Compared to sparrows in a nonmigratory state, migratory sparrows spent approximately two-thirds less time sleeping. Despite reducing sleep during migration, accuracy and responding on a repeated-acquisition task remained at a high level in sparrows in a migratory state. This resistance to sleep loss during the prolonged migratory season is in direct contrast to the decline in accuracy and responding observed following as little as one night of experimenter-induced sleep restriction in the same birds during the nonmigratory season. Our results suggest that despite being adversely affected by sleep loss during the nonmigratory season, songbirds exhibit an unprecedented capacity to reduce sleep during migration for long periods of time without associated deficits in cognitive function. Understanding the mechanisms that mediate migratory sleeplessness may provide insights into the etiology of changes in sleep and behavior in seasonal mood disorders, as well as into the functions of sleep itself.Twice a year, normally diurnal songbirds engage in long-distance nocturnal migrations between their wintering and breeding grounds. If and how songbirds sleep during these periods of increased activity has remained a mystery. We used a combination of electrophysiological recording and neurobehavioral testing to characterize seasonal changes in sleep and cognition in captive white-crowned sparrows  Neurophysiological and behavioural studies suggest that sleep- loss during the migratory season does not adversely affect cognitive capacity in captive white-crowned sparrows Seasonal behaviors occur in virtually all organisms, ranging from insects to mammals . Just asThe most extraordinary examples of seasonal migration occur in birds, many species of which regularly migrate thousands of kilometers. Given the long distances traversed during migration, much research has focused on the timing of migratory flights, navigation during migration, and the energetic costs of migration . One asp(Catharus ustulatus) flew on six of seven nights and traveled 1,500 km, with flights that occurred under favorable weather conditions lasting up to7 h  deprived of sleep also die, suggesting that sleep serves a function vital to survival in all animals , which migrates 4,300 km twice a year between Alaska and southern California , i.e., nocturnal activity, including hopping and wing flapping  from both hemispheres, as well as electromyographic activity (EMG) see . To contAll of the birds instrumented for EEG and EMG recording exhibited episodes of migratory restlessness starting between mid-July and mid-August, in synchrony with the other birds in the laboratory. Their behavior was indistinguishable from that displayed by the sparrows without electrophysiological implants. In three birds, bouts of migratory restlessness started prior to surgery or during postoperative recovery.The electrophysiological correlates of sleep and wakefulness in nonmigrating and migrating white-crowned sparrows were similar to each other, as well as to those previously described in other species of birds .Behavior during wakefulness included hopping and flying around the cage, feeding, drinking, feather preening, and actively scanning the room. Although movement artifacts obscured the EEG and EMG during gross movements (e.g., hopping and wing flapping), the recordings occurring immediately before and after such behaviors showed a low-amplitude, activated EEG in both hemispheres, and high EMG activity typical of alert wakefulness .Upon ceasing active wakeful behaviors, the sparrows entered drowsiness, a mixed state with behavioral and EEG characteristics of both wakefulness and slow-wave sleep (SWS) . SparrowIn contrast to the vigilant behaviors exhibited by sparrows in drowsiness, during SWS the birds were motionless, usually with closed eyes; the head was either pulled in toward the body and facing forward or resting on the bird's back . The ampAn activated EEG pattern similar to that occurring during wakefulness characterized REM sleep C. UnlikeDuring episodes of migratory restlessness, defined as active hopping and wing flapping at night interrupted by only brief pauses in motor activity, both eyes were completely open, and the EEG recorded from both hemispheres showed an activated pattern, indistinguishable from alert wakefulness D. There In nonmigrating sparrows, sleep and wakefulness patterns across the day were typical of those previously described in a songbird . The pron = 5) and migratory (n = 8) conditions. Total time spent sleeping was reduced by an average of 63% in migrating birds compared to nonmigrating birds  (t = 0.56, p > 0.1)  . Althoug0.1) see C.t = 2.68, p < 0.05) in migrating birds; drowsiness also increased during the dark phase, but this did not reach statistical significance (12.9% vs. 8.1%, t = 1.73, p > 0.1). On migratory nights, drowsiness usually occurred during the later half of night between bouts of migratory restlessness.Finally, despite the marked reduction in nocturnal sleep during migration, sleep did not occur during the light phase in migrating birds; as in nonmigrating birds, SWS and REM sleep were restricted to the dark phase of the LD cycle. Nonetheless, time spent in drowsiness increased significantly during the light phase  spectral analysis of the EEG. In mammals, SWS-related slow-wave activity (SWA) of 0.75- to 4.5-Hz appears to reflect sleep intensity, since arousal thresholds are positively correlated with the amount of SWA . SWA in Given the reduction in time spent sleeping and the apparent lack of a compensatory increase in sleep intensity during migration, we were interested in determining whether sparrows in a migratory state showed associated changes in cognitive function. Because we were interested in detecting seasonal changes in cognition, we used a test that could be administered repeatedly to the sparrows over long periods of time and without the potential confound of “practice” effects, a repeated-acquisition task; it has been widely used in humans and animals to provide repeated measures of the acute and chronic effects of neurotoxic insult on the ability to acquire a new sequence of operant responses . When coWe trained a group of eight sparrows not instrumented for electrophysiological recordings to respond under a multiple schedule of repeated-acquisition and performance in standard operant testing chambers see . In the To determine whether the task was sensitive to the effects of sleep restriction, as well as to provide a comparison for the effects of sleep loss occurring spontaneously during migration, sleep was restricted to the first 3 h of the dark phase on three consecutive nights during the nonmigratory (winter) season see . A 3-h sThe same group of eight sparrows was tested under the multiple schedule of repeated-acquisition and performance for one year, encompassing both spring and fall migrations. Because the sparrows did not all migrate at exactly the same time, we also examined the effect of migratory status on accuracy and responding more specifically by selecting for each bird two 3-wk periods with maximal nocturnal activity during the spring and fall migrations, and two 3-wk periods with minimal nocturnal activity during the summer and winter nonmigratory periods see . We chosEach spring and fall, songbirds switch from sleeping at night to migrating at night. Whether migrating songbirds sleep during flight, forgo sleep altogether, or compensate for night-time sleep loss by sleeping during the day has remained a mystery. Our EEG recordings of the white-crowned sparrow demonstrate a marked reduction in sleep, as well as distinct changes in sleep architecture during migration, including a shift in the timing of REM sleep to earlier in the night. Although an increase in drowsiness and a corresponding decrease in wakefulness were observed during the day, migrating sparrows did not compensate for sleep loss at night by sleeping more during the day or by increasing SWS intensity on migratory nights.Despite the apparent reduction in sleep occurring during migration, observations of songbirds in the wild suggest that they are fully capable of maintaining a high level of cognitive and physical function, including navigation during long-distance flights, foraging, and evading predators in novel environments. Our results from the repeated-acquisition and performance tasks also suggest that songbirds are not cognitively or physically impaired during episodes of migratory restlessness in the laboratory. Unlike sleep restriction during the nonmigratory season, which caused a decrease in accuracy and responding in both the acquisition and performance components of the task, accuracy and responding did not decrease during migratory periods , when compared to nonmigratory periods (summer and winter). In fact, responding was highest during the fall migration and lowest during the winter.We saw no evidence of sleep in active sparrows during periods of migratory restlessness in the laboratory setting, suggesting that songbirds do not sleep during migratory flights in the wild. Moreover, if birds have evolved the capacity to sleep while flying and depend upon it to avoid sleep deprivation, we would then expect them to be vulnerable to the effects of the sleep restriction resulting from migratory restlessness in the laboratory. The fact that birds exhibit migratory restlessness in captivity for periods of time similar to the duration of migration in the wild indicates an ability to withstand the effects of sleep restriction . Further(Tursiops truncates), northern fur seals , and cape fur seals  that swim in a coordinated manner while exhibiting unihemispheric SWS, a state characterized by SWS-related EEG in one hemisphere and EEG indistinguishable from wakefulness in the other hemisphere , however, rather than being phase advanced, the amplitude of the circadian rhythm is reduced during migration  deprived of sleep also show an increase in REM sleep during recovery sleep  did show a decline in SWS-related SWA (0.5- to 4.0-Hz) across the major sleep period, indicating that aspects of mammalian SWS regulation may be present in some birds  across the normal sleep period (SWS and REM sleep combined) when SWS was the predominant behavioral state, or an increase in SWA following 24 h of total sleep deprivation, suggesting a fundamental difference between sleep homeostasis in birds and mammals . Unlike me birds . In nonmAssuming that the decline in 1.25- to 2.5-Hz power in nonmigrating sparrows reflects SWS homeostasis, the apparent absence of an increase in this band during migration suggests that, unlike nonmigrating sparrows, migrating sparrows may require less SWS. A reduced need for SWS during migration may also be reflected in the shorter REM sleep latencies on migratory nights, since REM sleep latency is positively correlated with SWS need in humans . Alterna to migratory garden warblers (S. borin); the migratory species performed better on a long-term memory task simulating habitat selection, despite being trained during a period of migratory restlessness, when sleep was presumably restricted  were minimally intrusive. Regardless, birds in a migratory state were clearly able to maintain high levels of accuracy and responding during periods of spontaneous sleep loss occurring during migration. The only previous study to assess cognition in a migratory songbird compared the