Modelling Takenouchi-Kosaki syndrome using disease-specific iPSCs.

Takenouchi-Kosaki Syndrome (TKS) is a congenital multi-organ disorder caused by the de novo missense mutation c.191A > G p. Tyr64Cys (Y64C) in the CDC42 gene. We previously elucidated the functional abnormalities and thrombopoietic effects of Y64C using HEK293 and MEG01 cells. In the present study, we used iPSCs derived from TKS patients to model the disease and successfully recapitulated macrothrombocytopenia, a prominent TKS phenotype. The megakaryopoietic differentiation potential of TKS-iPSCs and platelet production capacity were examined using an efficient platelet production method redesigned from existing protocols. The results obtained showed that TKS-iPSCs produced fewer hematopoietic progenitor cells, exhibited defective megakaryopoiesis, and released platelets with an abnormally low count and giant morphology. We herein report the first analysis of TKS-iPSC-derived megakaryocytes and platelets, and currently utilize this model to perform drug evaluations for TKS. Therefore, our simple yet effective differentiation method, which mimics the disease in a dish, is a feasible strategy for studying hematopoiesis and related diseases.

Macrothrombocytopenia of Takenouchi-Kosaki syndrome is ameliorated by CDC42 specific- and lipidation inhibitors in MEG-01 cells.

Macrothrombocytopenia is a common pathology of missense mutations in genes regulating actin dynamics. Takenouchi-Kosaki syndrome (TKS) harboring the c.191A > G, Tyr64Cys (Y64C) variant in Cdc42 exhibits a variety of clinical manifestations, including immunological and hematological anomalies. In the present study, we investigated the functional abnormalities of the Y64C mutant in HEK293 cells and elucidated the mechanism of macrothrombocytopenia, one of the symptoms of TKS patients, by monitoring the production of platelet-like particles (PLP) using MEG-01 cells. We found that the Y64C mutant was concentrated at the membrane compartment due to impaired binding to Rho-GDI and more active than the wild-type. The Y64C mutant also had lower association with its effectors Pak1/2 and N-WASP. Y64C mutant-expressing MEG-01 cells demonstrated short cytoplasmic protrusions with aberrant F-actin and microtubules, and reduced PLP production. This suggested that the Y64C mutant facilitates its activity and membrane localization, resulting in impaired F-actin dynamics for proplatelet extension, which is necessary for platelet production. Furthermore, such dysfunction was ameliorated by either suppression of Cdc42 activity or prenylation using chemical inhibitors. Our study may lead to pharmacological treatments for TKS patients.

The use of endoillumination probe-assisted Descemet membrane endothelial keratoplasty for bullous keratopathy secondary to argon laser iridotomy.

PURPOSE: To report the first case of Descemet membrane endothelial keratoplasty (DMEK) for bullous keratopathy (BK) secondary to argon laser iridotomy (ALI). PATIENT: A 71-year-old woman presented with decreased visual acuity in her right eye due to BK secondary to ALI that was performed 10 years prior. RESULTS: Phacosurgery was performed first, followed by successful DMEK 4 months later. A DMEK shooter was used for donor insertion, which allowed for a stable anterior chamber during donor insertion, even when the anterior chamber was quite shallow. Also, removal of edematous epithelial cells and endoillumination probe-assisted DMEK was quite useful to visualize DMEK graft on the background of the dark brown iris seen in Asian eyes. The patient's best corrected visual acuity rapidly increased from 20/200 to 25/20 after 1 month, with complete resolution of corneal edema. CONCLUSION: We reported the first successful DMEK case for BK secondary to ALI. The use of a DMEK shooter for donor insertion and endoillumination assistance to visualize the DMEK graft was a useful technique for BK secondary to ALI.

Evaluation of internationally shipped prestripped donor tissue for descemet membrane endothelial keratoplasty by vital dye staining.

