Diffuse field loss and diffuse retinal nerve-fiber loss in glaucoma.

A total of 61 eyes of 61 patients were examined by light-sense, flicker and resolution perimetry. All fields were classified as being normal or showing diffuse loss, localized defects or diffuse plus localized loss. For the assessment of diffuse field loss, cumulative defect curves were derived. Retinal nerve-fiber layer (RNFL) loss was assessed by a semiquantitative scoring technique. In light-sense and flicker perimetry, the total diffuse and total overall RNFL scores for eyes with diffuse field loss were approximately twice those obtained for eyes without diffuse field loss. These differences were highly statistically significant (P = 0.0055 and 0.0087 for light-sense perimetry, P = 0.0033 and 0.0042 for flicker perimetry). In resolution perimetry, eyes with diffuse field loss also showed higher RNFL scores; the differences, however, were not statistically significant. The results show that diffuse field loss in light-sense and flicker perimetry is accompanied by diffuse nerve-fiber loss.


The selective effects of elevated intraocular pressure on temporal resolution.

Twenty-seven eyes of 27 patients with normal tension glaucoma (NTG), 68 eyes of 68 patients with primary open-angle glaucoma (POAG), and 11 eyes of 11 patients with special forms of high-tension glaucoma (SHTG) including (pigmentary glaucoma, angle-closure glaucoma, secondary glaucoma, etc.), were examined by automated light-sense and temporal resolution perimetry. Light-sense perimetry was performed with the Humphrey Field Analyzer, using program 30-2. Temporal resolution perimetry was conducted with a system developed by Lachenmayr. Global field indices were calculated for all visual fields and modalities: mean defect (MD) for light-sense perimetry and mean flicker defect (FD) for flicker perimetry. For each of the three glaucoma groups there was a significant linear correlation of FD over MD. The slope of the regression line (regression coefficient a) increased with increasing maximal pressure level of the glaucomatous group: for NTG a = 0.6671, for POAG a = -1.2413, and for SHTG a = -2.235. The differences of the regression coefficients between NTG and POAG and between NTG and SHTG were statistically significant (P = 0.0366 and P = 0.0046, respectively). The results of the present study provide evidence that the relative amount of damage to flicker compared to light-sense perimetry increases with increasing maximal pressure level of a glaucomatous population. Thus, flicker perimetry may be a tool for the identification of eyes with pressure-produced damage.


Pseudoexfoliation syndrome and glaucoma. Does glaucoma capsulare exist?

Pseudoexfoliation syndrome (PES) is seen in patients with a characteristic deposition of material on the anterior preequatorial region of the lens and on a variety of surfaces throughout the anterior segments of the eye. A high frequency of associated glaucoma has also been reported. In spite of numerous clinical and histological investigations, the mechanisms for the glaucoma are unclear. In a series of 5154 patients referred to our hospital for cataract extraction we found a prevalence of 6.1% (n = 314). In 51 of these patients (16.2%) glaucoma was diagnosed in contrast to 33 patients (10.9%) in an age-matched control group. The prevalence of glaucoma in PES patients and the control group was not statistically different, so our findings in cataract patients do not support the interpretation that there is a clinical entity called "glaucoma capsulare."


The pattern-electroretinogram in glaucoma and ocular hypertension. A cross-sectional and longitudinal study.

The pattern electroretinogram (PERG) is an indicator of retinal ganglion cell function. We studied the PERG in 65 normal eyes, in 52 eyes in early stages of glaucoma, and in 28 ocular hypertensive (OHT) eyes. The PERG was recorded using steady-state high contrast (98%) counterphasing checkerboard patterns at check sizes of 0.8 degrees and 15 degrees at 16 reversals/s and 98% contrast. Stimulation area was 27 degrees x 30 degrees. When compared to normals, in glaucoma eyes PERG amplitudes are reduced to 56 +/- 3.6%, (P < 0.0001) for 0.8 degrees and to 79 +/- 4.0% (P < 0.001) for 15 degrees check sizes. Preferential reduction for the small check size allows classification of patients on an individual basis: using discriminant analysis, normal and glaucoma eyes were classified with a sensitivity of 82.7% and a specificity of 90.8%. Nineteen of 28 OHT eyes were classified as pathological. Eyes with OHT were followed up to test whether the PERG can be used to predict visual field damage. At the repeat visit 5 to 35 months later (mean follow-up 20.2 +/- 8.2 months), in eyes with normal PERGs the average loss in mean sensitivity was -0.61 +/- 0.5 dB, while in eyes with pathological PERGs it was -2.6 +/- 0.7 dB (P = 0.05). Findings suggest that the PERG can be used to discriminate between OHT patients who will develop visual field loss and those who will not. Thus, the PERG may be a test of visual function helpful to decide which OHT patients require treatment before visual field deterioration and optic nerve damage have begun.


Size of glaucomatous optic discs.

In normal eyes, the optic disc size shows a high interindividual variability. In the diseased eye, some optic nerve anomalies and diseases occur preferentially in small optic discs, and some are more common in large optic nerve heads. We conducted a study to determine whether glaucoma subtypes are correlated with the optic disc size or not. Stereo optic disc photographs of 997 eyes with primary open-angle glaucoma, 44 eyes with normal-tension glaucoma, 129 eyes with pseudoexfoliative glaucoma, and 850 normal eyes were examined. Compared to eyes with primary open-angle glaucoma (mean optic disc area: 2.63 +/- 0.61 mm2), the optic disc was significantly larger in eyes with normal-tension glaucoma (2.96 +/- 0.73 mm2; P < 0.05), and it was smaller but not significantly smaller in eyes with pseudoexfoliative glaucoma (2.53 +/- 0.51 mm2). Eyes with primary open-angle glaucoma and normal eyes (2.69 +/- 0.69 mm2) did not differ significantly in optic disc size. The abnormally large optic disc in eyes with normal-tension glaucoma may be important diagnostically and pathogenetically.


Comparison of modulation transfer function and through focus response with monofocal and bifocal IOLs.

We analyzed modulation transfer function (MTF) and through focus response (TFR) of the TRUE VISTA bifocal IOL and monofocal IOLs and compared the experimental and clinical data. MTF and TFR were measured at different pupil sizes (2, 3, 4 mm), base powers (14, 21, 27 D), add powers (2, 3, 4 D), and distance zone diameters (1.25, 1.5, 1.75 mm). Standards were established to correlate MTF and contrast sensitivity, as well as TFR and the defocus curve measured clinically. MTF of TRUE VISTA decreased with increasing pupil size and base power. A near add of 4 D showed the best MTF at distant and near focus. MTF of TRUE VISTA at near focus was lower than at distant focus and MTF at distant focus was lower than MTF of monofocal IOLs. TFR and the defocus curve correlated closely. MTF and contrast sensitivity of TRUE VISTA at far and near focus also correlated closely. MTF and contrast sensitivity of monofocal IOLs and TRUE VISTA at distant focus did not correlate. MTF was considerably better with monofocal IOLs while contrast sensitivity was slightly better only at low contrast. However, MTF of monofocal IOLs was much higher than MTF of a normal eye while MTF of TRUE VISTA at distant focus was only slightly lower than the MTF of a normal eye. Using MTF of a normal eye as a threshold, our results demonstrated a close correlation between experimental and clinical findings. MTF and TFR may therefore reliably predict the performance of bifocal IOLs.


Ocular toxoplasmosis: the role of cellular immune defense in the development of recurrences. Results from animal experiments.

Five toxoplasmosis-free animals and ten animals that had been immunized intraocularly against toxoplasma organisms were used in a rabbit model of ocular toxoplasmosis. Five of the ten immune animals were immunosuppressed by cyclosporin A at a cellular level. Experimental toxoplasmic retinochoroiditis occurred in all animals, a process that in complete contradiction to experience reported previously. In the rabbit model, immunity after a past history of toxoplasma infection has thus far appeared to be a safe form of protection from experimental ocular toxoplasmosis. The concept of "immunological privilege of the eye" is discussed as the cause for the unexpected results.


The effects of basic fibroblast growth factor on bovine retinal pigment epithelium in vitro.

Basic fibroblast growth factor (bFGF) has been reported to initiate DNA synthesis in a variety of cells involved in the pathogenesis of proliferative vitreoretinal disorders, e.g., glial cells and fibroblasts. We analyzed the mitogenic effects of bFGF on cultured bovine retinal pigment epithelial cells in relation to time and dose response regulations and culture conditions. Maximum stimulatory effect of bFGF (+70% compared to control group) was found on day 3 following treatment of cultures with 80 ng/ml bFGF. The action of bFGF seems to depend on the serum concentration in the culture media, which means that cofactors may be present in the serum and potentiate the effects of bFGF on cell proliferation.


Effect of histamine on the membrane potential of cultured human nonpigmented ciliary epithelial cells.

The effect of histamine on the membrane potential of cultured human nonpigmented ciliary epithelial cells was examined using intracellular microelectrodes. The resting voltage was Vm = 42 +/- 0.7 mV (n = 41). Histamine (10(-6) mol/l) induced transient hyperpolarization (V = 19.4 +/- 1.2 mV, n = 35) and subsequent depolarization when applied for more than 1 min. The histamine effect was completely inhibited by the H1 antagonist diphenhydramine (10(-6) mol/l), but not influenced by the H2 antagonist cimetidine (up to 10(-3) mol/l). When histamine was applied repeatedly in the absence of external calcium, the amplitude of the hyperpolarization decreased until finally no response was observed. Ba2+ (2 mmol/l) and quinidine (1 mmol/l), both inhibitors of potassium conductance, significantly reduced histamine-induced hyperpolarization. It was also reduced when cells were depolarized by 40 mmol/l extracellular potassium and almost completely abolished in the presence of 80 mmol/l external potassium. We conclude that H1 receptors mediate hyperpolarization by increasing potassium conductance, probably via transient release of intracellularly stored calcium.


Fluorescence characteristics of indocyanine green in the normal choroid and in subretinal neovascular membranes.

