A review of 1000 referrals to Walsall's hospital eye service.

BACKGROUND: Referrals to ophthalmology are predominantly made from general practitioners (GPs) and optometrists. These two groups of referrers receive differing types and levels of training and are equipped with different instrumentation. The purpose of this study was to determine whether the quality of referrals to the hospital eye service (HES) differs between GPs and optometrists in Walsall. METHODS: Referrals into the HES were identified from Q1 2014 retrospectively until 1000 notes had been reached. Each record was scrutinized using a standard template. Data were analysed and summary statistics produced including positive predictive values and interobserver agreement. RESULTS: We achieved our target of auditing 1000 records. The false-positive rate (patients being discharged from HES with a 'normal vision' diagnosis) was 7.7% of referrals from GPs and 6.2% of referrals from optometrists. Concordance between referred condition and diagnosed condition at HES between optometrists and ophthalmologists was 76.1%, and between GPs and ophthalmologists was 67.2%. CONCLUSIONS: In view of findings from this study, it is important for commissioners in the new reconfigured National Health Service to ensure that enhanced ophthalmic services are commissioned only on the basis of hard evidence sourced from local data rather than opinion or on data from another geographical area.

Corneal collagen crosslinking for keratoconus or corneal ectasia without epithelial debridement.

PURPOSE: Corneal collagen crosslinking (CXL) is a relatively new technique to reduce the progression of keratoconus. The technique can be performed with or without complete debridement of the corneal epithelium. We describe a novel intermediate technique involving mechanical disruption of the epithelium, and evaluate its safety and efficacy. METHODS: The case notes of 128 eyes with progressive keratoconus or iatrogenic corneal ectasia who had undergone CXL using the epithelial disruption technique were retrospectively reviewed. Thin corneas were treated with hypotonic riboflavin. All others were treated with an isotonic solution. Note was made of preoperative and postoperative parameters, including uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), refraction, endothelial cell count, and corneal tomography. Occurrence of procedure-related complications was recorded. Statistical analyses were performed using the paired sample t-test and Wilcoxon signed-rank test, with a level of P<0.05 being accepted as statistically significant. RESULTS: At 12 months, 41.8% of patients treated with isotonic riboflavin had improved UCVA and 29.7% had improved BSCVA. Only 13.4% lost lines of UCVA and 14.9% lost BSCVA. Of the patients treated with hypotonic riboflavin, at 12 months, 75% demonstrated stability of BSCVA and 25% had stable Kmax. In addition, 25% showed improved visual acuity at 12 months, and 58.3% showed regression of their Kmax. Our rate of short-term complications was comparable to studies using complete epithelial removal. CONCLUSIONS: CXL with epithelial disruption is a safe and effective treatment for keratoconus or iatrogenic corneal ectasia, and may be better tolerated by patients than the epithelium-off technique.

Regulation of CD8 expression in mast cells by exogenous or endogenous nitric oxide.

We recently reported a novel CD8 molecule on rat alveolar macrophages and peritoneal mast cells (PMC). However, little is known about the regulation of CD8 expression and function on these cells. We investigated the regulation of CD8 expression on PMC by NO, because NO can regulate inflammatory responses and also because anti-CD8 Ab stimulates inducible NO synthase and NO production by PMC and alveolar macrophages. Ligation of CD8alpha on PMC with Ab (OX8) induced CD8alpha mRNA expression after 3-6 h, and flow cytometry demonstrated that OX8 treatment increased CD8alpha protein expression compared with PMC treated with isotype control IgG1. To test whether NO mediates the up-regulation of CD8alpha, we used the NO donor S-nitrosoglutathione (500 microM) and NO synthase inhibitors (N(G)-monomethyl-L-arginine and N(G)-nitro-L-arginine methyl ester; 100 microM). S-nitrosoglutathione up-regulated both mRNA and protein expression of CD8alpha in PMC compared with that in sham-treated cells, while NO synthase inhibitors down-regulated OX8 Ab-induced CD8alpha expression. To investigate how NO regulates CD8 expression on PMC, we examined the cGMP-dependent pathway using 8-bromo-cGMP (2 mM) and the guanylate cyclase inhibitor, 1H-oxadiazoloquinoxalin-1-one (20 microM). 8-Bromo-cGMP up-regulated CD8 expression, whereas 1H-oxadiazoloquinoxalin-1-one down-regulated its expression. Thus, ligation of CD8 up-regulates CD8 expression on PMC, a response mediated at least in part by NO through a cGMP-dependent pathway. The significance of this up-regulation of CD8alpha on mast cells (MC) is unclear, but since ligation of CD8 on MC with OX8 Ab can alter gene expression and mediator secretion, up-regulation of CD8 may enhance the MC response to natural ligation of this novel form of CD8.

