Dynamics of Corneal Swelling With Hypoosmolar Riboflavin After Induction During Corneal Collagen Crosslinking in Patients With Progressive Keratoconus.

PURPOSE: To evaluate the rate of corneal swelling induced by hypoosmolar riboflavin in patients with progressive keratoconus (KCN) with corneal thickness <400 µm after the induction phase using riboflavin with 20% dextran during epithelium-off corneal crosslinking (CXL). METHODS: Prospective, nonrandomized, single-center consecutive case series. Preoperative assessments included tomography, specular microscopy, and hysteresis. After epithelial debridement, riboflavin with 20% dextran (Photrexa Viscous; Glaukos, Burlington, MA) was applied at 2-min intervals during a 30-min induction phase. Eyes that dehydrated to a minimum corneal thickness (MCT) of <400 µm after induction (postinduction pachymetry) were recruited. Hypoosmolar riboflavin 0.146% (Photrexa; Glaukos) was used every 10 s to induce stromal swelling, with pachymetry performed every 30 s until the MCT was ≥400 µm (postswelling pachymetry). Corneal swelling rate was compared with variables using regression analysis. RESULTS: In 31 eyes of 31 patients, mean postinduction pachymetry was 338.4 ± 28.7 µm. Hypoosmolar riboflavin induced a postswelling pachymetry of 413.4 ± 15.0 µm over a mean of 5.2 ± 3.2 min, and the average stromal swelling rate was 10.3 ± 8.7 µm/30 s. All eyes reached a postswelling pachymetry MCT ≥400 µm and no cases were aborted. Eyes with highly severe KCN (Kmax >70 and Belin/Ambrosio enhanced ectasia display final D score >17) experienced quicker swelling (14.4 ± 12.8 µm/30 s and 14.9 ± 12.4 µm/30 s, respectively; P < 0.05 for both). A thicker postinduction pachymetry was moderately associated with a faster rate of swelling (rs = 0.389; P = 0.030). CONCLUSIONS: Hypoosmolar riboflavin 0.146% can be safely employed in thinner corneas, allowing for swelling to ≥400 µm for epithelium-off CXL. Associations between swelling rate, KCN severity, and postinduction pachymetry were determined, allowing for a more accurate prediction of procedure time during CXL.

Oxygen-supplemented and topography-guided epithelium-on corneal crosslinking with pulsed irradiation for progressive keratoconus.

PURPOSE: To investigate the effects of customized topography-guided epithelium-on crosslinking (epi-on CXL) with oxygen supplementation on procedural efficacy and corrected distance visual acuity (CDVA) in patients with progressive keratoconus (KC) at 1 year. SETTING: Private eye clinic, Brisbane, Australia. DESIGN: Retrospective, single-center, nonrandomized case series. METHODS: Topography-guided epi-on CXL using the Mosaic system was performed on patients with progressive KC. Oxygen goggles; transepithelial riboflavin; and pulsed, high UV-A irradiance (1 second on, 1 second off; 30 mW/cm2) were applied to enhance oxygen kinetics and bioavailabilities of riboflavin and UV-A. Guided by baseline topography, a higher UV-A dose (15 J/cm2) was applied to the area of steepest anterior curvature with decreasing fluence (as low as 7.2 J/cm2) toward the outer 9 mm. Postoperative CDVA and maximum keratometry (Kmax) were evaluated. RESULTS: 102 eyes (80 patients) were followed for 11.5 ± 4.8 months. At the latest follow-up, mean CDVA (logMAR), mean K, and Kmax (diopters [D]) improved from 0.18 ± 0.28, 46.2 ± 3.8, and 53.0 ± 5.67 at baseline to 0.07 ± 0.18, 45.8 ± 3.7, and 51.9 ± 5.56, respectively (P < .001). 3 eyes (3%) lost more than 1 CDVA line, and another 3 eyes (3%) had increased Kmax greater than 2 D. 43 eyes were followed for at least 12 months (n = 43): mean CDVA, mean K, and Kmax improved from 0.19 ± 0.33 logMAR, 46.5 ± 3.5 D, and 53.6 ± 5.67 D to 0.07 ± 0.17 logMAR, 46.0 ± 3.5 D, and 52.33 ± 5.49 D, respectively (P ≤ .002). No complications were observed. CONCLUSIONS: Tailoring oxygen-supplemented epi-on CXL with differential UV-A energy distributions, guided by baseline topography, in patients with KC seems to be safe and effective. At 1 year, study reports sustained improved CDVA and corneal stabilization.

