Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to Lamivudine.

Entecavir (ETV) exhibits potent antiviral activity in patients chronically infected with wild-type or lamivudine (3TC)-resistant (3TC(r)) hepatitis B virus (HBV). Among the patients treated in phase II ETV clinical trials, two patients for whom previous therapies had failed exhibited virologic breakthrough while on ETV. Isolates from these patients (arbitrarily designated patients A and B) were analyzed genotypically for emergent substitutions in HBV reverse transcriptase (RT) and phenotypically for reduced susceptibility in cultures and in HBV polymerase assays. After 54 weeks of 3TC therapy, patient A (AI463901-A) received 0.5 mg of ETV for 52 weeks followed by a combination of ETV and 100 mg of 3TC for 89 weeks. Viral rebound occurred at 133 weeks after ETV was started. The 3TC(r) RT substitutions rtV173L, rtL180M, and rtM204V were present at study entry, and the additional substitutions rtI169T and rtM250V emerged during ETV-3TC combination treatment. Reduced ETV susceptibility in vitro required the rtM250V substitution in addition to the 3TC(r) substitutions. For liver transplant patient B (AI463015-B), previous famciclovir, ganciclovir, foscarnet, and 3TC therapies had failed, and RT changes rtS78S/T, rtV173L, rtL180M, rtT184S, and rtM204V were present at study entry. Viral rebound occurred after 76 weeks of therapy with ETV at 1.0 mg, with the emergence of rtT184G, rtI169T, and rtS202I substitutions within the preexisting 3TC(r) background. Reduced susceptibility in vitro was highest when both the rtT184G and the rtS202I changes were combined with the 3TC(r) substitutions. In summary, infrequent ETV resistance can emerge during prolonged therapy, with selection of additional RT substitutions within a 3TC(r) HBV background, leading to reduced ETV susceptibility and treatment failure.

4-Thiazolidinones: novel inhibitors of the bacterial enzyme MurB.

4-Thiazolidinones were synthesized and evaluated for their ability to inhibit the bacterial enzyme MurB. Selected 4-thiazolidinones displayed activity against the enzyme in vitro. This activity, coupled with the design principles of the thiazolidinones, supports the postulate that 4-thiazolidinones may be recognized as diphosphate mimics by a biological selector.

Comparison of the D-glutamate-adding enzymes from selected gram-positive and gram-negative bacteria.

The biochemical properties of the D-glutamate-adding enzymes (MurD) from Escherichia coli, Haemophilus influenzae, Enterococcus faecalis, and Staphylococcus aureus were investigated to detect any differences in the activity of this enzyme between gram-positive and gram-negative bacteria. The genes (murD) that encode these enzymes were cloned into pMAL-c2 fusion vector and overexpressed as maltose-binding protein-MurD fusion proteins. Each fusion protein was purified to homogeneity by affinity to amylose resin. Proteolytic treatments of the fusion proteins with factor Xa regenerated the individual MurD proteins. It was found that these fusion proteins retain D-glutamate-adding activity and have Km and Vmax values similar to those of the regenerated MurDs, except for the H. influenzae enzyme. Substrate inhibition by UDP-N-acetylmuramyl-L-alanine, the acceptor substrate, was observed at concentrations greater than 15 and 30 microM for E. coli and H. influenzae MurD, respectively. Such substrate inhibition was not observed with the E. faecalis and S. aureus enzymes, up to a substrate concentration of 1 to 2 mM. In addition, the two MurDs of gram-negative origin were shown to require monocations such as NH4+ and/or K+, but not Na+, for optimal activity, while anions such as Cl- and SO4(2-) had no effect on the enzyme activities. The activities of the two MurDs of gram-positive origin, on the other hand, were not affected by any of the ions tested. All four enzymes required Mg2+ for the ligase activity and exhibited optimal activities around pH 8. These differences observed between the gram-positive and gram-negative MurDs indicated that the two gram-negative bacteria may apply a more stringent regulation of cell wall biosynthesis at the early stage of peptidoglycan biosynthesis pathway than do the two gram-positive bacteria. Therefore, the MurD-catalyzed reaction may constitute a fine-tuning step necessary for the gram-negative bacteria to optimally maintain its relatively thin yet essential cell wall structure during all stages of growth.

A new model of proliferative vitreoretinopathy.