Aerosolized Syk antisense suppresses Syk expression, mediator release from macrophages, and pulmonary inflammation.

Syk protein tyrosine kinase (PTK) is involved in signaling in leukocytes. In macrophages, Fcgamma-receptor cross-linking induces Syk PTK phosphorylation and activation, resulting in Syk-dependent events required for phagocytosis and mediator release. We hypothesized that Syk antisense oligodeoxynucleotides (ASO) delivered by aerosol to rat lungs in vivo would depress Syk PTK expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation. RT-PCR and RT-in situ PCR demonstrated that aerosolized Syk ASO administration reduced Syk mRNA expression from alveolar macrophages compared with cells isolated from sham-treated rats. Western blot analysis confirmed that Syk PTK expression was reduced after Syk ASO treatment. Compared with sham-treated rats (scrambled oligodeoxynucleotide), Syk ASO treatment suppressed Fcgamma-receptor-mediated nitric oxide (86.0 +/- 8.3%) and TNF (73.1 +/- 3.1%) production by alveolar macrophages stimulated with IgG-anti-IgG complexes. In contrast, Fcgamma-receptor-induced IL-1beta release was unaffected by Syk ASO treatment. Additionally, Syk ASO suppressed Ag-induced pulmonary inflammation, suggesting that Syk ASO may prove useful as an anti-inflammatory therapy in disorders such as asthma.

Activation of macrophage CD8: pharmacological studies of TNF and IL-1 beta production.

Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8 alpha (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1 beta and promoted TNF and IL-1 beta secretion. Similarly, OX8 Ab (CD8 alpha) stimulated NR8383 cells to secrete TNF, IL-1 beta, and NO. Activation of CD8 beta (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1 beta and stimulated secretion of TNF, but not IL-1 beta. Interestingly, anti-CD8 Abs did not stimulate IFN-gamma or PGE2 production, or phagocytosis by macrophages. OX8 (CD8 alpha)-induced TNF and IL-1 beta production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1 beta, stimulation by CD8 alpha is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1 beta production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8 alpha (OX8)-induced TNF and IL-1 beta production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1 beta expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.

Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages.

Direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) of selected mRNA expression in rat mast cells (MC) and alveolar macrophages (AM) was optimized. Rat peritoneal mast cells (PMC), rat cultured mast cells (RCMC), rat bronchoalveolar lavage cells (BALC) or rat cultured alveolar macrophages (NR8383) were studied for the detection of mRNA for beta-actin, TNF-alpha and/or CD8alpha. Each type of cell has unique optimal conditions for RT-in situ PCR. The following parameters were carefully evaluated for optimization: protease digestion, DNAse digestion, heparinase digestion, RT, PCR cycle number and signal development with chromagen. Heparinase digestion was required for PMC mRNA detection because they contain large amounts of heparin proteoglycan, which is a potent inhibitor of RT and Taq polymerase enzymes. Only a few PCR cycles were needed to produce a cytoplasmic signal for mRNA transcripts in RCMC, whereas other types of cells (PMC, BALC and NR8383) needed at least 20 cycles for mRNA detection. The mRNA signal in PMC was localized to the perinuclear region, whereas mRNA in other cell types (RCMC, BALC and NR8383) were detected throughout the cytoplasm. Furthermore, modified Southern blot analysis for TNF-alpha in RCMC treated with RT-in situ PCR demonstrated the specificity of amplification product. The modified and optimized protocols for this procedure were successfully applied to detect and localize several mRNA transcripts in rat MC and AM. The approach is valuable and can be used to further study selected gene expression in these and other cell types.

Novel CD8 molecule on macrophages and mast cells: expression, function and signaling.

