The variability in deceased organ donation registries worldwide has received little attention. We considered all operating registries, where individual wishes about organ donation were recorded in a computerized database. We included registries which recorded an individual's decision to be a donor (donor registry), and registries which only recorded an individual's objection (non-donor registry). We collected information on 15 characteristics including history, design, use and number of registrants for 27 registries (68%). Most registries are nationally operated and government-owned. Registrations in five nations expire and require renewal. Some registries provide the option to make specific organ selections in the donation decision. Just over half of donor registries provide legally binding authorization to donation. In all national donor registries, except one, the proportion of adults (15+) registered is modest (<40%). These proportions can be even lower when only affirmative decisions are considered. One nation provides priority status on the transplant waiting list as an incentive to affirmative registration, while another nation makes registering a donation decision mandatory to obtain a driver's license. Registered objections in non-donor registries are rare (<0.5%). The variation in organ donor registries worldwide necessitates public discourse and quality improvement initiatives, to identify and support leading practices in registry use. Organ donation registries are computerized databases that record member donation wishes. This is so a proper decision can be made on behalf of the deceased when the registry is checked by authorized personnel at the time of death. There are two distinct types of registries, donor and non-donor registries. Donor registries record an individual's decision to be a deceased donor. They are also used to promote organ donor awareness and evaluate public campaigns –3. This Whether donor registries effectively improve transplantation rates remains an open question. Nonetheless their use continues to expand, supported by the American Society of Transplantation and the public in many nations –11. ManyOur use of the term ‘donor’ registry refers to registries that record either affirmative decisions only or both affirmative decisions and objections. A ‘non-donor’ registry refers to registries that only record an objection to donation. The term ‘registries’ refers to both donor and non-donor registries.In an explicit consent (opt in) system an individual records their decision to become an organ donor in the event of their death. They then become an organ donor if their decision is registered in a donor registry or expressed by family members at the time of death. Donor registries can record legally binding ‘authorization’ or a non-binding expression of ‘intent’ by the deceased to donate. Some donor registries only record affirmative decisions while others record both affirmative decisions and objections.Non-donor (or objection-only) registries are used only in nations with presumed consent (opt-out) donation legislation . In suchWe considered all nations with active deceased organ donation programs. We collected information relevant to the design and use of each registry including: implementation date, ownership, operation level , minimum eligible age, expiration period of registered decision (if applicable), registration options , available methods of registration, priority status on transplant wait list, mandated choice, accessing the registry at the time of death , legal status of registered choices and registrant values (described according to registration choice). To calculate the registration proportions we defined the adult population as the total population 15 years of age and older using values from the Central Intelligence Agency World Factbook estimated for July 2011 . FifteenState and provincial registries operate in the USA and Canada respectively. When making generalizations about registry characteristics across these two nations, we described what the majority of states and provinces did. The only exception was for expirations; several states and provinces have registrations that must be renewed, and therefore warranted attention. For the total number of registrants, we used unweighted averages for both the USA and Canada. When providing registration information at the regional level, for American states the adult population was defined as the total population 18 years of age and older using values from the US Census Bureau . For CanData was collected from November 2010 until June 2011. A single author (AMR) first determined if there was an active registry by searching published literature and conducting Internet searches of ministries of health, nephrology and transplantation foundations’ websites. Relevant data was abstracted by this same author (AMR) into Excel 2007 . We sent our data to registry personnel for review and to supplement any missing fields. A second independent reviewer (AL) then reviewed all the data for accuracy including responses from nation representatives.The Global Observatory on Donation and Transplantation identifies 60 nations as having active deceased organ donation programs . LebanonCharacteristics of the registries included in this review are presented in Details on how each registry is accessed and used at the time of death is presented in Nation registry values are presented in Israel has the oldest registry, which was implemented in 1978. Registries have become more common in the last two decades, with a number of new registries in the last five years , and two Canadian provinces (New Brunswick and Quebec)).Nationally operated and government-owned registries are the most common . The three nations with regional registries are the USA, Canada and Iran. The USA and Canada have regional registries because deceased donation legislation falls under state/provincial legal jurisdiction. Also organ donation is linked to renewable government services managed at the state or province level, such as the department of motor vehicles and health insurance. Iran originally intended to have a national registry but changed to a regional system run by the 10 organ procurement organizations. This was done to provide more options for Iran's 31 provinces. Three nations  switched from having regional registries to a single national registry.Twenty-two nations (81%) have a minimum age requirement in order to register. In regions that do not, such as the UK, Sweden and about half of the American states, registrations can be made by individuals considered ‘minors’ if parental authorization is given at the time of registration and/or parents are responsible for making the final decision at the time of procurement.Five nations (19%) have registrations that expire. This often occurs in settings where the registrations are made through a driver's license or state identification card, with reaffirmation required when the license/card expires. In Belgium and Slovakia, registrations for individuals under the age of 18 that are made by a parent or guardian expire once the registrant turns 18.Eight nations have registries that record both ‘yes’ and ‘no’ responses , nine nations record only ‘yes’ responses, and eight nations record only ‘no’ responses. Two nations (Denmark and the Netherlands) are classified as ‘other’ because they offer more options than ‘yes’ or ‘no’. Registrants in Denmark can choose ‘yes’, ‘no’ or ‘unsure’. They may also add the condition ‘with next of kin approval’ to their registration. The Netherlands offers the choice of ‘yes’, ‘no’, ‘next of kin will decide’ or ‘a named individual will decide’. While Israel records affirmative choices only, individuals have the option of checking ‘yes’ or ‘yes with religious permission’.Seventeen nations (63%) allow registrants to specify which organs to include or exclude from donation. Five of these nations  are non-donor registries, so registrants choose which organs to include or exclude from their donation objection. The ability to specify organs to include or exclude in the donor designation is more common in nations with explicit consent (83%) than in nations with presumed consent (40%).Of the four most common registration modalities, in-person registration is the most frequently available (71%), followed by paper , online (50%), and telephone (29%). There are also some uncommon methods, including email (Austria) and select coffee shops (Israel). The UK has the most opportunities for registration, including applying online, and through telephone, driver's license, pharmacy advantage card, a physician, registration for a European Health Insurance Card and text message. None of the non-donor registries have online or telephone registration.Priority status is the practice of providing preference to individuals on the transplant waiting list who have registered to be deceased donors over those who have not. Israel is the only nation included in this study that has implemented this policy (in 2010). Priority is also extended to registrants’ first degree relatives and to non-directed living donors . A similMandatory or mandated choice is an approach in which individuals are required to register their donation choice. New Zealand is also the only nation where indicating one's donation decision is compulsory in order to obtain a driver's license.Health care providers for all nations with registries included in this review have a discussion with the next-of-kin about deceased donation as part of the organ procurement process. A registration can then be printed or verbally communicated by authorized personnel to the next-of-kin. All but two nations  indicated that they always consult their registry once a potential donor is referred and prior to discussion with the next-of-kin. Colombia's registry is more symbolic in nature, and is never used in the actual procurement process, while the New Zealand registry is only consulted if the next-of-kin requests that a search be made.Registries are most commonly accessed through a computer , and some systems offer additional telephone access. Computer access is less common in non-donor registries (50% compared to 79% of registries that include affirmative registrations).Individuals authorized to consult the registry vary by nation but mostly include individuals typically involved in the procurement process . Access is usually restricted to protect the privacy of registrants.While proof of registration may be used in the procurement consent process, not all registrations are considered legally binding. Registrations that fulfill the legal requirements for authorization and/or objection to deceased donation are valid legal documents and provide legal authorization for procurement to proceed. However, some nations still prefer to consider registrations as an indication of the deceased's intentions that are used in discussions with next-of-kin. Of the 19 donor registries that record affirmative registrations, 12 (63%) record decisions that are legally binding. Exceptions include two Canadian provinces (New Brunswick and Yukon) that record the intent to donate. Australia's donor registry was originally an ‘intention’ registry, but later changed to be legally binding. Australia records both legally binding authorizations and intent registrations. All registrations in the eight non-donor registries are considered legally binding objections to donation.Of the 19 donor registries, New Zealand (with mandatory choice) has the most registrants with 100% of their adult population registered (51% affirmative registrations). The Netherlands has the second highest proportion of registrations . None of the non-donor registries have proportions registered higher than 0.5%.When values in regional registries are considered, there are dramatic differences across American states and across Canadian provinces. In the USA only affirmative registrations are recorded. In the USA, the state of Alaska has the highest proportion registered (78%) while Vermont has a strikingly low value (0.3%). The later has been attributed to registration not being affiliated with its department of motor vehicles. In Canada, the province of New Brunswick has the most registrations (78% of adults), while Nova Scotia has the highest affirmative registrations (65%).To our knowledge, this is the most comprehensive and current global review of active donor and non-donor registries worldwide. We examined multiple characteristics covering the history, design, and use of registries, as well as the number of registrants. The results highlight the considerable variability in deceased donor registries worldwide. Most but not all registries are nationally operated and government owned. There is usually a specific minimum age requirement in order to register. Some registries provide registrants with the option to select specific organs to include and/or exclude from their donation decision. Most registries are consulted by health professionals involved in organ procurement through a computer after a donor referral and before discussion with the next-of-kin. Just over half of the donor registries are considered legally binding authorization to donation. In all national donor registries, except New Zealand, the proportion of adults (15+) registered (either affirmative decisions or objections) is modest (<40%), and is often even lower when only affirmative decisions are considered. Registered objections in non-donor registries are rare (<0.5%).There was also considerable variation amongst state and provincial donor registries in the USA and Canada, respectively. While this may be done for good reason, present inconsistencies may contribute to the large number of Americans and Canadians who indicate that they are confused about how to become an organ donor ,19.When contrasting national registries there a very important distinction between donor and non-donor registries. Both are primarily used to inform the procurement process and ensure a proper decision is made on behalf of the deceased. Donor registries are most often used in nations with explicit consent for deceased organ donation. These registries are used in the promotion of deceased donation, and can be used to target, measure and evaluate public awareness campaigns in support of organ donation. Non-donor registries in contrast are used in some presumed consent nations as a legal means for individuals to object to being a donor. They are not part of a strategy to improve deceased donation, and low proportions of their adult population registered  may be viewed positively by proponents of organ donation. Interesting, some registries operating in presumed consent nations record both objections and affirmative decisions. These types of registries were considered as donor registries for this review. However in some of these nations (such as Belgium), registries were originally conceived as objection to donation registries, and only later expanded to also record affirmative decisions.New Zealand is the only nation that was studied that makes registration compulsory in order to obtain a driver's license. The high proportion of adult registrants  suggests that this style of mandated choice helps overcome apathy to register a donation decision. However, some individuals may be unprepared when making their decision, making an uninformed or inaccurate choice. In New Zealand only half of the registrations are affirmative. In comparison, in the USA where all donor registries are affirmative only, some American states have proportions of adult registrants that exceed 70%.The strengths of our review include both the number of registries that were studied and the range of registry characteristics that were considered. The review extends previous studies which are smaller in scope and typically limited to European nations –22. ThesFuture studies are needed to investigate whether donor registries successfully improve the number of deceased donors. Direct comparisons need to be made between rates of deceased donation in nations with and without registries. There also needs to be an evaluation of individual registry design elements, so that specific recommendations for effective registry design can be made. This could be accomplished through studies that measure how much each design element (e.g. affirmative-only registry versus an affirmative and objecting registry) contributes to improved registration values and improved donation rates. Finally the influence of population preferences on the decision to register should be further investigated, as what constitutes effective design may vary by a nation's ideals and principles.In conclusion, we show registries are common around the world and that they vary in their objectives, design and use. This information can now be used to prompt public discourse and quality improvement initiatives amongst registry providers, to identify and support leading practices in registry use.