closely related nonmigratory Sardinian warbler stricted . Althougstricted , a proceThe apparent resistance to the effects of sleep restriction in migrating songbirds is unprecedented and clearly needs to be confirmed with further neurobehavioral testing. Future studies aimed at understanding the mechanisms underlying migratory sleeplessness may provide insight into the etiology and treatment of certain sleep disorders, as well as psychiatric disorders such as bipolar disorder, where similar seasonal bouts of sleeplessness with high levels of cognitive function are diagnostic of hypomania. Furthermore, an understanding of the mechanisms involved in migratory sleeplessness may lead to the development of methods to temporarily mitigate the effects of sleep deprivation that otherwise compromise performance in humans engaged in sustained operations where the maintenance of high levels of cognitive and physical function is critical. Finally, revealing the mechanisms through which migratory songbirds resist the effects of sleep deprivation may yield important clues as to the function of sleep in general.Sparrows used for the operant testing were captured in Alaska (lat 64°49′ N, long 147°52′ E) during June 2002. Sparrows used for the EEG recordings were captured on their wintering grounds in the Sacramento valley in California (lat 39°00′ N, long 122°00′ E) during November 2002. All birds were collected using mist nets under the authority of a United States Fish and Wildlife Service permit. Birds were transported to the University of Wisconsin-Madison where they were individually housed in galvanized wire cages (L: 35 cm × W: 25 cm × H: 32 cm) in environmentally controlled rooms . Each bird was in visual and auditory contact with other birds in the room. To simulate seasonal changes in photoperiod, operant birds were exposed to photoperiods ranging from 9.5:14.5 LD to 16.5:7.5 LD. Sparrows used for the EEG recordings were maintained under a 12:12 LD schedule; lights went on at 06:00 with an illuminance level of 540–640 lux measured at the level of the cage floor. Illuminance during the dark phase was less than 0.5 lux. Birds were fed a mixed-seed and provided water ad libitum, and their diet was supplemented daily with lettuce, dried insects, live mealworms, and grit. Birds involved in operant testing were food restricted as described below. All experimental protocols were approved by the University of Wisconsin-Madison Animal Care and Use Committee.http://www.minimitter.com) that logged the number of times that the bird crossed the infrared beam aimed across the center of the cage each 30-s interval. Although the infrared activity monitoring system may underestimate overall activity because it fails to quantify activity that does not result in a beam break, it nevertheless provides a rapid method for assessing gross seasonal changes in behavior. For the birds in which EEG was recorded, behavior was also continuously recorded using 16 infrared-sensitive cameras (two per bird) connected to a digital video storage system . Infrared illuminators provided lighting for the cameras during the dark phase.As in previous studies , gross a2). The bird's head was stabilized in a Kopf Instruments  stereotaxic device, using an adaptor developed for use with birds. A temperature-regulated heat pad set at 40 °C reduced heat loss during the procedure. After establishing a suitable anesthetic plane, the feathers overlying the cranium were clipped and the scalp was cleaned with 70% isopropyl alcohol. A longitudinal incision was made along the midline of the head to expose the cranium. After cleaning with 3% hydrogen peroxide and drying the cranium, four small holes were drilled through the cranium to the dura: two for the EEG electrodes, one for the reference electrode, and one for the ground electrode. To record the EEG from the left and right cerebral hemispheres, two holes were drilled 2 mm lateral of the midline, one over the left and one over the right Wulst, a brain region homologous to portions of the mammalian neocortex (http://www.dragonflyinc.com). The cable was attached to the bird's cranium using dental acrylic . To form a strong adhesion, the acrylic was allowed to infiltrate the porous cavity between the inner and outer layers of the cranium through small holes drilled only through the dorsal layer of the cranium (http://www.vpl.com).In July 2003, eight adult white-crowned sparrows approximately 13–14 mo of age were randomly selected from our captive population and surgically instrumented for chronic EEG and EMG recordings. All surgical procedures were performed under isoflurane anesthesia , digitized at 100 Hz, and visualized using Somnologica 3 software .After surgery, each bird was placed in the recording cage (L: 35 cm × W: 25 cm × H: 32 cm) for at least 10 d of postoperative recovery and adaptation to the recording cable. The recording cable was attached to a low-torque, six-channel mercury commutator (Dragonfly) designed for use with small birds , and theRepresentative 24-h periods occurring prior to and during migration were selected for behavioral state scoring. The migratory nights selected for scoring were preceded by several nights with similar amounts of migratory restlessness. The behavioral state was scored visually, using both electrophysiological (i.e., EEG and EMG) and video recordings, and categorized as either wakefulness, drowsiness, SWS, or REM sleep. For accurate detection of REM sleep, the duration of scoring epochs was set at 4 s, since episodes of REM sleep may be as brief as only several seconds in birds . As in pEEG power spectra of the left and right hemisphere derivation were computed for all 4-s epochs, as described previously, by a FFT routine  within the frequency range of 0.25–25.0 Hz . Values To encourage responding by the sparrows, their food was restricted to maintain 90% of their free-feeding weights during nonmigratory periods. In practice, however, it was difficult to maintain birds at this weight, especially during periods of premigratory fattening, when even food-restricted birds gained weight. Sparrows were tested for 60-min sessions once per day on 5–7 d per week from 1 February 2003 through 15 February 2004. Testing was always performed between 09:30 and 16:00, and the testing order was counterbalanced across days for each bird. Activity levels in the home cages were measured using the infrared activity monitoring system. The amount of active time during the dark phase was used to select for analysis two 3-wk periods when the birds were migrating  and two 3-wk periods when they were not migrating (winter and summer).Preliminary training for the repeated-acquisition task was described previously  and inclDuring acquisition components, the three response keys were illuminated at the same time with one of three colors: green, red, or white. Responding on the correct key in the presence of one color  changed the color of the key lights as well as the position for the next correct response. When the subject completed the response sequence by emitting three correct responses (i.e., one correct response in the presence of each color), the key lights were extinguished, and the stimulus light in the mixed-seed trough was illuminated for 0.05 s. Subsequently, the response keys were illuminated with the first color (i.e., green), and the sequence was reset. Within a given session, the correct response that was associated with a particular color did not change, and the same sequence (in this case, center-left-right [C-L-R]) was repeated during all acquisition components of a given session. Responding on this sequence was maintained by food presentation under a second-order FR3 schedule such that every third completion of the sequence resulted in the presentation of 5 s of access to mixed-seed. When sparrows responded on an incorrect key (in the example, the left or right key when the green lights were illuminated), the error was followed by a 5-s time-out. An incorrect response did not reset the three-response sequence (i.e., the stimuli and the position of the correct response were the same before and after a time-out).To establish a steady state of repeated acquisition, the sequence was changed from session to session. An example of sequences for five consecutive sessions was C-L-R, L-R-C, C-R-L, R-L-C, and L-C-R. The sequences were carefully selected to be equivalent in several ways, and there were restrictions on their ordering across sessions. Briefly, each sequence was scheduled with equal frequency, and consecutive correct responses within a sequence were scheduled on different keys. Occasionally, a correct sequence position for a given color was the same for two consecutive sessions (in the list of sequences above, L-R-C and C-R-L).During performance components, the response keys and the houselights were illuminated, and the sequence remained the same (R-C-L) from session to session. In all other aspects (color of the stimuli for each response in the sequence, second-order FR3 schedule of food presentation, 5-s time-out, etc.), the performance components were identical to the acquisition components. Experimental sessions always began with an acquisition component, which then alternated with a performance component after 20 reinforcers or 20 min, whichever occurred first. The performance component alternated back to the acquisition component after 10 reinforcers or 20 min, whichever occurred first. Each session terminated after 60 min.To determine whether accuracy and response rate on the repeated-acquisition and performance task were affected by sleep restriction during the nonmigratory season, sleep was restricted to the first 3 h of the dark phase (18:00–21:00) for three consecutive nights starting on 10 February 2004. Birds were deprived of sleep starting at 21:00 until the following day at 18:00 by experimenters who entered the housing room at least once every 5 min or sooner if behavioral signs of sleep were observed via closed-circuit cameras. Walking quietly past the cages was always sufficiently stimulating to keep the birds awake; we never had to handle the birds to induce wakefulness.t-test, with Welch correction for sample size, or ANOVA. All tests were performed using “R” (http://www.r-project.org). Prior to analysis two procedures were performed on the data for the cognitive testing. One consequence of counterbalancing the order of testing the birds was that the length of time from withdrawal of food to the onset of testing (and presumably one component of food motivation) varied on a 3-d schedule. For this reason a 3-d running average of the cognitive testing variables was computed. There was also a linear trend across the year, particularly in acquisition percentage correct (2r varied from 0.31 to as high as 0.67). This trend was removed before making seasonal comparisons.Comparisons were made using either Student's The spring and fall migratory periods and the summer and winter nonmigratory periods were determined as follows. For each bird, each date during the study was used to compute the average nighttime activity for the following 21 d. The periods in the spring (21 February–20 May) and fall (21 August–20 November) with the highest average activity for each bird were designated as peak migration times, and the periods in the summer and winter with the lowest activity for each bird were chosen as nonmigratory times.


