Hoplobatrachus rugulosus , were collected in Srakaew province, Thailand. For blood parasite examination, thin blood smears were made and routinely stained with Giemsa. The results showed that 70% of the frogs (98/140) were infected with 5 species of blood parasites, including a Trypanosoma rotatorium-like organism, Trypanosoma chattoni, Hepatozoon sp. a, Hepatozoon sp. b, and Lankesterella minima. Pathological examination of the liver, lung, spleen, and kidney of the frogs that were apparently infected with one of these blood parasites were collected and processed by routine histology and subsequently stained with haematoxylin and eosin. Histopathological findings associated with the Trypanosoma rotatorium-like organism and Trypanosoma chattoni-infected frogs showed no pathological lesions. Hepatozoon sp. a and Hepatozoon sp. b-infected frogs developed inflammatory lesions predominantly in the liver, demonstrating granuloma-like lesions with Hepatozoon sp. meronts at the centre. Tissue sections of Lankesterella minima-infected frogs also showed lesions. Liver and spleen showed inflammatory lesions with an accumulation of melanomacrophage centres (MMCs) surrounding the meronts and merozoites. It is suggested that Hepatozoon sp. a, Hepatozoon sp. b, and Lankesterella minima-infections are capable of producing inflammatory lesions in the visceral organs of rice field frogs, and the severity of lesions is tentatively related to levels of parasitemia.One hundred and forty adult rice field frogs,  Trypanosoma spp. (Euglenozoa: Kinetoplastida) are heteroxenous and vector transmitted through a variety of mechanisms. The classification of anuran trypanosomes is still in confusion because some species may be polymorphic [Trypanosoma rotatorium is a polymorphic species with a wide geographical distribution, while Trypanosoma chattoni is a monomorphic species that is found in Asia, Europe, and America [Hepatozoon spp. (Apicomplexa: Hepatozoidae) are often found in amphibians, appearing as large banana-shaped organisms in the cytoplasm of host erythrocytes. Hepatozoon species possess heteroxenous life cycles, with sexual reproduction and sporogony occurring in an arthropod definitive host. Transmission occurs when such an arthropod, infected with mature oocysts, often containing thousands of sporozoites, is consumed by a vertebrate host. Released sporozoites then migrate to the visceral organs, primarily to the liver, and undergo merogony. The meronts are released into the bloodstream where they form gamonts in erythrocytes. Lankesterella spp. (Apicomplexa: Lankesterellidae) are recognized as parasites of frogs and some other ectotherms. Merogony and sporogony occur in vascular endothelial cells in the visceral organs of the vertebrate hosts. Mature sporozoites are released into the bloodstream and invade erythrocytes. Intraerythrocytic sporozoites are taken up by vectors during feeding and it is believed that frogs became infected upon the digestion of sporozoite-bearing vectors. Leeches and mosquitoes have been shown to be capable vectors [Studies in several geographical regions have indicated anurans infected with variety of blood parasites, including viruses, rickettsiae, several species of protozoans, and microfilariae –6. Trypaymorphic –9. Trypa America , 10. HepTrypanosoma inopinatum were lethal to European green frogs and caused haemorrhages, swollen lymph glands, and anaemia [Trypanosoma rotatorium can be pathogenic in tadpoles and in heavy infections with trypanosomes accumulate in the kidneys [Hoplobatrachus rugulosus , is one of the most widely distributed frogs in this country and is of economic importance as a food source of the local population. In this paper, the blood parasites of naturally infected rice field frogs were examined, and a histopathological study of their visceral organs was undertaken.In Thailand, a survey of mainly blood parasites and helminthes of amphibians has been undertaken –12. Ther anaemia . Trypano kidneys . The ricHoplobatrachus rugulosus, from Wang Nam Yen district, Srakeaw province, Thailand were collected from July to October 2007 by a local commercial vendor. The frogs were bled by cardiac puncture, blood was preserved in heparinized tubes and then examined for hematocrit value. A few drops of fresh blood were smeared, fixed with 100% methanol, and stained with Giemsa. Each thin blood smear was examined and classified under a light microscope [One hundred and forty wild rice field frogs, croscope , 9, 13. Visceral organs such as liver, lungs, spleen, and kidney of the frogs that were apparently infected with just one species of blood parasite were then collected and processed by routine histology and subsequently stained with haematoxylin and eosin for histopathological examination. These slides were also observed with a light microscope.n = 98) and noninfected frogs (n = 42) at 22.64 ± 5.31 and 22.81 ± 5.82 (mean ± SD), respectively, (t = 0.167). The parasites were identified as 5 species: a Trypanosoma rotatorium-like organism (I–V forms), Trypanosoma chattoni, Hepatozoon sp. a, Hepatozoon sp. b, and Lankesterella minima , Trypanosoma chattoni , Hepatozoon sp. a ; Hepatozoon sp. b , and Lankesterella minima  respectively . The lung and kidney with merozoite stages also showed tissue damage with a mild degree inflammation. However, in infected frogs with average parasitemias below 0.01% of erythrocytes, no remarkable lesions could be recognized  were positive for blood parasites. Hematocrit values were not statistically different between the infected  and Trypanosoma chattoni observed in this study are similar to the observation of Werner [Hepatozoon was differentiated into species a and b by the characteristics of the parasitophorus vacuole [Lankesterella, sporozoites observed in the rice field frog were similar in size and morphology to those of Lankesterella minima, which is the most prevalent species reported from frogs in the USA, Europe, and Asia [Lankesterella in this report could be identified as Lankesterella minima. Blood parasites that were observed in wild rice field frogs, f Werner . The Hep vacuole . Howeverand Asia , 11, 12.Trypanosoma rotatorium-like organism and Trypanosoma chattoni, there was no evidence of pathological lesions in any visceral organs. It is suggested that the number of blood parasites found in those frogs with average parasitemias at or below 0.01% of erythrocytes did not affect frog health or produce any clinical signs. Hepatozoon sp. a and b meronts and Lankesterella minima meronts and merozoites produced pathological lesions predominantly in the liver with subacute to chronic inflammation [Lankesterella minima presented in various organs, that is, liver, lung, and kidney, producing mild degree inflammatory response [There was no statistical difference between the hematocrit value of infected and noninfected frogs. That could be because the blood parasites form part of the normal fauna of wild frogs. All the frogs were clinically normal and, thus, considered suitable for human consumption. In the case of the trypanosomes, including the ammation , 15. Thoammation . The merresponse . In the  Hoplobatrachus rugulosus, were infected with 5 species of blood parasites, including 2 species of trypanosomes, 2 of Hepatozoon, and one lankesterellid. Frogs apparently with just one of these infections showed pathological lesions in the visceral organs only in the case of Hepatozoon sp. a and b (meronts mainly in liver) and Lankesterella minima . No evidence of any inflammatory lesion was found in the various organs in the case of the Trypanosoma rotatorium-like organism and Trypanosoma chattoni. The severity of the lesions in the wild frogs infected with blood parasites ranged from severe to mild in the order Hepatozoon sp. a, Hepatozoon sp. b, Lankesterella minima, the Trypanosoma rotatorium- like organism and Trypanosoma chattoni, respectively. In addition, the severity of the pathological lesions appeared to relate to the levels of parasitemia (percentages of erythrocytes infected). However, the infected frogs did not show any clinical signs of sickness although blood parasites may be stress factors including immunosuppressive conditions that render hosts more susceptible to secondary infection. This information will be useful for health monitoring in the frog management industry and ecological conservation.From the results obtained, it can be concluded that wild rice field frogs,


This article reports on formative research into the context and practice of handwashing with soap by new mothers, which can substantially impact child morbidity and mortality. New mothers are an important target group for handwashing interventions: they are considered particularly susceptible to behaviour change and their actions can directly affect a child’s health.Twenty-seven mothers of infants (including neonates) from urban and rural sub-districts of Serang were recruited and filmed over a period of eight hours. Video footage was used to identify handwashing occasions and to understand the context in which behaviour took place. Each woman was subsequently interviewed.Handwashing with soap was found to be infrequent, typically occurring after eating, cooking and household chores or after cleaning a child’s bottom. Handwashing before preparing food or eating was rare. Pre-pregnancy routines were reported to have been disrupted. Advice on child care comes from many sources, particularly the midwife and new child’s grandmother.Developing interventions to change perceptions and practice of handwashing would seed an important behaviour and could save lives. New mothers represent an ideal target group for such an intervention. We suggest that interventions target an increase in handwashing with soap after contact with own and a baby’s faecal matter as part of the post-defecation hygiene routines. As the child’s grandmother is an authoritative source of information about parenting, interventions focussed on improving newborn care could target grandmothers as well as midwives. Handwashing with soap has been viewed as one of the most cost-effective ways of reducing the global infectious disease burden . The benInterventions that promote handwashing with soap are therefore vitally important to public health, yet changing behaviour is notoriously difficult. It may be possible to optimise the effectiveness of an intervention by concentrating efforts during a “teachable moment”, a concept from education describing a naturally-occurring event or life-stage that motivates a person to acquire new behaviours . In 2002It is also well known in marketing circles that major changes in life-status such as getting married, divorced, having a child or reaching pensionable age are associated with significant changes in the pattern of consumption ,10. MarkAs well as being a life-changing, emotionally-charged event, pregnancy is a time when women willingly adopt new behaviours to minimise health risks to both the mother and child; evidence suggests that other lifestyle change interventions can also be successfully implemented antenatally ,13. New http://clinicaltrials.gov/ct2/show/NCT01309321?id=10036&rank=2) which should strengthen the evidence base for this third assertion.As well as hypothesising that new and expectant mothers are particularly susceptible to behaviour change interventions, there are compelling public health reasons for focusing handwashing behaviour change interventions at this time in a woman’s life: 1) a mother’s hygiene behaviour directly impacts the health of her child, therefore early adoption of better hygiene practices that continue as the child grows could reduce morbidity and mortality from common diseases such as diarrhoea and respiratory infections ,14-16; 2Standard approaches to hygiene promotion - focused on educating people about germs and the value of using soap - have rarely resulted in positive, sustained behaviour change ,6,21-23.Indonesia has more than 230 million inhabitants spread over 17,000 islands . The forIn Indonesia the infant mortality rate is 34 per 1000 live births. Infant mortality is higher in Banten Province than the national average, at 46 per 1000 live births . Common Village midwife records from Community Health Centres in Kramatwatu and Pamarayan were used to identify a purposive sample of women who had given birth within the last year, including both primiparous and multiparous women, and infants as well as neonates. Potential participants were told the study aimed to learn how their life has changed since having children.Informed consent was obtained from all participants and their husbands whenever possible. Ethical approval was granted by LSHTM Independent Ethics Committee. The Maternal and Child Directorate of the Ministry of Health Indonesia also supported and approved the study.A range of tools can be employed for formative research. Due to our focus on how to introduce new hygiene behaviours into this teachable moment, we required good information about how new mothers currently engage in child-care and personal hygiene behaviours. This is very difficult to investigate through questioning. On the other hand, measuring behaviour accurately is also challenging; relying on self-report often produces implausible data ,31, whilWe used small hand-held video cameras (Panasonic SDR-S50) to directly observe behaviour. We piloted this method in India and Bangladesh and found it acceptable to participants and a valuable data source. Three local women were trained to carry out unobtrusive video recording. They were instructed to film the mother continuously during two periods: when she first woke (around 4 am) for five hours, and from 4 pm until after the evening meal . Filming at these times maximised the opportunity to observe periods of intense activity in the household such as food preparation, hygiene routines and eating at least one meal. To ensure filming began when the respondent woke up, the field worker stayed in a respondent’s home the previous evening, something found to be culturally acceptable in this setting. Although this was the primary reason for arriving the night before, gaining familiarity with the respondent in this way may possibly reduce reactivity among mothers when filming actually began  .Participants were not followed when they left the household compound, and any visitors to the home were not filmed unless they consented. Field workers were requested to keep the mother’s hands on camera whenever possible and to keep a record of any periods of absence from the compound to aid interpretation of video footage. Participants’ wishes for privacy were respected at all times, for example, by turning the camera away during breastfeeding and while in the bathroom.Semi-structured interviews were conducted the day after filming by four researchers  working in two teams, each consisting of one LSHTM and one Indonesian researcher . Participants were questioned about their handwashing practices and motivations for handwashing, how their life has changed since having children, their knowledge of health risks and their sources of information about child-care.how the young mothers’ daily lives had changed and suggested a variety of lines of questioning in interviews. The picture cards were also used as visual aids to help understand the flexibility of the daily routine, order of activities and to describe a typical day before having children.If we want to insert or change a particular behaviour in a person’s routine, it is first important to understand the particular sequence of activities that take place currently – the “script”. In a process called “script elicitation” (used in cognitive psychology ), we askVideos were reviewed daily so that unclear portions of film could be immediately discussed and interpreted together with the field worker and with the participant (in interview the following day). A detailed sequence of events was recorded using an Excel database. The database was used to identify occasions when hands were washed with and without soap and to document details of the physical environment. Previous work in this field has shown us that hands may well be washed with soap, but the activities that prompt handwashing (and soap use) are not usually times considered important from a public health perspective. We decided to categorise handwashing behaviour according to whether or not soap was used to wash hands and whether or not hands were washed at key times so we could better describe handwashing behaviour. Each participant was categorised into one of five categories: Washer ; Reactive Washer ; Rinser ; Reactive Rinser ; and Avoider (never observed to handwash). These categories best described their hand-washing behaviour based on the times when they washed their hands and whether or not they were ever seen to use soap to hand-wash on this occasion. Categorising individuals in this way allows the data to be viewed differently, illustrating the range of behaviours that take place and the differences between individuals in terms of whether and when soap is used to wash hands.We performed thematic analysis of interview transcripts to provide insights into practices and underlying motives and drivers of behaviour. This analysis allowed unexpected themes and concepts to emerge from the information provided by informants. Scripts (daily routines) from the different women were aggregated to find the most frequent sequence of daily events, thus generating a “master” routine that illustrates the course of life before and after giving birth to a first child. All aspects of this study conform to the RATS guidelines for qualitative research.Twenty-seven new mothers were filmed and interviewed, 15 in urban Kramatwatu and 12 in rural Pamarayan. Only two families were approached and refused to participate. Participants were aged between 18 and 39, all were literate and all had completed at least primary education, while two were university graduates. Monthly household income varied widely, from < US$115 to US$920. The mean monthly household income (collected in categories and converted from Indonesian Rupiah) was US$230-345 in urban households and < $115 in rural households, comparable with the general population. Half of the participants (n = 14) were first time mothers, eight of whom had returned to their maternal homes for an extended perinatal period as is customary in this region. The median age of the infant being cared for was two months (range 13 days to nine months); eleven were neonates  used soap on at least one occasion. Overall, nine participants were classified as “washers”, six were “reactive washers”, two were “rinsers”, two were “reactive rinsers” and eight were “avoiders” , and that they come into contact with dirty things or people when they go into town.In almost all households the latrine and handwashing station were in the bathroom so it was not possible to observe whether hands were washed post-defecation. On one occasion a mother who defecated in a defecation pond – a small pond with a hanging latrine behind the house - was observed to rinse her hands with water upon returning to the house Table .Handwashing following cleaning a child’s bottom after defecation was seen on film following 15 of 28 observed defecation events , preventing cord infection and the importance of breastfeeding. The instructions they give on preventing cord infection and breastfeeding mention handwashing. Immediately post-birth in some rural areas a traditional birth attendant may provide services such as washing the mother’s clothing and helping to care for and bathe the child. Women reported receiving conflicting advice from different sources, and despite the value placed on information given by midwives, family members, particularly a woman’s mother or mother-in-law are also important influences. Video footage showed grandmothers play an active role in preparing food and feeding young infants, and were frequently heard issuing instructions in the background on how to bathe the baby and clean up faeces.During interview women reported deliberately resting more and changing their diet during pregnancy (eating more vegetables and less spicy food) for the sake of their own health and that of their offspring. Mothers with neonates were observed on film to do very little other than care for their new charge, explaining in interview that they are supposed to spend extended periods of time sitting during the postpartum period to promote healing, and that household chores such as ironing and laundry are consequently taken over by other family members. Video footage and family structure Table  support Women have a wide range of beliefs concerning the health risks their child faces. They were particularly concerned about the child becoming ‘sprained’ when they are handled due to the child’s ‘floppy neck’, but also fear