Hemopoietic-specific Sf3b1-K700E knock-in mice display the splicing defect seen in human MDS but develop anemia without ring sideroblasts.

Heterozygous somatic mutations affecting the spliceosome gene SF3B1 drive age-related clonal hematopoiesis, myelodysplastic syndromes (MDS) and other neoplasms. To study their role in such disorders, we generated knock-in mice with hematopoietic-specific expression of Sf3b1-K700E, the commonest type of SF3B1 mutation in MDS. Sf3b1K700E/+ animals had impaired erythropoiesis and progressive anemia without ringed sideroblasts, as well as reduced hematopoietic stem cell numbers and host-repopulating fitness. To understand the molecular basis of these observations, we analyzed global RNA splicing in Sf3b1K700E/+ hematopoietic cells. Aberrant splicing was associated with the usage of cryptic 3' splice and branchpoint sites, as described for human SF3B1 mutants. However, we found a little overlap between aberrantly spliced mRNAs in mouse versus human, suggesting that anemia may be a consequence of globally disrupted splicing. Furthermore, the murine orthologues of genes associated with ring sideroblasts in human MDS, including Abcb7 and Tmem14c, were not aberrantly spliced in Sf3b1K700E/+ mice. Our findings demonstrate that, despite significant differences in affected transcripts, there is overlap in the phenotypes associated with SF3B1-K700E between human and mouse. Future studies should focus on understanding the basis of these similarities and differences as a means of deciphering the consequences of spliceosome gene mutations in MDS.

JAK2V617F mediates resistance to DNA damage-induced apoptosis by modulating FOXO3A localization and Bcl-xL deamidation.

The JAK2V617F mutation is found in most patients with a myeloproliferative neoplasm (MPN). This gain-of-function mutation dysregulates cytokine signaling and is associated with increased accumulation of DNA damage, a process likely to drive disease evolution. JAK2V617F inhibits NHE-1 upregulation in response to DNA damage and consequently represses Bcl-xL deamidation and apoptosis, thus giving rise to inappropriate cell survival. However, the mechanism whereby NHE-1 expression is inhibited by JAK2V617F is unknown. In this study, we demonstrate that the accumulation of reactive oxygen species (ROS) in cells expressing JAK2V617F compromises the NHE-1/Bcl-xL deamidation pathway by repressing NHE-1 upregulation in response to DNA damage. In JAK2V617F-positive cells, increased ROS levels results from aberrant PI3K signaling, which decreases nuclear localization of FOXO3A and decreases catalase expression. Furthermore, when compared with autologous control erythroblasts, clonally derived JAK2V617F-positive erythroblasts from MPN patients displayed increased ROS levels and reduced nuclear FOXO3A. However, in hematopoietic stem cells (HSCs), FOXO3A is largely localized within the nuclei despite the presence of JAK2V617F mutation, suggesting that JAK2-FOXO signaling has a different effect on progenitors compared with stem cells. Inactivation of FOXO proteins and elevation of intracellular ROS are characteristics common to many cancers, and hence these findings are likely to be of relevance beyond the MPN field.

Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.

BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Refractive versus corneal changes after photorefracive keratectomy for astigmatism.

PURPOSE: To define measures of assessing success and subsequent ways to improve excimer laser treatment of astigmatism. METHODS: We studied 97 eyes of 79 patients, followed for 12 months, that underwent photorefractive keratectomy (PRK) for myopia and astigmatism with a VISX 20/20 excimer laser. Preoperative spherical equivalent refraction at the corneal plane was between -1.00 and -15.00 D. Mean preoperative refractive astigmatism at the spectacle plan was -2.17 +/- 1.05 D (range, -1.25 to -6.00 D), which is -1.81 +/- 0.86 D (range -1.04 to -4.97 D) when calculated at the corneal plane. All patients were examined before and after surgery; examination included refraction, keratometry, and topography measurement. RESULTS: The success in treatment of astigmatism appeared measurably less than the treatment of sphere when analogous indices were used for assessment. Success in astigmatism surgery improved, as measured by all parameters, after an additional 20% was applied to astigmatism treatment magnitude indicated by the VISX computer algorithm. The sequential modes of treatment undercorrected astigmatism magnitude to a greater extent than elliptical, but equivalent success rates were present in view of the greater astigmatic changes attempted using the sequential mode. The elliptical mode tended to produce a greater undercorrection of associated sphere (p = 0.313). Results measured by refraction showed a larger change than those measured by topography and keratometry. CONCLUSION: During PRK with the VISX 20/20 laser, adjustment for undercorrection of astigmatism treatment achieves a fuller correction of astigmatism. When measuring astigmatic changes, results are different when comparing refractive astigmatism changes with corneal astigmatism changes measured by keratometry and topography.