Xenopus laevis, is widely used in studies of oogenesis, meiotic cell cycle and early embryonic development. However, in order to perform such studies, eggs are normally collected after the injection of hCG into the dorsal lymph sac of fully-grown female frogs following pre-injection of PMSF. Although this protocol is established and used as standard laboratory approach, there are some concerns over whether the injections could cause the transmission of deleterious microorganisms. Moreover, these injection protocols require a competent skilled worker to carry out the procedure efficiently.The African clawed frog, Recently, we established a novel method to induce fish ovulation by simply adding the natural maturation-inducing hormone of teleosts, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one , into the surrounding water. In the present study, we demonstrate how we can induce ovulation in frogs using the same methodology.in vitro. We then examined the ability of progesterone to induce ovulation in frogs. However treatment of frogs with progesterone alone only occasionally induced ovulation in vivo. The number of oocytes and the frequency of ovulation were significantly lower than that induced by hCG-injection. Thus, conditions were improved by using a combination of progesterone with estradiol and by pre-treating frogs with low concentrations of progesterone or estradiol. Finally, we established an efficient means of inducing ovulation in frogs which involved pre-treatment of frogs with salt solution followed by a mixture of estradiol and progesterone at high concentration. The frequency and numbers of oocytes obtained were identical to those resulting from PMSG-hCG induction. Fertilization rate of eggs ovulated by the new treatment method was comparable to eggs obtained by hCG-injection and juveniles developed normally.In frogs, progesterone was effective in the induction of oocyte maturation To conclude, we have successfully developed a novel method to induce ovulation in frogs but without the need for a potentially harmful injection strategy. In vitro maturation of oocytes in frogs has been shown to be induced by several types of steroid hormones -Progesterone (94 Ci/mmol) and -Estradiol-17β (84 Ci/mmol) were purchased from Amersham Pharmacia Biotechnology .Frogs  followed 24 to 30 hrs later by the injection of 500 I. U. human chorionic gonadotropin (hCG). Females were transferred into a glass bottle with screw cap of 12 cm in diameter which contained 1 L of 0.1 M NaCl per frog. Frogs remained in the bottle at 20°C until ovulation was completed .Spawning was induced in sexually mature in vivo by adding steroids directly into the water at 20°C . After incubation for 24 to 30 hrs, agents to induce ovulation were added and the frogs maintained at 20°C until ovulation was completed .Females were transferred into a same glass bottle used for hormonal injection which contained 1 L of 0.1 M NaCl per frog. For pre-treatment, females were exposed to steroids Ovulated eggs were collected into plastic dishes of 150 mm diameter and photographed by digital camera. Images were digitized and eggs visible on the images were scored using ImageJ software.3H]-Progesterone (3H-P) and [3H]-Estradiol-17β (3H-E) were added to stock solution of each steroid at 0.2 μM and 0.1 μM. To estimate the solubility of steroids in this experimental conditions, 100 μl of 3H-P containing 200 mM progesterone in ethanol added into 1 L of 0.1 M NaCl with 100 μl of 100 mM estradiol without 3H-E or 100 μl of 3H-E containing 100 mM estradiol in ethanol added into 1 L of 0.1 M NaCl with 100 μl of 200 mM progesterone without 3H-P. To estimate incorporation of steroids into frog tissues, frogs maintained in the bottle for 24 hrs. After anesthetized with Methyl p-Hydroxybenzoate, 0.1 g of blood, ovary and skeletal muscle from frogs were obtained. The radioactivity in the tissues was measured with a liquid scintillation counter (ALOKA LSC-6100).[in vitro insemination following methodology described previously [The fertilizing ability of eggs was assessed by eviously . FertiliXenopus were being maintained. Of the steroids examined, progesterone was shown to induce ovulation in vivo but only if applied at high concentration , these chemicals are normally crystallized when kept at 4°C. However, these stock solutions can be kept at room temperature for up to six months in order to avoid crystallization. To investigate how much amount of steroids incorporated into frog tissues in the treatment established in this study, we conducted biochemical determination of incorporation of steroids into tissues of frogs using Xenopus laevis ovary were classified into 6 stages [The fertilization rate of eggs obtained by the new induction method was comparable to those observed with eggs ovulated by standard hCG-injection methods. Moreover, juveniles developed normally. Females induced to ovulate by the new method appeared healthy and no damage was observed following treatment. Importantly, females could be re-used in further induction experiments 3-4 months after initial treatment. This is in line with timescales recommended for hCG-injection treatment in frogs. Consequently, we have successfully described a new method for the induction of frog ovulation without injection. Oocytes in 6 stages . hCG injHowever, basic questions were raised during the course of our study, which should be given due consideration. Firstly, we need to address the stimulatory effect of estradiol upon the induction of ovulation. Secondly, we need to address the stimulatory effect of pre-treating frogs with 0.1 M NaCl. These concerns are discussed below.in vivo and in vitro. In frogs, ovulation can be induced in vitro in dissected ovarian pieces (ovarian fragments) by adding PE into culture medium. Although the effects of steroids were only studied in conjunction with pituitary extracts in vivo, it was demonstrated that steroids alone can induce ovulation by using ovarian fragments in Xenopus and Rana pipiens [in vitro [in vivo. However, pre-treatment with estradiol reduced the rate of ovulation, while estradiol also inhibited PE-induced ovulation in vitro. We propose that estrogens possess stimulatory-activity upon the process of ovulation but will reduce the competence of oocyte maturation if used in pre-treatments. Recently, Pang et al. used the zebrafish model to demonstrate the down-regulation of the membrane progestin receptor (mPR) responsible for progestin-induced oocyte maturation in response to estrogen acting via the membrane estrogen receptor [Xenopus, it has been demonstrated that the mPRβ subtype is a receptor for progesterone [The effect of estradiol upon ovulation has been studied in several different types of frog. It has also been established that pituitary extracts (PE) exhibit ovulating-inducing activity, both  pipiens ,12. Of tin vitro . Estrogereceptor . In Xenoesterone . Thus, iesterone . It is pXenopus eggs is sticky in nature and tends to adhere to vessels. To avoid the absorption of eggs onto vessels, salt solution is normally applied to the water used to house frogs during ovulation. For these reasons, we also used salt water to collect and count the number of eggs. Surprisingly, pre-treatment with this solution alone caused a significant increase in the number of eggs ovulated. Generally, it is recommended to pass water activated charcoal to remove chlorine prior to exposure to frogs. However, there are no published reports of studies investigating the effect of chlorine upon ovulation in Xenopus. For the present study, we used ultra-pure water to prepare 0.1 M NaCl. We consider that the pre-treatment of frogs with water from which all traces of chlorine had been removed, caused an increase in the competency of oocytes to undergo maturation. Alternatively, it is possible that a hypertonic solution could cause physiological changes to frogs that may inadvertently induce ovulation. We were unable to find reports in the current scientific literature that connect high salt conditions with the induction of ovulation. However, abrupt changes in osmotic pressure could manifest in physiological changes. The precise reason for the induction of ovulation by salt solution remains to be addressed and should form the focus for further study.The stimulatory effect of pre-treatment with a mere salt solution is also surprising. The jelly layer surrounding ovulated Xenopus laevis which involved pre-treatment of frogs with salt solution followed by a mixture of estradiol and progesterone.We established an efficient means of inducing ovulation in The authors declare that they have no competing interests.AO, JD and YI carried out assay for ovulation. TT participated in the design of the study and drafted the manuscript. All authors read and approved the final manuscript.