PURPOSE: The aim of this study was to evaluate endothelial cell damage of internationally shipped prestripped donor tissue for Descemet membrane endothelial keratoplasty (DMEK) using vital dye staining. METHODS: Six internationally shipped prestripped DMEK donors were stained with trypan blue and were subsequently photographed before they were cut with a trephine. Quantitative analysis assessment of endothelial damage of the donor graft area (8.0 mm in diameter) was performed using Adobe Photoshop CS6 Extended software. Seven internationally shipped precut Descemet stripping automated endothelial keratoplasty (DSAEK) donors were used as controls. RESULTS: No statistical differences were noted between prestripped DMEK donors and precut DSAEK donors in mean donor age (67.7 vs. 56.4 years, P = 0.222), mean donor endothelial cell density (2687.3 vs. 2894.6 cells, P = 0.353), and death-to-preservation time (405.3 vs. 558.4 minutes, P = 0.173). However, the mean time of death-to-experiment time in DMEK donors was significantly longer than that of DSAEK donors (8.7 vs. 6.6 days, P = 0.031). Mean endothelial cell damage of prestripped DMEK donors was as low as 0.3%. However, DMEK donor endothelial damage (0.3%) was significantly higher compared with that of precut DSAEK donor tissue (0.01%, P = 0.029). CONCLUSIONS: Although endothelial damage of internationally shipped prestripped donor tissue for DMEK was higher than that of precut DSAEK donor, it was extremely low. Further evaluation using another vital dye and clinical studies may be needed to confirm this study.

Surgical therapies for corneal perforations: 10 years of cases in a tertiary referral hospital.

PURPOSE: To report surgical therapies for corneal perforations in a tertiary referral hospital. METHODS: Thirty-one eyes of 31 patients (aged 62.4±18.3 years) with surgically treated corneal perforations from January 2002 to July 2013 were included in this study. Demographic data such as cause of corneal perforation, surgical procedures, and visual outcomes were retrospectively analyzed. RESULTS: The causes of corneal perforation (n=31) were divided into infectious (n=8, 26%) and noninfectious (n=23, 74%) categories. Infectious causes included fungal ulcer, herpetic stromal necrotizing keratitis, and bacterial ulcer. The causes of noninfectious keratopathy included corneal melting after removal of a metal foreign body, severe dry eye, lagophthalmos, canaliculitis, the oral anticancer drug S-1, keratoconus, rheumatoid arthritis, neurotrophic ulcer, atopic keratoconjunctivitis, and unknown causes. Initial surgical procedures included central large corneal graft (n=17), small corneal graft (n=7), and amniotic membrane transplantation (n=7). In two cases the perforation could not be sealed during the first surgical treatment and required subsequent procedures. All infectious keratitis required central large penetrating keratoplasty to obtain anatomical cure. In contrast, several surgical options were used for the treatment of noninfectious keratitis. After surgical treatment, anatomical cure was obtained in all cases. Mean postoperative best corrected visual acuity was better at 6 months (logMAR 1.3) than preoperatively (logMAR 1.8). CONCLUSION: Surgical therapies for corneal perforations in our hospital included central large lamellar/penetrating keratoplasty, small peripheral patch graft, and amniotic membrane transplantation. All treatments were effective. Corneal perforation due to the oral anticancer drug S-1 is newly reported.

In vivo imaging of coin-shaped lesions in cytomegalovirus corneal endotheliitis by anterior segment optical coherence tomography.

PURPOSE: The aim of this study was to investigate in vivo corneal changes of coin-shaped lesions in cytomegalovirus corneal endotheliitis using anterior segment optical coherence tomography (AS-OCT). METHODS: Two eyes of 2 patients (69- and 71-year-old men), with polymerase chain reaction-proven CMV corneal endotheliitis presenting coin-shaped lesions, were included in this study. AS-OCT examination was performed on the initial visit and at follow-up visits by paying special attention to the coin-shaped lesions. Selected AS-OCT images of the cornea were evaluated qualitatively for changes in the shape and degree of light reflection. RESULTS: In both cases, coin-shaped lesions were observed at the corneal endothelial surface as clusters of fine precipitates using slit-lamp biomicroscopy. Using AS-OCT, high-resolution images of the putative coin-shaped lesions were successfully obtained in both patients as an irregularly thickened highly reflective endothelial cell layer. After anti-CMV treatment, the coin-shaped lesions were resolved as assessed by slit-lamp biomicroscopy and AS-OCT in both patients. CONCLUSIONS: High-resolution AS-OCT provides novel and detailed visual information of coin-shaped lesions in patients with CMV corneal endotheliitis. Visualization of coin-shaped lesions by AS-OCT may be a useful adjunct to the diagnosis and follow-up of CMV corneal endotheliitis.