Mini-haptics to improve capsular fixation of plate-haptic silicone intraocular lenses.

PURPOSE: To evaluate the effects of a new mini-haptic design on the strength and stability of capsular bag fixation of plate-haptic silicone intraocular lenses (IOLs) and determine whether this design encourages the growth of regenerating lens material or fibrous tissue around the haptic biomaterial and thus improves lens fixation in the capsular bag. SETTING: Center for Research on Ocular Therapeutics and Biodevices, Storm Eye Institute, Medical University of South Carolina, Department of Ophthalmology, Charleston, South Carolina, USA. METHODS: Six rabbits had bilateral continuous curvilinear capsulorhexis, phacoemulsification, and plate-haptic silicone IOL implantation. Each rabbit had a small-hole plate IOL (Chiron C10UB) implanted in the right eye and a mini-haptic plate IOL (Chiron C40UB) in the left eye. All rabbits were killed at 2 months. The force required to extract one haptic from the capsular bag was measured with a digital force gauge. Histopathologic analysis was performed on all specimens. RESULTS: The mini-haptic style IOLs required significantly more extraction force than the small-hole design (P = .011). Histopathologically, proliferating lens epithelial cells were observed growing circumferentially around the mini-haptics, causing a 360 degree synechia formation. This formation did not occur with the conventional small-hole plate IOLs used as the control. CONCLUSIONS: Lens epithelial cell proliferation around the mini-haptics significantly improved capsular bag fixation of the plate-haptic silicone IOL. This should decrease the incidence of clinical decentration and dislocation.

Three multizone photorefractive keratectomy algorithms for myopia. The Melbourne Excimer Laser Group.

OBJECTIVE: To compare the efficacy and complications of three different excimer laser algorithms for multizone photorefractive and photoastigmatic keratectomy. METHODS: Three different software algorithms were applied to treat myopia and myopic astigmatism with the VISX 20/20 excimer laser. Each algorithm had a maximum ablation zone of 6 mm but differed in the number of zones employed, the proportion of the total treatment allocated to each ablation zone, and the treatment of astigmatism. The Melbourne multizone technique equally divided myopia correction into a maximum of three ablation zones. The Pop multizone technique biased myopia treatment into the smaller diameter zones to a maximum of six ablation zones, with one central island pretreatment. The Alpins multizone technique equally divided myopia treatment through all zones up to a maximum of six, with one central island pretreatment. RESULTS: A total of 585 patients (780 eyes) were treated and 625 eyes (80%) were followed for more than 6 months. The mean baseline spherical equivalent refractive error was -5.63 D (-1.00 to -19.50 D). Between 71 and 79% of eyes were treated for astigmatism. There was no statistically significant differences in baseline refractive error or other characteristics among the three groups. At 6 months, the Alpins multizone algorithm had more eyes with a refractive error within +/- 1.00 D of emmetropia (p = 0.01) and more within +/- 2.00 D of emmetropia (p < 0.01). This new algorithm produced more eyes with an uncorrected visual acuity of 20/20 or better at 6 months (p < 0.01). When multiple logistic regression was used to correct for any differences in baseline myopia among the three groups, this algorithm also had a higher odds ratio for achieving 20/20 or better uncorrected visual acuity (OR = 1.58). CONCLUSION: At 6 months, all three algorithms were effective in the reduction of myopia. Significantly better visual acuity and refractive results were achieved with the Alpins multizone algorithm that spread the total treatment over a larger number of ablation zones, with an equal number of diopters of treatment in each zone.

