Pre-operative clinical assessment for anaesthesia and the effect of HIV infection.

BACKGROUND: HIV infection is common in South Africa, often remaining clinically latent and liable to be missed during clinical pre-operative assessment, despite the patient having a severe degree of immune compromise. OBJECTIVES: The primary objective was to determine the pre-operative physical status of patients presenting for anaesthesia, and to compare this with subsequent HIV tests and the CD4 counts of the HIV-positive patients. The secondary objective was to determine the prevalence of HIV infection in this group and in selected subgroups. METHOD: A sample of 350 adult patients presenting for anaesthesia at Chris Hani Baragwanath Hospital were interviewed pre-operatively, examined, and their American Society of Anesthesiologists physical status grading determined. In those who were confirmed HIV positive by blood sample, a CD4 count was checked. Further data were collected to determine trends in the characteristics of HIV-positive patients. RESULTS: HIV-positive patients were more likely to be classified as ASA 1 or 2 than ASA 3 or 4 (odds ratio (OR) 2.1). HIV-positive patients with CD4 counts >200 cells/microl were more likely to be ASA 1 or 2 (OR 3.88). Of HIV-positive patients with CD4 counts <200 cells/microl, significantly more were classified as ASA 1 or 2 than ASA 3 or 4 (p < 0.0001). Three patients with CD4 counts <50 cells/microl were classified as ASA 1 or 2. The overall prevalence of HIV infection was 29.4%. Females, patients presenting for obstetric surgery, and younger age groups had higher disease prevalence rates. Patients aged 30 - 39 years (43.0%) had the highest prevalence of HIV infection; the lowest was in patients aged 60 years or older (7.7%). CONCLUSIONS: Routine clinical pre-operative assessment in patients from a population with a high HIV prevalence rate may result in asymptomatic, severe immune compromise being missed in a significant number of patients.

Human retinal microglia express candidate receptors for HIV-1 infection.

BACKGROUND/AIMS: Microglia are the primary antigen presenting cells in the central nervous system and the retina, and can harbour viral antigens that may damage neural tissue via the release of neurotoxins. All cells bearing CD4 molecules and co-receptors (members of the chemokine receptor and Fcgamma receptor families) are potential targets for the human immunodeficiency virus (HIV). In this study, retinal microglia (in vitro and in situ) were investigated for the expression of candidate HIV-1 binding receptors. METHODS: Cultured human retinal microglia and frozen sections of human retinas were used. Immunohistochemistry was used to investigate expression of cell surface receptors necessary for HIV-1 infection: CD4, CC chemokine receptor 5 (CCR5), and Fcgamma receptors. RESULTS: Human retinal microglia expressed detectable levels of CD4, CD16, CD64, and CCR5 in vitro and Fcgamma receptor I (CD64) in situ. CONCLUSIONS: Human retinal microglia express several candidate receptors required for viral binding and as such may be a potential reservoir for HIV-1 infection.

Differential expression of GFAP in early v late AMD: a quantitative analysis.

BACKGROUND/AIMS: Glial fibrillary acidic protein (GFAP) is an established indicator of retinal stress; its expression in retinal astrocytes and Müller cells has been demonstrated to be modulated by cytokines and retinal pathology, including age related macular degeneration (AMD). This study aims to quantify the modulation of GFAP expression in retinas with drusen and atrophic AMD versus normal age matched controls. METHODS: Following a histopathological survey, 17 donor retinas were classified into four groups: drusen (n=5), geographic atrophy (GA) (n=6), aged normal (n=3), and young normal (n=3). Paramacular cryosections were immunolabelled with GFAP antibody, examined by confocal microscopy, and quantified by NIH digital image analysis. Groups were matched for potential confounding factors including age, sex, and postmortem delay. RESULTS: A significant increase in GFAP immunolabelling of macroglia was noted in aged normal compared with young normal retinas (p<0.04). Upregulation of GFAP immunoreactivity involving astrocytes was observed in drusen retinas compared with control retinas (p<0.03). GFAP was also upregulated in retinas with GA compared with controls (p<0.05) and in retinas with GA compared with drusen (p<0.04), both involving Müller cells. Discrete regions of GFAP upregulation in Müller cells were associated with drusen formation. In GA specimens atrophied retinal pigment epithelium (RPE) was substituted by GFAP immunoreactive Müller cell processes (gliosis). CONCLUSION: This study provides a quantitative assessment of GFAP modulation in ageing and AMD affected retinas. Morphological observations were consistent with quantitative analyses indicating differential modulation of GFAP immunoreactivity in inner and outer retina. Upmodulation of GFAP in inner retina and astroglial processes was predominantly associated with drusen, while in outer retina Müller glia upmodulation of GFAP was associated with disruption of the RPE and blood-retinal barrier.

