Acromyrmex leaf-cutting ants farm a fungal cultivar as their primary nutrient source. These ants also carry a Pseudonocardia Actinobacteria exosymbiont on their bodies that produces antifungal compounds that help inhibit specialized parasites of the ants' fungal garden. Major workers emerge from their pupal cases (eclose) symbiont-free, but exhibit visible Actinobacterial coverage within 14 days post-eclosion. Using subcolony experiments, we investigate exosymbiont transmission within Acromyrmex colonies. We found successful transmission to newly eclosed major workers fostered by major workers with visible Actinobacteria in all cases . In contrast, newly eclosed major workers reared without exosymbiont-carrying major workers did not acquire visible Actinobacteria . We further show that the majority of ants exposed to major workers with exosymbionts within 2 hours of eclosion acquired bacteria , while normal acquisition did not occur when exposure occurred later than 2 hours post-eclosion . Our findings show that transmission of exosymbionts to newly eclosed major workers occurs through interactions with exosymbiont-covered workers within a narrow time window after eclosion. This mode of transmission likely helps ensure the defensive function within colonies, as well as specificity and partner fidelity in the ant-bacterium association.Stable associations between partners over time are critical for the evolution of mutualism. Hosts employ a variety of mechanisms to maintain specificity with bacterial associates.  Riptortus pedestris, showed significantly greater acquisition of a horizontally transmitted Burkholderia midgut symbiont during the second instar larvae than other stages Hirudo verbana. Limited symbiont acquisition periods likely function to increase colonization specificity Symbiosis, the living together of unlike organisms Escovopsis spp.) of the ants' cultivar fungus Atta and Sericomyrmex, do not carry visible Actinobacteria Fungus-growing ants are associated with fungal mutualists that they have been farming for approximately 50 million years Acromyrmex leaf-cutting ants, young major workers are present in the fungus garden. These ants typically exhibit abundant coverage of Actinobacteria, while minor workers and foraging (older) major workers carry little to no visible exosymbiont Acromyrmex echinatior and Acromyrmex octospinosus hosted closely related actinobacteria in the genus PseudonocardiaA. echinatior is dominated by a single phylotype of Pseudonocardia, and that colonies appear to maintain the same strain for many years In Acromyrmex leaf-cutter ants, A. echinatior and A. octospinosus, using subcolonies with garden and leaf material. To determine if acquisition is a consequence of the presence of Actinobacteria-carrying major workers, we fostered newly-eclosed Acromyrmex major workers in the presence or absence of major workers carrying Actinobacteria, as well as with symbiont-free minor Acromyrmex workers, or symbiont-free Atta workers. We also fostered pupae with minor workers and a thorax dissected from a live major worker carrying a high level of exosymbiont coverage. Furthermore, to examine the time frame in which acquisition occurs, we reared newly-eclosed majors in subcolonies where Actinobacteria-carrying majors were introduced at different time points. Finally, specific conditions for within-colony transmission would help ensure long-term colony specificity in the ant-Actinobacteria association. To test for this, we isolated and sequenced 16 S rDNA of the Actinobacteria-symbiont Pseudonocardia from nine colonies, and compared these to sequences obtained when the same colonies were first collected 6–9 years earlier.Here we explore the mode of Actinobacterial transmission in two species of Acromyrmex echinatior: AP061104-01 Costa Rica; AeP5 & CC031209-02, CC031212-01 Panama; EC110523-1, EC110523-2 & EC110523-3 Costa Rica; Acromyrmex hispidus fallax UGM030327-02 Argentina; Acromyrmex laticeps UGM030330-04, CC030403-09, Argentina; Acromyrmex niger CC030327-02, Argentina; Acromyrmex octospinosus AL050505-11, CC031210-22, MP010908-1, SA010908-4, ST040116-01 & UGM020518-05 Panama; Atta cephalotes: AP061021-2 & AL050513-22 Costa Rica. Colonies were maintained at 24°C in the dark to mimic underground conditions, with overhead lights illuminated periodically for colony maintenance. Each ant colony was housed in a large outer plastic container (17.5 cm H×29 cm W×40 cm L) accommodating one smaller refuse dump container and one or two garden containers . Each container had a 1 cm diameter hole drilled to allow ants to move in and out. Mineral oil was applied regularly to the top 4 cm of the outer container to prevent ants from escaping. Colonies were provisioned maple (Acer sp.) and oak (Quercus sp.) leaves (frozen in the winter and fresh during summer) three times per week. Leaves were supplemented with oatmeal, rice and cornmeal. Wet cotton balls were stored in the outer box to increase humidity.The exosymbiont source and stability experiments were performed using the following colonies maintained at the University of Wisconsin-Madison: Acromyrmex octospinosus colonies  collected in Heredia, Costa Rica in 2011. These colonies were maintained in the laboratory at the La Selva Biological station at 24°C under ambient lab light conditions. Each colony was housed in a larger plastic container (28 cm H×40 cm W×56 cm L) accommodating one smaller plastic container for refuse dump and one to three smaller plastic containers  enclosing the colony fungus gardens. These colonies were provisioned with local flora, including leaves and flowers. Supplemental experiments were performed at the University of Wisconsin–Madison on the colonies mentioned above.Acquisition timing experiments were performed using 2 leaf fragment of pin oak  was added 24 hours or more after eclosion for the ants to cut and incorporate into the fungus garden. We started by establishing that Acromyrmex ants could acquire exosymbiont normally in a subcolony set-up consisting of a focal pupa, 0.1 g fungus, 2 major workers and 4 minor workers (n = 10 subcolonies) Leucoagaricus from Atta cephalotes, 2 major workers and 4 minor workers, n = 22 subcolonies; since pupae fail to eclose without assistance from older ants it was necessary to include ants in these set-ups]; 2) conspecific worker ants, via cross-fostering with another ant species with no visible Actinobacteria ; 3) minor workers , 4) major workers not carrying visible Actinobacteria , 5) major workers carrying visible Actinobacteria , and 6) minor workers with a thorax from a freshly dissected major worker with visible exosymbiont (changed every 48 hours to assure that bacteria were viable), to provide a bacterial source population . See We performed subcolony experiments to evaluate which colony components are necessary for successful exosymbiont transmission. Subcolonies were set up in small (4.0 cm H by 5.5 cm OD) clear plastic containers . After sterilizing the containers for at least 20 minutes using UV light, a Kimwipe moistened with distilled water was placed at the bottom to help provide humidity. A small (4.12 cm W×4.12 cm L×0.79 cm H) weigh boat  was placed on top of the Kimwipe, and then the fungus garden, ants and pupa were added. A∼1 cmPreliminary cross-fostering experiments suggested that the period when callow workers could acquire exosymbiont was less than 24 hours (data not presented). This narrow temporal window emphasized the importance of delineating the time of eclosion. Here, the relevant feature of eclosion is likely to be the time when a callow worker can first interact with the environment . Further, initial efforts to define a zero time point  revealed that the time it takes an individual to emerge from its pupal case is highly variable, ranging from minutes to hours. Attempts to visually verify the first holes in the pupal case via light microscopy lead to extremely high mortality. Given that pupae/callow workers often begin struggling to free themselves from their pupal case once this membrane is broken, as has been reported in fire ants To determine the time after eclosion during which acquisition would predominantly occur, we fostered pupae with four minor workers with no visible exosymbiont from their natal colony. Subsequently, we added two majors with visible exosymbiont collected from the ants' natal garden at different time points after focal ant eclosion. Once these majors were added, they remained in the subcolony until the end of the experiment. We selected major workers whose Actinobacterial coverage and colony location made it likely that they were carrying abundant exosymbiont loads and were involved in brood care Pseudonocardia from nine e-maintained colonies, and compared these to sequences obtained when colonies were first brought back to the from the field 6–9 years prior to this study. Other genera of Actinobacteria were not isolated at the time of colony collection, so we were unable to assess the stability of other potential members of the symbiont community. However, these are expected to occur in low frequency if at all Acromyrmex ants by scraping visible Pseudonocardia from their propleural plates onto chitin agar plates, allowing bacteria to grow for 3 weeks, then transferring colonies with Actinobacteria-like morphology  to yeast malt extract plates Pseudonocardia using the same methods. Subsequently, we assessed whether ant colonies maintained the same bacterial symbiont by comparing the 16 S ribosomal RNA gene sequences of the original isolates with those obtained in this study. Bacterial DNA was extracted using a cetyltrimethylammonium bromide (CTAB) protocol described previously A specific mode of within-colony transmission from older to younger major workers would suggest long-term stable association between individual ant colonies and their exosymbiotic bacterium. To test this we extracted and sequenced 16 S rDNA of the Actinobacteria symbiont Acromyrmex echinatior ants acquire Actinobacteria exosymbionts. Five of six focal pupae in control subcolonies  acquired exosymbiont normally, consistent with patterns of acquisition previously reported We explored the natural conditions under which newly eclosing Pseudonocardia, we conducted fungus-switching experiments. We fostered Atta and Acromyrmex pupae with majors and minors from their natal colonies, but with fungus garden fragments from a host ant of the other genera . Switching of the fungal garden resulted in high mortality among callow workers . No Acromyrmex echinatior ants lived longer than five days post-eclosion , so all results reported here are for Acromyrmex octospinosus . None of the five Atta ants acquired exosymbiont when raised with A. octospinosus fungus garden . When A. octospinosus and A. echinatior pupae were cross-fostered with Atta garden and Atta workers , no majors acquired visible exosymbiont coverage . These results indicate that the fungus garden is highly unlikely to be a significant source of exosymbiont inoculum.To test whether the ants' fungal garden could be the source of the Acromyrmex ants were raised with their own fungal garden and Atta workers , nor when pupae were raised with minors only  or minors only but with the presence of an Actinobacterial source population . In contrast, all major worker pupae raised with Acromyrmex major workers and fungus from either Acromyrmex or Atta colonies acquired visible exosymbiont . This indicates that major workers carrying visible levels of exosymbiont are necessary for normal acquisition. Further, our fostering of pupae in subcolonies with majors carrying no visible exosymbiont resulted in no workers acquiring exosymbionts . These results thus show that direct contact with major workers carrying visible exosymbiont is necessary for normal exosymbiont transmission  strains of Pseudonocardia symbionts from five colonies of Acromyrmex spp. ants following 8 years  and 9 years (one colony of A. octospinosus with UGM020518-05 with one independent re-isolate) of maintenance in the laboratory .Transmission of symbionts from older to newly-eclosed majors within a narrow time window suggests long-term propagation of specific Actinobacteria symbionts within individual ant colonies. This predicted long-term serial transmission of specific symbionts within individual ant colonies was supported by re-isolations of boratory . FurtherAcromyrmex, in addition to indicating that the opportunity for acquisition of symbionts by this mechanism is possible only during a narrow time window.Many, if not all, metazoans depend on beneficial microbial symbionts. These microbes provide their hosts with a range of services including nutrition and defence. In a number of these host-symbiont associations, a high degree of specificity exists between host species and/or populations and individual species or specific phylogenetic clades of closely related strains of microbes Acromyrmex leaf-cutting ants to acquire natural populations of cuticular Actinobacteria. This confirmed that newly eclosed major workers could acquire exosymbionts in small subcolonies with leaf fragments, fungal garden, and adult majors and minors present. Subsequent experiments fostering (within genus) and cross-fostering (across genera) callow ants in simplified subcolonies (lacking particular components such as minor workers) allowed us to eliminate the possibility that fungus garden fragments, minors, and freshly dissected cuticle with Actinobacteria from majors could provide a source of inoculum of the bacteria. Our findings indicate that the presence of live