Endothelial keratoplasty with infant donor tissue.

Here we report a case of endothelial keratoplasty with infant donor tissue obtained after brain death. A 52-year-old man with endothelial dysfunction of unknown cause in the right eye underwent non-Descemet stripping automated endothelial keratoplasty (nDSAEK) with tissue from an infant donor (2 years). Intraoperative and postoperative complications were recorded. Best corrected visual acuity and donor central endothelial cell density were recorded preoperatively and postoperatively. Infant donor tissue preparation with a microkeratome set at 300 µm was successful; the donor tissue was extremely elastic and soft compared with adult tissue. The central endothelial cell density of the infant donor tissue was as high as 4,291 cells/mm(2). No complications were observed during donor tissue (8.0 mm in diameter) insertion with the double-glide technique (Busin glide with intraocular lens sheet glide) or any of the other procedures. Best corrected visual acuity improved from 1.7 logMAR (logarithm of the minimum angle of resolution; 0.02 decimal visual acuity) preoperatively to 0.2 logMAR (0.6) after 6 months and 0.1 logMAR (0.8) after 1 year. The central endothelial cell density after 6 months was 4,098 cells/mm(2) (representing a 4.5% cell loss from preoperative donor cell measurements), and the central endothelial cell density after 1 year was 4,032 cells/mm(2) (6.0% decrease). Infant donor tissue may be preferably used for DSAEK/nDASEK, since it may not be suitable for penetrating keratoplasty or Descemet membrane endothelial keratoplasty.

In vivo imaging of radial keratoneuritis in patients with Acanthamoeba keratitis by anterior-segment optical coherence tomography.

PURPOSE: To investigate in vivo corneal changes of radial keratoneuritis in early-stage Acanthamoeba keratitis (AK) using anterior-segment optical coherence tomography (AS-OCT). DESIGN: Single-center, prospective clinical study. PARTICIPANTS: Four eyes (4 patients with a mean age of 28.5 years) with early-stage AK showing radial keratoneuritis were included in this study. Definitive diagnosis was made by confirmation of AK cysts using in vivo confocal microscopy and culture. METHODS: Anterior-segment OCT examination was performed on the initial visit and at follow-up visits paying special attention to radial keratoneuritis. MAIN OUTCOME MEASURES: Selected AS-OCT images of the cornea were evaluated qualitatively for the shape and degree of light reflection of abnormal neurons. RESULTS: With the use of AS-OCT, we successfully obtained high-resolution images of putative radial keratoneuritis in all patients as highly reflective bands or lines in the corneal stroma. The depth and width of the highly reflective bands/lines varied from case to case (anterior stroma to mid-stroma, from 20 to 200 µm). Some lines ran obliquely from the deep peripheral stroma toward the anterior stroma, and some were located at different depths (subepithelial and mid-stroma) and ran relatively parallel to the corneal layers. After appropriate treatment, radial keratoneuritis was resolved by both slit-lamp biomicroscopy and AS-OCT in all patients. CONCLUSIONS: High-resolution Fourier-domain AS-OCT provides novel and detailed visual information of radial keratoneuritis in patients with early-stage AK. Visualization of radial keratoneuritis by AS-OCT may be a useful adjunct to the diagnosis and follow-up of early-stage AK.

In vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta.

OBJECTIVE: To report the in vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta (OI) with special attention to the abnormality of Bowman's layer and sub-Bowman's fibrous structures (K-structures). PATIENTS AND METHODS: Two patients (67-year-old male and his 26-year-old son) with OI type I were included in this study. Slit lamp biomicroscopic and in vivo laser confocal microscopic examinations were performed for both patients. Central corneal thickness and central endothelial cell density were also measured. RESULTS: Although the corneas looked clear with normal endothelial density for both eyes in both patients, they were quite thin (386 µm oculus dexter (OD) (the right eye) and 384 µm oculus sinister (OS) (the left eye) in the father and 430 µm OD and 425 µm OS in the son). In both patients, slit lamp biomicroscopic and in vivo laser confocal microscopic examination showed similar results. Anterior corneal mosaics produced by rubbing the eyelid under fluorescein were completely absent in both eyes. In vivo laser confocal microscopy revealed an absent or atrophic Bowman's layer; a trace of a presumed Bowman's layer and/or basement membrane was barely visible with high intensity. Additionally, K-structures were completely absent in both eyes. CONCLUSION: The absence of K-structures and fluorescein anterior corneal mosaics strongly suggested an abnormality of Bowman's layer in these OI patients.