The seroprevalence of hepatitis C varies substantially between countries and geographic regions. A better understanding of the seroprevalence of this disease, and the risk factors associated with seropositive status, supply data for the development of screening programs and provide insight into the transmission of the disease. The purpose of this investigation was to determine the seroprevalence of hepatitis C and associated risk factors in an urban population in Haiti.A prospective survey for hepatitis C antibodies was conducted among an urban outpatient population in Cap-Haïtien, Haiti, with a sample size of 500 subjects. An anonymous 12 question survey, with inquiries related to demographic characteristics and risk factors for HCV acquisition, was concomitantly administered with testing. These demographic and behavioral risk factors were correlated with HCV antibody status using univariate and multivariate tests.The prevalence of positive HCV antibody was 22/500 (4.4%). Subjects that were anti-HCV positive had an average of 7 ± 8.6 lifetime sexual partners, compared to average of 2.5 ± 3.5 lifetime sexual partners among HCV-negative subjects (p = 0.02). In a multiple logistic regression model, intravenous drug use (OR 3.7, 1.52–9.03 95% CI) and number of sexual partners (OR 1.1, 1.04–1.20 95% CI) were independently associated with a positive HCV antibody result.A substantial number of subjects with HCV antibodies were detected in this population in Haiti. Further investigation into the correlation between the number of sexual partners and testing positive for hepatitis C antibodies is indicated. The seroprevalence of hepatitis C virus (HCV) varies substantially in different geographic regions throughout the world . Prior sRisk factors associated with HCV serologic status may be specific to a country or region. In particular, the role of sexual contact in the transmission of HCV appears to be influenced by characteristics and location of the population studied . TherefoThe study was approved by the Institutional Review Board at Brooke Army Medical Center (Fort Sam Houston, Texas). Subjects were recruited in the year 2000 from a healthy population utilizing hospital and clinic services in Cap-Haïtien, Haiti, which is the second largest city in the country. Subjects were recruited on presentation to the hospital laboratory for blood draws for routine laboratory tests. This laboratory was the only location in which subjects were recruited and samples obtained. Subject participation was not limited to a particular medical condition. The first 500 subjects that agreed to participate in the study were enrolled. These subjects were presenting to the hospital for services, and were not patients in a specific clinic. After informed consent was obtained, subjects completed a written 12 question survey in Creole, at the same location as where the blood was drawn. The subjects completed the survey by themselves. The questionnaire focused on demographic information and topic areas possibly associated with transmission of hepatitis C . Intravenous drug use and cocaine use was measured on a 0–3 scale (never-rare-frequent-daily). Blood transfusions, number of sexual partners, age of first sexual intercourse and number of tattoos were quantified. Serum was obtained for testing for HCV antibody, utilizing the Abbott HCV EIA 3.0 kit™ (Abbott Laboratories, Abbott Park, Illinois). The survey information and serum results were identified only by a subject identification number, with all other identifying information removed.Data were analyzed using univariate correlations with a Pearson's correlation coefficient. The number of sexual partners was compared between HCV-positive and negative subjects using an independent-sample t-test. Intravenous drug use between HCV-seropositive and seronegative subjects was compared using a Fisher's exact test. A multivariate logistic regression model was employed with stepwise backward elimination of non-significant variables, with HCV antibody status as the dependent variable. All of the variables from the survey were included in the model.A total of 500 subjects were recruited and had serum tested for HCV antibody. Only two of these subjects did not complete the survey. Most subjects who were informed of the study agreed to participate, but an exact number of subjects who refused participation in the study was not determined. The background characteristics of the patient population are displayed in Table The prevalence of positive HCV antibodies was 22/500 (4.4%, 95% CI 2.6–6.2%). Subjects that were anti-HCV positive had an average of 7 ± 8.6 lifetime sexual partners, compared to average of 2.5 ± 3.5 lifetime sexual partners among HCV-negative subjects (p = 0.02). There were no other statistically significant differences between the HCV antibody-positive and HCV antibody-negative groups in terms of demographic characteristics or topic areas associated with HCV infection/transmission. Among subjects with HCV antibodies, 4/22 subjects admitted intravenous drug use compared to 8/476 among HCV-antibody negative subjects (p < 0.001). A similar result was observed for rates of intranasal cocaine use among HCV-antibody positive (4/22) vs. HCV-antibody negative (8/474) subjects (p < 0.001). Of the 22 subjects with positive HCV antibodies, 12 subjects denied intravenous drug use, had no tattoos, and had either 1 or 2 lifetime sexual partners. Five subjects with positive HCV antibodies denied intravenous drug use and had >10 lifetime sexual partners.Intravenous drug use (r = 0.26, p < 0.001), intranasal cocaine use (r = 0.29, p < 0.001) and the number of lifetime sexual partners (r = 0.24, p < 0.001) were the only three variables with a statistically significant correlation with the presence of HCV antibodies on univariate analysis. The number of lifetime sexual partners had some correlation with intravenous drug use (r = 0.12, p = 0.009) and intranasal cocaine use (r = 0.13, p = 0.004). There was a very close correlation between intravenous drug use and intranasal cocaine use (r = 0.99, p < 0.001). Therefore, intranasal cocaine use was not incorporated into the multivariate model due to concerns about co-linearity. In the multiple logistic regression model with backwards elimination, intravenous drug use (OR 3.7, 1.52–9.03 95% CI) and number of sexual partners (OR 1.1, 1.04–1.20 95% CI) were independently associated with a positive HCV antibody result.We observed a prevalence of 4.4% of HCV antibodies in an urban population in Haiti. Additionally, we found that HCV antibodies were associated with intravenous drug use as would be expected, but also with increasing number of lifetime sexual partners. It is conceivable that subjects who reported higher number of sexual partners were more likely not to admit past intravenous drug usage. However, it is also possible that this finding represents sexual transmission of hepatitis C among subjects with multiple prior sexual partners. Finally, we observed HCV antibodies were present is some subjects who denied intravenous drug use and had less than 3 lifetime sexual partners.A recent survey of the influences on HIV preventive behaviors among youth in Haiti observed that 80% of males and 42% of females self-disclosed sexual activity . This suOnly a single prior study in Haiti suggested that HCV antibodies were rare . The mosIntravenous drug use does not exclusively explain the prevalence of HCV antibodies in the studied population. Although intravenous drug use was associated with an increased likelihood of having HCV antibodies (as has been thoroughly documented ), only fThe degree to which sexual transmission of HCV occurs is exceedingly controversial ,7. StudiOur findings are limited by the lack of information regarding active HCV infection. The presence of HCV antibodies only indicates prior exposure, and definitive documentation of active HCV infection requires detection of virus in the bloodstream utilizing HCV RNA polymerase chain reaction testing. However, since most patients exposed to hepatitis C develop chronic infection , HCV antIn conclusion, we provide seroprevalence data of HCV antibodies in an urban population in Haiti. These data are useful for understanding the risks of transfusion in Haiti if the blood has not been previously screened for HCV. This information contributes to our understanding of the worldwide prevalence of hepatitis C, which allows for informed decisions regarding the priorities of funding for the treatment and prevention of this infection. Additionally, we observed the number of sexual partners may be related to a greater likelihood of having HCV antibodies, but some subjects who denied intravenous drug use and had few lifetime sexual partners still had HCV antibodies. These findings could be utilized to foster consideration of new studies into some of the risk factors that are not clearly understood . These results suggest that further study into the mode of transmission of hepatitis C should focus on patients with a high number of lifetime sexual partners but no evidence of intravenous drug use.Matthew J. Hepburn, MD: no competing interests to declare.Eric J. Lawitz, MD: Dr. Lawitz has received research grants to conduct investigator-initiated research from Schering-Plough Corporation (Kenilworth, New Jersey).MH was involved in study design, data analysis, and manuscript preparation. EL was involved in study design, data collection, data analysis, and manuscript preparation.The pre-publication history for this paper can be accessed here:


Is aging inevitable or can it be "cured"? Recent work from many different fields of science is now providing clues into why we age and how long we might live The pathologist makes do with red wine until an effective drug is available, the biochemist discards the bread from her sandwiches, and the mathematician indulges in designer chocolate with a clear conscience. The demographer sticks to vitamin supplements, and while the evolutionary biologist calculates the compensations of celibacy, the population biologist transplants gonads, but so far only those of his laboratory mice. Their common cause is to control and extend the healthy lifespan of humans. They want to cure ageing and the diseases that come with it.“I would take resveratrol if it were feasible,” notes David Sinclair, assistant professor of pathology at Harvard Medical School in Boston, Massachusetts. In the meantime, he adds, “I do enjoy a glass of red wine about once a day.” It was Sinclair's laboratory, in association with a commercial partner, that revealed last August how the team had identified for the first time a group of simple organic molecules capable of extending lifespan. The most proficient of the group is resveratrol, the plant polyphenol found in red wine, and its discovery as a potential elixir to combat ageing represents another extraordinary advance in a decade of discoveries that have revolutionised the field.