fever, diarrhoea and infection from other children or from the cord. When asked explicitly what they could do to prevent illness, particularly diarrhoea, women felt there was little they could do and that childhood illness is related to the child passing through different developmental stages. Hygiene was rarely mentioned. Further questioning on whether handwashing could prevent illness was met with confusion or a “no”.This formative research was designed to capture detailed information on the lives and handwashing practices of new mothers in Serang, Indonesia. Handwashing with soap occurs at a low level, but is not constrained by water or soap availability. Hands are typically washed after eating, cooking, doing household chores and after cleaning a child: times when hands are visibly dirty, oily, smelly, sticky or otherwise uncomfortable. We divided the participants into handwashing ‘types’  to illustrate the range of hygiene behaviours that take place with respect to hands, and the differences between individuals in terms of whether and when soap is used to wash hands. We believe this categorization is insightful and may be true of other populations, and so could usefully guide program development on handwashing behaviour in future projects. This classification may also have implications for health promotion efforts, as it is possible that each of these types will require different kinds of intervention, since they appear to be motivated to use soap in different kinds of circumstances. While it is difficult to make specific recommendations for other handwashing programs without knowing their objectives, we believe that any ability to predict the types of handwashers likely to be found in a population can assist other programs, especially if they are not able to conduct research themselves. Knowing such types has program implications – for example, reactive washers are likely to be motivated by disgust at visible contamination on their hands, while avoiders seem not to respond to such cues to handwash, and so will require other means to change their behaviour.Emotional drivers of behaviour are also important determinants of handwashing . ObservaBoth the video footage and interviews indicate that handwashing is infrequent and does not seem to be prompted by having a new child, although “nurture” (desire to care for one’s offspring) has been previously demonstrated to drive maternal handwashing behaviour . FailureThe automaticity of existing handwashing behaviour  is one rFurther, rather than attempt to introduce handwashing with soap before breastfeeding (one of the current recommendation from midwives), which happens too frequently to be constantly interrupted by trips to a handwashing location, it is contact with faeces that should be the primary concern if the desire is to set a handwashing habit that will benefit the child’s health at a later date. In addition, we know from the videos that considerable time and care is already invested in cleaning and dressing babies in some households after a defecation event. The videos also showed us how variable handwashing practices were, even within the same individual, partially due to the presence of cues such as the physical setting, presence of an object, or visually dirty hands. Baskets for the various clothes and ointments needed at this time are a part of every new mother’s ‘kit’ for newborn childcare in Indonesia. It would probably be relatively easy to insert handwashing with soap soon after cleaning the baby’s bottom and before extensive further contact with the child. This is likely to be especially true if there is a handwash stand or other visual reminder present in a relevant location within the household as well. As we observed these cleaning and changing rituals being explicitly taught to young mothers by their mothers, it is natural to target these mothers-of-mothers as the appropriate channel for communicating the need to include handwashing with soap as part of normal child-care operations.This study had some obvious limitations. After every interview a discussion took place to clarify any points of confusion. However, we cannot exclude the possibility that the presence of “foreigners” during interviews may have influenced respondents, nor the fact that bias can also stem from local researchers’ perceptions of their own culture. The collection of video footage provided a rich data source, although the challenge of filming at first light and the inability to capture activity occurring inside a closed bathroom remained drawbacks, the latter a perennial difficulty in studies of handwashing behaviour ,42. AlthThis study used formative research methods to provide information on the lives and hygiene practices of new mothers, an important, yet under-exploited target group for handwashing interventions. Current handwashing rates are low, but handwashing facilities are available, and the ubiquity of women’s willingness to change other behaviours at this time bodes well for the possibility of incorporating handwashing with soap into their daily routines. If an intervention is developed it will also be important to decide when in the peri-natal period it should be delivered, as women are inundated with messages during pregnancy and the early stages of motherhood. We recommend efforts to promote handwashing focus on introducing handwashing into the newly-formed routine around baby defecation events, involving the grandmother(s) to provide support and authority.The authors declare that they have no competing interests.RA designed the study with contributions from EI. KG performed analysis and drafted the manuscript. All authors were involved in data collection and read and approved drafts of the manuscript, including the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/830/prepub


It was found that high-affinity anti-DNA antibodies were one of the major components of the intrathecal IgG response in multiple sclerosis (MS) patients . Recently we have shown that IgGs from the sera of MS patients are active in the hydrolysis of DNA. Here we have shown, for the first time, that average concentration of total proteins (132-fold), total IgGs (194-fold) and anti-DNA antibodies (200-fold) in the sera is significantly higher than that in the cerebrospinal fluid (CSF) of fifteen MS patients. The relative activities of total protein from sera and CSFs varied remarkably from patient to patient. It was surprising that the specific DNase activity of the total protein of CSF reparations were 198-fold higher than the serum ones. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We present first evidence showing that IgGs from CSF not only bind but efficiently hydrolyze DNA and that average specific DNase activity of homogeneous antibodies from CSF is unpredictably ∼49-fold higher than that from the sera of the same MS patients. Some possible reasons of these findings are discussed. We suggest that DNase IgGs of CSF may promote important neuropathologic mechanisms in this chronic inflammatory disorder and MS pathogenesis development. Multiple sclerosis (MS) is a chronic demyelinating pathology of the central nervous system presenting a serious medical and social problem. Its etiology remains unclear, and the most valid theory of its pathogenesis assigns the main role in the destruction of myelin-proteolipid shell of axons to inflammation related with autoimmune reactions  and natural abzymes are novel biological catalysts that have attracted much interest in the last years (reviewed in It has recently been shown that myelin basic protein (MBP)-hydrolyzing activity is an intrinsic property of IgGs, IgMs, and IgAs from the sera of MS patients At the same time, as mentioned above, anti-DNA Abs were found to be a major component of the intrathecal IgG response in MS patients In the present study we have used different approaches to provide, for the first time, a very strong direct evidence that DNase activity is intrinsic to IgGs from CSF of MS patients and compared some other parameters characterizing the MS CSF and sera.Fifteen patients (11 women and 4 men) satisfying the criteria for clinically or laboratory-supported definite MS according to It was interesting to compare some different indexes for CSF and sera of MS patients. Therefore, first we measured a relative concentration of total protein in CSF and sera of MS patients . The relIgGs from the serum and CSF were purified by affinity chromatography on protein-G Sepharose and IgG-protein concentration was estimated in the peaks eluted from the sorbent by an acidic buffer (see below). The relative concentration of total IgGs determined by two different methods was the same within the experimental error. Interestingly, the concentration of total protein in CSF was 6.9-fold higher than total IgGs, while this difference in the case of the serum (5.4-fold) was by a factor of 1.3 lower.450 units counting on a cerebrospinal fluid without dilution; average value 0.22±0.008 A450 units), while in the case of the serum (dilution 1000-fold) it was significantly varied . Since the relative concentration of anti-DNA antibodies in CSF was relatively low, we have removed all Abs from CSFs by passage through two columns: protein G-Sepharose and protein A-Sepharose. CSFs passed through the columns and controls containing only a standard buffer demonstrated the same A450 values, which were ≤5% in comparison with those for untreated CSFs. Thus, we have shown that the relatively low A450 values corresponding to untreated CSFs are caused by anti-DNA antibodies in these CSFs. In addition, we have shown that DNA-hydrolyzing activity is an intrinsic property of IgGs from CSF (see below).We estimated the relative concentration of anti-DNA antibodies in CSF and serum. The relative concentration of anti-DNA antibodies in the CSF of all patients was measured after dilution of CSFs 2-fold; the values for all CSFs were comparable  of all DNA-hydrolyzing enzymes and Abs in the sera and CSF of MS patients. Preparations of total protein were capable to hydrolyze supercoiled plasmid DNA (scDNA), forming single breaks in one strand of supercoiled DNA (relaxed DNA) and then multiple breaks yielding linear DNA. Finally, they hydrolyzed DNA into short and medium-length oligonucleotides. However, after such a deep hydrolysis of DNA, it was very difficult to estimate the relative activity of these preparations. The RAs of total protein from