REALMS study: real-world effectiveness and safety of fingolimod in patients with relapsing-remitting multiple sclerosis in Portugal.

BACKGROUND: Fingolimod, an oral sphingosine 1-phosphate receptor modulator, is approved by EMA for relapsing-remitting multiple sclerosis (RRMS). OBJECTIVES: To assess the effectiveness and safety of fingolimod in patients with RRMS in real-world clinical practice in Portugal. METHODS: Retrospective, multicentre, non-interventional study, reporting 3 years follow-up of data collected from October 2015 to July 2016. Sociodemographic data and previous treatments at baseline and data regarding disease evolution, including number of relapses, annualised relapse rates (ARR) and Expanded Disability Status Scale (EDSS), were collected. RESULTS: Two-hundred and seventy-five participants were enrolled in the REALMS study. Results showed that the main reason to switch to fingolimod was failure of previous treatment (56.7%) and only 3.6% were naïve patients. In the total population, there was a significant decrease in ARR of 64.6% in the first year of treatment, 79.7% in the second year and 82.3% in the third year, compared with baseline. More than 67.0% of patients had no relapses during the 3 years after switching to fingolimod. EDSS remained stable throughout the study. CONCLUSIONS: Therapy with fingolimod showed a sustained effectiveness and safety over the 3 years, particularly on patients switched from first-line drugs (BRACE). No new safety issues were reported.

A condition dependent link between testosterone and disease resistance in the house finch.

Testosterone has recently been proposed as a link between male quality and health and the expression of sexual traits. We investigated the relationship between testosterone and measures of the individual condition and health of males in a natural population of house finches (Carpodacus mexicanus). We also conducted a captive experiment in order to test for the effects of testosterone on resistance to coccidia, which is a common parasite of house finches. Free-living males in better condition had higher testosterone levels and lower corticosterone levels than free-living males in poor condition. In our captive experiment, increased testosterone accelerated the rate of coccidial infection as compared with sham-implanted or gonadectomized males. Although the differences were not significant, free-living males infected with coccidia had lower levels of testosterone and higher levels of corticosterone than males that were not infected. Thus, experimentally elevating testosterone levels in captive males resulted in a higher percentage of infected males, while free-living males with coccidial infection had low testosterone levels. This apparent discrepancy between captive and free-living males in the association of testosterone and disease may be explained by the condition dependence of testosterone. These results suggest that the testosterone-dependent sexual traits reliably indicate male overall condition and health and, thus, females could benefit from assessing potential mates based on these traits.

Responsiveness of the hypothalamo-pituitary-interrenal axis in an amphibian (Bufo terrestris) exposed to coal combustion wastes.

To assess the responsiveness of the interrenal axis to stress, we injected toads exposed to coal combustion wastes and toads from an unpolluted reference site with adrenocorticotropic hormone (ACTH), as well as the vehicle alone (saline). Initial circulating levels of corticosterone in toads captured at the polluted area were significantly higher than levels in toads from the reference site. Corticosterone levels in toads from the polluted site remained high even after 2 weeks of laboratory acclimation and injection with saline. The results may suggest disruption of hepatic enzymes responsible for the metabolic clearance of steroid hormones. Injection of toads from the polluted site with ACTH had no effect on plasma corticosterone levels, whereas a similar treatment of toads from the reference site stimulated a marked increase in corticosterone. Our study provides evidence that toads exposed to coal combustion wastes may be less efficient at responding to additional environmental stressors.

Chronic exposure to coal fly ash causes minimal changes in corticosterone and testosterone concentrations in male southern toads Bufo terrestris.

