Takeshi Kobayashi

Efficacy of commercial soft contact lens disinfectant solutions against Acanthamoeba

PurposeTo investigate the relative efficacy of Japanese commercial soft contact lens disinfectant solutions against Acanthamoeba trophozoites and cysts.Materials and methodsEight types of multipurpose solution (MPS), two types of hydrogen peroxide solution, and one povidone–iodine solution were evaluated to determine their effect against Acanthamoeba trophozoites and cysts (ATCC 50514). Acanthamoeba cysts were cultured in encystment medium for either 1 or 2 weeks (1 and 2-week-old cysts). The trophozoites and cysts were treated with each disinfectant solution for 0, 2, 4, 8, or 24 h. After performing four tenfold serial dilutions of each test solution, dilutions were cultured for 10 days. The number of surviving organisms was calculated using the trimmed Spearman–Karber method.ResultsAmong the MPS tested, only four were effective against trophozoites after treatment for 4 h, and none was effective against 2-week-old cysts. Hydrogen peroxide had a significant effect on trophozoites and 1-week-old cysts, but not on 2-week-old cysts. In contrast, povidone–iodine caused a 2.6 log reduction in 2-week-old cysts.ConclusionsMPS were found to have limited efficacy against trophozoites and no efficacy against 2-week-old cysts. Only povidone–iodine had any efficacy against 2-week-old cysts.

Involvement of P38MAPK in human corneal endothelial cell migration induced by TGF-β2

Because human corneal endothelial cells do not proliferate once the endothelial monolayer is formed, corneal wound healing is thought to be mediated by cell enlargement or migration rather than proliferation. However, the cellular mechanisms involved in corneal wound healing have not been fully determined. Because transforming growth factor-β(2) (TGF-β(2)) isoform is present in high concentrations in normal human aqueous humor, it may play a role in human corneal endothelial cell wound healing. The purpose of this study was to determine the effect of TGF-β(2) on the proliferation and migration of cultured human corneal endothelial cells (HCECs). To achieve this, we first examined the effect of TGF-β(2) on the wound closure rate in an inxa0vitro HCEC wound healing model. However, unexpectedly TGF-β(2) had no effect on the wound closure rate in this model. Therefore, a real-time cell electronic sensing (RT-CES) system and the BrdU incorporation assay were used to determine the effect of TGF-β(2) (0.1-10xa0ng/ml) on cultured HCEC proliferation during inxa0vitro wound healing. The specificity of this effect was confirmed by adding the TGF-β receptor I kinase inhibitor. TGF-β(2) inhibited the proliferation of HCECs in a dose dependent way and was blocked by TGF-β receptor I kinase inhibitor. Next, the Boyden chamber assay was used to determine how TGF-β(2) (10xa0ng/ml) affect HCEC migration. Exposure to TGF-β(2) increased cell migration, and a synergistic effect was observed when FGF-2 was added. To determine whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in the migration of HCECs, western blot analysis and Bio-Plex™ suspension array were used to detect phosphorylation of Erk1/2, p38, and JNK in HCECs stimulated by TGF-β(2) and/or FGF-2. The effect of the p38 MAPK inhibitor, SB239063 (10xa0μM), on TGF-β(2) and/or FGF-2-induced cellular migration was determined by the Boyden chamber assay. Both TGF-β(2) and FGF-2-induced p38 phosphorylation, and a synergistic effect was observed with exposure to both growth factors. SB 239063 inhibited TGF-β(2) and FGF-2-induced migration of HCECs. These results indicate that TGF-β(2) reduces proliferation but stimulates migration of cultured HCECs. In addition, TGF-β(2) and FGF-2 may have synergistic effects on the migration of HCECs mediated by p38 MAPK phosphorylation.