“These molecules will be useful for treating diseases associated with ageing, like diabetes and Alzheimer's.”Saccharomyces cerevisiae, the work suggests that researchers are only one small step from making a giant leap for humankind. “People imagined that it might have been possible, but few people thought that it was going to be possible so quickly to find such things,” says Sinclair. The field of ageing research is buzzing.Although the life-enhancing effects of Sinclair's polyphenols are so far confined to the baker's yeast Resveratrol stimulated a known activator of increased longevity in yeast, the enzyme Sir-2, and thereby extended the organism's lifespan by 70% . Sir-2 b“The study came out from a pretty big gamble,” recalls Sinclair, who used the human enzyme to screen and identify molecules that he expected would also stimulate those related enzymes in lower organisms. Unlike SIRT-1, these related enzymes are known to increase longevity when activated, usually by restricting the organism's calorie intake. Not only did they find “a whole collection of related polyphenols that activate ‘Sir-2 from humans,’ … but we put them onto yeast, justbeing the simplest model, and amazingly [they] did what we were hoping [they] would do,” recalls Sinclair. “But it was a real long shot.”Drosophila, which is a huge jump from a yeast cell,” says Sinclair. “So we're very encouraged by that.” Publication of these results is imminent. The team has also quickly broadened its horizons and is already testing the polyphenols on mouse disease models. “We think we may have tapped into a cell survival and defence programme [and] that these molecules will be useful for treating diseases associated with ageing, like diabetes and Alzheimer's,” says Sinclair. He hopes to publish the diabetes results by mid-2004 and those for Alzheimer's by the end of the year. Harvard and BIOMOL Research Laboratories, its commercial partner based in Pennsylvania, have already filed a patent application for the use of “synthetic related molecules” to combat diseases of ageing—an application, Sinclair adds, “very much linked to the [polyphenols] paper.”Now there's great eagerness in the Sinclair laboratory to complete and publish related research, notably by replicating the yeast work in higher organisms. “We have very promising results in There's been a radical shift in attitude towards ageing, says Sinclair. Before the 1990s, “people thought that we were a lot like cars, that we would just rust and breakdown—nothing we could do about it. The new idea is that there are pathways that can boost our defences against ageing—the ‘ageing-can-be-regulated’ discovery … that genes can control ageing [and] that there are pathways that [we can use to] slow down the process,” he says. “If that's true—and it really seems to be true for a lot of organisms—if it's true for us, it really means that there is hope that we will be able, one day, to find small molecules that can alter these pathways.”Sinclair expects to see such developments within his lifetime, but he ridicules the notion that humans will experience anything like the 70% extension to lifespan of his cultured yeast. “It'll be great if we can just give people an extra five years and have less disease in their old age and make it less painful,” he says. “We won't be seeing any Methuselahs around,” he insists.On his side are James Vaupel, one of Europe's leading demographers, and Marc Mangel, a mathematical modeller at the University of California at Santa Cruz. “Since 1840, life expectancy has been going up at 2.5 years per decade and will continue at this rate, maybe a little faster,” says Vaupel, head of the Laboratory of Survival and Longevity at the Max Planck Institute for Demographic Research in Rostock, Germany. Women in Japan currently have the highest average life expectancy of 85, he notes: “So the figure could be 100 in six decades, but not 500.” There's remarkably little people can do even if they want to live as long as possible, he says. “Give up smoking, lose weight, don't drive when drunk, install a smoke detector, take regular exercise,” suggests Vaupel, who insists he does them all, as well as taking vitamin supplements.“You look at these worms and think, ‘Oh my God, these worms should be dead.’ But they're not. They're moving around.”Mangel sees the problem of assessing the limitations of ageing research as fairly straightforward. Mathematical models, he says, could solve it by linking demographic properties and physiological developments. “We've had a separation of the biology of ageing and the demography of ageing, and they need to come together again,” notes Mangel, whose personal anti-ageing regime involves taking “a dose of anti-oxidant chocolate with a good feeling.”Caenorhabditis elegans, to show how long-lived mutant nematodes are as vigorous as normal young adults [But Cythnia Kenyon, whose laboratory reported in October that it had generated a 6-fold increase in the lifespan of its nematodes, is not so sure about the limitations. “You look at these worms and think, ‘Oh my God, these worms should be dead.’ But they're not. They're moving around…. Once you get your brain wrapped around that … then you start thinking, oh my goodness, so lifespan is something you can change—it's plastic. Then who knows what the limit is?” (Cynthia Kenyon has recorded video clips of the superstars of her lab, g adults .)Warming to the theme, Kenyon hypothesises: “If you'd asked me many generations ago, when we were actually common precursors of worms and flies, ‘Cynthia, you have a two-week lifespan, do you think that you could [live longer]?’ And if I'd told you, ‘Well, I think our descendants will live 1,000 times longer,’ you'd have said, ‘Oh, come on!’ But we do. It happened,” she notes.“Who knows what you could do in people?” Kenyon muses. “I don't want to go on record saying that it's not possible in people because I don't see why it wouldn't be…. I'm certainly not imagining that my company in the next few years is going to come up with a compound that can make people live to be 500. That seems just preposterous.” So the timescale is millions of years? “No, not necessarily,” she insists, “because once we understand the mechanism, then we can intervene and see what we can accomplish.”Yeast, as a model system for ageing, is at a distinct disadvantage. It lacks an endocrine system, and yet much research indicates that the key to longevity is control of hormones such as insulin and insulin-like growth factor 1 (IGF-1), as well as their downstream pathways and associated tissues, including the reproductive network.But David Sinclair, whose laboratory used yeast to show how an elixir might extend life, remains sanguine. “If it doesn't have an endocrine system, we can't understand cell-to-cell communication, but not all of ageing is just communication,” he says. “There are things that occur inside the cells that provide longevity, and that's where yeast can be applied.”Sinclair, assistant professor of pathology at Harvard Medical School, discovered a group of polyphenols that cause the human enzyme SIRT-1 and its homologues in lower organisms, including Sir-2 in yeast, to deacetylate the p53 protein and its homologues, notably the histones H3 and H4, in yeast.“Our findings are that the activation of the pathway downregulates p53's ability to cause cell death,” he notes. Although p53, the tumour suppressor, is known to be involved in programmed cell death, it is not known whether SIRT-1 has any role in ageing. So Sinclair “went straight back to yeast to prove the principle of longevity extension.”He found that deacetylisation of histones in yeast caused the DNA that's wrapped around them to become more compact and thus more stable. “DNA stability is key to longevity, and Sir-2 promotes that,” says Sinclair. “We don't know yet whether it's the same in humans.”Cynthia Kenyon, meanwhile, sees worms as the optimal model for helping to substantiate the links to humans. Besides the nematodes' being multicellular organisms with endocrine systems, she also notes that their short lifespan of around 20 days is a big advantage: “You can do lots of experiments with them.” Mice, which have short lifespans for mammals, still live two years, and long-lived mice for three or four years, she notes. And the advantage of fruit flies? “It's good to use more than one animal.”daf-2, whose receptor is similar to those for insulin and IGF-1 in humans and inhibits ageing, and daf-16, which promotes it.Kenyon, professor of biochemistry and biophysics at the University of California at San Francisco, has focused on decoding the role of genes in ageing, notably “The DAF-2 receptor activates a highly conserved PI-3-kinase, the PDK/Akt pathway, and that pathway affects ageing, at least in part, by inhibiting the activity of the DAF-16 transcription factor,” says Kenyon. “It does so by phosphorylating DAF-16 and inhibiting its entry into the nucleus.” She adds: “We think that the DAF-2 pathway has another way of influencing ageing … but we don't know what this other way is.”daf-2 receptor gene or in the genes encoding downstream signalling components, such as the PI-3-kinase, DAF-16's activity in the nucleus leads to the changes in expression of a wide variety of downstream genes, between 100 and 200, estimates Kenyon. Her studies show, she says, that a large number of those genes influence ageing.In the long-lived mutants, which are defective in the Kenyon, professor of biochemistry and biophysics at the University of California at San Francisco, is among the key contributors responsible for showing that a single gene, and subsequently many genes, can change an organism's lifespan.“It is inconceivable … that a life-extending therapy will ever be developed that is able to extend life independent of every other change.”daf-2 gene doubled the lifespan of the nematode C. elegans. daf-2 encodes a receptor that is similar to those for insulin and insulin-like growth factor-1 (IGF-1) in humans; this hormone receptor normally speeds up ageing in worms, but the mutations inhibit its action and enable the organisms to live longer. Before the results appeared, there was a “very negative attitude” towards ageing research, recalls Kenyon. Since then, and especially over the past few years in response to later findings, graduate students have been scrambling for a chance to work in her laboratory. “You can't believe the difference—there was such resistance to it,” she says. “daf-2 made a huge difference.” But then so did her subsequent research in the field.In a seminal paper published a decade ago, Kenyon's laboratory showed that mutations in the daf-2. “We discovered that in order for long-lived worms to live so long, they need another gene called daf-16,” recalls Kenyon. “daf-16 is kind of the opposite of daf-2, in the sense that it promotes longevity and youthfulness … so we call it ‘sweet 16.’” daf-16 encodes a transcription factor that controls the expression of more than 100 genes. “They don't do just one thing, they do many things,” says Kenyon. They can act as anti-oxidants , as chaperones (to prevent misfolded proteins from forming aggregates), as antimicrobials (to protect against bacteria and fungi), and as metabolic agents.Among her most significant findings is the identification of many more longevity genes; the results, published in July, derive directly from her early work on daf-2 and daf-16, the regulators, would be the conductors of an orchestra. They bring together the flutes and the violins and the French horns, each of which do different things, and they make them all work together in concert.”