More than 50% of the electricity in the United States is produced by coal-burning power plants. The byproduct of coal-burning plants is coal fly ash, which contains increased concentrations of trace metals and is disposed of in collection basins. Southern toads (Bufo terrestris) frequently use these basins for reproduction. Male toads were collected in spring 2001 and 2002 from an ash basin and a reference site and divided into four groups: toads collected at the control site and maintained on (1) control substrate and food or (2) ash and contaminated food and toads collected at the ash site and maintained in (3) control or (4) ash conditions. Blood was collected periodically during 5 months to determine testosterone and corticosterone concentrations. Reference to ash toads exhibited a significant, transient increase in corticosterone at 4 weeks, but neither corticosterone nor testosterone continued to increase beyond this time. In contrast, toads caught and maintained on ash did not exhibit increased corticosterone. Testosterone in these toads appeared to be unrelated to ash exposure. This unexpected lack of a corticosterone response and no effect on testosterone suggests that toads chronically exposed to trace metals can acclimate to a polluted environment, but they may still experience subtle long-term consequences.

Evolution of sex-biased maternal effects in birds: III. Adjustment of ovulation order can enable sex-specific allocation of hormones, carotenoids, and vitamins.

Overlap in growth of offspring should constrain the opportunity for sex-biased maternal effects, yet sex-specific allocation of maternal resources among simultaneously growing ova is often observed in vertebrates. In birds, such allocation can be accomplished either by temporal clustering of ova that become the same sex, resulting in sex-biased egg-laying order, or by follicle-specific delivery of maternal resources. Two house finch populations at the northern and southern boundaries of the species range have opposite ovulation sequences of male and female eggs, and thus, in the absence of sex differences in ova growth or sex-specific maternal strategies, would be expected to have opposite sex-specific accumulation of maternal products. We found that the populations had strong and similar gradients of steroid distribution in relation to ovulation order, whereas distribution of carotenoids and vitamins correlated with each follicle's accumulation of steroids. In both populations, temporal bias in production of sons and daughters within a clutch enabled strongly sex-specific acquisition of maternal products, and oocytes of the same sex were highly interdependent in their accumulation of steroids. Moreover, in nests where the sex-bias in relation to ovulation order deviated from population-specific patterns, eggs had highly distinct concentrations of steroids, carotenoids and vitamins. These results and previous findings of sex-specific yolk partitioning among oocytes suggest that oocytes that become males and females are temporally or spatially clustered during their ovarian growth. We discuss the implication of these findings for the evolution of sex-specific maternal resource allocation.

Effect of fall mating on ovarian development in the red-sided garter snake.

Although spring mating is an important factor in initiating vitellogenesis in female red-sided garter snakes (Thamnophis sirtalis parietalis), some females can become vitellogenic without having mated in the spring. Two hypotheses have been proposed to explain this phenomenon: 1) long-term storage of copulatory stimuli from mating the previous fall, or 2) additional cue(s) overcoming the lack of mating to stimulate vitellogenesis. Through oviductal biopsy, the presence of sperm in females returning to the hibernaculum in the fall was assessed. Laparotomies performed just before and 6 wk after hibernation indicated that although fall mating may stimulate slight ovarian development in the fall, there appears to be no effect of fall mating on spring ovarian recrudescence. Spring mating seems to be the most important factor in determining ovarian maturation. Therefore, it appears that there is no long-term storage of copulatory cues and that other as yet unknown cues are responsible for initiating vitellogenesis in spring unmated females.

Mating-induced ovarian recrudescence in the red-sided garter snake.

In the female red-sided garter snake (Thamnophis sirtalis parietalis) mating initiates a neuroendocrine reflex that has both a short-term (within hours) effect on circulating estrogen concentrations and a long-term (6-7 weeks) effect on ovarian development. The perception of mating appears at least facultative, if not obligatory, for the initiation and maintenance of vitellogenesis and hence successful reproduction.

Effects of arousal from hibernation and plasma androgen levels on mating behavior in the male big brown bat, Eptesicus fuscus.