sera and CSF varied remarkably from patient to patient. The relative specific DNase activity of total protein of the serum preparations was varied in the range 0.26–0.88 pmole DNA/1 mg of protein/1 h . It was surprising that the specific activity of the total protein of CSF reparations were 198-fold higher  than the serum ones . At the mix). The homogeneity of the 150 kDa IgGmix was confirmed by SDS-PAGE, which showed a single band before, and two bands corresponding to the H and L chains after Ab reduction with DTT (silver staining)  did not lead to the disappearance of Ab activity and the peak of activity tracked exactly with 150 kDa Abs  . To proveria mix ; b) gel- kDa Abs ; c) comp kDa Abs .mix using an in situ assay in SDS-PAGE gels containing DNA. After incubation, in order to allow DNase renaturation and staining with ethidium bromide, a sharp dark band at the position of DNA hydrolyzing proteins was revealed on a fluorescent background of DNA-bound ethidium bromide  but in none of 50 healthy donors We compared the RAs in the hydrolysis of DNA of IgG preparations from sera and CSFs of thirteen MS patients. The RAs of IgGs from sera and CSF varied markedly from patient to patient. It was surprising, but CSF IgGs possess significantly higher specific DNase activity than serum ones. Average specific DNase activity of serum IgGs  is 48.5-fold lower than that for CSF Abs  . At the There are no published data concerning CSF abzymes with any catalytic activities. Data reported in this paper provide strong evidence that DNase activity is an intrinsic property of IgGs present in CSF of MS patients: it is not due to copurifying enzymes. The presence of anti-DNA Abs and DNase IgGs in CSF of MS patients is in agreement with the detection of B-cells producing anti-DNA Abs directly in active plaques and periplaque regions in MS brain and cerebrospinal fluid of a patient with MS Overall, abzymes of MS patients may be significantly more active in the hydrolysis of DNA than what we have found . As it wAt the same time, an ever-growing number of observations suggest that autoimmune diseases originate from defects in hematopoietic stem cells We have estimated CCs between different characteristics of CSF and sera. It was shown that there was no good correlation between several identical indexes characterizing CSFs and sera of MS patients, CCs varying in the range of −0.12 to +0.57  and 3. TAn analysis of correlation between titers of Abs to DNA as well as to MBP and 13 different standard clinical parameters including Poser criteria  functions and sum of these characteristics) in the case of 49 patients with MS was carried out The groups of primary progressing remitting course and secondary progressing course of MS patients were not “homogenous” with respect to the patients’ characteristics, and their further subdivision using cluster and factorial analysis revealed high statistically significant correlation coefficients • radicals and osteopathin The absence of a definite dependence between titers of anti-DNA and anti-MBP Abs and these parameters with standard clinical indices may be due to several reasons. MS is an extremely multifactorial disease, in which similar pathomorphological and clinical indices manifested as MS may result from very different underlying processes and conditions Overall, all data obtained demonstrate that the DNase activity is an intrinsic property of IgGs deriving from CSF and sera of MS patients. These IgGs are polyclonal and may consist of extremely different repertoires of DNase subfractions in the case of CSF and sera. We have shown previously that the appearance of abzymes specifically hydrolyzing DNA is among the earliest and clear signs of autoimmune reactions in a number of autoimmune diseases when titres of Abs to DNA or other auto-antigens have not yet increased significantly and correspond to their ranges for healthy donors Most chemicals, proteins, Protein G-Sepharose, and the Superdex 200 HR 10/30 column were from Sigma or GE Healthcare. Fifteen consecutive MS patients  satisfying the criteria for definite MS according to the classification of McDonald At entry none of the patients had fever or other symptoms or signs of acute infections. Moreover, at the time of sample collection none of the patients had received any potential disease-modifying therapies during the 6 months before the study.The blood and CSF sampling protocols confirmed the local committee for medical ethics in research (Comitato Etico della Provincia di Ferrara) that approved our study in accordance with Helsinki ethics committee guidelines including written consent of patients confined to present of their blood and CSF for diagnostics of a possible disease and scientific purposes. The protocol was approved at 31 May 2007 and it was focused on the creation of a biological bank of CSF and serum samples, and related clinical data of patients with MS and other neurological diseases including: a) a study of potential markers  for diagnostic and prognostic significance in diseases of the nervous system; b) specific antibodies directed against antigens potential exogenous and/or endogenous; c) presence of pathogens (mostly viruses or bacteria) for association studies and pathogenesis; d) neurotransmitters and their metabolites; e) a study of different properties of different markers.CSF and serum samples were collected under sterile conditions and stored in aliquots at 80°C until assay. “Cell-free” CSF samples were obtained after centrifugation at room temperature of specimens taken by atraumatic lumbar puncture performed for purposes of diagnosis in the absence of contraindications. Serum samples derived from centrifugation of blood specimens withdrawn by puncture of an anterocubital vein at the same time of CSF extraction. Paired CSF and serum samples from MS patients were stored and measured under exactly the same conditions. Informed consent was given by all patients before inclusion and the study design was approved by the Regional Committee for Medical Ethics in Research. CSF and serum IgG levels were measured by immunochemical nephelometry with the Beckman Immage 800 Immunochemistry System  according to the procedure of Salden et al. In all cases, protein concentration in the intact CSF, sera of MS patients and final solutions of Abs was measured using Bradford assay with a bovine serum albumin standard. The concentration of IgGs after their purification by affinity chromatography on protein G-Sepharose (see below) was measured in the same way.Relative concentrations of IgGs in the intact CSF and the sera of MS patients were analyzed using special quantitative isoelectrofocusing and immunoblotting test system in Italy according to the standard manufacturer’s protocol and equipment . In addition, the relative concentrations of IgGs in the intact CSFs and the sera of MS patients were measured after Abs purification by affinity chromatography on protein G-Sepharose (see below).450) of the solutions was determined using a Uniskan II plate reader . The relative concentration of anti-DNA Abs in the samples was expressed as the difference in the relative absorbance at 450 nm (average of three measurements) between the experimental samples and the control samples containing no Abs. As additional controls, we have used preparations complete devoid of Abs after passage of CSFs through protein G-Sepharose and protein A-Sepharose. There was no difference in the relative absorbance at 450 nm of CSF preparation containing no Abs and controls containing a buffer only. Finally, A450 values were recalculated on respective biological fluids without dilution.The titers of anti-DNA Abs were determined using standard assay plates with immobilized double-stranded DNA, horseradish peroxidase-conjugated mouse Abs against human IgG, and tetraethyl benzidine as substrate according to the standard manufacturer’s protocol . The preparations of human blood serum and CSF were diluted respectively 1000 and 2 times and 100 µl of final solution was added to the strips. The reaction was stopped with sulphuric acid and optical density  containing 0.2 M NaCl for 20 min at 25°C. Separation of the IgGs under “acid shock” conditions was done by FPLC gel filtration on a Superdex 200 HR 10/30 column equilibrated with 50 mM glycine-HCl (pH 2.6) containing 0.1 M NaCl as previously described In some cases, electrophoretically homogeneous IgGs were chromatographed on Sepharose bearing immobilized monoclonal mouse Abs against light chains of human IgGs as in 2, 1 mM EDTA, 20 mM Tris-HCl (pH 7.5), and 0.003–0.2 mg/ml Abs or initial preparations of the sera or CSF  finally diluted respectively 1000- and 15-fold. Reaction mixtures were incubated for 0.5–3 h  at 37°C. The cleavage products were analyzed by electrophoresis in 1% agarose gel. The images of ethidium bromide-stained gels were captured on a Sony DSC-F717 camera and a relative amount of DNA in different bands was analyzed using ImageQuant v5.2 (Molecular Dynamics). The activities of IgG preparations were determined as a decrease in the percentage of DNA converted from the initial supercoiled form to the relaxed form, corrected for the distribution of DNA between these bands in the control (incubation of pBluescript in the absence of Abs). All measurements  were taken within the linear regions of the time courses (15–40% of DNA hydrolysis) and then recalculated to the standard conditions (see Tables).DNA-hydrolyzing activity of total protein and IgG preparations was analyzed using supercoiled (sc)DNA as earlier described for the analysis of DNase I and human serum catalytic antibodies SDS-PAGE analysis of Abs for homogeneity and for the polypeptide spectrum of the sera and CSF was performed in a 5–16% gradient gel containing 0.1% SDS (Laemmli system) as described in In situ, experiments DNase activity of IgGs after SDS-PAGE was analyzed in gel containing calf thymus DNA (5 µg/ml) under non-reducing conditions as in 2, and 0.2 mM CaCl2. To visualize the products of DNA hydrolysis, the gel was stained with ethidium bromide. The same ethidium bromide-stained or parallel longitudinal slices were used to detect the position of IgG in the gel by Coomassie Blue staining.The results are reported as mean ± S.E. of at least three independent experiments for each sample analyzed. Errors in the values were within 5–7%. The correlation coefficients (CC) between sets of different samples were analyzed.