“So the picture that emerges is that the way the insulin/IGF-1 hormone system produces these enormous effects on lifespan is by coordinating the expression of many genes that do different things to affect lifespan, each of which on its own has only a small effect,” notes Kenyon. “It's as though Kenyon is unequivocal about the bottom line: “Now we have a whole set of genes whose biochemical functions we can be working on to understand more about the actual mechanisms of ageing.” Complementary results in flies and mammals persuade her to be more explicit. “The common ancestor of worms, flies, and mice must have had an insulin/IGF-1-like hormone system that controlled ageing. And that ability has been maintained. So the question is, has [that ability] been lost in humans? I think it's quite likely that it will also function in humans, but there isn't a direct demonstration yet that that's the case.”Nevertheless, the discoveries about the role of the insulin/IGF-1 pathway in ageing have had a profound impact on her own lifestyle, which includes a tendency to discard the bread from sandwiches and eat only the toppings of pizzas . “I'm on“Caloric restriction extends lifespan of mice, and so does the insulin/IGF-1 pathway,” she notes. Indeed, starting a low-calorie diet at any point in adulthood appears to help fruit flies live longer, according to research in Britain published last September. “What we don't know for sure in mice,” Kenyon continues, “is whether the two pathways are different or the same.”While much ageing research focuses on these two influences, she says that there are another two areas of investigation. Her laboratory reported in December 2002 that inhibiting the respiration of mitochondria in developing worms increased longevity, but that it had no effect in adult worms, for reasons still unexplained, she says. Further microarray analysis is underway to pinpoint whether the cause simply lies downstream of the insulin/IGF-1 pathway or whether it is something different altogether.Then there's research looking at the effects on lifespan of changes to an organism's reproductive system. For Kenyon, such work often involves a battle to convince sceptics that longevity is not a trade-off with fertility. Four years ago, her laboratory reported that killing germ cells increases the lifespan of worms by 60%, but only because, she stresses, it affects endocrine signalling and not because it prevents reproduction. Further research, published last year, showed quite clearly, she says, that ageing and reproduction are controlled independently of one another. And as for her recent work on infertile worms, which lived six times as long as normal following the removal of their entire reproductive systems, she says: “If we could intervene in the hormone signalling pathways directly, we think the animals would still live six times as long as normal, but would be fertile as well.”Jim Carey is one of those “trade-off” sceptics. He is a population biologist at the University of California at Davis and his research, on the effect on life expectancy of replacing the ovaries of old mice with ovaries from younger mice, is intended to complement Kenyon's work. But he insists that “an honest discussion of lifespan extension must include consideration of tradeoffs.” Many manipulations that extend lifespan in model systems, whether genetic or dietary, for example, ignore or gloss over the side effects, such as permanent sterility, huge weight loss, distorted organ-to-body ratios, or major behavioural aberrations, he notes. “It is inconceivable to me that a life-extending therapy will ever be developed that is able to extend life independent of every other change,” he concludes. “All life systems are interlinked and hierarchically integrated at all levels, so to talk about life extension using analogies with a car warranty concept is wrong-headed.”Another “trade-off” sceptic takes a different tack. As Armand Leroi puts it: “During occasional periods of involuntary celibacy I have thought, well, I may not be getting laid, but at least I shall live to a miserable and solitary old age.” Leroi, an evolutionary biologist at Imperial College of Science, Technology, and Medicine in London, offers an optimistic appraisal of the chances of finding a cure for ageing in his new book about the effects of genetic variety on the human body. He sees ageing simply as a collection of curable diseases: “There is no obvious impediment to that advance, nothing to make us think that human beings have a fixed lifespan.”low carb—actually, low glycaemic index carbs,” she says. “That's not eating the kind of carbohydrates where the sugar gets into your bloodstream very quickly [and stimulates production of insulin].”Cynthia Kenyon's eating habits are defined by her ageing research on worms. “There's a lot of these diets … and what they all have in common is No desserts. No sweets. No potatoes. No rice. No bread. No pasta. “When I say ‘no,’ I mean ‘no, or not much,’” she notes. “Instead, eat green vegetables. Eat the fruits that aren't the sweet fruits, like melon.” Bananas? “Bananas are a little sweet.” Meat? “Meat, yes, of course. Avocados. All vegetables. Nuts. Fish. Chicken. That's what I eat. Cheese. Eggs. And one glass of red wine a day.”Kenyon, professor of biochemistry and biophysics at the University of California at San Francisco, has been on her diet for two-and-a-half years. “I did it because we fed our worms glucose and it shortened their lifespan.”But the diet is unproven, she cautions, and she's not recommending it for all. Nevertheless, she's pleased with its performance for her. “I have a fabulous blood profile. My triglyceride level is only 30, and anything below 200 is good.”Kenyon is angered by the general lack of nutritional knowledge: “It's a little bit embarrassing to say that scientists actually don't know what you should eat…. We can target particular oncogenes, but we don't know what you should eat. Crazy,” she says.Does her dieting represent a return to scientists experimenting on themselves? “I don't think so—you have to eat something, and you just have to make your best judgement. And that's my best judgement. Plus, I feel better. Plus, I'm thin—I weigh what I weighed when I was in college. I feel great —you feel like you're a kid again. It's amazing.”


Over the past two decades, social marketing programs have become an important element of the national family planning and HIV prevention strategy in several developing countries. As yet, there has not been any comprehensive empirical assessment to determine which of several social marketing models is most effective for a given socio-economic context. Such an assessment is urgently needed to inform the design of future social marketing programs, and to avoid that programs are designed using an ineffective model.This study addresses this issue using a database of annual statistics about reproductive health oriented social marketing programs in over 70 countries. In total, the database covers 555 years of program experience with social marketing programs that distribute and promote the use of oral contraceptives and condoms. Specifically, our analysis assesses to what extent the model used by different reproductive health social marketing programs has varied across different socio-economic contexts. We then use random effects regression to test in which socio-economic context each of the models is most successful at increasing use of socially marketed oral contraceptives and condoms.The results show that there has been a tendency to design reproductive health social marketing program with a management structure that matches the local context. However, the evidence also shows that this has not always been the case. While socio-economic context clearly influences the effectiveness of some of the social marketing models, program maturity and the size of the target population appear equally important.To maximize the effectiveness of future social marketing programs, it is essential that more effort is devoted to ensuring that such programs are designed using the model or approach that is most suitable for the local context. Social marketing programs in reproductive health all aim to improve reproductive health of the target population by using commercial approaches to promote healthy behaviors and/or to expand access to essential products and services. While improving reproductive health of the target population is the ultimate aim, donors recognize the potential of social marketing programs to build the sustainable delivery of reproductive health and family planning products and services in developing countries. Different types of social marketing models have been used to achieve these objectives. Social marketing programs have often been classified as either using the  model . -3. The N model . .While many social marketing variants have proven successful, it is generally assumed that the manufacturer's model is more feasible in middle-income countries with a fairly well developed commercial infrastructure, while NGO models are more appropriate in lower-income countries with less less-developed commercial infrastructure . AlthougThis paper analyzes a comprehensive database of annual social marketing statistics. The database is unique in that it includes annual data on social marketing programs in reproductive health in over 70 countries, which cumulatively represent 555 years of experience with social marketing programs. We analyze these data to provide empirical information on the extent to which different social marketing models have been used in different socio-economic contexts, and to identify the socio-economic context in which each model is most effective. Our analysis is restricted to programs that social market condoms or oral contraceptives. As only limited data on the features of social marketing programs are routinely collected, we focus on differences in management structure.In many countries, social marketing programs are an essential component of the national family planning and HIV prevention programs. At present, large-scale social marketing programs for reproductive health are in operation in over 60 countries worldwide. A growing body of evidence shows that social marketing programs can make an important contribution to the reproductive health of the target population -19.In 2002, social marketing programs in 69 countries sold almost 1.6 billion condoms .Social marketing tends to be a cost-effective approach to achieving widespread access to health products and services ,23. It usocial marketing foundations. This organization may receive technical assistance from an international social marketing organization but it operates as an independent entity. Implementation may also be by an existing organization, such as a family planning association (FPA) or another type of clinic-based organization. In such case an international NGO provides technical assistance to the local organization to develop and operate the social marketing program. Social marketing programs may also be implemented through a partnership between an international NGO and an existing commercial organization. In this case, the international NGO often helps to develop the market for the product through research and promotion, while the commercial partner handles all aspects of packaging, sales and distribution.Three different management structures are common in social marketing programs: 1) management by an affiliate of an international NGO, 2) management by local clinic-based or non-clinic-based organizations, and 3) partnerships with a commercial organization. The most commonly used management structure in health-oriented social marketing programs is management by an NGO. Typically this implies management by an affiliate of an international social marketing organization that provides an expatriate resident advisor. Several social marketing programs are managed by local organizations. For example, some social marketing programs are managed by a specially developed new organization that is independent of international affiliation. These are often called Not all management approaches are feasible in every context. For example, in countries with relatively high-income levels and a strong commercial sector there may be many opportunities for commercial partnerships, while in other countries there may be few such opportunities, if any . SimilarThis section analyses empirical data to study differences in social marketing models and their impact. Since data about program characteristics are scarce, we focus on differences in management structure. Specifically, our objectives are to 1) describe to what extent the type of social marketing management structure actually varies according to the socio-economic context in which the programs are implemented, and 2) to examine the effectiveness of various management structures in increasing socially marketed product