Joint punctures in children require adequate analgesia, sedation or both. Suitable for this are intravenous sedatives  and analgetics . The use of these substances is safe, but requires an intravenous line, monitoring and follow-up. Nitrous oxide/oxygen (N2O/O2) sedation has a long-standing history of safety and success. N2O/O2 has analgic and hypnotic quality characteristics. The mask inhalation of a fixed combination of nitrous oxide with 50% oxygen causes a mean analgesia without loss of consciousness or impairment of protective reflexes for the period of inhalation.We investigated the efficacy and safety of inhaled analgesia in 41 children compared to a control of 102 applications of an intravenous sedation with midazolam and ketamine under the guidelines of the American Academy of Pediatrics (AAP) and the American Society of Anesthesiologists (ASA) for sedation procedures.®) all patients met a ASA 1 or ASA 2 status. In total 41 patients were sedated with LIVOPAN ®. 35 applications were successful with good tolerance and adequate analgesia. No patient required a follow up. In 6 cases the procedure was aborted in 4 cases cause of insufficient analgesia, in 2 cases cause by respiratory irritation. After that we did sedation with midazolam and ketamine. 121 patients received sedation with midazolam and ketamine, there were no side effects and the tolerability was excellent. All patients required a follow up on the ward.From January 2010 till April 2011 121 children underwent the procedure of a joint puncture in our department. For patients between 7 and 17 years we offered the choice of intravenous sedation with midazolam/ketamin or sedation with Nitrous oxide/oxygen (LIVOPAN® is safe and efficient in short-term interventions with mean intensity of pain. Advantages are the fast and short application, the unneeded intravenous line and the unnecessary follow-up. Disadvantages are not always adequate analgesia and respiratory irritation. The atraumatic application, the early onset of analgesia within 3-5 minutes and the low side effects result in a high level of acceptance of the method in patients, parents and staff. The application of LIVOPAN ® is a good addition and allows a safe and painless joint puncture in older children.Inhalative analgesia with LIVOPAN


GM-CSF chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized EnvGM-CSF proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric EnvGM-CSF should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins.HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs) that target the envelope glycoprotein complex (Env). An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed Env Despite 20 years of research, a protective vaccine against HIV-1 is still elusive. Generating protective immunity against HIV-1 has proven to be extremely challenging and vaccines tested in several large-scale clinical trials induced no or very modest protection. An ideal HIV-1 vaccine should activate both the humoral and cellular arms of the immune system and the vaccines designed to stimulate only one arm have failed. The recent RV144 trial, designed to generate both B and T cell responses by combining an HIV-1 protein expressing pox-virus prime with a recombinant gp120 boost, yielded a modest 31.2% efficacy, providing hope that potent vaccine protection may eventually be achievable N-linked oligomannose glycans on Env can also facilitate suppression of dendritic cell-mediated immune activation and limit the induction of Ab responses Broadly neutralizing Abs (BrNAbs) can provide sterile immunity in non-human primates when administered passively, but no HIV-1 vaccine tested to date has been able to induce such BrNAbs. The HIV-1 envelope glycoprotein complex (Env), located on the outside of the virus particle, is the only relevant protein for the induction of BrNAbs, but a number of structural Env properties limit the induction of BrNAbs. Env contains surface loops that are highly variable in sequence between different HIV-1 isolates and these variable loops shield the conserved BrNAb targets GM-CSF, in which the first and second variable domains (V1V2) were replaced by GM-CSF, with the aim of improving immune responses against Env GM-CSF induced improved antibody and T helper responses against Env compared to unconjugated Env GM-CSF molecule was more immunogenic compared to Envwt, in vitro studies indicated that the GM-CSF domain within EnvGM-CSF was not as active as recombinant GM-CSF. Moreover, the insertion of GM-CSF in the V1V2 region resulted in subtle perturbation of the antigenicity of the CD4 binding site (CD4BS) and CD4-induced (CD4i) epitopes, which are positioned in close proximity to the inserted GM-CSF molecule.Env’s various defense mechanisms to evade humoral immunity forces us to search for unorthodox approaches to improve Env immunogenicity. We have constructed a chimeric Env protein, termed EnvGM-CSF immunogen by the following approaches: i) inserting of smaller GM-CSF domains; ii) modifying the disulfide bonded architecture of EnvGM-CSF; iii) removing the N-linked glycosylation sites of the GM-CSF domain; iv) optimizing the flexible linkers between the Env and GM-CSF domains; and v) embedding GM-CSF within cleaved Env trimers. The results show that the GM-CSF activity of EnvGM-CSF could be increased by 6-fold by extending the peptide linker between the GM-CSF and the Env. Interestingly, rendering EnvGM-CSF competent to proteolytic cleavage between gp120 and gp41 further improved the GM-CSF activity. The improved EnvGM-CSF variants drove the differentiation of primary monocytes into macrophages, testifying that these chimeric molecules are able to activate human immune cells. These optimized chimeric EnvGM-CSF variants should be useful HIV-1 vaccine candidates. Furthermore, these studies should assist the design of other chimeric molecules aimed at improving the immunogenicity of HIV-1 Env.In this study, we aimed to improve the GM-CSF activity and Env conformation of the EnvWe observed that replacement of the V1V2 domain by human GM-CSF had a minor effect on the exposure of the CD4 binding site (CD4BS), and decreased the exposure of CD4-induced (CD4i) antibody epitopes upon CD4 binding GM-CSF molecule by trimming the N- and C- terminal ends of GM-CSF molecule while maintaining the GM-CSF core that is essential for receptor binding ; and EnvGM-CSF3 (amino acids 33 to 112)  . The preimilarly .GM-CSF chimeras by ELISA. Binding of polyclonal Ig from pooled HIV-positive sera (HIV-Ig) was comparable for all EnvGM-CSF variants  did not bind to Envwt, but interacted efficiently with EnvhGM-CSF0 . TF-1 cells were cultured in the presence of the various EnvGM-CSF variants, and the number of living cells after three days of culturing was determined by FACS flow cytometry, as an indication of GM-CSF activity. As controls we used soluble recombinant GM-CSF (rhGM-CSF), the original EnvGM-CSF chimera, Envwt and mock medium. rhGM-CSF and the original EnvhGM-CSF0 chimera were able to induce survival of TF-1 cells, but none of the three truncated EnvGM-CSF variants were able to do so  at positions 37 and 47. Previous studies have shown that the functional activity of GM-CSF is increased when the N-linked glycans are removed, which could explain the higher activity of rhGM-CSF in our TF-1 stimulation assay N-linked glycosylation sites at positions 37 and 47 in GM-CSF might interfere with GM-CSF activity. We aimed to increase the potency of EnvGM-CSF by removing one or both glycans by substituting the asparagines for glutamines (N37Q and N47Q) resulting in EnvGM-CSF-N37Q, EnvGM-CSF-N47Q and the double mutant EnvGM-CSF-N37Q-N47Q  on either side of GM-CSF, were extended with three or six amino acids at both termini. The new linkers contained a positively charged lysine (K) in the N-terminal amino acid stretch and a negatively charged aspartic acid (D) on the opposite side in the C-terminal stretch to allow for stability by formation of a salt bridge. The inserted linkers were termed N1 (Lys-Ser-Gly) and N2 (Gly-Ser-Gly) at the N-terminus of GM-CSF; and C1 (Gly-Ser-Gly), C2 (Gly-Ser-Asp) at the C-terminus  (ab54429) to EnvF domain . In agreactivity . CollectGM-CSF0, EnvGM-CSF-N1N2-C1C2 and Envwt constructs  were expressed more efficiently than their uncleaved equivalents and insertion of GM-CSF did not affect the expression, regardless of the linker size (The cleavability of the Env trimers was measured by SDS-PAGE under reducing conditions. The cleavable Env (clEnv) and Envker size . AnalysiGM-CSF-N1N2-C1C2 resulted in a 5.3-fold higher GM-CSF activity compared to clEnvGM-CSF without additional linkers. Due to the