Characterization of mesenchymal progenitor cell populations from non-epithelial oral mucosa.

OBJECTIVES: The characteristics of cell populations extracted from oral mucosal non-epithelial tissues and their ability to differentiate were evaluated in vitro as a potential source of cells for mandibular and corneal regeneration. MATERIALS AND METHODS: Oral mucosal non-epithelial cells (OMNECs) were extracted from tissue samples and were studied by flow cytometry and RT-PCR. Cells differentiating into osteoblasts, adipocytes, chondrocytes, neurocytes, or keratocytes were characterized by RT-PCR and cell staining. RESULTS: OMNECs expressed CD44, CD90, CD105, CD166, and STRO-1 antigens, which are markers for mesenchymal stem cells. In addition, Oct3/4, c-Myc, Nanog, KLF4, and Rex, which are expressed by embryonic or pluripotent stem cells, were detected by RT-PCR. Expression of CD49d, CD56, and PDGFRα, proteins closely associated with the neural crest, was observed in OMNECs, as was expression of Twist1, Sox9, Snail1 and Snail2, which are early neural crest and neural markers. Specific differentiation markers were expressed in OMNECs after differentiation into osteoblasts, adipocytes, chondrocytes, or keratocytes. CONCLUSIONS: Populations of OMNECs may contain both mesenchymal stem cells and neural crest origin cells and are a potential cell source for autologous regeneration of mandibular or corneal stroma.

Phase I/II trial of definitive carbon ion radiotherapy for prostate cancer: evaluation of shortening of treatment period to 3 weeks.

BACKGROUND: The purpose of this study was to evaluate the feasibility of a new shortened 3-week treatment schedule of carbon ion radiotherapy (CIRT) for prostate cancer. METHODS: Beginning in May 2010, patients with T1b-T3bN0M0, histologically proven prostate adenocarcinoma were enrolled in the phase II trial of CIRT. Patients received 51.6 GyE in 12 fractions over 3 weeks (protocol 1002). The primary end point was defined as the incidence of late adverse events that were evaluated based on the Common Terminology Criteria for Adverse Events version 4.0. Biochemical failure was determined using the Phoenix definition (nadir +2.0 ng ml(-1)). RESULTS: Forty-six patients were enrolled, and all patients were included in the analysis. The number of low-, intermediate-, and high-risk patients was 12 (26%), 9 (20%), and 25 (54%), respectively. The median follow-up period of surviving patients was 32.3 months. Two patients had intercurrent death without recurrence, and the remaining 44 patients were alive at the time of this analysis. In the analysis of late toxicities, grade 1 (G1) rectal haemorrhage was observed in 3 (7%) patients. The incidence of G1 haematuria was observed in 6 (13%) patients, and G1 urinary frequency was observed in 17 (37%) patients. No ⩾G2 late toxicities were observed. In the analysis of acute toxicities, 2 (4%) patients showed G2 urinary frequency, and no other G2 acute toxicities were observed. CONCLUSIONS: The new shortened CIRT schedule over 3 weeks was considered as feasible. The analysis of long-term outcome is warranted.

Visual outcome in Japanese patients with Acanthamoeba keratitis.

PURPOSE: To identify prognostic factors affecting visual outcome in Acanthamoeba keratitis (AK) treated with topical chlorhexidine gluconate (CHG). METHODS: A total of 35 eyes in 34 patients with AK were treated with 0.02% topical CHG. Patients were divided into two groups according to the final visual outcome: Group 1, final visual acuity (VA) of 20/25 or greater (22 eyes); Group 2, less than 20/25 (13 eyes). We compared these groups and evaluated the effectiveness of topical CHG compared with outcomes in previous reports. RESULTS: Ring infiltrate was observed more often in Group 2 (4.5% vs 61.5%, OR 33.6, 95% confidence interval (CI) 3.4-333.9, P<0.01). The duration between onset and diagnosis of AK was significantly longer (24.9 days vs 48.4 days, OR 1.03, 95% CI 1.00-1.06, P = 0.04) and VA at initial examination (log MAR) significantly lower (0.47 vs 1.59, OR 25.5, 95% CI 3.4-186.7, P<0.01) in Group 2 (visual outcome <20/25). Multivariate analysis revealed that only VA at initial examination was independently associated with worse visual outcome (adjusted OR 24.5, 95% CI 1.9-312.6, P=0.01). Seventeen (85.0%) of the 20 eyes diagnosed within 1 month and 24 (82.8%) of 29 eyes diagnosed within 2 months achieved a VA of 20/40 or greater. CONCLUSION: VA at initial examination was the most predictive factors for final visual outcome in AK. Topical CHG was comparably effective to other treatments, including polyhexamethyl biguanide and propamidine isethionate.