Oxygen-supplemented transepithelial-accelerated corneal crosslinking with pulsed irradiation for progressive keratoconus: 1 year outcomes.

PURPOSE: To investigate the effects of combining oxygen supplementation with enhanced UV-A light and increased riboflavin permeability in improving the efficacy of epithelium-on crosslinking (epi-on CXL). SETTING: Private eye clinic in Brisbane, Queensland, Australia. DESIGN: Retrospective single-center nonrandomized uncontrolled longitudinal cohort case series. METHODS: Transepithelial CXL was performed on keratoconic eyes. Applications of an oxygen goggle and pulsed UV-A irradiation (1 second on, 1 second off) were used to enhance oxygen kinetics during epi-on CXL. Additional procedural modifications included the use of benzalkonium chloride and high UV-A irradiance level (30 mW/cm 2 ) to improve the stromal bioavailability of riboflavin and UV-A. The main efficacy outcomes were the changes in mean corrected distance visual acuity (CDVA) and safety over 12 months. Additional refractive and keratometry (K) outcomes were also observed. RESULTS: 53 eyes (38 patients) were included in this study. 12 months postoperatively, mean CDVA improved from a mean of 0.18 ± 0.2 at baseline to 0.07 ± 0.1 logMAR ( P < .0001). No statistically significant change was observed in maximum K (Kmax) and mean K, which were respectively 51.7 ± 5.8 diopters (D) and 46.4 ± 3.85 D at baseline and 51.2 ± 5.7 D ( P = .152) and 46.0 ± 3.84 D ( P = .06) 12 months postoperatively. Only 3 eyes experienced an increase of more than 2 D in Kmax; however, none of these eyes experienced a CDVA loss. There were no reported infections, corneal scarring, or other severe adverse effects. CONCLUSIONS: Performing supplemental oxygen epi-on CXL with accelerated, pulsed UV-A irradiation in conjunction with riboflavin permeability enhancers resulted in improved CDVA ( P < .0001) and stable keratometry up to 12 months postoperatively with a good safety profile.

Corneal collagen cross-linking: a review of 1-year outcomes.

PURPOSE: To review outcomes of corneal collagen cross-linking (CXL) for keratoconus (KC) or ectasia in a cornea subspecialty practice. METHODS: Results from controlled clinical trials at a single site cornea subspecialty practice, including 104 eyes (66 KC and 38 ectasia). Outcomes and the natural course of changes in postoperative parameters including maximum keratometry (KMax), uncorrected visual acuity (UCVA), and best-corrected visual acuity (BCVA) over 12 months are reviewed. In addition, corneal topography indices, wavefront higher-order aberrations, and the natural history of wound healing after CXL are discussed. Characteristics associated with CXL outcomes are reviewed as well. In predicting treatment outcomes for KMax and BCVA, the preoperative patient characteristics examined were gender, age, disease group, cone location, thinnest pachymetry, UCVA, BCVA, and KMax. RESULTS: At 1 year, an average of 1.7 diopter (D) flattening in KMax was found. Mean BCVA improved slightly more than 1 line (from 0.35±0.24 to 0.23±0.21 logMAR). All postoperative parameters similarly follow a trend of worsening between baseline and 1 month, and improvement thereafter. More specifically, quantitative improvements are typically seen at 3 months and may continue between 3 and 12 months. A review of baseline patient characteristics indicated that (1) eyes with preoperative KMax of 55 D or steeper were 5.4 times more likely to gain 2 D or more of KMax flattening at 1 year after CXL, and (2) eyes with preoperative BCVA of 20/40 or worse were 5.9 times more likely to gain 2 or more Snellen lines at 1 year after CXL. Conversely, no baseline characteristic was found to correlate with treatment complications of continual topographic steepening or loss of vision. CONCLUSIONS: Corneal collagen cross-linking seems to be effective in decreasing progression of KC, with improvements in optical measures in many patients. Postoperative parameters discussed within this review followed a seemingly reproducible trend in their natural course over 12 months. Generally, the trend observed was immediate worsening between baseline and 1 month, resolution at approximately 3 months, and improvement thereafter. In predicting outcomes after CXL, no patient characteristics showed correlations with negative treatment outcomes such as loss of vision or continual topographic steepening. However, steeper KMax (≥55 D) and poorer BCVA (≤20/40) at the time of treatment correlated with better postoperative KMax and BCVA outcomes at 1 year, respectively. These outcome predictors should be considered when offering CXL to patients with KC or postoperative corneal ectasia.