differences in expression of cleaved versus uncleaved constructs, no direct comparison between cleaved and uncleaved Env-GM-CSF constructs could be made, however tested in the same experiment the 5.3-fold increase in GM-CSF activity for cleaved Env trimers caused by the longer linker length was substantially higher than for uncleaved Env trimers (2.6-fold)  . This maGM-CSF chimeras on primary human immune cells, we isolated monocytes from human PBMC’s and cultured them with Env, EnvGM-CSF, EnvGMCSF-N1N2-C1C2, clEnv, clEnvGM-CSF, clEnvGMCSF-N1N2-C1C2 and mock supernatant with or without recombinant GM-CSF. After 5 days of culturing, we performed flow cytometry analysis to assess the differentiation of monocytes towards a macrophage-like phenotype. Based on their morphology (forward/side scatter characteristics), we observed higher percentages of macrophage-like cells in cultures supplemented with EnvGM-CSF variants compared to Env, and the levels were similar to these in cultures supplemented with recombinant GM-CSF .These results show that our EnvGM-CSF constructs can drive the maturation of monocytes into cells with a macrophage-like phenotype.In order to test the activity of Envnv alone . In agreGM-CSF) GM-CSF, combining optimal GM-CSF activity with an optimal antigenic Env structure and validated these molecules on primary human immune cells.So far, HIV-1 Env-based subunit vaccines have not been successful in providing immunity against HIV-1. Many intrinsic properties render HIV-1 Env protected against the induction of an adequate antiviral Ab response. Formulating subunit vaccines with adjuvants can improve their immunogenicity. A more sophisticated alternative or complementary approach to simply mixing the antigen and the adjuvant or costimulatory molecule is the direct conjugation or fusion of a co-stimulatory molecule to the antigen. This strategy ensures that the costimulatory molecule activates the exact same immune cells that interact with the antigen. HIV-1 Env has been fused at the N- or C-terminus to costimulatory molecules such as IFN-γ or TNF superfamily members such as APRIL, BAFF and CD40L Although GM-CSF embedded within Env was functional; it did not have the same activity of soluble rhGM-CSF GM-CSF with an additional six amino acids at both N- and C- termini resulting in 9 amino acids on each side (EnvGM-CSF-N1N2-C1C2) showed the highest GM-CSF activity. The longer linkers probably provide more conformational and/or steric flexibility that allows better binding to the GM-CSF receptor and formation of dodecameric ligand-receptor complexes. The improved GM-CSF function of EnvGM-CSF-N1N2-C1C2 may also be attributed to improved exposure of GM-CSF residues 21–31 and 77–94 which have been shown to be critical for GM-CSF activity Chimeric EnvN-linked glycans are present on GM-CSF, which can influence its systemic half-life, tissue distribution, biological activity and immunogenicity. Bacterially expressed non-glycosylated GM-CSF has been reported to be 10-fold more active than the glycosylated protein. The lower in vitro biological activity of glycosylated GM-CSF is thought to be due to reduced affinity for the receptor N-linked glycans from our EnvGM-CSF chimera, did not improve the GM-CSF activity. In fact, removal of the N-linked glycan at amino acid position 47 reduced GM-CSF activity dramatically, indicating that this glycan is required for GM-CSF folding and/or function in the context of EnvGM-CSF. Removing this glycan also reduced the recognition of conformational epitopes on Env, pointing at a global effect on EnvGM-CSF folding and/or conformation.Two GM-CSF was very similar to that seen with the corresponding, unmodified Env, with the exception of Abs targeting the CD4bs and CD4i epitopes of which the binding was reduced. The extension of the linkers between the Env and GM-CSF subdomains improved the binding of b12 (CD4bs) and 48d (CD4i), suggesting that there were less steric clashes between the GM-CSF subdomain and these Abs.An ideal Env antigen should expose most or all epitopes for BrNAbs. Ab binding to most Env epitopes in EnvGM-CSF was also not efficiently recognized by 48d when CD4 was added, indicating that the presence of GM-CSF in the place of V1V2 blocks the CD4-induced conformation changes, and indicating that properties intrinsic to the V1V2 allow the CD4-induced changes . Indeed we have successfully created EnvIL-2, EnvIL-4 and EnvIL-21 envokines by replacing the GM-CSF domain by these cytokines in the V1V2 domain. In particular the EnvIL-21 envokine is very potent in activating human B cells, which may be a desirable property for inducing a potent Env-directed B cell response. Furthermore, in parallel, we are currently investigating whether we can insert GM-CSF into other locations than the V1V2 domain to test the activity and antigenicity of the chimeric molecules side by side. In conclusion, we engineered chimeric Env-cytokine molecules that potently activate human immune cells yet retain an antigenic Env structure that should be favorable for inducing humoral immunity against HIV-1.Other cytokines have a similar four-helix bundle structure as GM-CSF and this allows us to design other chimeric EnvN/A.The plasmid expressing codon-optimized stabilized HIV-1 gp140 (SOSIP.R6-IZ) was used as the starting point for the constructs generated in this study . The EnvHIV-Ig was obtained through the AIDS Research and Reference Reagent Program (ARRRP), Division of AIDS, NIAID, NIH. MAb 2G12 was obtained from Hermann Katinger through the ARRRP. CD4-IgG2, sCD4 and anti-V3 gp120 MAb PA1 were gifts from Bill Olson . MAb b12 was donated by Dennis Burton . MAb 48d was a gift from James Robinson . The mouse monoclonal GM-CSF antibody  was purchased from Abcam . Recombinant human GM-CSF (rhGM-CSF) for TF-1 cell stimulation was obtained from Schering-Plough .293T cells were maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% heat inactivated fetal calf serum , MEM nonessential amino acids  and penicillin/streptomycin (both at 100 U/ml). TF-1 cells 293T cells were transiently transfected with plasmids expressing recombinant Env using linear polyethylenimine  as described elsewhere 2, and 2 mM MgCl2), serially diluted polyclonal HIV-Ig, Env specific monoclonal Abs, or CD4-IgG2, in TSM 5% BSA was then added for 2 h, with or without 1 µg/ml sCD4, followed by three washes with TSM, 0.05% Tween20. Horseradish peroxidase (HRP)-labeled goat-anti-human immunoglobulin G  was used as secondary Ab and the absorption at 450 nm was measured after the colorimetric reaction was stopped using H2SO4. Equal input levels were verified SDS-PAGE followed by western blot analysis.Env trimer ELISA was performed as previously described SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed as previously described 4 in 50 µl) were seeded in a 96-well plate. Serially diluted supernatants from Envwt, EnvGM-CSF variants, mock transfected 293T cells, or rhGM-CSF diluted in supernatant from mock transfected 293T cells were added to the cells. Equal input levels were verified SDS-PAGE followed by western blot analysis. Each sample was tested in quadruplicate wells. The cells were harvested on day five and the percentage of living cells was determined by flow cytometry analysis.Myeloid leukaemia TF-1 cells  were isolated from buffy coats of healthy blood donors using Lymphoprep  density gradient centrifugation. Monocytes were isolated by adherence to plastic and cultured in Iscove’s modified Dulbecco’s medium  supplemented with 10% (v/v) heat-inactivated human pooled serum (HPS) or 10% (v/v) heat inactivated fetal calf serum , penicillin , streptomycin  and ciproxin  for 5 days in the presence of Env constructs produced from 293T cells, GM-CSF , or medium alone at 37°C in a humidified atmosphere supplemented with 5% COCells were fixed with 1% paraformaldehyde for 10 min at room temperature and subsequently washed with PBS containing 0.5% BSA, 10% 0.13 M trisodiumcitrate, 2 mM EDTA and 0.01% Na-azide. Cells were stained with the following antibodies for 30 min at 4°C in the dark: anti-human conjugated to CD16-Alexa Fluor 647 , CD64-Alexa Fluor 488 , CD80-PE-Cy5 , CD163-PE (BD Biosciences), CD206-APC-Cy7 (Biolegend). Mouse IgG1κ conjugated to FITC, PE, PerCP-Cy5.5, APC and APC-Cy7 was used as isotype control (BD Biosciences). Flow cytometry was performed with the FacsCanto II (BD Biosciences) and the results were analyzed in FlowJo, version 9.4.3 .Percentages of macrophage-like cells were assessed by analyzing the SSC vs. FSC plot in flow cytometry. The statistical significance of differences was tested using a paired student’s T-Test (GraphPad Prism 5).