Interleukin-1ß causes long-term potentiation deficiency in a mouse model of septic encephalopathy.

Sepsis induces multiple organ dysfunction syndrome including septic encephalopathy (SE), which results in cognitive impairment. However, an effective treatment for SE remains unknown. We determined the role of interleukin-1ß (IL-1ß) in long-term potentiation (LTP) deficiency after SE. At first, endotoxin level in the blood was increased at 24 h after cecum ligation and puncture (CLP) (i.e. SE model). Second, the expression of IL-1ß and its receptor in the hippocampus was determined by immunohistochemistry and immunoblotting. The number of Iba1-positive cells and their expression of IL-1ß were enhanced by CLP with disruption of the blood brain barrier. Also, Iba1, IL-1ß, and occludin protein expressions were consistent with immunohistochemical results. Third, we used an electrophysiological technique and observed the LTP deficiency, a hallmark of learning and memory, in the slices of hippocampus after CLP. Since type 1 interleukin-1 receptors (IL-1R1s) on neuronal cells were increased in the hippocampus, we utilized IL-1R1 antagonist. Pre-incubation with IL-1R1 antagonist for 30 min before recording of field excitatory post-synaptic potentials (fEPSPs) in the hippocampus canceled LTP deficiency after CLP. These results suggest the novel importance of IL-1ß in synaptic plasticity deficiency associated with sepsis-induced brain inflammation. In a mouse model of SE, IL-1R1 inhibition is important in protecting synaptic function of the hippocampus after induction of SE.

Surgical management of corneal perforation secondary to gonococcal keratoconjunctivitis.

AIMS: To report five cases of gonococcal keratoconjunctivitis with severe corneal involvement treated with therapeutic keratoplasty. DESIGN: Retrospective case series. METHODS: Five consecutive cases of gonococcal keratoconjunctivitis treated with keratoplasty for corneal perforation, with a mean age of 21.2 years, were analysed by patient's history, surgical approaches, and clinical outcomes, corrected visual acuity at initial visit and last follow-up. RESULTS: All adult cases were originally diagnosed as epidemic keratoconjunctivitis by elsewhere, and corneal perforation occurred with a mean duration of 11 days after development of conjunctivitis. While laboratory tests revealed Neisseria gonorrhoeae in all five cases, three patients showed resistance to ofloxacin. Intensive medical treatment using penicillins and/or cephems was initiated. Two patients had peripheral corneal perforations, one had a paracentral perforation, and another, a large corneal perforation with stromal melting. One case had a central microcorneal perforation. In all cases, the anterior chamber was flat. Corneal perforations were treated with lamellar or penetrating keratoplasty using cryopreserved or fresh corneal grafts. All grafts remained clear during the mean follow-up period of 34.9 months. Final best-corrected visual acuity ranged from 20/60 to 20/16. CONCLUSIONS: Severe gonococcal keratoconjunctivitis can benefit from intensive surgical and medical intervention resulting in satisfactory visual rehabilitation.

Tear function and lipid layer alterations in dry eye patients with chronic graft-vs-host disease.

PURPOSE: To investigate the changes in the tear film lipid layer in haematopoietic stem cell transplantation (HSCT) patients with dry eye (DE) associated with chronic graft-vs-host disease (cGVHD) and compare with HSCT recipients without DE. METHODS: We performed a prospective study in 10 HSCT patients with DE associated with cGVHD and 11 HSCT recipients without DE. We performed Schirmer's test, tear film break up time examinations, ocular surface dye staining and meibum expressibility test and DR-1 tear film lipid layer interferometry. DR-1 interferometry images of the tear film surface were assigned a 'DR-1 grade' according to the Yokoi severity grading system. The DR-1 grades were analysed according to the presence or absence of DE, conjunctival fibrosis and systemic cGVHD. RESULTS: The mean DR-1 severity grade in patients with DE related to cGVHD (DE/cGVHD group; 3.9+/-0.9) was significantly higher than in patients without DE after HSCT (non-DE/non-cGVHD group; 1.3+/-0.6; P<0.05). The DR-1 grade for HSCT recipients with conjunctival fibrosis was significantly higher than in patients without conjunctival fibrosis (P<0.05). When DE severity was graded according to the recommendation of the 2007 Dry Eye Workshop Report, our results showed a correlation between the severity of DE and DR-1 grades (r=0.8812, P<0.0001). CONCLUSION: DR-1 interferometry may be applicable to diagnosing DE and evaluating its progression subsequent to HSCT.