Effects of triamcinolone acetonide on microglial morphology and quantitative expression of MHC-II in exudative age-related macular degeneration.

Animal models, in vitro assays and pilot clinical studies suggest that intravitreal triamcinolone acetonide may be useful in the treatment of age-related macular degeneration. The present case study reports the effect of intravitreal triamcinolone acetonide injection on a subretinal neovascular lesion, microglial morphology and quantitative expression of MHC-II antigens. Triamcinolone acetonide significantly decreased MHC-II expression consistent with immunocytochemical observations which revealed condensed microglial morphology. The modulation of subretinal oedema and microglial morphology correlates with in vitro observations suggesting that downregulation of inflammatory markers and endothelial cell permeability are significant features of the mode of action of triamcinolone acetonide.

Glyt-1 expression in cultured human Müller cells and intact retinae.

In this study, we demonstrate that Müller cells cultured from human retinas are capable of strongly expressing the glycine transporter Glyt-1 as assessed by immunocytochemistry. By contrast, intact normal and pathological human retinas exhibit Glyt-1 immunoreactivity only in neurons. These data suggest that Glyt-1 expression in cultured Müller cells is an epiphenomenon associated with culturing in vitro, rather than a normal physiological or even pathophysiological phenomenon in vivo.

Immunological and aetiological aspects of macular degeneration.

Aetiological and immunological aspects of AMD, a leading cause of blindness in Western countries, have been reviewed. Developmental studies suggest that anatomical features unique to the fovea result in a critical relationship between metabolic demand and blood supply at the macula, which is maintained throughout life. Recent studies show a sufficient degree of consistency in the link between smoking and both dry and wet AMD to regard it as causative. Dry AMD is considered to be the natural endstage of the disease; epidemiological and morphological studies point to choroidal vascular atrophy as the causative event and it is suggested that signals associated with acute vascular compromise lead to the development of subretinal neovascularisation. The relationship between sub-pigment epithelial deposits, including basal laminar deposit, and the pathogenesis of AMD is examined. Much of the literature is consistent with a choroidal origin for the constituents of drusen. The blood-retinal barrier preserves the physiological environment of the neural retina and limits inflammatory responses. The factors, including cytokines, adhesion molecules and the presence of resident immunocompetent cells (microglia), which determine the immune status of the retina are considered. Historical descriptions of the involvement of inflammatory cells are provided, evidence implicating inflammation in the pathogenesis of AMD involving macrophages, giant cells and microglia has been derived from observations of human and animal subretinal neovascular lesions. The role of humoral factors such as anti-retinal autoantibodies and acute phase proteins together with clinical observations has been surveyed. Taken together these data demonstrate the involvement of both cellular and humoral immunity in the pathogenesis of AMD. It remains to be determined to what degree the influence of immunity is causative or contributory in both wet and dry AMD, however, the use of anti-inflammatory agents to ameliorate the condition further indicates the existence of an inflammatory component.

Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1 (ICAM-1) expression of the ECV304 cell line: implications for macular degeneration.