Transglutaminase 2-dependent deamidation of glyceraldehyde-3-phosphate dehydrogenase promotes trophoblastic cell fusion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein as well as a classic glycolytic enzyme, and its pleiotropic functions are achieved by various post-translational modifications and the resulting translocations to intracellular compartments. In the present study, GAPDH in the plasma membrane of BeWo choriocarcinoma cells displayed a striking acidic shift in two-dimensional electrophoresis after cell-cell fusion induction by forskolin. This post-translational modification was deamidation of multiple glutaminyl residues, as determined by molecular mass measurement and tandem mass spectrometry of acidic GAPDH isoforms. Transglutaminase (TG) inhibitors prevented this acidic shift and reduced cell fusion. Knockdown of the TG2 gene by short hairpin RNA reproduced these effects of TG inhibitors. Various GAPDH mutants with replacement of different numbers (one to seven) of Gln by Glu were expressed in BeWo cells. These deamidated mutants reversed the suppressive effect of wild-type GAPDH overexpression on cell fusion. Interestingly, the mutants accumulated in the plasma membrane, and this accumulation was increased according to the number of Gln/Glu substitutions. Considering that GAPDH binds F-actin via an electrostatic interaction and that the cytoskeleton is rearranged in trophoblastic cell fusion, TG2-dependent GAPDH deamidation was suggested to participate in actin cytoskeletal remodeling.

Descemet's stripping and non-Descemet's stripping automated endothelial keratoplasty for microcornea using 6.0 mm donor grafts.

BACKGROUND: The purpose of this paper is to report our experience of Descemet's stripping and non-Descemet's stripping automated endothelial keratoplasty (DSAEK/nDSAEK) for microcorneas using 6.0 mm donor grafts. METHODS: Three eyes of two patients (a 56-year-old woman and a 59-year-old woman) with microcornea and suffering from bullous keratopathy were treated with either DSAEK or nDSAEK. A small donor graft (6.0 mm) was inserted into the anterior chamber using a double glide (Busin glide and intraocular lens sheet glide) donor insertion technique. Both patients were followed for at least 12 months. Clinical outcomes, including intraoperative and postoperative complications, visual acuity, and endothelial cell density were evaluated. RESULTS: In all three cases (100%), no intraoperative complications were noted. In one case with a flat keratometry value (32.13 D), a partial donor detachment was noted one day postoperatively, but it was reattached by rebubbling. In another case, rejection was noted 8 months postoperatively, but treatment with systemic corticosteroids was successful. A clear cornea remained in all three cases (100%), with best-corrected visual acuity greater than 20/100 (mean 20/50) at 12 months. Mean postoperative endothelial cell counts were 2,603 ± 18 cells/mm(2) at 6 months (7.4% decrease from preoperative donor cell counts) and 1,799 ± 556 cells/mm(2) at 12 months (36.5% decrease). CONCLUSION: We report for the first time the successful use of a small donor graft (6.0 mm) for DSAEK/nDSAEK in cases of microcornea. Additional stud ies using a large number of patients are required to evaluate fully the potential advantages and drawbacks of small diameter donor grafts for microcornea.

In vivo laser confocal microscopy findings of radial keratoneuritis in patients with early stage Acanthamoeba keratitis.