Measurement of fornix depth and area: a novel method of determining the severity of fornix shortening.

PURPOSE: To measure the depth and area of the fornix in normal adults and patients with symblepharon. METHODS: The fornix depth of normal eyes (n=20) and patients with symblepharon (Stevens-Johnson syndrome, n=4) was measured at six locations in each eye using non-invasive, dull-edged stainless steel metric scale. Before and after conjunctival reconstruction surgery, the depths of fornix were also measured by the same method. The overall area was assessed from those fornix depth at six points. RESULTS: The mean depths of the superior nasal and temporal, inferior nasal and temporal, fornices as well as mean depths of medial nasal and temporal canthi fornices in normal eyes were 14.1+/-2.5, 14.1+/-2.5, 10.0+/-2.1, 10.4+/-1.8, 3.0+/-0.9, and 5.2+/-1.2 mm, respectively. The mean overall area of the conjunctival fornix in normal subjects was 909.6+/-162.2 mm(2). All these parameters were significantly lower in the eyes of patients. Both depth and area after surgery were significantly deeper and larger than before. CONCLUSIONS: This estimation may prove useful when evaluating the severity of symblepharon and determining the post-surgical prognosis.

Combination treatment of intravenous immunoglobulin and cultivated oral mucosal epithelial transplantation for ocular cicatricial pemphigoid.

Ocular cicatricial pemphigoid (OCP) is a progressive cicatrising autoimmune disease affecting the skin and mucous membranes, including ocular surface epithelia. Although systemic immunosuppression is advocated, both progression of ocular surface inflammation and systemic side effects remain a problem. For the treatment of an active OCP, both control of autoimmune reaction and ocular surface reconstruction are necessary. We believe that the combination of intravenous immunoglobulin (IVIg) and cultivated oral mucosal epithelial transplantation (COMET) is a powerful treatment modality while minimising the risk of postoperative consequences inevitably associated with immunosuppression. A patient with OCP successfully treated by a combination of IVIg and COMET is described here.

Alterations of the ocular surface epithelial MUC16 and goblet cell MUC5AC in patients with atopic keratoconjunctivitis.

BACKGROUND: An increased understanding of the ocular surface at cellular level in the conjunctiva and the cornea may help explain the pathogenesis and the subsequent clinical appearance of atopic ocular allergies, which may be potentially blinding. PURPOSE: To investigate the MUC16 and MUC5AC alterations, tear function and the ocular surface disorder in patients with atopic keratoconjunctivitis (AKC). METHODS: Thirty-six eyes of 18 AKC patients as well as 28 eyes of 14 age- and sex-matched normal subjects were studied. The subjects underwent corneal sensitivity measurements, Schirmer test, tear film break-up time (BUT), fluorescein and Rose-Bengal staining of the ocular surface, conjunctival impression cytology and brush cytology. Impression cytology samples underwent periodic acid schiff and immunohistochemical staining with MUC16 and MUC5AC antibodies. Brush cytology specimens underwent evaluation for inflammatory cell numbers and quantitative real-time polymerase chain reaction (PCR) for MUC16 and MUC5AC mRNA expression. RESULTS: The mean corneal sensitivity and BUT values were significantly lower in patients with AKC, compared with controls (P < 0.001). Brush cytology specimens from AKC patients revealed significantly higher numbers of inflammatory cells (P < 0.001). Specimens from patient eyes showed positive staining for MUC5AC and MUC16. MUC16 mRNA expression was significantly upregulated with significant downregulation of MUC5AC mRNA expression in eyes with AKC compared with the eyes of control subjects. CONCLUSION: Ocular surface inflammation, decline in corneal sensitivity, tear film instability, changes in conjunctival epithelial MUC5AC and MUC16 mRNA expressions were thought to be important in the pathogenesis of atopic ocular surface disease.

Alterations of the tear film and ocular surface health in chronic smokers.