BACKGROUND: We previously identified, using flow cytometry and in situ RT-PCR, a novel CD8 molecule on rat alveolar macrophages (AM) and mast cells (MC). RT-PCR also demonstrated that mouse AM express CD8 mRNA. Functional studies on rat AM determined that ligation of CD8 alpha- and beta-chains induced inducible nitric oxide synthase (iNOS) upregulation, nitric oxide (NO), TNF-alpha and IL-1beta (CD8alpha only) secretion. However, CD8 did not induce AM phagocytosis of IgG-opsonized or unopsonized particles. Rat MC stimulated through CD8 secreted NO and TNF-alpha, but not histamine. Because of its potential role in regulating cell function, we investigated the signaling pathways involved in macrophage CD8 stimulation. METHODS AND RESULTS: Inhibitor of src family kinases (PP1) significantly (p<0.05) inhibited CD8alpha (OX8 antibody)-induced iNOS upregulation, NO, TNF-alpha and IL-1beta production in rat AM. In addition, Ro 31-8220 (a PKC inhibitor) inhibited OX8-induced iNOS upregulation, NO and IL-1beta production, but did not inhibit TNF-alpha production. Using Syk antisense, we further determined that OX8 stimulation of NO is Syk kinase dependent. CONCLUSION: Studies on the signaling mechanisms of CD8 determined that src family kinases, PKC, and Syk kinase are involved in CD8 signaling. Additionally, CD8 may have differential signaling pathways, as an inhibitor to PKC downregulated OX8-induced IL-1beta, but not TNF-alpha release. Our studies demonstrate that AM CD8 is similar to T lymphocyte CD8 in that src kinases are involved in CD8-mediated signaling. However, p56(lck), which is expressed in T lymphocytes, has not been found in macrophages, suggesting that other src family kinases may be involved in AM and MC CD8 signaling.

Mast cells express novel CD8 molecules that selectively modulate mediator secretion.

CD8, a marker largely restricted to subsets of T lymphocytes and NK cells, was detected on freshly isolated rat peritoneal mast cells (PMC). Using flow cytometry, Percoll-enriched rat PMC (> or = 98% purity) were positive for the hinge region of CD8alpha (67.5 +/- 9.5%; Ab OX8) and CD8beta (27.8 +/- 2.3%; Ab 341). CD8+ PMC consisted of two populations, CD8alpha+ (22.5%) and CD8alpha+ beta+ (15.9%). Interestingly, G28, an Ab that identifies the IgV-like region of CD8alpha on T lymphocytes, did not bind PMC, suggesting that PMC CD8alpha is distinct from that on T lymphocytes. Moreover, a similar pattern of Ab positivity for CD8 was observed on a rat mast cell line, RBL 2H3. The presence of CD8alpha immunoreactivity on rat PMC was further confirmed by confocal microscopy. In situ reverse-transcription PCR and reverse-transcription PCR analysis demonstrated that PMC contained mRNA transcripts encoding CD8alpha. In functional studies of CD8 on PMC, both TNF-alpha and nitric oxide production were induced by OX8 (CD8alpha) and 341 Ab (CD8beta) in a dose-dependent manner. However, neither OX8 nor 341 induced histamine secretion from PMC. Ag-induced secretion of TNF-alpha, nitric oxide, and histamine was not affected by OX8 or 341 Abs, suggesting that there are distinct signaling mechanisms mediated by CD8 and Fc epsilonRI. These results indicate that rat PMC express functional CD8 molecules that may be distinct from those of T lymphocytes. The difference suggests there is a ligand other than MHC class I for mast cell CD8.

Mechanisms of macrophage stimulation through CD8: macrophage CD8alpha and CD8beta induce nitric oxide production and associated killing of the parasite Leishmania major.

Prior studies demonstrated that rat macrophages express CD8, which differs from T lymphocyte CD8 within the ligand binding domain. We investigated whether stimulation of macrophage CD8 could induce mediator release and regulate host defense. Cross-linking either CD8alpha (OX8, 5 microg/ml) or CD8beta (341, 10 microg/ml) stimulated nitric oxide (NO) production, which correlated with an up-regulation of inducible NO synthase protein. Cell signaling inhibitors were used to elucidate the pathways of CD8alpha and CD8beta stimulation. Genistein (broad spectrum protein tyrosine kinase inhibitor, 10 microg/ml), PP1 (src family kinase inhibitor, 5 microg/ml), polymyxin B (protein kinase C (PKC) inhibitor, 100 microg/ml), and Ro 31-8220 (PKC inhibitor, 1 microM) significantly inhibited anti-CD8alpha- and anti-CD8beta-stimulated NO production and inducible NO synthase up-regulation, suggesting that tyrosine kinase(s) (src family) and PKC are involved in CD8 signaling. In addition, cross-linking CD8alpha stimulated NO-dependent macrophage killing of the parasite Leishmania major. For the first time, this work demonstrates that the beta-chain of macrophage CD8, in addition to the alpha-chain, can regulate mediator release. These results further illustrate the importance of this molecule and support our previous data demonstrating differences between macrophage and T lymphocyte CD8. Additional studies on the signaling mechanisms and possible ligand(s) for macrophage CD8 will lead to a greater understanding of inflammation and host defense.