Actinobacteria-carrying majors is necessary for successful transmission to occur, making this source the primary, likely sole, route of within-colony transmission for these two Acromyrmex species. Other fungus-growing ant genera carry Actinobacteria on the same locations of the cuticle as AcromyrmexOlder major workers had previously been proposed as the source of Actinobacterial transmission Buckholderia gut-symbiont during a single phase of larval development Our work indicates that successful exosymbiont transfer requires worker-to-worker contact occurring less than 2 hours after worker eclosion. The mechanisms responsible for this narrow time window for successful exosymbiont transfer are unknown, but are likely linked to the physiology of the ants, which actively maintain the bacteria on so-far unidentified cuticular secretions A. echinatior from Panama are dominated by a single Pseudonocardia bacterium from either of two specific phylogenetic clades, consistent with previous findings Pseudonocardia exosymbionts from nine Acromyrmex colonies that had been maintained in the lab for up to 9 years. We found that 16 S DNA sequences were identical, or near identical, to those of the Pseudonocardia symbionts originally isolated from colonies at the time of collection. Five of these colonies were from the same Panamanian population as those in Andersen and colleagues Acromyrmex species  collected in Argentina. While not all re-isolated sequences were identical to the original isolates, the sequence differences were fewer than what could be expected due to routine Sanger sequencing errors, confirming the findings from Poulsen and colleagues Acromyrmex species, in addition to suggesting that these patterns of colony-level specificity and long-term stability with Pseudonocardia exist across the genus Acromyrmex. Future work sampling workers from field colonies over several years would be desirable to confirm our findings in the presence of the potential selective pressure imposed by the horizontally transmitted pathogen Pseudonocardia.A narrow time window for active transmission of specific Actinobacteria to newly eclosed major workers suggests that specific bacterial symbionts are continuously inoculated onto new individuals within ant colonies and therefore maintained over the lifetime of the colony. Andersen and colleagues Pseudonocardia strains within colonies. However, these results could reflect stable associations of ant colonies with bacteria from the same phylogenetic clade, not a single specific strain. The reason for this is that 16 S is not a reliable marker to distinguish between closely related strains of bacteria . Nevertheless, given the specific mechanisms involved in transmission identified in this study, it is likely that individual nests in fact do form stable associations with a single dominant Pseudonocardia strain. This is congruent with the work by Poulsen and colleagues (2005) and Caldera & Currie EF-Tu, a more variable phylogenetic marker, and six house-keeping genes, respectively.Our results, in combination with those of Andersen and colleagues (2013), support stable long-term maintenance of specific Pseudonocardia and do not engage in colony founding) and the bacterial mutualist. This default vertical transmission mode, between host generations via virgin queens, should produce patterns of host-symbiont co-diversification, which to a large extent have been observed, only disrupted by events of horizontal transmission and acquisitions of free-living Pseudonocardia to the symbiosis Pseudonocardia in Acromyrmex leaf-cutting ants has the potential to mediate long-term stability and host-symbiont specificity of the mutualism.The specific conditions for within-colony transmission described are expected to both ensure the efficient acquisition of the symbiont to new workers and to decrease the opportunity for other microbes to exploit this niche. This would benefit the ants by ensuring the association with their beneficial symbiont, and the symbiont by ensuring its transmission within the colony. This mode of transmission from older to newly eclosed adults is likely to be equivalent for the inoculation of virgin queens that are known to carry Actinobacterial exosymbiont on their cuticle during colony foundation Figure S1Images of subcolony setup and ant colonization. Experimental subcolony setup (a) with symbiotic  and aposymbiotic  ants. a) A subcolony with newly eclosed worker (lighter ant) and two major workers (darker ants), fungus garden and leaf fragments; images contrasting symbiotic  with aposymbiotic ants ; c, d photos of adult ants, e-g dissecting microscope images of workers 14 days post-eclosion, h-k environmental scanning electron micrographs of workers 21 days post-eclosion. Note characteristic morphology of Actinobacteria in symbiotic ants and absence of these features in aposymbiotic ant. Photos b and c ©Alex Wild (used by permission), other images by Sarah Marsh.(DOCX)Click here for additional data file.Table S1Experimental design overview. The first column outlines each experiment, with subsequence columns providing the components in each subcolony: species of pupae  and adult ants; number of exosymbiotic and aposymbiotic majors, and minors; fungus source colony ant species, and finally results .(DOCX)Click here for additional data file.


The STOPAH trial was a double-blind, 2x2 factorial, phase III randomised clinical trial assessing the treatment of prednisolone and pentoxifylline in patients with severe alcoholic hepatitis; a study that aimed to evaluate 28-day mortality .At the time of trial development, the benefits/harms of pentoxifylline in this patient population were unknown. Prednisolone had a significant though uncertain evidence base. No treatment interaction was assumed yet there was uncertainty about its effect. Therefore, pre-specified stopping rules to assist decision making in stopping treatment arms were required.There was no published methodology on stopping rules for factorial trials, so a design based on the Peto-Haybittle  rule wasAn independent data monitoring committee assessed these stopping rules after 200, 400 and 800 patients had reached the primary endpoint. The stopping rules were not met at any of the three interim looks. The results of the STOPAH trial have recently been published .


Schistosoma haematobium is primarily stimulated by antigens from dying worms. Praziquantel treatment kills adult worms, boosting antigen exposure and protective antibody levels. Current schistosomiasis control efforts use repeated mass drug administration (MDA) of praziquantel to reduce morbidity, and may also reduce transmission. The long-term impact of MDA upon protective immunity, and subsequent effects on infection dynamics, are not known. A stochastic individual-based model describing levels of S. haematobium worm burden, egg output and protective parasite-specific antibody, which has previously been fitted to cross-sectional and short-term post-treatment egg count and antibody patterns, was used to predict dynamics of measured egg output and antibody during and after a 5-year MDA campaign. Different treatment schedules based on current World Health Organisation recommendations as well as different assumptions about reductions in transmission were investigated. We found that antibody levels were initially boosted by MDA, but declined below pre-intervention levels during or after MDA if protective immunity was short-lived. Following cessation of MDA, our models predicted that measured egg counts could sometimes overshoot pre-intervention levels, even if MDA had had no effect on transmission. With no reduction in transmission, this overshoot occurred if protective immunity was short-lived. This implies that disease burden may temporarily increase following discontinuation of treatment, even in the absence of any reduction in the overall transmission rate. If MDA was additionally assumed to reduce transmission, a larger overshoot was seen across a wide range of parameter combinations, including those with longer-lived protective immunity. MDA may reduce population levels of immunity to urogenital schistosomiasis in the long-term (3–10 years), particularly if transmission is reduced. If MDA is stopped while S. haematobium is still being transmitted, large rebounds (up to a doubling) in egg counts could occur.Previous studies suggest that protective immunity against  Urogenital schistosomiasis, caused by schistosome blood flukes, infects more than 100 million people in sub-Saharan Africa. Current control efforts involve regularly treating all school-aged children with the drug praziquantel, which kills schistosome worms. Earlier work by our group suggests that protective immunity against schistosomes is mainly stimulated by dying worms, and that in the short term, praziquantel treatment boosts immunity through killing worms. The longer-term impact upon the development of protective immunity is unknown. In this paper, we used a mathematical model which was able to replicate short-term patterns of infection and antibody to predict the long-term changes in antibody and infection levels that would occur during and after a 5-year treatment programme. We found that the longevity of protective immunity was particularly influential. Short-lived protective immunity was associated with levels of protective antibody declining below pre-treatment levels in the long term, and also with an increase in measured infection levels (eggs in urine) to peak above pre-treatment levels after the treatment programme finished. Antibody declines and infection peaks post-treatment were also predicted if treatment programmes reduced schistosome transmission. These results highlight the possible negative consequences of ceasing mass treatment programmes once they have commenced. Schistosoma haematobium) remains a prevalent tropical disease, infecting over 100 million people in sub-Saharan Africa Urogenital schistosomiasis , as suggested by earlier model fitting S-transferase We used a stochastic individual-based model which describes changes in worm burden, egg output and a protective antibody response with age for people living in an area with endemic schistosome infection. This model has been fully described previously S. haematobium egg counts and specific antibody responses from several rural sites in Zimbabwe with endemic infection In earlier work, this model was parameterised using data from studies in Zimbabwe and elsewhere, and fitted to population data on pre- and post-treatment A population of 175 individuals was simulated, with 5 individuals in each yearly age group from 1 to 34 years old at the time of the baseline survey. Individuals were simulated up to their respective ages before the initial baseline egg count and antibody levels were recorded and the first round of treatment applied. Individuals were then simulated for a further 15 years after this initial survey, during and after MDA (see next section for treatment schedules). When individuals reached the age of 34, they were replaced by 1 day old infants with no worms or antibody, to maintain a constant population size.Six treatment schedules were used which vary treatment frequency, target population, coverage and reduction in transmission . For theIn each of the other treatment schedules, one parameter was changed from the standard schedule . In scheFor all treatment schedules (1–6), treatment was applied over a five year period and each treatment was applied the day after egg counts and antibody levels were recorded. Treatment was applied randomly across the eligible population at the required coverage level (75% or 90%) at each round of treatment, meaning that an individual's chance of being treated in each round was independent of whether they had been treated in previous rounds. Treatment was assumed to be given independently of worm burden or egg output, in line with the usual MDA strategy of giving treatment to all school-aged children For each parameter set, 200 repeat simulations of the whole population were run, and mean levels of egg output and antibody for the whole population aged 6–34 years old were calculated pre-treatment and at yearly intervals during and after the simulated treatment regime, averaged over the 200 repeat simulations. This age range was used in order to capture the changes in egg counts and antibody levels in treated individuals as they aged over the long follow-up period. Egg output and antibody dynamics were studied to see how quickly they returned to pre-treatment levels. The conditions  under which protective antibody levels fell below pre-treatment levels or egg counts overshot pre-treatment levels were identified.relative changes in antibody levels. Actual antibody levels are shown for comparison in supplementary We found that protective antibody dynamics were mainly determined by immune decay rate and worm life span – this is illustrated with selected parameter sets which reflect the general patterns seen (exceptions are noted in the section on overshoots in egg output). −1). Models with a longer parasite life span showed both a higher boost and a more substantial drop in antibody levels than those with shorter parasite life span (−1) and worm life span was short (3 years) (ife span . Antibod3 years) . Egg out3 years) .