PURPOSE: To design a new model of proliferative vitreoretinopathy (PVR) that would not rely on the addition of exogenous cells. The release of endogenous cells from surrounding attachments seems to be an early event in the pathogenesis of PVR. Because the proteolytic enzyme dispase dissociates tissues, the hypothesis was that an intraocular injection of dispase could trigger events that would cause PVR. The requirement for a surgical retinal break at the time of dispase injection was also examined. METHODS: One eye of Dutch Belted rabbits was injected with 0.003 U to 1.0 U dispase in the subretinal space or vitreous cavity. Control rabbits received a saline injection. An intentional retinal tear was created in animals in some groups. Observations were made for at least 10 weeks after surgery. RESULTS: Proliferative vitreoretinopathy developed in response to subretinal or intravitreal dispase, with or without creation of a controlled retinal break. Increased severity of PVR correlated with increasing doses of dispase. Evidence of PVR included preretinal membranes, distortion of myelin wings and retinal vessels, fixed retinal folds, and traction retinal detachment. Proliferative vitreoretinopathy did not develop in saline-treated control animals. CONCLUSIONS: Dispase initiated the development of PVR without the addition of exogenous cells, growth factors, or cytokines typically found in PVR membranes. A cascade of events was probably triggered by dispase, causing native cells and factors to produce PVR. The dispase model of PVR was technically easy to perform, permitted a clear view of the retina, and had a high success rate in development of PVR.

Laser-cured fibrinogen glue to repair bleb leaks in rabbits.

OBJECTIVE: To determine whether laser-cured fibrinogen glue can close bleb leaks in rabbits. METHODS: Full-thickness filtration surgery with intraoperative mitomycin and a sutured limbus-based conjunctival flap was performed in 1 eye each of 19 New Zealand albino rabbits. On the second postoperative day, a 2- to 3-mm hole was made in the bleb. In 9 rabbits, the hole was glued using fibrinogen glue with indocyanine green dye added. The glue was "cured" with a diode laser. Eyes that had been glued and developed a subsequent leak had the glue reapplied on the day the leak was detected. RESULTS: The glue remained on the conjunctiva for an average (mean+/-SD) of 1.9+/-1.8 days (range, 0-5 days). The last day of bleb leak for the rabbits with glued eyes was 1.6+/-2.4 days; for the control rabbits, it was 8.0+/-4.4 days (P=.001, Mann-Whitney U test). CONCLUSION: Laser-cured fibrinogen glue is effective in closing bleb leaks in rabbits.

Pressure measurements during injection of corticosteroids.

Corticosteroid injection into the orbit, eyelid and larynx is a common treatment for inflammation and neoplasm. Complications include embolisation into the ocular circulation resulting in permanent loss of vision. The overall aim of the reported research is to develop an injection cannula and monitoring system which can prevent inadvertent embolisation into the ocular circulation during injection of corticosteroids. To that end, a special cannula was designed that allows simultaneous estimation of pressure at the tip of the cannula and flow rate during injection. The cannula was tested with backpressures corresponding to physiological ranges of 0 to 125 mmHg and injection flow rates of 3 to 11 cm3 min-1. The estimated pressure at the tip of the cannula during injection of corticosteroids was compared with direct pressure measurements. The results show that the mean estimated pressure is linearly related to the mean measured pressure with a slope of 0.99 and correlation coefficient of 0.99. Statistical analyses show that with standard error of estimate (SEE) of 2.14 mmHg, the estimated pressure is well within the 95% prediction interval limits of the measured values. The estimation of pressure from the cannula and monitoring system was accurate and warrants further testing in animal models.

Optic nerve neovascularization in incontinentia pigmenti.

PURPOSE: To describe the presence of optic disk neovascularization in a child with incontinentia pigmenti. METHODS: A 6-month-old infant with incontinentia pigmenti developed optic disk neovascularization in addition to vascular abnormalities in the peripheral retina. Panretinal photocoagulation was performed. RESULT: The optic disk neovascularization regressed after panretinal photocoagulation. CONCLUSIONS: Optic disk neovascularization is another notable retinal manifestation of incontinentia pigmenti. When optic disk neovascularization is detected, retinal ablation should be considered as treatment.

Diagnosis and treatment of an ophthalmic artery occlusion during an intralesional injection of corticosteroid into an eyelid capillary hemangioma.