sales in different contexts.For the purpose of this study, we compiled a database that comprises data on most of the major social marketing programs that were in operation at any time during the period between 1988 and 2001. Many, but not all, of these programs were still in operation in 2001. The database contains annual data on several program characteristics and on indicators of the local socio-economic context. The main sources of information for data on program characteristics were DKT International's "Contraceptive Social Marketing Statistics," Population Services International's (PSI) MIS database, and published data on Futures Group International (TFGI) programs . These tFor each social marketing program, the database contains one case for each year of operation within the 1988–2001 study period. In other words, our unit of analysis is the "program-year." Using "program-years" as our unit of analysis has the advantage that all indicators, including program characteristics, can change over time. In addition, it implies that in the analyses programs that have been in operation for a longer period of time are given more weight than newer programs. For example, a social marketing program that has been operating since 1988 will contribute 14 program-years to the database (one case for each year of operation); a program that started in 1995 and was terminated in 1997 will contribute three program-years, and a program that started in 2001 will contribute only one program-year.After restricting the database to those social marketing programs that have a reproductive health component, the database contains information on social marketing programs in over 70 countries, which represent a total of 555 years of program experience. This includes 508 years of experience with condom social marketing and 208 years of experience with OC social marketing.Our measures of market potential are based on the indicators used by Michigan State University's Center for International Business Education and Research . Specifi• Population size• Percentage of population living in urban areas• Per capita GNI in current international dollars. This indicator measures the GNI converted in international dollars using purchasing power parity. An international dollar has the same purchasing power as a dollar in the U.S. [World Bank 2003]• Telephone mainlines per 1,000 inhabitants• Television sets per 1,000 inhabitantsThe database also includes selected indicators of the characteristics of the social marketing programs, including:• Management structure • Program maturity/years of operation (less than 3 years, 4–6 years, and 7+ years)• Number of reproductive health products social marketed (one vs. two or more). Only condoms, oral contraceptives, injectables and IUDs are considered.In addition to these indicators of the socio-economic context and of the characteristics of the programs, information is available on the following outcome measures:• Per capita condom sales• Per capita OC salesWhile some social marketing programs distribute and/or promote injectables or IUDs, their number is not sufficiently large to allow multivariate analyses. Hence, data on sales of injectables and IUDs are not examined in this study. Other indicators of program effectiveness, such as user-prevalence rates are not collected on an annual basis.Ideally, one would also want to examine the cost-efficiency of implementing different social marketing models. A commonly used approach to estimate cost-efficiency is to conduct a cost-effectiveness analysis. By definition, any cost-effectiveness analysis requires obtaining data on both the effectiveness of the program, i.e. the impact of the program, and on the cost of implementing the program ,34. UnfoIn the next sections, we use cross-tabulations to examine to what extent management structure varies by socio-economic context. Since our unit of analysis consists of program-years of operation, these tabulations show the percentage of program-years that had each management type.We also test the extent to which different indicators of socio-economic context affect effectiveness of social marketing programs with different management structures. For this purpose, we use multiple regression analyses to assess the effect of indicators of socio-economic context on per capita condom sales for different management structures. Because each social marketing program has multiple entries in the data set (one for each year of operation), the standard errors in ordinary linear regression are incorrect. We use random-effects regression to solve the problem that observations for a given country-program are not independent .For each type of management structure, we assess the net effect of socio-economic context on per capita condom sales, after controlling for the number of reproductive health products marketed, program maturity, time period (1995–2001 vs. earlier). Similar analyses are conducted for per capita OC sales.We now analyze our database to illustrate to what extent different management structures have been used in social marketing programs . In larger countries, other management structures have been somewhat more common.Breakdown by level of urbanization confirms that the range of management strategies used increased with socio-economic status. The percentage of social marketing program-years managed by NGO affiliates ranged from 86% when urbanization was low to 47% when urbanization was high. In highly urbanized populations, 31% of program-years were managed by existing local organizations and 22% by commercial partnerships.Social marketing programs in countries with a low per capita GNI (<$1,000) have been managed predominantly by NGO affiliates (97% of program-years), with local organizations being a distant second (3%). In countries with higher GNI levels, a wider range of management strategies has been used. When the per capita GNI levels are medium-low, management through NGO affiliates was still the most common . However, in medium-low GNI countries local organizations accounted for 18% of program years and commercial partnerships for 13%. Finally, when the GNI exceeded $3,000 per capita, NGO affiliates accounted for only 52% of program-years, followed by local organizations (25%) and commercial partnerships (23%).Our indicators of the level of development of the commercial infrastructure show that the NGO model has dominated in settings with a relatively poorly developed commercial infrastructure. For example, NGO affiliates accounted for roughly 87% of program years in countries with fewer than 5 phone mainlines per 1,000 inhabitants, and for 88% of program years in countries with fewer than 20 television sets per 1,000 in habitants. Management through existing local organizations and commercial partnerships is most common in settings with over 30 phone mainlines and over 100 television sets per 1,000 inhabitants.Management structures have also changed over time. For example, the percentage of program-years managed through NGO affiliates has steadily increased from 56% in 1986–93 to 86% in 1998–2001. Simultaneously, management through existing local organizations and commercial partnerships has gradually declined.The last panel of Table We now use random-effect GLS regression analyses to examine to what extent market size, market intensity, commercial infrastructure, and program features  affect per capita condom sales and per capita OC sales. The variable "number of television sets per 1,000 inhabitants" was removed from the multivariate analyses as it correlated too much with some of the other predictor variables. We conduct separate analyses for social marketing programs managed by NGO affiliates, commercial partnerships, and local organizations. Due to the small number of cases, we are unable to differentiate between existing organizations and new organizations.The second column in Table As expected, social marketing programs managed through an NGO affiliate tend to be more effective, as measured by per capita condom sales, in countries with smaller populations (β = -.006). Per capita condom sales do not vary with the level of urbanization, per capita Gross National Income (an indicator of market intensity), nor with the number of phone mainlines .The third column in Table Table The third column in Table Table The third column in Table Several studies have commented on the differences between social marketing programs that use the non-governmental organization model and those that use the manufacturer's model -3. AlthoThis paper has used a database of annual data on social marketing programs in over 70 countries to verify the extent to which the different social marketing models that have been used vary across socio-economic contexts, and to test in which socio-economic context each of the models is most successful at increasing use of socially marketed products.A rigorous analysis of the relative effectiveness of different social marketing models would require detailed data about the characteristics of the program, including data about financial structures, branding, pricing (and level of subsidies), distribution, and promotion.Because data on the features of social marketing programs are limited, we focused on differences in the programs' management structure. Ideally, we would also like to examine are range of outcome measures, including per capita sales, the user-prevalence, and the unit-cost for each method. Since such prevalence and unit-cost data are unavailable, we have focused on per capita condom and OC sales.We have used empirical analyses to obtain a better understanding of the variations that exist in social marketing programs. Specifically, we have analyzed a database of annual statistics on social marketing programs in over 70 countries, accounting for a total of 555 years of program experience. The database contains annual data on the management structure, program maturity, the number of reproductive health products marketed, and on product sales. The database also contains indicators of the local socio-economic context , including the population size, level of urbanization, per capita GNI, number of telephone mainlines and television sets per 1,000 inhabitants.Our analyses have shown that of the 555 years of social marketing program experience covered by the database, over 70% has used the NGO management structure. Nevertheless, there are considerable variations in management structure. The NGO affiliate management structure has been the dominant model when the per capita GNI is below $3,000 and the level of urbanization below 50%. The data also show that use of the NGO management structure has increase dramatically over the course of our study period.The findings support the theory that there may be more opportunities for successful partnerships with the private sector in countries with a high per capita GNI. The highest percentage of such partnerships (23%) was found in countries with a GNI exceeding $3,000 per capita. This percentage decreases to 13% in countries with a per capita GNI between $1,000 and $3,000. None of the commercial partnerships in our database were in countries with a per capita GNI below $1,000. One of the reasons for this pattern may be that there is more commercial activity and a wider range of possible partners in countries with a higher per capita income. Users in those countries are also more likely to be able to afford the commercially sustainable brands that are marketed under this approach.Although the program characteristics usually appear to be appropriate for the local socio-economic context, it is questionable whether this has always been the case. For example, over 50% of program-years in countries with a medium-high GNI (over $3,000) used the NGO model, even though countries with such income levels typically provide opportunities for