[Clinical analysis of corneal interface infection].

Objective: To analyze the clinical features of corneal interface infection. Methods: A retrospective case series study was conducted to explore the clinical features of interstitial corneal infection. The data of eight patients (eight eyes) who were diagnosed with interstitial corneal infection after undergoing corneal transplant or corneal refractive surgery and visited Beijing Tongren Eye Center from January to December 2018 were collected, including two male and six female patients aged between 18 and 55 years (median age, 27 years). The patients' general information, surgical type, onset time, and clinical manifestations were recorded. The lesions were examined by in vivo corneal laser confocal microscopy (IVCM), and microbial cultures and drug sensitivity tests were performed. Results: Among the 8 patients, 4 had undergone small-incision lenticule extraction (SMILE), 2 had undergone lamellar keratoplasty, and 2 had undergone endothelial keratoplasty. The onset of infection occurred between 2 and 30 days after surgery, with a mean of 9.8 days. Among the 3 patients who had undergone SMILE, the treatment outcome was corneal haze or opacity, while the remaining 5 cases required corneal transplantation for interstitial infections. The pathogens of the 4 cases of interstitial infection after corneal transplantation were all Candida species. Under the IVCM, patients with corneal interstitial bacterial infections showed a large amount of necrotic tissue with no normal tissue structure in the corneal stroma, with infiltration of inflammatory cells and local aggregation of inflammatory cells, but no typical pathogen was observed. Patients with fungal infections showed fungal hyphae under the corneal cap (filamentous fungal infection) or dense, punctate, high-reflection structures in the corneal interstitial space (yeast-like fungal infection). Conclusions: Corneal interlayer infection is difficult to diagnose early and has a poor prognosis. IVCM can assist in early diagnosis. The pathogen spectrum of corneal interlayer infection may differ from that of corneal infection caused by trauma.

[Clinical manifestations of 1 015 cases of herpes simplex virus keratitis].

Objective: To explore the demographic distribution, clinical signs, and clinical types of herpes simplex virus keratitis (HSK). Methods: Retrospective case series. The data of 1 015 cases of HSK (1 054 eyes) diagnosed in Beijing Tongren Eye Center, Beijing Tongren Hospital of Capital Medical University from January 2010 to June 2019 were collected. The patients included 613 males and 402 females, and the age was 47.43±16.79 years. Information of the patients such as age, sex, the season of onset, eye laterality, and clinical signs was assessed. Slit-lamp microscopy and corneal fluorescein staining were used to locate the anatomical position of lesions. HSK was classified into epithelial type, neurotrophic type, stromal type, endothelial type, and mixed type. The distribution data was compared by the Chi-square test or Fisher's exact test. Results: There were 41 children (≤14 years old; 4.04%), 338 youth (15-44 years old; 33.30%), 374 middle-aged (45-59 years old; 36.85%), and 262 elderly (≥60 years old; 25.81%) patients. The type was epithelial in 246 cases (24.24%), neurotrophic in 27 cases (2.66%), stromal in 372 cases (36.65%), endothelial in 274 cases (26.99%), and mixed in 96 cases (9.46%). There was statistically significant difference in clinical typing among the different age groups (χ2=30.197, P=0.003). Epithelial HSK was found in 141 males (57.32%) and 105 females (42.68%), neurotrophic HSK in 16 males (59.26%) and 11 females (40.74%), stromal HSK in 226 males (60.75%) and 146 females (39.25%), endothelial HSK in 171 males (62.41%) and 103 females (37.59%), and mixed HSK in 59 males (61.46%) and 37 females (38.54%). There was no statistically significant difference in clinical classification of keratitis between genders (χ2=1.519, P=0.823). Among the cases of mixed type, there were 21 cases of epithelial-stromal type (21.88%), 30 cases of epithelial-endothelial type (31.25%), 37 cases of stromal-endothelial type (38.54%), 1 case of epithelial-neurotrophic type (1.04%), and 7 cases of neurotrophic-stromal type (7.29%). Conclusions: HSK occurs mainly in middle-aged and young adults, but rarely in children. The proportion of males is higher than that of females. The proportion of stromal HSK is highest, and 9.46% of patients present mixed HSK.