PURPOSE: To demonstrate the usefulness of simultaneous indirect ophthalmoscopy in the diagnosis and treatment of embolization of the ocular circulation during intralesional injection of corticosteroids into capillary hemangiomas. METHODS: A 4-month-old infant had an ophthalmic artery occlusion during an intralesional injection of corticosteroids into a right upper eyelid capillary hemangioma. The injection was discontinued immediately and a paracentesis was performed. Fluorescein angiography was performed 20 minutes and three weeks after the ophthalmic artery occlusion. RESULTS: Fluorescein angiography after the paracentesis showed delayed retinal and choroidal filling and large areas of retinal and choroidal ischemia. Three weeks after treatment, the angiographic abnormalities had resolved, and the retinal and choroidal circulations were normal. Twenty-eight months after treatment, the visual acuity was 20/20 in each eye. CONCLUSION: Ophthalmic artery occlusion can occur during intralesional injection of corticosteroids into capillary hemangiomas. Simultaneous indirect ophthalmoscopy allows the surgeon to discontinue the injection and provide treatment to allow for the best possible visual outcome after this complication.

Attempted autoenucleation.

PURPOSE: A 24-year-old man had visual acuity of no light perception in the left eye after attempted autoenucleation. METHODS: An urgent lateral canthotomy was performed, followed by treatment with high-dose intravenous corticosteroids. RESULTS: Visual acuity improved to L.E.: 20/30. Visual field testing disclosed recovery of the central visual field with persistent arcuate visual field defects. CONCLUSION: Visual acuity of no light perception after attempted autoenucleation does not preclude the return of good visual acuity.

BMS-182123, a fungal metabolite that inhibits the production of TNF-alpha by macrophages and monocytes.

A fungal metabolite, BMS-182123, which inhibited bacterial endotoxin-induced production of tumor necrosis factor (TNF-alpha) in murine macrophages and human peripheral blood monocytes (in vitro), was isolated from the culture broth of Penicillium chrysogenum strain V39673. The effective BMS-182123 concentration (IC50) resulting in 50% inhibition of lipopolysaccharide-induced TNF-alpha production in murine macrophages and human monocytes was 600 ng/ml and 4.0 microgram/ml, respectively. BMS-182123 suppressed the lipopolysaccharide-induced TNF-alpha promoter activity and did not affect the stability of posttranscriptional mRNA. Addition of hydrophobic resin, Amberlite XAD-8 (1%), to the fermentation enhanced the production of BMS-182123 by 5.5 fold. A total of 577 mg pure BMS-182123 was recovered from a 250-liter fermentation supplemented with 1% Amberlite XAD-8.

Photodynamic therapy of experimental choroidal neovascularization using lipoprotein-delivered benzoporphyrin.

OBJECTIVE: To investigate photodynamic therapy of experimental choroidal neovascularization using benzoporphyrin derivative monoacid (Verteporfin). METHODS: Photodynamic therapy using benzoporphyrin derivative monoacid was investigated in cynomolgus monkeys. Following intravenous injection of benzoporphyrin derivative monoacid (1 to 2 mg/kg) complexed with low-density lipoprotein, the eyes were irradiated with 692-nm light at a fluence of 50 to 150 J/cm2 and irradiance of 150 to 600 mW/cm2. Choroidal neovascularization was documented before photodynamic therapy and closure was demonstrated by fundus photography, fluorescein angiography, and light and electron microscopic examination. RESULTS: Following photodynamic therapy, vessels within choroidal neovascularization were occluded, and there was damage to the choroidal neovascularization endothelium and the subjacent choriocapillaris. Damage to the retinal pigment epithelium and photoreceptors was also observed. CONCLUSION: Photodynamic therapy with lipoprotein-delivered benzoporphyrin derivative monoacid was effective in this animal model of choroidal neovascularization and may be a promising, potentially selective, therapy for choroidal neovascularization.

Erbium:YAG laser surgery of the vitreous and retina.