OBJECTIVE: To investigate in vivo corneal changes of keratoneuritis in early stage Acanthamoeba keratitis (AK) using in vivo laser confocal microscopy. DESIGN: Single-center, prospective, clinical study. PARTICIPANTS: Thirteen eyes (12 patients; 5 men and 7 women; mean age ± standard deviation, 22.3 ± 4.2 years) with keratoneuritis resulting from early stage AK were included in this study. TESTING: In vivo laser confocal microscopy was performed, paying special attention to keratoneuritis. MAIN OUTCOME MEASURES: Selected confocal images of corneal layers were evaluated qualitatively for shape and degree of light reflection of abnormal cells and deposits. RESULTS: In all patients, Acanthamoeba cysts were observed clearly in the basal epithelial cell layer as highly reflective round particles with a diameter of 10 to 20 µm. Bowman's layer infiltration of Acanthamoeba cysts was observed in only 1 case, and no cases showed stromal or nerve infiltration of Acanthamoeba cysts. In the stroma, all cases showed highly reflective activated keratocytes forming a honeycomb pattern; these changes were significant around the keratoneuritis. Infiltration of inflammatory cells, possibly polymorphonuclear cells, was observed along with keratocyte bodies in all cases. Numerous highly reflective spindle-shaped materials were observed around the keratoneuritis. Most notably, highly reflective patchy lesions were observed around the keratoneuritis in 11 cases (84.6%). Inflammatory cells also were observed in the endothelial cell layer in 4 cases (30.8%). CONCLUSIONS: In vivo laser confocal microscopy identified consistent corneal abnormalities around keratoneuritis in early stage AK patients, of which highly reflective patchy lesions may be characteristic of keratoneuritis. Further morphologic studies of corneas with early stage AK in a larger number of patients may elucidate the clinical significance of radial keratoneuritis and may help us to understand the interaction between Acanthamoeba organisms and host corneal cells or nerves.

Clinical features of single and repeated globe rupture after penetrating keratoplasty.

BACKGROUND: In this paper, we report our experience of the clinical features of single and repeated globe rupture after penetrating keratoplasty. METHODS: We undertook a retrospective analysis of single and repeated globe ruptures following keratoplasty in eight eyes from seven consecutive patients referred to Kanazawa University Hospital over a 10-year period from January 2002 to March 2012. We analyzed their ophthalmic and demographic data, including age at time of globe rupture, incidence, time interval between keratoplasty and globe rupture, cause of rupture, complicated ocular damage, and visual outcome after surgical repair. RESULTS: Five patients (71.4%) experienced a single globe rupture and two patients (28.6%) experienced repeated globe ruptures. Patient age at the time of globe rupture was 75.4 ± 6.8 (range 67-83) years. Four of the patients were men and three were women. During the 10-year study period, the incidence of globe rupture following penetrating keratoplasty was 2.8%. The time interval between penetrating keratoplasty and globe rupture was 101 ± 92 months (range 7 months to 23 years). The most common cause of globe rupture in older patients was a fall (n = 5, 79.8 ± 3.7 years, all older than 67 years). Final best-corrected visual acuity was >20/200 in three eyes (37.5%). In all except one eye, globe rupture involved the graft-host junction; in the remaining eye, the rupture occurred after disruption of the extracapsular cataract extraction wound by blunt trauma. CONCLUSION: Preventative measures should be taken to avoid single and repeated ocular trauma following penetrating keratoplasty.

In vivo laser confocal microscopy after Descemet's membrane endothelial keratoplasty.

OBJECTIVE: To investigate the in vivo corneal changes in patients with bullous keratopathy who underwent Descemet's membrane endothelial keratoplasty (DMEK) with the use of in vivo laser confocal microscopy. DESIGN: Single-center, retrospective clinical study. PARTICIPANTS: Five eyes of 4 patients (3 men, 1 women; mean age, 61.3 ± 9.6 years) with bullous keratopathy who had undergone successful DMEK were enrolled in this study. TESTING: In vivo laser confocal microscopy was performed before and 1, 3, and 6 months after DMEK. MAIN OUTCOME MEASURES: Selected confocal images of corneal layers were evaluated qualitatively and quantitatively for the degree of haze and the density of deposits. Subepithelial haze, donor-recipient interface haze, donor-recipient interface particles, and host stromal needle-shaped materials were graded on a scale of 4 categories (grade 0 = none, grade 1 = mild, grade 2 = moderate, grade 3 = severe) at each time point. Time trends of the outcomes were graphically displayed and evaluated with Mantel-Haenszel trend test. RESULTS: The following were observed preoperatively in all patients: slight corneal epithelial edema, moderate subepithelial haze, keratocytes in a honeycomb pattern, and tiny needle-shaped materials in the stroma. After DMEK, moderate subepithelial haze persisted during the follow-up period. Needle-shaped materials had a tendency to decrease after DMEK. Most notably, donor-recipient interface haze and donor-recipient interface particles were barely noticeable after DMEK as early as 1 month postoperatively. CONCLUSIONS: In vivo laser confocal microscopy can identify subclinical corneal abnormalities after DMEK, such as subepithelial haze, host stromal needle-shaped materials, and minimum donor-recipient interface haze/particles. These abnormalities seemed subtle compared with Descemet stripping automated endothelial keratoplasty; this may explain the superior postoperative visual acuity after DMEK. Further studies with this technology in a large number of patients and long-term follow-up are needed to fully understand the long-term corneal changes after DMEK. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Rock-dependent calponin 3 phosphorylation regulates myoblast fusion.