[Diagnostic value of fungal fluorescence staining on corneal scrapings for fungal keratitis].

Objective: To analyze the sensitivity and specificity of fungal fluorescent staining in the diagnosis of fungal keratitis, and to compare it with conventional fungal culture, in vivo confocal microscopy (IVCM) and Giemsa staining. To explore its value of clinical application. Methods: Prospective case-control study. A total of 105 consecutive patients (105 eyes) diagnosed with infectious keratitis at Beijing Tongren Hospital from August 2017 to April 2018 were included. Patients with infectious keratitis were divided into fungal keratitis (FK) group and non-fungal keratitis (NFK) group by slit lamp microscopy, corneal in vivo confocal microscopy (IVCM) examination, and the results of Giemsa staining, fluorescent staining and pathogenic culture of corneal scraping from ulcer. The sensitivity and specificity of the above-mentioned examination methods for the diagnosis of fungal keratitis were analyzed. The receiver operating characteristic curve (ROC curve) and Area Under Curve (AUC) values were calculated to determine the diagnostic value of fungal fluorescent staining for fungal keratitis. Results: Among the 105 patients with infectious keratitis, 66 were fungal keratitis, 39 were non-fungal keratitis (29 cases of bacterial keratitis and 10 cases of acanthamoeba keratitis). Isolation from fungal keratitis were mainly Fusarium spp. (43.5%), followed by Alternaria spp. (21.7%) and Aspergillus spp. (19.6%). After fluorescent staining of the ulcer smear, the background of tissue demonstrated homogeneous black or weak blue fluorescence. The cell wall of fungi showed bright blue-violet to blue fluorescence, and the morphology, structure and hyphal density were easily recognized. The sensitivity of different methods for the diagnosis of corneal fungal infection were smear fluorescence staining (97.0%), IVCM (87.9%) , Giemsa staining (86.7%), and fungal culture (69.7%); the specificity of fungal culture was the highest (100%), followed by IVCM and Giemsa staining (94.9%), and fluorescent staining (87.2%). The ascending order of AUC values was: fungal culture (0.848)

[Etiological and drug sensitivity analysis of lacrimal canaliculitis].

Objective: To analyze the etiology and drug sensitivity of lacrimal canaliculitis. Methods: Retrospective study of case series. The general information, culture results and drug sensitivity results of 52 patients (including 10 males and 42 females with an average age of 60.3 years) clinically diagnosed with lacrimal canaliculitis during 2011 and 2016 at Beijing Tongren Hospital of Capital Medical University have been analyzed. The enumeration data have been tested with Chi-square method. Results: The positive rate of bacterial culture was 78.8%, and the fungal culture tests of all cases showed negative results. Sixty strains of bacteria were isolated from 41 patients whose bacterial culture tests showed positive results, Gram-positive bacteria have been confirmed as the main among the isolated bacteria with Streptococcus (18.3%), Propionibacterium (18.3%), and Streptococcus (15.0%) identified as the three common genera. Thirteen cases (25.0%, all the 13 patients were female) involved with mixed infection, 13.3% (8/60) of the isolated strains were multi-drug resistant bacteria. The drug sensitive rate of the bacteria to fluoroquinolones antibiotics(79.3%, 230/290) was higher than that to cephalosporins(62.1%, 36/58) and aminoglycoside antibiotics(56.3%, 98/174), and such differences are of statistical significance (χ(2)=7.977, 27.738, P<0.05). Except for the fact that gram-positive bacteria are mostly sensitive to vancomycin, the sensitive rate of the bacteria to gatifloxacin was the highest and that to tobramycin was the lowest. Conclusion: Lacrimal canaliculitis tend to affect women and elderly patients. Staphylococcus, Propionibacterium, and Streptococcus are the three most common genera. Gatifloxacin may be the preferred antibiotic. Antibiotics combination therapy should be applied for multi-drug resistant bacteria. (Chin J Ophthalmol, 2018, 54: 111-114).