PURPOSE: These studies evaluated an erbium:YAG laser for transection of vitreous membranes, retinotomy, and incision and ablation of epiretinal membranes. METHODS: Elevated vitreous membranes, detachments, and epiretinal membranes were induced in rabbit eyes. An erbium:YAG laser, emitting at a wavelength of 2.94 microns and equipped with a flexible fiber and endoprobes with tips ranging from 75 to 375 microns, was used to perform vitreous membrane transections, retinotomies, and epiretinal membrane incisions and ablations in nontransmitting aqueous media with the endoprobe in proximity to the tissue. Ablations of epiretinal membranes also were performed in transmitting media, including air and perfluoro-N-octane with the endoprobe elevated above the membrane. RESULTS: Twenty-five vitreous membrane transections were made in 16 eyes at distances ranging from 0.5 to 4.5 mm from the retina with radiant exposures ranging from 2 to 50 J/cm2 with nonhemorrhagic retinal damage in a single transection. Sharp, linear retinotomies were created successfully in five eyes. Epiretinal membrane ablations were performed with radiant exposures ranging from 1.8 to 22.6 J/cm2. In aqueous media, results of microscopic examination showed partial- to full-thickness ablation with a maximum lateral thermal damage of 50 microns. In air- and perfluoro-N-octane-filled eyes, there was increased lateral damage with desiccation of residual tissue. In 12 aqueous-filled eyes, 18 linear incisions were successfully performed, with retinal nonhemorrhagic damage in 2 eyes and hemorrhage in 5. CONCLUSION: The erbium:YAG laser may provide new approaches to maneuvers performed in vitreoretinal surgery.

Experimental transient exudative retinal detachment in the rat.

OBJECTIVE: To establish a new model of exudative retinal detachment in the rat. METHODS: Photothrombosis was produced in a single retinal vein using direct treatment with a dye laser operating in the yellow wavelength (577 nm). Control eyes received identical laser applications, but treatment was placed alongside the vessel and photothrombosis was not produced. Eyes were examined at intervals during the subsequent week with ophthalmoscopy, photography, fluorescein angiography, and light microscopy. RESULTS: In 13 (41%) of 32 eyes with photothrombosis, bullous retinal detachments developed 1 day after laser treatment, and continued occlusion of the vein was confirmed with fluorescein angiography. Detachments persisted for 2 to 4 days and spontaneously resolved; resolution coincided with restored venous patency at 5 to 7 days. None of the 20 control eyes developed bullous detachments (P < .005). CONCLUSIONS: Laser photothrombosis in the rat offers a simple and accessible model of transient exudative retinal detachment without the need for exogenous chromophores.

The early natural history of subfoveal neovascular membranes in eyes with age-related macular degeneration.

During the course of age-related macular degeneration, the visual prognosis is abruptly changed when the presence of a subretinal neovascular membrane (SRNVM) is established by intravenous fluorescein angiography (IVFA). Patients who have been alerted to this condition may notice distortion on the Amsler grid and report within days of the onset of symptoms. Unfortunately most patients either do not notice the early changes in central vision or incorrectly attribute these symptoms to the development of cataracts and defer proper retinal evaluation for several months. We reviewed our records to determine the natural history of those SRNVMs which on initial IVFA either originated or extended into the foveal portion of the macula. Our results clearly indicate that treatment must be undertaken early in the course of this disease to maintain useful visual function for as long as possible.

Treatment of Eales' disease with scatter laser photocoagulation.

Eales' disease is an extremely uncommon vasoproliferative retinal disease predominantly affecting otherwise healthy young men. The etiology of this condition is unknown, but its progression and clinical appearance are similar to other vasoproliferative diseases of the retina such as diabetic and sickle cell retinopathies. These similarities make Eales' disease a good prospect for inclusion in a model pattern of retinal ischemic disease and suggest that treatment by panretinal photocoagulation should be effective in this condition. We had good results in treating eight eyes diagnosed as having Eales' disease with scattered laser photocoagulation.

Characterization and comparative immunoreactivity of antibody to newt (T. cristatus) globins.

Rabbit antisera to newt (T. cristatus) globin were produced by repeated injections of globin and antiglobin antibodies purified by chromatography on globin-Sepharose 4B. Ouchterlony and SDS PAGE analysis indicated that the material eluted from the affinity column was rabbit IgG. The antiglobin antibodies tested by immunodiffusion and ELISA cross-reacted with native hemoglobin and globin from T. cristatus and to varying extents with globins of N. viridescens, R. pipiens and X. laevis, but not with human globin. The degree of cross-reactivity appeared to parallel the evolutionary relatedness of these species, suggesting common antigenic determinants among globins of various vertebrate species.

Iris arteriolar diameters in hypoxia and hyperoxia: a photographic study in albino guinea pigs.

The irides of albino guinea pigs were photographed, and the iris arteriolar diameters were measured with the animal breathing room air, 10% O2/90% N2, or 100% O2. The iris arterioles constrict significantly in hyperoxia and dilate in hypoxia. The average constriction in 100% O2 was 27% +/- 8%, and the average dilatation in 10% oxygen was 34% +/- 20%.