Indocyanine green angiography with scanning laser ophthalmoscopy is a new supplementary method studying subretinal neovascular membranes. However, it is still controversial as to whether of not exudation of this dye occurs from regular choroidal vessels or such membranes. Fluorescence of the macula area, a parapapillary retinal vein, and of venous blood and plasma was therefore measured with a picture analysis system in five healthy young volunteers. Furthermore, the angiograms of 150 subretinal neovascular membranes were reviewed. Membrane fluorescence was measured in ten exemplary cases and compared with the background fluorescence. The results show that macula fluorescence is detectable much longer than the fluorescence of retinal vessels. However, a comparison with the fluorescence of blood and plasma samples revealed that this late fluorescence must be caused by retinal and choroidal capillaries with a significantly reduced hematocrit. It is not caused by apposition or exudation of indocyanine green. The same was true for the majority of subretinal neovascular membranes. Exudation was rarely detected in membranes that were regularly surrounded by exudates but could be induced by laser coagulation. These observations indicate that exudation occurs only after massive destruction of the endothelial barrier. The results are important for the interpretation of indocyanine green angiograms and for possible photodynamic therapy with this dye.


Congenital grouped albinotic spots: a rare anomaly of the retinal pigment epithelium.

We describe the fundus findings of a 6-year-old girl with congenital grouped albinotic retinal pigment epithelial spots distributed over the fundus including the macular area. Apart from the fundus changes functional and electrophysiologic findings were normal, and the family history was uneventful. At follow-up 3 years later, no progression was noted.


Cone dystrophies: clinical and electrophysiological findings.

We analyzed the clinical and electrophysiological findings of 77 patients suffering from progressive cone or cone-rod dystrophies. The onset of symptoms was at the average age of 19.7 +/- 19.4 years. In some patients, the disease started within the 5th decade. The mean visual acuity was 0.19 +/- 0.2, while in 38%, the visual acuity was lower than 0.1. Color vision defects and visual field defects were found in most patients. The electrooculogram was recorded in 59 patients and was normal in only 19. On the electroretinogram (ERG), 60 patients had a reduction of the 30-Hz flicker amplitude and of the responses at maximum stimulus intensity when dark and light adapted. The ERG alterations showed a correlation to the visual field defects and to the reduction of the light rise on the electrooculogram. No correlation existed between the ERG amplitudes and visual acuity or color vision. Ophthalmoscopically, the posterior pole was normal in 25 patients. In the remaining patients, fundus changes ranged from mild pigment irregularities to severe pigment clumping. No correlation between fundus changes and functional findings existed.


Sclerokeratoplasty in recurrent pterygium.

There are many methods for the treatment of recurrent pterygium, and lamellar keratoplasty seems to be the most effective of them. A new method has been developed for the surgical treatment of recurrent pterygium. The most important steps in the procedure are: total removal of the cicatrix and then covering the bare corneoscleral surface with a single piece of corneoscleral tissue taken from a cadaver bulb. The operation was carried out in 11 cases. The follow-up period varied from 10 months to 4 years, the average being 28.7 months. No recurrences were observed. Pseudopterygium developed following the ninth removal of the recurring pterygium; however, even 2 years after the operation, it did not reach the area of the pupil.


Transscleral suture fixation of posterior chamber intraocular lenses through the ciliary sulcus: endoscopic comparison of different suture techniques.

Two technical difficulties exist in transscleral suture fixation of posterior chamber intraocular lenses (PCL) in the ciliary sulcus: first, exact needle penetration of the sulcus and second, exact positioning of the PCL haptics in the sulcus. We used, for the first time, intraocular endoscopy to visualize the site of needle penetration and the final location of the haptics in patients. It turned out that with our previously described standard techniques, precision was far less than anticipated. Thus, new technical ways had to be sought to improve the precision of positioning. In secondary implantation, we achieved best results when the needle was passed from the outside before opening the eyeball, taking advantage of a precisely prepared blue-white line. Passing the needle in an already hypotonic eyeball gives much less precise results. In combination with perforating keratoplasty, passing the needle from the inside by feeling one's way into the sulcus with the needle tip gives the best results. Correct positioning of the PCL haptics is at least as difficult as is needle penetration, a fact which up to now has mostly been ignored. More suitable lens designs may offer a solution for this problem. Their clinical value has to be established by intraocular endoscopy.


Asthenopia: frequency and objective findings.

Fifty young adults were interviewed about the asthenopic symptoms experienced in the preceding 6 months by means of a questionnaire; they then underwent ophthalmic investigation. We established the frequency of occurrence of single asthenopic symptoms and looked for their prevalence in subgroups determined by objective findings such as phorias, different accommodation widths, different fusional vergence widths, and others. The frequencies of single complaints were: headache 42/50 (84%), ocular pain 17/50 (34%), foreign body sensation 25/50 (50%), red eyes 22/50 (44%), photophobia 24/50 (48%), double vision 5/50 (10%), difficulties when changing fixation distance 21/50 (42%). We did not find any obvious preference for one of the symptoms in any of the subgroups mentioned above.


Diffuse field loss and central visual function in glaucoma.

Eighty-two eyes of 82 patients with different types of glaucoma were examined with program 30-2 of the Humphrey Field Analyzer. Forty-six of 82 eyes (56.1%) had diffuse field loss and 36/82 (43.9%) eyes had no diffuse field loss according to the cumulative defect curves. Various foveal threshold criteria including temporal transfer and color vision were tested in all eyes. All criteria showed a loss of sensitivity in the presence of diffuse field loss. The loss of sensitivity was statistically significant for the high-frequency end of the foveal temporal contrast-sensitivity function (P = 0.0498), for foveal flicker-fusion frequency (P = 0.0275) and for foveal and parafoveal blue-on-yellow sensitivity (fovea: P = 0.0009; 4 parafoveal points: P = 0.0001; 16 points in the central 10 degrees: P < 0.0001). The loss in sensitivity was not statistically significant for the low and intermediate temporal frequencies of the foveal temporal contrast-sensitivity function and for the FM 100-Hue loss score. All temporal threshold criteria showed a low sensitivity (25-37%) and a low negative predictive value (48-50%) for the presence of diffuse field loss. Their specificity (75-94%) and positive predictive value (65-83%) are high, however. Thus, whenever one of the foveal temporal threshold criteria as used in the present study is abnormal, the probability is very high that there is some diffuse field loss.


Contact cw-Nd:YAG laser cyclophotocoagulation for treatment of refractory glaucoma.

Contact cw-Nd:YAG laser transscleral cyclophotocoagulation was performed on 42 patients (44 eyes) with glaucoma who were not responding to medical treatment or had failure of prior surgical procedures. No selection was made as to the type of glaucoma (congenital, primary, secondary due to trauma or inflammation). We applied between 16 and 60 exposures 0.5-2 mm posterior to the limbus. Prior to cyclophotocoagulation the mean intraocular pressure (IOP) was 40.4 mm Hg (+/- 8.0). As a result of the treatment, the mean IOP was lowered to 21.1 mm Hg (+/- 5.2). In eyes with congenital, neovascular, and inflammatory glaucoma, a therapeutic effect could only be achieved by repeated coagulations. The patients were followed for a mean of 4.8 months (3-7 months). One eye developed a transient choroidal detachment. In two eyes, iris hemorrhages were noted. Contact cyclophotocoagulation is generally well tolerated. Randomized studies are to be carried out to confirm whether cyclophotocoagulation is an effective tool in the treatment of glaucoma. Until then, cyclophotocoagulation should be restricted to cases not responding to conventional therapy.


The properties of perimetric thresholds in normal and glaucomatous eyes.

In perimetry, threshold is defined as the stimulus intensity at which the frequency of perception is 50%. Around the threshold, there is a transition zone in which the frequency of perception turns from 0% to 100%. We examined the frequency-of-seeing-function in 21 persons with and without pathological damage at various locations in the visual field. We used the classic method of constant stimuli: 12 different stimulus intensities were presented 25-50 times each. The results were: (1) The frequency-of-seeing curve showed the typical S-shaped transition zone as is known from other psychophysical thresholds. The slope of the curve and thereby the size of the transition zone varied by a factor of 17. The 'threshold coefficient,' which is the difference between the intensities that provoke a 50% and a 84% frequency-of-seeing, ranged from 1.13 to 19.83 dB. (2) The threshold coefficient was strongly correlated with the threshold level itself (r2 = 0.596). (3) Other influencing factors such as pathological damage or peripheral location appeared to be covariant, because they influenced the threshold level. A substantial influence of these factors on the threshold coefficient could not be found. Different fluctuations of the threshold are therefore mainly an effect of the threshold level. This may serve as a anticipatory knowledge that allows the optimization of staircase measurement procedures and enables the improvement of the statistical analysis of visual fields.


Number of stimuli as a reliability parameter in perimetry.

Catch trials test patient performance during automated, static perimetry, but their adequacy to estimate reliability is uncertain even though up to 10% of the test time is reserved for catch trials. The 308 visual fields (program G1, all 3 phases, Octopus 201) of 308 eyes of 308 glaucoma, suspected glaucoma, and normal subjects were studied. The 108 visual fields (mean sensitivity > 10 dB; corrected loss variance < 50 dB2) without false responses to catch trials were considered reliable. A multiple linear regression analysis of these 108 fields was performed and revealed the following result (r2 = 0.751): Number of stimuli = 480 + (40.short-term fluctuation) + (8.8.the square root of the index corrected loss variance) - (2.2.mean sensitivity). This equation was used to estimate the number of stimuli required of a reliable subject to complete an examination. Excess stimuli would thus be a sign of reduced reliability. The difference between the estimated and the actual number of stimuli was called the 'stimulus discrepancy'. In 169 fields with false-positive and 58 fields with false-negative responses, the false-positive and false-negative responses correlated with the 'stimulus discrepancy' (r = 0.19, P = 0.014; r = 0.29, P < 0.026, respectively). The number of stimuli depends not only on reliability but also on the software and hardware of the perimeter. 'Stimulus discrepancy' may be an additional useful perimetric reliability parameter which does not require extra testing time.


Multiple-stimulus presentation and voice control in automated perimetry.

In all, 55 eyes of 55 patients were examined prospectively in random order with the Humphrey field analyzer [central field 76 points, full threshold strategy, single-stimulus presentation, response-button control (HFA 1); central field 76 points, defect-depth strategy, response-button control (HFA 2)] and the Dicon TKS-4000 [central field 76 points, defect-depth strategy, multiple-stimulus presentation, response-button control (DIC 1); central field 76 points, defect-depth strategy, multiple-stimulus presentation, voice control (DIC 2)]. Some 26 patients (47%) had glaucomatous field defects, 7 patients (13%) had lesions of the visual pathway, 5 patients (9%) had normal fields. The other 17 patients (31%) had field defects due to vascular lesions of the retina or the optic nerve, retrobulbar neuritis, cataract, dysthyroid optic neuropathy, disorders of the macula, or human immunodeficiency virus (HIV) retinopathy. The mean testing time for the whole study population was 5.2 +/- 2.7 min for DIC 1. The difference from the mean testing time for HFA 2 (6.4 +/- 2.7 min) is statistically significant (p = 0.0013). DIC 2 reduces the mean testing time to 4.9 +/- 2.6 min. The difference from DIC 1 is not statistically significant (p = 0.8110). A multiple-stimulus presentation and voice control seem to be useful methods to reduce the testing time in automated perimetry without a loss of accuracy. Voice control, as used in the DICON TKS 4000, still has to be improved, however.


