Chemical force microscopy using functionalized ZnO whisker probe tips.

We have developed an atomic force microscope-based technique utilizing a zinc oxide whisker crystal as a probe tip. This technique was used for measuring interactions between the chemically modified tip and oxidized silicon substrates as a function of solution pH. Surfaces terminating in amine and methyl functional groups were prepared by covalently modifying the silicon substrate with self-assembled monolayers of (3-amino-propyl)triethoxysilane and n-butyltrichlorosilane, respectively. Patterned samples prepared by microcontact printing consisted of amine-terminated square regions surrounded by a methyl-terminated background. The contrast in lateral force images of the patterned samples obtained with amine-functionalized tips was seen to strongly depend on solution pH. High friction was observed between the attractive and strongly interacting functional groups in the methyl-terminated regions at low pH values and the amine-terminated regions at intermediate pH values. Low friction was observed between repulsive and weakly interacting functional groups. In addition, interacting force measurements on approach showed long-range attractive forces between the amine-terminated surfaces at intermediate pH values. Adhesive force measurements showed a dependency on the state of ionization of the amine groups, which was controlled by varying the pH. The pKa value estimated from the measurements was shifted to a lower pH than that of primary amine groups in solution. These results show that functionalized ZnO whisker probe tips have great potential for chemically sensitive imaging.

Comparison of LogMAR Eye charts with angular vision for visually impaired: the Berkeley rudimentary vision test vs LogMAR One target Landolt ring Eye chart.

BACKGROUND: It is not common to quantify visual acuity worse than 2.0 logarithm of the minimal angle resolution (logMAR) (commensurate with decimal visual acuity 0.01) at ophthalmology clinics. Recently, the Berkeley rudimentary vision test (BRVT) was developed as a simple measurement tool of logMAR with angular vision for quantifying poor levels of visual acuity. We compared the difference between BRVT and conventional Landolt ring logMAR chart with angular vision measured by the logMAR one target Landolt ring eye chart (LogMAR LEC). METHODS: We reviewed 110 patients with best-corrected visual acuity (BCVA) in the better eye from light perception (LP) to 0.8 logMAR measured by LogMAR LEC. The reproducibility of the log MAR LEC and BRVT was evaluated on 39 eyes from 20 patients, and 33 eyes from 20 patients respectively. The comparison of logMAR between BRVT and logMAR LEC was evaluated by surveying 61 eyes from 70 patients. In addition, regardless of their BCVA, the eyes from patients with worse than 2.0 logMAR by LogMAR LEC were re-evaluated by BRVT. RESULTS: The logMAR of patients examined by BRVT or logMAR LEC did not show any significant difference between the first and second examinations, and there was a strong correlation between the examinations in both eye charts. The BRVT significantly produced better logMAR compared with logMAR LEC, and the strong correlation was shown between both eye charts. Although 35 eyes from 28 patients among 110 patients could not be quantified by logMAR LEC, 18 eyes of 35 eyes could be quantified logMAR by BRVT. CONCLUSIONS: The BRVT and logMAR LEC are reliable visual acuity measurement tools. Moreover, the BRVT is potentially effective in quantifying visual acuity of the more severe visually impaired patients.

[Four-digit allele genotyping of HLA-A and HLA-B genes in Japanese patients with Behçet's disease (BD) by a PCR-SSOP-luminex method and stratification analysis according to each major symptom of BD].

PURPOSE: High resolution (four-digit) allele genotyping was used to determine the association of the HLA-A and -B alleles with Behçet's disease (BD) in Japanese patients. We also analyzed our results for the association of these alleles with the individual clinical features of BD. SUBJECTS AND METHODS: We enrolled 389 Japanese BD patients and 254 healthy controls in this study. Genotyping of the HLA-A, -B alleles was performed by the PCR-SSOP-Luminex method and the phenotype frequencies of the HLA-A, and -B alleles were estimated. RESULTS: Some HLA-A and -B alleles were significantly associated with BD. When we recalculated the phenotype frequencies for the HLA-B*51-negative subjects to exclude the effects of the linkage disequilibrium with the HLA-B*51 allele, HLA-A*2601 was most strongly associated with BD. In addition, we observed a significant association between several clinical features and some alleles, including HLA-A*2602. CONCLUSION: The significant increase of HLA-A* 26 in the BD patients without HLA-B*51 suggests that this allele itself might be one of the primary susceptibility genes involved in the development of BD independently of HLA-B*51.

