Reprint of ""Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury""

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields alpha-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at 1 week after surgery. (C) 2009 Elsevier Ltd. All rights reserved.

Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields a-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at I week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at I week after surgery. (C) 2009 Elsevier Ltd. All rights reserved.

Ranolazine Exerts Potent Effects on Atrial Electrical Properties and Abbreviates Atrial Fibrillation Duration in the Intact Porcine Heart

Introduction: In vitro studies and ambulatory ECG recordings from the MERLIN TIMI-36 clinical trial suggest that the novel antianginal agent ranolazine may have the potential to suppress atrial arrhythmias. However, there are no reports of effects of ranolazine on atrial electrophysiologic properties in large intact animals. Methods and Results: In 12 closed-chest anesthetized pigs, effects of intravenous ranolazine (similar to 9 mu M plasma concentration) on multisite atrial effective refractory period (ERP), conduction time (CT), and duration and inducibility of atrial fibrillation (AF) initiated by intrapericardial acetylcholine were investigated. Ranolazine increased ERP by a median of 45 ms (interquartile range 29-50 ms; P < 0.05, n = 6) in right and left atria compared to control at pacing cycle length (PCL) of 400 ms. However, ERP increased by only 28 (24-34) ms in right ventricle (P < 0.01, n = 6). Ranolazine increased atrial CT from 89 (71-109) ms to 98 (86-121) ms (P = 0.04, n = 6) at PCL of 400 ms. Ranolazine decreased AF duration from 894 (811-1220) seconds to 621 (549-761) seconds (P = 0.03, n = 6). AF was reinducible in 1 of 6 animals after termination with ranolazine compared with all 6 animals during control period (P = 0.07). Dominant frequency (DF) of AF was reduced by ranolazine in left atrium from 11.7 (10.7-20.5) Hz to 7.6 (2.9-8.8) Hz (P = 0.02, n = 6). Conclusions: Ranolazine, at therapeutic doses, increased atrial ERP to greater extent than ventricular ERP and prolonged atrial CT in a frequency-dependent manner in the porcine heart. AF duration and DF were also reduced by ranolazine. Potential role of ranolazine in AF management merits further investigation. (J Cardiovasc Electrophysiol, Vol. 20, pp. 796-802, July 2009).

Dynamics of Parathyroid Hormone Secretion After Total Parathyroidectomy and Autotransplantation

Secondary hyperparathyroidism is a common complication in uremic patients. Total parathyroidectomy combined with partial autotransplantation into brachioradialis muscle has been the preference among the options for surgical treatment. This study was designed to evaluate the reserve and ability of suppression of autotransplanted parathyroid tissue using dynamics tests. We studied, prospectively, 12 patients in recent (RP) and late (LP) postoperative of total parathyroidectomy with autotransplantation. For analysis of the secretory reserve capacity, we induced hypocalcemia by ethylenediaminetetraacetic acid (EDTA) infusion. Furthermore, for analysis of the ability for parathyroid hormone (PTH) suppression, the hypercalcemia test was used, by intravenous administration of calcium in LP. In RP, there was a decrease in the average serum levels of PTH, phosphorus, and alkaline phosphatase, which ranged from 13 to 231 (87 +/- A 65) pg/ml, 2.3 to 6.2 (3.3 +/- A 1.1) mg/dl, and 77 to 504 (250 +/- A 135) U/L, respectively, similar to that observed in LP. The analysis of the average curve of variations in PTH during testing of the stimulus with EDTA showed lack of secretion in RP and partial response in LP. Impaired suppression ability of the graft in LP was observed in the test with intravenous calcium. Total parathyroidectomy followed by partial autotransplantation was effective in reducing PTH serum levels in patients with terminal kidney disease. The elevation of serum calcium during the suppression test was not able to inhibit the autograft gland secretion of PTH. The assessment of parathyroid graft function demonstrated an inability to respond to the stimulus of hypocalcemia induced by EDTA, although there was a partial recovery, in late postoperative period.