Whilst animal studies and a pilot clinical trial suggest that intravitreal triamcinolone acetonide (TA) may be useful in the treatment of age-related macular degeneration (AMD), its mode of action remains to be fully elucidated. The present study has investigated the capacity of TA to modulate the expression of adhesion molecules and permeability using a human epithelial cell line (ECV304) as a model of the outer blood-retinal barrier (BRB). The influence of TA on the expression of ICAM-1 and MHC-I was studied on resting and phorbol myristate acetate (PMA)- or interferon-gamma (IFN-gamma)- and/or tumour necrosis factor-alpha (TNF-alpha)-activated cells using flow cytometry and immunocytochemistry. Additionally, ECV304 cells were grown to confluence in uncoated Transwell chambers; transepithelial resistance (TER) across resting and PMA-activated cells was monitored. TA significantly decreased the paracellular permeability of ECV304 cells and down-regulated ICAM-1 expression, consistent with immunocytochemical observations. PMA-induced permeability changes were dose-dependent and TA decreased permeability of both resting and PMA-activated monolayers. MHC-I expression by ECV304 cells however, was not significantly affected by TA treatment. The modulation of TER and ICAM-1 expression in vitro correlate with clinical observations, suggesting re-establishment of the BRB and down-regulation of inflammatory markers are the principal effects of intravitreal TA in vivo. The results further indicate that TA has the potential to influence cellular permeability, including the barrier function of the retinal pigment epithelium (RPE) in AMD-affected retinae.

Pre-admission clerking of urology patients by nurses.

Pre-admission clerking by nurse specialists is as effective as that by medical staff in preventing cancellation of elective surgery. Patients assessed at a pre-admission clinic by nurses developed fewer postoperative complications.

The human hyaloid system: cell death and vascular regression.

The present study had investigated the roles of apoptosis and necrosis in the regression of the human fetal hyaloid vasculature. Normal human fetal hyaloid specimens (n = 67) ranging from 10 to 20 weeks' gestation were studied. Specimens were either immunolabeled with anti-von Willebrand factor and major histocompatibility complex class I antibodies or investigated using the terminal-deoxyribonucleotidyl transferase-mediated dUTP-biotin DNA nick-end labeling technique. A fluorescent DNA-binding dye acridine orange/ethidium bromide mixture was also applied to unfixed flat mounts of hyaloid vasculature and some specimens were processed for transmission electron microscopy. Vascular regression including cell loss in the connecting vessels, stretching and thinning of the vasa hyaloidea propria, tunica vasculosa lentis and the pupillary membrane was clearly evident after 13 weeks' gestation. Cresyl violet staining revealed condensed cells and pyknotic bodies throughout the hyaloid system; cell death occurred either in single cells or along small capillary segments associated with vascular regression. Acridine orange/ethidium bromide staining showed DNA condensation at early and late stages of cell death. Similarly, DNA nick-end labeling was positive in endothelial cells, pericytes and vessel and non-vessel associated hyalocytes. The observation of hyalocytes juxtaposed to cytolysed endothelial cells may indicate a role for these cells in vascular regression. Features of apoptosis were more evident during early vascular regression whilst necrosis was increasingly evident at later stages.

Are neuronal transporters relevant in retinal glutamate homeostasis?

Exposure of isolated retinas to 30 microM D-aspartate, which is a substrate for all high affinity glutamate transporters, for 30 min, resulted in the accumulation of such D-aspartate into Müller glial cells but not glutamatergic neurons as evinced by immunocytochemistry for D-aspartate. Further incubation of such loaded retinas in physiological media, in the absence of D-aspartate, resulted in the slow release of accumulated D-aspartate from the Müller cells and its accumulation into populations of photoreceptors and bipolar cells. This result indicates that after initial transport into Müller cells, reversal of direction of transport of D-aspartate, and thus by inference glutamate, by GLAST, readily occurs. D-aspartate released by Müller cells was strongly accumulated into cone photoreceptors which are known to express GLT-1, and into rod photoreceptors which we demonstrate here to express the retina specific glutamate transporter EAAT5 (excitatory amino transporter 5). Populations of glutamatergic bipolar cells, which express GLT-1 also exhibited avid uptake of D-aspartate. We conclude that the Müller cell glutamate transporter GLAST is responsible for most of the initial glutamate clearance in the retina after its release from neurones. However, some glutamate is also returned from Müller cells, to neurons expressing GLT-1 and EAAT5, albeit at a slow rate. These data suggest that the role of neuronal glutamate transporters in the retina may be to facilitate a slow process of recycling glutamate back from Müller cells to neurons after its initial clearance from perisynaptic regions by GLAST.

Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas.

PURPOSE: To determine whether MT1-MMP and MMP-2 are expressed in normal and keratoconic corneas, and to investigate the ability of MT1-MMP, expressed on cultured keratocytes after stimulation with concanavalin A, to activate pro-gelatinase A (pro-MMP 2). METHODS: Specimens of keratoconus corneas (n = 20), removed at corneal transplantation, were obtained from pathology archives, sections were cut, and were stained with an antibody to MT1-MMP, using peroxidase immunohistochemistry. Eye banked corneas served as controls (n = 14). Normal human keratocyte cultures were initiated from eye bank corneas, and after stimulation with con A, MMPs in the media were examined using gelatin zymography and immunoblotting, and MT1-MMP expression was analysed by flow cytometry and immunoblotting. RESULTS: All corneas showed some expression of MT1-MMP and MMP-2, although the degree of staining varied greatly. The MMPs were present in the epithelium, endothelium and stroma. Expression of MT1-MMP, but not MMP-2, in the epithelium and stroma, was significantly elevated in keratoconus, compared to normal corneas. In vitro, keratocytes stimulated with con A expressed MT1-MMP and produced active MMP-2, detected by zymography. These responses to con A were concentration-dependent and MT1-MMP expression and MMP-2 activation correlated significantly (p = 0.0003) In addition, MMP inhibitors abolished MMP-2 activation, providing further evidence that MT1-MMP activated MMP-2. CONCLUSION: The observation that MT1-MMP expression may be up-regulated in keratoconus corneas, taken together with the demonstration that human corneal cells can express this enzyme, which in turn can activate latent MMP-2, provide evidence for a possible role for MT1-MMP in the pathogenesis of keratoconus.

Astrocyte proliferation during development of the human retinal vasculature.

Wholemounts of human fetal retinas were labeled with antibodies to Ki67 or proliferating cell nuclear antigen, to map the distribution of proliferating cells in the developing primary vasculature and neural retina. Double labeling was used to determine the relative proportions of endothelial cells (CD34), astrocytes (glial fibrillary acidic protein - GFAP) and microglia (major histocompatability complex class II) associated with the developing vessels. The differentiated region of neural retina (cold spot) was 3.5 mm(2)at 15 weeks gestation (WG), centred on the incipient fovea, and increased in size with age to 80.5 mm(2)by 23-24 WG. Ki67 immunoreactive cells were distributed throughout the developing vasculature at all ages. The mean density of dividing cells in the neural retina increased with gestational age from 146 mm(-2)at 15 WG, to 624 mm(-2)at 23-24 WG. By 20 WG proliferation in the vasculature overlapped the margins of the cold spot, which was almost completely vascularized by 23-24 WG, except for a narrow strip on the horizontal meridian, which included the incipient fovea. Counts of CD34/Ki67 immunoreactive cells indicated that 15-52% of proliferations in the developing vasculature at 18 WG are endothelial cells. In contrast, in the fellow retina 65-85% cells were Ki67/GFAP immunoreactive, indicating proliferation of astrocytes in situ. No dividing microglia were observed. The findings suggest that large numbers of proliferating astrocytes accompany the developing vessels as they migrate across the primate retina.

UTI in patients with urethral catheters: an audit tool.

This article presents an audit tool for the evaluation of practice relating to urinary tract infection in hospital patients with indwelling urethral catheters. It has been formulated primarily because urinary tract infection is a known complication of catheterization, and because studies have shown practitioners' knowledge in this area to be poor. Although health professionals have an obligation to ensure their practice is evidence based, this requires substantial time and skills in critical appraisal. The standard presented here is based on evidence from an extensive literature review on how best to minimize urinary tract infection during catheter insertion, meatal hygiene and management of the drainage system. The audit tool offers the potential for improved practice and demonstration of clinical effectiveness through measurable reduction in rates of urinary tract infection. Moreover, it provides an ideal opportunity for nurses to take the lead in clinical audit activity, which is so often medically led. The supplementary information will also provide a useful guide for nurses to undertake and initiate clinical audit activity in the future.