Visfatin regulates genes related to lipid metabolism in porcine adipocytes.

Visfatin is a visceral adipose tissue-specific adipocytokine that plays a positive role in attenuating insulin resistance by binding to the insulin receptor. Visfatin has been suggested to play a role in the regulation of lipid metabolism and inflammation; however, the mechanism remains unclear. We investigated the effects of visfatin on the regulation of gene expression in cultured porcine preadipocytes and differentiated adipocytes. In preadipocytes, the mRNA abundance of lipoprotein lipase and PPARgamma were significantly increased by visfatin or insulin treatment after 8 d (all P < 0.05). In the presence of insulin, the mRNA abundance of adipocyte fatty acid-binding protein was 24.7-fold greater than in the untreated group (P < 0.05), whereas visfatin alone had no effect on adipocyte fatty acid-binding protein mRNA abundance. Adipocyte differentiation was induced by insulin treatment for 8 d. In differentiated porcine adipocytes, exposure to insulin or visfatin for 24 h increased (P < 0.05) fatty acid synthase mRNA abundance but had no effect on the expression of sterol regulatory element binding-protein 1c mRNA. We also found a 5.8-fold upregulation of IL-6 expression in porcine adipocytes after 24 h of treatment with visfatin (P < 0.05). These results demonstrated that visfatin upregulated lipoprotein lipase expression in preadipocytes, potentially facilitating lipid uptake, and increased the gene expression of fatty acid synthase in differentiated adipocytes to potentially enhance lipogenic activity. Furthermore, visfatin can upregulate IL-6 expression in differentiated porcine adipocytes. The information presented in this study provides insights into the roles of visfatin in lipid metabolism in pigs.

Pharmacokinetics of terbinafine in the rabbit ocular tissues after topical administration.

OBJECTIVE: To determine the distribution of terbinafine in the cornea and aqueous humor after topical administration. METHODS: A corn oil ointment of terbinafine 0.2% (resolved in sterile corn oil) was applied to the conjunctival sac of albino rabbits twice (with a 5-min interval). The concentration of terbinafine was determined with high-performance liquid chromatography (HPLC) 5, 15, 30, 60, 120 and 240 min after administration of terbinafine. RESULTS: After topical administration, the concentration of terbinafine increased gradually, reached a peak (1.39 microg/ml at 30 min in the cornea and 82.9 ng/ml at 30 min in aqueous humor, respectively) and then decreased. The concentration was 0.18 microg/g at 240 min in the cornea, but terbinafine could not be tested at 120 min in aqueous humor. CONCLUSIONS: Topical ophthalmic terbinafine 0.2% could penetrate into the cornea and aqueous humor at concentrations adequate for inhibition of fungus.

Identifying substrates for endothelium-specific Tie-2 receptor tyrosine kinase from phage-displayed peptide libraries for high throughput screening.