Differences Between Niche Cells and Limbal Stromal Cells in Maintenance of Corneal Limbal Stem Cells

PURPOSEnTo investigate the differing characteristics of limbal niche cells (LNCs) and limbal stromal cells (LSCs) in the maintenance of limbal epithelial stem/progenitor cells in the cornea.nnnMETHODSnLimbal niche cells were obtained from direct dissection of the human corneal limbus, and LSCs were obtained from explant cultures of limbal stromal tissues under the same culture conditions. The resulting cultures were examined for their ability to support the growth of limbal stem/progenitor cells in colony-forming capacity, stratified epithelial cell sheet formation, maintenance of limbal epithelial stem/progenitor cell characteristics, and gene expression levels of factors that supported the limbal epithelial stem/progenitor cells.nnnRESULTSnThe colony-forming efficiency of limbal epithelial stem/progenitor cells in the LNC group (6.57 ± 1.54%) was significantly higher than that in the LSC group (1.43 ± 0.47%). The epithelial cell sheets in the LNC group stratified into four or five layers compared with two or three stratified layers in the LSC group. Staining of both the colonies and the epithelial cell sheets in the LNC group showed a higher intensity of the limbal stem cell marker ΔNp63 than in the LSC group. Moreover, reverse transcription polymerase chain reaction analysis revealed that compared with the common expression of EGF and so on, the LNCs showed a higher expression level of E-cadherin and a lower expression level of neurotrophin-3 (NT3) than the LSCs.nnnCONCLUSIONSnLNCs have a different role compared to LSCs in their ability to support epithelial stem/progenitor cells and epithelial cellular sheet formation.

Degradation of Tributyltin in Microcosm Using Mekong River Sediment

The degradation of tributyltin (TBT) and changes of bacterial number and community structures were investigated in microcosms using the sediment collected from the Mekong River, Vietnam. Concentrations of TBT in sediments were less than 0.62xa0ng/g (dry wt), lower than those reported from other areas. TBT-resistant bacteria were found in the three sampling sites, and the occurrence rates were 11–16% out of the total viable count. In this microcosm experiment, initial concentration of TBT [1.0–1.4xa0μg/g (dry wt)] decreased to 0.6xa0μg/g (dry wt) during 150xa0days, whereas that in the control microcosm with autoclaved sediment did not change, indicating that Mekong River sediment contains high TBT-degrading activity by microorganisms. The occurrence of TBT-resistant bacteria and the bacterial community structures monitored by denaturing gradient gel electrophoresis were almost the same between test and control groups, indicating that the addition of TBT had little influence on microbial community structure. Mekong River sediment seems to have a stable microbial community against TBT pollution.

Corneal Epithelial Wound Healing Impaired in Keratinocyte-Specific HB-EGF–Deficient Mice In Vivo and In Vitro

PURPOSEnTo study the role played by HB-EGF in corneal epithelial wound healing.nnnMETHODSnA 2-mm corneal epithelial wound was created in keratinocyte-specific, HB-EGF-deficient mice--HB(lox/lox):K-5Cre (HB(-/-))--and the speed of wound healing was compared with that in wild-type (WT) mice. Cultured confluent mouse corneal epithelial cells (MCECs) from WT and HB(-/-) mice were scraped, and the bare area was measured. The proliferation of MCECs was determined by BrdU incorporation. The degree of attachment of WT and HB(-/-) MCECs was also determined. The mRNA expression of EGF family and cell adhesion molecules was determined by real-time PCR.nnnRESULTSnCorneal epithelial wound healing was significantly delayed in HB(-/-) mice, and the expression of HB-EGF was detected at the leading edge of the wound in HB(lox/+):K5-Cre (HB(+/-)) mice by the presence of lacZ staining. The wound closure was significantly impaired in HB(-/-) MCECs and was improved by adding HB-EGF. The number of BrdU-positive MCECs of WT and HB(-/-) mice was not significantly different, and both increased to different degrees when HB-EGF was added. The adhesion of isolated HB(-/-) MCECs was lower than that of WT MCECs, but the degree of adhesion was restored by adding HB-EGF. The expression of epiregulin was upregulated in HB(-/-) MCECs, and α6- and β1-integrin were upregulated by adding HB-EGF.nnnCONCLUSIONSnHB-EGF plays an important role in corneal epithelial cell healing by enhancing cellular attachment and part of cell proliferation.