Influence of third-generation oral contraceptives on the complexity analysis and symbolic dynamics of heart rate variability

Objective To evaluate the influence of oral contraceptives (OCs) containing 20 mu mu g ethinylestradiol (EE) and 150 mu mu g gestodene (GEST) on the autonomic modulation of heart rate (HR) in women. Methods One-hundred and fifty-five women aged 24 +/-+/- 2 years were divided into four groups according to their physical activity and the use or not of an OC: active-OC, active-non-OC (NOC), sedentary-OC, and sedentary-NOC. The heart rate was registered in real time based on the electrocardiogram signal for 15 minutes, in the supine-position. The heart rate variability (HRV) was analysed using Shannon`s entropy (SE), conditional entropy (complexity index [CInd] and normalised CInd [NCI]), and symbolic analysis (0V%, 1V%, 2LV%, and 2ULV%). For statistical analysis the Kruskal-Wallis test with Dunn post hoc and the Wilcoxon test (p < 0.05 was considered significant) were applied. Results Treatment with this COC caused no significant changes in SE, CInd, NCI, or symbolic analysis in either active or sedentary groups. Active groups presented higher values for SE and 2ULV%, and lower values for 0V% when compared to sedentary groups (p < 0.05). Conclusion HRV patterns differed depending on life style; the non-linear method applied was highly reliable for identifying these changes. The use of OCs containing 20 mu mu g EE and 150 mu mu g GEST does not influence HR autonomic modulation.

Determination of Cyclophosphamide Enantiomers in Plasma by LC-MS/MS: Application to Pharmacokinetics in Breast Cancer and Lupus Nephritis Patients

This article describes the enantioseleclive analysis of cyclophosphamide (CPA) in human plasma using LC-MS/MS. CPA enantiomers were extracted from plasma using a mixture of ethyl acetate and chloroform (75:25, v/v). The enantiomers were separated on a Chiralcel(R) OD-R column, with the mobile phase consisting of a mixture of acetonitrile and water (75:25, v/v) plus 0.2% formic acid. The protonaled ions and their respective product ions were monitored using two functions, 261 > 141 for CPA enantiomers and 189 > 104 for the internal standard (antipyrine). Recovery rates were higher than 95% and the quantification limit was 2.5-ng/ml plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of intra- and interassay precision and accuracy were less than 10%. The method was applied for the investigation of the enantioselective pharmacokinetics of CPA in a lupus nephritis patient treated with 1 g CPA infused over 2 h and in a breast cancer patient treated with 0.9 g infused over 1 h. No stereoselectivity in the pharmacokinetic parameters was observed for either patient. Clearance values of 2.63 and 2.93 l/h and of 3.36 and 3.61 l/h for (-)-(S) and (+)-(R)-CPA were obtained for the breast cancer and lupus nephritis patient., respectively. Chirality 21:383-389, 2009. (C) 2008 Wiley-Liss, Inc.

Stereoselective Analysis of Labetalol in Human Plasma by LC-MS/MS: Application to Pharmacokinetics

Labetalol is clinically available as a mixture of two racemates (four stereoisomers). The stereoisomer (R,R) has as main activity the beta(1)-antagonism and the stereoisomer (S,R) is highly selective for the alpha(1) adrenoceptor and is responsible for most of the alpha-blocker activity. In the present investigation, a method for the analysis of labetalol stereoisomers in human plasma was developed and applied to pharmacokinetic studies. Plasma samples (0.5 ml) were extracted with methyl tert-butyl ether at pH 9.5. The four labetalol stereoisomers were analyzed by LC-MS/MS on a Chirobiotic (R) V column using a mobile phase consisting of methanol, acetic acid, and diethylamine, with a recovery of more than 90% for all four. The quantitation limit was 0.5 ng/ml and linearity was observed at 250 ng/ml plasma for each stereoisomer. Studies of precision and accuracy presented coefficients of variation and percentage inaccuracy of less than 15%, indicating that the method is precise and accurate. The method was applied to the study of the kinetic disposition of labetalol over a period of 12 h after oral administration of a single 100 mg dose to a hypertensive pregnant woman. The clinical study revealed stereoselectivity in the pharmacokinetics of labetalol, with a lower plasma proportion for the active stereoisomers (R,R)-labetalol and (S,R)-labetalol. The stereoselectivity observed after oral administration is due to the hepatic metabolism and the first pass effect, with an AUC((R,R))/AUC((S,S)) ratio of 0.5. Chirality 21:738-744, 2009. (C) 2008 Wiley-Liss, Inc.