The peptide substrate specificity of Tie-2 was probed using the phage display method in order to identify efficient substrate for high throughput screening. Two random peptide libraries, pGWX3YX4 and pGWX4YX4, were constructed, in which all twenty amino acid residues were represented at the X positions flanking the fixed tyrosine residue Y. A fusion protein of GST and the catalytic domain of human Tie-2 was used to perform the phage phosphorylation. The phosphorylated phage particles were enriched by panning over immobilized anti-phosphotyrosine antibody pY20 for a total of 5 rounds. Four phage clones (3T61, 3T68, C1-90 and D1-15) that express a peptide sequence that can be phosphorylated by the recombinant catalytic domain of human Tie-2 were identified. Synthetic peptides made according to the sequences of the 4 selected clones from the two libraries, which had widely different sequences, were active substrates of Tie-2. Kinetic analysis revealed that D1-15 had the best catalytic efficiency with a k(cat)/K(m) of 5.9x10(4) M(-1) s(-1). Three high throughput screening assay formats, dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), radioactive plate binding (RPB) and time-resolved fluorescent resonance energy transfer (TR-FRET) were developed to assess the suitability of these phage display selected peptides in screening Tie-2 inhibitors. Three out of four peptides were functional in the DELFIA assay and D1-15 was functional in the TR-FRET assay.

Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library.

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide library containing 2 x 10(8) independent recombinants. A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1', P2', and P3' subsites of the peptide substrates. Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2, 4-dinitrophenyl-GPLGMRGL-NH(2) (CP), has a k(cat)/K(m) value of 4.22 x 10(6) m(-)(1) s(-)(1) for hydrolysis by collagenase 3. CP was synthesized as a consensus sequence deduced from the preferred subsites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-1, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan. Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type IV collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type II and type IV collagens.

[Study of depyrogen deactivation of syringes by using 3 efficacious pyrogen deactivator methods].

2% three-efficacious pyrogen deactivator (TEPD) was put into ultrasound washer for syringe washing. A total of 370 syringes were divided into three groups. Each group was washed by using a different program of sterilization, decontamination, and depyrogen. The results showed that the following program was not only effective for depyrogen but also convenient and inexpensive: (1)5 minutes' ultra-washing by using ultrasound washer with TEPD; (2)suspension over 1 hours; (3) another 5 minutes' ultra-washing; (4)ordinary washing and sterilization.

16S rDNA analysis of Butyrivibrio fibrisolvens: phylogenetic position and relation to butyrate-producing anaerobic bacteria from the rumen of white-tailed deer.

Complete 16S rDNA sequences of six strains of Butyrivibrio fibrisolvens, including the type strain (ATCC 19171), were determined. The type strain was found to have less than 89% sequence similarity to the other isolates that were examined. The five plasmid-bearing strains formed a closely related cluster and three of these strains (OB156, OB157 and OB192) were very highly related (> 99%), indicating that they are isolates of the same genomic species. The phylogenetic position of Butyrivibrio was found to be within the subphylum Clostridium, of Gram-positive bacteria. The closest relatives to the type strain were Eubacterium cellulosolvens and Cl. xylanolyticum and the closest relatives to the separately clustered strains were Roseburia ceciola, Lachnospira pectinoschiza and Eubacterium rectale.

Analysis by surface plasmon resonance of the influence of valence on the ligand binding affinity and kinetics of an anti-carbohydrate antibody.

The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and single-chain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (koff approximately 0.25 s-1). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 x 10(5) M-1 s-1. Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the onrate was the same as that obtained with Fab and scFv monomer.

Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

Structure of a single-chain antibody variable domain (Fv) fragment complexed with a carbohydrate antigen at 1.7-A resolution.

We describe here the 1.7-A resolution structure of a single-chain antibody variable domain (scFv) molecule, based on the carbohydrate-binding antibody Se155-4, complexed with the trisaccharide ligand alpha-D-Gal(1-->2)[alpha-D-Abe(1-->3)]alpha-D-Manp1-->OMe, where Abe is abequose. The scFv expressed in Escherichia coli has the variable region light chain to heavy chain polarity with the domains connected by a 19-residue linker. Although the linker is partially disordered in the crystal, the packing of the molecules suggests a monomeric state of the scFv. The carbohydrate adopts a different conformation about the Man-Gal linkage than was observed previously in the Fab-trisaccharide complex. Instead of a direct hydrogen bond between O2Abe and O2Gal, these two atoms are bridged by a water molecule in the present complex.

Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display.

A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, five of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77-->Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties.