Inactivation of Bacillus anthracis and Bacillus atrophaeus spores on different surfaces with ultraviolet light produced with a low-pressure mercury vapor lamp or light emitting diodes.

AIMS: To obtain quantitative efficacy data of two ultraviolet light (UVC) technologies for surface inactivation of Bacillus anthracis Ames and Bacillus atrophaeus spores. METHODS AND RESULTS: Spores were deposited onto test coupons and controls of four different materials, via liquid suspension or aerosol deposition. The test coupons were then exposed to UVC light from either a low-pressure mercury vapor lamp or a system comprised of light emitting diodes, with a range of dosages. Positive controls were held at ambient conditions and not exposed to UVC light. Following exposure to UVC, spores were recovered from the coupons and efficacy was quantified in terms of log10 reduction (LR) in the number of viable spores compared to that from positive controls. CONCLUSIONS: Decontamination efficacy varied by material and UVC dosage (efficacy up to 5·7 LR was demonstrated). There was no statistical difference in efficacy between the two species or between inoculation methods. Efficacy improved for the LED lamp at lower relative humidity, but this effect was not observed with the mercury vapor lamp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study will be useful in determining whether UVC could be used for the inactivation of B. anthracis spores on different surface types.

HIF signalling: The eyes have it.

The hypoxia inducible factors (HIFs) promote changes in gene expression in response to hypoxia, and mediate key physiological responses such as angiogenesis. They play important roles in development and normal physiology, as well as in ischaemic and other pathologies. The human eye is a complex organ, with tight regulation of vascularisation and oxygen delivery, with the highly specialised retina containing both highly vascularised and avascular regions. This review, written to honour the significant contribution of Lorenz Poellinger to this field, covers the role of the HIFs in normal development of the eye, specifically the vasculature, as well as their roles in numerous retinal pathologies, including ischaemic retinopathies, and age-related macular degeneration (AMD). The characterisation of the HIFs in the eye has improved our understanding of the development, function, and numerous pathologies of the eye, and should inform future therapeutic approaches.

A simple decontamination approach using hydrogen peroxide vapour for Bacillus anthracis spore inactivation.

AIMS: To evaluate the use of relatively low levels of hydrogen peroxide vapour (HPV) for the inactivation of Bacillus anthracis spores within an indoor environment. METHODS AND RESULTS: Laboratory-scale decontamination tests were conducted using bacterial spores of both B. anthracis Ames and Bacillus atrophaeus inoculated onto several types of materials. Pilot-scale tests were also conducted using a larger chamber furnished as an indoor office. Commercial off-the-shelf (COTS) humidifiers filled with aqueous solutions of 3 or 8% hydrogen peroxide (H2 O2 ) were used to generate the HPV inside the mock office. The spores were exposed to HPV for periods ranging from 8 h up to 1 week. CONCLUSIONS: Four- to seven-day exposures to low levels of HPV (average air concentrations of approx. 5-10 parts per million) were effective in inactivating B. anthracis spores on multiple materials. The HPV can be generated with COTS humidifiers and household H2 O2 solutions. With the exception of one test/material, B. atrophaeus spores were equally or more resistant to HPV inactivation compared to those from B. anthracis Ames. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple and effective decontamination method is another option that could be widely applied in the event of a B. anthracis spore release.

Whole-building decontamination of Bacillus anthracis Sterne spores by methyl bromide fumigation.