Molecular evidence for the ancient origin of the ribosomal protection protein that mediates tetracycline resistance in bacteria

The ribosomal protection proteins (RPPs) mediate the resistance to tetracycline (TC) in Gram-positive and Gram-negative bacteria. The RPPs display sequence similarity to translation elongation factors, EF-G/EF-2 and EF-Tu/EF-1α. To determine the evolutionary origin of the RPPs, we constructed a composite phylogenetic tree of the RPPs, EF-G/EF-2 and EF-Tu/EF-1α. This tree includes two universal trees for the EF-G/EF-2 and EF-Tu/EF-1α, which form clusters corresponding to the respective two groups of proteins from three superkingdoms. The cluster of RPPs was placed at a point between the EF-G/EF-2 and EF-Tu/EF-1α clusters. The branch length (substitutions/site) between the node for the RPP cluster and the primary divergence of the RPPs was statistically shorter than that between the node for this cluster and the primary divergence in the EF-G/EF-2 cluster. This indicates that the RPPs derived through duplication and divergence of the ancient GTPase before the divergence of the three superkingdoms. Furthermore, this suggests the RPPs’ extant function occurred before the streptomycetes that include the TC-producing strains. Therefore, the RPPs evolved independent of the presence of TCs and serve a function other than antibiotic resistance. The RPPs may provide ribosomal protection against other chemical substances in the environment.

Various pAQU plasmids possibly contribute to disseminate tetracycline resistance gene tet(M) among marine bacterial community.

Emergence of antibiotic-resistant bacteria in the aquaculture environment is a significant problem for disease control of cultured fish as well as in human public health. Conjugative mobile genetic elements (MGEs) are involved in dissemination of antibiotic resistance genes (ARGs) among marine bacteria. In the present study, we first designed a PCR targeting traI gene encoding essential relaxase for conjugation. By this new PCR, we demonstrated that five of 83 strains isolated from a coastal aquaculture site had traI-positive MGEs. While one of the five strains that belonged to Shewanella sp. was shown to have an integrative conjugative element of the SXT/R391 family (ICEVchMex-like), the MGEs of the other four strains of Vibrio spp. were shown to have the backbone structure similar to that of previously described in pAQU1. The backbone structure shared by the pAQU1-like plasmids in the four strains corresponded to a ~100-kbp highly conserved region required for replication, partition and conjugative transfer, suggesting that these plasmids constituted “pAQU group.” The pAQU group plasmids were shown to be capable of conjugative transfer of tet(M) and other ARGs from the Vibrio strains to E. coli. The pAQU group plasmid in one of the examined strains was designated as pAQU2, and its complete nucleotide sequence was determined and compared with that of pAQU1. The results revealed that pAQU2 contained fewer ARGs than pAQU1 did, and most of the ARGs in both of these plasmids were located in the similar region where multiple transposases were found, suggesting that the ARGs were introduced by several events of DNA transposition into an ancestral plasmid followed by drug selection in the aquaculture site. The results of the present study indicate that the “pAQU group” plasmids may play an important role in dissemination of ARGs in the marine environment.

Use of 5-Cyano-2,3-Ditolyl-Tetrazolium Chloride Staining as an Indicator of Biocidal Activity in a Rapid Assay for Anti-Acanthamoeba Agents

