Estimating energetics in cetaceans from respiratory frequency: why we need to understand physiology.

The accurate estimation of field metabolic rates (FMR) in wild animals is a key component of bioenergetic models, and is important for understanding the routine limitations for survival as well as individual responses to disturbances or environmental changes. Several methods have been used to estimate FMR, including accelerometer-derived activity budgets, isotope dilution techniques, and proxies from heart rate. Counting the number of breaths is another method used to assess FMR in cetaceans, which is attractive in its simplicity and the ability to measure respiration frequency from visual cues or data loggers. This method hinges on the assumption that over time a constant tidal volume (VT) and O2exchange fraction (ΔO2) can be used to predict FMR. To test whether this method of estimating FMR is valid, we measured breath-by-breath tidal volumes and expired O2levels of bottlenose dolphins, and computed the O2consumption rate (V̇O2 ) before and after a pre-determined duration of exercise. The measuredV̇O2 was compared with three methods to estimate FMR. Each method to estimateV̇O2 included variable VT and/or ΔO2 Two assumption-based methods overestimatedV̇O2 by 216-501%. Once the temporal changes in cardio-respiratory physiology, such as variation in VT and ΔO2, were taken into account, pre-exercise restingV̇O2 was predicted to within 2%, and post-exerciseV̇O2 was overestimated by 12%. Our data show that a better understanding of cardiorespiratory physiology significantly improves the ability to estimate metabolic rate from respiratory frequency, and further emphasizes the importance of eco-physiology for conservation management efforts.

Ocular penetration of levofloxacin, ofloxacin and ciprofloxacin in eyes with functioning filtering blebs: investigator masked, randomised clinical trial.

BACKGROUND/AIMS: To compare the penetration of levofloxacin, ofloxacin and ciprofloxacin in the aqueous humour of eyes with functioning filtering blebs. METHODS: In this investigator-masked study, 48 patients with functioning filtering blebs requiring cataract surgery were randomised into six groups of eight patients. Groups 1, 2 and 3 received topical ofloxacin 0.3% (Ocuflox), ciprofloxacin 0.3% (Ciloxan) and levofloxacin (Quixin) respectively; Groups 4, 5 and 6 received the same treatment with the corresponding oral dose of ofloxacin 400 mg (Floxin), ciprofloxacin 400 mg (Cipro) and levofloxacin 250 mg (Levaquin). Aqueous antibiotic levels were determined by mass spectrometry of aqueous samples from each patient. RESULTS: The mean aqueous level for topical levofloxacin was significantly higher than those achieved by topical ofloxacin or ciprofloxacin (p value = 0.02 and 0.01, respectively). The combination of topical and oral levofloxacin was significantly higher than topical levofloxacin alone (p = 0.05) and the ciprofloxacin combination (p = 0.003) but not significantly higher than the ofloxacin combination therapy. CONCLUSIONS: Topical levofloxacin penetrates better than ofloxacin or ciprofloxacin into the aqueous of eyes with functioning filtering blebs. The combination of topical and oral levofloxacin may be preferable in the treatment of bleb-associated infections (NCT 00392275; Clinical trials.gov).

Intraocular pressure-lowering efficacy of bimatoprost 0.03% and travoprost 0.004% in patients with glaucoma or ocular hypertension.