−1); for models with an antigen threshold, the parameter combinations all had rapid immune decay (0.8 year−1), a low antigen threshold (25 antigen units) and moderate antibody strength (0.256 units per plasma cell). Of all the unique parameter sets explored, 11/293 parameter sets for models including an antigen threshold, and 4/12 without an antigen threshold, gave rise to an infection overshoot. Models without an antigen threshold predicted higher overshoots (>70% above pre-treatment levels) than models which included an antigen threshold , some parameter combinations were identified which gave rise to overshoots in measured egg output after MDA ceased . The oveThe impacts of separately varying the frequency of treatment and the coverage and age-range of the target population (treatment schedules 2–4) are shown for single parameter combinations , but demWith biennial treatment (schedule 2), protective antibody declined and egg output increased following non-treatment years, but their levels approached those seen with annual treatment (schedule 1) one year after each treatment . The ove−1; The effects of assuming that transmission is reduced during MDA are shown for one parameter set , but theS. haematobium infection will be substantially reduced by repeated MDA, while levels of protective antibody will be initially boosted by treatment, in agreement with patterns seen in the field. We predict that, in the long-term, levels of antibody could fall below pre-treatment levels after or even during MDA. More rapid declines in protective antibody levels are predicted with more rapid immune decay, shorter worm life span or reduced transmission. After the initial increased exposure to dying worms that treatment brings about, the reduced worm burden leads to a subsequent reduction in exposure to dying worms, leaving antibody levels strongly influenced by immune decay rates. Reduced transmission further reduces antigen exposure. Baseline antigenic exposure rates are expected to be lower in models with a longer worm life span, and so the reduced antigenic exposure following treatment will have a more rapid effect in models with short worm life span, leading to more rapid declines in antibody.Several MDA programs for schistosomiasis are currently underway in Africa S. mansoni) suggest that schistosome-specific antibody levels may decline below pre-treatment levels following an initial boost, behaviour predicted for medium- or short-lived antibody responses in our model We found that measured egg output could rebound to levels exceeding pre-treatment levels after cessation of MDA. Our finding that this was very likely to happen if treatment temporarily reduced transmission confirms findings from earlier work using different models Our results suggests that MDA might disrupt the build-up of protective immunity (or may disrupt existing immunity) against schistosomes, despite short-term boosting of this protective response. Interestingly, a reduction in antibody levels below pre-treatment levels during MDA did not necessarily correspond with overshooting of egg output after treatment ceased. It should be noted that, even when overshoots in egg output occur after treatment stops, the overall impact of the intervention on egg output (taking the overshoots into account) may still be positive; the reductions in egg output during the control programme may be sufficient to offset the increases seen after treatment stops.S. haematobium in Ghana S. japonicum in China We found that increasing the coverage of treatment of school children from 75% to 90% only increased population antibody levels and decreased measured infection levels by a very small amount. The random allocation of treatment at each round meant that even at 75% coverage, the chances of an individual never being treated over the five rounds of treatment were very small, which may account for the comparatively small coverage effect. Two previous modelling studies looking at Previous modelling studies which considered the effects of acquired immunity on the impact of MDA suggest that the strength and duration of protective immune responses play an important role in determining infection dynamics Our results support the long-term maintenance and monitoring of existing MDA programmes, to ensure that treatment continues while transmission is still ongoing. In addition to MDA, other measures to reduce transmission should also be strengthened, including the provision of safe water and sanitation facilities, and good health education S. haematobium worms, repeated MDA will boost protective immunity initially, but antibody levels could decline below pre-treatment levels during or after MDA. In some circumstances, we also predict that post-MDA egg output could exceed pre-intervention levels. Field data are not currently available to test these predictions, but they have been made using a calibrated model which reproduces robust patterns seen in short-term pre- and post-treatment studies of S. haematobium infection In conclusion, our models predict that, with protective immune responses stimulated by dying Figure S1Dynamics of protective antibody and egg output during and after treatment, by immune decay rate: Absolute values. The results from −1; for all parameter sets, worm life span is 6.5 years.(TIF)Click here for additional data file.Figure S2Dynamics of protective antibody and egg output during and after treatment, by worm life span, for different immune decay rates. Similar results to −1,  0.008 year−1.(TIF)Click here for additional data file.


Congenital high airway obstruction syndrome (CHAOS) is a rare life-threatening condition characterised by complete or near-complete developmental obstruction of the foetal airway. Although antenatal imaging findings have been described, the postmortem MRI findings have not been reported.To present postmortem MRI features of CHAOS.We retrospectively reviewed our hospital pathology and imaging databases for cases of CHAOS over a 2-year period.We identified two cases of CHAOS. In both cases, postmortem plain radiographs demonstrated gross abdominal distension with distortion and splaying of the rib cage. Both foetuses had characteristic postmortem MRI findings including large-volume fluid-filled lungs on T2-weighted imaging, diaphragmatic eversion, fluid-filled airway dilatation below the level of obstruction, centrally positioned and compressed heart, and massive ascites. One foetus had an associated limb abnormality.Postmortem MRI in foetuses suspected of having CHAOS allows confirmation of the diagnosis, determination of the anatomical level of the atresia or stenosis, and identification of associated abnormalities without the need for invasive autopsy. Congenital high airway obstruction syndrome (CHAOS) is a rare life-threatening condition characterised by complete or near-complete intrinsic developmental obstruction of the foetal airway. It is usually fatal, and the classic features are detectable by antenatal US examination. Most cases occur sporadically, with no associated gene abnormality, although about 50% of cases have additional structural abnormalities.Typical prenatal sonographic findings include large hyperechoic lungs with inverted diaphragm, ascites and dilated fluid-filled lower airways, and in some cases the level of the obstruction can be identified , 2. It iAlthough antenatal imaging findings have been described, the postmortem MRI findings have not been reported. Postmortem confirmation of the diagnosis provides potentially important information, both for the parents and for improved understanding of the underlying pathogenesis, and hence optimisation of management for future cases . HoweverWe retrospectively searched our hospital pathology and imaging databases for cases of CHAOS who had undergone postmortem imaging as part of the autopsy procedure. Informed consent was provided for postmortem examination and imaging, and the use of non-identifiable autopsy data for research was approved by the local research ethics committee.Postmortem MRI is offered as part of a less-invasive autopsy service at our institution, a tertiary referral children’s hospital, to parents who decline a full autopsy. We searched our databases over the 2-year period since the introduction of postmortem imaging at our institution, 2012–2014, identifying parents of two foetuses with CHAOS who consented to both postmortem MRI and autopsy.Before postmortem MRI, bodies were stored in a mortuary at 4°C. All scans were performed on a 1.5-T MR scanner  using whole-body 3-D T2-weighted turbo spin echo (TSE) sequences , 3-D T1-weighted volumetric interpolated breath-hold examination (VIBE) sequences  and 3-D constructive interference in the steady state (CISS) sequences .Postmortem MR images were reported by a paediatric radiologist with 5 years’ experience (O.J.A.) prior to a minimally invasive, laparoscopy-assisted autopsy performed by a perinatal pathologist with more than 10 years’ experience (N.J.S.). At the time of this report, only the antenatal reports were available to the radiologist and pathologist, with antenatal imaging correlation only available after all investigations had been completed.Laparoscopy-assisted autopsy was performed via a 30-mm lower sternal incision to allow abdominal and thoracic organ sampling . The larThere was no significant medical or obstetric history. Following an unremarkable initial antenatal course, routine US at about 20 weeks of gestation demonstrated a male foetus with an apparently dilated trachea with bilateral enlarged echogenic lungs, inverted diaphragm, compressed heart and severe ascites. No additional abnormalities were noted. An amniocentesis was performed for karyotyping (46 XY) and the parents opted for termination of pregnancy at 24 weeks’ gestation. Postmortem examination including postmortem MRI was offered as part of routine clinical care.Postmortem skeletal radiography showed gross soft-tissue swelling of thorax and abdomen, with splaying of the ribcage Fig.  but no bFollowing an uncomplicated initial antenatal course and low-risk screening for Down syndrome, a routine US examination at 20 weeks of gestation revealed a male foetus with bilateral enlarged echogenic lungs and ascites. An amniocentesis was carried out (46 XY). A second sonogram at a regional foetal medicine unit confirmed these findings along with generalised subcutaneous oedema, diaphragmatic eversion and bilateral talipes equinovarus. The parents opted for termination of pregnancy at 24 weeks, and postmortem examination including postmortem MRI was offered as part of routine clinical care.Postmortem plain radiographs demonstrated gross abdominal distension with distortion and splaying of the rib cage, with right fibular hemimelia and talipes equinovarus Fig. . The lefIn both cases, similar external appearances were noted, and laparoscopic assisted autopsy was performed including sampling of the larynx and trachea, which histologically confirmed almost complete occlusion at the level of the larynx, consistent with failure of recanalisation Fig. .Both foetuses had characteristic postmortem MRI findings of CHAOS, which was confirmed at autopsy. Classic imaging features included high signal intensity large-volume fluid-filled lungs on T2-W imaging, eversion of the diaphragm, airway dilatation (with fluid) below the level of obstruction, centrally positioned and compressed heart, and massive ascites as well as moderate subcutaneous oedema, and pleural and pericardial effusions. Autopsy tissue sampling allowed more specific determination of laryngeal anatomy, but this additional information did not affect patient management or counseling; the potential advantage of a postmortem MR-based approach is to offer parents an alternative to full conventional autopsy. We demonstrate that postmortem imaging, along with minimally invasive tissue sampling if required, confirms the diagnosis and might be an acceptable choice for parents who decline conventional autopsy.Numerous reports of prenatal sonographic findings in foetuses affected by CHAOS show characteristic bilateral, enlarged and echogenic lungs associated with diaphragmatic eversion and ascites, with about 130 cases described –12. In CCauses of CHAOS include laryngeal atresia, laryngeal or subglottic stenosis, webs, laryngeal cysts and tracheal atresia or stenosis. Imaging is useful to define the features of the sequence, to investigate the level of obstruction  , 13, andAlthough most cases of CHAOS are sporadic, about 50% of foetuses with CHAOS in other series have associated congenital abnormalities, particularly of the musculoskeletal system or digits . One of Because they are less invasive, imaging-based methods of investigation after death are becoming increasingly important, particularly for parents who do not agree to traditional postmortem examination for moral, religious, social or other reasons , 10. HowOne limitation of postmortem imaging in these cases is that we did not specifically adapt the imaging protocols to try to better delineate the level of the obstruction. It is possible that more advanced imaging techniques, or the installation of contrast agents  could delineate more precisely the level of obstruction. We thought this was unnecessary because more precise localisation of the level of obstruction has little prognostic significance. We also acknowledge that normal postmortem changes can contribute to the fluid accumulation seen in the lungs at postmortem imaging, although the contribution in this particular condition is likely to be minimal, given the close correlation between pre-mortem and postmortem imaging findings.Postmortem MRI in foetuses with suspected CHAOS allows confirmation of the diagnosis, determination of the anatomical level of the atresia/stenosis, and identification of associated abnormalities without the need for invasive autopsy.