AIMS: To evaluate the field inactivation of Bacillus anthracis Sterne spores with methyl bromide (MB) using commercial fumigation techniques. METHODS AND RESULTS: Eighty-seven wood and 87 glass coupons each containing ca. 1 × 10(6) B. anthracis Sterne spores, were placed in 22 locations inside a 1444 m(3) conference building. Four additional 12-coupon sets (six wood, six glass) were removed from the building at 16, 24, 32 and 40 h during fumigation. The building was sealed under two tarpaulins and fumigated with MB at ≥225 g m(-3) mean concentration for 48 h at 28°C and 83% RH. All B. anthracis spores fumigated for more than 16 h were inactivated. A single wood coupon from the 16-h set yielded ca. 2 × 10(3)  CFU. No damage to the building or its contents was observed. CONCLUSIONS: MB fumigation is a rapid, economical and effective whole-structure decontamination method for B. anthracis spores. SIGNIFICANCE AND IMPACT OF THE STUDY: MB fumigation offers a method of whole-structure B. anthracis decontamination without removal of materials, damage to sensitive electronics, costly indoor retrofitting.

Fumigation of a laboratory-scale HVAC system with hydrogen peroxide for decontamination following a biological contamination incident.

AIMS: To evaluate hydrogen peroxide vapour (H2 O2 ) for its ability to inactivate Bacillus spores within a laboratory-scale heating, ventilation and air-conditioning (HVAC) duct system. METHODS AND RESULTS: Experiments were conducted in a closed-loop duct system, constructed of either internally lined or unlined galvanized metal. Bacterial spores were aerosol-deposited onto 18-mm-diameter test material coupons and strategically placed at several locations within the duct environment. Various concentrations of H2 O2 and exposure times were evaluated to determine the sporicidal efficacy and minimum exposure needed for decontamination. For the unlined duct, high variability was observed in the recovery of spores between sample locations, likely due to complex, unpredictable flow patterns within the ducts. In comparison, the lined duct exhibited a significant desorption of the H2 O2 following the fumigant dwell period and thus resulted in complete decontamination at all sampling locations. CONCLUSIONS: These findings suggest that decontamination of Bacillus spore-contaminated unlined HVAC ducts by hydrogen peroxide fumigation may require more stringent conditions (higher concentrations, longer dwell duration) than internally insulated ductwork. SIGNIFICANCE AND IMPACT OF THE STUDY: These data may help emergency responders when developing remediation plans during building decontamination.

Environmental persistence of vaccinia virus on materials.

Smallpox is caused by the variola virus, and ranks as one of the most serious diseases that could originate from a biological weapon. However, limited data exist on the persistence of variola and related viruses on materials (that may act as fomites), under controlled environmental conditions. To fill these data gaps, we determined the persistence of the vaccinia virus (an established surrogate for the variola virus) as a function of temperature, relative humidity and material. Experiments were conducted with vaccinia virus in a freeze-dried form, using four materials under four sets of environmental conditions. After elapsed times ranging from 1 to 56 days, the virus was extracted from small coupons and quantified via plaque-forming units (PFU). The vaccinia virus was most persistent at low temperature and low relative humidity, with greater than 10(4) PFU recovered from glass, galvanized steel and painted cinder block at 56 days (equivalent to only a c. 2 log reduction). Thus, vaccinia virus may persist from weeks to months, depending on the material and environmental conditions. This study may aid those responsible for infection control to make informed decisions regarding the need for environmental decontamination following the release of an agent such as variola.

Laboratory evaluation of large-scale decontamination approaches.

AIMS: To evaluate the effectiveness of two spray-based decontamination methods for surface contamination reduction and to determine the potential for contamination spread by these methods. METHODS AND RESULTS: Material coupons (treated plywood and concrete) were contaminated with c. 1 × 10(7) spores of Bacillus atrophaeus by aerosol deposition. Decontaminants (pH-adjusted bleach or Spor-Klenz(®) RTU) were applied to coupons by either backpack sprayer or gas-powered sprayer. Contact time, reapplication frequency and rinse method were also varied. In addition to surface removal efficacy, partitioning of contamination between the rinsate and aerosol fractions was determined. Results indicated that pH-adjusted bleach was effective (≥6 logs reduction) when two applications and a 30 min contact time were administered, regardless of the decontaminant application method or material. Spor-Klenz(®) RTU was effective on wood, but achieved ≤3 logs reduction on concrete. A shortened application procedure with pH-adjusted bleach resulted in lower efficacy on wood, and a greater apparent potential for contamination spread. CONCLUSIONS: Consideration of material surface type is important when selecting a decontaminant. Also, achieving conditions that effectively inactivate surface biological contamination are critical to preventing the spread of contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Results presented here are intended to help development of remediation plans following a biological contamination incident.