commercial partnerships.Our multivariate analyses identified the factors that affect per capita condom and OC sales for each of the four social marketing models. The reader is cautioned, however, that condom and OC sales may also be influenced by other factors that were not measured in our data base, such as HIV prevalence and overall levels of contraceptive prevalence. Bearing these cautions in mind, the analyses yielded the following key findings:• Per capita condom sales are higher in countries with smaller populations. This may be influenced by the difficulty of achieving high distribution coverage in large, mostly rural countries (e.g., India, Indonesia, Vietnam, Nigeria) and/or the time it takes to large such large populations with social marketing campaigns. Also, social marketing programs in countries with a larger population may have more competition (e.g., India, South Africa). By contrast, several small countries have a high HIV/AIDS prevalence and/or limited method choice (e.g., sub-Saharan Africa);• Per capita condom sales are higher for more mature programs and those that market more than one product. This probably reflects improvements in distribution capability over time, as well as the cumulative effect of behavior change programs that stimulate demand. More developed programs with stronger distribution are also more likely to market several products;• Per capita condom sales do not vary with the level of urbanization, per capita income, or commercial infrastructure. These variables are more likely to affect access-related factors, such as product access and ability to pay. Other factors to look for that may affect sales are related to demand: level of effort of the social marketing program (which reflects funding levels), health context  and competition from the public sector and other NGOs that may cancel out the higher market potential in the more developed countries;• None of the factors examined had any significant influence on the per capita OC sales of social marketing programs managed through an NGO affiliate.• Per capita condom sales tend to be higher in countries with smaller populations and with a lower per capita growth. At first glance, this finding is counter-intuitive. That is, initially one would expect sales for commercial partnerships to increase with population and per capita GNI, because commercial program need both a high demand and ability to pay. However, as population size and income increase sales may decline due to increased competition from commercial and other suppliers. Several countries with middle-high incomes (Turkey, the Philippines, and Jordan) or large populations (Turkey, the Philippines, and Indonesia) have strong public sector programs that offer free contraceptives, as well as many commercial suppliers. Also, demand for condoms in these countries has been very low, given that HIV prevalence is low that other contraceptive methods are available. By contrast, smaller countries such as Haiti and Zimbabwe have high HIV rates, less choice of methods, and probably less competition from other suppliers.• Programs in countries with larger populations have significantly higher per capita condom sales. One likely explanation for this pattern is that local organizations tend to have good distribution networks, which pays off in countries with a larger population. Some of the larger countries (e.g., Bangladesh and Colombia) have well-established organizations with a big market share. For example, in Bangladesh SMC provides almost all condoms and the demand is very high;• OC sales are significantly higher for programs that have been in operation for seven or more years. The longer a program operates, the more developed its distribution is likely to be. Social marketing communication efforts are also more likely to pay off over the long run;• Programs in countries with a higher level of urbanization have lower per capita condom sales. This was anticipated, given that NGOs and family planning associations have a strong track record of serving users in peri-urban and rural areas. In addition, when urbanization increases, access to commercial products sold in pharmacies and other private sector outlets increases as well, which allows commercial suppliers to grow their market share and compete with social marketing and public sector products;• The level of commercial infrastructure, as measured through the number of telephone mainlines per 1,000 inhabitants has a small negative effect on per capita OC sales. This finding may reflect that a better commercial infrastructure implies more competition from commercial suppliers.Our analyses of records on 555 years of experience with social marketing programs have illustrated the tendency to design social marketing programs with a management structure suitable for the local context. However, the evidence also shows examples where this has not been the case. While socio-economic context clearly influences the effectiveness of some of the social marketing models, program maturity and the size of the target population appear equally important. Consequently, it is crucial that social marketing programs are designed using the model or approach that is most suitable for the local context.Dr. Meekers is the former Research Director of PSI (1996–2001), and has been a paid consultant for other PSI research projects.Dr. Meekers developed the study design, assisted with the analysis, and contributed to the writing of the final manuscript. Mr. Rahaim collected data, conducted literature reviews, and contributed to the editing of the paper.The pre-publication history for this paper can be accessed here:


Vpr virion packaging was normal when amino acid #1–23 of the Gag p6 domain was preserved. Most importantly, Ala substitutions for Phe15, Arg16 and Phe17 in the context of amino acid #1–23 of the Gag p6 domain abolished Vpr virion packaging. Single Ala substitutions for Phe15 and Phe17 also abolished Vpr virion packaging, whereas Ala substitution for Arg16 had no effect. Our studies have revealed a novel signal sequence for Vpr packaging into the HIV-1 virion. The 15FRFG domain in p6 resembles the FxFG repeat sequences commonly found in proteins of the nuclear pore complex. These results have provided novel insights into the process of virion packaging of Vpr and suggest for the first time that Vpr may recognize the FxFG domain for both virion packaging and association with nuclear pores.The auxiliary regulatory protein Vpr of HIV-1 is packaged in the virion through interaction with the Gag C-terminal p6 domain. Virion packaging of Vpr is critical for Vpr to exert functions in the HIV-1 life cycle. Previous studies suggest that Vpr interacts with a (Lxx)4 domain in p6 for virion packaging. In the present study, mutational analysis of HIV-1 Gag p6 domain was performed in the context of the HIV-1 genome to examine the effect on virion packaging of Vpr. Surprisingly, Ala substitutions for Leu Vpr is a 15 kDa auxiliary regulatory protein of HIV-1 produced in the late phase of the viral life cycle and packaged in the virion . Thus, VTransiently expressed Vpr localizes in the nucleus, and specific nuclear localization signals have been identified in Vpr . Vpr nucVirion packaging of Vpr is through interaction with the Gag C-terminal p6 domain . With va15FRFG domain in HIV-1 Gag p6, 4 amino acid residues downstream of the Tsg101-binding domain, is found critical for Vpr virion packaging. Since FxFG domains also occurs in nuclear pore proteins, the current finding also suggests for the first time that Vpr may recognize the same FxFG domain for both virion packaging and association with nuclear pores. Thus, the FxFG domain appears to be a favorite signal for in vivo recognition by Vpr. We discuss the impact of this finding in the context of the HIV-1 life cycle.In this study, sequences in HIV-1 Gag p6 domain required for Vpr virion packaging was dissected in the context of the HIV-1 genome. Surprisingly, the previously identified (Lxx)4 domain in p6 is shown non-essential for Vpr virion packaging. Instead, a -/R+ and p89.6/Pol-/R- constructs were described before [-/R+ and p89.6/Pol-/R- to generate p89.6/HA-Ub/R+ and p89.6/HA-Ub/R-constructs, respectively a/R+ constructs were prepared by inserting a PstI/StuI digested PCR DNA fragment into the PstI/BalI sites of p89.6/HA-Ub/R+. For p89.6/Pr-/R+, PCR was performed with the p89.6/5' clone as the template [-(LF)a/R+, PCR was performed with the same template and primer 3 and primer 5 CTGACCAGGCCTCCCGGGTTATTTTATTGTGACGAGGGGTCGTTGCCTGCGGC TGATCTGAGGGAAGC. For constructs p89.6/Pr (Lxx)-/R+, p89.6/Pr-(1–23)/R+ and p89.6/Pr (FRF)a/R+, the PCR DNA was digested with PstI/SmaI and ligated into the PstI/SmaI sites of p89.6/Pr- (LF)a/R+. For p89.6/Pr- (Lxx)-/R+, PCR was performed with the p89.6/5' template and primer 3 and primer 6 GTACTACCCGGGAGGCCTTTATTCCTTGTCTATCGGCTCCTGC. For p89.6/Pr-(l-23)/R+, PCR was performed with primer 3 and primer 7 GTACTACCCGGGAGGCCTTTATTGAGTTGTTGTCTCCTCCCCAAACC. For p89.6/Pr- (FRF)a/R+, PCR was performed with primer 3 and primer 8 GTACTACCCGGGAGGCCTTTATTGAGTTGTTGTCTCCTCCCCGGCCGCGGCGC TCTCTGCTGG. The construct p89.6/Pr-F15A/R+, p89.6/Pr-R15A/R+, and p89.6/Pr-F17A/R+ were prepared in the same way as p89.6/Pr-(1–23)/R+, except that the PCR was performed with primer 3 and a new primer instead of primer 7: primer 9 (for p89.6/Pr-F15A/R+) ACTCGACCCGGGAGGCCTTTATTGAGTTGTTGTCTCCTCCCCAAACCTGGCGC TCTCTGCTGG, primer 10 (for p89.6/Pr- R16A/R+) ACTCGACCCGGGAGGCCTTTATTGAGTTGTTGTCTCCTCCCCAAACGCGAAGC TCTCTGC, and primer 11 (for p89.6/Pr- F17A/R+) ACTCGACCCGGGAGGCCTTTATTGAGTTGTTGTCTCCTCCCCGGCCCTGAAGC TCTC. The construct p89.6/Pr- (l-23)/R+/Δ Ub was prepared by removing the BamHI-BamHI fragment, encoding the HA-tagged Ub gene, from the p89.6/Pr-(1–23)/R+ construct.The p89.6/Prtemplate , and pri+ genome , no Vpr was detected in the virion (lane 2). We subsequently prepared a protease-truncated construct based on the R+ genome, named Pr-/R+, and observed normal Vpr virion packaging a/R+ construct (lane 4), which contains L44A/F45A double mutations in the Gag p6 domain -/R+ construct, and again normal Vpr packaging was detected -/R+ construct still maintains a 15FRFG domain in p6 which resembles the FxFG domain frequently observed in resident proteins of the nuclear pore [-(1–23)/R+ a/R+ construct /R+ construct. As shown, removal of HA-Ub had no effect on Vpr virion packaging 4 repeat domain in Gag p6 is essential for Vpr virion packaging [15FRFG domain or the (Lxx)4 domain for Vpr packaging. It is noticeable that although the 15FRFG domain is highly conserved among different HIV-1 strains, it is replaced with 15FRSG in the HIV-1 Hxb2 strain (GenBank accession number K03455) and 15VRFG in the Yu-2 strain (GenBank accession number AF287352). Future studies may reveal if an engineered FRFG domain in these HIV-1 strains can allow Vpr packaging in the absence of the (Lxx)4 domain.In this report we provide evidence that HIV-1 Vpr is packaged into the virion through the previously unrecognized ackaging ,22. The ackaging  or on th15FRFG domain of p6 resembles the FxFG domains of certain nucleoporins with respect to both the FxFG core and the following hydrophilic residues rich in Ser/Thr residues (Fig. Significantly, the ues Fig. . Thus, VVpr interaction with nucleoporins has been reported before . In part15 in the 15FRFG domain of p6 abolishes Vpr virion packaging (Fig. It is likely that Vpr recognizes the FxFG domain and not other types of FG repeats. Single Ala substitution for PheMLV: murine leukemia virus; Ub: ubiquitin.None declared.HZ and HJ participated in the construction of mutant HIV-1 genomes, cell culture, transfection, and Western blot analyses. LZ conceived of the study and participated in its design, coordination and execution. All authors read and approved the final manuscript.