Nanoparticles, microparticles, and other biomaterials are advantageous in vaccination because these materials provide opportunities to modulate specific characteristics of immune responses. This idea of “tuning” immune responses has recently been used to combat infectious diseases and cancer, and to induce tolerance during organ transplants or autoimmune disease. Lymph nodes and other secondary lymphoid organs such as the spleen play crucial roles in determining if and how these responses develop following vaccination or immunotherapy. Thus, by manipulating the local microenvironments within these immunological command centers, the nature of systemic immune response can be controlled. This review provides recent examples that harness the interactions between biomaterials and lymph nodes or other secondary lymphoid organs to generate immunity or promote tolerance. These strategies draw on mechanical properties, surface chemistry, stability, and targeting to alter the interactions of cells, signals, and vaccine components in lymph nodes. While there are still many unanswered questions surrounding how best to design biomaterial-based vaccines to promote specific structures or functions in lymph nodes, features such as controlled release and targeting will help pave the way for the next generation of vaccines and immunotherapies that generate immune responses tuned for specific applications. Vaccination has produced one of the greatest impacts on human health in history . No otheIn the last several years, the impact of nanoparticles (NPs), microparticles (MPs), and other biomaterial vaccine and immunotherapy carriers on LNs has been an intriguing area of focus. These studies reveal the potential of biomaterials to program the local LN microenvironment to control systemic immune response. The broad potential of biomaterials for vaccination and immunotherapy has recently been reviewed –4. This The innate immune system is composed of first-response defense mechanisms including (i) skin that creates a physical barrier against pathogens, (ii) immune cells that home to and engulf pathogens or other immunogenic structures, and (iii) receptors that detect broad classes of molecular patterns absent in mammals but present in viruses and bacteria. In contrast, adaptive immunity involves the generation of immune responses specific for a particular molecule, termed an antigen. Generation and control of these antigen-specific responses require complex interactions between immune cells, antigens, and soluble factors in secondary lymphoid organs (SLOs) ,6. TheseLNs are found throughout the body, concentrating antigens from a network of lymphatic vessels that continually sample tissue for antigens or other immune signals ,8. SolubLNs are bean-shaped structures surrounded by a collagen-rich fibrous capsule Fig. . AntigenDCs and T cells comprise an interior region of LNs called the paracortex (“T cell zone”), while B cells and specialized follicular dendritic cells (FDCs) make up a surrounding cortex called the “B cell zone” Fig.  10,11).,11.10,11+ T cells are activated by DCs that have encountered, processed, and are presenting the antigen these T cells are specific for   that migrate from LNs to destroy host cells expressing the target antigen . A similar process occurs in DCs presenting antigen in MHC-II to CD4+ helper T cells that play an important role in the activation of B cells to produce antibodies.The major classes of adaptive responses include i) cell-mediated immunity, through which cytotoxic T lymphocytes directly destroy infected host cells, and ii) antibody-mediated immunity, which involves binding, neutralization, and clearance of antigens by circulating antibodies specific for these pathogens. In the simplest sense, cell-mediated immunity removes intracellular pathogens such as viruses, while antibodies are able to address extracellular toxins and pathogens . Cell-mediated and antibody-mediated responses develop following the activation of naive, antigen-specific T cells and B cells, respectively ,13. Thesen) Fig. . Importa+ T cells with the same antigen specificity migrate toward the B cell zone following activation by DCs. B cells are activated at the edge of the cortex by these helper CD4+ T cells, then move back into the cortex and proliferate to form a germinal center (GC). In GCs, the affinity of the proliferating B cell for the cognate antigen is increased through interaction with resident FDCs that deliver survival signals to B cells that strongly bind antigens presented by FDCs. These processes involve somatic mutation and affinity maturation and are detailed in recent reviews . In. In16). The physicochemical properties of biomaterials can act as intrinsic immune signals that help shape immunity. Some of the properties which have been studied along these lines—reviewed in –4—includBiomaterials provide a unique platform for vaccination and immunotherapy. Interestingly, these materials can mimic some features of clinically approved adjuvants , for example, by condensing or encapsulating antigen or other immune signals into particulate structures with sizes ranging from tens of nanometers to several microns. This size range allows efficient uptake by APCs. Biomaterials can also be used to passively or actively target immune tissues such as LNs. Lastly, these materials allow co-delivery of multiple cargos  and controlled release of vaccine and immunotherapy components. This last feature of co-delivery is becoming increasingly important in modulating the types of responses that are generated for a specific vaccine or therapy. The sections below will provide specific examples of how these properties are being harnessed to enhance “traditional” immune responses aimed at arming the body to destroy infectious pathogens or cancer.Both traditional  and biomaterial-based vaccine components must reach LNs to generate adaptive immune responses. Several groups have carefully controlled the size of NPs and other vaccine carriers to passively target LNs and the DCs residing in these tissues. For example, by altering particle size, the effectiveness of drainage through the lymphatics and the retention time within LNs can be changed. In studies conducted by Reddy et al., poly(propylene sulfide) NPs with defined sizes were injected intradermally into the tail of mice and the particle drainage through the lymphatics to LNs was monitored ,35. TwenThe route of injection also helps define if and how NPs of a given size will reach LNs. For example, injection of 90 nm virus-like particles via multiple different injection routes  resulted in unique particle drainage patterns to the inguinal, lumbar, popliteal, and sciatic LNs . Notably+ T cell-mediated adaptive response instead of MHC-II molecules which is the traditional route for phagocytized antigens. In addition to particle size, recent studies have investigated the role that the geometry of vaccine carriers plays in immunogenicity. These studies demonstrate that the shape and aspect ratio of synthetic carriers play an important role in modulating T cell activation , and others include emulsions (MF59 and AS03), liposomes (AS01), and synthetic DNA and RNA sequences (polyI:C and CpG). Though there is still some debate as to the mechanism by which alum or other adjuvants enhance vaccination, these materials often persist at the injection site to serve as a depot  and increase antigen phagocytosis and presentation that enhances DC activation . In a ditivation . Thus, fIn addition to passive targeting by size, LNs and LN-resident cells are being actively targeted by conjugating NPs and MPs with specific ligands or receptors. One of the molecules that has been targeted is DEC-205 (CD205), a transmembrane protein found primarily on DCs. Monoclonal antibodies specific to DEC-205 have been used to decorate acid-degradable polymer and liposome vaccines loaded with model antigens  and B16-melanoma antigens ,39. Trea+ T cells and functional inflammatory cytokines ) was observed, resulting in tumor regression and prolonged survival in immunized mice. These highly promising outcomes are fundamentally a result of the higher concentrations (targeting) and retention (exposure time) in LNs, important characteristics that motivate the discussion below on the role that duration and concentration of antigen and immune signals in LNs play in driving immunity.Liu et al. recently used albumin as a shuttle to direct lipid-based vaccines to LNs . Albumin+ T cells  one bolus dose, (ii) regularly spaced, equivalent doses, (iii) regular injections with exponentially decreasing doses, or (iv) regular injections with exponentially increasing doses Fig. . Across lls Fig. . Functiolls Fig. . These r+ T cells and antigen-specific serum antibody levels, respectively. Mice immunized and boosted subcutaneously with ICMV formulations loaded with malaria antigens (VMP-ICMV) generated high levels of malaria-targeted antibodies compared to vaccination with alum as an adjuvant. Strikingly, this effect persisted for more than 400 days after inoculation  –43. ICMVculation . Since GSeveral other approaches using conventional or biomaterial-based vaccines have sought to induce GCs by delivery of multiple adjuvants or TLR agonists. The Pulendran lab has studied the effect of NPs loaded with multiple TLRs in individual particles compared with co-loading these signals in the same particle . In thesRecently, vaccines composed of chitosan and heparin have been used to mimic specialized molecules called granules . GranuleThe examples highlighted thus far share the aim of generating effective immune responses against foreign pathogens. However, another prominent goal of biomaterial-based vaccines and immunotherapies is centered on treating cancer. Cancer cells and the tumor microenvironment exhibit a number of characteristics that hinder the ability of the immune system to fight cancer . Notably+ T cells modified with NPs then injected into mice maintained the ability to home to tumor cells, carrying particles and cargo to these sites. This unique approach allowed efficient delivery of cytokine-loaded NPs to melanoma tumors and resulted in rapid proliferation of tumor-specific CD8+ T cells in LNs. Mechanistically, T cells conjugated with NPs polarized CD8+ T cells toward a central memory phenotype which is more effective at breaking tumor immunosuppression. Thus, mice treated with these tumor-specific T cells modified with NPs eradicated tumors, while untreated mice and mice treated with soluble drugs and T cells all succumbed.Stephan et al. approached cancer immunotherapy with biomaterials by modifying the surface of T cells with liposomes or polymeric NPs loaded with cytokines or adjuvants . This ap+ T cells in tumor-draining LNs also expressed high levels of PD-L1 and PD-1, respectively. Binding of PD-1 on CD8+ T cells to the cognate ligand (PD-L1 on APCs) negatively regulates T cell proliferation and pro-inflammatory cytokine secretion, leading to suppression of T cell function. This suppression supports tumor growth during cancer. In these studies, it was shown that while the tumor-draining LN was immunosuppressed, these tissues also contained more antigen-specific T cells, likely due to the proximity to the tumor  (PPS) NPs in tumor-draining LNs and distal LNs  for cancer therapy . This wohe tumor .