The effects of arousal from hibernation and presence of plasma androgen on the expression of mating behavior in male big brown bats (Eptesicus fuscus) were tested in a captive population exposed to seminatural conditions in central Alabama. In the mild winter of 1994-1995, flight cage temperatures never fell below 10 degrees C. Bats were never observed to enter sustained (over 2 d) torpor. They were also never observed to mate. Unmanipulated, sham-operated, and gonadectomized males and unmanipulated females were exposed to 6 d of 4 degrees C. All individuals appeared torpid, and body temperatures of monitored bats fell at least 15 degrees-20 degrees C. Plasma androgen levels of torpid unmanipulated, sham-operated, and gonadectomized males averaged 25.4 +/- 9.2, 19.7 +/- 9.1, and 1.5 +/- 0.25 ng/mL, respectively, and did not differ significantly from levels for the same groups 1 mo previous to induced torpor. When animals were returned to 23 degrees C, 57% of unmanipulated, 40% of the sham-operated, and 33% of gonadectomized males displayed mating behavior upon arousal. Almost all matings occurred within 48 h of arousal, the majority in the first 3 h. Males not exposed to low temperatures were not observed to mate. Although individuals from all three treatments mated, gonadectomized males averaged fewer mounts and copulations per individual. Androgen levels declined significantly from torpor levels in all groups 48 h after arousal. Thus, an extended period of low body temperature and arousal appeared to be a short-term activator of sexual behavior in the big brown bat; unmanipulated males were more strongly affected by this stimulus than gonadectomized males.

Seasonal cycles in gonadal activity and plasma gonadotropin in the musk turtle, Sternotherus odoratus.

Plasma gonadotropin cycles were examined in relation to the seasonal gonadal cycle in freshly captured musk turtles, Sternotherus odoratus, from South Carolina. Acute and chronic stress effects of captivity on testicular growth and circulating plasma testosterone (T) and follicle-stimulating hormone (FSH) also were examined. Monthly mean FSH levels in freshly captured males were correlated significantly with plasma T. Both T and FSH were minimal in spring when testes were small, and highest in the fall in males that had begun spermiation and whose testes had begun to regress. Individual plasma T and FSH values were significantly correlated during the fall when both hormones were at elevated levels. In contrast, plasma luteinizing hormone (LH) remained undetectable throughout the year in males. Females had nondetectable levels of both plasma LH and FSH throughout the year. There was a seasonal pattern in plasma 17 beta-estradiol which was correlated with follicular growth. Holding males in captivity (outdoors) reduced plasma T by 35-60% within 24 hr of capture (depending on season) and plasma T remained at these lower levels after 2 weeks. However, FSH was not significantly affected by such short-term "stress." Testicular weight and FSH cycles of captive males were similar to those of males in the field. Monthly plasma T of long-term captives also exhibited a seasonal cycle but at a level significantly lower than in the field and with a delayed peak. Thus, while the data suggest that FSH may be important to both seasonal testicular growth and androgen secretion, fluctuations in FSH alone cannot account for all observed patterns or changes in spermatogenesis or plasma androgen levels.

Photothermal effects on the testicular cycle in the musk turtle, Sternotherus odoratus.

A series of photothermal treatments was initiated at different times of year to examine the relative contributions of exogenous and endogenous factors affecting the timing of the testicular cycle of the kinosternid turtle, Sternotherus odoratus. Elevated temperature appeared to be the major factor stimulating testicular recrudescence (spermatocytogenesis, spermiogenesis, and, to some extent, androgen secretion). However, depending on the time of year, short or long daylengths, in conjunction with elevated temperatures, enhanced testicular responses (recrudescence or regression). Also, high temperatures are not requisite for recrudescence. Spermatogenic activity and testis growth differ in their temperature sensitivity and may become dissociated, and an endogenous mechanism may have affected peak androgen secretion. Testicular regression exhibited a dependence on an interaction among temperature, photoperiod, and endogenous regulation. Turtles with testes in advanced stages of spermiogenesis underwent accelerated regression when exposed to even slightly reduced temperatures, especially at short daylengths. However, regression was delayed by a combination of high temperature and short daylength in fall. Results are compared to other experimental studies of photothermal effects on turtles and discussed in terms of the described latitudinal variations in the timing of the testicular cycle of S. odoratus.

Comparison of reproductive parameters in male yellow-blotched map turtles (Graptemys flavimaculata) from a historically contaminated site and a reference site.