Horner's syndrome: a retrospective analysis of 90 cases and recommendations for clinical handling.

The records of 90 cases of oculosympathetic paresis (1982-1991), 39 women and 51 men aged between 3 months and 82 years, were evaluated. The mean baseline anisocoria was 0.92 mm but did not exceed 2.4 mm. The mean difference in the position of the upper eyelid was 2.3 mm. Enophthalmus of 1 mm or more was found in only 25% and exophthalmus of 1 mm or more, in 18%. Exophthalmus or enophthalmus of more than 2 mm was not encountered. The cocaine test (5% solution in most cases) was performed in 85 cases and could be quantified in 65 cases. The average dilation of the involved pupil was 0.52 mm, whereas the normal pupil dilated 2.14 mm. The average postcocaine anisocoria was 2.54 mm. Hydroxyamphetamine 1% dilated the involved pupil in cases with presumed preganglionic lesions slightly more than the normal fellow pupil (2.39 mm and 2.09 mm respectively). The difference was significant (P < 0.05). In postganglionic lesions, the hydroxyamphetamine dilation was 0.57 mm. The hydroxyamphetamine test had a specificity of 90% for postganglionic lesions and 88% for preganglionic. An underlying acquired disease could be identified in 53 cases; 6 cases were congenital. In 37 cases (including the congenital ones), no cause was found. Among the 33 preganglionic lesions, only one malignant tumor was found, whereas 6 malignant tumors were encountered among the 20 postganglionic cases. Additional ocular motor palsies or other local signs were present in these 6 cases. Goiter was frequently associated with preganglionic Horner's syndrome.


Diffuse loss of rod function in autosomal dominant retinitis pigmentosa with pro-347-leu mutation of rhodopsin.

There is considerable variety among the clinical features of autosomal dominant retinitis pigmentosa (ADRP). This is probably at least in part due to genetic heterogeneity. Recently, various mutations of the rhodopsin gene have been detected in some ADRP families. We report on six patients from two families with ADRP who were investigated by means of psychophysical and electrophysiological methods. All displayed the same rhodopsin gene mutation at codon 347, which exchanges the amino acid proline for leucine (pro-347-leu). The patients had early-onset night blindness and impaired side vision as of the end of their second life decade. They produced monophasic dark-adaptation curves, showing a lack of rod function and elevated cone thresholds. Dark-adapted two-color threshold perimetry using 500- and 650-nm stimuli revealed a diffuse loss of rod function and centrally preserved cone function. The electroretinogram was nonrecordable at the age of about 30 years. A certain variability of visual function loss was noted among patients in the overall severe course of the disease, but the clinical findings of this genotype corresponded to type 1 ADRP of Massof and Finkelstein in all cases.


Fluorescence characteristics of drusen during indocyanine-green angiography and their possible correlation with choroidal perfusion.

The fluorescence of drusen and their correlation with choroidal perfusion was investigated using indocyanine-green videoangiography in 37 patients (mean age, 66 +/- 16 years) with juvenile and senile drusen. Most senile drusen (89%) blocked choroidal indocyanine-green fluorescence, and only three patients had small, weakly hyperfluorescent drusen at the vascular arcade. In contrast, all juvenile drusen (patients aged 31-52 years) demonstrated strong hyperfluorescence. Impaired choroidal perfusion was not detected. In 33% of the patients, all of them older than 60 years, moderate choroidal hypofluorescence could be explained by the presence of confluent drusen and/or a thickening of the pigment epithelium-Bruch's membrane complex. The results of the present study demonstrate that indocyanine-green angiography enables the differentiation of juvenile drusen from senile drusen, which could be an indication of a differing pathogenesis for the two diseases. Indocyanine-green hyperfluorescence of drusen must be caused by the transmission of choroidal fluoresence. This jeopardizes the hypothesis that the fluorescein fluorescence of drusen is invariably caused by storage of the dye. Our results contradict previous fluorescein studies that have demonstrated impaired choroidal perfusion in patients with age-related macular degeneration.


Central subretinal hemorrhage from a retinal arterial macroaneurysm--two-step treatment with laser and vitreoretinal surgery.

Central subretinal hemorrhage can be a complication of arterial retinal macroaneurysm. Subretinal bleeding may lead to mechanical and toxic damage of photoreceptors, causing persisting central scotomas. Prophylactically, we perform a two-step therapy using an argon laser and vitreoretinal surgery to achieve coagulation of the bleeding source and drainage of subretinal blood.


Lipoprotein (a): a risk factor for retinal vascular occlusion.

The protein moiety of lipoprotein (a) consists of apoB-100 and apolipoprotein (a). Lipoprotein (a) is an independent risk factor for premature atherosclerosis. Apolipoprotein (a) and plasminogen are structurally homologous. Through interference with the fibrinolytic system, lipoprotein (a) may act as a thrombogenic factor. In the present study, we determined lipoprotein (a) concentrations in 84 patients (60 men and 24 women) with retinal vascular occlusion (RVO) and in 2 groups of healthy volunteers (n = 40 and 46). In all, 29% of the patients had Lp (a) levels of above 300 mg/l. In the two reference groups, only 10% and 9% of the subjects exceeded this level. According to the chi-square test, the association between Lp (a) levels and RVO was significant. Lp (a) concentrations did not differ between patients with arterial occlusion and those with venous occlusion. No difference in the total cholesterol, triglyceride, or LDL-cholesterol values was observed between patients and controls. We therefore conclude that Lp (a) represents an independent risk factor for RVO.


A method of lens extraction for the injection of liquid intraocular lenses.

A method for the injection of liquid intraocular lenses into the capsular bag would offer many benefits in the treatment of cataracts. The corneoscleral incision could be made even smaller, the lens diaphragm would be conserved more physiologically, and a method of therapy for presbyopia might eventually be possible. As a prerequisite, a method of endocapsular lens removal is needed. We have developed such a method for our experiments on the injection of liquid intraocular lenses in the enucleated pig eye. We use a bimanual technique through two opposing 1-mm corneoscleral incisions and two openings measuring 1 mm in diameter in the peripheral anterior capsule. By using curved needles and suction tips, we could remove the capsular contents completely. The injected material was polymerized in situ by exposure to blue light and resulted in an optically clear cast of the lens capsule of surprisingly good optical quality.


Light-induced endocapsular polymerization of injectable lens refilling materials.

The use of advanced phacoemulsification techniques enables the surgeon to remove opacified lens material while preserving a nearly complete capsular bag. For this reason, several groups are presently working on lens refilling techniques. We have developed a method for the injection of a liquid monomer into the lens capsule. The material can be polymerized by exposure to cold light (wavelength, 400-500 nm) within the capsular bag. Using cell-culture experiments, we selected an optically clear material that has cytotoxic properties when placed in direct contact with cells but does not release cytotoxic substances into the surrounding medium. We have shown that reflux of the liquid monomer into the anterior chamber can be avoided by using a material that can be polymerized within the capsule. Initial experiments in rabbits indicate that the technique can be used without inducing a significant postoperative inflammatory response or leading to morphological changes in the cornea or the retina.


Tear secretion in dry eyes as assessed by objective fluorophotometry.

To date, little information has been available about the actual amount of tear secretion in dry eyes. In the present study tear secretion was studied in 20 consecutive dry eye patients [40 eyes; Schirmer test after topical anesthesia < 5.5 mm/5 min; at least moderate squamous metaplasia of the conjunctival epithelium; bengal rose staining (Bijsterveld score > 4); severe dry eye complaints]. Tear secretion was evaluated by automated, computerized objective fluorophotometry and compared with that in an age-matched healthy control group. Tear secretion of the dry eyes examined was found to have decreased drastically (0.2 +/- 0.2 microliters/min) compared with that in healthy control eyes (1.2 +/- 0.5 microliters/min; P < 0.001). None of the dry eyes had a tear secretion of > 0.4 microliters/min.


Clinical use of recombinant plasminogen activator for intraocular fibrinolysis.

In a preliminary clinical study, recombinant tissue plasminogen activator (rt-PA) was injected into the anterior chamber of 18 eyes and into the vitreous cavity of 2 eyes in a total of 20 patients. The clinical indications were severe fibrin formation after filtering operations for glaucoma (8 eyes), diabetic neovascular glaucoma (3), fibrin and hemorrhage after vitrectomy (3), keratoplasty (3), posterior chamber lens implantation (1), trauma (1), and endophthalmitis (1). Complete fibrinolysis within a minimum of 2 h was achieved in 81% of 26 injections. Although doses of about 12 micrograms rt-PA were generally given, in our observations, 6 micrograms proved to be sufficient for rapid fibrinolysis in selected cases. Injections of rt-PA should not be considered before the 3rd postoperative day so as to avoid rebleeding and recurrent fibrin formation. No toxic intraocular side effect was observed after the use of rt-PA in this study.


Block excision of congenital and infantile nonpigmented epithelial iris cysts. Report on eight infants.

Spontaneous congenital and acquired nonpigmented epithelial iris cysts are rare unilateral lesions of the anterior uvea. From 1980 to 1991, eight infants with progressive nonpigmented epithelial iris cysts were successfully treated by block excision. This technique consists of simultaneous removal of the adjacent iris, the pars plicata of the ciliary body, and all layers of the sclera and cornea in contact with the lesion that serve as a shell for the proliferating epithelium. The resulting defect is covered with a tectonic corneoscleral graft. Five of the children, whose ages ranged from 1 month to 7 years (average, 3.7 years), presented with a congenital nonpigmented epithelial iris cyst and three infants presented with a cystic epithelial ingrowth following perforating injury. On histopathological examination, all but one patient revealed epithelial involvement of the surface of the ciliary body. All patients were followed for an average of 45.5 months. The long-term visual acuity of our very young patients was encouraging, ranging from 20/200 to 20/20. No recurrence of iris cysts was noted and no enucleation was necessary. Our results indicate that block excision may be the treatment of choice for congenital and infantile nonpigmented epithelial iris cysts.