+ T cells specific for the corresponding antigens. Targeting the tumor-draining LN also led to a decrease in the number of immunosuppressive cells ) compared with mice injected on another limb to target the non-tumor-draining LN  or a model antigen (OVA) were injected in the footpad on the same side as tumor induction. This approach resulted in targeting of NPs to the tumor-draining LN. Mice treated in this manner with NPs containing CpG and TRP2 or OVA reduced tumor growth in a melanoma model and in an OVA-expressing lymphoma model. These improvements correlated with an increase in CD8ining LN . A relatining LN . TogetheAs discussed earlier, once NPs reach LNs, there are also opportunities to direct immunity by delivering multiple signals or by controlling the release of antigens, adjuvants, and other signals. Below, new direct approaches to achieve LN delivery are discussed.Building on the idea that the kinetics and combinations of immune signals delivered to LNs play an integral role in the development of cell-mediated and antibody-mediated immunity, an intriguing area of fundamental and clinical research has focused on direct injection of vaccines to LNs. In humans, intra-LN (i.LN.) injection generally involves injection of soluble vaccine components to LNs using ultrasound guidance, whereas preclinical studies in mice utilize tracer dyes or surgical procedures to access the LN for injection –54. SeveAlong these lines, Mohanan et al. tested the delivery of common particle formulations  along intradermal, intramuscular, subcutaneous, and i.LN. routes. The response to particles laden with OVA antigen or OVA and adjuvants was then assessed by antibody titers and cytokine secretion across injection routes. Formulations injected i.LN. resulted in the highest antigen-specific IgG2a antibody titers regardless of whether or not a TLR agonist  was present in particles. This approach also increased the secretion of INF-γ from splenocytes in the presence and absence of adjuvants .+ T cells circulating in blood 1 week after a single i.LN. injection, an effect that was not observed with soluble vaccine formulations —is incorrectly recognized as foreign by lymphocytes and antibodies –68. Thisolerance ,4,69.One of the most fundamental ways in which biomaterials can be harnessed to promote tolerance is as a carrier of drugs or other immune signals to LNs or other immunological sites –79. In LREGS in mice treated with the liposomes  . Uptake iposomes . ImportaOne intriguing approach being developed to promote antigen-specific tolerance is based on design of NPs decorated with complexes of self-antigen loaded in MHC molecules . As discREG function, along with reductions in activity of inflammatory T cells   by the stromal conduits, macrophages and other APCs in the SCS engulf and process larger particles to support presentation of antigenic fragments from these materials. These differences have been exploited to promote antigen-specific tolerance by conjugating 500-nm polystyrene beads (PSB) or PLGA particles with a myelin peptide (MYE)—the self-antigen attacked by the immune system in MS ,81. Follivation) . These s+ helper T cell proliferation in LNs, but PLGA promoted regulatory function and reduced hypersensitivity reactions while TMC-TPP stimulated antibody responses. Although the mechanisms of these differences are under investigation, potential contributing factors may include size  or the duration over which these particles release antigen  (PEI), a cationic polymer, were rapidly trafficked to the follicular and marginal zones of LNs and the spleen. These complexes stimulated enzyme pathways that promoted DCs and T cells with regulatory characteristics, effects not observed when either PEI or bacterial DNA was administered alone . Togethe+ molecules expressed on helper T cells  (PLA) conjugated with an immunosuppressant (cyclosporine A (CsA)) ,88. CsA- T cells . These p earlier .+ T cells in LNs when mice were treated with ERY1 conjugated to T1D antigens, but not when soluble T1D antigens were administered. Together, these approaches demonstrate that a diverse set of cell modification approaches can alter how APCs and T cells function, as well as harness natural apoptotic clearance and tolerance mechanisms to regulate or redirect inflammatory immune reactions.Significant fractions of erythrocytes  are rapidly produced and destroyed on a daily basis in healthy individuals. In these cases, cell destruction occurs through a non-pathogenic mechanism of cell death, apoptosis . This meBiomaterials have already demonstrated great potential in the field of immunology. In coming years, continuing to improve our understanding of how material properties impact molecular signaling will remain an important issue, as this knowledge will lead to more rational design of vaccines. Employing biomaterials as tools to study new fundamental questions will also provide new opportunities to inform vaccine and immunotherapy design. An exciting avenue of research centers on engineering biomaterials with specific properties that allow for precise interactions with LNs and LN-resident cell populations that effectively modify the structure of these tissues. Some of these questions may also focus on understanding how the kinetics and combinations of immune signals in LNs impact stromal function , and if these changes can be induced or exploited to direct immunity. Another interesting approach on the horizon is the design of artificial LNs or SLOs that could locally recapitulate the functions of these tissues, perhaps eliminating the targeting challenges facing many vaccines and treatments. Biomaterials offer unique opportunities to address each of these areas, and the answers to these questions will continue to push the forefront of what may become possible in modulating immune function.


The discovery of Epidermal Growth Factor Receptor (EGFR) mutations in Non Small Cell Lung Cancer (NSCLC) launched the era of personalized medicine in advanced NSCLC, leading to a dramatic shift in the therapeutic landscape of this disease. After ten years from the individuation of activating mutations in the tyrosine kinase domain of the EGFR in NSCLC patients responding to the EGFR tyrosine kinase inhibitor (TKI) Gefitinib, several progresses have been done and first line treatment with EGFR TKIs is a firmly established option in advanced EGFR-mutated NSCLC patients. During the last decade, different EGFR TKIs have been developed and three inhibitors have been approved so far in these selected patients. However, despite great breakthroughs have been made, treatment of these molecularly selected patients poses novel therapeutic challenges, such as emerging of acquired resistance, brain metastases development or the need to translate these treatments in earlier clinical settings, such as adjuvant therapy.The aim of this paper is to provide a comprehensive review of the major progresses reported so far in the EGFR inhibition in this molecularly-selected subgroup of NSCLC patients, from the early successes with first generation EGFR TKIs, Erlotinib and Gefitinib, to the novel irreversible and mutant-selective inhibitors and ultimately the emerging challenges that we, in the next future, are called to deal with. AfteHowever, despite great breakthroughs have been made, treatment of these molecularly selected patients poses novel therapeutic challenges, such as emerging of acquired resistance, brain metastases development or the need to translate these treatments in earlier clinical settings, such as adjuvant therapy.The aim of this paper is to provide a comprehensive review of the major progresses reported so far in the EGFR inhibition in this molecularly-selected subgroup of NSCLC patients, from the early successes with first generation EGFR TKIs, Erlotinib and Gefitinib, to the novel irreversible and mutant-selective inhibitors and ultimately the emerging challenges that we, in the next future, are called to deal with.wild type receptor, since some of the regulatory proteins that balance the EGFR pathway present altered expression in cancer [The Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase (TK) receptor that is activated upon binding to the Epidermal Growth Factor and other growth factor ligands, triggering several downstream pathways, including RAS/MAPK, PI3K/Akt and STAT that regulate different cellular processes, including DNA synthesis and proliferation. EGFR signaling is commonly deregulated in cancer through different mechanisms, including genetic mutations of the receptor , 3. Mutan cancer .in vitro sensitivity to Gefitinib and with clinic-pathological characteristics preliminary associated with clinical activity [activating mutations” as they are constitutive activated and oncogenic. These mutant kinases present a reduced affinity for the ATP, accounting for the increased sensitivity to EGFR TKIs compared with the wild type counterparts, since these inhibitors compete with ATP for binding to the catalytic site [In 2004 two different groups simultaneously identified the presence of somatic mutations in the tyrosine kinase domain of the EGFR in a small group of patients with NSCLC responding to the EGFR tyrosine kinase inhibitor (TKI) Gefitinib , 6. Thesactivity , 8: Asiaactivity . These stic site –12.With the exception of PI3KCA mutations , the majSome authors have suggested a differential sensitivity to EGFR TKIs for exon 19 deletions and exon 21 L858R point mutations, with the former associated with longer overall survival (OS) and progression-free survival (PFS) , 15. Thewild type tumors, including a more frequent liver involvement at the diagnosis [wild type [Oncogene addicted tumors, such as EGFR mutated NSCLCs, may present peculiar patterns of metastatization compared with iagnosis , higher iagnosis –27 and hiagnosis  diffuse iagnosis , 29. Howild type  or signiild type .Moreover, some authors have suggested a possible interaction between EGFR mutation type and site of metastatization. For instance, Sekine et al. reported that patients harboring exon 19 deletions present a peculiar pattern of brain metastatization that resemble to that of miliary brain metastases, with multiple and small brain tumors with minimal peritumoral edema .common mutations” ~10% of patients with EGFR mutations may ehibit mutations in codons other than 19 and 21. Rare mutations could be detected in a small fraction of patients including exon 18 mutations, predominantly at G719 (~3–4%), exon 20 mutations (~4–5%) as well as uncommon mutations in exon 19  and 21 (i.e. L851Q/R), and finally compound mutations. The clinical significance of these rare mutants is not always fully understood with some variants associated with response to EGFR TKIs  other associated with resistance (i.e. exon 20 T790M) and other with uncertain clinical significance [In addition to classic “ificance –35. The ificance .The presence of EGFR mutations defines a specific molecular subgroup of NSCLC (~15% of Caucasian patients with adenocarcinomas) , 38 thatwild type clones may proliferate, altering the relative proportion of EGFR-mutated/EGFR-wild type cells within the tumor mass. A direct observation of increased sensitivity to chemotherapy is the fact that patients with EGFR mutations usually exhibit increased ORR to first-line chemotherapy [Several phase III trials demonstrated the superiority of EGFR TKIs over chemotherapy in EGFR-mutated NSCLC in the first line setting in terms of overall response rate (ORR) and progression free survival (PFS), but not in overall survival (OS) due to the extensive cross-over between treatment arms, although recently a pooled analysis of patients harboring common mutations enrolled in the LUX-Lung-3 and -6 trials reported a survival advantage also . Since notherapy . These sbranched evolution” hypothesis [In 2012 in a seminal paper Gerlinger and coll. reported evidence of intratumor heterogeneity and spatial separation of subclones in metastatic renal cancer, establishing the “pothesis , suggestpothesis . In contpothesis . In addipothesis .Further studies are required to throw a light on this controversial issue and therefore use of a single-site biopsy should continue to be the standard in clinical practice.The clinical development of EGFR inhibitors in NSCLC started before the discovery of EGFR mutations, therefore initial studies with Gefitinib and Erlotinib were conducted in unselected patients in both pretreated –54 and iThe extension of approval of Erlotinib for patients harboring EGFR mutations, in addition to the previous indication for unselected patients with NSCLC after at least one chemotherapeutic line, was obtained after the publication of two large phase III trials conducted in Asia  and Euroin vitro NSCLC models and some have also been confirmed in patients. Some of these mechanisms seem to be mutually exclusive, although distinct mechanisms of resistance may be operative in the same tumors [second generation” inhibitors) has been historically one of the most extensively studied. Despite promising preclinical evidences of activity against EGFR-mutated cell lines harboring the gatekeeper T790M mutation [wild type EGFR. Instead, their role is more defined in the front-line treatment of NSCLC patients harboring EGFR activating mutations. Recently, Afatinib received regulatory approval in the first line setting after the publication of the LUX-Lung-3 and -6 trials [Unfortunately, despite initial responses, virtually all patients, ultimately, progress because of the acquisition of resistance. In addition, a proportion of patients with EGFR mutations exhibits de novo resistance and does not respond to TKIs treatment. Several mechanisms of primary and acquired resistance to EGFR TKIs , 68 havee tumors , 70. Seve tumors , 72 and mutation –75 the mmutation , 70, neimutation  nor Neramutation  nor Dacomutation  demonstr6 trials  and anot6 trials . OngoingHowever, the role of second generation EGFR TKIs in acquired resistance is not definitely thrown over, since combining these agents with other targeted agents, such as the dual combination Afatinib plus Cetuxumab  and Nerawild type EGFR, WZ4002 is a covalent pyrimidine EGFR inhibitor specifically designed against EGFR T790M that is 30–100 fold more potent against EGFR T790M, and up to 100 fold less potent against wild type EGFR than quinazoline based EGFR inhibitors in vitro and is effective in murine models of lung cancer driven by EGFR T790M [in vivo impressive activity in patients with acquired resistance to EGFR TKIs. Rociletinib  is structurally related to WZ4002 and is the first drug of its class in clinical development for the treatment of T790M-positive NSCLC. In vitro and in vivo studies demonstrated that Rociletinib irreversibly and selectively inhibits mutant EGFR, including T790M mutation, with minimal activity against wild type EGFR, suggesting that drug tolerability to this agent may be superior to first and second generation EGFR inhibitors, for which major toxicities  are attributed to wild type EGFR blockage. Interestingly, NSCLC cells resistant to Rociletinib do not exhibit secondary mutations of the EGFR or amplification of the EGFR gene, but increased expression of genes involved in the epithelial-mesenchimal transtition (EMT) and Akt pathway [in vitro, with lower activity against wild type EGFR lines, with profound and sustained tumor regression in EGFR mutant tumor xenograft and transgenic models [In vitro data suggests the potential to target both HER2 and HER4 kinase activity, a property that may be important as HER2 amplification may mediate acquired resistance to EGFR TKI in 5–12% of cases [In attempt to overcome the limits of second-generation EGFR inhibitors, a novel class of mutant-selective inhibitors has been developed. The first in class third generation inhibitor, named WZ4002, was discovered in 2009 , throughFR T790M . WZ4002  pathway . Recentlc models . In vitrof cases , 88. AZDof cases . AZD9291of cases . Three oPreliminary results of a dose-escalating phase I study evaluating a third mutant-selective inhibitor, HM61713, has been recently presented. This inhibitor was well tolerated and reported a promising activity in EGFR-mutated NSCLC patients previously treated with an EGFR-TKI with a more prominent activity in T790M-positive tumors  than T790M-negative  [The emergence of brain metastases (BM) represent a major issue in clinical practice and is associated with a dismal prognosis. Traditionally, systemic treatments were considered mostly ineffective on BMs, with RR of 23–50% with platinum-based combinations . The lowHowever, the degree of Blood Brain Barrier (BBB) penetration of EGFR TKI may be influenced by factors patients, such as the extent of central nervous (CNS) disease and prior CNS-directed therapies, explaining why the CNS may still act as a partial sanctuary site, with approximately 20% of patients with EGFR-mutant tumors developing BMs while on EGFR TKI therapy . DiffereThe recent results, albeit preliminary, with third generation EGFR inhibitors with responses seen even in the CNS are encouraging  and deseThe use of EGFR TKIs is the standard of care in advanced/metastatic NSCLC with activating EGFR mutations, however their role in early-stage lung cancer is far less defined. Two large randomized trials evaluated the role of EGFR TKIs in unselected resected stage IB-IIIA NSCLC. The BR.19 study started in 2002 when adjuvant chemotherapy was not considered standard of care in resected NSCLC. Therefore, in 2003 the study was amended to allow adjuvant chemotherapy, so only 17% of patients received adjuvant chemotherapy and 5% adjuvant radiotherapy. The BR.19 randomized patients to receive Gefitinib 250 mg/d versus Placebo for 2 years . After tp = 0.039) [p = 0.001) and a trend toward improved OS  for patients with resected NSCLC treated with Erlotinib or Gefitinib with or without adjuvant chemotherapy [The detrimental effect observed even in EGFR-mutated patients may be attributable to the low mutation rate observed in the study, that could not have a sufficient powerful effect on the study results . The res= 0.039) , 101. Mootherapy .Ongoing studies in resected EGFR-mutated NSCLC  will definitely provide evidence of efficacy of this strategy, as observed in other solid malignancies, such as Imatinib in GIST and Trastuzumab in HER2-positive Breast Cancer.Exon 19 in-frame deletions and point-mutation L858R of the exon 21 account for more than 90% of all EGFR mutations and are associated with sensitivity to EGFR TKIs.de novo T790M mutations or exon 20 insertions presented in general inferior ORR and shorter PFS and OS compared with patients harboring other uncommon mutations, such as exon 18 G719X and exon 21 L861Q, that exhibited a range of sensitivity similar to that of common mutations. However, in some cases with exon 20 insertions or T790M mutations durable control of the tumor was observed [However, efficacy data for the remaining ~10% of EGFR mutations are lacking and mostly from retrospective analyses , 103. Daobserved . Data frobserved .wedge” that lock the C helix of the EGFR in its inward, active position. Therefore, the clinical resistance of this mutant is not due to steric hindrance with inhibitor binding, but simply not sensitizing to EGFR TKIs inhibition [A more extensive comprehension of the mechanisms underlying EGFR TKI-sensitivity of these mutations should help to provide more effective strategies for patients harboring these rare mutants. Recently, Yasuda et al. described the chemical structure of the exon 20 mutations D770_N771insNPG, showing that the three amino-acid insertion forms a “hibition . These fhibition .magic bullet that can cure cancer by blocking a single signaling pathway, however combining these inhibitors each other or with other no overlapping drugs and a better comprehension of inherited mechanisms of resistance to these targeted agents might provide more durable therapeutic successes with the hope, in a not so far future, to cure lung cancer.Since the discovery of EGFR mutations in 2004, the therapeutic landscape of advanced NSCLC has dramatically changed with unprecedented results in this usually disappointing disease. However, the clinical success of this strategy is limited by several challenges with which we are called to deal, such as emergence of acquired resistance or treatment of rare and compound mutations. To date three different EGFR TKIs have been approved and no prospective data are available to guide optimal treatment selection in clinical practice. Ongoing comparative randomized studies between reversible and irreversible EGFR TKIs and the recent development of novel mutant-selective inhibitors in patients with acquired resistance will definitely provide definitive conclusions about the optimal therapeutic strategy in this molecularly selected subgroup of patients. Probably, there is no