[Case of neurosarcoidosis with rapid visual field defect progression].

OBJECTIVE: To report a case of neurosarcoidosis with rapid progression of visual field defects. CASE: A 28-year-old woman presented with bilateral uveitis and was diagnosed as having sarcoidosis after skin and cervical lymph node biopsy. Since bilateral excavations of the optic nerve head and visual field defects were observed, endocranial lesion was suspected. However, a computed tomography (CT) scan of the head detected nothing abnormal. It was regarded as a case of sarcoidosisaccompanied by normal-tension glaucoma and treatment was initiated with latanoprost. Four months later, the patient's visual field deteriorated rapidly. A CT scan showed a pituitary mass. Neurologicalfindings and hypopituitarism were found which improved with systemic prednisolone therapy. Diabetes insipidus developed after the start of treatment, and was treated with intranasal desmopressin therapy. After 6 weeks, head magnetic resonance imaging (MRI) showed a remarkable reduction of the enhanced regions. CONCLUSIONS: Although ocular sarcoidosis is often accompanied by secondary glaucoma or optic nerve atrophy, the progression of neurosarcoidosis can lead to visual field defects. Central nervous system (CNS) sarcoidosis is rare, but a precise examination with enhanced MRI should be considered when the visual field defect progresses rapidly.

Single nucleotide polymorphisms of Ficolin 2 gene in Behçet's disease.

BACKGROUND: Genetic susceptibility to Behçet's disease (BD) is well documented for HLA-B51 positivity. However, BD is not a simple hereditary disease and it is exaggerated by exogenous stimuli such as microorganisms' infections. Ficolin 2 is a lectin that binds to the surface of microbial cells and kills microbial cells through the activation of complement system. Novel single nucleotide polymorphisms (SNPs) of human Ficolin 2 gene (FCN2 gene) have been recently identified in Caucasian people. OBJECTIVE: The aim of the study was to elucidate the contribution of FCN2 gene in the pathogenesis of BD. METHODS: The frequencies of genotypes and alleles of FCN2 gene SNPs in the promoter regions (-987, -602, -557, -64, -4) and exon 8 (+6359, +6424) were examined in 83 patients with BD and 64 healthy controls by genotyping with a DNA sequencing method. RESULTS: There were no significant differences in genotype and allele frequencies of FCN2 gene SNPs between BD patients and healthy controls. No significant differences in genotype and allele frequencies of FCN2 gene SNPs were detected among different clinical subgroups in BD patients. Significant differences in allele frequencies of FCN gene SNPs at both -557 and -64 sites in the promoter regions were found between HLA-B51 positive groups and HLA-B51 negative groups of BD patients. CONCLUSION: The significant differences in allele frequencies of FCN2 gene SNPs in the promoter lesions (-557 and -64 sites) among HLA-B51 positive BD patients may reveal the possibility that ficolin may contribute to the innate immunity of BD among HLA-B51 haplotypes in BD patients.

Induction of regulatory T cells by the immunomodulating cytokines alpha-melanocyte-stimulating hormone and transforming growth factor-beta2.

Recently, we have reported that the cytokines alpha-melanocyte-stimulating hormone (alpha-MSH) and transforming growth factor-beta2 (TGF-beta2) work in synergy to induce the activation of regulatory T (Treg) cells. When we used alpha-MSH and TGF-beta2 to generate ocular autoantigen-specific Treg cells and adoptively transferred them into mice susceptible to experimental autoimmune uveoretinitis (EAU), there was suppression in the incidence and severity of EAU. Specificity to a retinal autoantigen was required for the Treg cells to suppress EAU. When stimulated, these Treg cells produced TGF-beta1, and their production of interferon-gamma, interleukin (IL)-10, and IL-4 was suppressed. Also, the Treg cells are suppressed in their proliferative response. Our results demonstrate that alpha-MSH with TGF-beta2 induce Treg cells that can subdue a tissue-specific autoimmune response. This also promotes the possibility of using these immunomodulating cytokines to purposely induce antigen-specific Treg cells to prevent and suppress autoimmune disease.