Myogenesis occurs during embryonic development as well as regeneration following postnatal muscle fiber damage. Herein, we show that acidic calponin or calponin 3 (CNN3) regulates both myoblast cell fusion and muscle-specific gene expressions. Overexpression of CNN3 impaired C2C12 cell fusion, whereas CNN3 gene knockdown promoted skeletal myosin expression and fusion. CNN3 was phosphorylated at Ser293/296 in the C-terminal region. The basal inhibitory property of CNN3 against myoblast differentiation was enhanced by Ser293/296Ala mutation or deletion of the C-terminal region, and this inhibition was reversed by Ser293/296Asp mutation. Ser293/296 phosphorylation was required for CNN3 to bind actin and was dependent on Rho-associated kinases 1/2 (ROCK 1/2). Gene knockdown of ROCK1/2 suppressed CNN3 phosphorylation and impaired myoblast fusion, and these effects were partially attenuated by additional CNN3 overexpression of Ser293/296Asp CNN3. These findings indicated that CNN3 phosphorylation by ROCK blunts CNN3's inhibitory effects on muscle cell differentiation and fusion. In muscle tissues, satellite cells, but not mature myofibrils, expressed CNN3. CNN3 was also expressed and phosphorylated during myotube induction in isolated muscle satellite cells. Taken together, these results indicate that CNN3 is a downstream regulator of the ROCK signaling pathway for myogenesis.

Identification of cytomegalovirus and human herpesvirus-6 DNA in a patient with corneal endotheliitis.

PURPOSE: To report the case of a patient with unilateral corneal endotheliitis in which both cytomegalovirus (CMV) and human herpesvirus-6 (HHV6) DNA was identified in the aqueous humor. CASE: A 67-year-old man with corneal endotheliitis OD was referred to us for decreased visual acuity. Local corneal stromal edema, pigmented keratic precipitates, a coin-shaped lesion and minimal anterior chamber reaction were observed by slit-lamp biomicroscopy. Cells with owl's eye appearance in the endothelial cell layer were observed by in vivo laser confocal microscopy. The patient had rheumatoid arthritis, which was treated by oral prednisolone and intravenous abatacept. Polymerase chain reaction analysis of aqueous humor samples detected both CMV and HHV6 DNA, but not other HHVs. Treatment with topical ganciclovir and systemic valganciclovir resulted in a clear cornea. CONCLUSIONS: A patient with corneal endotheliitis had both CMV and HHV6 DNA identified in the aqueous humor. Although both viruses were identified in this case, clinical manifestations resembled CMV corneal endotheliitis, and it was unclear whether HHV6 could affect the clinical course. Systemic abatacept and corticosteroid therapy might play a positive role in cases with both CMV and HHV6 DNA in this corneal endotheliitis.

Ex vivo laser confocal microscopy findings of cultured Acanthamoeba trophozoites.