Rana sylvatica, from Interior Alaska survives freezing at –16°C, a temperature 10–13°C below that tolerated by its southern conspecifics. We investigated the hepatic freezing response in this northern phenotype to determine if its profound freeze tolerance is associated with an enhanced glucosic cryoprotectant system. Alaskan frogs had a larger liver glycogen reserve that was mobilized faster during early freezing as compared to conspecifics from a cool-temperate region . In Alaskan frogs the rapid glucose production in the first hours of freezing was associated with a 7-fold increase in glycogen phosphorylase activity above unfrozen frog levels, and the activity of this enzyme was higher than that of frozen Ohioan frogs. Freezing of Ohioan frogs induced a more modest (4-fold) increase in glycogen phosphorylase activity above unfrozen frog values. Relative to the Ohioan frogs, Alaskan frogs maintained a higher total protein kinase A activity throughout an experimental freezing/thawing time course, and this may have potentiated glycogenolysis during early freezing. We found populational variation in the activity and protein level of protein kinase A which suggested that the Alaskan population had a more efficient form of this enzyme. Alaskan frogs modulated their glycogenolytic response by decreasing the activity of glycogen phosphorylase after cryoprotectant mobilization was well under way, thereby conserving their hepatic glycogen reserve. Ohioan frogs, however, sustained high glycogen phosphorylase activity until early thawing and consumed nearly all their liver glycogen. These unique hepatic responses of Alaskan R. sylvatica likely contribute to this phenotype’s exceptional freeze tolerance, which is necessary for their survival in a subarctic climate.The wood frog,  Rana sylvatica, is one of several amphibians known to tolerate the freezing of its body fluids as an adaptation to survive winter’s cold The wood frog, Freeze tolerance in wood frogs is based in part on a glycemic response that is initiated once freezing begins. Glucose, generated from liver glycogen, is quickly exported from the liver and transported to corporal tissues before circulation ceases Freezing mobilization of glucose in the wood frog involves β-adrenergic stimulation of hepatocytes, which results in a rapid activation of protein kinase A  in the first minutes of freezing The capacity for freeze tolerance depends in large part on the cryoprotectant levels that can be reached in the corporal tissues The purpose of this study was to examine the glucosic cryoprotectant system of a wood frog population from near the northern limit of this species’ range. Frogs from Interior Alaska tolerate much lower temperatures than frogs from more temperate locations, such as Ohio and Pennsylvania R. sylvatica were collected in late winter  in Adams County, south-central Ohio, USA , on private land with the permission of the landowner, under a permit issued by the Ohio Division of Wildlife. Frogs were collected by hand or using a dip net. Experimental frogs were euthanized by double-pithing before being dissected. Rearing and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Miami University (research protocol number 812).Wood frogs were collected near Fairbanks, Alaska, USA , on public land, during early August, 2011, under a permit issued by the Alaskan Department of Fish and Game. Additional Alaskan frogs were held in a programmable environmental chamber  and exposed over 5 weeks to dynamic, diel cycles of temperature and ambient light, which, based on long-term records of weather , were seasonal and appropriate to their origin. Initially, temperature varied daily from 17 to 8°C and the photophase was 16.5 h, but by the end of acclimatization, in mid September, temperature varied daily from 13 to 3°C and the photophase was 13.3 h. Throughout, frogs were fed three times weekly with crickets that were dusted with a vitamin supplement . Following acclimatization, frogs were kept at 4°C, in darkness, in simulated hibernation until used in mid November.2 pen at the Ecology Research Center , Miami University, until autumn. Frogs had access to a pool of water and were fed vitamin-fortified crickets three times weekly, and this diet was supplemented by a host of arthropods that was attracted to a “black light” hung in the pen. Feeding was suspended in late October, and in November, the frogs, on the verge of dormancy, were recaptured and kept at 4°C, in darkness, in simulated hibernation until used in January.Ohioan frogs were kept, unfed, on damp moss within darkened plastic boxes (4°C) for 3 weeks after collection from the field. Thereafter they were kept outside in a 48-mWe aimed to sample only adult males to eliminate potential gender- and age-based differences in physiology. This objective was largely achieved in the Ohioan frog samples. However, as secondary sex characteristics were not evident in August, Alaskan frogs collected and used in this study comprised about 37% females, which were randomly distributed amongst treatment treatments.Tb) was recorded at 30-s intervals on a multichannel data logger . Tubes containing these frogs were submerged to the cap in a refrigerated bath  containing ethanol. After each frog had supercooled slightly (Tb∼–1°C), freezing was initiated by inoculating the skin with small ice crystals created by applying aerosol coolant to the exterior of the tube. Initiation of freezing was confirmed by the occurrence of a freezing exotherm that resulted from the change in the physical state of body water. The temperature of the bath decreased 0.05°C h–1 to the target temperature, –2.5°C, which was reached after 30 h; some frogs were kept frozen for an additional 18 h, remaining frozen for a total of 48 h. Groups of frogs (N = 5–6) were removed from the bath and sampled at 2, 6, 30, and 48 h after freezing commenced. Other frogs were frozen for 48 h and sampled after thawing at 4°C for either 6 h (N = 5–6), or 120 h . A reference group of unfrozen frogs (N = 7–8) was sampled directly from their holding cups (or plastic boxes) in the 4°C chamber.Frogs used in this experiment were frozen and thawed following an established protocol that facilitates cryoprotective responses, promotes survival, and mimics natural freezing and thawing episodes 2. Initial water concentration in the tissue, expressed as mg water g–1 dry matter, was determined by dividing the mass lost upon drying the sample by its dry mass. Liver samples were stored at –80°C before enzyme assays and metabolite analyses were carried out.Frogs were weighed to 0.1 g, double-pithed, and dissected at 4°C. The liver was excised rapidly  and a small sample of tissue was blotted to remove excess moisture, weighed to 0.01 mg, and placed in a 65°C oven for 3–5 d, to determine tissue dry mass. The remaining tissue, destined for enzymatic, protein, and metabolite analyses, was immediately frozen in liquid N−1) at 40°C for 2 h in a 0.2 M sodium acetate buffer, pH 4.8. After incubation, the reaction was stopped by adding cold 7% (w/v) perchloric acid and the free glucose in the sample was determined using a colorimetric assay kit ; glycogen concentration was expressed as µmol glucosyl units g−1 dry liver tissue after subtraction of initial free glucose, assayed as above, in the initial homogenate. We also expressed glycogen levels on a µmol liver−1 basis, calculated by multiplying the liver glycogen concentration by the dry mass of the entire liver, thus obtaining an estimate of the organ’s total glycogen content. We estimated the cumulative glucose output from the liver during freezing by subtracting the mean liver glycogen remaining at each sample time from the mean liver glycogen content of unfrozen frogs.Deproteinized extracts of liver samples were prepared by homogenization in cold 7% (w/v) perchloric acid. Liver samples were assayed for glycogen using an enzymatic procedure PKA activity was measured in liver extracts using a non-radioactive assay kit  that determines phosphotransferase activity with fluorescent kemptide (PepTag® A1 peptide) as the substrate. Frozen liver tissue was homogenized (1∶10) on ice in cold Tris-HCl buffer (pH 7.4) and centrifuged at 4°C , per the manufacturer’s instructions. To determine activity of the free catalytic subunit of PKA (PKAc activity), samples were assayed without cAMP; additionally, samples were assayed with 1 µM cAMP to release the catalytic subunits bound to the regulatory subunits, thereby providing a measure of total PKA activity 10) for total PKA activity. Enzyme activity was expressed as U mg−1 of total soluble protein, where one Unit was defined as the number of nanomoles of phosphate transferred to the substrate per minute.PKA activity was assayed at 22°C for 30 min, after which the reaction was stopped by boiling the samples at 95°C for 10 min. Phosphorylated and non-phosphorylated PepTag® A1 peptides were separated by 0.8% agarose gel electrophoresis. The fluorescence of the phosphorylated peptides was recorded with an Alpha Inotech Imager  and the bands were analyzed by densitometry with the use of a standard curve of fluorescent kemptide present in each gel. Each liver extract was run in duplicate and the average of the activity values was taken to represent the individual. The percentage of PKA present as the free catalytic subunit was calculated by dividing the activity of PKAc by the total PKA activity and multiplying the ratio by 100. Liver extracts from the unfrozen group were also assayed for total PKA activity at 0°C. The reaction was set up as described above, except that the incubation was done for 30 min on ice. We used the activities measured at 22°C and at 0°C to compute the temperature coefficient  with bovine serum albumin (BSA) as the standard.Liver tissue collected from unfrozen, 48 h-frozen, and 120 h-thawed frogs was homogenized whilst frozen in RIPA buffer (1∶10), on ice, containing a protease-inhibitor cocktail . The homogenate was centrifuged  for 5 min at 4°C. Following centrifugation, the supernatant was assayed for protein concentration using the Bio-Rad protein assay (Bio-Rad), with bovine serum albumin (BSA) as the standard, aliquoted, and frozen at −80°C.Total soluble protein  was mixed with Laemmli sample buffer (containing 5% β-mercaptoethanol) and heated at 95°C for 5 min. SDS-PAGE of the protein samples was performed using a 4–15% Tris-HCl gradient gel (Bio-Rad). To allow comparisons among different gels, we loaded into all gels a standard relative to which all protein bands were normalized.