Recurrent respiratory papillomatosis (RRP) is a primary benign disease, which is characterized by papillomatous growth in the respiratory tract. Malignant transformation occurs in only 3–5% of cases, however, local growth of the benign papillomas is interpreted as clinically malignant in a markedly higher proportion of patients. Local surgical or endoscopic interventional debulking or excision is currently the commonly selected treatment method and antiviral therapy is a potential adjuvant approach. However, the long-term management of RRP patients, who commonly require multiple procedures over numerous years, is challenging and the overall therapeutic armamentarium remains unsatisfactory. The administration of systemic bevacizumab treatment in a series of five patients with long histories of RRP, who required repeated local interventions to control papilloma growth is evaluated. Treatment with the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab was administered at a dose of 5 mg/kg (n=1), 10 mg/kg (n=3) or 15 mg/kg (n=1) intravenously to the five RRP patients, who were clinically classified as exhibiting progressive disease. Endoscopic evaluations were performed prior to the first infusion of bevacizumab and intermittently at variable time points during the course of therapy. Histopathological analyses were performed using pre- and post-treatment papilloma biopsies, including immunohistochemical analyses of VEGF and phosphorylated VEGF receptor (VEGFR)-2 expression. The patients received between three and 16 courses of bevacizumab . The first course was initiated when progression following the previous intervention was observed. An immediate response to bevacizumab treatment was demonstrated in all five RRP patients. While the cumulative number of interventions in the five patients was 18 throughout the 12 months prior to the initiation of bevacizumab treatment, only one patient required interventional treatment due to a malignant transformation during the 12 months following treatment with bevacizumab . Histopathological analyses revealed regressive perivascular edema and normalization of the vascular structure, however, immunohistochemical analyses of the VEGF and phosphorylated VEGFR-2 expression did not demonstrate any changes following therapy. Due to the limited number of alternative treatments, VEGF-targeted therapies may represent a promising novel strategy in the treatment of RRP, which may have the potential to modify the current treatment standards, particularly in patients with poorly accessible papilloma lesions, however, this requires further investigation in clinical trials. Recurrent respiratory papillomatosis (RRP) is a potentially devastating, incurable disease that is caused by infection with the human papilloma virus (HPV), predominantly HPV-6 and -11. Primary benign tumorous manifestations of RRP occur throughout the respiratory tract and may lead to laryngeal, tracheal or bronchial obstructions, and pulmonary nodes and cystic lesions due to valve effects . Furtheret al  presented at the Department of Medicine  with histories of RRP ranging between 6 months and 37 years . The patient characteristics are presented in Written informed consent was obtained from each patient. Bevacizumab administration was initiated at a dose of between 5 and 15 mg/kg intravenously every 2–3 weeks. Treatment intervals were subsequently extended after achieving the maximum response as demonstrated by endoscopy and/or bronchoscopy. Endoscopy was performed prior to and between days three and seven following treatment initiation. Follow-up endoscopies were performed prior to every subsequent course of bevacizumab. Digital endoscopic imaging documentation was conducted using a routine documentation systems . Computed tomography (CT) was performed in two patients to follow up the RRP involvement of the lung parenchyma or paranasal sinuses.Papilloma tissue samples were obtained for standard histological and immunohistological analyses prior to and following the first infusion of bevacizumab in two patients. For immunohistochemistry, formalin-fixed and paraffin-embedded tissues were analyzed using primary polyclonal rabbit anti-human antibodies specific to VEGF , phosphorylated VEGFR-2  and the alkaline phosphatase anti-alkaline phosphatase double-bridge technique . Briefly, tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Following antigen retrieval in a microwave oven at 450 W  the primary antibodies were applied to the tissue sections overnight at 4°C. Subsequent steps were performed according to the manufacturer’s instructions. The Fast-Red substrate (DakoCytomation) was used for detection of phosphatase activity and sections were counterstained with hematoxylin and eosin .To investigate apoptosis in the histology specimen, the In Situ Cell Death Detection kit, AP  was used, which is based on the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-reaction. TUNEL-positive apoptotic cells were counted in three representative independent microscopic fields  per tumor .For statistical analysis the Wilcoxon signed-rank test was used. P-values were two-sided and P<0.05 was considered to indicate a statistically significant difference.1bN0M0, TNM Classification for Malignant Tumors)  .Four weeks later, a follow-up bronchoscopy demonstrated progression of the tracheal papillomatosis and clinical stridor was detected . At thatThis 49-year-old male was diagnosed with severe laryngeal RRP in January 2011. At the initial diagnosis local papilloma debulking was achieved using laser therapy. However, 6 months later, the papillomatosis relapsed and required a second laser-based intervention. The disease progressed again and bevacizumab treatment was initiated . An endoscopy prior to treatment revealed exophytic papilloma growth in the right supraglottic area . PatholoThis patient was a 56-year-old female, who was diagnosed with laryngeal and glottal RRP in 2009. Clinical symptoms predominantly included hoarseness, an irritative cough and dyspnea. Throughout the subsequent 3 years a total of 16 laser ablation therapies were necessary at increasingly shorter intervals, with the final interventions only leading to a short-term improvement of the symptoms. Systemic therapy with bevacizumab was initiated in March 2012. An endoscopy seven days after the first dose of bevacizumab (10 mg/kg) revealed a partial response (PR) of the supraglottic papilloma, while the manifestation on the right vocal cord remained stable. After the second cycle, a further reduction of the supraglottic lesion was observed. Clinically, the patient’s voice became increasingly powerful and dyspnea on exertion significantly improved shortly after treatment initiation. Bevacizumab therapy was paused following four cycles and the patient’s clinical condition remained stable for >9 months . After tA 2-year-old female, who was diagnosed in 2007 with HPV-11-associated RRP involving the larynx, trachea, deeper bronchi, and the lung parenchyma required a tracheostomy in August 2008 and multiple laser ablation therapies until January 2012. Adjuvant treatment attempts with interferon-alpha, cidofovir and celecoxib did not result in disease control. In January 2012 laser therapy of the large tracheal RRP manifestations was performed. A 32-year-old male was diagnosed with nasopharyngeal papillomatosis and paranasal sinus papilloma in 2010. Over 24 months several interventions, including laser vaporization and radical surgery via midfacial degloving had been necessary. In February 2012 an attempt to treat the sinonasal inverted papilloma with radiotherapy was made. However, three months later additional laser therapy was required and was complicated by chronic bleeding from the nasopharynx, which led to severe anemia . A CT scan performed in July 2012 showed inverted papilloma in all of the paranasal sinuses, including a complete obstructed right frontal sinus, a subtotal obstruction of the left frontal sinus and a complete obstruction of the left maxillary sinus. An endoscopy revealed recurrent papilloma growth at the velum  while thHistopathological analyses of pre- and post-therapeutic papilloma biopsies showed regressive edema and normalization of the vascular structures . HoweverThe clinical efficacy of systemic bevacizumab treatment is summarized in The induction of papilloma regression is presented in five consecutive patients with progressive RRP who, following various previous treatment attempts, were administered with systemic bevacizumab therapy at our institution. Previously, systemic administration of bevacizumab has been hypothesized as an effective adjuvant treatment modality following local intervention. It was reported that bevacizumab may have delayed relapse in one case of RRP  and was An immediate and sustained therapeutic effect of systemically administered bevacizumab was observed in all five RRP patients in the present case series. Notably, it was possible to document the response to treatment bronchoscopically as early as within a few days following the first infusion of bevacizumab. Continued anti-VEGF treatment resulted in sustained PR or VGPRs of tracheal or laryngeal papilloma manifestations. Remission has been sustained in one patient via administration of bevacizumab at prolonged treatment intervals of 3–4 months. Three patients, who exhibited disease progression following discontinuation of bevacizumab, subsequently responded again following the second cycle of treatment. Notably, none of the patients showed papilloma progression whilst treatment was ongoing. Patient 2 required a laryngectomy due to malignant transformation. The condition of patient 3 was clinically stable for 9 months following bevacizumab treatment, however, papilloma regrowth was observed following treatment discontinuation. After this re-growth, re-treatment led to a complete resolution of the supraglottic lesions. In this patient, an infraglottic lesion did not respond to the same extent as the other lesions and was abrased; a diagnosis of malignant transformation was excluded. In patient 4, with former multilocular tracheobronchial and lung parenchyma involvement, a solitary manifestation, which was clinically interpreted as granuloma associated with mechanical irritation by the distal end of the tracheal cannula, had to be abrased. However, histological assessment indicated that this particular manifestation was papilloma growth.At the time of writing the longest period of progression-free survival observed during follow-up is 27 months in patient 1, who is receiving maintenance therapy. Notably, in the present case series, it was possible to document the responses to bevacizumab therapy with regard to lung parenchyma involvement and papillomas of the paranasal sinuses, which are usually not accessible to local interventions .The rapid response to bevacizumab was accompanied by a disappearance of the perivascular edema in papilloma lesions as revealed by histopathological analyses of the pre- and post-treatment papilloma lesions. This finding is consistent with the antibody-mediated inhibition of VEGF-induced vascular permeability . DNA fraIn conclusion, the experience of the five present patients suggests that systemic bevacizumab presents a highly effective treatment option for RRP, offering a unique opportunity even for patients with non-accessible bronchial lesions, lung parenchyma involvement, papillomas of the paranasal sinuses, or for patients at high risk for voice mutilation by interventional therapies. These findings indicate that systemic bevacizumab treatment may have the potential to alter the current management of RRP. Whether the treatment will have an impact on the frequency of malignant transformation in papillomatosis remains unclear and VEGF-targeted therapeutic strategies require further investigation in clinical trials.