Flomoxef sodium and levofloxacin concentrations in aqueous humor.

AIMS: We intravenously administered flomoxef sodium (FMOX) 120 minutes before cataract surgery, topically administered levofloxacin (LVFX) into the eyes four times at 30-minute intervals before surgery, and measured the aqueous humor concentrations of these agents to investigate their penetration into the aqueous humor and their efficacy in the prevention of postoperative endophthalmitis. METHODS: Sixty-eight patients who underwent cataract surgery at the Department of Ophthalmology, Yokohama City University School of Medicine, or its affiliate, Kanazawa Hospital, Yokohama, were enrolled in this study. They received one or both of the following: 1.0 g FMOX via a 20-minute intravenous drip and LVFX ophthalmic solution applied four times at 30-minute intervals, both beginning two hours before the operation. Aqueous humor was aspirated from the anterior chamber and assayed for FMOX and LVFX concentrations using high-performance liquid chromatography (HPLC). RESULTS: The mean intraoperative FMOX and LVFX concentrations in the patients' aqueous humor were 1.21 +/- 0.63 microg/ml and 0.69 +/- 0.47 microg/ml, respectively. These concentrations sufficiently exceeded the MIC90 values against Staphylococcus epidermidis, S. aureus, and Propionibacterium acnes. CONCLUSIONS: The FMOX and LVFX concentrations in the aqueous humor sampling were adequate to kill bacteria in vitro. These drugs may have efficacy in the prevention of postoperative endophthalmitis in patients undergoing cataract surgery.

Effects of astaxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo.

PURPOSE: Astaxanthin (AST) is a carotenoid that is found in marine animals and vegetables. Several previous studies have demonstrated that AST exhibits a wide variety of biological activities including antioxidant, antitumor, and anti-Helicobacter pylori effects. In this study, attention was focused on the antioxidant effect of AST. The object of the present study was to investigate the efficacy of AST in endotoxin-induced uveitis (EIU) in rats. In addition, the effect of AST on endotoxin-induced nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor (TNF)-alpha production in a mouse macrophage cell line (RAW 264.7) was studied in vitro. METHODS: EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). AST or prednisolone was administered intravenously at 30 minutes before, at the same time as, or at 30 minutes after LPS treatment. The number of infiltrating cells and protein concentration in the aqueous humor collected at 24 hours after LPS treatment was determined. RAW 264.7 cells were pretreated with various concentrations of AST for 24 hours and subsequently stimulated with 10 microg/mL of LPS for 24 hours. The levels of PGE2, TNF-alpha, and NO production were determined in vivo and in vitro. RESULTS: AST suppressed the development of EIU in a dose-dependent fashion. The anti-inflammatory effect of 100 mg/kg AST was as strong as that of 10 mg/kg prednisolone. AST also decreased production of NO, activity of inducible nitric oxide synthase (NOS), and production of PGE2 and TNF-alpha in RAW264.7 cells in vitro in a dose-dependent manner. CONCLUSIONS: This study suggests that AST has a dose-dependent ocular anti-inflammatory effect, by the suppression of NO, PGE2, and TNF-alpha production, through directly blocking NOS enzyme activity.

Effect of human cationic antimicrobial protein 18 Peptide on endotoxin-induced uveitis in rats.