AIM: To evaluate the efficacies of bimatoprost and travoprost for lowering of intraocular pressure (IOP) for the treatment of glaucoma and ocular hypertension. METHODS: Prospective, randomised, investigator-blinded, parallel-group clinical trial. After completing a washout of all glaucoma drugs, patients (n = 157) were randomised to bimatoprost or travoprost for 6 months. Visits were at baseline, 1 week, and 1, 3 and 6 months. IOP was measured at 09:00 h at each visit and also at 13:00 and 16:00 h at baseline and at 3 and 6 months. RESULTS: No significant between-group differences were observed in IOP at baseline, at 09:00, 13:00 or 16:00 h (p> or =0.741). After 6 months, both drugs significantly reduced IOP at every time point (p< or =0.001). After 6 months, mean IOP reduction at 09:00 h was 7.1 mm Hg (27.9%) with bimatoprost (n = 76) and 5.7 mm Hg (23.3%) with travoprost (n = 81; p = 0.014). At 13:00 h, mean IOP reduction was 5.9 mm Hg with bimatoprost (25.3%) and 5.2 mm Hg (22.4%) with travoprost (p = 0.213). At 16:00 h, the mean IOP reduction was 5.3 mm Hg (22.5%) with bimatoprost and 4.5 mm Hg (18.9%; p = 0.207) with travoprost. Both study drugs were well tolerated, with ocular redness the most commonly reported adverse event in both treatment groups. CONCLUSIONS: Bimatoprost provided greater mean IOP reductions than travoprost.

Determination of threshold values for determining the size of the fraction of steroid hormone receptor-positive tumor cells in paraffin-embedded breast carcinomas.

BACKGROUND: For 4 years we used a multiparameter DNA flow cytometric (MP-FCM) technique to assess steroid hormone receptor expression in the diagnostic workup of routinely processed formalin-fixed, paraffin-embedded breast carcinomas as an alternative to immunohistochemistry (IHC) for the quantification of hormone receptor-positive cells. In all cases a positive fraction of hormone receptor-expressing epithelial cells was detected. This observation raised the question of what the cutoff value might be to distinguish receptor-negative from receptor-positive tumors. METHODS: In our search for a possible threshold value of positivity for estrogen receptor (ER) and progesterone receptor (PR) in MP-FCM, we developed four steps. First, we compared IHC results in our own laboratory with the results obtained by MP-FCM on a small series of breast tumors (n = 42). Second, after collecting our first 843 tumors, we made a comparison with the literature of the distribution of receptor positivity according to age classes. Third, using the most likely threshold that resulted from this comparison, we compared a subset of 340 node-negative tumors for their combined ER/PR profiles with the data from a similar group of node-negative tumor cases from the National Cancer Institute's Surveillance, Epidemiology and End-Result (SEER) study. Fourth, with the results of these comparisons, we prospectively collected IHC data and MP-FCM results of the same tumor samples for a period of 1 year. In this way, we collected data for an additional 180 tumors. RESULTS: The first step in this process resulted in an previous publication where 20% of steroid hormone receptor-positive cells seemed to be an acceptable cutoff point for positivity. However, the second step provided the best correlation at approximately 35% of ER reactive cells in the cytokeratin-positive cell population. With this cutoff, the distribution of combined ER/PR profiles in our patient population of node-negative breast cancers also showed a distribution similar to the data from the SEER study. The fourth step, using the 35% threshold value, resulted in a good correlation (r = 0.85, P < 0.0001) for ER and PR between IHC and MP-FCM in the 180 tumors investigated. CONCLUSION: By comparing in-house data with those from large external data collections in the literature, a threshold percentage can be defined that distinguishes steroid hormone receptor-negative from hormone receptor-positive tumors. As a result, information about DNA content and cell cycle distribution can be obtained. This observational study provides additional support to our opinion that MP-FCM is an alternative for IHC determination of ER and PR positivity. It is more objective and quantification can be done more appropriately. The additional value of this approach is that we generate continuous variables of ER/PR content instead of categorical classes, which can be used at different threshold levels for evaluation of clinical relevance.

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer.