−1 Tris, 500 mmol l−1 NaCl, and 0.1% Tween-20; pH 7.5). Goat primary antibody for GP  was used to detect total GP in liver samples; rabbit primary antibody for PKAc  was used to detect PKAc in liver samples. Nitrocellulose membranes were incubated in anti-GP antibody solution (1∶2000 dilution) or anti-PKAc antibody solution (1∶3000) overnight, at 4°C, with constant oscillation. Secondary antibodies were horseradish peroxidase-linked donkey anti-goat for GP (Santa Cruz Biotechnologies) and goat anti-rabbit for PKAc (Sigma-Aldrich). All primary and secondary antibodies were diluted in a 5% non-fat milk TBS-T solution. Following three 15-min washes in TBS-T, membranes were incubated in secondary antibody (1∶3000) for 2 h at room temperature with constant oscillation. Membranes were thrice washed in TBS-T for 15 min, incubated for 2 min in ECL (enhanced chemiluminescence) detection reagents , and exposed to autoradiography film.Following electrophoresis, proteins were transferred to a nitrocellulose membrane . After the transfer, each membrane was stained with 0.2% (w/v) Ponceau S (Sigma-Aldrich) containing 5% (v/v) acetic acid to verify uniformity of protein transfer; these membranes were digitized for densitometry analysis of the total protein loaded. After the membranes were digitally scanned, they were destained using 0.1 M NaOH, rinsed for 3 min with ultrapure water, and blocked overnight in 10% non-fat milk in TBS-T buffer (10 mmol lPKAc and GP antibodies were exposed to their respective antigenic peptides in a competition assay to determine the specificity of the bands visible in the blot (results not shown). We analyzed only the bands that were specific to each antibody.t = 2.683, P = 0.028, data not shown); thus, we instead elected to use total protein as a loading control. Following Aldridge et al. Bands from digitally scanned radiography films were semi-quantified using AlphaView spot densitometry (ProteinSimple). We had intended to use β-tubulin as a loading control but found that the β-tubulin concentration in livers of unfrozen Alaskan frogs was about double that of the unfrozen Ohioan frogs ; significance was accepted at P<0.05.Means ± standard error of the mean (SEM) are the descriptive summaries used for the variables measured. Mean values for samples of unfrozen, frozen, and thawed frogs were compared within each population using a one-way ANOVA; post-hoc Dunnett’s test distinguished the mean for each group of frozen or thawed frogs from that for unfrozen frogs. A two-way ANOVA was used to examine the mean response of the variables as a function of two factors, population (Alaskan and Ohioan) and sample treatment , or the interaction of these two factors. If a significant interaction between the factors was found, Bonferroni post-hoc test was used to distinguish between the two population means for each sample treatment. Mean values for the enzyme activity at 0°C and for the thermal coefficient of enzyme activity were compared between Alaskan and Ohioan frogs using the Student’s Exotherms were observed in the temperature recordings of all frogs subjected to experimental freezing, confirming that frogs from both populations were successfully frozen. Upon dissection, Alaskan frogs contained relatively little ice, even after 48 h of freezing, which was present beneath the skin ventral to the submaxillary and rectus abdominis muscles. Frozen Ohioan frogs had large, subcutaneous ice crystals present both dorsally and ventrally along the body, in the coelom, and also between muscle fibers of the hind limbs. Overall, Alaskan frogs were much more pliable as compared to frozen Ohioan frogs, which were more rigid. Survival was assessed in the 120 h-thawed groups before they were dissected for tissue sampling. All frogs survived experimental freezing and thawing except for one Alaskan frog, which was eliminated from the study.F6,35 = 13.06, P<0.0005; Ohioan, F6,29 = 11.52, P<0.0005; P<0.01) noticeably in Alaskan frogs as early as 6 h of freezing, but glycogenolysis slowed as freezing progressed, and these frogs consumed only 63% of their initial glycogen store by 48 h of freezing. On the other hand, Ohioan frogs first showed a significant (P<0.0005) decrease in liver glycogen by 30 h of freezing, and by 48 h had consumed 90% of their initial glycogen store. Overall, Alaskan frogs had a higher  liver glycogen content than Ohioan frogs, indicating that their potential to mobilize glucose during freezing could be greater if GP activity is also comparatively higher during early freezing. The pattern of glucose output, deduced from the time course of glycogen depletion, showed that Alaskan frogs mobilized glucose more rapidly in the early stages of freezing, although by 48 h both populations had produced identical amounts of glucose , although no sample treatment had an activity level that differed from that of the unfrozen frogs  with freezing/thawing in the Ohioan frogs, decreasing to 12% of the values of unfrozen frogs by 30 h; however, activity levels were restored to basal values by 120 h of thawing  activity of PKAc.Activity of PKAc in the Alaskan frogs varied significantly , but the values were substantially higher  in the Alaskan frogs , with Ohioan frogs showing an earlier change; however, the percentage of enzyme present as PKAc returned to basal levels during thawing in both populations  between populations, and the values, 0.36±0.05 U mg−1 for Alaskan frogs and 0.25±0.02 U mg−1 for Ohioan frogs, were ca. 66% lower than those determined for preparations incubated at 22°C. The resultant Q10 values for Alaskan and Ohioan frogs, 1.6±0.09 and 1.8±0.10, respectively, were not statistically distinguishable .Total activity of PKA in enzyme preparations incubated at 0°C did not differ (P = 0.025) 1.5-fold with freezing but returned to basal values by 120 h of thawing  in PKAc protein levels. However, these frogs had approximately twice the amount of PKAc protein as did Alaskan frogs .Incubation with primary antibody for PKAc yielded in all samples a band of ca. 42 kDa, and some samples showed an additional band of ca. 60 kDa. In the competition assay, in which the antibody was pre-incubated with its antigenic peptide prior to incubation with the samples, the 42 kDa band disappeared, whereas the 60 kDa band retained equal intensity; thus, we analyzed only the former, which was specific to the antibody. Immunoblotting results showed that the quantity of PKAc in the liver of Alaskan frogs increased , increasing 7-fold over values for unfrozen frogs within 2 h, but subsequently fell such that it was no longer distinguishable from basal levels by 48 h of freezing , displaying a 4-fold increase over basal values within 2 h; however, this increased activity was maintained throughout the freezing exposure and even during the early hours of thawing  between population and sample treatment. Specifically, GPa activity was similar (P>0.99) in unfrozen frogs from both populations, but by 30 h of freezing was about two-fold higher (P = 0.007) in Alaskan frogs than in Ohioan frogs. This trend was reversed later in the freezing exposure and by 6 h of thawing Ohioan frogs had the higher (P<0.0005) activity  and total GP activity  during early freezing, but by 6 h of thawing, values for Alaskan frogs had returned to basal levels. In contrast, Ohioan frogs showed increases in both the percentage of GPa  and total GP activity , and these only returned to basal values by 120 h of thawing .Overall, changes in both the percentage of enzyme present as GPa and total GP activity reiterated the GPa response observed in both populations. Alaskan frogs showed an increase in both the percentage of GPa  between populations. These activity values, 0.017±0.004 and 0.019±0.004 U mg−1 for Alaskan and Ohioan frogs, respectively, were ca. 90% lower than those determined for preparations incubated at 22°C. The resultant values for Q10, 3.2±0.24 for Alaskan frogs and 3.3±0.14 for Ohioan frogs, respectively, were not statistically distinguishable .Total GP activity in enzyme preparations incubated at 0°C was not different  throughout the experiment. However, in Ohioan frogs, GP levels did vary  with freezing and thawing, although none of the sample groups differed from values for unfrozen frogs. GP levels in frogs sampled at 48 h of freezing were 1.5-fold higher in Ohioan frogs as compared to Alaskan frogs  R. sylvaticaEurosta solidaginisLittorina littorea) Orconectes virilis) and an anoxia-tolerant turtle (Trachemys scripta elegans) –1, respectively; data from R. sylvaticaAs freezing progresses and glycogenolysis proceeds, it becomes unnecessary to sustain high PKAc activity R. sylvatica derives from variation in the PKAc gene or a post-translational modification of the protein R. sylvatica to colonize Interior Alaska, where subnivian habitats are particularly cold and the demand for rapid, copious production of cryoprotectant is especially great R. sylvatica populations would be exemplary among known cases of intraspecific enzymatic adaptation along a latitudinal gradient Livers of Alaskan frogs had a comparatively higher total PKA activity  and yet Pseudacris triseriata) maintains a constituitively high activity of GPa (ca. 21 U g−1 fresh liver) that is not further increased upon freezing R. sylvatica, however, basal activity of this enzyme is much lower (ca. 1 U g−1 fresh liver), but increases substantially, to ca. 15 U g−1 fresh liver, upon freezing R. sylvatica from southern Canada, are on the same order as those we recorded for frogs from both Alaskan and Ohioan populations.One common physiological response to freezing in many freeze-tolerant animals is the almost instantaneous activation of liver GP, with a large amount of the enzyme shifting from the inactive form, GPb, to the active form, GPa, through phosphorylation by PhK Freezing induced a large increase over unfrozen frogs in the activity of GPa  in both Alaskan and Ohioan frogs. However, in early freezing the activity of this enzyme was significantly higher in Alaskan frogs, as compared to the southern phenotype, for specimens frozen for 2 h or 30 h . TherefoIn Alaskan frogs, GPa activity  returned to basal levels by 48 h of freezing, whereas, in Ohioan frogs it remained elevated through the early hours of thawing. Preservation of the hyperglycemic state during freezing is achieved through a bypass of the usual glucoregulatory mechanisms and maintenance of elevated GP activity in the liver Because the cryoprotective glycemic response is triggered by the onset of freezing 10 values for total PKA activity and total GP activity showed that the thermal sensitivity of both enzymes was similar between populations. The Q10 values for total PKA activity in liver homogenates from Alaskan and Ohioan frogs were 1.6 and 1.8, respectively, similar to that determined for PKA purified from R. sylvatica10 for this enzyme, known from other ectotherms, ranges from 2.5 to 10 10 values for total GP activity from our Alaskan and Ohioan frogs were 3.2 and 3.3, respectively. A study of hepatic glycogenolysis rate as a function of temperature showed the Q10 for the process was 1.98 10 for total GP activity. Thus, there must be cellular mechanisms that compensate for the relatively high thermal sensitivity of GP, since the overall glycogenolytic pathway seems to be less affected by temperature than this enzyme, which is essential to glycogenolysis.Comparing QThe ability to change the expression levels of certain enzymes and other proteins is an essential response conferring local adaptation and stress tolerance R. sylvatica, freezing-responsive genes include ones involved in metabolic suppression, cell repair, and antioxidation, as well as those encoding novel proteins Changes in gene and protein expression are common responses in freezing adaptation in myriad organisms. In R. sylvatica, which can survive freezing at temperatures at least as low as –16°C R. sylvatica likely play an important role in this phenotype’s exceptional freeze tolerance, which is essential for the survival of this species in regions where winter conditions are so extreme.In this study, we determined whether the enhanced freeze tolerance of Alaskan