Efficacy of liquid spray decontaminants for inactivation of Bacillus anthracis spores on building and outdoor materials.

AIMS: To obtain data on the efficacy of various liquid and foam decontamination technologies to inactivate Bacillus anthracis Ames and Bacillus subtilis spores on building and outdoor materials. METHODS AND RESULTS: Spores were inoculated onto test coupons and positive control coupons of nine different materials. Six different sporicidal liquids were spray-applied to the test coupons and remained in contact for exposure times ranging from 10 to 70 min. Following decontamination, spores were recovered from the coupons and efficacy was quantified in terms of log reduction. CONCLUSIONS: The hydrogen peroxide/peracetic acid products were the most effective, followed by decontaminants utilizing hypochlorous acid chemistry. Decontamination efficacy varied by material type. SIGNIFICANCE AND IMPACT OF THE STUDY: The study results may be useful in the selection of technologies to decontaminate buildings and outdoor areas in the event of contamination with B. anthracis spores. These results may also facilitate selection of decontaminant liquids for the inactivation of other spore-forming infectious disease agents.

Optimizing acidified bleach solutions to improve sporicidal efficacy on building materials.

AIMS: We evaluated whether lowering pH (with acetic acid) and raising free available chlorine (FAC) levels in bleach solutions would improve efficacy in inactivating Bacillus spores on different materials. We also determined how varying pH and FAC levels affected bleach stability. METHODS AND RESULTS: Acidified bleach solutions with pH levels of 4.5, 6 and 7.5 and FAC levels between 5000 and 10,000 ppm were evaluated for decontamination efficacy against Bacillus subtilis spores inoculated onto test coupons made from wood, ceramic and galvanized steel. Lowering the pH or increasing the FAC level improved efficacy in some of the tests, but depended on the material, which significantly affected decontamination efficacy. The acidified bleach at pH of 7.5 was significantly less effective than bleach at a pH of 4.5 or 6. The FAC levels in the bleach were the most stable at pH 4.5, and stability at pH 4.5 was not significantly affected by the initial FAC level. CONCLUSIONS: It may be advisable to use bleach solutions with lower pH (rather than high FAC levels) in light of both the decontamination efficacy and bleach stability results. For wood materials, use of sporicides other than acidified bleach may be warranted. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may be useful in preparing acidified bleach solutions for decontamination of materials contaminated with spores such as Bacillus anthracis.

Dry thermal resistance of Bacillus anthracis (Sterne) spores and spores of other Bacillus species: implications for biological agent destruction via waste incineration.

AIMS: To obtain needed data on the dry thermal resistance of Bacillus anthracis spores and other Bacillus species for waste incinerator applications. METHODS AND RESULTS: Tests were conducted in a pilot-scale incinerator utilizing biological indicators comprised of spores of Geobacillus stearothermophilus, Bacillus atrophaeus and B. anthracis (Sterne) and embedded in building material bundles. Tests were also conducted in a dry heat oven to determine the destruction kinetics for the same species. In the pilot-scale incinerator tests, B. atrophaeus and G. stearothermophilus demonstrated similar thermal sensitivity, but B. anthracis (Sterne) was less thermally resistant than G. stearothermophilus. For the dry heat oven tests conducted at 175°C, the D-values were 0·4, 0·2 and 0·3 min for B. atrophaeus, B. anthracis (Sterne) and G. stearothermophilus, respectively. CONCLUSIONS: Bacillus anthracis (Sterne) possesses similar or less dry heat resistance compared to B. atrophaeus and G. stearothermophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous studies have demonstrated conditions under which bacterial spores may survive in an incinerator environment. The data from this study may assist in the selection of surrogates or indicator micro-organisms to ensure B. anthracis spores embedded in building materials are completely inactivated in an incinerator.