The human hyaloid system: cellular phenotypes and inter-relationships.

We have investigated the expression of leucocyte markers, phenotypic characteristics and cellular relationships of the normal human fetal hyaloid vasculature using immunohistochemistry, light and electron microscopy. Antibodies against von Willebrand Factor, alpha-smooth muscle actin, glial fibrillary acidic protein, vimentin, major histocompatibility complex classes-I and -II, CD45 (leucocyte-common antigen) and calcitonin gene-related peptide were used to identify the cellular constituents of the hyaloid vasculature in whole mounts. Additional morphological features were described at the ultrastructural level. Endothelial cells throughout the hyaloid system were immunoreactive to von Willebrand Factor and major histocompatibility complex class-I antibodies. Pericytes were immunoreactive to alpha-smooth muscle actin antibody; labeled cells were distributed along large branches of the hyaloid artery, vasa hyaloidea propria, tunica vasculosa lentis and pupillary membrane but no immunoreactivity was detected on small connecting capillaries. Vessel and non-vessel-associated hyalocytes on the hyaloid artery, vasa hyaloidea propria, tunica vasculosa lentis, pupillary membrane and vitreous were immunoreactive to major histocompatibility complex classes-I and -II, CD45 and calcitonin gene-related peptide antibodies. Anti-glial fibrillary acidic protein reactivity was detected on Bergmeister's papilla but not on the hyaloid artery. Cells immunoreactive for vimentin were present throughout the hyaloid vasculature including small connecting capillaries. Ultrastructural observations of the hyaloid vasculature revealed junctional complexes, including zonulae adherens, macula adherens and possible zonulae occludens, between adjacent endothelial cells. Fenestrae were not observed in the gestational ages included in the present study. The use of whole mounts in conjunction with specific antisera has provided novel immunohistochemical definitions of the structure and cellular constituents of the human hyaloid. The results indicate that hyalocytes are a heterogeneous population of leucocyte-lineage cells.

Sodium butyrate modulates p53 and Bcl-2 expression in human retinoblastoma cell lines.

Sodium butyrate (SB) is a potent biological modifier that can induce diverse effects including growth inhibition, differentiation, or apoptosis of many cell types including retinoblastoma (Rb), and modulation of genes such as c-fos and p53. In this study we assessed the effects of SB on cell growth and expression of p53, critical for cell cycle control, and Bcl-2, an inhibitor of apoptosis, in two human Rb cell lines (Y79 and WERI-Rb1). Attachment cultures were treated with 1 mM SB for up to 5 days and immunocytochemistry was used to examine for the expression of neural cell adhesion molecule (NCAM), p53, and Bcl-2. Suspension cultures of both cell lines were also treated with 1 and 4 mM SB, and at selected times cell extracts were prepared and the expression of p53 and Bcl-2 proteins determined by Western blot analysis. Treatment with 1 mM SB of both cell lines for 5 days inhibited growth and induced morphological changes including extension of neurite-like processes. Up to 12 h after 1 mM SB treatment, p53 and Bcl-2 expressions were similar to control levels, then gradually decreased to very low levels at 5 days. SB (4 mM) also inhibited growth associated with cell death, which was apparent at 24 h posttreatment. Expressions of p53 and Bcl-2 were decreased below control levels at 4 h, and by 24 h only very low levels of protein were detected. SB-induced modulation of p53 and Bcl-2 expression may have implications for controlling Rb growth, particularly in combination with chemotherapy drugs, which are increasingly used in the treatment of Rb.

Exudative macular degeneration and intravitreal triamcinolone: 18 month follow up.