AIM: To investigate whether multiparameter flow cytometry (MP-FCM) can be used for the detection of micrometastasis in sentinel lymph nodes (SLNs) in breast cancer. METHODS: Formalin fixed, paraffin wax embedded sentinel lymph nodes (n = 238) from 98 patients were analysed. For each lymph node, sections for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for cytokeratin (MNF116) were cut at three levels with a distance of 500 microm. The intervening material was used for MP-FCM. Cells were immunostained with MNF116, followed by an incubation with fluorescein isothiocyanate (FITC) labelled goat antimouse immunoglobulin. DNA was stained using propidium iodide. From each lymph node 100,000 cells were analysed on the flow cytometer. RESULTS: Thirty eight of the 98 patients with breast carcinoma showed evidence of metastatic disease in the SLN by one ore more of the three methods. In 37 of 38 cases where metastatic cells were seen in the routine H&E and/or IHC, more than 1% cytokeratin positive cells were detected by MP-FCM. In 24 patients, metastatic foci were more than 2 mm (macrometastasis) and in 14 these foci were smaller than 2 mm (micrometastasis). In three of these 14 cases, MP-FCM revealed positive SLNs, although this was not seen at first glance in the H&E or IHC sections. After revision of the slides, one of these three remained negative. However, MP-FCM analysis of the cytokeratin positive cells showed an aneuploid DNA peak, which was almost identical to that of the primary breast tumour. Duplicate measurements, done in 41 cases, showed a 99% reproducibility. In five of 14 patients with micrometastasis, one or two metastatic foci were found in the non-SLN. However, in 15 of 24 macrometastases multiple non-SLNs were found to have metastatic tumour. All micrometastases except for the remaining negative one mentioned above showed only diploid tumour cells, despite the fact that their primary tumours contained both diploid and aneuploid tumour cells. In primary tumours with more than 60% aneuploid cells, predominantly aneuploid macrometastasis were found, whereas diploid primary tumours only showed diploid micrometastases or macrometastases in their SLN. Aneuploid SLN macrometastases were associated with non-SLN metastases in five of seven patients, whereas diploid cases showed additional non-SLN metastases in only seven of 16 patients. CONCLUSION: In all cases, MP-FCM was sufficient to detect micrometastatic tumour cells in a large volume of lymph node tissue from SLNs. In some cases it was superior to H&E and IHC staining. Approximately 30% of SLN micrometastases are accompanied by additional non-SLN metastases. The size of the aneuploid fraction (> 60%) in the primary tumour may influence the risk of having both SLN and non-SLN metastases.

Measurement of iris color using computerized image analysis.

PURPOSE: To develop a standardized method for measuring from iris photographs, light and dark segments of areas and densities of iris color. METHODS: Computerized image analysis was used to measure the iris photographs. The reproducibility of this method was studied in 30 normal eyes with three different colored irides, green-brown, blue-grey brown and yellow-brown. Three photographs were taken of each iris with a slit lamp camera at three different exposures at baseline. The photographs were repeated with exposure providing for the best reproducibility at 6.5 +/- 1.7 months as a first follow-up after baseline and 3.6 +/- 0.8 months following the first follow-up visit. At least one measurement was made for each photograph. RESULTS: The mean percent coefficient of variation (standard deviation of triplicate measurements/mean x 100) ranged from 1.0 to 4.1% for area and density measurements. Furthermore, the range of mean percent differences between baseline and follow-up visits ranged from 1.2 to 6.3%. CONCLUSION: We have developed a standardized method which appears suitable for measuring changes over time in iris color.

Penetration of ofloxacin and ciprofloxacin into the aqueous humor of eyes with functioning filtering blebs: a randomized trial.

OBJECTIVE: To determine concentrations of ofloxacin and ciprofloxacin hydrochloride in aqueous humor after topical or combined topical and oral administration in eyes with filtering blebs. DESIGN: A prospective, investigator-masked, randomized, controlled comparative study involving 36 eyes of 34 patients with functioning filtering blebs who were to undergo cataract surgery. Treatment groups received either topical ofloxacin or topical ciprofloxacin (instillation of 0.3% ophthalmic solution every 30 minutes for 4 hours before surgery), or a combined topical plus oral regimen (ciprofloxacin hydrochloride, four 100-mg tablets, or ofloxacin, one 400-mg tablet, administered 24-26, 12-14, and 2 hours preceding surgery). The main outcome measure was antibiotic concentration measured by chromatographic separation and mass spectrometry of aqueous samples obtained during surgery. RESULTS: Topical antibiotic treatment yielded mean concentrations of ofloxacin, 0.75 microg/mL, and ciprofloxacin, 0.21 microg/mL, in aqueous. With combined topical and oral therapy, significantly more ofloxacin was measured than ciprofloxacin (3.84 microg/mL vs 0.35 microg/mL [P<.001]). The combination regimen produced significantly greater ofloxacin levels than did topical therapy alone (P =.007). CONCLUSIONS: Ofloxacin penetrates better than ciprofloxacin into the aqueous of eyes with filtering blebs, particularly after combined topical and oral administration, by which ofloxacin reaches more than a 10-fold greater concentration than does ciprofloxacin. Combined topical and oral therapy with ofloxacin may be beneficial in the treatment of bleb-associated infections.