PURPOSE: The purpose of the current study was to investigate ex vivo laser confocal microscopic findings of cultured Acanthamoeba trophozoites obtained from Acanthamoeba keratitis patients. METHODS: Eight cultured samples of Acanthamoeba trophozoites from eight eyes of seven patients (mean age, 26.9 years; age range, 18-52 years) were used. Seven samples were from corneal scrapings of Acanthamoeba keratitis patients and one sample was from the solution in a soft contact lens case. Ex vivo laser confocal microscopy was performed to qualitatively evaluate the shape and degree of light reflection of the living Acanthamoeba trophozoites. RESULTS: Ex vivo laser confocal microscopy demonstrated highly reflective, high-contrast Acanthamoeba trophozoites with no walls (mean size, 25.4 µm; range, 17.1-58.5 µm). The shapes of the trophozoites were highly pleomorphic, and some showed characteristic acanthopodia by laser confocal microscopy. CONCLUSION: Ex vivo laser confocal microscopy was effective in demonstrating cultured Acanthamoeba trophozoites of various shapes and sizes. The observations of the current study may be helpful when similar structures are identified under in vivo conditions.

Rationale for performing penetrating keratoplasty rather than DSAEK in patients with bullous keratopathy in Japan.

BACKGROUND AND OBJECTIVE: To analyze the rationale for performing penetrating keratoplasty (PK) rather than Descemet's stripping automated endothelial keratoplasty (DSAEK) in patients with bullous keratopathy (BK) in Japan. PATIENTS AND METHODS: A total of 136 eyes of 130 patients with consecutive BK were enrolled. Patients treated by DSAEK were categorized as the DSAEK group. The remaining patients were considered unsuitable for DSAEK due to the presence of risk factors, and were treated by PK (PK group). In both groups, the number of the patients and the causes of BK were analyzed. Also, specifically in the PK group, the reasons for not performing DSAEK were analyzed. RESULTS: The causes of BK differed significantly between the two groups (P < .001). Risk factors considered unsuitable for DSAEK include significant stromal scarring, iris abnormalities, and lens abnormalities. CONCLUSION: For successful DSAEK, risk factors and contraindications should be carefully evaluated before surgery.

Bowman's layer encystment in cases of persistent Acanthamoeba keratitis.

BACKGROUND: The purpose of this study was to report Acanthamoeba encystment in Bowman's layer in Japanese cases of persistent Acanthamoeba keratitis (AK). METHODS: Laser confocal microscopic images of the cornea were obtained in vivo from 18 consecutive eyes from 17 confirmed AK patients. Retrospectively, 14 cases treated over 4 months were categorized as a nonpersistent group and three cases that required prolonged therapy for more than 6 months were categorized as a persistent group. Clinical outcomes based on final best-corrected visual acuity were retrospectively analyzed, and selected confocal images were evaluated qualitatively for abnormal findings. RESULTS: The final best-corrected visual acuity was significantly lower (P < 0.01) for patients in the persistent group compared with that in the nonpersistent group. At the initial visit, in vivo confocal microscopy demonstrated Acanthamoeba cysts exclusively in the epithelial layer in both the nonpersistent group (80%) and the persistent group (100%). At a subsequent follow-up visit, numerous Acanthamoeba cysts were observed in the epithelial cell layer and in Bowman's layer in all patients with persistent AK, but Acanthamoeba cysts were undetectable in all cases with nonpersistent AK tested. CONCLUSION: Invasion of cysts into Bowman's layer was characteristically observed in patients with persistence of AK. This finding suggests that invasion of Acanthamoeba cysts into Bowman's layer may be a useful predictor for a persistent clinical course.

Calponin 3 regulates actin cytoskeleton rearrangement in trophoblastic cell fusion.

Cell-cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted BeWo cell fusion. CNN3 at the cytoplasmic face of cytoskeleton was dislocated from F-actin with forskolin treatment and diffused into the cytoplasm in a phosphorylation-dependent manner. Phosphorylation sites were located at Ser293/296 in the C-terminal region, and deletion of this region or site-specific disruption of Ser293/296 suppressed syncytium formation. These CNN3 mutants were colocalized with F-actin and remained there after forskolin treatment, suggesting that dissociation of CNN3 from F-actin is modulated by the phosphorylation status of the C-terminal region unique to CNN3 in the CNN family proteins. The mutant missing these phosphorylation sites displayed a dominant negative effect on cell fusion, while replacement of Ser293/296 with aspartic acid enhanced syncytium formation. These results indicated that CNN3 regulates actin cytoskeleton rearrangement which is required for the plasma membranes of trophoblasts to become fusion competent.