Silicone oil adhesion to intraocular lenses: an experimental study comparing various biomaterials.

PURPOSE: To perform an in vitro experimental study comparing the degree of adherence of silicone oil to various rigid and foldable intraocular lens (IOL) designs and to the human lens capsule. SETTING: Center for Research on Ocular Therapeutics and Biodevices, Department of Ophthalmology, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA. METHODS: Seven IOL styles comprising various biomaterials were studied: fluorine-treated (Fluorlens), heparin-surface-modified (HSM), hydrogel, Memory-Lens, Poly(methyl methacrylate) (PMMA), soft acrylic, and silicone lenses; the human crystalline lens was also studied. Each lens was immersed in silicone oil for 12 hours, than photographed, studied by scanning electron microscopy (except the crystalline lens), and subjected to computer-generated image analysis to determine the silicone oil coverage. RESULTS: Silicone oil coverage of dry silicone lenses was 100% and of lenses immersed in normal saline, 82.5%. The least coverage was on the heparin-surface-modified lens (mean score 9.4%). Coverage of the other four lenses ranged from approximately 15.1% to 33.7%. Mean coverage of the human lens capsule was 10.9%. CONCLUSION: Although a silicone IOL shows maximal adherence to silicone oil, other lens biomaterials are not immune to this complication. Silicone oil coverage was related to the dispersive energy component of the surface charge of the IOL biomaterial. Low dispersive energy materials had less silicone oil coverage, while those with higher dispersive energy had more oil coverage.

Effect of surface photorefractive keratectomy and laser in situ keratomileusis on the corneal endothelium.

PURPOSE: To investigate endothelial cell loss in pairs of fresh human autopsy globes following high-diopter myopic photorefractive keratectomy (PRK) or laser in situ keratomileusis (LASIK). SETTING: Center for Research on Ocular Therapeutics and Biodevices and Magill Laser Center for Vision Correction, Storm Eye Institute, Charleston, South Carolina, USA. METHODS: In the first part of the study, 12 globes had either -10 diopters (D) multizone surface PRK or -10 D single-zone LASIK. In the second part, three groups of 5 globes each had -15 D, -20 D, or -25 D multizone-blend LASIK procedures. Fellow globes in both groups were used as untreated controls. Corneoscleral buttons were excised from all globes. Following 7 days in corneal organ culture, the endothelial surface was stained with two vital dyes: calcein-AM and ethidium homodimer. Fluorescence microscopy was used to obtain endothelial cell counts. RESULTS: The mean dead cells per square millimeter (cells/mm2) were 0.94 in the -10 D PRK treated corneas compared with 0.91 in the fellow untreated eyes (P = 0.06(. The mean dead cells/mm2 in the -10 D single-zone LASIK-treated corneas and in the fellow untreated eyes were 0.61 (P = 0.88). The mean dead cells/mm2 in the -15 D, -20 D, and -25 D multizone-blend LASIK-treated corneas were 3.08, 2.33, and 5.55, respectively, compared with 3.49, 1.92, and 5.01 in the fellow untreated eyes (P = 0.276, P = 0.339, and P = 0.427, respectively). Dead cell counts for treated and control paired corneas were highly correlated in all treatment groups. CONCLUSIONS: No significant endothelial cell loss occurred after -10 D PRK or LASIK corrections up to -25 D. Although this study has limitations that prevent direct extrapolation to the clinical situation, it does afford a comparable clinical correlate for endothelial cell toxicity following a typical excimer laser ablations.

Security of capsular fixation: small-versus large-hole plate-hepatic lenses.