Anti-NMDA-encephalitis is caused by antibodies against the N-methyl-D-aspartate receptor (NMDAR) and characterized by a severe encephalopathy with psychosis, epileptic seizures and autonomic disturbances. It predominantly occurs in young women and is associated in 59% with an ovarian teratoma.We describe effects of cerebrospinal fluid (CSF) from an anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis patient on in vitro neuronal network activity (ivNNA). In vitro NNA of dissociated primary rat cortical populations was recorded by the microelectrode array (MEA) system.The 23-year old patient was severely affected but showed an excellent recovery following multimodal immunomodulatory therapy and removal of an ovarian teratoma. Patient CSF (pCSF) taken during the initial weeks after disease onset suppressed global spike- and burst rates of ivNNA in contrast to pCSF sampled after clinical recovery and decrease of NMDAR antibody titers. The synchrony of pCSF-affected ivNNA remained unaltered during the course of the disease.Patient CSF directly suppresses global activity of neuronal networks recorded by the MEA system. In contrast, pCSF did not regulate the synchrony of ivNNA suggesting that NMDAR antibodies selectively regulate distinct parameters of ivNNA while sparing their functional connectivity. Thus, assessing ivNNA could represent a new technique to evaluate functional consequences of autoimmune encephalitis-related CSF changes. Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a recently described disorder characterized by psychosis, epileptic seizures, inflammatory cells in cerebrospinal fluid (CSF) and NMDAR-binding antibodies detectable in serum and CSF. InitialThe diagnostically defining anti-NMDAR antibodies bind to the surface of CNS neurons. It was shown that the binding epitope is part of the NR1-subunit of the NMDAR on postsynaptic dendrites in the forebrain and hippocampus. After cHere, we report that CSF from a young woman with anti-NMDAR encephalitis suppressed in vitro-neuronal network activity in the acute stage of the disease and that this effect was reversible when the patient recovered upon removal of an ovarian teratoma and subsequent immunosuppression. Network suppression became manifest in significantly reduced spike and burst rates while network synchrony was preserved. Interestingly, this reaction pattern is also observed when functional neuronal networks are exposed to a specific NMDA receptor antagonist.In April 2010, a previously healthy 23-year old woman was admitted to our hospital because of a first generalized epileptic seizure. The family of the patient reported about a slight personality change with inappropriate distrustful and jealous behavior that had occurred in previous 4 weeks before admission. Neurological examination was unremarkable and there were no signs of a marked neuropsychiatric or mental disorder. Cerebral magnetic resonance imaging (MRI) was normal. CSF examination revealed a pleocytosis of 72 white blood cells (WBC) /μl, an intrathecal synthesis of immunglobulin M and positive oligoclonal bands (OCBs). All polymerase chain reaction (PCR) -tests against neurotropic viruses including herpes simplex virus 1+2, cytomegaly, Epstein-Barr virus, human herpes virus 6, varicella zoster virus, adenovirus, JC virus, and coxsackie virus remained negative. The electroencephalogram (EEG) showed bi-frontal slowing but no epileptic discharges. During the following 12 days the patient progressively developed a dysexecutive syndrome and exhibited a bizarre and disorganized behavior despite treatment with high doses of valproate (1200 mg/day) and lorazepam (2 mg/day)  and a reduction of the anti-NMDAR antibody titer in serum to 1:320 and in CSF to 1:100.At follow-up investigation three months later, the patient did not present any neurological or psychiatric abnormalities. Neuropsychological examination revealed a significant improvement Figure. CSF parAnti-NMDAR antibodies in both, CSF and serum, were measured by antigen-antibody-reaction with indirect immunofluorescence on NR1-transfected Human Embryonic Kidney cells (HEK cells) as well PCR-tests for neurotropic viruses were performed with the standard technique “TaqMan realtime” and revealed no presence of herpes simplex virus 1+2, cytomegalovirus, Epstein-Barr virus, human herpes virus 6, varicella zoster virus, adenovirus, JC virus, and coxsackie virus in the cerebrospinal fluid.3/cm2 onto poly-D-lysine- and laminin-coated MEAs and cultured in a humidified atmosphere (5% CO2 / 95% air) at 37°C. Medium was replaced twice a week. After approximately 3 weeks in vitro, cultures developed a dense synaptically connected neuronal network and reached stable electrophysiological properties including population bursting. Functional neuronal networks were used for recordings when they reached a minimum bursting activity of 20 bursts per minute. MEA experiments were performed between 26 and 64 days in vitro (see below), a time window of preserved synchronous population bursting. Earlier immunocytochemical experiments of identical cell preparations[Cortical cells were freshly prepared from embryonic day 18 Wistar rats. Cell preparation and animal care were performed in compliance with the German Animal Protection law . Permission/Ethic approval was granted by the Institutional Review Board of Düsseldorf University (O/5/2009). Dissociated cortical cells were isolated and collected in ice cold N2 medium  and trypsinized for 10 minutes. After dual centrifugation for 1 minute at 2000 and 5 minutes for 1500 repeats per minute, cells were resuspended in N2-supplemented Neurobasal medium (Invitrogen) containing 5% fetal calf serum  and plated at a density of 150 × 10arations demonstr2, 3 mM KCl, 1 mM MgCl2, 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 10 mM glucose, pH 7.4; Sigma, St. Louis, MO). Network activity was then recorded for the next 15 minutes and averaged for one data point. Then, aCSF was replaced against pure pCSF or cCSF and network activity was again measured for 15 minutes and averaged. The pH was adjusted (HEPES buffer) to 7.4 in all solutions  prior to every experiment and remained constant (+/− pH 0.1) for at least 30 minutes outside the incubator. In FigureTo investigate the potential effect of the patients´ CSF (pCSF) on electrophysiological neuronal network parameters like global network activity and the synchrony of population bursts, pure pCSF of 3 different time points during the course of the disease was administered to at least 4 independent microelectrode arrays , respectively. Control experiments with independent MEAs (n=4) were performed using CSF of a healthy person (cCSF). In detail, for MEA experiments that were all continuously performed outside the incubator for 30–40 minutes, cell culture medium was first replaced against aCSF  were used. Signals from all 60 electrodes were simultaneously sampled at 25 kHz, visualized and stored using the standard software MC_Rack provided by Multi Channel Systems. Spike and burst detection was performed off-line by custom-built software . Details of data collection are given elsewhere. TemporaPatient CSF taken 4 days and 5 weeks after admission (both with NMDAR antibody titers of 1:100) induced a significant suppression of global activity markers (spike rate and burst rate) in spontaneously bursting in vitro-cortical neuronal networks in comparison to CSF from a healthy control person (FigureWe present a case with severe paraneoplastic anti-NMDAR encephalitis and complete recovery after surgical removal of the ovarian teratoma and multimodal immunomodulatory therapy. High-dose methylprednisolone treatment, plasma exchange and administration of immunoglobulins rapidly reversed the downhill course and allowed the patient to recover. Most likely, B-cell depletion with rituximab sustained the subsequent complete recovery. Following the complete clinical recovery, OCBs were no longer detectable. Disappearance of OCBs from the CSF after treatment is highly unusual. As NMDAR antibody titers, also at the late time point, were still measurable, it is unlikely that OCBs represented a specific anti-NMDAR immune response.It was previously found that NMDAR auto-antibodies lead to receptor internalization and, consecutively, a hypo-functional state including decreased NMDAR currents. Here, wThe pCSF-related effects observed in this study resolved in parallel to clinical improvement and a reduction of the antibody titer to 1:10. We could also demonstrate a fast suppressive effect of pCSF on network activity within the first 15 minutes after pCSF application in comparison to aCSF suggesting that next to NMDAR capping and internalization, pCSF might additionally induce a rapidly evolving dysfunction of neuronal