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PubMed1996

Gene family use and somatic mutation in primary and secondary fluorescein-specific IgM antibody responses.

A comparative analysis of the DNA sequences of primary and secondary IgM, fluorescein-specific antibodies was performed. These antibodies were secreted by hybridomas generated following fusion of immunized BALB/c mouse lymphocytes and SP2/0 myeloma cells. Our results show that primary and secondary fluorescein-specific IgM antibodies use a variety of segments from the variable region of the immunoglobulin heavy chain locus (VH), with members of the J558 and 7183 VH gene families predominating in both populations. D regions from the DF116 and DSP2 families were used exclusively in our primary antibody sample and predominated in the secondary response. In the primary antibodies, 15 out of 18 definable D regions were transcribed in reading frame one, but in the secondary antibodies the three reading frames were used stochastically. Secondary IgM antibodies showed a higher frequency of somatic mutation than their primary counterparts, but we could detect no evidence of selection for mutations in the complementarity determining regions as compared with the framework regions. It appears that fusion of secondary cells, 3-6 days after immunization, is able to 'capture' the IgM-producing population of B cells at a stage in their development following mutation but prior to antigenic selection.

Amino Acid Sequence↗
PubMed2002

A comparative study to assess the clinical use of Fluorescein Meniscus Time (FMT) with Tear Break up Time (TBUT) and Schirmer's tests (ST) in the diagnosis of dry eyes.

INTRODUCTION: The clinical diagnosis of dry-eye is confirmed by a suitable test of tear production and the technique commonly used today to diagnose dry eye is the Schirmer's test (ST). Although the ST is easy to perform it gives variable results, poor reproducibility and low sensitivity for detecting dry eyes. Another test, the tear break up time (TBUT) is used to assess the stability of tears which if abnormal may also cause symptomatic dry-eye. We present the results of both these tests and a new test, which shows greater sensitivity than the ST in detecting aqueous tear deficiency. The fluorescein meniscus time (FMT) is a new test developed in conjunction with one of the authors (CL) and the Department of Ophthalmology at the University Hospital of Wales. The FMT is a measure of the rate at which a fluorescent tear meniscus is formed using 2% sodium fluorescein, a stopwatch and suitable illumination with a slit lamp. METHOD: An open controlled study in 62 patients and 51 controls was conducted to compare the ability of ST, FMT and TBUT to detect dry-eye in a group of patients diagnosed with rheumatoid arthritis and symptomatic dry eyes for a minimum period of 6 months. A separate control group of 15 subjects was tested on three separate occasions to assess the reproducibility of the FMT test. RESULTS: All three tests showed a statistically significant difference between the patient and control populations; Mann-Whitney P < 0.001. There was a correlation between the right and left eye for all three tests in the control group (ST r(2) = 0.77, FMT r(2) = 0.98, TBUT r(2) = 0.94). This correlation was markedly reduced for FMT and TBUT in the patient population and was in keeping with the symptoms reported as being worse on one side in a proportion of the patients (FMT r(2) = 0.52, TBUT r(2) = 0.54, ST r(2) = 0.75). A correlation with age was also observed for all the three tests in the control group (ST r(2) = 0.74, FMT r(2)= 0.92, TBUT r(2) = 0.51), but not in the patient population (ST r(2) = 0.06, FMT r(2) = 0.18, TBUT r(2) = 0.03). A significant correlation was observed between the ST and FMT in both the control (ST vs FMT r(2) = 0.65) and patient population (ST vs FMT r(2) = 0.44). There was no value greater than 200 seconds for FMT recorded in the control group. Using this value to define an abnormal FMT, 85% of the patients (72% of the eyes tested) had an abnormal result. This was in contrast to 35% of patients (26% of the eyes tested) with abnormal results detected by ST. Using ANOVA and Student's paired t-test, there were no significant differences between the three sets of values recorded serially over 3 months to assess the reproducibility of the FMT. The average standard error of the mean was 2.72% and the average co-efficient of variation 4.07%. CONCLUSION: Our study suggests that the FMT is a more sensitive test with good reproducibility compared to the Schirmer's test. The FMT correlates with the ST and suggests that both tests measure aqueous tear deficiency. The FMT therefore is a better alternative to ST currently being used to test aqueous tear deficiency.

Adolescent↗
PubMed2005

Retinal artery macroaneurysms: clinical and fluorescein angiographic features in 34 patients.