PURPOSE: To evaluate the safety and efficacy of intravitreal triamcinolone after 18 months of follow up in patients with age-related macular degeneration and subfoveal or juxtafoveal choroidal neovascularization considered unsuitable for laser photocoagulation. METHODS: Thirty eyes of 28 patients, referred from general eye clinics as well as the private clinic of one of the authors to a hospital-based retinal out-patient clinic, were treated with an intravitreal injection of triamcinolone (4 mg). The primary outcome measure was the proportion of eyes with loss of six or more lines on a Bailey-Lovie Chart. The incidence of adverse events associated with treatment was also observed. RESULTS: Of the 20 eyes with initial visual acuity (VA) of 6/60 or better, the vision was maintained (+/-1 Bailey-Lovie lines) in 11 eyes (55%), while six eyes (30%) suffered severe visual loss (six or more lines). The VA improved by five to six lines in three of 10 eyes with initial vision of 3/60 or worse. Three of four eyes receiving a second injection suffered either progressive cataract or elevated intra-ocular pressure (IOP) requiring cataract surgery and/or filtering surgery. One of 26 eyes (3%) receiving a single injection showed progression of cataract and elevation of IOP within 6 weeks of treatment and required anti-glaucoma medication for 6 weeks. Progression of nuclear sclerosis 8-12 months after treatment was observed in six of 26 eyes (23%) receiving a single injection. CONCLUSIONS: The results of the present study suggest that a single intravitreal injection of 4 mg triamcinolone is reasonably well tolerated by the human eye. The rate of development of severe visual loss was less than reported for historical controls. Because the results are preliminary and uncontrolled, the treatment should not be used routinely until its benefit to patients is established by a prospective, randomized controlled study.

Disposable bedpads for incontinence: predicting their clinical leakage properties using laboratory tests.

A multi-centre project has been run to identify laboratory tests capable of predicting the leakage performance of disposable incontinence bedpads. Each of 95 subjects tested each of six products for a week in turn and reported whether or not they and/or their carers found the leakage performance of each product acceptable. In addition, carers noted the severity with which individual used bedpads had leaked so that, when they had been weighed, their leakage performance could be determined as a function of urine weight. These clinical data were compared with results from the 16 different laboratory tests used routinely for bedpad evaluation in three hospital laboratories. Each test was evaluated by seeing how well the data it yielded correlated with the clinical test data. No individual test was very successful at predicting the performance of bedpads when used as sole protection but a combination of an absorption capacity test and an absorption time test predicted the percentage of users/carers finding leakage performance acceptable, accurate to within +/- eight percentage points for all six test products. A different absorption capacity test proved most successful for bedpads used as back-up to body-worn products. It predicted the percentage of users/carers finding leakage performance acceptable, accurate to +/- five percentage points for all six products.

Isolation, culture and characteristics of human foetal and adult retinal pigment epithelium.

BACKGROUND: Current methods for the isolation and culture of adult retinal pigment epithelium (RPE) provide successful primary culture. However, most methods result in contamination with other cell types and low cell yields. The isolation and culture of human foetal RPE presents further problems associated with the limited size of the eye cup and adherence among cells. Reliable methods are necessary for the culture of human RPE and subsequent functional studies. METHODS: The present procedure is based on mechanical peeling of the whole RPE layer under the dissecting microscope. Dissected pieces are subsequently explanted to a 35 mm culture dish and are cultured with Dulbecco's modified Eagle's medium containing 10% foetal bovine serum. Peroxidase immunohistochemical methods were used to investigate cell phenotypes. RESULTS: Primary cultures were obtained within 10-14 days with high yields, good viability and purity in subsequent culture. Cultured cells were vimentin and cytokeratin positive and CD31 negative. CONCLUSIONS: This mechanical dissection technique is recommended for the isolation of foetal and young adult RPE cells, while the enzyme digestion method is preferred for aged adult tissue.

Modulation of the resistance of a human endothelial cell line by human retinal glia.

BACKGROUND: Human umbilical endothelial cells were used to model the vascular component of the blood-retinal barriers and to examine the capacity of glial cultures to modulate endothelial cell resistivity in vitro. METHODS: Endothelial cell resistivity was monitored with and without cocultured human retinal glia. Immunohistochemistry indicated that both macroglia and microglia were present in one culture, while only macroglia were detectable in the second culture. RESULTS: Both cocultures produced increased resistivity in the target endothelial cells; however, a further significant increase in resistivity was noted with the glial coculture containing microglia. The results suggest that the presence of microglia significantly increases the capacity of astrocytes and Müller cells to modulate endothelial cell resistivity.