A colour-flicker analysis of visual function in patients with retinal detachment.

Patients with a history of retinal detachment were examined by the following tests: (1) setting of an isoluminant match between yellow and green constituent squares of a checkerboard and (2) measurement of the flicker fusion frequency for a contrast-reversal of this yellow-green pattern. The results suggest that the test may provide useful data for assessing the severity of any damage to a given part of the retina. The fusion frequencies range from over 10 Hz (normal) to about 2 Hz for a severely impaired retina. The data for the relative amounts of red and green light needed to achieve a match rarely show any abnormality; thus, a standard anomaloscope test or pseudoisochromatic plates would not detect these deficiencies.


Mucinous sweat-gland adenocarcinoma of the eyelid.

A case of mucinous sweat-gland adenocarcinoma of the eyelid, clinically diagnosed as a cyst, is presented. This lesion, although accounting for a small proportion of all sweat gland tumors, has a predilection for the eyelid. It is locally aggressive, has metastatic potential, and wide local excision is recommended as the primary procedure. This case illustrates the clinical, macroscopic, and histopathological features of a rare primary malignancy of the eyelid.


Acute leukemias with the t(4;11)(q21;q23).

The t(4;11)(q21;q23) has been associated with marked lineage heterogeneity. Most of the reported cases were classified as acute lymphoblastic leukemia (ALL). The t(4;11) is one of the commonest specific chromosomal translocations in ALL, occurring in 2% of childhood and 5% of adult cases. In childhood ALL, this translocation is associated with female sex, age less than 1 year, hyperleukocytosis, CD10-/CD19+ B-precursor cell immunophenotype, and myeloid-associated antigen (CD15) expression. There also appears to be an age-related difference in treatment outcome. Adults had the worst prognosis, and children aged 1 to 9 years appeared to have a better outcome than infants or adolescents. Reported cases of acute myeloid leukemia (AML) or secondary leukemia with the t(4;11) have not been well characterized. It is intriguing that virtually all of the reported cases with secondary leukemia had received epipodophyllotoxins or doxorubicin, agents that affect topoisomerase II and are associated with secondary AML characterized by 11q23 abnormalities. Identification of the involved gene(s) in the t(4;11) will provide a molecular approach permitting more accurate classification of these cases.


Measurement of proliferation indices in non Hodgkin's lymphoma--is it useful?

In this review we discuss the possible value of additional information concerning growth characteristics of NHL's for the prognosis of the individual patient. Techniques for measuring cell cycle kinetic parameters (e.g. S-phase assessment) and proliferation related antigens (e.g. anti-transferrin receptor and Ki67) are shortly reviewed. This is followed by an overview of proliferation studies in NHL. Until now in most of these studies overall measurements of tumour tissue have been performed. Moreover the parameters that have been assessed are related to proliferation state rather than proliferation rate. None of the methods used has a convincing superiority with regard to clinical outcome. In the future, tumour specific assessment (by means of double-labeling techniques) of proliferation rate parameters might provide information, more relevant to the prognosis of the individual patient with NHL.


Sinus histiocytosis with massive lymphadenopathy.

To date, the morphological aspects of sinus histiocytosis with massive lymphadenopathy (SHML) have been fully described. The disease is characterized by an enlargement of lymph nodes in which the sinuses are dilated and infiltrated by histiocytes, often phagocytosing lymphocytes. Even if the prognosis is usually benign and not requiring therapy, several fatal cases have been reported. The etiology is still obscure and the biology is not yet completely clear. Recent immunophenotypical studies suggest that histiocytes may belong to the T-zone associated histiocyte lineage. They may be cytologically homogeneous, but can express different antigenic patterns according to their stage of differentiation. Cytogenetic and molecular aspects of the disease have only been sporadically investigated. In order to better understand the pathogenesis of SHML, which seems to be a disorder lying in between the fields of infections, immunological disease and neoplasia, it is considered very useful to systematically employ a variety of immunophenotypical, cytogenetic and molecular techniques to study the disease, particularly in cases which are clinically atypical or with a more aggressive evolution.


F-MACHOP in advanced aggressive lymphoma.

The results of our ten years experience indicate that F-MACHOP, conceptually designed to exploit cell kinetic and synergistic principles, is an effective regimen with acceptable toxicity in a large proportion of adults with advanced stages of diffuse large cell or small non-cleaved cell lymphoma. More than 60% of all treated patients all expected to achieve long-term EFS. Patients achieving CR within 3 courses have a very high chance of being cured of their disease and do not require, in our opinion, intensification with other regimens. On the other hand the identification of a group of patients with poorer outcome (i.e. those failing to achieve an early CR) enabled testing of the potential efficacy of superintensive salvage regimens. Together with other 6 Italian hematologic institutions, we have recently participated in a prospective comparative randomized trial in which F-MACHOP was compared to MACOP-B in adults with advanced stages of diffuse large cell or small non-cleaved cell lymphoma. The preliminary results of this trial appear to confirm that F-MACHOP is an effective and tolerable regimen in such patients and there is a suggestion that its efficacy is superior to that of MACOP-B. This trial was closed to accrual in September 1991 and we look forward to the first complete analysis in March 1992.


A phase I/II study of dose and administration of non-glycosylated bacterially synthesized G-M CSF in chemotherapy-induced neutropenia in patients with non-Hodgkin's lymphomas.

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) derived from E. coli was administered to 24 previously untreated patients with non-Hodgkin's lymphoma following the first cycle of CHOP chemotherapy. Four dose levels were examined, 1.5, 3.0, 5.5 and 11 micrograms/kg and patients were randomized to receive the drug either once or twice daily subcutaneously (s.c.). During rhGM-CSF treatment, the leucocyte counts increased up to 3-4 fold in 20/24 patients, reaching a peak 24-48 (mean 35) hours after initiation of rhGM-CSF. The leukopenic period in cycle one of the CHOP chemotherapy with rhGM-CSF, was shorter than after the course of chemotherapy without rhGM-CSF and also shorter when compared to cycle one of CHOP in the 127 historical controls (p < 0.05 and p < 0.001 respectively). Similar results were observed for neutrophil counts. No effect was seen on platelet counts at nadir but a significant, although moderate increase occurred in the recovery period on days 15 and 22 when compared to control cycles and historical controls. When dose levels were compared, there was only a trend to higher WBC counts at the higher dose groups (5.5 and 11 micrograms/kg) when compared to the two lower dose groups (1.5 and 3.0 micrograms/kg). In the overall evaluation there was no statistical significant difference in results between patients treated s.c. once daily versus twice daily. However when only the two highest dose levels (5.5 + 11 micrograms/kg) were compared, s.c. administration of rhGM-CSF twice daily led to higher leucocyte counts than once daily in the recovery period on day 15 (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Childhood acute lymphoblastic leukemia immunophenotypes and their prognostic significance: experience of the IGCI-study in 389 children. International Society for Chemo-immunotherapy (IGCI-Vienna) Cooperative Group.

The prognostic significance of immunophenotype and other features including sex, age, anaemia, WBC, FAB type, and PAS staining were analysed in a group of 389 children newly diagnosed as acute lymphoblastic leukemia (ALL) and treated according to the BFM 1981/1983 protocol. The CR rate was higher (82-94%) in immunophenotypic subgroups defined as 'non-B' compared with B-ALL (54%). The probability of being in CCR at the end of follow up was 0.68 (median. observation, 3 years). Using the stepwise Cox regression analysis the following independent factors predictive of duration of CCR were selected (relative risk in brackets): 1. WBC (> 25G/1:< 25G/1 = 2.0, P = 0.0008), 2. age (> 10y:2-10y = 1.3, P = 0.04), 3. CALLA positivity (neg.:posit. = 2.4, P = 0.04), 4. CALLA within B-cell progenitor ALL (pre;preB,Calla-:Calla+ = 1.7, P = 0.007). T-ALL appeared to have a worse prognosis than U-ALL and B-progenitor derived ALL but it did not retain independent prognostic significance in multivariate analysis.


The role of IL-4 in human myeloid leukemia: stimulation of RNA synthesis and transduction of differentiation signals through an IL-4 receptor leads to functional and HLA positive HL-60 cells.

The ectopic expression of lineage markers on irrelevant cell types may be of importance in the differentiation pathway(s) of these cells. One example, that is the subject of this study, is the presence of the interleukin-4 (IL-4) receptor on the surface of the human HL-60 myeloid leukemia cell line. The presence of such a receptor, that at first seems to be a simple genetic misprogramming, has an unusual biological function: It serves as a bridge to link the B cell growth factor IL-4 in order to transduce a number of differentiation signals in this M2 acute myeloid leukemia (AML) population. Signal transduction is followed by stimulation of RNA synthesis and subsequent induction of differentiation. Daily administration of low IL-4 dose yields proliferative senescent cells that exhibit 66% of growth inhibition in a 5-day tritiated thymidine incorporation assay. These cells clearly exit from the standard M2 morphology and show more mature characteristics as assessed by the Giemsa-Wright staining technique, followed by a 2-fold increase of the monocyte-granulocyte-specific Mac-1 surface antigen. Cellular function is also affected positively since phagocytosis of latex beads increases considerably after IL-4 treatment. Finally, as reported for normal human and murine monocytes and macrophages, the receptor-ligand interaction augments the levels of the class I and class II antigenic determinants by approximately 60%. Our results suggest that ectopic expression of markers may be a "distinct" event required during a short period in the differentiation of certain hemopoietic cells leading to mature and normal phenotypes.


EoL-1, a human eosinophilic cell line.

EoL-1 cells, a recently established human eosinophilic leukemia cell line, have cytological features of myeloblasts under normal culture conditions, and differentiate not only phenotypically but also functionally into eosinophils by a number of stimuli. EoL-1 cells are particularly useful for analyzing leukemic cell differentiation and the properties of malignant eosinophils. EoL-1 cells are also a useful in vitro model for studying human eosinophil functions and their regulation.


High incidence of myeloproliferative disorders in Ashkenazi Jews in northern Israel.