From May to September of 1998, we collected monthly plasma samples from male yellow-blotched map turtles captured at two sites in the Pascagoula River drainage, Mississippi. One site (Vancleave) has a documented history of pollution from industrial sources (principally 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD). Fish consumption advisories at the Vancleave site were lifted in 1996 and current impacts appear minimal. However, the yellow-blotched map turtle, a federally protected species, continues to decline in numbers. To determine if endocrine disruption could be a factor in the low reproductive rates observed in Vancleave turtles, we examined levels of plasma testosterone (T) and estradiol-17beta (E(2)) from males at this site and a second site (Leaksville), which has no known source of industrial pollution. Plasma was also tested for vitellogenin (VTG), which, in males, can be a biomarker of exposure to estrogenic contaminants. No males had detectable plasma VTG nor did mean monthly E(2) levels differ between sites. However, 10% of males from the historically polluted site were found to have high levels of E(2) (equivalent to levels found in females) and T was significantly lower for males captured at this site for 3 of 5 months. Our data suggest that the current impact of contaminants on reproduction in this population is limited. However, a portion of the population may have been affected developmentally, as represented by differences in reproductive parameters detected between sites.

Effects of ovariectomy and estrogen replacement on attractivity and receptivity in the red-sided garter snake (Thamnophis sirtalis parietalis).

Activation of courtship behavior in male red-sided garter snakes, Thamnophis sirtalis parietalis, is independent of the presence of sex steroids. The only consistent treatment that stimulates courtship behavior in males is prolonged exposure to low temperature followed by subsequent warming, mimicking the emergence from hibernation. We investigated whether attractivity and receptivity in female red-sided garter snakes is similarly steriod independent. Female red-sided garter snakes are attractive when they emerge from hibernation and are courted by males; most mate within an hour of emergence. In a series of experiments, groups of females were either ovariectomized (OVEX) in the late spring, fall or while in hibernation. They were tested for attractivity and receptivity upon emergence from hibernation. Females OVEX in the spring were unattractive whereas those OVEX in fall or while in hibernation were attractive. Thus, attractivity appears determined the year before emergence and is dependent on the presence of the ovaries. All OVEX females were unreceptive upon emergence. OVEX females were also given replacement estradiol (E) treatment (either in Silastic capsules or single injections) at various points of their annual cycle. The only treatment that resulted in reinstating receptivity in OVEX females was the injection of E (20 micrograms) one hour prior to emergence. The effectiveness of E in reinstating receptivity was time dependent: the longer the period between emergence and injection, the less effective the same dosage was in stimulating receptive behavior. These experiments suggest that sexual behavior in female red-sided garter snakes is, unlike males, dependent on the presence sex steroid hormones. Although E is naturally at its lowest seasonal level upon emergence, the concentration is sufficient to stimulate receptivity. However, it appears that temperature regulates a time-limited window of sensitivity to E.

Control of attractivity and receptivity in female red-sided garter snakes.

Female red-sided garter snakes emerge from their hibernacula in the spring attractive and receptive to males. Attractivity is communicated by a pheromone released through the female's skin and is a consequence of ovarian recrudescence the previous summer. Receptivity, on the other hand, is stimulated by ovarian estrogen secretion during emergence itself. Mating renders females both unattractive and unreceptive. Another "mating" pheromone of male origin is important in making females unattractive after mating. To investigate the role of cloacal stimulation in the loss of attractivity and receptivity we injected a local anesthetic (lidocaine or tetracaine) in the cloacal region of females before mating. This does not prevent mating, although it blocks neural transmission of copulatory sensory stimuli. The time course of transition from attractive and receptive states was then observed. Females treated with local anesthetic as well as control females were unattractive within 15 min of mating. However, when retested 2-3 and 24 h after mating, a significantly higher proportion of treated females regained their attractivity, while mated control females remained unattractive. This restorative effect was transient, though, as treated females retested 48 h after mating were as unattractive as the controls. Both anesthetized and control females were unreceptive when tested following mating and did not regain receptivity with time. Last, the mating-induced surge in circulating concentrations of prostaglandin was diminished in females that received a local anesthetic prior to mating. Taken together these results indicate that the loss of attractivity and receptivity following mating in the red-sided garter snake is due to combined effects of a mating pheromone and a physiological, neurally mediated response to the sensation of stimuli associated with the act of mating.

