In Vitro Evaluation of Platelet/Biomaterial Interactions in an Epifluorescent Video Microscopy Combined with a Parallel Plate Flow Cell

Abstract: Suitable evaluation systems are critical for ranking various biomaterials in order to develop a method to design and synthesize nonthrombogenic biomaterials. We have recently developed an in vitro test system to evaluate platelet/biomaterial interactions in whole blood. The system consists of a parallel plate flow cell and epifluorescent video microscopy (EVM). A glass coverslip coated with a polymer was incorporated into the flow cell, and blood was perfused using a syringe pump via a polymer–coated PVC tubing connected to the flow cell. Whole human blood was anticoagulated with heparin (2 U/ml), and the platelets were labeled with the fluorescent dye mepacrine (5 μM). This system permitted real–time and dynamic observations of platelet/biomaterial interactions in whole blood under a defined flow condition. In order to evaluate the feasibility of this system, two different segmented polyether–polyurethanes (SPEUs), PU–PTMG(650) and PU–PTMG(2000), were chosen as test polymers. Surface characteristics verified with electron spectroscopy for chemical analysis (ESCA) and contact angle measurements showed similar results in both SPEUs. Blood was perfused at a wall shear rate of 200 s^–1 for 20 min. Excitation light was applied for 2 s at 1 min intervals. The real–time image was then analyzed at each time point for the percentage of surface area of platelet coverage. Plasma β–thromboglobulin (β–TG) levels were also measured before and after each run. PU–PTMG(650) showed a significantly higher number of adhered platelets than PU–PTMG(2000) at each time point. β–TG levels of PU–PTMG(650) were also higher than those of PU–PTMG(2000), which is comparable to the results of EVM. Thus, this EVM system has been proven to be an excellent and highly sensitive in vitro analytical method for evaluating platelet/biomaterial interactions.     

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.     

Surgical removal of subfoveal hard exudates in diabetic maculopathy

PURPOSE: To examine the efficacy of surgical removal of subfoveal hard exudates during surgery in diabetic maculopathy. PATIENTS AND METHODS: This study was done on 60 patients (66 eyes) that all underwent surgical removal of subfoveal hard exudates under the same surgeon. Thirty-two men (37 eyes) and 28 women (29 eyes) were included in this study. The average age of the patients was 60 years (range, 30-77 years). The average follow-up period was 21 months (range, 12-48 months). All eyes were classified into 3 groups: 13 postoperative massive type eyes, 31 massive type eyes, and 22 scatter type eyes. We evaluated the visual results of these types. RESULTS: Preoperative and postoperative mean visual acuity was as follows: postoperative massive type 0.11, 0.22, massive type 0.12, 0.29, and scatter type 0.19, 0.33. Postoperatively visual acuity improved significantly in all types. There were no subfoveal hard exudates postoperatively in any eyes. CONCLUSION: Surgical removal of massive subfoveal hard exudates is effective, but its effects on vision are limited. Surgical removal of deposited subfoveal hard exudates may prevent massive subfoveal hard exudates postoperatively and improve visual prognosis.     

Reconstruction of right ventricular outflow tract with stentless xenografts in Ross procedure

To investigate whether the use of a stentless porcine aortic xenograft can be an alternative for right ventricular outflow tract (RVOT) reconstruction during the Ross procedure, 9 patients underwent the Ross procedure and RVOT reconstruction with a stentless xenograft since January 2000. After the aortic valve was replaced with a pulmonary autograft, a stentless xenograft with a xeno- pericardial roll was implanted in the RVOT. One patient required subsequent aortic valve replacement because of severe regurgitation of the pulmonary autograft. All patients recovered well from the operation. The right ventricle-pulmonary arterial pressure gradient was 18 +/- 7 mm Hg at discharge and was not significantly increased during the 2-year follow-up period. Although 1 patient died of ventricular arrhythmia 5 months after, his cardiac function was normal, and transpulmonary valve pressure was 19 mm Hg in the follow-up. The other 7 patients are currently in New York Heart Association functional Class I. Although long-term follow-up is required to explain the durability, the stentless xenograft with a pericardial roll is considered to be an alternative for reconstruction of the RVOT within 2 years after the Ross procedure.     

Invasive pulmonary aspergillosis associated with influenza B

Invasive pulmonary aspergillosis (IPA) usually occurs in immunocompromised patients. However, rarely, this infection can occur in normal hosts. This review of the literature identified 13 cases of IPA associated with influenza, of which 12 had influenza A and the type of influenza was not mentioned in the other case. Reported here is a case of IPA, which was associated with influenza B, in a 63-year-old immunocompetent woman. Her lungs showed gross invasion and she was treated with itraconazole and amphotericin B. She required mechanical ventilation for about 5 months but recovered completely. This is the first reported case of IPA associated with influenza B.     

Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2002

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 899 strains of Gram-positive bacteria, 1500 strains of Gram-negative bacteria, and 158 strains of anaerobic bacteria obtained from 28 medical institutions during 2002 was measured. The results were as follows; 1. MEPM was more active than other carbapenem antibiotics against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MIC90 of MEPM against Pseudomonas aeruginosa was the lowest of the drugs tested. MEPM showed low cross-resistant rate against both imipenem-resistant P. aeruginosa and ciprofloxacin-resistant P. aeruginosa. MEPM was active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE). 2. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (4 strains) in Escherichia coli and 1.9% (2 strains) in Klebsiella pneumoniae. Carbapenems including MEPM were active against these ESBL strains. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem; at present, 7 years after available for commercial use.     

Subretinal washout for subtle subfoveal hard exudates in diabetic macular edema

PURPOSE: To evaluate the efficacy of subretinal washout for subtle subfoveal hard exudates in diabetic macular edema. METHODS: This study was done retrospectively on a series of patients with diffuse diabetic macular edema accompanied with subtle subfoveal hard exudates and operated on by one surgeon(NO). Patients ranged in age from 30 to 76 years(mean, 59 years). The postoperative follow-up interval ranged from 12 to 76 months, with a mean of 35 months. Two groups were identified. The first group contained all 26 eyes that had vitreous surgery with subretinal washout. This was compared with a second group of 51 eyes without subretinal washout. We compared the rate of occurrence of postoperative massive foveal hard exudates, visual acuity results, and complications with and without subretinal washout. RESULT: There was no significant difference in base line demographics between the two groups. Massive foveal hard exudates did not occur in eyes with subretinal washout, but occurred in 29 (57%) of the eyes without subretinal washout(p < 0.0001) and in 15 eyes which had undergone reoperation with subretinal washout. Visual acuity improved in 54% of the subretinal washout eyes and 45% of the eyes without it. Visual acuity improved to 20/30 or better in 23% of the eyes with subretinal washout and in 8% of the eyes without subretinal washout. There was no serious complication related to subretinal washout. CONCLUSION: Subretinal washout for subtle subfoveal hard exudates in diabetic macular edema may prevent massive subfoveal exudates and improve visual results. Further study is needed to investigate the pathogenesis.     

Strabological findings after macular translocation surgery with 360 degrees retinotomy

PURPOSE: To examine the strabological findings after macular translocation surgery with a 360 degrees retinotomy. METHODS: Thirty-two patients who underwent macular translocation surgery were divided into three groups based on their responses to the Bagolini striated lenses test: fusion, ignoring the image, and diplopia. The relevant factors affecting binocularity were compared among the three groups. RESULTS: Five patients had peripheral fusion and three of these had gross stereopsis. Fifteen patients ignored the second image, and 12 patients had diplopia. The objective angle of macular rotation was smaller in the patients with peripheral fusion (15.0 +/- 6.1 degrees) than in those with diplopia (32.7 +/- 11.7 degrees). The subjective angle of cyclotorsion in those with peripheral fusion (6.0 +/- 4.2 degrees) was smaller than in those who ignored the image ("ignoring" group; 20.5 +/- 9.19 degrees) and the diplopia group (30.7 +/- 12.8 degrees). The amount of torsional sensory compensation in patients with diplopia (2.08 +/- 3.83 degrees) was significantly smaller than in those with peripheral fusion (9.00 +/- 7.42 degrees) and in the ignoring group (6.73 +/- 3.86 degrees). Patients with peripheral fusion were significantly younger (54.2 +/- 14.3 years) than those in the ignoring group (67.7 +/- 10.0 years) and those with diplopia (68.0 +/- 5.4 years). CONCLUSIONS: Adaptive mechanisms are activated to reduce the surgically induced objective angle of cyclotorsion, and a cyclodeviation of 15 degrees was the critical angle separating those who had peripheral fusion from those who did not. This value corresponds to the cyclofusional amplitude in normal adults.     

Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis

It has been shown that osteopontin (OPN) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). However, the molecular mechanism of OPN action is yet to be elucidated. Splenic monocytes obtained from arthritic mice exhibited a significant capacity for cell migration toward thrombin-cleaved OPN but not toward full-length OPN. Migratory monocytes expressed alpha9 and alpha4 integrins. Since cleavage of OPN by thrombin exposes the cryptic epitope recognized by alpha9 and alpha4 integrins, we investigated the role of the cryptic epitope SLAYGLR in a murine RA model by using a specific antibody (M5) reacting to SLAYGLR sequence. The M5 antibody could abrogate monocyte migration toward the thrombin-cleaved form of OPN. Importantly, M5 antibody could inhibit the proliferation of synovium, bone erosion, and inflammatory cell infiltration in arthritic joints. Thus, we demonstrated that a cryptic epitope, the SLAYGLR sequence of murine OPN, is critically involved in the pathogenesis of a murine model of RA.