High-dose cyclophosphamide, BCNU, and VP-16 (CBV) conditioning before allogeneic stem cell transplantation for patients with non-Hodgkin's lymphoma.

Allogeneic stem cell transplantation (SCT) has been shown to be a curative therapy for some patients with non-Hodgkin's lymphoma (NHL). Total-body irradiation and high-dose cyclophosphamide combinations are the most established conditioning regimens used in this setting. We examined the efficacy and toxicity of cyclophosphamide, BCNU, and VP-16 (CBV) as a suitable chemotherapy-only regimen for NHL patients. In total, 18 patients, median age 42 years, with NHL were treated with CBV followed by allotransplant. Patients had received a median of two prior chemotherapy regimens. Median times to neutrophil and platelet recovery were 19 and 15 days, respectively. Interstitial pneumonitis occurred in one patient. There have been four relapses after a median follow-up of 39 months. Overall, there were four deaths, one because of relapse. The 2-year estimates of relapse-free and overall survival are 56 and 76%, respectively. CBV is a safe and an effective alternative to TBI-containing regimens before allogeneic SCT for NHL.

Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas.

Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.

Identification and immunolocalization of actin cytoskeletal components in light- and dark-adapted octopus retinas.

Photoreceptors in the octopus retina are of the rhabdomeric type, with rhabdomeres arising from the plasma membrane on opposite sides of the cylindrical outer segment. Each rhabdomere microvillus has an actin filament core, but other actin-binding proteins have not been identified. We used immunoblotting techniques to identify actin-binding proteins in octopus retinal extracts and immunofluorescence microscopy to localize the same proteins in fixed tissue. Antibodies directed against alpha-actinin and vinculin recognized single protein bands on immunoblots of octopus retinal extract with molecular weights comparable to the same proteins in other tissues. Anti-filamin identified two closely spaced bands similar in molecular weight to filamin in other species. Antibodies to the larger of the Drosophila ninaC gene products, p174, identified two bands lower in molecular weight than p174. Anti-villin localized a band that was significantly less in molecular weight than villin found in other cells. Epifluorescence and confocal microscopy were used to map the location of the same actin-binding proteins in dark- and light-adapted octopus photoreceptors and other retinal cells. Antibodies to most of the actin-binding proteins showed heavy staining of the photoreceptor proximal/supportive cell region accompanied by rhabdom membrane and rhabdom tip staining, although subtle differences were detected with individual antibodies. In dark-adapted retinas anti-alpha-actinin stained the photoreceptor proximal/supportive cell region where an extensive junctional complex joins these two cell types, but in the light, immunoreactivity extended above the junctional complex into the rhabdom bases. Most antibodies densely stained the rhabdom tips but anti-villin exhibited a striated pattern of localization at the tips. We believe that the actin-binding proteins identified in the octopus retina may play a significant role in the formation of new rhabdomere microvilli in the dark. We speculate that these proteins and actin remain associated with an avillar membrane that connects opposing sets of rhabdomeres in light-adapted retinas. Association of these cytoskeletal proteins with the avillar membrane would constitute a pool of proteins that could be recruited for rapid microvillus formation from the previously avillar region.

Thiol redox modulation of doxorubicin mediated cytotoxicity in cultured AIDS-related Kaposi's sarcoma cells.

The chemotherapeutic, doxorubicin, is currently used empirically in the treatment of AIDS- related Kaposi's sarcoma (AIDS-KS). Although often employed in a chemotherapeutic cocktail (doxorubicin, bleomycin, vincristine) single-agent therapy has recently been attempted with liposome encapsulated doxorubicin. Although doxorubicin's mechanism of action against AIDS-KS is unknown, we hypothesized that doxorubicin's ability to undergo redox cycling is associated with its clinical efficacy. The current study was conducted to investigate the effects of doxorubicin on selected xenobiotic-associated biochemical responses of three cellular populations: KS lesional cells, nonlesional cells from the KS donors, and fibroblasts obtained from HIV- aged matched men. Our results show that during doxorubicin challenge, there are strong positive correlations between cellular glutathione (GSH) levels and viability (r = 0.94), NADPH levels and viability (r = 0.93), and GSH and NADPH levels (r = 0.93), and demonstrate that as a consequence of their abilities to maintain cellular thiol redox pools HIV- donor cells are significantly less susceptible to doxorubicin's cytotoxic effects relative to AIDS-KS cells. Additional studies further supported the contribution of reduced thiols in mediating doxorubicin tolerance. While pretreatment with the GSH precursor, N-acetylcysteine was cytoprotective for all cell groups during doxorubicin challenge, GSH depletion markedly enhanced doxorubicin's cytotoxic effects. Studies to investigate the effects of a hydroxyl scavenger and iron chelator during doxorubicin challenge showed moderate cytoprotection in the AIDS-KS cells but deleterious effects in the HIV control cells. Inactivation of the longer lived membrane generated ROI in the cytoprotective deficient AIDS-KS cells, as well as an impairment of endogenous defenses in the HIV- donor control cells, may account for these scavenger and chelator associated findings. In summary, our findings show that doxorubicin mediates, at least in part, its AIDS-KS cellular cytotoxic effects by a redox related mechanism, and provides a biochemical rationale for doxorubicin's clinical efficacy in AIDS-KS treatment.

Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphorylation in response to colony-stimulating factor 1/c-fms signaling.

An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate-early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1-stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.

Thiol redox modulation of tumor necrosis factor-alpha responsiveness in cultured AIDS-related Kaposi's sarcoma cells.

Both clinical and experimental evidence indicates that AIDS-related Kaposi's sarcoma (AIDS-KS) has a multifactorial pathogenesis with factors such as HIV viral load, latent virus induction, and opportunistic infections contributing to disease progression. However, a consistent feature that unites these apparently diverse putative etiologic agents is sustained serum elevations of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). While virtually every cell responds to TNF-alpha with gene activation, the extent of TNF-alpha-mediated cellular signaling is regulated by a delicate balance between signal activation and signal arresting events. Reactive oxygen intermediates (ROI), which are generated as a consequence of TNF-alpha membrane interaction, are part of this TNF-alpha-initiated cellular activation cascade. Previous studies in our laboratory have shown that AIDS-KS cells possess impaired oxygen intermediate scavenging capacities, thereby establishing conditions permissive for the intracellular retention of ROI. In this study, we used cellular capacity to upregulate the cytoprotective enzyme superoxide dismutase (SOD) to address the extent of cellular response to TNF-alpha. Concurrent with the SOD analyses, nucleotide profiles were obtained to assess cellular bioenergetic responses during TNF-alpha challenge. Proliferative growth levels of mitochondrial (Mn)SOD activities showed an activity spectrum ranging from lowest activity in AIDS-KS cells, to intermediate levels in matched, nonlesional cells from the AIDS-KS donors, to highest activities in HIV normal fibroblasts. In contrast, following TNF-alpha challenge, the AIDS-KS and KS donor nonlesional cells showed a 11.89- and 5.86-fold respective increase in MnSOD activity, while the normal fibroblasts demonstrated a 1.35-fold decrease. Subsequent thiol redox modulation studies showed that only the normal fibroblast cultures showed a potentiation of TNF-alpha-mediated MnSOD upregulation following GSH depletion. In addition, provision of the GSH precursor, N-acetylcysteine during TNF-alpha challenge only diminished MnSOD activity and mitochondrial compartmentalization in the AIDS-KS cells, a finding that likely reflects the lower levels of reduced thiols in this cellular population. Our data, which show that a perturbation in their cellular thiol redox status accentuates AIDS-KS cellular responsiveness to TNF-alpha, suggest a biochemical rationale for the recognized TNF-alpha AIDS-KS clinical correlation.