We have analysed epidemiological parameters in 339 patients with myeloproliferative disorders (MPD) diagnosed in northern Israel between 1975 and 1989 as having polycythemia vera (191 patients), agnogenic myeloid metaplasia (AMM) (113) and essential thrombocythemia (ET) (36). Mean average annual incidence was 11.4 per 1 million residents for polycythemia vera, 6.5 for AMM and 2.1 for ET. For all three diseases the average annual incidence increased with age and was 10 times higher in patients over 65 years compared to those less under the age of 45 years. Four percent of all patients had relatives with MPD. Incidence of MPD in Jews was 10 fold higher than expected compared to Arabs and this difference was noted for all 3 diseases. The incidence in Ashkenazi Jews originating from eastern and central Europe, was 10 and 20 folds higher than in Sephardic Jews and Arabs respectively. Mean age at diagnosis of MPD in Arabs and Sephardic Jews was lower than in Ashkenazi Jews (52 and 56 years compared to 64 years P < 0.05). Likewise, mean age at diagnosis was lower in the 11.5% of MPD patients with prior exposure to biological or chemical hazards compared to unexposed individuals (58 years versus 63 years, P < 0.02). These data demonstrate a cluster of MPD in Ashkenazi Jews in northern Israel and emphasize the importance of genetic predisposition possibly interacting with acquired factors in the pathogenesis of these disorders.


Sequencing of PCR-amplified DNA.

Alternatives for sequencing of PCR products essentially fall into one of two categories; generation of single-stranded DNA for sequencing or the direct sequencing of double-stranded product. Of the two alternatives, sequencing of double-stranded PCR products is likely to be of greatest immediate significance in terms of general applicability and rapidity. Double-stranded sequencing allows the use of the PCR product for other purposes either prior to or subsequent to generation of sequence data. The single-stranded sequencing methods generally require some prior decision regarding sequencing of the product. Assisted by automated workstation development, sequencing of single-stranded DNA PCR products generated either during thermal cycling or following affinity-capture strand separation may have significant future utility, particularly in genome mapping and routine clinical diagnosis. Despite template type and protocol differences, in all situations the purity and concentration of PCR-amplified DNA template used remains the most critical factor determining the efficiency and reliability of nucleotide sequencing methods.


Quantitative determination of rare mRNA species by PCR and solid-phase minisequencing.

We present a new method for quantification of mRNA, in which the limitations of the current quantitative PCR methods can be overcome. A known amount of a synthetic RNA standard differing from the mRNA to be quantified by a single nucleotide is reverse-transcribed and amplified together with the mRNA template using a biotinylated primer. The biotinylated PCR product is immobilized on a streptavidin-coated solid support and denatured. The ratio between the two amplified sequences is determined by separate "mini-sequencing" reactions, in which a detection step primer annealing immediately adjacent to the site of the variable nucleotide is elongated by a single labeled dNTP complementary to the nucleotide at the variable site. The ratio between the incorporated labels accurately determines the ratio between the two sequences in the original RNA sample. We applied this method to quantify the mRNA of human aspartylglucosaminidase (AGA) in tissues and cultured cells. AGA is a lysosomal enzyme participating in the degradation of glycoproteins. A mutation in the AGA gene abolishes the enzyme activity and leads to aspartylglucosaminuria (AGU), a recessively inherited metabolic disorder. The mRNA quantification revealed that the normal and mutant genes are expressed at similar levels in kidney, liver, and cultured fibroblast, whereas the amount of AGA mRNA in normal placenta and brain is significantly higher than that found in the corresponding samples from AGU patients. The method presented here is generally applicable for PCR-based quantification of rare mRNAs and DNA as well.


Preferential PCR amplification of alleles: mechanisms and solutions.

The preferential PCR amplification of one allele relative to another in a heterozygous sample could result in an incorrect or ambiguous genetic typing of that sample. There are several mechanisms that could potentially lead to such preferential PCR amplification. First, preferential amplification can result from significant GC% differences between alleles if the conditions of the reaction (denaturation temperature (Tden), duration at the Tden' salt and co-solvent concentrations, etc.) allow the denaturation of one allele but not the other (differential denaturation). For example, the DQa1.1, -1.2, and -1.3 alleles of the HLA-DQa locus do not amplify at a Tden < 89 degrees C; these same conditions still allow amplification of the DQa2, -3, and -4 alleles. However, no differences in amplification efficiency were found between the different HLA-DQa alleles when the Tden was set at the recommended Tden of 94 degrees C, even after as many as 102 cycles of amplification. Second, for PCR-based genetic typing systems in which the PCR products from different alleles differ in length, preferential amplification of the shorter allelic product can occur. Experiments in which the variable number tandem repeat (VNTR) marker D17S5 (YNZ22) was amplified under various conditions suggest that the smaller allelic products are amplified preferentially when Taq polymerase is limiting. Preferential amplification of VNTR alleles can also occur if the target DNA is sufficiently degraded. Third, when the initial number of genomes sampled is very small, stochastic fluctuation in the number of copies of each allele can result in what appears to be preferential amplification. Finally, less efficient priming of DNA synthesis of one allele versus another can occur because of mismatches between the primer and the specific allelic template, resulting in preferential amplification of the other allele. General strategies to avoid preferential amplification are discussed.


The rat genome contains a p53 pseudogene: detection of a processed pseudogene using PCR.

The p53 gene is the most frequently mutated gene in human cancer. Our investigation of this gene in radiation-induced tumors led to the discovery of a processed pseudogene in the rat genome. We amplified eight coding exons of the p53 gene using rat liver DNA as template, and, in each case, one major amplification product was apparent on agarose gels. When we selected primers to amplify fragments containing more than one exon, two major products were apparent. In each case, the size of the larger amplification product was consistent with that of the expected p53 fragment. The sizes of the shorter amplification products suggested that these fragments are amplified from a processed p53 pseudogene. When the blotted fragments were probed with sequences internal to the amplification primers, both the gene and putative pseudogene fragments were seen. Sequences of the shorter coamplicons have high homology with the p53 cDNA and cross intron splice junctions. These findings suggest that the rat genome contains a processed p53 pseudogene. The data demonstrate the usefulness of the polymerase chain reaction for revealing processed pseudogenes, and suggest that the pseudogene can be used as an internal control when amplifying the rat p53 gene.


Simplified template preparation and improved direct sequencing using Taq polymerase.

A streamlined version of direct dideoxy sequencing is presented that includes template preparation as well as sequencing protocols. The method is used routinely to sequence double-stranded PCR products after minimal purification with one of the primers used in amplification. Either 35S or 32P labeling can be used with equally good results.


Quantitative RNA and DNA gene amplification can rapidly monitor HIV infection and antiviral activity in cell cultures.

We have developed a quantitative gene amplification procedure to assess the replication of human immunodeficiency virus (HIV) in cell cultures and evaluate the effect of drugs on viral replication. Increases in HIV gag RNA and DNA in phytohemagglutinin-stimulated normal peri-pheral blood mononuclear cells (PBMC) infected with HIV at very low multiplicity of infection paralleled the production of HIV p24 antigen in culture supernatants. Quantitative gene amplification was able to monitor the accumulation of viral nucleic acids in control cultures and demonstrate the effect of various concentrations of azidothymidine (AZT) on the replication of both AZT-sensitive and -resistant strains of HIV. The sensitivity of patient-derived virus strains to AZT could also be successfully measured by these procedures. The results of our studies suggest that quantitative measurement of HIV gag RNA and DNA can be used to monitor the kinetics of viral replication, antiviral activity, viral drug resistance, and mechanism of drug action.


Detection of influenza A and B in respiratory secretions with the polymerase chain reaction.

Influenza A and B are RNA-containing viruses that frequently infect humans. Currently, sensitive detection of these viruses requires fresh respiratory secretions and special facilities for culture. To facilitate diagnosis of influenza, the polymerase chain reaction (PCR) was used in the present studies to detect DNA produced by reverse transcription of influenzal RNA in vaccines, tissue culture fluids, and stored respiratory secretions. Primers were directed at targets on the highly conserved segment 7 (matrix gene) of influenza A (212-bp product) and B (365-bp product). The primers were completely type specific. Critical variables in the assay were the concentration of pleotropic salts used during preparation of samples, the use of carrier RNA and RNase inhibitors during sample preparation, and the use of optimum K+ and Mg2+ levels at each step. Studies of 33 patients with symptoms of viral respiratory infection whose nasal washes had been cultured and frozen for up to 1 year before assay showed that PCR provided type-specific detection of influenza with a sensitivity comparable to that of culture of the fresh secretions. The assay offers a powerful test for detection of devitalized influenza viruses and may be useful in both diagnostic work and epidemiological studies of influenza.


A novel method for site-directed mutagenesis using PCR and uracil DNA glycosylase.

A novel method for site-directed mutagenesis of DNA sequences based on the use of the PCR is described. The method uses two oligonucleotide primers that contain the desired sequence change and overlap at their 5' ends. In addition, the thymine residues in the overlap region have been substituted with deoxyuracil. Amplification of the template plasmid by PCR results in incorporation of the primers and the desired mutation into the PCR product. Excision of the deoxyuracil residues in the PCR products by uracil DNA glycosylase (UDG) destablizes base-pairing at the ends of DNA molecules and thus generates 3' protruding ends in the opposite strand. Due to overlapping nature of the primers, the resulting 3' protruding ends are complementary and can anneal rapidly after treatment with UDG. When the entire plasmid is amplified, a linear mutant PCR product is generated that circularizes after treatment with UDG. Circularized molecules can then be transformed into competent cells without ligation, generating transformants with the mutant genotype. Alternatively, the gene of interest is amplified in two segments using overlapping mutant primers and cloned in the desired orientation into pAMP1 by UDG cloning. Application of this method to site-specific mutagenesis of the lacZ alpha gene and the human c-raf oncogene was demonstrated. The accuracy of the mutations was confirmed by nucleotide sequence analysis as well as phenotypic assays. The method is rapid, highly efficient (> 99%), and applicable to genes cloned in any vector as well as to genomic DNA or RNA.(ABSTRACT TRUNCATED AT 250 WORDS)


A lock-docking oligo(dT) primer for 5' and 3' RACE PCR.

We describe a method that can be used to obtain and sequence 3' and 5' ends of cDNA transcripts directly from PCR products. The method employs a modified oligo(dT) primer that enables it to "lock-dock" at the junction of gene-specific cDNA sequence and a natural (3') or appended (5') poly(A) tail. As a result, discrete, first-round PCR products are obtained that are easily isolated and sequenced directly.


Improved blood sample processing for PCR.

To simplify our procedure for blood sample processing for PCR, we introduced a simpler, shorter, and more cost-effective method for the separation of peripheral blood lymphocytes (PBL) and DNA extraction for amplification with Taq polymerase. By this method, blood samples are processed in two simple 15-min steps: (1) separation of PBLs from whole blood by red blood cell lysis with the Roche Specimen Washing Solution, and (2) DNA extraction by heat-detergent treatment of separated PBLs. This new method is simpler than the standard Ficoll-Hypaque method for PBLs separation and Proteinase K digestion for DNA extraction. It is not inhibitory to DNA amplification and it allows effective processing of blood samples even after prolonged storage (as long as 8 days) at room temperature.


