Junko Yoshida

Mutations in the quinolone resistance determining region in Staphylococcus epidermidis recovered from conjunctiva and their association with susceptibility to various fluoroquinolones

Background: Staphylococcus epidermidis is one of the prominent pathogens in ocular infection. The prevalence of mutations in the quinolone resistance determining region (QRDR) area in S epidermidis isolated from the ocular surface and its association with fluoroquinolone resistance has not been fully elucidated. Methods: Mutations in the QRDR of gyrA, gyrB, parC, and parE genes of 138 isolates of S epidermidis recovered from the human conjunctival flora were analysed. The minimal inhibitory concentrations (MICs) of four fluoroquinolones (levofloxacin, gatifloxacin, moxifloxacin and tosufloxacin) against these isolates were also determined using agar dilution methods. Results: The MIC90 values of levofloxacin, gatifloxacin, moxifloxacin and tosufloxacin were 3.13, 1.56, 0.78 and 3.13 μg/ml, respectively. The MIC values of all fluoroquinolones showed a bimodal distribution (susceptible strain and less susceptible strain). Mutations with amino acid substitution in the QRDR were present in 70 (50.7%) isolates. 19 different combinations of mutations were detected: 3 isolates (2.2%) had four mutations, 8 (5.8%) had three mutations, 43 (31.2%) had double mutations and 16 (11.6%) had single mutations. Isolates with mutations in the QRDR of both gyrA and parC (n = 53) were less susceptible to fluoroquinolones. Conclusions: The present findings show that approximately half the S epidermidis isolates from the normal human conjunctiva have mutation(s) in the QRDR. The presence of mutations in both gyrA and parC is strongly associated with reduced susceptibility to fluoroquinolones.

Development and Evaluation of Porcine Atelocollagen Vitrigel Membrane With a Spherical Curve and Transplantable Artificial Corneal Endothelial Grafts

PURPOSE To develop a collagen vitrigel (CV) optimized as a corneal endothelial cell (CEC) carrier and create an artificial corneal endothelial graft. METHODS We first developed a flat-shaped collagen vitrigel for regenerative medicine (CV-RM) using porcine atelocollagen and ultraviolet (UV) irradiation. The optimal UV amount was determined by measuring the CV-RM transparency under various irradiating conditions. The collagen vitrigel for corneal endothelial regenerative treatment (CV-CERT), a transparent porcine atelocollagen with a curved shape, was made using spherically curved molds and UV irradiation. The membrane permeability of the CV-CERT was tested in vitro. The biocompatibility, transparency, and adhesiveness of the CV-CERT were evaluated in rabbit eyes. We also developed a culture technique for distributing human CECs on the curved CV-CERT. RESULTS The optimal amount of UV irradiation for CV-RM transparency was 2400 mJ/cm(2). Membrane permeability of CV-CERT at day 5 was higher than that of commercially available CV (P = 0.032). The CV-CERT was transparent and biocompatible in rabbit corneas for up to 4 months. The CV-CERT remained attached to the rabbit corneal posterior surface, whereas the flat-shaped CV-RM, differing only in shape from the CV-CERT, dislocated soon after surgery. Human CECs seeded on the CV-CERT using our technique were evenly distributed with a single layer structure and a mean cell density of 2650 ± 100 cells/mm(2). CONCLUSIONS We developed a transparent and biocompatible porcine-derived atelocollagen vitrigel membrane with a spherical curvature. A transplantable artificial endothelial graft was created by combining cultured human CECs and the CV-CERT.

Differential binding patterns of three antibodies (VOBM1, VOBM2, and VOM2) in the rat vomeronasal organ and accessory olfactory bulb

Immunohistochemical properties of monoclonal antibodies raised against the rat vomeronasal epithelium were examined in adult rats. Three monoclonal antibodies, VOBM1, VOBM2, and VOM2, reacted specifically to the luminal surface of the sensory epithelium of the vomeronasal organ. In addition, the reactivities of VOBM1 and VOBM2 were detected in the vomeronasal nerve layer and the glomerular layer of the accessory olfactory bulb. Electron-microscopic study revealed differential patterns of the immunoreactivity of the three antibodies to the microvilli of vomeronasal sensory epithelium. VOBM1 immunoreactivity was found on the microvilli of the supporting cells, whereas VOBM2 immunoreactivity was found on those of the sensory cells. VOM2 immunoreactivity was observed on the microvilli of both the sensory and supporting cells. These results suggest that the three antibodies recognize different antigens on the vomeronasal sensory epithelium. In particular, VOBM2 antibody appears to react to an antigen specific to the microvilli of the vomeronasal sensory cells.

In vitro susceptibilities of bacterial isolates from conjunctival flora to gatifloxacin, levofloxacin, tosufloxacin, and moxifloxacin.