Comparison of the clinical success and quality-of-life impact of brimonidine 0.2% and betaxolol 0.25 % suspension in patients with elevated intraocular pressure.

BACKGROUND: Brimonidine tartrate 0.2%, a selective alpha2-adrenergic receptor agonist, and betaxolol 0.25% suspension, a cardioselective beta1-adrenergic receptor antagonist, are used in the treatment of elevated intraocular pressure (IOP). OBJECTIVE: This study compared the clinical success and quality-of-life impact of 4 weeks of treatment with brimonidine 0.2% BID compared with those of 4 weeks of treatment with betaxolol 0.25% suspension BID in patients with elevated IOP. METHODS: This was a multisite, double-masked, comparative clinical trial in patients with glaucoma or ocular hypertension. Patients were randomly assigned to receive either brimonidine or betaxolol BID. Morning IOP was measured at baseline and at weeks 1 and 4 using Goldmann applanation. Efficacy was determined by reduction in IOP from baseline. Patients experiencing a > or =20% reduction in IOP were considered to have a successful IOP-lowering response. The Glaucoma Disability Index questionnaire was administered at week 4 to assess quality-of-life factors and the incidence of adverse events. Ophthalmic examinations were conducted at each visit. RESULTS: One hundred fifty-nine patients were randomized to treatment and completed the study, 81 receiving brimonidine and 78 receiving betaxolol. The majority were white (77.4%) and female (61.6%), and had a diagnosis of open-angle glaucoma (56.0%). After 4 weeks of treatment, both brimonidine and betaxolol effectively lowered IOP from baseline (mean IOP reductions: brimonidine, 5.96 mm Hg; betaxolol, 5.07 mm Hg; P = NS). However, a significantly higher percentage of brimonidine patients (52/81 [64.2%]) than betaxolol patients (37/78 [47.4%]) had a > or =20% reduction in IOP (P = 0.033). No serious adverse events were reported with either study medication. On the quality-of-life assessments, more betaxolol patients reported hyperemia (P = 0.011), and the reported hyperemia was significantly more severe in betaxolol patients (P = 0.009). CONCLUSIONS: After 4 weeks of treatment, brimonidine 0.2% BID was clinically successful in significantly more patients and was better tolerated than 4 weeks of treatment with betaxolol 0.25% BID in this population.

Cataract surgery after tube shunts.

PURPOSE: To determine the effect of cataract surgery on glaucomatous eyes with functioning tube shunts. METHODS: Retrospective analysis of 11 eyes of 11 patients with functioning tube shunts who underwent cataract extraction. RESULTS: The mean follow-up after cataract extraction was 21 +/- 27 months (range, 4-97 months). There was no statistically significant difference between the mean preoperative intraocular pressure and mean postoperative intraocular pressure (17.4 +/- 3.7 mm Hg vs 17.8 +/- 5.9 mmHg; P = 0.85, paired t test). Most patients exhibited a statistically significant rise or drop in pressure, but in none was there a clinically significant change. The mean number of preoperative and postoperative antiglaucoma medications was also not significantly different (1.5 +/- 1.1 vs 1.7 +/- 1.2; P = 0.44, paired t test). Snellen visual acuity improved at least 2 lines in 6 eyes (55%). Complications after cataract extraction included corneal edema in three eyes, one of which had subsequent loss of control of intraocular pressure. CONCLUSIONS: Eyes with a functioning tube shunt undergoing cataract extraction can maintain control of intraocular pressure while achieving visual improvement.