Detection of mutation delta F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry.

A method to detect the main cystic fibrosis (CF) mutation delta F508 from dried blood spots, whole blood, or saliva using the polymerase chain reaction (PCR) and time-resolved fluorometry (TRF) is described. Samples are treated by boiling in mild alkaline solution, after which two allele-specific PCR reactions are performed. Allele-specific primers and a common biotinylated primer are used in the amplification reactions. To detect the PCR product, an europium-labeled oligonucleotide, complementary to the biotinylated strand of the PCR product, is used in a solution hybridization. Hybridization is done in streptavidin-coated microtitration wells, making the detection easy to perform. After a washing step, the bound label is detected using a time-resolved fluorometer. To analyze function of the assay, 20 dried blood spot samples were tested. PCR amplification of the deletion region combined with gel retardation assay was used as a control method. In the initial testing, 2 samples giving discrepant results in the two assays were found. In addition, 17 samples from known CF patients together with 6 normal control samples were analyzed. Among these patient samples, 10 homozygotes and 6 carriers for mutation delta F508 were found.


Detection of the most common mutations causing beta-thalassemia in Mediterraneans using a multiplex amplification refractory mutation system (MARMS).

A rapid, simple, cost-effective, non-radioactive method for detection of the most common mutations causing beta-thalassemia in Mediterranean people has been developed by combining multiplexing with the amplification refractory system. This approach, the multiplex amplification refractory mutation system (MARMS), provides an easy assay for direct detection of normal and mutant beta-globin genes in homozygotes and heterozygotes. The strategy involves multiplex PCR of four of the five regions of interest within the beta-globin gene in a single reaction containing a common oligoprimer and either the normal or mutant oligonucleotides corresponding to IVS-1 nucleotide 1 or IVS-1 nucleotide 6, IVS-1 nucleotide 110, codon 39, and IVS-2 nucleotide 1 regions. Primers are chosen so that the sizes of the four PCR products differ, thereby facilitating detection on agarose gels following amplification. Patient samples are primed with either four normal or four mutant oligonucleotide mixtures and the common oligoprimer, and PCR products run in parallel on gels to detect band presence or absence. This approach simplifies mutation detection and shows promise for automation employing fluorescent-tagged primers.


Immunomagnetic recovery of Chlamydia trachomatis from urine with subsequent colorimetric DNA detection.

There is need for integrated systems that provide rapid and sensitive diagnosis of Chlamydia trachomatis. Here we describe for the first time that specific antibodies to a C. trachomatis surface protein can be used for magnetic recovery of bacteria directly from crude urine samples. The combination of immunomagnetic separation (IMS), where the bacterial cells are both purified and concentrated, with a colorimetric PCR assay makes the method sensitive and suitable for routine clinical screening in microtiter format. No filtrations, centrifugations, extractions, or electrophoretic techniques are required throughout the whole procedure. Using magnetic beads as the solid phase, the colorimetric detection of immobilized amplified nucleic acids (DIANA) also allow direct DNA sequencing of positive samples for more detailed analysis. Of the 27 urine samples analyzed, eight were found to be positive using this method. Only five out of these eight were determined to be positive by cell culture.


Estradiol-promoted accumulation of receptor in nuclei of porcine endometrium cells. Comparison of the retention of receptor in nuclei during subcellular fractionation of untreated and hormone-treated cells.

Nuclei were isolated from porcine endometrium of castrated pigs either unexposed or exposed to estradiol in vivo by two techniques, one of which included a hypotonic step. Aliquots were analyzed for estradiol content. Receptor was extracted from buffered, Surfynol-stabilized suspensions by either (a) KCl alone, (b) in combination with dithiothreitol, or (c) by dithiothreitol with polypentosanesulfate and addition of KCl. The yields rose from a-->c. The same proportional gains with increasing extractant efficacies were obtained from nuclei of unstimulated and estradiol-treated cells. Receptor recovery with extractant "c" rose linearly over the range of 9-80 x 10(6) nuclei/mL and was independent of the technique used for isolation. Nuclear fractions isolated using steroid-free solutions contained more estrogen receptor than estradiol; the numerical excess in control nuclei persisted in the nuclei of stimulated cells featuring a stoichiometric rise of ligand and receptor contents. The increase of receptor contents in nuclei isolated from hormone-stimulated cells coincided with a decline in the cytoplasmic fractions. An excess of hormone over receptor was seen only when nuclei were isolated from untreated cells with media containing 10 nM estradiol. Our data strengthen earlier notions of an estradiol-promoted receptor translocation into the nucleus and are not compatible with the ligand-filling hypothesis of preexisting nuclear binding sites.


Estradiol-promoted accumulation of receptor in nuclei of porcine endometrium cells. Immunogold electron microscopy of resting and estradiol-stimulated cells.

Endometrium was collected by curettage from castrated pigs, either untreated or exposed to estradiol in vivo by intrauterine injection, and processed for electron microscopy. The resin LR Gold was used for embedding, and sections were floated on droplets of 10 nm diameter gold particles, coated with the immunoglobulin-G1 (IgG1) fraction or its Fab2 fragment of a monospecific polyclonal antiserum raised in goats against the C-terminal half of the estradiol receptor. On average, only one gold particle per microns 2 became attached in the cytoplasmic area of untreated cells, whereas four were found over the nuclear area. These figures rose to 2-3/microns 2 and 15-26/microns 2, respectively, within 10 min after exposure to estradiol. The labeling intensities of nuclei in cell clusters and of coprocessed nuclei released from cells ruptured during curettage were identical in all situations. Nuclear pores were frequently tagged after estradiol treatment. The proportions of tagging densities in nuclei of untreated and estradiol-exposed cells corresponded to those of receptor contents measured in extracts of isolated nuclei by ligand binding. This correlation was not seen for the cytoplasmic compartment of untreated cells, the scarce tagging of which is interpreted by hidden antigenic determinants. Our morphological analyses support the conclusions drawn from biochemical data (Sierralta et al., 1992) of an estradiol-promoted translocation of receptor from the cytoplasm into the nucleus.


Short-term in vivo regulation of prolactin receptors in the liver, testes, kidneys, and mammary gland of rats.

Infusion of 17-beta-estradiol for 3 h caused a more than 100% increase in the number of PRL receptors (PRL) in the microsomal liver fraction of male rats without affecting the hormone-receptor affinity. A small but significant increase in PRL receptors also was found in testes but not kidneys. Testosterone infusion to female virgin rats for a similar time period resulted in some nonsignificant decreases in PRL receptors in the liver but not the mammary gland or kidneys. Testosterone infusion to males or 17-beta-estradiol infusion to females did not change the number or affinity of PRL and growth hormone receptors. The level of serum PRL was not influenced during infusion with both hormones.


Microsomal steroid receptors in target tissues.

Target tissues contain microsomal receptors for steroid hormones. High-affinity, low-capacity binding sites for steroids are located in the endoplasmic reticulum and do not bind DNA. Studies by diverse groups on the nature and function of these receptors using biochemical and morphological approaches are discussed. The findings indicate that microsomes can be the site of receptor synthesis. Microsomes can also play a role in the control of receptor recycling and/or receptor processing after the complexes exit the nucleus of target cells. Moreover, microsomal receptors may be involved in posttranscriptional actions of steroid hormones.


Ophthalmic drugs and anesthesia interactions.

Ophthalmic medications are often overlooked in the preoperative evaluation for patients experiencing surgery and anesthesia as "just eye drops." Both acute and chronic administration of these agents affects the anesthesia practitioner. Virtually every patient experiencing anesthesia for cataract extraction will have other nonanesthetic medications administered perioperatively. Medications are dispensed throughout the age continuum for the treatment of glaucoma on a chronic basis. All of these medications have the potential to cause morbidity and mortality by themselves or in combination with other drugs, to include anesthetic agents. This article focuses on the more common agents the anesthetist will encounter.


Ambulatory ophthalmic surgery and the Health Care Financing Administration's Outpatient Surgery Generic Quality Screen Guidelines.

The quality of care rendered in ambulatory ophthalmic surgical centers is subject to the standards outlined in the Health Care Financing Administration's "Outpatient Surgery Generic Quality Screen Guidelines." Ophthalmic Surgery accounts for 28.2% of all ambulatory surgery performed in the United States. Diabetes, respiratory disorders, renal failure, and hypertension are a few of the clinical problems that are handled in a nontraditional fashion in ophthalmic surgery facilities throughout the United States with excellent results and low incidence of complications. Trends in the clinical management of patients in ophthalmic surgical centers are described by data obtained in a survey conducted by the anesthesia and surgical staff of the Parris-Castoro Cataract and Laser Center in Bel Air, MD. The survey addressed the current trends in obtaining history and physicals, electrocardiograms, chest radiographs, and laboratory studies before outpatient ophthalmic surgery.


Periocular regional block: experience with a two-point injection technique.

As an alternative to traditional retrobulbar needle placement during regional anesthesia of the eye, a two-point injection technique has been successfully employed which uses less aggressiveness in needle tip approach to key structures and affords good lid akinesia without need of separate blockade of facial nerve branches. While this technique is more closely related to so-called "peribulbar" approaches in terms of needle direction and volume of anesthetic administered, it is recognized that final needle tip placement with any technique may, at times, be difficult to ascertain. This emphasizes the need for more generic terminology in block description such as "periocular" or "ocular regional blockade."


A review of anesthesia for the open globe.

There are many factors, both physiological and pharmacological, that influence IOP during anesthesia. The choice of anesthetic technique must consider factors other than IOP such as the patient's general condition, concomitant injuries, and the nature of the operation. At this time, there is no ultrarapidly acting neuromuscular blocking agent to allow succinylcholine to be completely abandoned and no method of succinylcholine pretreatment is completely effective in blocking the increase of IOP. The anesthesia provider must balance the overall risk to the patient with the risk to the injured eye when deciding if succinylcholine is to be used. The controversy regarding the actual risk that succinylcholine presents to the open eye has gone on for years and can be expected to continue.


Retrobulbar block for open-eye injuries: a report of 19 cases.

In open-eye injuries, which are often emergency procedures where the patient may have a full stomach or be in precarious health, retrobulbar anesthesia offers definite advantages over general anesthesia. A "low-volume" technique is described which gives adequate anesthesia without dangerously increasing intraocular pressure. A table containing the salient description and outcome of 19 anesthetics given in this manner is included.


Propofol-fentanyl versus midazolam-fentanyl: a comparative study of local sedation techniques for cataract surgery.

Administering intravenous sedation in conjunction with intraoperative monitoring to cataract surgery patients is a widely accepted technique. Numerous articles report local sedation techniques for cataract surgery that are, in essence, abbreviated general anesthetic techniques for insertion of the retrobulbar block (RBB). Because of variations in levels of consciousness, a number of complications have been encountered with this specific patient population, ie, movement upon insertion of the RBB, intraoperative patient movement, confusion, hypotension, respiratory depression, and respiratory arrest. In an attempt to meet the specific needs of this patient population, a study comparing propofol-fentanyl with midazolam-fentanyl was initiated. Seventy-five (ASA 1 to 3) patients were randomly assigned to two groups: propofol-fentanyl (P/F) or midazolam-fentanyl (M/F). The mean age of patients in the P/F group was 71.1 +/- 13 SD, and the mean age in the M/F group was 74.4 +/- 8.8 SD. All patients entered the operating room unpremedicated. Before the RBB, patients in both groups were given a single intravenous dose of 50 micrograms fentanyl. Propofol (mean dose, 24.7 mg) or midazolam (mean dose, 1.58 mg) was then titrated to slurred speech or nystagmus. Patients' responses to the RBB were evaluated and recorded by an objective observer. The amnestic properties of both agents were evaluated by patient questioning at 10 minutes and 24 hours. Levels of discomfort were evaluated on a scale of 1 to 5, with 1 being extremely uncomfortable and 5 being noticeable without pain. Respiratory depressant effects of both techniques was assessed via continuous pulse oximetry. Results were analyzed using the chi 2 test, rank t test, and SD.(ABSTRACT TRUNCATED AT 250 WORDS)


Artificial blood substitutes.

Investigations directed toward the development of an effective artificial blood substitute are continuing as concerns remain regarding the safety and adequacy of the nation's blood supply. Perfluorochemical compounds, modified hemoglobin solutions, and microencapsulated hemoglobin represent the major areas of research. If a successful red cell substitute can be developed, many problems inherent in current blood transfusion practices could be alleviated, particularly in emergency situations.


Anesthetic concerns and complications during suspension microlaryngoscopy procedures.

The use of lasers during microsurgery on the larynx is becoming more commonplace in the operating rooms of many hospitals. Because of the intense cardiovascular response to suspension microlaryngoscopy and the short duration of these cases, the anesthesia provider is faced with one of the most challenging anesthetic cases. This article includes a brief introduction to suspension microlaryngoscopy, a case study, a description of suspension microlaryngoscopy, and the anesthetic concerns and possible complications of importance during suspension microlaryngoscopy.


Anesthetic implications for the surgical patient prescribed psychotropic medications.

The anesthetist must be aware of anesthesia implications for the surgical patient who is taking prescribed psychotropic medications. The number of patients taking psychotropic medications who present for surgery is increasing. Psychotropic medications combined with anesthesia can produce serious complications. This article reviews commonly known psychotropic medications and introduces the anesthetic implications of two new drugs, fluoxetine and buspirone.


Total intravenous anesthesia with a continuous propofol-alfentanil infusion.

A total intravenous anesthetic using propofol and alfentanil was evaluated to determine if it would provide a shorter recovery-room stay than a more traditional balanced anesthetic using isoflurane and alfentanil. Forty-three ASA I or II patients between 17 and 50 years of age undergoing major abdominal or orthopedic procedures were studied. The propofol group received alfentanil 50 mg/kg followed by propofol 1 mg/kg for anesthesia induction. Continuous propofol infusion was initiated at induction using 170 mg/kg/min for 10 minutes, followed by 130 mg/kg/min for 10 minutes, then maintained at 100 mg/kg/min until 10 minutes before the end of surgery. Ventilation was supported with an air-oxygen mixture. The group receiving balanced anesthesia received alfentanil 1 mg/kg, and anesthesia was induced with sodium thiopental 4 mg/kg. A 1% isoflurane inhalation with air-oxygen was initiated immediately upon induction. Both groups received a continuous infusion of alfentanil titrated to maintain heart rate within 10% of preinduction levels. Recovery from anesthesia was measured using a subjective pain assessment, a verbal fluency test, and a short-term memory test. No differences were detected in the rate of recovery at 30 minutes or 60 minutes postextubation. Hemodynamic stability during induction and intubation was slightly better in the propofol group than in the isoflurane group. One episode each of intraoperative awareness and delayed eye opening occurred in the propofol group. Total intravenous anesthesia using propofol and alfentanil is just as effective as a balanced inhalation anesthetic and provides equally rapid recovery. However, practitioners are cautioned to include an amnestic adjuvant when using propofol as the sole anesthetic agent.


Carbon dioxide lasers in anesthesia.

Carbon dioxide lasers have been used in medicine since the 1960s. They have created new challenges for the anesthesia provider. A review of laser physics, laser characteristics, potential complications, and anesthetic techniques is presented.


The legislative scheme of pre-trial destruction or lawful disposal of narcotic drugs and psychotropic substances in India.

The present article provides a description of the practice of pre-trial destruction of seized narcotic drugs and psychotropic substances in India as provided for in the Code of Criminal Procedure, 1973, and the Customs Act, 1962, and in the context of the United Nations Convention against Illicit Traffic in Narcotic Drugs and Psychotropic Substances of 1988. It includes explanations of why, as well as how, the pre-trial destruction of such substances should be carried out.


Drug abuse and illicit trafficking in Italy: trends and countermeasures, 1979-1990.

The author begins with a discussion on trends in drug abuse and trafficking in Italy in the period 1979-1990, when the country was experiencing major heroin-related problems and the situation with regard to cocaine trafficking and abuse was rapidly deteriorating. He then describes the organization of the anti-drug services in Italy and identifies strategies pursued by the Italian Government with a view to intensifying its activities in international drug control.


Law enforcement approaches and measures used in countering illicit drug problems in Thailand.

The Government of Thailand, which has long recognized the serious threat posed by illicit drugs, has implemented stringent law enforcement measures aimed at suppressing illicit drug trafficking by dismantling clandestine laboratories, intercepting essential chemicals, effecting significant seizures and eradicating illicit crops. In addition, the Government has taken steps to initiate the enactment of legislation providing for the confiscation of proceeds derived from illicit drug trafficking activity. Furthermore, it has maintained and strengthened its already close bilateral and multilateral cooperation in the field of drug abuse control.


Analysis of heroin seized in France.

Because of the increase in heroin trafficking in France, it was decided that data on samples of heroin seized in the country would be filed in a computer in the Interregional Forensic Science Laboratory at Lyon. The computer file has made it possible to identify certain physical and chemical similarities among samples of seized heroin. According to the data in the computer file, the colour, purity, diluents etc. of the heroin offered on the street has changed significantly since 1987. The Laboratory will be in a better position to identify the geographical sources of heroin seized in France once samples of seized heroin have been obtained directly from their countries of origin.


Drug control: policies and practices of the International Criminal Police Organization.

Drug trafficking and drug abuse are among the major social issues that the world has to grapple with. The far-reaching consequences of drug abuse in humanitarian, social and economic terms have led to intensive action on the part of Governments, intergovernmental and non-governmental organizations. The International Criminal Police Organization (ICPO/Interpol) plays a key role as the central command for the joint efforts of the national law enforcement agencies of its 158 member States. The present paper outlines the role of ICPO/Interpol, particularly its Drugs Sub-Division, in international drug control efforts. It includes a discussion of ICPO/Interpol policies and practices related to drug control and of recent developments in its information and communication capabilities.


Drug control policies in some parts of Asia.

The Asian region has experienced drug problems ever since opium was exported to China in the eighteenth century. Today, in some parts of the region, high priority is placed on drug control policies relating, for example, to the eradication of opium poppy cultivation, criminal sanctions against drug trafficking, and treatment programmes for drug-dependent persons. Some good results have been achieved in recent years: according to estimates, opium cultivation in at least one country in the region has decreased; the number of new cases involving drug-dependent persons is falling in a number of countries; and major drug trafficking syndicates have been disbanded.


Patterns of drug trafficking and countermeasures: the personal view of a veteran.

In the present paper, a law enforcement official with nearly 40 years of experience in combating drug trafficking provides insight that may prove useful to persons in other organizations and/or cultures. He examines, in particular, the development, at the national level, of drug control legislation, a drug law enforcement strategy and a drug intelligence unit.


Vascular endothelium.

Endothelium lines all blood vessels and the cardiac cavities and has a central role in cardiovascular homeostasis. It releases several potent substances which mediate the control of vascular tone and cardiac contraction. Endothelial dysfunction is implicated in many disease states. Elucidation of the underlying mechanisms is likely to lead to novel therapeutic strategies.


Advances in the management of gastroenteritis in children.

Gastroenteritis continues to be an important problem in developed communities, although less so than in the past. Most children can be managed at home with oral rehydration therapy. Only those children with severe dehydration require hospital admission and intravenous therapy.


Cutaneous manifestations of malignant disease.

This review discusses the more common cutaneous disorders associated with internal malignancy. Some rare but specific entities are included, and some recently recognized associations are highlighted. Particular emphasis is given to the key physical signs, which can alert the physician to the possibility of underlying neoplastic disease.


Management of an abnormal smear.

Women with a smear that suggests the presence of moderate or severe dyskaryosis should be referred for colposcopic examination. This article outlines briefly the principles of colposcopy and treatment of these abnormalities using large loop excision of the transformation zone.


Assessment of students.

Assessment can use the written word, the spoken word or practical activity. Often the learner has little idea of the purpose of the assessment or the expectations. Here I present a brief outline for designing assessments and balancing assessment techniques in relation to the purposes and content of the learning experience.


Magnetic stimulation of the nervous system.

Magnetic brain stimulation is a painless method of exciting cortical neurons and provides a measure of corticospinal tract function. Its clinical role is currently limited though advances in coil design may lead to wider clinical applications and to greater understanding of how the cerebral cortex functions in vivo.


Streetlife and delinquency.