Purpose. To evaluate and compare the in vitro susceptibilities of various fluoroquinolones (i.e., gatifloxacin, levofloxacin, tosufloxacin, and moxifloxacin) against conjunctival bacterial flora. Methods. Two hundred sixty-six bacterial isolates were collected from the conjunctival sacs of 251 eyes of 224 patients (118 females and 106 males) ranging in age from 6 to 91 years old, who were scheduled for intraocular surgery at National Tokyo Medical Center. The minimum inhibitory concentration (MIC) was determined by broth dilution testing. Results. Of 266 isolates, 258 (97.0%) strains were gram-positive bacteria and eight (3.0%) strains were gram-negative bacteria. The MIC90 values of gatifloxacin, levofloxacin, tosufloxacin, and moxifloxacin against &agr;-hemolytic streptococci were 0.39 &mgr;g/mL, 1.56 &mgr;g/mL, 0.39 &mgr;g/mL, and 0.20 &mgr;g/mL, respectively. The MIC90 values of gatifloxacin, levofloxacin, tosufloxacin, and moxifloxacin against Staphylococcus aureus were 1.56 &mgr;g/mL, 3.13 &mgr;g/mL, 0.78 &mgr;g/mL, and 0.78 &mgr;g/mL, respectively. The MIC90 values of gatifloxacin, levofloxacin, tosufloxacin, and moxifloxacin against Staphylococcus epidermidis were 1.56 &mgr;g/mL, 3.13 &mgr;g/mL, 3.13 &mgr;g/mL, and 0.78 &mgr;g/mL, respectively. Conclusions. Although the clinical usefulness and efficacy of newer fluoroquinolones remains to be defined by clinical outcomes, the current study provides data for predicting relative in vivo potency among the fluoroquinolones.

Stromal bed quality and endothelial damage after femtosecond laser cuts into the deep corneal stroma

Aim To evaluate the stromal bed quality and endothelial damage after femtosecond laser (FSL) cuts into the deep corneal stroma. Methods Using a 150-kHz FSL, a lamellar cut was aimed at a depth of 100, 300, or 500 μm in porcine corneas. Stromal bed smoothness was graded from light microscopy and scanning electron microscopy images. Rabbit corneas were cut at remaining thicknesses of 70, 100 and 150 μm using the FSL. The effects of peeling off the corneal flap and the distance between laser spots (2 or 4 μm) were examined. Results The ratio of damaged cells in the group with a remaining depth of 70 μm was significantly larger than that in the groups with a remaining depth of 150 μm. The ratio of damaged cells in the group with a 4-μm spot separation and the flap peeled off was significantly larger than that in the group with a 4-μm spot separation and the flap not peeled off. Conclusions Corneal endothelial damage is likely to increase when the remaining depth is less than 70 μm, and peeling off the flap damages corneal endothelial cells when the remaining depth is less than 100 μm.

Transplantation of Human Corneal Endothelial Cells Cultured on Bio-Engineered Collagen Vitrigel in a Rabbit Model of Corneal Endothelial Dysfunction

ABSTRACT Purpose: To evaluate the effect of transplanting bioengineered corneal endothelial grafts in a rabbit model of corneal endothelial failure. Methods: Human corneal endothelial cells (HCECs) were seeded on a vitrigel carrier. After Descemet’s membrane was removed from the eyes of rabbits, transplantation was done with a vitrigel/HCEC graft or vitrigel alone without cells, or the eyes were left untreated. Slit lamp examinations and measurement of the central corneal thickness (CCT) were performed for 14 days postoperatively. Results: HCECs cultured on vitrigel were strongly positive for ZO-1 and Na+/K+ ATPase. On day 14, the cornea showed mild edema and the pupil margins were visible through the grafts in the vitrigel/HCEC graft group. HCECs completely covered the grafts on day 14. In contrast, there was severe corneal edema and the pupil margins were undetectable on day 14 after transplantation of the vitrigel carrier alone or no transplantation. Proliferation of host cells was not observed in these groups. On day 14, the mean CCT was significantly thinner in the vitrigel/HCEC graft group than in the other two groups (p = 0.0008). Conclusions: Transplantation of a vitrigel/HCEC graft was effective for reducing the corneal thickness and restoring corneal transparency, suggesting the usefulness of vitrigel as a carrier for corneal endothelial cells.

1242 Regeneration of the vomeronasal nerves in adult rat after transection

Masumi Ichikawa, Junko Yoshida Transection of the axons of vomeronasal (VN) receptor cells (VN nerves) in adult rat results in degeneration of receptor cells followed by replacement of the receptor cell population. An axonal tracing analysis was made to determine the growth and termination of regenerating VN nerves in the olfactory bulb (OB) following the unilateral transection of the nerves in adult rat. HRP-WGA was injected into the VN organ at recovering time of 1, 8, 12, 16 weeks after the transection. No HRP labeled nerves was observed in the OB at 1 week recovering time. At 8 weeks, the labeled nerves were recognized in the surface of medial wall in olfactory bulb. The nerves did not enter the OB. At 12 and 16 weeks, the labeled nerves were recognized in the accessory olfactory bulb (AOB) of 20-30% of HRP injected rats.The volume of the nerves in the AOB was very low comparing to the control side. This study confirms that regenerating VN nerves are capable of termination in the AOB..

Immunocytochemical Study of the Vomeronasal Epithelium of the Infant Rat

The vomeronasal organ (VNO) is thought to be a detector of chemical signals, i.e., pheromones [1,2]. The primordium of the organ is first observed on embryonic day 11 and then gradually increases in size with growth [3]. The shape of the VNO in the neonatal rat is similar to that in the adult, and its histological maturation is assumed to be completed by the 3rd postnatal week [4]. Past immunohistochemical studies indicate that the VNO has unique characteristics which distinguish it from the olfactory organ [5–7]. Its function, however, remains to be clarified. Recently, monoclonal antibodies (mAbs) VOM2, VOBM1, and VOBM2 have been generated against rat vomeronasal epithelium by Osada et al. [8]. The reactivities of these mAbs were observed only in the sensory epithelium, and VOM2 immunoreactivity (IR) was especially limited on the luminal surface of the sensory epithelium. Here we report the developmental changes in the IR of these three MAbs for the sensory epithelium of the VNO.