Drosophila. The regulation of signaling specificity remains largely obscure. We show that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/β-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, we propose that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways.The Frizzled  and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/β-catenin pathway or Fz/planar cell polarity (PCP) signaling in  Differential subcellular localization of Fz1 and Fz2 receptors contributes to signaling specificity within the Wnt/Fz signaling pathways Pattern formation in multicellular organisms relies on inductive signaling events. Several evolutionarily conserved ligand–receptor combinations and associated signal transduction pathways are used again and again during development to induce tissue- and cell-type-specific responses. Thus, context-dependent signaling specificity is an important prerequisite for patterning and differentiation. Although for many signaling pathways the flow of information is largely established, the underlying signaling specificity mechanisms remain unclear.Drosophila; reviewed in Drosophila and vertebrates (Drosophila. For example, in wing cells the PCP response is the formation of an actin spike (the wing “hair”) that points distally, and in the eye PCP is manifest in the regular ommatidial arrangement in the anteroposterior and dorsoventral axes (reviewed in Members of the Frizzled (Fz) family of seven-pass transmembrane proteins act as receptors for the Wnt family of secreted ligands . In mosttebrates . PCP is Drosophila, Fz (for clarity we will refer to it as Fz1) and Fz2 are functionally redundant receptors for Wg, activating the canonical Wg/Arm cascade  -Fz(s) combinations are associated with either canonical or Fz/PCP signaling. However, the specificity is not simple. For example, although Wnt5a and Wnt11 cause embryonic phenotypes associated with the Fz/PCP-like pathway , coexpredppGal4 control) is localized throughout the apical–basal axis of larval imaginal disc epithelia, and extracellular Wg binds to Fz2 predominantly at the basolateral membrane . Similarly, overexpressed Fz2 . In addition, we show that the seven-pass transmembrane region contains elements that are critical for PCP signaling. Based on our data, we propose a model in which subcellular localization, possibly through the association of Fz with other membrane proteins such as coreceptors, is a critical aspect in regulating the signaling readout and specificity within the Wnt/Fz signaling pathways.dpp-Gal4 control also displays a strong enrichment in the apical region of the disc epithelium  in wing imaginal discs. Fz1 and Fz2 were subdivided into three parts: (1) the N-terminal Wnt-interacting cysteine-rich domain (CRD), (2) the remaining proximal extracellular domain and 7 transmembrane region and loop region (collectively referred to as 7-TM), and (3) the intracellular C-tail. All chimeric proteins were Myc-tagged between the CRD and 7-TM region . In sumdpp-Gal4 control in wing imaginal discs caused wing cell hairs to point away from the expression domain  phenotype ; Table 1n domain B. This in domain . Expressn domain ; Table 1n domain ; Table 1Taken together, these experiments demonstrate that (1) apical Fz1 localization correlates with higher levels of Fz/PCP signaling activities and (2) the 7-TM region of Fz1 is critical for effective PCP signaling.Next we wished to determine which part of the C-tail of Fz1 or Fz2 is responsible for the difference in subcellular localization. The protein sequences of the Fz1 and Fz2 C-tails are homologous over the first 29 amino acids (45% identity), but Fz2 is longer by an additional 61 amino acids . The apiFirst, we mutated several Fz1–1-1–specific residues to those of Fz2, or deleted conserved amino acid stretches within the Fz1–1-1 C-tail see , and wheSecond, we tested whether sequences within the extended Fz2 C-tail have an effect on localization or PCP signaling. We introduced a stop codon after the L633 residue of Fz2 (corresponding to the position of the stop codon in Fz1) in Fz1–1-2 and Fz1–2-2 chimeras A and 2E.These same chimeras were tested in the functional assay for PCP signaling activity. Strikingly, expression of Fz1–1-2S caused a phenotype very similar to that of Fz1–1-1 H, but diTo test whether the extension within the Fz2 C-tail can more generally block apical localization, we added the Fz2 extension on to Fz1–1-1 E, resembtubulin promoter [tub] ) were also tested for their ability to rescue the −fz eye and wing PCP phenotype. tub-Fz1–1-1 and tub-Fz1–1-2S  fully rescue the −fz loss-of-function (P21fz/R52fz) phenotype in both the eye and wing   . Similaron (APF) . These don (APF) , suggestE59fmi clones in larval wing imaginal discs. Fz1–1-1 is localized apically in E59fmi mutant cells in third instar imaginal discs, indistinguishable from its localization in wild-type tissue  often causes wing notching and loss of wing margin bristles , with 65% of wings showing large areas of margin bristles missing and severe wing notching  we observe very strong wing notching effects and a general reduction of the posterior wing compartment , suggesting that Fz1–1-2 contains substantial PCP signaling activity.Is the apical localization of Fz required for PCP signaling? The Fz1–1-2 chimera, which is distributed ubiquitously within the apical–basolateral membrane, only partially rescues the ling see D. In conssay see . Taken t−fz in the eye), the relative difference might be sufficient for the partial rescue.Because both GOF and loss-of-function studies indicate that the Fz1 7-TM region is critical for Fz1 function, Fz1–1-2 is expected to have Fz/PCP signaling activity, although with altered subcellular distribution. Thus, the remaining PCP signaling activity of Fz1–1-2 seen is probably due to the presence of some of this protein in apical regions. It is difficult to determine how much of Fz1–1-2 is actually localized to this membrane region. Since the immunohistochemical staining indicates that it is not excluded apically, we assume that Fz1–1-2 has enough apical localization to participate when PCP signaling is initiated. It has been suggested that wing cell orientation does not depend on absolute Fz levels, but instead depends on relative Fz/PCP activity differences in a Fz activity gradient across a field . Thus, atub-fz1–1-2 rescues the −fz phenotype better in the wing than in the eye, whereas there is no apparent difference in rescue activity between the eye and the wing for tub-fz1–1-1 or tub-fz1–1-2S. The difference could be due to the observed nonautonomous PCP signaling effects in the wing , and it36 h APF , suggestfmi dependent. This result suggests that Fmi and Fz1 get recruited to apical junctions independently. During later stages, Fmi and Fz1 then affect each other's localization through PCP signaling. At this point, it remains unclear which molecules initially recruit Fz1 into the apical junctional region.We showed here that initial apical localization of Fz1, preceding both stages of PCP signaling, is not Secreted Wg mainly binds to Fz2 at basolateral membrane regions of the wing epithelium , indirecfz, fz2 double mutant phenotype . UAS-Fz1–2-1 was constructed by combining the Fz1 CRD (Fz1 residues 1–166) with the Myc tag, the Fz2 7-TM region (amino acids 220–617), and the Fz1 C-tail (amino acids 558–585). A HindIII site (generated in vitro) was used to combine Fz1 CRD with the Fz27-TM region. An XhoI site was used to link the Fz2 7-TM region with the Fz1 C-tail. C-tail mutation constructs of Fz1 were generated through PCR-based site-directed mutagenesis . Fz1–1-2S and Fz1–2-2S were generated by introducing a stop codon after residue L633 of Fz2. Fz1–1-1C2 was generated by introducing a BsiWI site at the residues R574–T575 of Fz1 and R626–T627 of Fz2 (the RT residues remain the same by this mutagenesis). This added the Fz2 amino acids 627–694 to the Fz1 C-tail at the RT residues. The respective UAS transgenic flies were generated by standard procedures.dpp-Gal4, en-Gal4, or omb-Gal4 (tub-promoter-driven Fz chimeric constructs were generated by cloning the respective Fz1/2 constructs into the Casper4-tub vector (containing a 2.4-kb tub promoter fragment in Casper4—a kind gift from Stephen Cohen). P21fz and R52fz are null alleles of fz  in 0.1% Triton X-100 PBS for about 30 min or longer, and then mounted in 80% Glycerol PBS. Eye embedding and sectioning was performed as described by