The correlation between class and delinquency often observed in areal studies and assumed in prominent sociological theories is elusive in studies of individuals commonly used to test these theories. A restricted conceptualization of class in terms of parental origins and the concentration of self-report survey designs on adolescents in school have removed from this area of research street youth who were once central to classic studies of delinquency. We argue that street youth experience current class conditions that cause serious delinquency, and that life on the street is an important intervening variable that transmits indirect effects of control and strain theory variables, including parental class origins. Data gathered from nearly 1000 Toronto school and street youth are analyzed with important implications for the conceptualization of class and delinquency, testing and integrating sociological theories of delinquency, the measurement of delinquency, and the use of cross-sectional and longitudinal research designs. Our findings especially encourage incorporation of street-based samples into research on class-based aspects of theories of delinquency.


Striatal extracellular dopamine in conscious vs. anesthetized rats: effects of chloral hydrate anesthetic on responses to drugs of different classes.

Many investigations using the microdialysis technique have been performed in anesthetized animals, both in this laboratory and elsewhere. Concern arises with this preparation that the anesthetic may compromise neuronal function, or that it may interact with test drugs affecting neurotransmitter overflow. In addition, in these studies the microdialysis probe typically is introduced into the brain on the day of testing, and data collection commences within an hour or two following probe insertion. It has been suggested that transmitter recovered in the perfusate probably represents leakage due to tissue damage as well as exocytotic release, and may not accurately reflect neuronal responses to the manipulations of interest. Such potential confounds present important implications for the interpretation of data from these studies. The present investigation examined the effects of chloral hydrate anesthetic on (1) basal dopamine (DA) overflow in the anterior striatum, and (2) DA responses to systemically delivered drugs of two different classes known to influence DA activity. Three putative indices of impulse-dependent release were measured: (a) the time course and stability of basal DA overflow over several hours; (b) sodium channel involvement by adding tetrodotoxin (TTX) to the artificial CSF; and (c) calcium channel involvement using magnesium (Mg) in a calcium-free perfusate. Basal DA levels became stable in both conscious and anesthetized preparations by the second hour after probe insertion. Levels of recovered DA overflow in the anterior striata of conscious rats were approximately double those in chloral hydrate-anesthetized rats. Consistent with other findings, this suggests a general depression of CNS function by chloral hydrate.(ABSTRACT TRUNCATED AT 250 WORDS)


Behavioral and neurochemical indices of barrel cortex-basal ganglia interaction.

Previous experiments from our laboratory have shown a wide variety of time-dependent lateralized changes in behavior and nigrostriatal function following unilateral manipulation of the mystacial vibrissae of rats. The present experiment investigated the effects of unilateral radiofrequency lesion of the cortical vibrissae representation (the barrel fields) in light of these results. We measured lateralized changes in behavior as well as tissue monoamines in neostriatum and substantia nigra, between 1 and 16 days post-lesion. Short-term asymmetries in exploratory behavior (thigmotactic scanning) and neostriatal serotonin metabolism that lasted up to day 6 were seen. In substantia nigra, time-related asymmetries in dopamine concentrations were found with higher ipsilateral values on day 3 and higher contralateral values on day 6. After day 6, the animals had recovered from these acute effects and thereafter, neostriatal dopamine metabolism became asymmetrical. Also during this time, they showed a directional bias in spontaneous and apomorphine-induced turning. Finally, neostriatal serotonin was bilaterally elevated on day 16. These results parallel some of the effects previously seen following unilateral removal of the vibrissae, indicating that the barrel cortex is a critical link in the functional interaction between the vibrissae and basal ganglia.


Unilateral neostriatal kainate, but not 6-OHDA, lesions block dopamine agonist-induced ascorbate release in the neostriatum of freely moving rats.

Unilateral kainate lesions of the neostriatum and 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle were used to assess the role of neostriatal and ascending dopaminergic neurons, respectively, on dopamine-agonist induced release of neostriatal ascorbate as measured voltammetrically in freely moving rats. Electrochemically modified, carbon-fiber electrodes recorded the effects of direct (a combination of 10 mg/kg SKF-38393 and 1.0 mg/kg quinpirole) as well as indirect (2.5 mg/kg D-amphetamine or 20.0 mg/kg GBR-12909) dopamine agonists. Relative to controls, kainate, but not 6-OHDA, lesions abolished the ability of both direct and indirect dopamine agonists to induce neostriatal ascorbate release. These results suggest that unlike dopaminergic afferents, neostriatal output pathways play a critical role in the modulation of neostriatal ascorbate levels.


Antiparkinsonian drugs and in vitro excitotoxicity.

N-Methyl-D-aspartate (NMDA) receptor activation has been implicated in the pathogenesis and clinical expression of Parkinson's disease. Because some antiparkinsonian drugs have NMDA antagonist properties, we examined their effects on NMDA toxicity, measured by lactate dehydrogenase (LDH) release, in neuron-enriched cerebrocortical cultures. Amantadine reduced NMDA toxicity with half-maximal reduction at approximately 30 microM, while trihexphenidyl, L-3,4-dihydroxyphenylalanine (L-DOPA), bromocriptine and selegiline were ineffective, and benztropine was itself toxic. Amantadine and related drugs could not only reduce parkinsonian symptoms, but also modify underlying neurodegenerative processes.


Visualization of dopamine nerve terminals by positron tomography using [18F]fluoro-beta-fluoromethylene-m-tyrosine.

[18F]-6-Fluoro-beta-fluoromethylene-m-tyrosine ([18F]FFMMT) was evaluated as a potential imaging agent for dopamine nerve terminals using positron emission tomography (PET). Biodistribution and time course of this tracer in mice after i.p. injection was consistent with the distribution of dopamine. PET imaging studies involving rhesus macaques showed specific uptake in the dopamine-rich caudate-putamen region. This specific localization was blocked by inhibiting the enzyme L-aromatic amino acid decarboxylase and the transport of the tracer into brain was shown to be stereospecific. These results show the promise of L-[18F]FFMMT as a PET tracer in monitoring degeneration of the CNS dopamine system.


Single retinal ganglion cells sending axon collaterals to the bilateral superior colliculi: a fluorescent retrograde double-labeling study in the Japanese monkey (Macaca fuscata).

Single retinal ganglion cells projecting bilaterally to the superior colliculi (SC) by way of axon collaterals were revealed in the Japanese monkey (Macaca fuscata). After injecting Fast blue into the SC on one side and Diamidino yellow into the SC on the opposite side, some retinal ganglion cells were double-labeled with both tracers. Most of them were large cells (more than 25 microns in diameter), and were localized in a narrow strip around the vertical meridian of the retina on each side. This retinal area roughly corresponds to the reported strip of nasotemporal overlap, where both crossed and uncrossed retinofugal projections arise.


Serotonin-dependent cerebral activation: effects of methiothepin and other serotonergic antagonists.

In scopolamine-treated (5.0 mg/kg, s.c.) rats hippocampal rhythmical slow activity (RSA) and neocortical low voltage fast activity (LVFA) occur only in close correlation with head movements, spontaneous changes in posture, or locomotion (Type I behavior). Previous work indicates that such scopolamine-resistant RSA and LVFA are dependent on ascending serotonergic projections. A test of 9 serotonergic antagonists (methiothepin; ritanserin; ketanserin; pizotifen; mianserin; pirenperone; ICS-205-930; metoclopramide; methysergide) showed that methiothepin produces a partial reduction in RSA and LVFA in scopolamine-treated rats, while the other antagonists are completely inactive over a wide range of doses. It may be that serotonergic cerebral activation depends on both 5-HT1 and 5-HT2 receptors.


Inhibitory effect of xylamine on the uptake of [3H]norepinephrine into primary astrocyte cultures.

Primary astrocyte cultures from neonatal rat brains show a Na+-dependent, desipramine-sensitive uptake of [3H]norepinephrine ([3H]NE). Xylamine, a nitrogen mustard, attenuated this uptake of [3H]NE into the astrocytes (IC50 = 0.8 microM). The dose-dependent reduction of [3H]NE uptake by xylamine indicated competitive kinetics. However, xylamine lost the effect if the active transport inhibitor, ouabain or iodoacetate, was also incubated during the pretreatment period of if Na+ was absent. The activity of Na(+)-K(+)-ATPase in astrocytes was not modified by xylamine at the concentrations sufficient to block the uptake of [3H]NE. These findings suggest that xylamine has the ability to compete with the transport of [3H]NE into astrocytes through an effect on the carrier instead of an action on enzymatic activity.


Magnocellular neurosecretory neurons with ferritin-like immunoreactivity in the hypothalamic supraoptic and paraventricular nuclei of the rat.

Immunohistochemistry for rat liver ferritin (FRT) revealed an intensive labeling in some structures of the rat brain. In the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei, almost all neurosecretory neurons with vasopressin (AVP)-like immunoreactivity were immunostained with FRT. After water deprivation, a marked enlargement of cell body and an immunoreactivity to transferrin receptors were found in AVP-, FRT- and double (AVP+FRT)-labeled neurons in the SON and PVN.


Perforated-patch method reveals extracellular ATP-induced K+ conductance in dissociated rat nucleus solitarii neurons.

Extracellular adenosine 5'-triphosphate (ATP)-induced responses were investigated in acutely dissociated rat nucleus tractus solitarii (NTS) neurons, using nystatin perforated-patch and conventional whole-cell patch-clamp recordings. Extracellular application of ATP could evoke the inward currents with both methods. An additional outward current was observed only with the perforated patch method. The outward current resulted in an increase of K+ conductance which was activated by Ca2+ influx through the ATP receptor-channel complex.


Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned aversive stimuli.

The protein product of the c-fos proto-oncogene was immunocytochemically localized in forebrain regions of adult male Lewis rats subjected to a physically aversive footshock stimulus or a Pavlovian-conditioned, non-aversive, auditory stimulus. Animals receiving the conditioned stimulus were first conditioned by repeatedly pairing electric footshock, the unconditioned stimulus (US), with an auditory cue, the conditioned stimulus (CS). These animals were later tested with the CS in the absence of the US, a procedure which, like footshock itself, suppresses immune function. In animals exposed to the conditioned or unconditioned stressor, c-Fos was strongly expressed in cells of the paraventricular nuclei (PVN) of the hypothalamus, some of which contain corticotropin-releasing hormone (CRH), and other forebrain areas directly associated with autonomic function, the ventral lateral septal nuclei (LSV), the medial amygdaloid nuclei (AME), the sensorimotor cortex, the basal ganglia and thalamic nuclei. Control animals exhibited very little or no c-Fos in the above areas. The identified forebrain nuclei can now be targeted for further study aimed at elucidating their role in stress-induced immune alteration.