P = 0.505). Based on patient diaries, the lengths of “good time periods” were comparable . However, generic ropinirole therapy achieved almost 3.0 hours shorter on time without dyskinesia  and 2.5 hours longer on time with slight dyskinesia  than the branded ropinirole did. Except for gastrointestinal problems, nonmotor symptoms were similarly controlled. Patients did not prefer either formulation. Although this study has to be interpreted with limitations, it demonstrated that both generic and branded ropinirole administration can achieve similar control on most symptoms of PD.The aim of this study was to compare the efficacy of the branded and a generic extended-release ropinirole formulation in the treatment of advanced Parkinson's disease (PD). Of 22 enrolled patients 21 completed the study. A rater blinded to treatment evaluated Unified Parkinson's Disease Rating Scale, Fahn-Tolosa-Marin Tremor Rating Scale, Nonmotor Symptoms Assessment Scale, and a structured questionnaire on ropinirole side effects. Besides, the patients self-administered EQ-5D, Parkinson's Disease Sleep Scale (PDSS-2), and Beck Depression Inventories. Branded and generic ropinirole treatment achieved similar scores on all tests measuring severity of motor symptoms (primary endpoint, UPDRS-III: 27.0 versus 28.0 points,  Parkinson's disease (PD) is a neurodegenerative disease having major burdens on both the families and the healthcare system. Although most symptoms of PD have an excellent response to dopamine replacement therapies  in the early phase, the response to medication may become progressively inconsistent as the disease progresses. As the therapeutic window becomes narrower, long-term complication of dopaminergic treatment develops. The pharmacological management of wearing off, fluctuations, and dyskineasias (both chorea and dystonia) may be challenging. Therefore, patients with advanced PD usually require complex drug combinations, more frequent dosing, and usually increased dosages , 2.Although dopaminergic medications are not considered as very expensive compared to other pharmacological agents, the long treatment duration and the frequent need to use complex combinations  makes thMedicinal products are considered to be pharmaceutically equivalent if they contain the same amount of the same active substance(s) in the same dosage forms that meet the same or comparable standards. Pharmaceutical equivalence does not necessarily imply bioequivalence as differences in the excipients and/or the manufacturing process can lead to faster or slower dissolution and/or absorption.On the basis of European Medicines Agency (EMA) recommendations, two medicinal products containing the same active substance are considered bioequivalent if they are pharmaceutically equivalent or pharmaceutical alternatives  and their bioavailabilities (rate and extent) after administration in the same molar dose lie within acceptable predefined limits . The curCmax) must fall within 80–125% [Based on the assumption that bioequivalence equals therapeutic equivalence, generic and branded drugs can be used interchangeably. The regulatory limits applied in bioequivalence studies require that the areas under the drug concentration (AUC) versus time curves must be within 90% confidence intervals and the maximum plasma concentrations  correlate with the appearance of both motor and nonmotor symptoms PD  and consBased on this assumption and the previously described correlation between the late dopaminergic side-effects and plasma concentrations, one can argue that dopaminergic drugs licensed for advanced PD should be considered as narrow therapeutic index drugs (NTIDs), in which group the acceptance interval for AUC is tightened by the current regulations to 90.00–111.11% in bioequivalence studies , these rFurther possible issues in the generic drug prescription for advanced PD might be raised. One possible concern might be that currently bioequivalence measurements for dopaminergic medications are not performed on PD patients. Whereas the development of a branded medication demands the establishment of pharmacokinetics, efficacy, safety, and tolerability on both healthy and target patient population, the licensing of a new generic drug requires only the demonstration of bioequivalence with the branded counterpart, which is done only in healthy subjects . TherefoThere are very few available reports from either patients or physicians that address the question of generic drug-usage in PD from clinical perspectives . The majRecently, a study compared the pharmaceutical quality of seven generic levodopa/benserazide hydrochloride combination products marketed in Germany with the original product (Madopar) . All genEven less data is available on the generic formulations of DAs. Because modified-release  formulations of various DAs are available on the market, another issue might be raised concerning the length of clinical action. Whereas branded extended-release dopamine-agonists are claimed to have beneficial effects lasting for 24 hours, EMA and other regulators do not require any clinical evaluation of duration of action before licensing new extended-release generic drug formulations. Instead of clinical studies, the performance of much cheaper dissolution tests is required .The aim of the present study was to compare the clinical efficacy of the branded extended-release ropinirole  with an also extended-release generic comparator . We assumed that any significant differences between these two formulations could produce altered severity of either motor or nonmotor symptoms of PD.Twenty-two Parkinson's disease (PD) patients with fluctuation were enrolled to our study . All of them had advanced PD with long-term dopaminergic side-effects (wearing off and fluctuation) and received levodopa (423.8 ± 249.3 mg) and ropinirole (15.0 ± 5.6 mg) combination therapy. Each patient took extended-release (retard) formulation of ropinirole once-a-day, in the morning.fulfillment of the UK Brain Bank clinical diagnostic criteria for PD ,receiving stable (≥8 mg/day) dosage of extended-release ropinirole >3 months,stable symptoms of PD before enrollment,signed written consent according to the approval of Regional Ethical Board.Inclusion criteria for participation were the following:presence of other disorders capable of producing tremor ,suspicion of any Parkinson Plus syndromes or secondary parkinsonism,abnormal brain MRI ,implanted deep brain stimulator,levodopa/carbidopa intestinal gel infusion therapy,history of drug abuse,presence of any contraindications for ropinirole administration,presence or history of impulsive control disorder,suspicion of psychogenic symptoms,presence of any disorders capable of interfering with the study .Exclusion criteria were the following:Patients were examined at Department of Neurology, University of Pécs  and Kaposi Mór County Hospital . The study protocol was approved by the Regional Ethical Board (4230.316-2470/KK15/2011). Our examination was an investigator initiated and noncommercial study. Requip modutab and Ralnea medication were obtained from GSK and Krka pharmaceutical companies, respectively. None of the manufacturers had any influence on the present study.Our a priori hypothesis was that the generic ropinirole has noninferiority compared to the branded one. The study had a rater-blinded and crossover design lasting for 3 months. At enrollment and 1 month later (Visits 1 and 2), the patients received stable dosage of branded ropinirole (Requip modutab). After completing the tests of Visit 2, the patients switched to same dosage of generic ropinirole; therefore, we evaluated the clinical effects of Ralnea at Visits 3 and 4. Except for switching from Requip to Ralnea, the patients received unchanged medication and dosing throughout the study.Before enrollment, neuropsychological tests  were applied to exclude demented patients . We usedAt each visit, the motor symptoms  were tested at the same part of the day. These tests were scheduled 1.5 hours after the intake of last levodopa dose. We videotaped the application of these motor tests according to our video protocol . After tA trained Parkinson nurse obtained the rest of UPDRS , FTMTRS (parts B and C), Nonmotor Symptom Assessment Scale (NMSS), activity of daily living , a structured questionnaire on ropinirole side-effects, and Montgomery Asberg Depression Rating Scale (MADRS). Besides, the patients also self-administered the EQ-5D quality of life instrument , ParkinsWe asked our patients to keep patient diaries at least 2 days before each visit to assess ON-time without dyskinesia, ON-time with slight dyskinesia, ON-time with severe dyskinesia, OFF-time, and daytime sleeping time . “Good tBecause most of the variables did not follow the normal distribution, nonparametric tests were applied. Kruskal-Wallis test was assessed to evaluate any differences between Visits 1–4 except for HYS where Chi-square test was calculated by using SPSS Software . Wilcoxon's signed rank test was used during the comparison of two visits for continuous variables, whereas McNemar test for dichotomous variables and Chi-square for ordinal variables. The level of statistical significance was set at 0.05.Twenty-one patients completed the study; only their data were used for further analyses. One patient withdrew from the study because he moved to another city and consequently became unable to carry on with the protocol.The result of Mini-Mental Status Examination was 26.6 ± 2.6 points, whereas the average score on Mattis Dementia Rating Scale  was 133.Before further analyses, we compared the results of Visit 1 with Visit 2. Because none of the examined variables changed significantly between Visits 1-2, we assumed that our patients did not experience any changes (either worsening or improvement) during the Requip modutab phase. Similarly we did not identify any changes during the stable Ralnea phase (between Visits 3-4).P > 0.05, As far as the motor symptoms were concerned, we did not observe any differences between each visit . Consequently, the length of “good time periods” remained unchanged between the branded and generic ropinirole treatment phase  from 9.5 hours to 6.5 hours after switching from branded to generic ropinirole .Among the nonmotor tests  only the gastrointestinal section of NMSS showed worsening during generic ropinirole treatment  achieved similar control on the motor and nonmotor symptoms of PD and both formulations had similar safety and side-effect profile with the exception of gastrointestinal problems. Although the NMSS score on gastrointestinal problems significantly increased during the generic ropinirole treatment phase, the patient reported side-effect profile was similar in both generic and branded ropinirole treatment phases.This basically positive outcome may also be verified by the clinical practice of the authors, because we have not observed any serious clinical issues with this generic ropinirole formulation since their introduction to the Hungarian market in 2011. Despite similar number of patients choosing the branded and the generic formulations of ropinirole therapy immediately after completion of the study, 6 months later somewhat more patients were on generic formulation medication. In the background of this tendency, the increased financial burden of patients may be suspected. As the generic ropinirole formulations spread into the Hungarian market, the reimbursement of the branded formulation was lowered by the government-based healthcare system. Therefore the end-user price of branded ropinirole became increased compared to its comparators. In our opinion, the increased patients' costs might have been the main reason why increasingly more patients chose the generic ropinirole treatment.Although studies of generic versus branded products are very difficult to perform across a large population of patients, the authors are aware of the major limitations of the study: because our study was not a randomized and double-blind examination, the patients' preference and beliefs might have biased the results. Theoretically we could have applied overcapsulation to ensure double-blind evaluation, but we were unable to organize such study without appropriate funding. Therefore, we applied a blinded-rater approach and planned four visits (2 with branded and 2 with generic drug usage) to minimize possible biases.Another limitation of the study is the lack of pharmacokinetic data. Because our study was a nonsponsored and investigator-initiated examination, we did not have any opportunity to study the pharmacokinetics of the branded and generic ropinirole.


Antihistamines (Histamine receptor antagonists) are widely prescribed medicines in the treatment of allergic disorders, especially the symptoms of hypersensitivity reactions, mainly blocking the activity of vasoactive amines to their receptors. Drug adverse reactions such as sleepiness and dry mouth are frequently encountered. However, drug hypersensitivity provoking itchy hives by antihistamines were rarely reported. A 41-year-old female patient visited allergy clinic for generalized itchy hives from time to time, which had been aggravated 3 months before. Whenever she was exposed to antihistamines for treatment, she felt her hives got immediately full-blown. As a screening, she was tested with various antihistamines on her skin, then skin test-negative antihistamines were orally administered. Finally we failed to choose a safe antihistamine for the treatment of her symptoms. We report a case of drug hypersensitivity to various antihistamines with cross-reactions in a patient with chronic urticaria.