ABSTRACT The usefulness of 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) staining to determine the respiratory activity of Acanthamoeba was evaluated in this study. Acanthamoeba trophozoites and cysts have a red fluorescence after staining with CTC. To determine the effectiveness of CTC staining as a CTC biocidal assay for Acanthamoeba, the trophozoites and cysts of Acanthamoeba castellanii (ATCC 5037) were treated with serial concentrations of disinfectant solutions, namely, polyhexamethylene biguanide (PHMB) and commercial soft contact lens (SCL) disinfectant solutions. The treated Acanthamoeba organisms were stained with CTC, and their respiratory activity was determined by the intensity of fluorescence in a fluorescence microplate reader. The survival rates of the same samples were determined by a culture-dependent biocidal assay using the Spearman-Karber method. Our results showed that the respiratory activities determined by the CTC biocidal assay and the survival rates determined by the culture-dependent biocidal assay for Acanthamoeba trophozoites and cysts decreased in a dose-dependent way after PHMB treatments, and the results were significantly correlated (r = 0.83 and P < 0.01 for trophozoites; r = 0.60 and P < 0.01 for cysts; Spearman rank correlation test). The respiratory activities in the trophozoites and cysts treated with SCL disinfectant solutions were significantly correlated with the survival rate (r = 0.70 and P < 0.01 for trophozoites; r = 0.64 and P < 0.01 for cysts; Spearman rank correlation test). The significant correlation of the results indicated that the CTC biocidal assay can be used as an alternative method to a culture-dependent biocidal assay. The CTC biocidal assay is a rapid and simple method to test the effectiveness of disinfectant solutions against Acanthamoeba trophozoites and cysts.

Important role of epiregulin in inflammatory responses during corneal epithelial wound healing.

PURPOSEnTo investigate the role played by epiregulin in corneal epithelial wound healing in vivo in epiregulin-knockout (KO) mice and cultured mouse corneal epithelial cells (MCECs).nnnMETHODSnA 2-mm diameter central epithelial wound was created in epiregulin-KO and wild-type (WT) mouse corneas. The size of the unhealed area and the epithelial cell proliferation and migration were examined. Myeloperoxidase assay was performed to determine the number of polymorphonuclear (PMN) cells infiltrating corneal stroma. Real-time PCR was used to determine expression of the mRNA of inflammatory cytokines in the corneal epithelial cells. Expression of chemokine (C-X-C motif) ligand 2 (CXCL2) response to IL-1β was examined in MCECs with or without recombinant mouse epiregulin. Repetitive injuries were created to determine the effect of inflammation in healing in epiregulin-KO mice.nnnRESULTSnAfter a single injury, corneal epithelial wound healing and cell migration and proliferation were unimpaired. However, corneal opacities and a larger number of infiltrating PMN cells were observed in epiregulin-KO mice. Expression levels of IL-1β, IL-6, CXCL1, and CXCL2 were higher in epiregulin-KO than in WT corneal epithelia cells. The addition of epiregulin significantly reduced the expression of CXCL2 in response to IL-1β in MCECs. In response to repetitive injuries, a significant delay in healing and more severe opacities were observed in epiregulin-KO mice than in WT mice.nnnCONCLUSIONSnOur results indicate that during wound healing, epiregulin may regulate the expression of cytokines and chemokines to reduce an excessive accumulation of PMN cells, which will cause corneal opacity and persistent epithelial defects.

Rapid detection of Acanthamoeba cysts in frozen sections of corneal scrapings with Fungiflora Y

Aims: To evaluate the usefulness of serial frozen sections of corneal scrapings stained with Fungiflora Y (FFY) to diagnose Acanthamoeba keratitis (AK). Methods: Eight patients with suspected AK were studied. Serial frozen sections were made from part of the corneal epithelial scrapings and stained with FFY. The remaining corneal epithelial scrapings were submitted for laboratory culture. Results: The FFY stained frozen sections were completed within an hour, and Acanthamoeba cysts were detected under a fluorescence microscope in all eight patients. The same sections were examined with a light microscope, and Acanthamoeba cysts were confirmed to be present from their morphological characteristics. Five of the eight patients had positive laboratory cultures for Acanthamoeba. Conclusion: FFY staining of frozen sections of corneal scrapings is a rapid and reliable technique which can be used to make an early diagnosis of AK.