network activity. The reversibility and functional dynamics of neuronal network activity under the influence of pCSF shown here and the course of anti-NMDAR antibody titers in our patient strengthen the notion that anti-NMDAR antibodies mediate this neuropsychiatric syndrome. However, it remains to be elucidated if antibodies can indeed exert such a fast functional effect on NMDA receptors as antibody binding usually needs some time. Our report has several shortcomings: First, we only investigated one pCSF. Second, as a variety of different drugs were administered in the course of the disease, additional drug-related effects cannot be completely excluded. However, as benzodiazepines were administered only during the second collection of lumbar CSF, we consider benzodiazepine effects on neuronal network activity unlikely. Third, we were not able to directly attribute the observed effects to NMDAR autoantibodies as the limited volume of the CSF probes rendered further pCSF-based investigations impossible. Thus, further experiments with a higher number and larger volume of CSF probes from different NDMAR encephalitis patients are needed.We described a method to assess functional consequences of pathologically altered CSF specimens from a NMDA receptor encephalitis patient. As new limbic encephalitis antibodies against surface epitops are continuously discovered newly, this method can serve to investigate CSF specimens from so far antibody-negative limbic encephalitis patients.AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; aCSF: artificial cerebrospinal fluid; CSF: cerebrospinal fluid; EEG: electroencephalography; GABA: gamma-aminobutyric acid; HEK cells: human embryonic kidney cells; ivNNA: in vitro neuronal network activity; MEA: microelectrode array; mEPSCs: miniature excitatory post synaptic currents; MRI: magnetic resonance imaging; NMDA: N-methyl-D-aspartate; NMDAR: N-methyl-D-aspartate-receptor; OCB: oligoclonal bands; PCR: polymerase chain reaction; pCSF: patient´s cerebrospinal fluid; WBC: white blood cells.SUJ, DS and RJS were responsible for the clinical course of the patient. MD and SU.J wrote the manuscript. CW was responsible for the neuropsychological examinations and corresponding parts in the manuscript. SF, KQ, SI and MD are responsible for the examination of the CSF by the MEA technology. H-PH, RS and MD revised the manuscript for intellectual contents. All authors read and approved the final manuscript.


Chiropractors commonly provide care to people with acute low-back pain (LBP). The aim of this survey was to determine how chiropractors intend to support and manage people with acute LBP and if this management is in accordance with two recommendations from an Australian evidence-based acute LBP guideline. The two recommendations were directed at minimising the use of plain x-ray and encouraging the patient to stay active.This is a cross sectional survey of chiropractors in Australia. This paper is part of the ALIGN study in which a targeted implementation strategy was developed to improve the management of acute LBP in a chiropractic setting. This implementation strategy was subsequently tested in a cluster randomised controlled trial. In this survey phase of the ALIGN study we approached a random sample of 880 chiropractors in three States of Australia. The mailed questionnaire consisted of five patient vignettes designed to represent people who would typically present to chiropractors with acute LBP. Four vignettes represented people who, according to the guideline, would not require a plain lumbar x-ray, and one vignette represented a person with a suspected vertebral fracture. Respondents were asked, for each vignette, to indicate which investigation(s) they would order, and which intervention(s) they would recommend or undertake.Of the 880 chiropractors approached, 137 were deemed ineligible to participate, mostly because they were not currently practising, or mail was returned to sender. We received completed questionnaires from 274 chiropractors (response rate of 37%). Male chiropractors made up 66% of respondents, 75% practised in an urban location and their mean number of years in practice was 15. Across the four vignettes where an x-ray was not indicated 68% : 64%, 71%) of chiropractors responded that they would order or take an x-ray. In addition 51%  indicated they would give advice to stay active when it was indicated. For the vignette where a fracture was suspected, 95%  of chiropractors would order an x-ray.The intention of chiropractors surveyed in this study shows low adherence to two recommendations from an evidence-based guideline for acute LBP. Quality of care for these patients could be improved through effective implementation of evidence-based guidelines. Further research to find cost-effective methods to increase implementation is warranted. Low back pain (LBP) is a common and costly problem in high income countries like Australia. At any one time, approximately one in five Australians has LBP, and four out of five Australians will experience it at some time in their lives . The dirIn 2004, an evidence-based clinical practice guideline for acute LBP ,6, and aThis paper reports on one phase of the ALIGN study. The ALIGN study explored barriers and enablers to the uptake of the Australian acute LBP guideline in order to develop a targeted implementation strategy for use in a chiropractic setting which was subsequently tested in a cluster randomised controlled trial . Other aThis study is a cross sectional survey of chiropractors in Australia.The sample size was calculated to address the primary aim of the survey which was to identify factors (e.g. knowledge) that were predictive of intention to perform a particular behaviour, and then to use this information to develop the ALIGN implementation strategy. The sample size was calculated to detect a 0.5 difference in intention to perform a particular behaviour, measured on a 7-point Likert scale, between dichotomised factors , with 90% power. To detect this difference, a sample of 440 chiropractors was required, assuming a 5% significance level and standard deviation of 1.6  in the study (and not be contacted further about this study). It was therefore considered that a return mail card, as per the Total Design Method, was not necessary in this study.Double data entry was performed independently by two researchers (MP and KB) and discrepancies were resolved via discussion with a third researcher.Descriptive statistics were used to describe demographic details of the chiropractic respondents. The percentage of chiropractic respondents indicating which investigation(s) and which intervention(s) they would undertake for each vignette was calculated. Confidence intervals were calculated adjusting for the stratification variable. Exact binomial confidence intervals were calculated when the normal approximation was not valid . ConfideHuman research ethics approval for this study was granted by Monash University (CF07/1060 - 2007000274).The survey was administered in April 2009. One-hundred and thirty seven 16%) chiropractors approached were deemed ineligible to participate, mostly because they were not currently practising, or mail was returned to sender. We received completed questionnaires from 274 of 743 eligible chiropractors, resulting in a response rate of 37%. Male chiropractors made up 66% of respondents, 75% practised in an urban location and their mean number of years in practice was 15  of chiropractors responded that they would order or take an x-ray, and 51%  indicated they would give advice to stay active. For the vignette where a fracture was suspected, 95%  of chiropractors would order an x-ray Table . In addiRegarding treatment and management options that the chiropractors would employ 76%  of chiropractors indicated that they would perform spinal manipulation/adjustment for vignettes 1 to 4 range 63% to 92%), and 51%  of chiropractors indicated they would give advice to stay active when it was indicated. Other treatments and care that chiropractors indicated they would provide for each vignette are detailed in Table % to 92%,The aim of this survey was to determine if management intended by chiropractors is in accordance with two key recommendations in the Australian evidence-based acute LBP guideline  and moreWhere an x-ray was not recommended according to the guideline 68% of chiropractors responded that they would order or take an x-ray, indicating an overall x-ray guideline adherence of 32%. It has been reported previously that chiropractors use plain x-rays at a high frequency for people with acute non-specific LBP, with rates varying from 12% to 63% -22. Our Across all five vignettes approximately 25%  of chiropractors would take, or order, a full