OBJECTIVE: To investigate the effects of chronic smoking on ocular surface and tear functions. METHODS: Fifteen right eyes of 15 healthy chronic smokers (9 men, 6 women; age range: 36-47 years) who smoked 20 cigarettes per day for 20 years and 20 eyes of 20 control non-smokers (12 men, 8 women; age range: 38-43 years) were included in this prospective study. All subjects underwent measurements of breath and haemoglobin CO concentration, tear lipid layer interferometry, evaporimetry, tear film break-up time (TBUT), Schirmer's I test, corneal fluorescein staining, conjunctival impression, and brush cytology. RESULTS: The mean Hb CO level was significantly higher in smokers compared to non-smokers. TBUT was also significantly shorter in smokers. Tear lipid layer showed significant slowing in spread over the tear film with a concomitant significant increase in tear evaporation rate. Conjunctival impression cytology revealed significant loss of goblet cells and squamous metaplasia in smokers. Brush cytology showed significant conjunctival neutrophil infiltration in smoker subjects. CONCLUSION: Chronic smoking induced distinctive quantitative and qualitative disturbances on the ocular surface health.

Ocular surface and tear functions after topical cyclosporine treatment in dry eye patients with chronic graft-versus-host disease.

We investigated the effect of 0.05% topical cyclosporine (Cys) on the ocular surface and tear functions in dry eye patients with chronic GVHD (cGVHD) in a prospective comparative study. Thirty eyes of 15 patients refractory to baseline treatment were recruited and the patients assigned for topical Cys treatment group (14 eyes of 7 patients) and control group (12 eyes of 6 patients) respectively. Two patients dropped out because of intolerable irritation while using topical Cys eye drops. Visual analog scale symptom scores, corneal sensitivity, Schirmer I test value, tear film break-up time (TBUT), tear evaporation rate and ocular surface vital staining scores were recorded at baseline and at the end of the following one month. Conjunctival impression and brush cytology were performed before and after the treatment. After topical Cys treatment, significant improvements were found in symptom scores, corneal sensitivity, tear evaporation rate, TBUT, vital staining scores, goblet cells density, conjunctival squamous metaplasia grade, inflammatory cell numbers and the MUC5AC expression. Our study suggests that 0.05% topical Cys may be an effective treatment for dry eye patients with cGVHD. The improvements in the ocular surface and tear functions resulted presumably from the decreased inflammation, increased goblet cell density and MUC5AC mRNA expression. Bone Marrow Transplantation (2008) 41, 293-302; doi:10.1038/sj.bmt.1705900; published online 5 November 2007.

Alterations of the ocular surface epithelial mucins 1, 2, 4 and the tear functions in patients with atopic keratoconjunctivitis.

BACKGROUND: An increased understanding of the ocular surface alterations at the cellular level in the conjunctiva and the cornea, may help explain the pathogenesis and the subsequent clinical appearance of atopic ocular allergies, which may be potentially blinding. PURPOSE: To investigate MUC 1, 2 and 4 alterations, tear function and the ocular surface disorder in patients with atopic keratoconjunctivitis. METHODS: Twenty-eight eyes of 14 atopic keratoconjunctivitis patients as well as 22 eyes of 11 age-and sex-matched normal subjects were studied. The subjects underwent corneal sensitivity measurements, Schirmer's test, tear film break-up time (BUT), fluorescein and Rose Bengal staining of the ocular surface, conjunctival impression cytology and brush cytology. Impression cytology samples underwent periodic acid-Schiff and immunohistochemical staining with MUC 1, 2 and 4 antibodies. Brush cytology specimens underwent evaluation for inflammatory cell numbers and quantitative real-time-PCR for MUC 1, 2 and 4 mRNA expression. Patient eyes with fluorescein and Rose Bengal scores greater than four points were regarded to have significant epithelial disease in this study. RESULTS: The mean corneal sensitivity and BUT values were significantly lower in atopic patients with significant epithelial disease, compared with patients with insignificant epithelial disease and controls (P < 0.01). Brush cytology specimens from patients with significant epithelial disease revealed significantly higher numbers of inflammatory cells (P < 0.01). Specimens from patient eyes showed positive staining for MUC 1, 2 and 4. MUC 1, 2 and 4 mRNA expressions were significantly higher in eyes with significant epithelial disease compared with eyes with insignificant epithelial disease and eyes of control subjects. CONCLUSION: Ocular surface inflammation, decline in corneal sensitivity, tear film instability, changes in conjunctival epithelial MUC 1, 2 and 4 mRNA expressions were thought to be important in the pathogenesis of atopic ocular surface disease.

Microkeratome assisted deep lamellar keratoprosthesis.