AIM: To describe the clinical and fluorescein angiographic features of patients with macroaneurysms (MAs) attending an ophthalmology department over 8 years. METHODS: All the patients between 1997 and 2004 with a diagnosis of retinal artery MA were identified from hospital records. Photographs and fluorescein angiograms were examined, and the MAs classified according to their clinical (haemorrhagic or exudative) features, and angiographic (saccular or fusiform) features. Baseline data of the patients were taken including visual acuity and associated ocular and medical conditions. RESULTS: A total of 38 MAs were identified in 34 patients. The age at presentation ranged from 36 to 93 years, with a mean of 73.7 years. Of them, 24/34 (70%) were female patients, and 10/34 (30%) were male patients. There was a history of hypertension in 9/14 patients (64.3%). Of 38 MAs, 32 were identifiable as either saccular or fusiform. Of these, 22/32 (68.75%) were saccular, the rest fusiform. In all, 72.7% of the saccular MAs showed haemorrhage as the predominant feature. Of the 10 fusiform MAs, 90% showed haemorrhage as the predominant feature. CONCLUSION: Retinal artery macroaneurysm is a rare condition that may mimic a variety of other conditions. Most MAs do not require treatment. We suggest guidelines for diagnosis and propose a treatment algorithm for this condition.

Adult↗
PubMed2001

Retinopathy of Great Pyrenees dogs: fluorescein angiography, light microscopy and transmitting and scanning electron microscopy.

OBJECTIVE: Investigation of the pathogenesis of Great Pyrenees retinopathy. ANIMALS: One male and two female puppies of parents who were affected with Great Pyrenees retinopathy and one 4-year-old affected adult male Great Pyrenees dog. PROCEDURE: The puppies were examined daily from 7 weeks of age by indirect ophthalmoscopy and their fundi were photographed until the lesions were static. Fluorescein angiography was completed at 7 weeks of age, within 24 h of detection of ophthalmoscopic lesions, and then weekly. The eyes of a 4-year-old and two 20-week-old puppies were examined with light microscopy, and transmitting and scanning electron microscopy. RESULTS: Blocked choroidal fluorescence was detected at 7 weeks of age. The blocked fluorescence enlarged, when the characteristic serous retinal detachments developed at 11 weeks of age. The detachments enlarged in size and number until the puppies were approximately 20 weeks old. Fluorescein pooling confirmed the presence of transient retinal pigment epithelial detachments. Leakage of dye into serous retinal detachments was not detected in this study. Light microscopy and transmitting and scanning electron microscopy confirmed the presence of multifocal serous retinal detachments with focal retinal degeneration that extended to the inner nuclear layer in a 4-year-old dog. The retinal detachments were accompanied by hypertrophy, hyperplasia, increased pigmentation, and vacuolation of the retinal pigment epithelium. CONCLUSIONS AND CLINICAL RELEVANCE: Great Pyrenees retinopathy is manifested by multifocal serous retinal and retinal pigment epithelial detachments. These detachments are similar to those noted with central serous retinopathy of humans. The sudden development of multifocal retinal and retinal pigment epithelial detachments, and the serous nature of these detachments, supports a theory that they develop secondary to focal secretion and absorption defects in retinal pigment epithelial cells. Given the age of the puppies when the blocked choroidal fluorescence was noted and maturation of the dog retina at 8 weeks postpartum, this retinopathy is considered to be a retinal pigment epithelial dysplasia. This unique inherited retinopathy offers an opportunity to study retinal pigment epithelial secretion.

Animals↗
PubMed2005

Simultaneous fluorescein and indocyanine green angiography for exudative macular degeneration.

BACKGROUND: The present study investigates how often a simultaneous fluorescein and indocyanine green (ICG) angiography had therapeutic consequences and if it is useful as a clinical routine diagnostic tool. PATIENTS AND METHODS: 502 consecutive simultaneous angiographies in eyes with exudative macular degeneration were retrospectively studied. RESULTS: A classic extra- or juxtafoveolar choroidal neovascularisation (CNV) was found in 3.5 % of the eyes. A subfoveal predominantly classic CNV was present in 19 % of the angiographies. ICG angiography showed a vascular network in 3 % of the eyes with occult CNV in fluorescein angiography. A neovascularisation supplied by retinal vessels (retinal angiomatous proliferation) was found in 9 % and a polypoidal choroidal vasculopathy (PCV) in 6 %. Other plaques or hot spots were visible in 4 %. In 11 eyes with progressive exudation from PCV and threatening of the fovea, laser treatment was successfully performed. CONCLUSIONS: Combined angiography identifies treatable PCV. Advantages of a combined procedure (easier logistics, no missing of treatable cases) and arguments for a two step procedure with ICG angiography only in selected cases (lower costs, lower rate of adverse reactions) must be weighed against each other.