Graft versus host disease (GVHD) is an uncommon complication after orthotopic liver transplantation (OLT) with an incidence of 0.1–2%, but an 80–100% mortality rate. Patients can present with skin rashes, diarrhea, and bone marrow aplasia between two to eight weeks after OLT. Diagnosis of GVHD is made based on clinical and histologic evidence, supported by chimerism studies showing donor HLA alleles in the recipient bone marrow or blood. Several therapeutic approaches have been used for the management of GVHD after OLT including increased immunosuppression, decreased immunosuppression, and cellular therapies. However, success rates have been low, and new approaches are needed. The first successful human orthotopic liver transplant (OLT) was performed in 1967 by a surgical team led by Dr. Thomas Starzl in Denver, Colorado. Today more than 6000 liver transplants are performed each year in the United States with a one year survival rate of 80–85% [Acute graft versus host disease (GVHD), a frequent complication after hematopoietic cell transplantation (HCT), occurs infrequently after solid organ transplantation . However9/L. A bone marrow biopsy showed a markedly hypocellular marrow with no evidence of dysplasia. Flow cytometry showed less than 0.1% CD34 positive progenitors consistent with severe myelosuppression. Bone marrow chimerism studies showed 80% liver donor and 20% host cells. Cytogenetic analysis was limited to one metaphase, which showed a 46 XY karyotype, consistent with male donor origin. Due to the pancytopenia and documented donor chimerism, the working diagnosis was GVHD restricted to the bone marrow. She had no nausea, diarrhea or skin rash suggestive of gastrointestinal or skin GVHD, and liver GVHD would not be expected given the OLT. She was treated with rabbit anti-thymocyte globulin (ATG) 1.5 mg per kg for three consecutive days and alefacept 30 mg per dose, twice weekly for three doses. Tacrolimus, mycophenolate mofetil and prednisone were discontinued. HLA-typing of her two siblings was initiated.A 53-year-old woman had a 14 year history of primary biliary cirrhosis and received a liver transplant from a cadaveric male donor. Transplantation was uncomplicated and she received 3 doses of rabbit antithymocyte globulin (ATG) induction peritransplant. Post-transplant immunosuppression was with tacrolimus and mycophenolate mofetil. She was discharged from the hospital on post-operative day (POD) 14. Prednisone 5 mg daily was added on POD 20 for additional immunosuppression. On POD 38 she was readmitted with tremors, forgetfulness, headache and difficulty with word finding. Her absolute neutrophil count was 0.09 × 10Follow-up bone marrow biopsy 12 days after administration of ATG showed continued aplasia with < 0.1% CD34+ cells. Bone marrow chimerism was 27% donor and 73% host, while unsorted peripheral blood chimerism showed 98% donor and 2% host cells. No further bone marrow studies were done.On POD 53 after OLT and day 7 after starting ATG, she developed an erythematous, pruritic and painful skin rash, most prominent on her hands but also extensively involving her back, arms and legs. Skin biopsy was consistent with GVHD, and chimerism studies showed male (donor) lymphocytes in the biopsy specimen. She was treated with topical steroids for these lesions given the high risk of infection and the fact that the lesions were not progressing.Her subsequent clinical course was complicated by prolonged vancomycin-resistant enterococcal bacteremia, which was treated with multiple antibiotics and granulocyte transfusions. Weekly rituximab was administered for EBV viremia and abdominal CT showing retroperitoneal lymphadenopathy, suggestive of post-transplant lymphoproliferative disorder.Liver function tests were normal throughout her course. HLA-typing showed that the liver donor shared a single DQB1 antigen with the patient, but the donor was heterozygous at all loci. Thus, the HLA typing of donor and patient would not have predicted a high risk for graft-versus-host disease.Due to the profound bone marrow failure, the plan was to proceed with allogeneic HCT. However, the patient’s clinical condition deteriorated rapidly, and she died POD 86 from septicemia, pulmonary aspergillosis and respiratory distress syndrome before a donor was identified.In 1988, Burdick and colleagues first described acute GVHD after OLT, and more than 80 cases have been reported since then . Graft-vDiarrhea is most commonly the result of absorptive function loss caused by lymphocyte infiltration and destruction of the intestinal mucosa, and is more evident as the post-transplant period lengthens. Biopsy specimens of gastric, duodenal or colonic mucosa demonstrate apoptosis and infiltration by donor lymphocytes . In case9–1010 donor-derived leukocytes including monocytes, T-cells and natural killer cells are transplanted with the liver allograft [+ cells and B lymphocytes [In OLT, about 10llograft , approxillograft ,13, and phocytes . Lymphocphocytes . HoweverEngraftment of donor lymphocytes can be confirmed by chimerism studies and identification of donor HLA alleles in the recipient bone marrow or blood using a polymerase chain reaction (PCR) test . A DNA sDomiati-Saad and colleagues reported serial single tandem repeat results in 49 patients during the first two months after OLT. In 38 of the 49 patients, donor T cell chimerism was present on POD 2, and remained positive by the end of the first week in 23 patients. Donor CD8+ T-cell chimerism was less than CD3+ T cell chimerism measured on the same sample, but in the two patients with acute GVHD the level of CD8+ T-cell chimerism was generally equal to or greater than the CD3+ T-cell chimerism. These two patients had characteristic signs of acute GVHD. Donor CD3+ fractions above 10–20% of lymphocytes correlated strongly with acute GVHD .Shared HLA antigens between donor and recipient are the most important risk factors for the induction of GVHD. Use of an HLA-homozygous donor, resulting in one way HLA matching in the GVHD direction, significantly increases the risk of developing GVHD after OLT .Kamei and colleagues investigated the incidence of fatal GVHD following living donor liver transplantation (LDLT) in a retrospective study of 906 donor-recipient pairs. They analyzed the ages of the recipient and donor, donor relationship, original disease, initial symptoms, onset and course of GVHD, Glucksberg stage, and donor/recipient HLA type. Eight cases (1%) of fatal GVHD after LDLT were identified with skin rash as the most common initial manifestation. Seven cases had unidirectional HLA matching at 3 loci  in the GVHD direction (donor homozygous). Case 8 had unidirectional HLA matching at 2 loci (HLA-A and –DR) in the GVHD direction with the B locus matched because of recipient homozygocity. Among these 8 cases, four donors were children, three were parents and one was a sibling. The authors suggested that the risk of fatal GVHD following LDLT may depend on the number of loci with donor-dominant one-way HLA matching, and was highest with mismatching in the GVHD direction at all 3 loci  .Key and colleagues reported a single-center analysis of 412 consecutive adult OLT recipients where HLA typing data were available. Their data showed that HLA matching at HLA-B, but not HLA-A or –DR was a significant risk factor for the development of GVHD. Of 14 donor-recipient pairs matched at HLA-B, three 21%) developed GVHD [% developChan and colleagues studied 205 OLT patients surviving more than 30 days. Four patients (2%) developed GVHD, and three of them had received a donor liver with steatosis graded mild or more severe. This analysis did not identify HLA-matching as a risk factor for GVHD, and could not confirm cryptogenic cirrhosis (as an indication for liver transplantation) or older recipient age (>40 yrs) to be predictors of GVHD. Alcoholic liver disease, particularly in combination with hepatocellular carcinoma and glucose intolerance, conferred a very high risk for GVHD. They concluded that immunodeficient states due to diabetes mellitus type I and II, various autoimmune diseases and hepatocellular carcinoma increased the risk for developing GVHD . InfectiAlthough GVHD after OLT usually appears two to eight weeks after OLT, similar to what is seen in hematopoietic cell transplantation, Pollack et al reported a case of a 52-year-old liver transplant recipient who developed gastrointestinal symptoms and severe aplasia 8 months after OLT. After elimination of all other possible causes of these symptoms, chimerism analysis was performed and showed that 96% of the peripheral blood mononuclear cells were of liver-donor origin. An allogeneic peripheral blood HCT was carried out, but the patient died of septicemia 5 days after transplantation . Severe Several therapeutic approaches have been proposed in the literature. One strategy has been to decrease immunosuppression, in an effort to shift the balance in favor of recipient immunity, but most investigators have tried to increase immunosuppression.The literature describes cases where patients were treated with corticosteroids as monotherapy because of their well-known ability to induce apoptosis in lymphocytes and profound anti-inflammatory effect. However, high doses of corticosteroids did not seem to improve response rates and may increase the risk for these patients to die of complications of GVHD, infection and multi-organ failure due to side effects of high doses of corticosteroids.Treatment with immunosuppression beyond steroids has been very heterogeneous and has included antimetabolites, alkylating agents, anti-T cell antibodies, anti-B cell antibodies, intravenous immunoglobulin, cytokine inhibitors, immunostimulants and cellular therapy, including T cell infusions and hematopoietic stem cell transplantation.The literature may reflect heavy publication bias, where successful cases are reported, but unsuccessful treatment attempts are not. In cases of GVHD-associated marrow failure, standard treatments for GVHD have not been successful.The Table Anti-T cell antibodies have shown some success in treating corticosteroid resistant GVHD, but are associated with an increased risk for development of post-transplantation lymphoproliferative disease and viral reactivation. Weekly monitoring by PCR for CMV and EBV reactivation is recommended after starting antilymphocyte therapy.The second most common approach is to administer cytokine inhibitors, most commonly against IL2 and TNF-α. Anti-interleukin-2 receptor antibodies daclizumab and basiliximab have shown some benefits if introduced early after high-dose corticosteroids. Infliximab is a chimeric mouse/human immunoglobulin-G antibody directed against soluble and transmembrane forms of human TNF-α. Some authors suggest that infliximab should be introduced earlier after the diagnosis of steroid refractory acute GVHD, and if possible, before ATG to avoid increased risks of infection and subsequent lymphoproliferative disorders . EtanercThere is one report of a haploidentical transplant for GVHD-associated pancytopenia, with documented transient engraftment of the haploidentical donor cells followed by rejection of the donor graft and recovery of recipient hematopoiesis. The haploidentical donor was related to the recipient (family member) and different from the liver donor .Another report showed successful harvest and ex vivo expansion of recipient T cells, which upon re-infusion were successful in combating GVHD . Thus, aIt is clear that more successful approaches to treating OLT GVHD need to be developed. Such advances will require a coordinated effort among transplant centers to systematically test potential treatment algorithms given the rarity of this complication.GVHD after OLT is a rare problem, but one associated with a greater than 80% mortality. Rapid diagnosis, immunosuppressive treatment, and aggressive supportive care are key to potentially reversing this otherwise fatal condition. Our patient presented with symptoms five weeks after OLT, which is similar to previously reported cases. In cases of pancytopenia due to GVHD, rapid evaluation for possible HCT may offer the best option for survival. In conclusion, the diagnosis and treatment of GVHD after solid organ transplantation is difficult because the early symptoms are nonspecific, and no standard treatment is available. Reducing the risk factors for development of GVHD, identifying the disease promptly, preventing infection, and starting treatment as early as possible may improve the dismal outcome associated with this disease. It would be helpful to report cases to registries and world data banks that collect data from organ transplant centers in order to better understand this complication and allow for the design and testing of treatments to try to improve survival.The authors declare that they have no competing interests.IMR and SJL designed research and wrote the paper, JD revised the paper. All authors read and approved the final manuscript.


