent ATPase activity was related to the prevention of all calcium-dependent, but not calcium-independent, reactions with both nucleotides.

Adenosine Triphosphate↗
PubMed1991

[A new method for the determination of circulating blood volume--erythrocyte marking with sodium fluorescein].

The knowledge of circulating blood volume extends the possibility of judgement of the circulation status and is a valuable help for the management of volume therapy. Blood volume measurements are rarely done in clinical practice in underestimation of their value for the therapy because of their high technical expense, long duration of examination, bad repeatability and radiation exposure. A method for blood volume determination which possesses more favorable abilities than known radioactive methods has been developed by staining red cells with fluorescein sodium. This paper presents an insight into the method and examines the reliability of measurement by means of repeated determinations in vivo.

Adult↗
PubMed1991

Measurement of different human microvascular dimensions by combination of videomicroscopy with Na-fluorescein (NaF) and indocyanine green (ICG) in normals and patients with systemic sclerosis.

Using a double tracer technique with the two dyes indocyanine green (ICG) and Na-fluorescein (NaF) it becomes possible to measure diameters of the capillary, red blood cell column plasma layer and pericapillary halo. 12 healthy controls and 12 patients with systemic sclerosis were studied. The first three mentioned diameters were significantly enlarged in the patient group on the arteriolar, venular and apical side of the nailfold capillaries (p less than 0.03-0.001). Halo diameter was only significantly increased at the apex and not at the two capillary limbs. This finding confirms the visual impression of "dwarf hat formation" or "apical capping" after NaF injection as an important sign for microangiopathy due to collagen vascular disease. It corresponds to preferential halo enlargement at the loop apex. Since ICG couples almost completely to plasma proteins it permits delineation of the inner capillary contours and visualization of capillary aneurysms even when they are completely filled by plasma.

Adult↗
PubMed1991

Clearance of sodium fluorescein incorporated into microspheres from the vitreous after intravitreal injection.

The clearance of microsphere-bound fluorescein from the vitreous was studied in rabbits to evaluate microspheres as a controlled-release drug delivery system. Microspheres of different combinations of copolymers of lactic and glycolic acid were injected intravitreally in three groups of rabbits. High concentrations were detected in the vitreous of all groups within 24 hours. By 5 days, the levels had gradually decreased, but they remained measurable up to 16 days in the rabbits in which lactide microspheres had been injected. Clearance was more rapid in the lactide/glycolide combination group than in the lactide group.

Animals↗
PubMed1990

AIDS and hepatitis B and C: contamination risk at transurethral resection. A study using sodium fluorescein as a marker.

The human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) are all transmitted by minute amounts of infected blood. Surgeons are at risk of contracting these infections by a number of routes including splashes of blood on to the mucous membranes of eyes, nose and mouth, needle stick injuries and spillage of blood on chapped ungloved hands. The endoscopic surgeon is quite unaware of nearly all instances of facial contamination because the splashes are frequently both minute and dilute. Sodium fluorescein, an indelible fluorescent dye detectable in dilutions of one to two parts per million, has been used in this prospective study as a marker of irrigating fluid used during transurethral resection. In 17 out of 20 consecutive operations its presence on the face of the surgeon was revealed by photography, although only a few splashes were visible to the naked eye. There was a random distribution of splashes and also a recurring pattern of contamination of the orbit of the eye that looks through the telescope and the tip of the nose. Vaccination against hepatitis B is recommended for all endoscopic surgeons, together with the use of protective glasses and a mask that covers both the nose and mouth. The greatest danger to the health care team is from an infectious patient who is an unknown risk. It is recommended that high risk patients presenting for transurethral resection should first be screened for the presence of HBV, HCV and HIV antigens.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

[The pharmacokinetics of fluorescein, hematoporphyrin and its derivative during the development of granuloma].

In experiments on rats with granuloma reproduced by subcutaneous implantation of foreign material there was shown a slowing of excretion from the organism as well as a considerable redistribution between the organs of fluorescein, hematoporphyrin and its derivative as compared with intact animals. The noted changes practically did not depend on the time of granuloma development.

Animals↗
PubMed1990

Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.

Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

Amino Acid Sequence↗
PubMed1990

Comparative study of three fluorescent dyes for angiography: sodium fluorescein, carboxyfluorescein, and calcein.

To compare the characteristics of carboxyfluorescein (CF) and calcein (Calc) with those of sodium fluorescein (Naf), the only fluorescent dye currently in clinical use, we performed angiography in rabbits and primates using these three dyes. The circulation decay time of all dyes was longer in primates than in rabbits. In primates, CF and Calc had longer decay times than Naf. Calc produced the greatest contrast between the choroidal and retinal vasculature. Tissue staining and dye leakage into the vitreous immediately after retinal photocoagulation were minimal with Calc, moderate with CF, and marked with Naf. The limited leakage and longer circulation time of Calc may permit simultaneous angiography and photocoagulation therapy without obscuring the fundus view with leaking dye from the photocoagulated structure.

Angiography↗
PubMed1985

Identification of recurrent idiotypes within the unrestricted anti-fluorescein immune response.

The BALB/cV immune response to fluorescein (F1) was analyzed for expression of serologically defined idiotypes (Id) present on two anti-F1 hybridoma proteins, 3-13 and 3-17. After a I degree, II degree, or III degree immunization with F1-KLH, Id 3-13 and 3-17 were expressed in 15 of 28 and 22 of 28 mice, respectively. Solid-phase Id inhibition assays, performed by using pooled or individual anti-F1 antibody, indicated Id 3-13 and 3-17 represented less than 1.0% of the I degree, II degree, or III degree serum anti-F1 repertoire. Hybridoma proteins 3-13 and 3-17 had similar but distinct active sites as indicated by similar patterns of Id inhibition, fine specificity of binding, and affinity for F1. Analyses of 11 anti-F1-AECM-Ficoll hybridoma proteins (predominantly mu, lambda) indicated 4 of 11 clones were weakly cross-reactive with 3-13 and 3-17. Anti-F1-KLH II degree antibody from mice of various Igh allotypes was also screened and indicated these Id were expressed in each strain but at lower levels than in BALB/cV mice and were not strictly allotype associated. Id 3-13 and 3-17 represent the first demonstration of repertoire-specific recurrent Id expressed during a T-dependent immune response lacking a dominant Id.

Animals↗