The chemical stability and sterility of sodium thiopental after preparation.

UNLABELLED: Manufacturer's instructions recommend discarding unused portions of sodium thiopental 24 h after reconstitution. Heeding this recommendation may result in the disposal of a large proportion of prepared thiopental. Although thiopental is relatively inexpensive, the volume prepared by many anesthesia departments could make this waste significant. To address this possibility, we investigated the chemical stability and sterility of thiopental in pharmacy-prepared, prefilled syringes. Stock solutions of thiopental were mixed and drawn into syringes under sterile conditions by pharmacists or pharmacy assistants. Fifty-six samples were stored under refrigeration (3 degrees C); the remaining 56 samples were stored at room temperature (22 degrees C). Each day for 7 days, eight samples from each group were analyzed by using high-performance liquid chromatography for chemical stability and cultured for microbiological colonization. Differences in thiopental concentration between the room temperature and the refrigerated samples were measured over time by using repeated-measures analysis of variance (P < or = 0.05). Three positive culture samples (S. epidermidis and S. hemolyticus) most likely represent laboratory contamination and not colonization. At 22 degrees C, thiopental remains stable and sterile for 6 days and well beyond 7 days at 3 degrees C. IMPLICATIONS: This study examines the shelf life of the anesthetic drug thiopental in pharmacy-filled syringes stored at either room temperature or under refrigeration. The results justify the use of prepared solutions beyond the package insert recommendation of 24 h.

Changes in body temperature after administration of antipyretics, LSD, delta 9-THC, CNS depressants and stimulants, hormones, inorganic ions, gases, 2,4-DNP and miscellaneous agents.

This survey concludes a series of complications of data from the literature, primarily published since 1965, on thermoregulatory effects of antipyretics in afebrile as well as in febrile subjects, LSD and other hallucinogens, cannabinoids, general CNS depressants, CNS stimulants including xanthines, hormones, inorganic ions, gases and fumes, 2,4-dinitrophenol and miscellaneous agents including capsaicin, cardiac glycosides, chemotherapeutic agents, cinchona alkaloids, cyclic nucleotides, cycloheximide, 2-deoxy-D-glucose, dimethylsulfoxide, insecticides, local anesthetics, poly I:poly C, spermidine and spermine, sugars, toxins and transport inhibitors. The information listed includes the species used, route of administration and dose of drug, the environmental temperature at which the experiments were performed, the number of tests, the direction and magnitude of body temperature change and remarks on the presence of special conditions such as age or lesions, or on the influence of other drugs, such as antagonists, on the response to the primary agents.

Changes in body temperature after administration of acetylcholine, histamine, morphine, prostaglandins and related agents.

This survey, the second in a series, presents extensive tabulations of literature, primarily since 1965, on thermoregulatory effects of cholinergic agonists and antagonists, histamine and H1- and H2-receptor antagonists, narcotic analgesics and antagonists in both non-tolerant and tolerant subjects and of prostaglandins and related agents. The information listed includes the species used, route of administration and dose of drug, the environmental temperature at which the experiments were performed, the number of tests, the direction and magnitude of body temperature change and remarks on the presence of special conditions, such as age or lesions, or on the influence of other drugs, such as antagonists, on the response to the primary drug.

Changes in body temperature after administration of adrenergic and serotonergic agents and related drugs including antidepressants.

This survey, the third in a series, presents extensive tabulations of literature, primarily since 1965, on thermoregulatory effects of adrenergic and serotonergic agonists and their antagonists including ergot alkaloids, amphetamines, tryptamines, monoamine oxidase inhibitors, tricyclic and other antidepressants, a variety of other agents which alter presynaptic aminergic mechanisms including reserpine, 6-hydroxydopamine, p-chlorophenylalanine, alpha-methyltyrosines, cocaine, guanethidine and bretylium. The information listed includes the species used, route of administration and dose of drug, the environmental temperature at which the experiments were performed, the number of tests, the direction and magnitude of body temperature change and remarks on the presence of special conditions, such as age or lesions, or on the influence of other drugs, such as antagonists, on the response to the primary drug.