PURPOSE: Human cationic antimicrobial protein 18 (hCAP18, 18 kDa) was originally identified in leukocytes on the basis of its antimicrobial activity. The peptide composed of the 27 C-terminal amino acids of hCAP18 (hCAP18(109-135)) binds lipopolysaccharide (LPS). The purpose of the present study was to investigate the effects of hCAP18 peptide on endotoxin-induced uveitis (EIU) in rats. METHODS: EIU was induced by footpad injection of LPS. Each rat was injected intravenously with 1, 10, or 100 micro g hCAP18 peptide in 0.1 mL of PBS immediately after LPS injection in male Lewis rats. At 24 hours after LPS injection, enzyme-linked immunosorbent assay was performed to evaluate concentrations of protein, nitric oxide (NO), tumor necrosis factor (TNF)-alpha, prostaglandin (PG)-E2, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-2 in aqueous humor. Also, EIU was evaluated by counting inflammatory cells in aqueous humor. RESULTS: hCAP18 peptide at 10 and 100 micro g significantly suppressed an LPS-induced increase in the number of inflammatory cells and the levels of protein, NO, TNF-alpha, PGE2, MCP-1, and MIP-2. The anti-inflammatory effect of 10 micro g hCAP18 peptide was as strong as that of 100 micro g hCAP18 peptide. Treatment with 1 micro g hCAP18 peptide did not suppress EIU, compared with the LPS group. CONCLUSIONS: The present results indicate that hCAP18 peptide suppresses development of EIU. A possible mechanism for the ocular anti-inflammatory effect of hCAP18 peptide is that it suppresses onset of LPS-triggered inflammatory reactions by binding directly to LPS.

Relation of aortic valve sclerosis to carotid artery intima-media thickening in healthy subjects.

We studied 252 healthy adults by echocardiography and carotid ultrasonography to determine the relation between early subclinical aortic valve sclerosis (AVS) and carotid intima-media thickness (IMT). Carotid IMT was significantly greater in subjects with AVS than in those without AVS. There was a significant correlation between the grade of AVS and carotid IMT.

Anti-inflammatory effects of alpha-melanocyte-stimulating hormone against rat endotoxin-induced uveitis and the time course of inflammatory agents in aqueous humor.

PURPOSE: We examined the effects of the immunosuppressive neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) on rat endotoxin-induced uveitis, and to measure the expression of inflammatory cytokines and chemokines with and without the alpha-MSH treatment over the course of the disease. METHODS: We injected Lewis rats once with Salmonella typhimurium lipopolysaccharide (LPS) to induce uveitis. The rats were given intravenous injections of 250, 500 or 1000 microg of alpha-MSH. The eyes were examined over the next 24 h for inflammation. Aqueous humor was collected 6, 12 and 24 h after endotoxin injections and the number of infiltrating cells were counted in anterior chamber. In addition, we assayed the concentration of protein, nitric oxide, TNF-alpha, IL-6, MCP-1 and MIP-2. RESULTS: Rats injected with alpha-MSH showed a significant decrease in the number of infiltrating cells in anterior chamber. Moreover, alpha-MSH-treated rats with endotoxin-induced uveitis (EIU) showed significantly lower concentrations of protein, nitric oxide, proinflammatory cytokines and chemokines in their aqueous humor. Even the early stages of EIU were suppressed by the injection of alpha-MSH. CONCLUSIONS: Our results demonstrate that the immunosuppressive neuropeptide alpha-MSH inhibits the early induction events of endotoxin-induced inflammation in the eye; therefore, suppresses the subsequent infiltration of cells and intraocular production of inflammatory cytokines and chemokines in eyes. alpha-MSH has a possibility of being a therapeutic strategy for anterior uveitis.

Correlation between occult neoplastic cells in the lymph node sinuses and recurrence in patients with Dukes' C colorectal cancer.

The aim of this study was to investigate whether it was possible to select patients with Dukes' C primary colorectal cancer who had a high risk of metastasis and recurrence. Among 434 patients who underwent surgical resection of primary colorectal cancer, 115 patients (26.5%) had Dukes' C cancer. Among them, 35 patients (30.45%) suffered from metastasis/recurrence postoperatively. Thirty-two patients with recurrence for whom complete follow-up was possible (recurrence group) and 32 patients who survived for at least 5 years without recurrence (non-recurrence group) were compared by immunohistochemical staining of resected lymph nodes for cytokeratin using two antibodies (AE1/AE3 and CAM 5.2). AE1/AE3 staining was positive in 93.85% of the recurrence group versus 68.85% of the non-recurrence group (p=0.0250), while CAM 5.2 staining was positive in 84.45% vs. 53.15%, respectively (p=0.0152). The occult neoplastic cell count (mean +/- SD) in the peripheral lymph node sinuses was 6.28+/-5.17 vs. 2.38+/-3.03 (p=0.0002) in the AE1/AE3-positive patients from the recurrence and non-recurrence groups, while it was 5.13+/-4.84 vs. 1.53+/-2.37 (p=0.0003) in the CAM 5.2-positive patients in each group. Accordingly, the immunohistochemical positivity rate for both AE1/AE3 and CAM 5.2, as well as the occult neoplastic cell count, were significantly higher in the recurrence group. These results suggest that patients with Dukes' C primary colorectal cancer who have a higher risk of recurrence can be selected by immunostaining of resected lymph nodes for cytokeratin.