Canine population dynamics: the potential effect of sterilization campaigns

Objective. To analyze, through mathematical modeling, the potential ability of sterilization campaigns to reduce the population density of pet dogs. Methods. Mathematical models were constructed to simulate the canine population dynamics and project the results of control strategies based on several sterilization rates. Results. Even at high sterilization rates (for example, 0.80 year(-1)), it would take approximately 5 years to reduce density by 20%. Even so, other sources of population growth, such as the importing of dogs from other geographic areas, could outweigh the effects of a sterilization program. Conclusions. A program`s effectiveness is contingent upon not only on the sterilization rate, but also the rate of population growth. Sterilization campaigns may potentially reduce population density, but this reduction may not be immediately evident.

Modeling the Dynamics of Viral Evolution Considering Competition Within Individual Hosts and at Population Level: The Effects of Treatment

We consider two viral strains competing against each other within individual hosts (at cellular level) and at population level (for infecting hosts) by studying two cases. In the first case, the strains do not mutate into each other. In this case, we found that each individual in the population can be infected by only one strain and that co-existence in the population is possible only when the strain that has the greater basic intracellular reproduction number, R (0c) , has the smaller population number R (0p) . Treatment against the one strain shifts the population equilibrium toward the other strain in a complicated way (see Appendix B). In the second case, we assume that the strain that has the greater intracellular number R (0c) can mutate into the other strain. In this case, individual hosts can be simultaneously infected by both strains (co-existence within the host). Treatment shifts the prevalence of the two strains within the hosts, depending on the mortality induced by the treatment, which is, in turn, dependent upon the doses given to each individual. The relative proportions of the strains at the population level, under treatment, depend both on the relative proportions within the hosts (which is determined by the dosage of treatment) and on the number of individuals treated per unit time, that is, the rate of treatment. Implications for cases of real diseases are briefly discussed.

Dynamics and Control of Stray Dog Populations

The population dynamics of stray dogs is simulated to assess the effects of sterilization and euthanasia. From simulations representing less than 5 years, sterilization is less efficient than euthanasia to reduce the stray dog population, considering similar rates, but the total number of sterilized dogs is less than the total number of euthanized dogs per km(2) per year. Over 20 years, both strategies have similar efficiency. Beyond a certain rate of dog abandonment, both strategies are inefficient.

Parasitic dynamics of gastrointestinal nematode infection in the periparturient period of beef cattle in the State of Para

The experiment was conducted to investigate the dynamics of infection by gastrointestinal nematodes during the periparturition period in cows. One hundred and six beef cows were divided into two groups: G I was formed by 42 cows of one and two parturitions, and G2 by 76 cows of three or more parturitions. From the 120 days pre partum until the 90 days post partum, feces were collected for faecal egg counts (EPG) while blood was collected to determine the packed cell volume and hemoglobin levels of each animal, with monthly intervals. In the same intervals the body condition scores (BCS) were evaluated. The mean values standard deviation of the EPG for Cl were equal to 19.4 +/- 42.9, and for G2 31.1 +/- 68.0. No significant differences were observed between Cl and G2 in relation to EPC; and hematological parameters, which remained within normal patterns for both groups. The two groups had higher counts of EPG in the post partum period than in the pre partum period, with averages of 32.5 +/- 55.5 and 51.5 +/- 84.8 for groups Cl and G2, respectively. A significant difference (p < 0.05) in the parameters was observed when comparing the pre and post partum within each group studied resulting in declining values of blood and body score and an increase in EPG in the post partum. The results suggest that the cows may be more susceptible to infection by nematodes from giving birth up to 90 days post partum. However, adult cows, when well-managed, are not an important factor in the epidemiology of gastrointestinal nematodes, even in the post partum period.

