Comparative sensitivity of fluorescent antibody staining of conjunctival scrapings and irradiated McCoy cell culture for the diagnosis of hyperendemic trachoma.

The sensitivity of an indirect fluorescent antibody (FA) staining technique for detecting chlamydial inclusions in scrapings from the whole conjunctiva (upper tarsus, upper fornix, and lower lid) was compared with the sensitivity of culture in irradiated McCoy cells for the diagnosis of hyperendemic trachoma. In a group of 211 patients with various grades of active trachoma from the Bandar Abbas area of Southern Iran 42 patients were positive for chlamydiae by either method. There was little difference between the rates of positivity of FA staining of the scrapings from the whole conjunctiva (28 positives) and culture in irradiated McCoy cells (32 positives). In the patients included in this study chlamydial inclusions were detected in 15 eyes by examination of FA stained scrapings taken from the upper tarsal conjunctiva, whereas inclusions were detected in 40 eyes by the additional examination of scrapings taken from the upper fornix and lower lid (P less than 0.001). The examination of FA stained scrapings taken from the whole conjunctiva and spread as a single but larger smear may provide a satisfactory alternative to cell culture methods for the diagnosis of trachoma, particularly for field studies when cell culture facilities are not available.     

Isolation of Chlamydia trachomatis from different areas of conjunctiva in relation to intensity of hyperendemic trachoma in school children in Southern Tunisia.

The simplified one-passage technique of culture in irradiated McCoy cells, in conjunction with certain other developments in technique, was used to isolate Chlamydia trachomatis from specimens collected from 78 children with trachoma in Douz, Southern Tunisia. The results show that C. trachomatis is not confined to the upper tarsal area of the conjunctiva in hyperendemic trachoma. The higher isolation rate and corresponding increase in the number of inclusions obtained from swabbings of the upper fornix and lower lid in addition to the conventional collection from the upper tarsus show the superiority of collecting specimens for culture from the whole conjunctiva. Specimens could be collected from the whole conjunctiva by using 1 swab for each eye and pooled for subsequent inoculation, so that the laboratory incurred no additional work. A close correlation was observed between isolation rate, together with the number of inclusions, obtained in cell culture, and intensity of inflammatory disease in hyperendemic trachoma. The sensitivity and practicability of this cultural test should provide a valuable laboratory index for use in epidemiological and therapeutic studies of trachoma.     

Characterization of free-floating spheres from human trabecular meshwork (HTM) cell culture in vitro

It has been observed in several tissues that direct isolation of cells in serum-free media and on nonadhesive substrates results in the formation of spherical clusters of cells known as free-floating spheres. Such free-floating spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Our goal was to isolate and characterize such free-floating spheres from HTM cell primary cultures. For this purpose, HTM cells were incubated in serum-free media and on a nonadhesive substrate. Individual free-floating spheres generated in these conditions were isolated in 96-well plates, and their proliferative capacity was evaluated by monitoring their size increase over time. The expression of the TM markers, MGP and CHI3L1, was examined using recombinant adenoviruses containing the respective promoters. Morphology of the free-floating spheres was analysed in semithin sections, and the gene expression profile was obtained using Human Genome U133 Plus 2.0 Affymetrix microarrays. HTM cells incubated in serum-free media and on nonadhesive substrate generated free-floating spheres that could be grown for more than 3 months. Addition of serum to the culture media promoted the attachment of the spheres to the substrate, migration of cells from the spheres, and differentiation into cells phenotypically similar to normal TM cells. Gene profiling analysis demonstrated strong similarities between the gene expression profiles of the spheres and HTM cell monolayers. Both infection with the recombinant adenoviruses and gene array analysis demonstrated the expression of CHI3L1 and MGP, indicating that free-floating spheres likely originate from HTM cells. Gene array analysis also showed expression of the marker for neural precursor cells nestin, as well as leukemia inhibitory factor, a gene involved in the maintenance of the undifferentiated state of progenitor cells. Analysis of semithin sections indicated that these TM free-floating spheres were highly dynamic structures demonstrating a distinct radial gradient of cell proliferation, survival, and apoptosis. Extensive up- and down-regulation of gene expression was associated with the processes of sphere attachment and cell migration after the addition of serum. These results suggest that HTM primary cultures might contain relatively undifferentiated or progenitor cells. The availability of TM progenitor cell cultures could constitute a useful tool to investigate cell therapy approaches targeting the TM in glaucoma.     

Long-term studies of visual field changes by means of computerized perimetry (Octopus 201) in eyes with glaucomatous field defects after normalization of the intra-ocular pressure

126 eyes with open-angle glaucoma and defects of the visual field were studied over five years (±1.3) by repeated perimetries with Octopus Perimeter, Program 31, after the IOP had been normalized from 26.49 mm Hg to 19 mm Hg.All patients had suffered visual field defects in the period with increased pressure preceeding our study and had many perimetric examinations before. The aim of the study was to check the behaviour of the visual field defects after pressure normalization.For evaluation of the visual fields, the upper most line of the test-points and the 6 test-points surrounding the blind spot were disregarded. The evaluation of each field and the comparison with preceding fields (no change, better, or worse) were done by the symmetry test of Bowker instead of the Delta Program, taking into account each of the 61 test-points with a significance level of 5%. In each patient the eye with the heavier field loss was evaluated.Results: 67% of all eyes had no change during the observation time, 12% became better, and 21% worse. Improvement of the visual fields occurred even in old age: 10% became better in the age-group over 70 years. The total loss decreased in the 15 eyes which improved by 269 dB. In eyes which deteriorated, the total loss increased by 282 dB.The good news of this study is that normalization of IOP can stop the further decay of the visualfield in 67% and an improvement of the field loos is possible even in old age of with heavy field loss.Finally, the case of a 14 years old boy is reported who had late developmental glaucoma and who improved from a total loos of 1200 dB in his only eye of 500 dB after his IOP was normalized.     

Comparison of pharmacological agents (aspartate vs. aminophosphonobutyric plus kynurenic acids) to block synaptic transmission from retinal photoreceptors in frog.

The combination of aminophosphonobutyric plus kynurenic acids (APB/Kyn) was compared to aspartate with respect to its ability to block synaptic transmission from photoreceptors. Like aspartate, APB/Kyn blocks photoreceptor synaptic transmission, as monitored by the b- and d-waves of the electroretinogram, by the proximal negative response and M-wave of the proximal retina, and by the light-evoked increase in extracellular K+ concentration in the inner plexiform layer. Unlike aspartate, APB/Kyn has relatively minor effects on retinal resistance, light-evoked changes in K+ and Ca2+ concentrations in the subretinal space, light-evoked changes in subretinal space volume, resting extracellular concentrations of K+ and Ca2+ in the proximal and distal retina, and the c-wave. Effects of APB/Kyn are generally more reversible than effects of Asp. A disadvantage of APB/Kyn is that the a-wave usually becomes smaller and slower. Overall, APB/Kyn disrupts the retina less than aspartate. Therefore, in some situations in which blockade of photoreceptor synaptic transmission is desired, the use of APB/Kyn may be preferable to that of aspartate.     

An introduction to analysis of variance (ANOVA) with special reference to data from clinical experiments in optometry.

This article is aimed primarily at eye care practitioners who are undertaking advanced clinical research, and who wish to apply analysis of variance (ANOVA) to their data. ANOVA is a data analysis method of great utility and flexibility. This article describes why and how ANOVA was developed, the basic logic which underlies the method and the assumptions that the method makes for it to be validly applied to data from clinical experiments in optometry. The application of the method to the analysis of a simple data set is then described. In addition, the methods available for making planned comparisons between treatment means and for making post hoc tests are evaluated. The problem of determining the number of replicates or patients required in a given experimental situation is also discussed.