Correlation between occult neoplastic cells in the lymph node sinuses and recurrence in patients with curatively resected Dukes' B colorectal cancer.

This study investigated whether it is possible to detect patients who have a high risk of metastasis and recurrence after resection of stage II Dukes' B primary colorectal cancer. Among 434 patients who underwent curative resection of primary colorectal cancer, 167 (38.5%) had Dukes' B cancer. Among them, 19 patients (11.4%) suffered from postoperative metastasis or recurrence. In 17 patients with recurrence who could be followed-up completely (recurrence group) and 17 other patients who survived for at least 5 years without recurrence (non-recurrence group), immunohistochemical staining of resected lymph nodes for cytokeratin (AE1/AE3 and CAM 5.2) was performed. AE1/AE3 was positive in 76.5% and 47.1% of the patients from the recurrence and non-recurrence groups, respectively, while CAM 5.2 was positive in 52.9% and 17.6%, respectively. There were no significant differences of either AE1/AE3 or CAM 5.2 positivity between the groups. However, the occult neoplastic cell count (mean +/- SD) floating in the lymph node sinuses was significantly higher in patients from the recurrence group who were positive for AE1/AE3 or CAM 5.2 than in patients from the non-recurrence group (6.12+/-6.00 vs. 0.59+/-0.71; p=0.0019 and 3.94+/-5.06 vs. 0.29+/-0.69; p=0.0098, respectively). These results suggest that patients with Dukes' B primary colorectal cancer who have a higher risk of recurrence can be selected by immunostaining of resected lymph nodes for cytokeratin.

Selection criteria for high risk and low risk groups of recurrence and metastasis in patients with primary colorectal cancer.

Among 371 patients with primary colorectal cancer, 54 patients suffered from recurrence/metastasis (recurrence group) and 317 survived without recurrence for at least 5 years (non-recurrence group). The clinicopathological characteristics of the 2 groups were compared and occult neoplastic cells (ONCs) in the lymph node sinuses were detected by cytokeratin immunohistochemistry. There were significant differences of the following factors: venous invasion (v-) vs. (v+) for Dukes' A patients (p=0.0315); harvested lymph nodes (LN) or=15 for Dukes' B patients (p=0.0388); (v-) vs. (v+) (p=0.0059), lymphatic invasion (ly-) vs. (ly+) (p=0.0435) for Dukes' A and B patients combined; D>n vs. D=n (p=0.0033), depth of tumor invasion or=se/a2 (p=0.0329) for Dukes' C patients. When the detection of >or=3 ONCs was defined as positive, the sensitivity, specificity, PPV, and NPV were respectively 77%, 100%, 100% and 71% in Dukes' B patients, as well as 75%, 72%, 73% and 74% in Dukes' C patients. The high-risk groups for recurrence/metastasis were identified by the following criteria: (v+) and (ly+), or=se/a2, and ONCs (+) of those with >or=2 factors for Dukes' C patients (selection rate; approximately 21.2-37.5%). These factors seem to be appropriate for separating patients into high-risk and low-risk groups of colorectal cancer recurrence/metastasis.

[Survey of smoking status among preoperative patients and characteristics of smokers].

Smoking is an independent risk factor for both pulmonary and nonpulmonary perioperative complications. For safer anesthetic management, it is important to encourage and support the cessation of smoking in the preoperative period. As a first step to design a preoperative smoking cessation program, we conducted a survey of preoperative patients about smoking status and characteristics of smokers at an outpatient clinic for an anesthesiology department. The percentages of male and female smokers were 42% and 19%, respectively. Percentages of preoperative smokers considered to be in the preparation stage of smoking stage were 26% in male and 19% in female, as compared to 3% in male and 5% in female in general smokers. Twenty four % of both male and female patients were strongly committed to achieving smoking cessation. These trends in smoking characteristics indicate that preoperative smokers are more likely to quit smoking without heavy support and encouragement than general smokers. In designing a preoperative smoking cessation program, these results must be taken into consideration.