AIMS: To establish a keratoprosthesis (Kpro) surgical technique that maintains an intact superficial corneal layer. METHODS: A manual microkeratome (Moria LSK-1) was used to create a 130 mum flap of approximately 10 mm diameter in the right eye of Japanese white rabbits. The stoma beneath the flap area was dissected before the removal of a 5.0 mm stromal disc. A 5.0 mm collagen I immobilised poly(vinyl alcohol) (COL-PVA) disc was placed on the exposed posterior stroma close to Descemet's membrane. The flap was repositioned and fixed using 10-0 nylon sutures, which were removed 2 days following surgery. The corneas were followed clinically by slit lamp microscopy and photographs. Rabbits were sacrificed after 6 months, and the transplanted corneas were examined histologically by haematoxylin and eosin staining and immunohistochemistry against vimentin and alpha-smooth muscle actin (alpha-SMA). RESULTS: The transplanted COL-PVA discs remained transparent throughout the study, with no complications related to the flap or overlying epithelium. The interface between COL-PVA and Descemet's membrane remained clear without signs of opacification caused by scarring or cellular deposition. Pathology revealed the intact COL-PVA polymer in the posterior stroma, with minimal cellular infiltration along the anterior and posterior interfaces. Immunohistology shows vimentin and alpha-SMA staining at levels comparable to lamellar keratoplasty control. CONCLUSIONS: Microkeratome assisted deep lamellar keratoprosthesis may be a safe technique for the transplantation of artificial hydrogels for therapeutic purposes.

Preliminary repair of eyelids for the treatment of opaque corneas caused by burns.

The treatment of opaque cornea as a result of a burn is challenging, because the cornea often re-opacifies even after transplantation. One of the main risk factors is the deformity of the eyelids, and we have developed a new treatment for this. Preliminary repair of the lower eyelid is done by an auricular composite graft before transplantation, so that the grafted materials can be safely protected by the eyelids. The results of treatment since have greatly improved. Preliminary repair of the eyelid is an effective way to secure successful transplantation.

Transplantation of corneal endothelium with Descemet's membrane using a hyroxyethyl methacrylate polymer as a carrier.

AIMS: To evaluate the histology and function of Descemet's membrane transplanted with intact endothelium. METHODS: Japanese white rabbits and human eyebank eyes were used as donors and recipients of Descemet's membrane transplantation. Donor endothelium was hydrodissected by injecting indocyanine green from a limbal incision, and then processed as a corneal scleral button. A 6 mm diameter donor sheet was trephined, and folded in half using a 6 mm diameter polymer as a carrier. Recipient endothelium was also hydrodissected from the limbus using trypan blue to stain the Descemet's membrane. Continuous curvilinear descemetorhexis (CCD) was performed to remove a circular section of the Descemet's membrane using a 27 gauge cystotome. Donor tissue was inserted into the anterior chamber through a 5 mm limbal incision and apposed to the host stroma. Polymers were removed following transplantation. Similar surgical procedures were performed in both rabbits and eyebank eyes. Haematoxylin eosin stains were performed after 28 days in rabbits, and eyebank eyes were fixed immediately following surgery for endothelial cell counts. RESULTS: Rabbit control eyes demonstrated stromal oedema caused by loss of Descemet's membrane, whereas transplanted eyes had clear corneas. The mean (standard deviation) pachymetry of operated eyes was 376.6 (SD 32.5) mum compared with 389.6 (SD 25.1) mum in the unoperated eye. Mean endothelial density immediately following surgery in eyebank eyes was 2749 (SD 288) cells/mm(2). CONCLUSIONS: Transplantation of Descemet's membrane by CCD produces a functional graft with an optically clear interface similar to control cornea.

Efficacy of core vitrectomy preceding triple corneal procedure.

AIMS: To evaluate the effectiveness of core vitrectomy preceding triple corneal procedure (penetrating keratoplasty, extracapsular cataract extraction, and intraocular lens (IOL) implantation). METHODS: Thirty one consecutive eyes of 31 patients with indication for triple corneal procedure were randomly assigned to either triple procedure with core vitrectomy (vitrectomy group) or without vitrectomy (control group). The success rate of IOL implantation, IOL positioning, intraoperative and postoperative complications, endothelial cell loss, and best corrected visual acuity (BCVA) were compared. Follow up period was six months. Factors that may contribute to vitreous pressure elevation were also investigated in each case. RESULTS: There was no statistically significant difference in each clinical parameter examined except a tendency of facilitating IOL implantation (p = 0.11). There were two cases of vitreous loss in the control group. Retinal detachment was not seen in any of the cases. The body mass index and age were related to higher vitreous pressure (p<0.05). CONCLUSION: Core vitrectomy preceding triple corneal procedure is not necessary for all cases.