Increased circulating levels of testosterone and corticosterone in southern toads, Bufo terrestris, exposed to coal combustion waste.

This study describes an interrenal stress response in adult toads, Bufo terrestris, after exposure to coal combustion waste (characterized by a variety of trace elements). In the first portion of this study, free-ranging male toads captured at the coal ash polluted site exhibited significantly higher circulating levels of corticosterone (B) in both June/July and August than conspecifics captured at uncontaminated sites. In addition, both calling and noncalling males from the polluted site had higher B levels than conspecifics engaged in the same behaviors at reference sites. Testosterone levels were elevated in toads from the polluted site, regardless of capture month or behavioral state, suggesting altered androgen production, utilization, and/or clearance. In the second portion of this study, male toads from reference sites were transplanted to enclosures at the polluted site or an uncontaminated site. Toads held at the polluted site exhibited significant increases in B after 10 days of exposure compared to toads held at the reference site. B levels remained significantly elevated in toads transplanted to the polluted site after 12 weeks. We hypothesize that high concentrations of various trace elements in the polluted site are responsible for these hormonal responses.

Induction of sexual receptivity in the female broad-headed skink, Eumeces laticeps, by estradiol-17 beta.

Sexual receptivity in the female scincid lizard Eumeces laticeps occurs naturally only during the spring breeding season, which is also when maximal follicular development occurs. The presumption that high estrogen levels are coincidentally present and the need for a reliable method of inducing sexual receptivity for behavioral studies prompted tests of the hypothesis that estrogen induces sexual behavior. A series of experiments established that estradiol-17 beta induces sexual behavior. A series of experiments established that estradiol-17 beta induces sexual receptivity within 4 days when injected every other day at 2.0 micrograms in 20 microliters peanut oil in intact or ovariectomized females. In behavioral tests conducted during August, all control females (intact or ovariectomized injected with vehicle only) rejected courtship whereas all females receiving estrogen copulated. Estrogen injections also induced a statistically significant change from rejection to receptivity within individuals. Initial attempts to implant estradiol-17 beta in Silastic tubes killed all females so treated.

Courting and noncourting male red-sided garter snakes, Thamnophis sirtalis parietalis: plasma melatonin levels and the effects of pinealectomy.

Previous studies found that pinealectomy of male Canadian red-sided garter snakes (Thamnophis sirtalis parietalis) in the autumn, before prolonged exposure to low temperatures (hibernation), significantly impaired the expression of courtship behavior upon emergence in the spring. Additionally, pinealectomized animals with a disrupted diel cycle of plasma melatonin did not court while those exhibiting a more typical diel pattern did. These results suggested that the pineal gland functions in the transduction of a temperature cue which stimulates courtship. To test this hypothesis, we pinealectomized males in the spring after they had undergone a normal hibernation but were still courting. Pinealectomy of courting males in the spring, in each of the 3 years of study, had no effect on courtship. This result suggests that once the cue is transduced, the pineal gland no longer has a modulatory effect on courtship behavior. Finally, we took advantage of the fact that, in the laboratory, there is always a small percentage of males that do not court upon emergence. Pinealectomy of these noncourters greatly increased the percent of males expressing courtship behavior in each of the study years. Plasma melatonin levels of unmanipulated courting and noncourting males was measured after emergence in successive years. In both years, courters had a typical pattern of melatonin secretion (low in the photophase, high in the scotophase) while persistent noncourters displayed the opposite pattern.

Seasonal changes and annual variability in daily plasma melatonin in the red-sided garter snake (Thamnophis sirtalis parietalis).

