Accuracy of retinal thickness measurements obtained with Cirrus optical coherence tomography.

AIM: To report the frequency and severity of retinal thickness measurement errors in a Fourier domain optical coherence tomography (FDOCT) device, Cirrus OCT. METHODS: Data from 209 eyes undergoing Cirrus OCT imaging with the Macular Cube protocol were collected. For each eye, the position of the automated retinal boundary lines used by the Cirrus OCT software for thickness calculations was assessed using a 6-point categorical scale. The presence of errors was correlated with various parameters including: retinal morphological features and disease diagnosis. RESULTS: Errors of retinal boundary detection were observed in 57.5% of eyes but were severe in only 9.6% of eyes. The identification of subretinal fluid, subretinal tissue, pigment epithelium detachment or a diagnosis of choroidal neovascularisation was associated with more severe errors. Retinal cysts or a diagnosis of retinal vascular disease were less likely to be associated with significant error. CONCLUSIONS: Retinal thickness measurement errors appear to occur less frequently with Fourier domain OCT (Cirrus OCT), but segmentation errors remain a concern, particularly in assessment of eyes with structurally complex retinal disease. With the recent release of multiple FDOCT systems, assessment of segmentation error may be an important factor in determining the relative merits of these systems.

Comparison of the optical coherence tomographic features of choroidal neovascular membranes in pathological myopia versus age-related macular degeneration, using quantitative subanalysis.

AIM: To compare the retinal morphological characteristics of eyes with choroidal neovascularisation (CNV) secondary to pathological myopia versus eyes with CNV secondary to age-related macular degeneration (AMD), using quantitative optical coherence tomography (OCT) subanalysis. METHODS: Twenty-one eyes of 21 patients newly diagnosed as having CNV secondary to pathological myopia, and 43 consecutive cases of eyes with newly diagnosed subfoveal CNV secondary to AMD were retrospectively collected. In all patients, StratusOCT images and fluorescein angiograms (FA) were available for analysis. StratusOCT images were analysed using custom software (termed "OCTOR"), which allowed calculation of the thickness/volume of the neurosensory retina, subretinal fluid (SRF), subretinal tissue (SRT) and pigment epithelial detachments (PEDs). FA images were used to calculate CNV leakage area and CNV lesion size for each eye. RESULTS: The total volume of neurosensory retina in the pathological myopia group was significantly less than in the AMD group (7.10 (SD 0.50) mm3 vs 7.76 (0.93) mm3, p = 0.004). The total volume of SRF in the pathological myopia group was less than in the AMD group, but the difference was not statistically significant (0.33 (1.38) mm3 vs 0.55 (0.82) mm3, p = 0.434). The total volume of SRT in the pathological myopia group was less than in the AMD group, but the difference was not statistically significant (0.16 (0.15) mm3 vs 0.36 (0.60) mm3, p = 0.144). The total volume of PED in the pathological myopia group was markedly less than in the AMD group (0.01 (0.03) mm3 vs 1.09 (1.89) mm3, p<0.001). On FA, the total leakage of CNV in the AMD group was significantly greater than in the pathological myopia group (4.17 (3.29) DAs vs 0.53 (0.58) DAs, p<0.001). CONCLUSIONS: CNV lesions in pathological myopia were associated with considerably less retinal oedema, SRF and SRT compared with CNV associated with AMD. PEDs were almost negligible in myopic lesions compared with AMD. These findings are consistent with previous clinical and angiographic descriptions of myopic CNV as relatively small lesions with modest exudation.

Evidence for functionally significant polymorphism of human glutamate cysteine ligase catalytic subunit: association with glutathione levels and drug resistance in the National Cancer Institute tumor cell line panel.

Glutamate-cysteine ligase (GCL) is the first and rate-limiting enzyme involved in the biosynthesis of glutathione (GSH). The GCL heterodimer is encoded by two genes: GLCLC, which directs synthesis of the catalytic subunit, and GLCLR, which encodes the regulatory subunit. We have previously identified a polymorphic GAG/CTC trinucleotide repeat within the 5' untranslated region of GLCLC. Here we report the further characterization of GLCLC polymorphism and the existence of five GLCLC alleles as defined by the trinucleotide repeat, which exhibits a range of 4 to 10 uninterrupted repeats. Significant variation in GLCLC allele frequencies was observed in four different ethnic populations examined. Interindividual variation in the capacity to produce GSH due to GLCLC polymorphism is hypothesized to influence the cellular response to environmental toxicants and chemotherapeutic agents. To test this hypothesis, the 60 tumor cell lines of the National Cancer Institute drug screening panel were genotyped for the GLCLC trinucleotide repeat, and the association of GLCLC genotype with GSH levels and drug sensitivity/resistance data was examined. Here we demonstrate an association between certain GLCLC alleles and GSH levels and/or drug sensitivity, providing evidence that suggests polymorphism of human GLCLC is functionally significant.

Population-based studies reveal differences in the allelic frequencies of two functionally significant human interleukin-4 receptor polymorphisms in several ethnic groups.

PURPOSE: The presence of functionally significant human interleukin-4 receptor sequence variants, Gln551Arg and Ile50Val, was examined in four anonymous New York State populations defined by ethnic origin. These variants were studied because they are associated with atopy or atopic asthma whose prevalence varies in different populations. METHODS: PCR/RFLP (Ile50Val) and PCR/allele-specific oligonucleotide hybridization (Gln551Arg) assays were developed to detect both polymorphisms in 855 newborn screening specimens. RESULTS: Arg551 was most frequently found in Blacks (allele frequency of 68%). However, the Ile50 allele was most common in Whites (allele frequency, 87%). Significantly more Blacks had chromosomes bearing both of the "enhanced signaling" variants (Ile50/Arg551). CONCLUSIONS: Enhanced IL-4R signaling is associated with increased IgE production (atopy). Therefore, our data suggest that the African American population may be at increased risk for diseases, including asthma, which are associated with atopy. These data also emphasize the importance of determining the frequencies of single nucleotide polymorphisms in different populations before drawing conclusions from allele association studies, since the background allele frequencies may be disparate between different populations.

Cell separation based on the reversible interaction between calmodulin and a calmodulin-binding peptide.

A cell separation system based on the calcium-dependent interaction of calmodulin (CM) with a calmodulin-binding peptide (CBP) has been developed. The prototype of this system utilizes an indirect method to label the target cell population. Cells are first labeled with a primary monoclonal antibody directed to a specific cell surface antigen, then with a secondary affinity reagent, consisting of a polyclonal goat anti-mouse IgG (GAM-IgG) that has been cross-linked to a CBP derived from the sequence of the rabbit skeletal muscle myosin light chain kinase. In the presence of Ca2+, the CBP on the cells labeled with GAM-IgG-CBP binds to biotinylated calmodulin (CM-Biotin) with high affinity. The target cells are then captured with a solid-phase streptavidin. The unbound non-target cells are washed away and the immobilized target cells are released by chelating Ca2+ with EGTA. The specificity of the GAM-IgG-CBP and CM-Biotin and the feasibility of using this system to separate cells was demonstrated using the KG-1 human acute myelogenous leukemia cell line. KG-1 cells were fractionated on the basis of cell surface expression of HLA-DR. The cell selection reagents and the cell separation process did not affect KG-1 cell viability while cells selected by this procedure were 90% pure with a yield of 75%. This cell separation system also was used for rare cell isolation from normal human peripheral blood mononuclear cells. T cells expressing the Vbeta5 T cell receptor, which represent < 5% of the unfractionated cells, were isolated with 89% viability, 72% purity, 80% yield, and retained the ability to respond to activation signals as measured by blast transformation. The results from this study show that a cell selection system based on the reversible interaction between CM and a CBP can be applied to gently and efficiently isolate cells from a heterogeneous starting population that are free of the solid matrix without exposure to the stresses of mechanical or enzymatic release.

Long-term exposure to methotrexate induces immunophenotypic changes, decreased methotrexate uptake and increased dihydrofolate gene copy number in Jurkat T cells.

Methotrexate (MTX) treatment of rheumatoid arthritis may require increasing doses to maintain clinical efficacy. An overall plateau of clinical response is reached after only six months of treatment. To study the immunologic, biochemical and genetic effects of MTX on T cells, the Jurkat T cell line was made MTX-resistant by serial addition of methotrexate sodium into culture medium. Cells proliferated and divided successfully in MTX concentrations ranging to 15 microM. MTX resistance of Jurkat T cells in vitro was accompanied by significantly (P < 0.05) decreased expression of CD2, CD3, CD4, CD28, and CD69, IL-2 production, and MTX uptake assessed by cell association or disassociation of 3[H]-MTX or fluoresceinated MTX (FMTX), respectively. In addition, there was DHFR gene amplification and increased levels of DHFR in all resistant cell lines. Both permanent and transient phenotypic changes developed in resultant cell lines exposed to increasing concentrations of MTX in vitro. Expression of CD4 and CD25 and sensitivity to MTX returned to near-parental levels after removal of MTX from culture medium, whereas expression of CD26 and MTX uptake were significantly increased. Expression of CD2, CD3, CD69 and IL-2 production as well as the DHFR levels did not return to the parental phenotype after removal from MTX. We conclude that MTX-cultured cells express depressed levels of cell-surface markers vital for T cell function and activation. The return of enhancement of these cell-surface markers critical to T cell activation suggests a possible mechanism for the severe flares experienced by rheumatoid arthritis patients when drug treatment is discontinued.

Hydroquinone resistance in a murine myeloblastic leukemia cell line. Involvement of quinone reductase and glutathione-dependent detoxification in nonclassical multidrug resistance.

A hydroquinone-resistant derivative of the M1 cell line, designated M1HQ, was generated and used to evaluate the biochemical mechanism responsible for resistance to oxidative stress-inducing agents. The hydroquinone concentrations that were cytotoxic to 50 and 90% of the parental M1 cell line in 48 hr were 25 and 90 microM, respectively, whereas exposure to 500 microM hydroquinone did not decrease M1HQ viability significantly. M1HQ cells grew slower than M1 cells and exhibited significantly higher resistance to colchicine, doxorubicin, hydrogen peroxide, 4-hydroperoxycyclophosphamide, and 1,3-bis (2-chloroethyl)-1-nitrosourea but not to benzoquinone, vinblastine, or gamma-radiation. M1HQ cells possessed significantly higher levels of total thiols, glutathione, glutathione peroxidase, glutathione reductase, quinone reductase, and gamma-glutamyl transpeptidase than the parental M1 cell line. Steady-state gamma-glutamylcysteine synthetase mRNA expression also was 1.6-fold higher in M1HQ cells. P-glycoprotein transcripts were detectable in both M1 and M1HQ cells, but were 2-fold higher in M1HQ. Multidrug resistance-associated protein transcripts were not detectable in either M1 or M1HQ. Hydroquinone resistance in M1HQ cells was partially reversible with a combination of inhibitors of quinone reductase, gamma-glutamylcysteine synthetase, glutathione peroxidase, and the multidrug resistance-associated protein, but not with inhibitors of P-glycoprotein, gamma-glutamyl transpeptidase, or glutathione-S-transferase. When treated with [14C]hydroquinone, M1HQ cells did not generate significant hydroquinone-protein adducts but did release an adduct similar to N-acetylcysteinyl-benzoquinone. In contrast, numerous [14C]hydroquinone-protein adducts were produced in M1 cells, while the N-acetylcysteinyl-benzoquinone-like molecule was undetectable. Thus, hydroquinone resistance in M1HQ cells appeared to result from a glutathione-dependent detoxification and export mechanism.

Influence of oxygen partial pressure on human and mouse myeloid cell line characteristics.

Mouse and human cells have been reported to have different thiol characteristics (J. M. Messina and D. A. Lawrence, 1992, Int. J. Immunopharmacol. 14, 1221-1234). In addition, when cells are undergoing active growth, they usually have an increased thiol content. In an attempt to determine whether a mouse and a human cell line with similar characteristics can be induced to change their phenotype simply by being cultured at a lower oxygen partial pressure (pO2), the human KG-1 and mouse M1 myeloid cell lines were cultured at 5 and 20% oxygen. It is important to note that 5% O2 is close to the physiological pO2, whereas the percentage of O2 usually employed for most in vitro methods is atmospheric pO2. After long-term culturing at 5% (L cell lines) versus 20% O2 (H cell lines), the thiol content of the original (H) cell lines significantly changed. The amount of total and surface thiols was lower in both L cell lines, but only significantly different on the M1 lines, whereas the glutathione content was significantly lower in the L cell lines of KG-1 and M1. The mouse lines showed the greatest sensitivity to pO2 changes; however, the mouse cell lines were not more sensitive to hydrogen peroxide than the human cell lines even though they possess significantly less total thiols and glutathione. Interestingly, cell lines maintained at lower pO2 (physiological pO2) were more sensitive to hydrogen peroxide than their parental counterparts that were maintained at atmospheric oxygen levels.

Genetic mapping of GLCLC, the human gene encoding the catalytic subunit of gamma-glutamyl-cysteine synthetase, to chromosome band 6p12 and characterization of a polymorphic trinucleotide repeat within its 5' untranslated region.

The first and rate-limiting step in the formation of glutathione is catalyzed by gamma-glutamylcysteine synthetase (glutamate-cysteine ligase, E.C. 6.3.2.2). Herein, we describe a trinucleotide repeat polymorphism located in the 5' untranslated region of the human gene, GLCLC, that encodes the catalytic subunit of this enzyme, and we genetically map GLCLC to band 6p12, 1.6 cM distal to D6S295.

Glutathione depletion in human T lymphocytes: analysis of activation-associated gene expression and the stress response.

Glutathione depletion achieved by continuous exposure of mitogen-activated human T lymphocytes to L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, leads to a marked inhibition of the proliferative response. Concanavalin A-activated T cells treated with buthionine sulfoximine failed to exhibit the increase in glutathione content normally observed in activated T cells and were depleted of cellular glutathione over 4 days of culture. On Day 3 of culture, DNA synthesis was inhibited by greater than 75%. In addition, total RNA synthesis was dramatically reduced in the glutathione-depleted cells being inhibited by 26, 61, and 82% on Days 2, 3, and 4, respectively. Despite this global reduction in RNA synthesis, no specific effects on mRNA expression of a number of critical T cell genes required for activation and/or proliferation were detected. In contrast to a recent report of GSH depletion leading to down-regulation of ras mRNA expression in a number of transformed cell lines, glutathione depletion did not influence N-ras mRNA expression in T lymphocytes. No influence of glutathione depletion on the induction of histone mRNA expression was observed. However, consistent with previous studies on regulation of histone mRNA expression, histone transcript levels were reduced when DNA synthesis was markedly inhibited. A cellular stress response, characterized by an increase in mRNA levels of the two stress response genes, HSP70 and gadd 153, was evident in glutathione-depleted unstimulated cells. Additionally, in these cells at 48 hr, we observed a 3.5-fold increase in the steady-state level of mRNA encoding the catalytic subunit of gamma-glutamylcysteine synthetase, the enzyme inhibited by buthionine sulfoximine.

N-ras mRNA expression is unaffected in glutathione-depleted cells of hematopoietic origin.

Glutathione (GSH) depletion in mitogen-stimulated T lymphocytes has been shown to markedly inhibit their proliferative response. This block in proliferation is associated with a significant reduction in total RNA and DNA synthesis; however, the specific mechanism involved in this inhibition of proliferation is unknown. Miller et al. have reported that lowering intracellular GSH levels by greater than 30%, in murine and human tumor cell lines of non-hematopoietic origin, leads to down-regulation of HA-, Ki- and N-ras oncogene expression [Miller. A.C., Gafner, J., Clark, E.P. and Samid, D. (1993) Mol. Cell Biol., 13, 4416-4422]. The reduction in ras transcript levels correlated with the extent of GSH depletion and was independent of the specific mode of oncogene activation. Since the activity of p21(ras) is thought to be involved in pathways of T cell activation, we set out to determine whether down-regulation of ras expression in T cells could be the mechanism by which T cell proliferation was inhibited in GSH-depleted T lymphocytes. Despite reducing the GSH level of concanavalin A-activated human peripheral blood mononuclear cells by 66%, no effect on ras mRNA expression was observed. Similarly, no reduction of ras transcript levels were detected in a human T cell line (Jurkat) or in a human monocytic cell line (THP-1) depleted of glutathione. Our results demonstrate that the mechanism by which GSH depletion inhibits T cell proliferation does not appear to involve a decrease in ras mRNA expression. In addition, our results suggest that differences in the regulation of ras mRNA expression may exist between lymphoid/monocytic cells of non-hematopoietic origin.

A fiberoptic pick-manipulator for vitreoretinal surgery.

We have developed a new multifunction instrument for use during bimanual vitreoretinal surgery. The pick-manipulator provides the functions of a vitreoretinal pick, fiberoptic illumination, bipolar diathermy, and irrigation-aspiration of fluids and tissues in a single 19- or 20-gauge instrument.