Prospective evaluation of extraocular motility following double-plate molteno implantation.

OBJECTIVE: To determine the incidence and type of extraocular motility disturbance after double-plate Molteno implantation. METHODS: In a prospective clinical series, we evaluated preoperative and postoperative ocular motility at 3 and 6 months in 24 eyes of 24 patients undergoing double-plate Molteno implantation. Visual acuity, motility testing, and subjective and objective diplopia were evaluated at each examination. RESULTS: Within the first 6 months postoperatively, new or worse strabismus developed in 11 (46%) of the 24 study patients. Three of the 11 patients had a generalized restriction of the superior rectus and the superior oblique muscles, all of which persisted 6 months after surgery. Four patients had clinical features consistent with an acquired Brown syndrome, and 6 months after surgery, 3 of the 4 patients had a residual deviation, although the deviation in 1 patient resolved. A superior oblique palsy developed in 3 patients, and a lateral rectus palsy developed in 1 patient. All 4 of the muscle palsies resolved or were resolving during the follow-up period, which ranged from 6 to 12 months. CONCLUSIONS: Extraocular motility disturbances are not rare after double-plate Molteno surgery. Muscle palsies, acquired Brown syndromes, and generalized restrictions occurred in similar proportions. CLINICAL RELEVANCE: Patients should be counseled before Molteno surgery concerning the risk of strabismus and diplopia.

Mitomycin C in higher risk trabeculectomy: a prospective comparison of 0.2- to 0.4-mg/cc doses.

PURPOSE: This randomized, masked, prospective study was conducted to compare the outcome of filtering surgery using doses of 0.2 mg/cc or 0.4 mg/cc of mitomycin C (MMC) in eyes that were at higher risk from previous conjunctival incisional surgery. METHODS: Eyes of 50 consecutive patients with primary open-angle, pseudoexfoliation, or pigmentary glaucoma requiring trabeculectomy who had previously undergone either limbal cataract surgery or trabeculectomy were enrolled. Patients received an intraoperative dose of either 0.2 or 0.4 mg/cc MMC for 2 minutes (n = 25 in each study group). Intraocular pressure (IOP), logMAR visual acuity, and complications were monitored at regular intervals for 1 year. Unpaired student t tests were used to compare percent decrease in IOP in both study groups at each interval. RESULTS: The percent decrease in IOP was not significantly different between groups at 1 day, 1 week, 1 month, 3 months, 6 months, or 1 year after surgery. LogMAR visual acuity was not significantly different between groups at 1 month, 6 months, or 1 year. Treatment failure occurred in seven patients in the 0.2 mg/cc group (28%) and seven patients in the 0.4 mg/cc group (29.2%). Postoperative hypotony, choroidal effusions and hemorrhages, and wound leaks occurred in both groups, but occurred more often in the group receiving 0.4 mg/cc MMC. CONCLUSION: Filtering surgery performed on higher risk eyes was as effective using a lower dose (0.2 mg/cc) of MMC as that using a higher dose (0.4 mg/cc). Incidence of complications and treatment failures was slightly higher in the group receiving high-dose MMC.

The effect of mitomycin C on Molteno implant surgery: a 1-year randomized, masked, prospective study.