Adult↗
PubMed1983

[Macular edema due to posterior vitreous detachment--observations with fluorescein angiography].

Description of five characteristic cases of macular edema caused by senile posterior vitreous detachment. Fluorescein angiography revealed no abnormalities and the macula remained devoid of significant changes. In cases where a fluorescein leakage in the macular area was found, a thick preretinal fibrosis occurred after several months with a decrease in visual acuity, which was initially good.

Aged↗
PubMed1983

[Evaluation of fluorescein angiograms using the multitemporal technic].

If there are differences in the density of black-and-white photographs of the fundus oculi taken at different times, they may be transformed into color differences by means of the multitemporal technique. A multitemporal picture is obtained by additive color mixing of black-and-white photographs transilluminated at the same time with one primary color each. Since, in a serial angiogram, the interrelation between the film density and the passage of the fluorescein concentration in the fundus oculi is known, it is possible to draw conclusions from the multitemporal picture regarding phase differences and changes in the curve shape of the fluorescein concentration.

Diagnosis, Differential↗
PubMed1980

[Physiological side differences in fluorescein inflow into eyes (author's transl)].

Rapid-sequence fluorescein angiography was performed simultaneously on both eyes of each of 241 healthy individuals in order to analyze and compare dye inflow patterns in the eyes of each subject. The many uncertainties of successive investigations resulting from uncontrollable changes in systemic circulation parameters between the two angiographies were thus eliminated. In over 90% of the subjects there was no difference between the two eyes in respect of arm-fundus time nor the filling pattern of choroid and retina. A few subjects did show a difference, but this lasted for only one exposure and was normalized on the second exposure of the sequence. It is therefore concluded that side differences of the fluorescein inflow pattern on simultaneous bilateral angiography are highly suggestive of unilateral circulatory disturbances in the cervical, retinal or choroidal vessel. Such disturbances are practically certain if side differences last for more than just one exposure. In contrast, the filling pattern in cilioretinal arteries was very variable and over one third of the subjects presented with side differences between their two eyes. Therefore the filling pattern of these vessels cannot be used for diagnostic purposes.

Adolescent↗
PubMed2000

The conformational activation of antithrombin. A 2.85-A structure of a fluorescein derivative reveals an electrostatic link between the hinge and heparin binding regions.

Antithrombin is unique among the serpins in that it circulates in a native conformation that is kinetically inactive toward its target proteinase, factor Xa. Activation occurs upon binding of a specific pentasaccharide sequence found in heparin that results in a rearrangement of the reactive center loop removing constraints on the active center P1 residue. We determined the crystal structure of an activated antithrombin variant, N135Q S380C-fluorescein (P14-fluorescein), in order to see how full activation is achieved in the absence of heparin and how the structural effects of the substitution in the hinge region are translated to the heparin binding region. The crystal structure resembles native antithrombin except in the hinge and heparin binding regions. The absence of global conformational change allows for identification of specific interactions, centered on Glu(381) (P13), that are responsible for maintenance of the solution equilibrium between the native and activated forms and establishes the existence of an electrostatic link between the hinge region and the heparin binding region. A revised model for the mechanism of the allosteric activation of antithrombin is proposed.

Amino Acid Sequence↗
PubMed2000

A remarkably stable phosphorylated form of Ca2+-ATPase prepared from Ca2+-loaded and fluorescein isothiocyanate-labeled sarcoplasmic reticulum vesicles.

After the nucleotide binding domain in sarcoplasmic reticulum Ca2+-ATPase has been derivatized with fluorescein isothiocyanate at Lys-515, ATPase phosphorylation in the presence of a calcium gradient, with Ca2+ on the lumenal side but without Ca2+ on the cytosolic side, results in the formation of a species that exhibits exceptionally low probe fluorescence (Pick, U. (1981) FEBS Lett. 123, 131-136). We show here that, as long as the free calcium concentration on the cytosolic side is kept in the nanomolar range, this low fluorescence species is remarkably stable, even when the calcium gradient is subsequently dissipated by ionophore. This species is a Ca2+-free phosphorylated species. The kinetics of Ca2+ binding to it indicates that its transport sites are exposed to the cytosolic side of the membrane and retain a high affinity for Ca2+. Thus, in the ATPase catalytic cycle, an intrinsically transient phosphorylated species with transport sites occupied but not yet occluded must also have been stabilized by fluorescein isothiocyanate (FITC), possibly mimicking ADP. The low fluorescence mainly results from a change in FITC absorption. The Ca2+-free low fluorescence FITC-ATPase species remains stable after addition of thapsigargin in the absence or presence of decavanadate, or after solubilization with dodecylmaltoside. The remarkable stability of this phosphoenzyme species and the changes in FITC spectroscopic properties are discussed in terms of a putative FITC-mediated link between the nucleotide binding domain and the phosphorylation domain in Ca2+-ATPase, and the possible formation of a transition state-like conformation with a compact cytosolic head. These findings might open a path toward structural characterization of a stable phosphorylated form of Ca2+-ATPase for the first time, and thus to further insights into the pump's mechanism.

Biological Transport, Active↗
PubMed1999

Progressive changes in the fluorescein and indocyanine green angiogram in acute idiopathic maculopathy.

AIMS/BACKGROUND: To report progressive changes in the fluorescein and indocyanine green angiograms of a patient with acute idiopathic maculopathy (AIM). METHODS: Over a two-year period, the patient underwent repeated ophthalmoscopic examinations and fluorescein (FA) and indocyanine green (ICG) angiography. RESULTS: The patient presented with subretinal neovascularization in his right eye. He developed recurrences after laser photocoagulation and surgical removal of the neovascular complex. One year later, he experienced a sudden loss of vision in his left eye with a maculopathy consistent with AIM. The maculopathy resolved after two weeks with poor vision. During the acute stage, FA showed lobular hyperfluorescence in the early phase and pooling in the late phase of the angiogram. In the resolved stage of the disease, FA showed irregular window defects and blockage. ICG revealed late hyperfluorescence of the macula in the acute stage. In the resolved stage of the disease, early hypofluorescence was noted in the ICG, which persisted throughout the late phase. CONCLUSION: This patient had poor vision in his right eye as a result of subretinal retinal neovascularization and poor vision in his left eye from a severe form of AIM. FA and ICG differed markedly during the acute and resolved stages of AIM. All cases of idiopathic subretinal neovascularization should be carefully evaluated to exclude AIM as the primary disease.

Acute Disease↗
PubMed1978

Fluorescein colchicine. Synthesis, purification, and biological activity.

The synthesis of a fluorescent colchicine derivative permits the localization of colchicine-binding receptors in cells. Fluorescein colchicine (FC) was prepared by the addition of fluorescein isothiocyanate to deacetyl colchicine. The product, FC, was separated from the reactants by thin-layer chromatography (TLC). The purity of FC was demonstrated by TLC, UV spectral analysis, and analysis of the kinetics of photodecomposition. FC inhibited [3H] colchicine binding to purified brain tubulin. The biological activity of FC was compared to the activity of unlabeled colchicine on mitosis, motility, secretion, and myogenesis. The effects of FC were identical to those of unlabeled colchicine in all biological systems tested. The results demonstrate that FC may be substituted for colchicine in biological experiments without significant loss in specificity or effectiveness.

Colchicine↗
PubMed1976

Antifluorescein affinity columns. Isolation and immunocompetence of lymphocytes that bind fluoresceinated antigens in vivo or in vitro.

A new method for the isolation of specific immunocompetent lymphocytes has been described in which lymphocyte populations are exposed to fluoresceinated antigens (FLAGs) in vivo or in vitro, and the FLAG-binding cells retained on antifluorescein affinity columns. Specific cells are then eluted with an unrelated FL-labeled protein and shown to be fully immunocompetent. This methodology has been applied successfully in diverse antigenic systems including polymerized flagellin and TNP-specific B cells and alloantigen-reactive cytotoxic T lymphocytes. The method is rapid, inexpensive (requiring only antifluorescein beads), and can be applied to any antigens (or antibodies) in which a fluorescein group can be introduced.

Animals↗
PubMed1988

Ocular fluorescein clearance in patients with hypotony secondary to chronic uveitis.

The development of hypotony is not uncommon in patients with uveitis. Recent evidence suggests that prostaglandins may cause reduction in intraocular pressure by increasing uveoscleral outflow. Ocular fluorescein clearance was measured in patients with hypotony due to chronic uveitis. The mean fluorescein clearance was found to be 0.44/hr., which is consistent with the results found by other investigators in normal subjects.

Administration, Topical↗
PubMed1997

Fibroblasts contracting collagen matrices form transient plasma membrane passages through which the cells take up fluorescein isothiocyanate-dextran and Ca2+.

When fibroblasts contract collagen matrices, the cells activate a Ca(2+)-dependent cyclic AMP signaling pathway. We have found that contraction also stimulates uptake of fluorescein isothiocyanate-dextran molecules from the medium. Our results indicate that fluorescein isothiocyanate-dextran enters directly into the cell cytoplasm through 3- to 5-nm plasma membrane passages. These passages, which reseal in less than 5 s in the presence of divalent cations, also are likely sites of Ca2+ uptake during contraction and the first step in contraction-activated cyclic AMP signaling. The formation of plasma membrane passages during fibroblast contraction may reflect a general cellular response to rapid mechanical changes.

Binding Sites↗
PubMed1978

Conjugation of coxsackievirus type B1-B6 immunoglobulins with fluorescein isothiocyanate by a "reversed" dialysis method.

Immunoglobulins from antisera to coxsackievirus B types 1--6 were labeled with fluorescein isothiocyanate by a "reverse" dialysis method of conjugation. Conjugates thus obtained were labeled at reproducible ratios of fluorescein to protein with weight-weight ratios ranging from 5.0 to 7.1 and estimated molar ratios ranging from 2.1 to 2.9. Conjugates were tested on cover-slip HeLa cell cultures by the direct method of staining. Two types of specific immunofluorescence were observed: intensely fluorescent perinuclear masses and discrete foci of bright fluorescence scattered throughout the cytoplasm. Titers of homotypic conjugates varied from 1:40 to 1:160. Heterotypic staining, consisting of a diffused hazy fluorescence, was observed only at the highest concentrations of conjugate and not above the 1:10 dilution. This technique proved to be specific and sensitive for the detection of group B coxsackievirus antigens in HeLa cells but failed in specific detection of the same antigens in samples of the myocardium obtained by biopsy of three patients clinically ill with idiopathic myocarditis.

Antibodies, Viral↗
PubMed1998

First clinical implications of perioperative red cell volume measurement with a nonradioactive marker (sodium fluorescein).

UNLABELLED: We measured red cell volume (RCV) with the nonradioactive marker sodium fluorescein (SoF) in 30 patients undergoing gynecological operations. Sixteen patients underwent preoperative isovolemic hemodilution (PIHD). RCV measurements were performed before and after PIHD and at the end of the operation. All RCVs were related to corresponding hematocrit (hct) levels. We report a simplified method for its clinical application by reducing the number of blood samples required. To validate our method, we compared RCV within the PIHD bags (bag RCV) with the difference of the patients' RCV before and after PIHD. Bag RCV obtained during PIHD (mean 399+/-81 mL) was measured with a precision of 4.2% by using SoF. There was a significant difference (mean 286+/-401 mL; P < 0.05) between intraoperatively estimated and measured blood loss. The blood loss tended to be underestimated and, in some cases, was underestimated or overestimated substantially. Preoperative and postoperative hct values only offered an imprecise estimation of the patients' RCV. We conclude that RCV measurement using SoF is a precise method for monitoring changes in RCV during PIHD and surgical operation. IMPLICATIONS: We measured red cell volume changes of 30 patients with the nonradioactive marker sodium fluorescein before and after hemodilution and postoperatively with a high precision. We frequently found large differences between intraoperatively estimated and measured blood loss. Preoperative hematocrit values offered an imprecise estimation of the patients' red cell volume.

Adult↗
PubMed1999

Retinal artery air embolism in dogs: fluorescein angiographic evaluation of effects of hypotension and hemodilution.

UNLABELLED: Cerebral air embolism can cause cerebral complications after open heart surgery. The duration of cerebral artery occlusion by air embolism is thought to vary depending on the conditions. However, no study has evaluated factors affecting the duration of occlusion. In this study, we examined the effects of blood pressure and hemodilution on the duration of retinal artery occlusion caused by air embolism in dogs. The duration of retinal artery occlusion caused by the injection of 0.6 mL of air into the common carotid artery was measured by fluorescein angiography and compared among the following three periods: a control period, during which the mean blood pressure (MBP) was maintained at 80 mm Hg; a hypotension period, during which MBP was decreased to 60 mm Hg by exsanguination; and a hypotension plus hemodilution period, during which an additional exchange of blood with hydroxyethyl starch solution was performed and MBP was maintained at 60 mm Hg. When MBP was lowered from 80 to 60 mm Hg, the duration of retinal artery occlusion was prolonged from 34+/-39 to 166+/-90 s (P < 0.01). In dogs with MBP of 60 mm Hg, hemodilution (12.0+/-0.9 to 7.3+/-0.5 g/dL hemoglobin concentration) shortened the duration from 166+/-90 to 75+/-50 s (P < 0.05). Our results demonstrate that hypotension prolongs and hemodilution shortens the duration of retinal artery occlusion caused by air embolism. IMPLICATIONS: We evaluated the effects of blood pressure and hemodilution on the duration of retinal artery occlusion caused by air embolism by retinal fluorescein angiography. Hypotension prolonged and hemodilution shortened the duration of retinal artery occlusion caused by air embolism.

Animals↗