High prevalence of mutations in the EYS gene in Japanese patients with autosomal recessive retinitis pigmentosa.

PURPOSE: To screen for disease-causing mutations in the Eyes shut homolog (EYS) gene in Japanese patients with retinitis pigmentosa (RP). Methods. Blood samples were obtained from 68 RP patients and 68 controls. Genomic DNA was extracted from the blood samples and used for screening of mutations in the coding exons by direct sequencing. Each patient underwent a detailed clinical examination. RESULTS: Nine nucleotide sequence variations causing amino acid changes were observed in homozygous or heterozygous alleles in 26 patients but not in 68 controls. Seven truncating mutations were found in 21 (32.8%) of 64 patients with nonsyndromic RP composed of 23 autosomal recessive RP (arRP) and 41 sporadic cases. The most abundant mutation was p.S1653Kfs*2, which was generated by a single adenine insertion into exon 26 (c.4957dupA) and was carried by 15 patients. The mutation p.Y2935*, produced by a single nucleotide substitution (c.8805C>A) in the last exon, was carried by five patients. These two truncating mutations were probably founder mutations because each was carried by the particular haplotype. The patients with homozygous or compound heterozygous truncating mutations showed a severe decline in visual acuity, whereas those with a single truncating mutation showed a mild decline. CONCLUSIONS: One-third of Japanese patients with nonsyndromic arRP carried probable pathogenic mutations in the EYS gene, including two founder mutations. Because the genotype was correlated with the phenotype, genotyping in the EYS gene could be a valuable tool for predicting long-term prognoses of Japanese patients with arRP and thus could be useful for genetic counseling and future gene therapy.

Reconsideration of the most appropriate criterion in the lowest classification of vision disability in Japan.

PURPOSE: To verify the current Japanese classification of vision disability in regard to visual acuity. METHODS: A questionnaire was sent to 100 ophthalmology services in Japan. Each service was asked to extract 300 of their outpatient records. From these records, patients who had a sum of corrected visual acuity in both eyes of less than or equal to 0.62 were selected for the questionnaire. The questionnaire consisted of items related to prevalence, age, sex, with or without vision-disabled certification at any grade, the corrected visual acuity of each eye and the name of any disease the subject may have had. RESULTS: Sixty-five services responded, and, of 20,235 total records reviewed, 971 patients were eligible for the questionnaire. The average age was 66.9 ± 20.0 years, and 74.6% were over 60 years old. The distribution of corrected visual acuity showed three categories. CONCLUSIONS: Our analysis indicates that a new candidate criterion for vision-disabled certification is needed for the sixth grade, which, at present is defined as, "The sum of the corrected visual acuity of both eyes is more than 0.2, but less than or equal to 0.4."

Clinical Course before and after Cataract and Glaucoma Surgery under Systemic Infliximab Therapy in Patients with Behçet's Disease.

PURPOSE: Patients with Behçet's disease often need intraocular surgeries for the treatment of secondary cataract or glaucoma. This study aims to report the clinical course before and after the intraocular surgeries of 5 patients who were systematically treated with infliximab. METHODS: Retrospective case series. RESULTS: Seven eyes of 5 male patients with Behçet's disease, who underwent intraocular surgery while under systemic infliximab therapy at Yokohama City University Hospital from 2007 to 2009, were included in the study. The mean age at surgery was 44.2 years. Phacoemulsification was performed on 4 eyes, and trabeculectomy was done on the remaining 3 eyes. The mean duration since the onset of the ocular symptoms was 107 months. Control of the ocular attacks with the use of other systemic medications was difficult for all patients; however, the use of infliximab enabled adequate control of the attacks. The visual acuity status during the preoperative stage did not worsen during the postoperative period. No infectious complication was observed in all cases. CONCLUSIONS: Our results suggest that infliximab treatment does not complicate any subsequent intraocular surgery. Patients with Behçet's disease in need of intraocular surgery can benefit from control of attacks with infliximab treatment.