Drosophila (dKCNQ) when mutated show decrements in associative short- and long-term memory, with KCNQ function in the mushroom body α/βneurons being required for short-term memory. Ethanol disrupts memory in wildtype flies, but not in a KCNQ null mutant background suggesting KCNQ maybe a direct target of ethanol, the blockade of which interferes with the plasticity machinery required for memory formation. We show that as in humans, Drosophila display age-related memory impairment with the KCNQ mutant memory defect mimicking the effect of age on memory. Expression of KCNQ normally decreases in aging brains and KCNQ overexpression in the mushroom body neurons of KCNQ mutants restores age-related memory impairment. Therefore KCNQ is a central plasticity molecule that regulates age dependent memory impairment.In humans KCNQ2/3 heteromeric channels form an M-current that acts as a brake on neuronal excitability, with mutations causing a form of epilepsy. The M-current has been shown to be a key regulator of neuronal plasticity underlying associative memory and ethanol response in mammals. Previous work has shown that many of the molecules and plasticity mechanisms underlying changes in alcohol behaviour and addiction are shared with those of memory. We show that the single KCNQ channel in  KCNQ1 loss of function mutations result in the most common form of cardiac arrhythmia, Long QT syndrome, while gain of functions mutations cause Short QT and atrial fibrillation KCNQ1 mutations also result in adult onset type II diabetes KCNQ2 or KCNQ3 loss-of-function mutations result in a developmental form of epilepsy called Benign familial neonatal convulsions KCNQ4 loss-of-function mutations are a common cause of autosomal dominant deafness and age-dependent hearing impairment KCNQ (Kv7) channels mediate a range of important physiological functions and are a hotspot of genetic diseases and therefore target for existing and novel drug treatments. In human cardiac muscle, Drosophila has a single KCNQ (dKCNQ) channel that is most highly expressed in the nervous system dKCNQ loss-of-function mutant flies showing arrhythmias similar to those seen in aged wildtype flies, whose hearts shows age dependent reduction in dKCNQ expression KCNQ-RNAi in Drosophila neurons increased neural excitability, while KCNQ overexpression decreased excitability in vivo. dKCNQ loss-of-function mutant flies increased ethanol sensitivity and tolerance with acute activation of dopaminergic neurons by heat-activated TRP channel or KCNQ-RNAi expression shown to produce ethanol hyperexcitability dKCNQ mutants on memory, showing that dKCNQ expression decreases in the brain with aging and is linked to age-dependent cognitive deficits.KCNQ deletion mutant contained an imprecise excision of the EP2074 element (186KCNQ) that removes all the 5′ and transmembrane regions of the channel and therefore is a null KCNQ control was a precise excision of the element (97KCNQ) leaving the gene completely intact uas-KCNQ flies allowed Gal4 promoter driven overexpression of KCNQuas-KCNQ-RNAi (Bloomington stock 27252) allowed Gal4 targeted knockdown of the channel. The KCNQ stocks were kind gifts of Dr Rolf Bodmer. Elav-Gal4, uas-mCD8-GFP and OK107-Gal4tsOK107-Gal4, Gal80c305a-Gal4MB247-Gal4Amn(c316)-Gal4Canton S w- (CSw-) from a stock previously maintained in the Waddell lab. All KCNQ mutant, Gal4 and uas lines were out crossed with the relevant CSw- line prior to behavioural analysis. All genotypes and all other crosses were raised on corn-meal agar medium at 22±2°C and 60±10% humidity under 12∶12 hr light-dark cycle.The 3, 115 mM sucrose, 4 mM MgCl2 5 mM trehalose, 1.5 mM CaCl2, and 5 mM HEPES, pH 7.3) and isolated brains were fixed in 4% paraformaldehyde in HL3.1 for 30 min before being washed in HL3.1 Drosophila DLG (PDZ1-2) a protein known to be highly expressed in these memory-related structures KCNQ expression pattern was determined by visualising membrane targeted GFP expressed using KCNQ-Gal4 reporter lines .Adult fly brains were dissected in HL3.1 ; Carolina biological supply company, Burlington, NC, USA) containing 10% ethanol in water containing a small amount of blue dye (0.05% Bromophenol blue) ∼12 hr before testing. The controls were given the same water-blue dye solution but lacking ethanol. The blue dye was used to monitor whether the flies had actually drunk the ethanol solution; this was confirmed as all the flies had blue abdomens prior to the test. For tsOK107-Gal4, Gal80 experiments KCNQ transgene expression 1–2 days prior and during behavioural testing. Olfactory acuity was quantified by exposing naive flies to the odour versus air in the T-maze during a 2 min test trial. The performance index was calculated by counting the number of flies avoiding odour divided by total number flies. Shock reactivity was quantified by placing grids in each arm of the T-maze, and applying shock via the grid in one arm of the maze during a 2 min test trial. The performance index was calculated by counting the number of flies avoiding shock divided by total number flies All experiments were performed at 25°C and 70% humidity under red light using the olfactory aversive conditioning protocol KCNQ mRNA was measured using a TaqMan Kit(KCNQ - Dm01846741_g1) and was normalised to Rpl23 (Rpl23-Dm02151827_g1) mRNAas a control allowing standardisation between the samples for aging experiments. The cDNA concentration was measured using Roche’s Light Cycler system and using multiplexing on a Stratagene Mx3000P system (Stratagene). All statistical analysis of data were performed and plotted with Graphpad Prism software.Age-matched flies were frozen in liquid nitrogen and decapitated by vortexing. Heads were collected and an equal number of heads from each genotype were homogenised. Trizol was added directly in to the homogenised heads and RNA was extracted according to manufacturer’s instructions (Invitrogen). RNA was DNAase treated (Ambion Inc) and reverse-transcribed . Drosophila memory include those of the mushroom body that are labelled by OK107-Gal4MB247-Gal4  and can be labelled by amn(c316)-Gal4 that expresses in the DPM Gal4 promoter enhancer trap within the KCNQ gene to drive GFP expression  assessed 1 hr after training . KCNQ o or 1 hr . We alsoo normal . Acute rcontrols , showingn in LTM . No diffn in LTM –B or shon in LTM  was obseKCNQ expression decreases in old fly hearts leading to age related cardiac impairments KCNQ expression in heads over the lifespan of Drosophila  and caused deficits in associative memory. Kv channels have also been implicated in plasticity underlying fly memory KCNQ in the mushroom body α/β neurons was sufficient to reduce memory, while expressing KCNQ in the same neurons in a fly otherwise completely lacking KCNQ rescued the KCNQ mutant memory phenotype to normal  flies similarly (p>0.05) avoided OCT A. and MCH B. odour used in the memory assay. C. Experimental and control (CSw- wildtype or KCNQ control) flies similarly (p>0.05) avoided the arm of the T-maze delivering 60 V DC electric shock. Data in A-C were analysed by 1-way ANOVA with Bonferroni post-hoc test.A. KCNQ channel mutants display normal olfactory acuity and shock reactivity. Experimental and control ((TIFF)Click here for additional data file.