LC-MS/MS quantitation of plasma progesterone in cattle

Quantitation of progesterone (P(4)) in biological fluids is often performed by radioimmunoassay (RIA), whereas liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been used much less often. Due to its autoconfirmatory nature, LC-MS/MS greatly minimizes false positives and interference. Herein we report and compare with RIA an optimized LC-MS/MS method for rapid, efficient, and cost-effective quantitation of P(4) in plasma of cattle with no sample derivatization. The quantitation of plasma P(4) released from three nonbiodegradable, commercial, intravaginal P(4)-releasing devices (IPRD) over 192 h in six ovariectomized cows was compared in a pairwise study as a test case. Both techniques showed similar P(4) kinetics (P > 0.05) whereas results of P(4) quantitation by RIA were consistently higher compared with LC-MS/MS (P < 0.05) due to interference and matrix effects. The LC-MS/MS method was validated according to the recommended analytical standards and displayed P(4) limits of detection (LOD) and quantitation (LOQ) of 0.08 and a 0.25 ng/mL, respectively. The high selective LC-MS/MS method proposed herein for P(4) quantitation eliminates the risks associated with radioactive handling; it also requires no sample derivatization, which is a common requirement for LC-MS/MS quantitation of steroid hormones. Its application to multisteroid assays is also viable, and it is envisaged that it may provide a gold standard technique for hormone quantitation in animal reproductive science studies. (C) 2011 Elsevier Inc. All rights reserved.

Effects of gonadotropin-releasing hormone at initiation of the 5-d timed artificial insemination (AI) program and timing of induction of ovulation relative to AI on ovarian dynamics and fertility of dairy heifers

Two experiments evaluated the effects of the first GnRH injection of the 5-d timed artificial insemination (AI) program on ovarian responses and pregnancy per AT (P/AI), and the effect of timing of the final GnRH to induce ovulation relative to AT on P/AI. In experiment 1, 605 Holstein heifers were synchronized for their second insemination and assigned randomly to receive GnRH on study d 0 (n = 298) or to remain as untreated controls (n = 307). Ovaries were scanned on study d 0 and 5. All heifers received a controlled internal drug-release (CIDR) insert containing progesterone on d 0, a single injection of PGF(2 alpha),, and removal of the CIDR on d 5, and GnRH concurrent with timed AT on d 8. Blood was analyzed for progesterone at AI. Pregnancy was diagnosed on d 32 and 60 after AI. Ovulation on study d 0 was greater for GnRH than control (35.4 vs. 10.6%). Presence of a new corpus luteum (CL) at PGF(2 alpha),, injection was greater for GnRH than for control (43.1 vs. 20.8%), although the proportion of heifers with a CL at PGF(2 alpha) did not differ between treatments and averaged 87.1%. Progesterone on the day of AT was greater for GaRH than control (0.50 +/- 0.07 vs. 0.28 +/- 0.07 ng/mL). The proportion of heifers at AI with progesterone <0.5 ng/mL was less for GURH than for control (73.8 vs. 88.2%). The proportion of heifers in estrus at AI did not differ between treatments and averaged 66.8%. Pregnancy per AI was not affected by treatment at d 32 or 60 (GnRH = 52.5 and 49.8% vs. control = 54.1 and 50.0%), and pregnancy loss averaged 6.0%. Responses to GnRH were not influenced by ovarian status on study d 0. In experiment 2, 1,295 heifers were synchronized for their first insemination and assigned randomly to receive a CIDR on d 0, PGF(2 alpha) and removal of the CIDR on d 5, and either GnRH 56 h after PGF(2 alpha) and AI 16 h later (OVS56, n = 644) or GnRH concurrent with AI 72 h after PGF(2 alpha) (COS72; n = 651). Estrus at AI was greater for COS72 than for OVS56 (61.4 vs. 47.5). Treatment did not affect P/AI on d 32 in heifers displaying signs of estrus at AI, but COS72 improved P/AI compared with OVS56 (55.0 vs. 47.6%) in those not in estrus at AI. Similarly, P/AI on d 60 did not differ between treatments for heifers displaying estrus, but COS72 improved P/AI compared with OVS56 (53.0 vs. 44.7%) in those not in estrus at AI. Administration of GnRH on the first day of the 5-d timed AI program resulted in low ovulation rate and no improvement in P/AI when heifers received a single PGF(2 alpha) injection 5 d later. Moreover, extending the proestrus by delaying the finAI GnRH from 56 to 72 h concurrent with AI benefited fertility of dairy heifers that did not display signs of estrus at insemination following the 5-d timed AI protocol.