A novel CD8 molecule expressed by alveolar and peritoneal macrophages stimulates nitric oxide production.

Macrophages play an essential role in host defense, and we have identified a novel CD8 molecule, on alveolar and peritoneal macrophages, that may be involved in regulating this function. Flow cytometric analysis of bronchoalveolar lavage from normal rats identified a large number of CD8-positive cells that could not be accounted for by T lymphocytes. Within the scatter profile region in which the majority of cells were alveolar macrophages (OX41; 89 +/- 1%), 63 +/- 5% of the cells stained positively for CD8alpha (OX8) and 52 +/- 3% for CD8beta (341). Double-staining of lavage cells confirmed the presence of CD8 on alveolar macrophages. Interestingly, flow cytometry showed differences between CD8 on alveolar macrophages and on T lymphocytes within the ligand binding domain for MHC class I. Reverse transcription-PCR analysis on FACS-enriched alveolar macrophages showed the presence of CD8alpha mRNA, determining that macrophages synthesize CD8. Further studies identified both the alpha (49 +/- 8%)- and beta (37 +/- 4%)-chains of CD8 on peritoneal lavage cells (86 +/- 3% macrophages (OX42, CD11b)). As with alveolar macrophages, there were differences within the ligand-binding domain of CD8 on peritoneal macrophages compared with T lymphocytes. Functional studies determined that anti-CD8alpha (OX8) stimulated a dose-dependent release of nitric oxide, indicating that CD8 can directly regulate macrophage function. Thus, macrophages express an unusual CD8 molecule that differs within its ligand-binding domain, compared with T lymphocytes, and these findings suggest hitherto unknown ligand(s) for CD8. These findings will lead to a greater understanding of macrophage function and regulation.

Human tear film pre-rupture phase time (TP-RPT)--a non-invasive technique for evaluating the pre-corneal tear film using a novel keratometer mire.

A non-invasive method of assessing the stability of the human pre-corneal tear film is described using a novel keratometer mire designed in the form of a grid pattern (HIR-CAL Grid). In a laboratory environment, the technique measures with ease and little variability the time taken for the tear film to loose stability and enter the pre-rupture phase (TP-RPT) before rupturing (BUT). This technique may prove useful in evaluating the stability or otherwise of the pre-corneal tear film of prospective contact lens wearer and the pre-contact lens tear film stability on differing current and new contact lens materials.

Conjunctival cytology in hard and soft contact lens wear.

The conjunctival cytological profiles of asymptomatic healthy contact lens wearers are described. An impression biopsy technique (5 h after insertion of contact lenses) was used. Significantly higher cell counts (neutrophils and lymphocytes) were found amongst the soft lens wearers', biopsies compared to hard lens wearers' biopsies or control subjects' biopsies. It is postulated that conjunctival cytological examination may reveal early, subclinical, cytotoxic effects attributable to the preservatives and chelating agents in soft contact lens care systems.

Diurnal variation of some cytological characteristics of the conjunctiva.

Variation of conjunctival cells of healthy non-contact lens wearing subjects is described using an impression biopsy technique over a period of nine waking hours. The total cell count was found to be highest on waking, falling to about half that value during the first 3 h, attributable almost exclusively to the change in the neutrophil count. A further slower decrease and finally a small increase in some cellular components was noted.

Some clinically observed phenomena in extended contact lens wear.

Two groups of volunteer subjects wearing extended-wear soft lenses were monitored over a period of 20 weeks. Of 6 factors measured only 1, corneal touch threshold, was found to show evidence of progressive change. However, a high incidence of lens surface deposits was encountered.

Corneal thickness in extended wear of soft contact lenses.

The continual wearing of a soft contact lens for a period of 20 weeks is shown to produce no significant evidence of corneal swelling, although the use of soft lenses in conjunction with a topically applied solution is shown to produce evidence of transient swelling. The extent of corneal thinning during waking hours is also shown to be reduced among wearers of contact lenses for long periods.

Thickness of human cornea measured by topographic pachometry.

In the hands of trained user, the topographic pachometer proves to be an instrument of acceptable clinical precision (standard deviation = 0.006mm). An examination of corneal thinning during waking hours revealed a rate of thinning which was greatest upon lid opening and which declined uniformly throughout the day (p = 0.01). An investigation into menstrually related change in corneal thickness revealed that at least 122 subjects would be required to investigate this change on an acceptable statistical basis (p = 0.05).