PURPOSE: The authors assess whether adjunctive mitomycin C improves Molteno tube shunt surgery in terms of intraocular pressure (IOP), visual acuity, and complication rates. PATIENTS AND METHODS: Twenty-five eyes of twenty-five consecutive patients undergoing double-plate Molteno implant surgery were randomized to receive either mitomycin C (MMC) 0.4 mg/mL for 2 minutes or a control balanced salt solution in a masked, prospective study. Intraocular pressure, visual acuity, and complications were recorded 1 week and 1, 3, 6, and 12 months after surgery. A repeated measures analysis of variance (ANOVA) model was used to test the overall effect of the drug on IOP and percent change from preoperative IOP. RESULTS: Thirteen eyes received balanced salt solution and 12 eyes received MMC. There was no difference between the groups with respect to age, preoperative IOP, log mean angle of resolution (LogMar) visual acuity, or number of preoperative medications. Except for week 1, there were no differences between the groups at any of the clinic visits with respect to IOP and percent change from baseline IOP. Analysis of the visual acuity (LogMar) showed reduction in vision for both groups. Complications were similar in each group, as were number of postoperative hypotensive agents required. CONCLUSIONS: Adjunct MMC does not demonstrate a significant difference in outcomes compared with placebo in pressure-ridged Molteno implant surgery. Results of this study are limited by a small number of patients in each group and a fixed dose of MMC.

Comparison of T cell responses in patients with a long-term surviving renal allograft versus a long-term surviving liver allograft. It's a different world.

The aim of the present study was to analyze whether acquired transplantation tolerance had developed in patients with a long-term surviving renal or liver allograft. Analysis of antidonor cytotoxic T cell precursor frequencies was performed in 31 renal allograft recipients and 9 liver allograft recipients with good graft function 2 years after transplantation. The results demonstrated that, before transplantation, normal antidonor T cell responses were generated in both groups of patients. Two years after transplantation, donor-specific CTL nonresponsiveness had developed in a minority of the renal transplant recipients. In contrast, 8 out of 9 liver transplant recipients showed donor-specific mixed lymphocyte culture and CTL nonresponsiveness. These findings indicate that development of donor-specific T cell nonresponsiveness is not a common event after kidney transplantation, whereas liver transplantation seems to induce, at least in vitro, a state of donor-specific T cell nonresponsiveness.

Nonresponsiveness to the male antigen H-Y in H-2 I-A-mutant B6.C-H-2bm12 is not caused by defective antigen presentation.

The B6.C-H-2bm12 (bm12), H-2 I-Ab mutant, originated in the C57BL/6 (B6, H-2b) strain, is a nonresponder to the male antigen H-Y in both T cell proliferation and cytotoxic T lymphocyte (CTL) responses. Defective H-Y presentation by bm12-adherent cells, as a cause of the CTL nonresponsiveness, can be excluded, because 1) CTL from primed responder/nonresponder F1 female mice (B6/bm12 F1) were activated by H-Y antigen on antigen-presenting cells (apc) from either parent; 2) T cells from primed nonresponder bm12 females did not generate CTL to H-Y presented by responder B6 apc, but conversely, CTL from B6 females could be activated by antigen on bm12 apc; and 3) adherent cell-depletion experiments indicated that male adherent cells are necessary for generation of optimal H-Y-specific CTL responses, male adherent bm12 cells being equally as efficient as male adherent B6 cells in presentation to F1 T cells. The need for T helper cell activation by H-Y-presenting adherent cells during secondary in vitro restimulation can be circumvented by interleukin 2 (IL 2), because IL 2 completely restored the H-Y-specific CTL response of B6/bm12 F1 cells in cultures depleted of adherent cells. However, addition of IL 2 during in vitro restimulation of bm12 cells did not result in an H-Y-specific CTL response, indicating that H-Y-specific, I-A-restricted T helper cells are probably needed in vivo during priming for the generation of sufficient numbers of Db-restricted CTL memory cells. The data are compatible with the concept that H-Y antigen is presented in the context of the I-A alpha, beta molecule on the surface of adherent cells to T helper cells and in the context of the Db molecule on the surface of nonadherent as well as adherent cells to CTL precursors. The most likely explanation for the difference in H-Y response between B6 and bm12 include positive selection of the responder phenotype by the I-Ab alpha, beta molecule of the B6 strain or generation of suppressor T cells in the bm12 strain.