Heparin-protamine balance after neonatal cardiopulmonary bypass surgery.

Essentials Heparin-protamine balance (HPB) modulates bleeding after neonatal cardiopulmonary bypass (CPB). HPB was examined in 44 neonates undergoing CPB. Post-operative bleeding occurred in 36% and heparin rebound in 73%. Thrombin-initiated fibrin clot kinetic assay and partial thromboplastin time best assessed HPB. SUMMARY: Background Neonates undergoing cardiopulmonary bypass (CPB) are at risk of excessive bleeding. Blood is anticoagulated with heparin during CPB. Heparin activity is reversed with protamine at the end of CPB. Paradoxically, protamine also inhibits blood coagulation when it is dosed in excess of heparin. Objectives To evaluate heparin-protamine balance in neonates undergoing CPB by using research and clinical assays, and to determine its association with postoperative bleeding. Patients/Methods Neonates undergoing CPB in the first 30 days of life were studied. Blood samples were obtained during and after surgery. Heparin-protamine balance was assessed with calibrated automated thrombography, thrombin-initiated fibrin clot kinetic assay (TFCK), activated partial thromboplastin time (APTT), anti-FXa activity, and thromboelastometry. Excessive postoperative bleeding was determined by measurement of chest tube output or the development of cardiac tamponade. Results and Conclusions Of 44 neonates enrolled, 16 (36%) had excessive postoperative bleeding. The TFCK value was increased. By heparin in neonatal blood samples, but was only minimally altered by excess protamine. Therefore, it reliably measured heparin in samples containing a wide range of heparin and protamine concentrations. The APTT most closely correlated with TFCK results, whereas anti-FXa and thromboelastometry assays were less correlative. The TFCK and APTT assay also consistently detected postoperative heparin rebound, providing an important continued role for these long-established coagulation tests in the management of postoperative bleeding in neonates requiring cardiac surgical repair. None of the coagulation tests predicted the neonates who experienced postoperative bleeding, reflecting the multifactorial causes of bleeding in this population.

A hypothesis to suggest that light is a risk factor in glaucoma and the mitochondrial optic neuropathies.

The authors propose that light entering the eye interacts with retinal ganglion cell (RGC) axon mitochondria to generate reactive oxygen intermediates (ROI) and that when these neurons are in an energetically low state, their capacity to remove these damaging molecules is exceeded and their survival is compromised. They suggest that in the initial stages of glaucoma, RGCs exist at a low energy level because of a reduced blood flow at the optic nerve head and that in the mitochondrial optic neuropathies (MONs), this results from a primary, genetic defect in aerobic metabolism. In these states RGCs function at a reduced energy level and incident light on the retina becomes a risk factor. Preliminary laboratory studies support this proposition. Firstly, the authors have shown that light is detrimental to isolated mitochondria in an intensity dependent manner. Secondly, light triggers apoptosis of cultured, transformed RGCs and this effect is exacerbated when the cells are nutritionally deprived. Detailed studies are under way to strengthen the proposed theory. On the basis of this proposal, the authors suggest that patients with optic neuropathies such as glaucoma or at risk of developing a MON may benefit from the use of spectral filters and reducing the intensity of light entering the eye.

Alpha-lipoic acid protects the retina against ischemia-reperfusion.

The aim of this study was to examine whether the antioxidant alpha-lipoic acid protects retinal neurons from ischemia-reperfusion injury. Rats were injected intraperitoneally with either vehicle or alpha-lipoic acid (100 mg/kg) once daily for 11 days. On the third day, ischemia was delivered to the rat retina by raising the intraocular pressure above systolic blood pressure for 45 min. The electroretinogram was measured prior to ischemia and 5 days after reperfusion. Rats were killed 5 or 8 days after reperfusion and the retinas were processed for immunohistochemistry and for determination of mRNA levels by RT-PCR. Ischemia-reperfusion caused a significant reduction of the a- and b-wave amplitudes of the electroretinogram, a decrease in nitric oxide synthase and Thy-1 immunoreactivities, a decrease of retinal ganglion cell-specific mRNAs and an increase in bFGF and CNTF mRNA levels. All of these changes were clearly counteracted by alpha-lipoic acid. Moreover, in mixed rat retinal cultures, alpha-lipoic acid partially counteracted the loss of GABA-immunoreactive neurons induced by anoxia. The results of the study demonstrate that alpha-lipoic acid provides protection to the retina as a whole, and to ganglion cells in particular, from ischemia-reperfusion injuries. alpha-Lipoic acid also displayed negligible affinity for voltage-dependent sodium and calcium channels.

Induction of apoptosis in cultured human retinal pigment epithelial cells is counteracted by flupirtine.

PURPOSE: The aim of the study was to determine whether flupirtine can counteract the induction of apoptosis in cultured retinal pigment epithelium (RPE) cells. METHODS: Confluent cultures were subjected to experimental ischemia (medium free of serum, glucose, and oxygen) with or without various substances for specific periods. The cells were then examined for breakdown of DNA by the TUNEL procedure and agarose gel electrophoresis. Moreover cells were processed for the localization of oncogene proteins (bcl-2, TIAR, ICH-1t) associated with apoptosis. The effect of flupirtine on reactive oxygen species also was determined. RESULTS: When RPE cells were subjected to ischemia for 72 hours approximately 65% of cells remained attached to the coverslips and approximately 65% of their nuclei showed clear fragmentation of DNA by TUNEL. Most of the cells exhibited a shrunken appearance typical of apoptosis. Fragmentation of the DNA from cells given ischemia for 72 hours was also confirmed by agarose gel electrophoresis. Inclusion of flupirtine (flupirtine gluconate, 100 microM) or 10% fetal calf serum in the medium prevented ischemia-induced apoptosis occurring after 72 hours. Neither N-methyl-D-aspartate (NMDA) (100 microM) nondeferoxamine (100 microM) nor the NMDA antagonists dextromethorphan (100 microM), memantine (100 microM), and MK-801 (10 microM) had a similar effect. NMDA, and to a lesser extent memantine, induced apoptosis independently. Treatment of RPE cells in serum-free medium with flupirtine (flupirtine gluconate, 100 microM) for 72 hours caused an upregulation of bcl-2 protein. In contrast, the oncogene proteins for TIAR and ICH-1t, were lower in flupirtine-treated cells than in control cells. Flupirtine, like deferoxamine, prevents iron-ascorbate-induced reactive oxygen species formation in retinal cells, but only flupirtine prevents ischemia-induced apoptosis in RPE cells. CONCLUSIONS: The combined data demonstrate that flupirtine is an effective agent in preventing death by apoptosis. Flupirtine reduces formation of reactive oxygen species in retinal dissociates and causes changes in various oncogene products in RPE cultures, which may explain its action in preventing apoptosis induced by ischemia. The current results also suggest that NMDA receptors are not involved in the induction of ischemia-induced apoptosis in RPE, cells.

Melatonin counteracts ischemia-induced apoptosis in human retinal pigment epithelial cells.

PURPOSE: To investigate whether the neurohormone melatonin can prevent apoptosis caused by deprivation of oxygen, glucose, and serum (experimental ischemia) in cultured human retinal pigment (RPE) cells. METHODS: Cultures of human RPE cells established from a variety of donors were grown to passage four and then subjected to experimental ischemia, with or without various substances, for up to 72 hours. Cells were examined for morphologic changes and breakdown of DNA, assessed by TdT-dTUP terminal nick-end labeling (TUNEL) and agarose gel electrophoresis. Changes in transcription and translation of various proto-oncogenes (bcl-2, TIAR, ICH-1S/1) were assessed by analysis of mRNA and protein levels, respectively. The effect of various substances on the iron-ascorbate-induced formation of reactive oxygen species (ROS) in chick retinal dissociates was also investigated. RESULTS: Cultured human RPE cells on coverslips that were incubated in serum-free medium, glucose, and oxygen remained viable for up to 40 hours. Thereafter, there was a steady decrease in cell numbers and an increase in the number of cells labeled by the TUNEL method. By 72 hours 65% of cells remained attached to the coverslips, of which approximately 65% were TUNEL positive. Furthermore, most of the experimental ischemia-treated cells exhibited a shrunken appearance typical of apoptosis. Fragmentation of the DNA from cells in which ischemia was induced for 72 hours was also confirmed by agarose gel electrophoresis. Inclusion of 100 microM melatonin significantly decreased the amount of apoptotic cell nuclei after ischemia, but the effect was mild compared with that of fetal calf serum, which almost completely counteracted cell death. The action of melatonin was not prevented by 0.01 mM to 1 mM luzindole, a melatonin receptor antagonist. In addition, 100 microM ascorbate did not counteract ischemia-induced apoptosis. Treatment of RPE cells with 100 microM flupirtine gluconate for 72 hours caused an upregulation of the proto-oncogene protein Bcl-2 and a decrease in TIAR and ICH-1L proteins compared with that in control cells. Melatonin at 100 microM had no such effect. The levels of the mRNA transcripts for ICH-1L relative to those for ICH-1S were significantly decreased in cultures treated with 100 microM flupirtine or 100 microM melatonin when compared with levels in control cells. However, the effect of flupirtine was greater than that of melatonin. Ten micromolar ascorbate and 5 microM iron stimulated the formation of ROS in chick retinal cell dissociates. Ascorbate, melatonin, and flupirtine (all at 100 microM) blunted this response in the order flupirtine > melatonin >> ascorbate. Luzindole had no effect, alone or in the presence of melatonin. CONCLUSIONS: The presented data show that melatonin counteracted ischemia-induced apoptosis in human RPE cells by a process that seemed to be independent of melatonin receptors. Moreover, melatonin and flupirtine counteracted iron-ascorbate-induced ROS formation and decreased the ratio of mRNA for ICH-1L and ICH-1S. However, melatonin was less potent than flupirtine in its action in each case, which suggests that either the two compounds act on different signaling pathways or that they act on the same pathway with differing potency. The failure to detect an effect of melatonin on the levels of Bcl-2, ICH-1L, and TIAR proteins when compared with the effect of flupirtine was probably caused by the sensitivity of the procedures. It is suggested that substances that can prevent ROS formation can potentially nullify apoptotic cell death, but this is difficult to detect experimentally when the substance has only a mild effect, such as in the case of ascorbate.

The influence of zinc on caspase-3 and DNA breakdown in cultured human retinal pigment epithelial cells.

OBJECTIVES: To investigate the role of extracellular zinc on the death process of cultured human retinal pigment epithelial (RPE) cells. METHODS: Confluent cells on borosilicate glass coverslips were treated with substances in serum-free growth medium for various times and were analyzed for death by means of changes in morphologic features, numbers of attached cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) procedure. Some cultures were also exposed to experimental ischemia (defined as a lack of oxygen, glucose, and serum). Electrophoresis and Western blotting and enzyme assays were used to investigate changes in expression of the protease enzyme, caspase-3. RESULTS: Experimental ischemia caused death of RPE cells. Zinc sulfate had no effect on these cells at low concentrations (100 pmol/L to 10 nmol/L), but protected them at higher concentrations (< or = 10 micromol/L) and appeared to exacerbate cell death at still greater concentrations. Moreover, zinc compounds (>10 micromol/L) also induced death of cells in control cultures that could be blocked by zinc chelators and partially by the caspase-3 inhibitor, DEVD-FMK. Zinc also increased the amount of the active form of caspase-3 in RPE cells. CONCLUSIONS: Zinc salts protect RPE cells from experimental ischemia-induced death at low concentrations (100 pmol/L-10 nmol/L). However, at higher concentrations, zinc causes cell death and alters the cellular level of caspase-3. These observations are consistent with the death process being apoptosis. CLINICAL RELEVANCE: Zinc supplements are taken by many individuals. Low doses of zinc can protect RPE cells against ischemic-type insults as may occur in certain ocular complaints. Furthermore, high concentrations of zinc can damage RPE cells. Because zinc ions are known to be taken up by RPE cells from the choroidal circulation, the actual therapeutic dose taken by patients is critical.

Prevention of glutathione depletion-induced apoptosis in cultured human RPE cells by flupirtine.

We have recently reported that the non-opiate analgesic, flupirtine, counteracts apoptosis in cultures of human retinal pigmented epithelial (RPE) cells induced by deprivation of serum, oxygen and glucose (experimental ischaemia). In the present study, human RPE cells grown on coverslips were treated with buthionine sulphoxamine (BSO), a compound that inhibits glutathione biosynthesis. BSO caused a dose-dependent reduction in culture density and an increase in the number of cell nuclei that were positively labelled by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) procedure. These data show that reduction of glutathione levels causes apoptosis in the RPE cultures. When flupirtine gluconate was co-incubated with BSO, it dose-dependently prevented the induction of apoptosis. The most effective concentration of flupitine found to inhibit cell death caused by BSO (1 micro M - 1 mM) was 100 micro M. The presence of serum (2% or 10%) in the culture medium did not have any effect on the outcome of apoptosis and overall cell death caused by BSO. Futhermore, melatonin, also known to reduce experimental ischaemia-induced overall cell death and apoptosis of cultured RPE cells had only a mild protective effect at 1 mM. The combined data suggest that flupirtine prevents apoptosis by increasing the cellular levels of reduced glutathione and/or protects the cells against the damaging effects of reactive oxygen species (ROS) that are produced subsequent to inhibition of glutathione production.

Induction of apoptosis in cultured human retinal pigmented epithelial cells: the effect of protein kinase C activation and inhibition.

The presence of the non-selective protein kinase C (PKC) inhibitors, staurosporine (100 nM) and polymyxin B (100 microM) in cultured human RPE cells for more than 24 h triggers apoptotic death. Apoptosis is characterized by a diminishing number of cells, a labelling of nuclei by the TUNEL method and by observable morphological changes. An inhibitor of PKC and cyclic nucleotide-dependent protein kinases, 1-(5-isoquinolinesulphonyl)-2-methyl piperazine (H-7; 100 microM), was without effect, as was the specific PKC inhibitor, calphostin C (100 nM). The PKC-activating phorbol esters, phorbol-12-myristate-13-acetate (PMA; 1 microM) and phorbol-12,13-dibutyrate (PDB; 1 microM) and the non-tumour-promoting phorbol ester, 4 alpha-PMA (1 microM) were without effect, as was the diacyl glycerol analogue, 1,2-dioctanoyl-snglycerol (DOG; 10 microM). The PKC activators did not attenuate the apoptosis induced by staurosporine or polymyxin B. Furthermore, deprivation of glucose and oxygen (simulated ischemia) for 72 h induced apoptosis: this could be prevented by inclusion of 10% (v/v) foetal bovine serum (FBS) but not by a variety of PKC activators. Six PKC isoenzymes were shown to be present in RPE cells (alpha, beta 1, beta 2, delta, epsilon, E) and only the calcium-dependent cPKC levels changed after treatment with staurosporine or simulated ischaemia. Since only the less selective inhibitors of PKC induced apoptosis, it is suggested that PKC is not involved directly in the induction process of apoptosis in RPE cells. It is possible that the staurosporine and polymyxin B-induced effects of apoptosis in RPE cells are triggered by an unknown kinase-dependent pathway, but whether the 'ischaemia'-induced death is related to this same process remains to be elucidated.

Retinal protein kinase C.

The protein kinase C (PKC) family of serine/threonine kinase isoenzymes are universally expressed in vertebrate tissues where they control vital cellular functioning. PKC comprises twelve currently identified mammalian isoenzymes, described in three distinct groups according to their need for different effector stimulation. Immunological localisation studies in various vertebrate retinas have indicated the presence, so far, of eight of the PKC subspecies, each with a unique cellular distribution in this tissue. Use of these immunological probing techniques with antibodies raised to the individual PKC family members by immunohistochemistry and western blotting, along with biochemical tools such as the potent activators, the tumour-promoting phorbol esters can hopefully lead to elucidation of the roles of these enzymes in the neural retina. Research work to date has pinpointed a number of roles for PKC in this tissue including control of dopamine release, modulation of glutamate receptor function (probably by a process of direct receptor phosphorylation), phosphorylatory modulation of GABAC-receptor function, an involvement in the retinal ischaemic cascade process (the relevance of which is unknown as yet), involvement in control of cytoskeletal interactions by cytoskeletal element-kinase action and feedback control of enzymes involved in the process of inositol phosphate signalling. PKC has been shown to have an important regulatory role in the process of phototransduction: many of the enzymes and proteins making up the phototransduction cascade act as in vitro and in vivo substrates for PKC-dependent phosphorylation and can have their normal function modified in this way. Also, PKC has been implicated in the control of spinule formation in the retina, a process involved in retinal synaptic plasticity and functioning. All of this work has been described, herein. Collation and utilisation of knowledge of all of the work described here may help us to determine the exact roles for individual isoenzymes in the retina. This in turn may help us to understand and further to prevent pathological conditions leading to inappropriate retinal functioning and possible blindness. Furthermore, understanding the roles of PKC in the neural retina may lead us to vital clues in the understanding of the functioning of this important group of enzymes in the nervous system as a whole and eventually to the prevention of many major neuropathological disorders.

Methamphetamine exacerbates the toxic effect of kainic acid in the adult rat retina.

The recreational use of the psychoactive drug, methamphetamine has increased markedly over the last three decades. It has long been known that this drug has detrimental effects upon the mammalian brain monoaminergic system, but the long- or short-term effects on the retina, a neurological extension of the central nervous system, have received little attention. The aim of this study was, therefore, to determine whether intraocular injection of methamphetamine (MA) is toxic to the healthy adult rat retina and to analyse its effects on the compromised retina after an injection of the ionotropic glutamate receptor agonist, kainate, which is known to cause retinal neuropathology. The equivalent of 1 mM (in the vitreous humour) MA and/or kainate (40 microM) were injected intravitreally. Flash electroretinograms (ERGs) were recorded before and 2 and 4 days after treatment. Five days after treatment, animals were killed and the retinas analysed either for the immunohistochemical localisation of various antigens or for electrophoresis/Western blotting. Some animals were kept for 19 days after treatment and the retinas analysed for tyrosine hydroxylase immunoreactivity. No differences could be found between vehicle- and MA-treated retinas with respect to the nature or localisation of either tyrosine hydroxylase immunoreactivity after 5 or 19 days or other antigens after 5 days. Moreover, the normal ERG and GFAP and calretinin protein antigens were unaffected by MA. Kainate treatment, however, caused a change in the ERGs after 2 and 4 days, an alteration in every antigen localised by immunohistochemistry and an increase in the retinal levels of calretinin and GFAP proteins. Significantly, the changes seen in the b-wave amplitude and implicit time of the ERG after 4 days and the increased level of GFAP protein after 5 days following kainate treatment were enhanced when MA was co-injected. Intravitreal injection of methamphetamine had no detectable detrimental effect on the normal adult rat retina but exacerbated the damaging effects of kainic acid. Such data suggest that a neurotoxic effect of MA may be more obviously illustrated when the tissue is already compromised as occurs in, for example, ischemia.