We report seasonal and annual variation in the daily cycle of plasma melatonin levels in male red-sided garter snakes, Thamnophis sirtalis parietalis. In autumn of 1989 and 1990, levels averaged a maximum of 210 pg/ml during scotophase and a minimum of 45 pg/ml during photophase and had a similar diel pattern. Under hibernation conditions (4 degrees, 0:24 L:D), melatonin was undetectable and a diel cycle could not be determined. In Spring 1990, melatonin levels rose rapidly and precipitously within an hour of emergence (while in photophase), peaked at levels significantly higher than those seen in the autumn (approximately 900 pg/ml) and remained significantly high for 24 hr after emergence (though the majority of animals did have decreased levels at the 0400 sample). By the 10th day after emergence, a diel cycle was reestablished and absolute melatonin levels had decreased. The next spring (1991), melatonin again rose within an hour after emergence, while in photophase, but not as high as the previous year. Also unlike the previous year, a diel cycle was observed within 24 hr of emergence. Melatonin levels at emergence were significantly higher than those observed 10 days later. Disruption of a diel rhythm of plasma melatonin (by pinealectomy the previous autumn) inhibits courtship behavior by males the next spring, implying a role for melatonin in the stimulation of sexual behavior. Males in 1991 (with quickly established melatonin cycles) courted much sooner after emergence than did males in 1990. Therefore, the initial day/night difference in melatonin levels at emergence (i.e., establishment of a normal diel cycle) may function in synchronizing and modulating reproductive behavior in male red-sided garter snakes.

Pinealectomy, melatonin, and courtship behavior in male red-sided garter snakes (Thamnophis sirtalis parietalis).

Activation of courtship behavior in male red-sided garter snakes is independent of androgens. Only exposure to extended periods of low temperature with subsequent warming stimulates courtship in males. The pineal gland is thought to transduce temperature as well as photoperiodic information in reptiles. Therefore, we explored the relationship of the pineal and melatonin to sexual behavior in this species. Pinealectomy of male garter snakes disrupted sexual behavior upon emergence from a 17-week period of low temperature in approximately 60% of treated individuals in each of the 3 years of study. However, 40% of the males were unaffected by the pinealectomy, engaging in vigorous courtship. Administration of exogenous, chronic melatonin did not significantly modulate the effect of pinealectomy. Upon pinealectomy in the autumn (before hibernation), plasma levels of melatonin fell. However, upon emergence from hibernation, melatonin levels in pinealectomized (PINX) and sham-treated (SHAM) animals were equivalent, indicating extrapineal source(s) of melatonin. However, PINX males did not exhibit a diel cycle in melatonin levels upon emergence. Instead, melatonin remained elevated through the subsequent 24-hr period. SHAMs did exhibit a diel cycle. Ten days after emergence, PINX animals either had a disrupted/abnormal melatonin cycle and were non-courters or had a cycle similar to SHAM males and courted. Therefore, a normal diel cycle of melatonin appeared necessary for the proper expression of courtship behavior. These results suggest that the pineal in snakes 1) is part of a complex, multi-oscillator system as it is in birds and lizards and 2) may play a role in maintaining polymorphism in timing of reproductive behavior.

Changes in hormone levels in relation to breeding behavior in male bullfrogs (Rana catesbeiana) at the individual and population levels.

Fluctuations in plasma androgen (testosterone and 5 alpha-dihydrotestosterone), corticosterone (B), and luteinizing hormone (LH) of male bullfrogs in central California were measured during the spring mating season. Androgen and LH levels generally increased in the population prior to the initiation of chorusing and establishment of territories, whereas plasma B peaked in a 2-week period at the start of heavy chorusing; this coincided with a transitory, 1 week decline in androgen. Individual males showed fluctuations in plasma androgen and LH levels throughout the breeding season, often within 1 day, but there was no clear correlation between changes in the two hormones. No time of day effect was apparent on any of the hormones. B and androgen levels were significantly but weakly correlated, (r = 0.35) but LH and androgen were not. Although the seasonal trend of increasing androgen corresponded with the start of intense chorusing (and presumably sexual activity) by the population, behavior of individuals and their circulating androgen levels did not correlate. After acquiring territories, males showed no overall trend of increased plasma androgen. Moreover, males that showed no vocal or territorial behavior had significantly higher androgen and lower B levels than calling males. Increased B levels suggest that territorial behavior and especially direct agonistic encounters represent stresses that could have an inhibitory effect on androgen secretion.