PURPOSE: To assess the effect of relatively large positioning holes on the security of capsular bag fixation of plate-haptic silicone intraocular lenses (IOLs). SETTING: Center for Research on Ocular Therapeutics and Biodevices, Department of Ophthalmology, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA. METHODS: This study tested the hypothesis that larger holes allow ingrowth of lens material, fibrous tissue, or both through them, which helps fixate the lens more firmly in the capsular bag. Five rabbits had bilateral continuous curvilinear capsulorhexis, phacoemulsification, and implantation of a plate-haptic silicone IOL. An IOL with a small, round positioning hole (Staar AA-4203V) was implanted in the right eye in each rabbit, and a large-hole IOL (Staar AA-4203VF) was implanted in the left eye. After 2 months, all rabbits were killed. The force required to extract one haptic from the capsular bag was measured with a digital force meter. All eyes had histopathological analysis. RESULTS: It was slightly more difficult to extract a large-hole IOL from the capsular bag, although this trend was not statistically significant. However, histopathological analysis consistently showed 360 degree synechia formation through the holes, showing that the IOL could be securely fixed in position. CONCLUSIONS: Proliferation of lens epithelial cells through a large positioning hole in a plate-haptic silicone IOL may improve the long-term security of capsular bag fixation. This will help reduce the incidence of IOL decentration and dislocation.

Irreversible silicone oil adhesion to silicone intraocular lenses. A clinicopathologic analysis.

PURPOSE: To report a newly defined complication of foldable intraocular lenses (IOLs), namely silicone oil-silicone IOL interaction. This is a complication not generally seen by the implanting cataract surgeon but, rather, at a later stage in a patient's postoperative course, by a vitreoretinal surgeon. METHODS: Three clinical case histories, including two explanted silicone IOLs, were submitted for analysis. The submitted silicone lenses were photographed under water, and the nature of the silicone oil coating was documented. RESULTS: In each instance, the silicone coating was manifest as a thick coating with droplet formation on the lens surface that was tenaciously adherent and could not be dislodged by instruments or injection of viscoelastics. CONCLUSION: The use of silicone IOLs in patients with current vitreoretinal disease or those who are at high risk for future vitreoretinal disease that may require silicone oil as part of the therapy should be reconsidered. The authors recommend that information regarding the existence and significance of this complication be printed on all silicone oil and silicone IOL packages and inserts (if not as a warning, at least as an informative comment regarding the existence of this condition). This is a rare but clinically significant complication that will affect the occasional patient treated with both of these modalities.

Endophthalmitis in Auckland 1983-1991.

A review was made of 58 cases of suspected endophthalmitis presenting to Auckland Public Hospital between January 1983 and July 1991 (103 months). Thirty culture positive cases were found comprising 13 early onset postoperative, six delayed onset postoperative, five bleb-associated, four post-traumatic and two endogenous cases. The most frequent organism grown in postoperative endophthalmitis was Staphylococcus epidermidis. No conclusion could be reached as to the effectiveness of early versus delayed vitrectomy. Endophthalmitis due to S. epidermidis usually had a good prognosis while streptococcal endophthalmitis had a poor prognosis. Delayed-onset postoperative endophthalmitis most often had a good prognosis while bleb-associated endophthalmitis had a uniformly poor visual outcome. Two isolates responsible for cases of delayed-onset postoperative endophthalmitis, Propionibacterium acnes and Rhodococcus species, did not grow on agar plates until the seventh day of culture. Significant bacterial resistance to cephazolin was identified with 33% of Gram-positive and 75% of Gram-negative isolates resistant. Prevention of postoperative endophthalmitis is discussed.

Trial of a disc lens for intercapsular cataract extraction.

The preliminary results are presented of a prospective trial of a new 'disc' intraocular lens. (Pharmacia Model CD801A). Forty-four lenses were implanted into the capsular bag using an intercapsular cataract extraction technique. The lens is a one-piece all-polymethylmethacrylate (PMMA) lens with a 6 mm biconvex optic. The optic sits within a 9 mm diameter flexible PMMA ring. Two haptic stalks link this flexible haptic ring to the optic. The lens was assessed for its ease of insertion, its rate of decentration, the incidence of posterior capsular opacity and its effects on pupillary function. Results of the trial at three months follow-up are presented. They show that the lens is easy to insert into the capsular bag even through small pupils. It is very easy to dial, placing no stress on the bag when doing so. The incidence of decentration of 1 mm or greater was 5%, and the incidence of posterior capsular fibrosis at three months was 2%. As predicted, the lens sits well back in the eye, clear of the pupil.