spine plain x-ray. This was despite the lack of information in the vignettes about any symptoms or examination findings at the other regions of the spine. There is no evidence that full spine x-rays are warranted in these vignettes and this finding is troubling considering the radiation dose that the patient would receive ,25.Around one third of chiropractors surveyed  would perform spinal manipulation of the spine in a patient with a likely fracture. We did not investigate the reasons for this, however it may be that a) the potential for fracture diagnosis in the vignette was not considered, or b) the chiropractor intended to apply manipulation to regions above or below the fracture site.The findings above have implications for patients' safety and health resource use. First, a full spine x-ray will expose the patient to needless ionising radiation, and manipulation of a spinal fracture may worsen the fracture with potentially serious consequences. Second, the findings have implications for third party payments for these extensive and expensive diagnostic imaging.Over half the chiropractors  indicated that in the first four vignettes they would advise the patient to stay active, while 60%  responded they would give exercises  to people seen who were similar to the first four vignettes. In this sample of chiropractors, there appears to be a need to encourage the giving of advice to patients with acute LBP to stay active, regardless of any exercises they would recommend to the patient in order to bring chiropractic management in line with evidence-based practice.The response rate was low (37%) and therefore the results have an increased potential for selection bias where those who choose to participate differ in important characteristics with those who do not choose to participate . It may Patient vignettes are limited by a lack of detail that may be gleaned during a patient consultation, including visual cues relating to pain during an examination. Further, patient vignettes may not be sensitive enough to pick up the fine nuances of practice that would better guide the practitioner . TherefoThe current intention of a significant number of Australian chiropractors who responded to this survey and who manage people with acute LBP is not in accordance with evidence-based recommendations. Quality of care for these patients could potentially be improved through effective implementation of evidence-based guidelines. Further research to find cost-effective methods to increase implementation is warranted.Bruce Walker is Editor in Chief and Simon French is an Associate Editor of Chiropractic & Manual Therapies. All decisions on this manuscript were made by another editor. All other authors declare that they have no competing interests.All authors contributed to the design and conduct of the study. SF wrote the first draft of this manuscript with comments from BW. JM undertook the statistical analysis. BW undertook further revisions of the manuscript with feedback from all authors. All authors agreed to the final manuscript.Patient vignettes. This file contains the five patient vignettes that were used in the survey.Click here for fileInvestigations and intervention options for the five patient vignettes. This file shows the options available to the survey respondents for investigations and interventions for each of the patient vignettes.Click here for file


The recombinant expression vector was transformed into Pichia pastoris GS115, and the recombinant strains were screened by SDS-PAGE. As expected, the 3DHSA-G-CSF showed high binding affinity with HSA antibody and G-CSF antibody, and the natural N-terminal of 3DHSA was detected by N-terminal sequencing. The bioactivity and pharmacokinetic studies of 3DHSA-G-CSF were respectively determined using neutropenia model mice and human G-CSF ELISA kit. The results demonstrated that 3DHSA-G-CSF has the ability to increase the peripheral white blood cell (WBC) counts of neutropenia model mice, and the half-life of 3DHSA-G-CSF is longer than that of native G-CSF. In conclusion, 3DHSA can be used to extend the half-life of G-CSF.Protein fusion technology is one of the most commonly used methods to extend the half-life of therapeutic proteins. In this study, in order to prolong the half-life of Granulocyte colony stimulating factor (G-CSF), the domain III of human serum albumin (3DHSA) was genetically fused to the N-terminal of G-CSF. The 3DHSA-G-CSF fusion gene was cloned into  CHU-2 cells and showed specific activity on stimulation of mainly granulocyte colony formation [Human Granulocyte colony stimulating factor (G-CSF) gene was first reported in 1986, and G-CSF is a 174 amino acid glycoprotein belonging to the family of hematopoietic cytokine [ In clinical practice, recombinant human G-CSF has been widely used for neutropenia caused by chemotherapy and radiotherapy . RecentlP. pastoris [Several approaches have been used to extend the half-life of drugs, and protein fusion technology is one of the most commonly used methods to prolong the half-life of protein and peptide drugs. Based on the development of molecular biology and genetic engineering, some natural proteins with long half-life have been used as fusion partners to enhance the circulating half-life of drugs, such as IgG-Fc, transferrin, and human serum albumin (HSA) –11. Therpastoris . Many efpastoris . In genepastoris . Furtherpastoris . So 3DHSpastoris . HoweverP. pastoris GS115. To our knowledge, this is the first report of 3DHSA-G-CSF expression in any expression system. The biological activity and pharmacokinetic property of the purified fusion protein were also studied utilizing neutropenia model mice and normal mice, and the results indicated that 3DHSA-G-CSF may be considered as a drug candidate of neutropenia for further research and development.In this study, in order to prolong the half-life of G-CSF, we genetically fused it with 3DHSA and then successfully expressed the 3DHSA-G-CSF fusion protein in E. coli DH5α, P. pastoris GS115, and pPICZαA vector were purchased from Invitrogen. Both HSA and G-CSF genes were synthesized by Invitrogen . Male 7~8-week-old BALB/c and ICR mice were purchased from the Comparative Medical Center of Yangzhou University . The animals used in this study were under a 12 h light-dark cycle, and the environmental temperature was maintained at 25°C. Animal experiments in this study were performed in compliance with the Guidelines for Animal Experimentation issued by the Chinese Association for Laboratory Animals Science and the Standards Relating to the Care and Management of Experimental Animals.CTCGAGAAGAGAGTGGAAGAGCCTCAGAATTT-AAT-3′  and (P2) 5′-CCAGCGGGGTCAAACCCAAAGCAGCTTGAG-3′. G-CSF gene was used as template for cloning H10-G-CSF with (P3) 5′-TTTGGGTTTGACCCCGCTGGGACCGGCAAG-3′ and (P4) 5′-CGTCTCGAGTTAGGGCTGGGCAAGGTGGC-3′. The C-terminal of the 3DHSA-G10 sequence was genetically fused with the H10-G-CSF to construct 3DHSA-G-CSF fusion gene by overlap PCR with (P1) and (P4). The fusion gene was inserted into the XhoI site of pPICZαA along with the open reading frame of the α-factor signal under the control of the AOX1 promoter. The KEX2 cleavage site, located between the signal peptide and the 3DHSA-G-CSF sequences, enables the release of mature 3DHSA-G-CSF with a predicted molecular weight of 42 kD. The constructed recombinant plasmid named pPIC-3DHSA-G-CSF was confirmed by PCR, single restriction, and nucleotide sequencing.HSA gene was used as template for cloning 3DHSA-G10 with the following primers: (P1) 5′-TCTSal I and transformed into competent P. pastoris host strain GS115 by electroporation as Wu and Letchworth reported [Pichia Expression Kit . At first, protein samples were checked by 12% SDS-PAGE to screen the recombinant strains, and then the sample of highest expression was transferred to nitrocellulose (NC) filter membranes. The Western blotting was carried out with mouse monoclonal antibody, respectively, against human G-CSF and HSA as primary antibodies and horseradish peroxidase-conjugated goat anti-mouse Ig antibody as secondary antibody for detection of the expressed fusion protein.The positive recombinant expression vector was linearized with reported . The bacThe recombinant strain was cultured in BMGY medium, and methanol was added every 24 h at the final concentration of 1% (v/v) to induce 3DHSA-G-CSF secreted into BMMY medium at 30°C. 3DHSA-G-CSF was purified by Cibacron Blue F3G-A Sepharose and Butyl-Sepharose 4B , and the4HCO3). A MALDI TOF MS spectrometer was used to record and identify the intense peaks. Submit the peptide peak lists to Mascot.3DHSA-G-CSF was loaded on SDS-PAGE and stained with Coomassie Blue R250. Then the target bands were excised from the gels and digested by Trypsin  membrane (Millipore) and subsequently stained with ponceau stain solution. Compared with the prestained protein marker, the corresponding PVDF bands were cut and sequenced using Edman degradation method to analyze the first 10 amino acids of N-terminal.The CD spectra of 3DHSA-G-CSF with the concentration of 0.2 mg/mL in 5 mM sodium acetate buffer (pH 5.0) were detected by a Jasco-720 spectropolarimeter over a range of wavelength of 190–250 nm using a 0.2 cm cell.As G-CSF is a hematopoietic cytokine that acts on neutrophil lineage cells and activates mature neutrophils, recombinant G-CSF has been widely used for adjuvant chemotherapy. The biological activity of 3DHSA-G-CSF can be evaluated by determining G-CSF activity of increasing WBC counts of neutropenia murine which was induced by subjecting with cyclophosphamide (CTX) which is a commonly used drug for chemotherapy. Thirty BALB/c mice were intraperitoneally injected with CTX at a single dose of 200 mg/kg to induce neutropenia . The micA preliminary pharmacokinetic study was performed using male ICR mice (7~8 weeks old). Sixty mice were randomly divided into two groups, which were, respectively, injected subcutaneously (S.C) with 1 mg/kg of G-CSF and 2.33 mg/kg of 3DHSA-G-CSF . For G-CSF group, blood was collected at 0.25, 0.5, 1, 2, 4, 8, 12, 16, and 24 h after injection, and, for 3DHSA-G-CSF group, blood was collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 36 h after injection. Three mice of the two groups were sacrificed at each time point. Blood samples were centrifuged at 3000 rpm for 15 min to obtain serum and then stored at −80°C until analysis. The serum concentrations of G-CSF and 3DHSA-G-CSF were determined by human G-CSF ELISA kit . Plasma concentration data was analyzed with PKSolver .pPICZαA to generate a recombinant plasmid pPIC-3DHSA-G-CSF. Details of the plasmid pPIC-3DHSA-G-CSF and the cloning sites are schematically shown in The 547 bp and 647 bp fragments corresponding to 3DHSA-G10 and H10-G-CSF were amplified by RT-PCR as described above. The full length 3DHSA-G-CSF gene of 1164 bp in size was obtained through overlap PCR, and the purified fusion protein gene was inserted into the expression vector The positive strains expressing fusion protein 3DHSA-G-CSF were screened by 12% SDS-PAGE. As shown in After optimization of the culture conditions, the secretion level of 3DHSA-G-CSF in the broth was 199.5 mg/L. 3DHSA-G-CSF was purified by the combination of affinity chromatography (Cibacron Blue Sepharose Fast Flow column) and hydrophobic chromatography (Butyl Sepharose 4B Fast Flow column), and the products were analyzed by SDS-PAGE. A clear single target band for the fusion protein was shown in The peptide mass mapping was adopted to confirm the primary structure of 3DHSA-G-CSF, and the result showed that the peptide fragments were consistent with the theoretical protein sequence, such as KVPQVSTPTLVEVSR and AGGVLVASHLQSFLEVSYR . MeanwhiThe secondary structure of 3DHSA-G-CSF was evaluated by circular dichroism, and the waveform of 3DHSA-G-CSF was shown in P < 0.01).Neutropenia model mice were injected with native G-CSF and 3DHSA-G-CSF as described in P < 0.01). The data indicated that 3DHSA could be used to extend the half-life of G-CSF.Plasma concentration data of G-CSF and 3DHSA-G-CSF were shown in pPIC-3DHSA-G-CSF expression vector, and the 3DHSA-G-CSF fusion protein was expressed in P. pastoris system. P. pastoris has been developed as an attractive expression platform for heterologous protein production as it grows rapidly and has the ability to accomplish some complex posttranslational modification, such as protein glycosylation, processing, and correct folding. What is more, the very low amount of endogenous proteins secreted by P. pastoris represents one of the major advantages of this expression system and serves as the first purification step [P. pastoris and G-CSF/IgG-Fc fusion protein in COS-1 cells, 3DHSA-G-CSF was successfully secreted into the supernatant and effectively avoided soluble aggregation product during the fermentation process [E. coli [In the present study, protein fusion technology was adopted to prolong the half-life of G-CSF. 3DHSA was fused with G-CSF to construct recombinant ion step . Compare process , 22. In  process , 24. Mor process . This ma process . The yie[E. coli , 28. Thi[E. coli . in vivo activity of 3DHSA-G-CSF using the neutropenia model mice. We observed that both G-CSF and 3DHSA-G-CSF could significantly increase the WBC counts in neutropenia murine model. The result of 3DHSA-G-CSF demonstrated that 3DHSA as a fusion partner did not change the bioactivity of G-CSF and also gave an important evidence for characterization of correct structure of 3DHSA-G-CSF. We next determined the preliminary pharmacokinetic of 3DHSA-G-CSF and G-CSF using normal mice. The half-life of G-CSF is about 2.1 h, while the half-life of 3DHSA-G-CSF is about 3.4 h. We can find that the half-life of G-CSF was prolonged by fusing with 3DHSA. The result also well agrees with the report that 3DHSA is necessary and sufficient for the long serum persistence of albumin [In order to confirm 3DHSA-G-CSF fusion protein keeps the bioactivity of G-CSF, we determined the  albumin , 30. We  albumin . From thP. pastoris is an efficient host for 3DHSA-G-CSF expression. 3DHSA-G-CSF is uniform chemical entity which comprising 3DHSA fused to G-CSF, so it may be simpler to manufacture and applied for both research and industrial purpose. Further studies showed that the 3DHSA-G-CSF retained the bioactivity of G-CSF, and its half-life was longer than G-CSF. These data indicated that 3DHSA has the promise to extend the half-life of G-CSF. More importantly, this study provides an experimental evidence for 3DHSA to be applied as a fusion partner to extend the half-life of some other recombinant peptides and proteins in further research.In conclusion, we successfully expressed 3DHSA-G-CSF fusion protein for the first time. The strategy established above suggested that


Introduction. Donor workup in renal transplantation is extensive. Despite this, chyle leakage following donor nephrectomy, a rare complication, has been reported in the literature. We encountered two cases of chyle leak in kidney donors in our series of open donor nephrectomies. Summary of Cases. After complete workup, standard open retroperitoneal donor nephrectomy with drain placement was performed in 684 living renal donors. We encountered chyle leak in two cases. The first case was a 33-year-old female who underwent an otherwise uneventful left donor nephrectomy but continued to have high drain output (upto 300–400 mL/24 hrs) in the postoperative period. The drain fluid was milky, raising the suspicion of chyle which was confirmed on biochemical analysis. The second case was a 42-year-old female with a similar case history. Both were managed conservatively with low-fat diet. The leak subsided spontaneously in three weeks and one week in the first and second patients, respectively. The drain was removed, and the patients remained symptom-free on followup. Conclusions. Both of our cases of chyle leak following open donor nephrectomy were managed successfully with conservative management. The management options and the experience of other centers are reviewed and discussed. Donor workup in renal transplantation is extensive with the aim of ensuring donor safety at all costs and at the same time providing an adequately functioning kidney to the recipient. However, complications may occur. One of them, although rare, is postoperative chyle leak. We reviewed our experience of 684 living renal donors since 1991 to date. They had undergone standard open retroperitoneal donor nephrectomy after complete preoperative workup. At the time of closure, a drain was placed in the renal fossa which was removed when the drainage became less than 50 mL/day. We encountered chyle leak in two cases.A 33-year-old female, resident of nonfilaria endemic area, was a voluntary kidney donor for her husband. She underwent an otherwise uneventful left retroperitoneal donor nephrectomy. After an initial fall, drain output started to increase and became milky after postoperative day 3, after commencement of oral feeds . SuspiciShe was managed aggressively and was started on TPN for a week. Drain output reduced, and subsequently oral feeds were restarted comprising of high-protein, low-fat (mainly medium-chain triglyceride) diet. Leak gradually subsided spontaneously in total of three weeks, and at that time the drain was removed . A 42-year-old female was with a similar case history as the first case. She also had undergone left open donor nephrectomy with an uneventful intraoperative period. She had milky fluid in drain after start of oral diet (max output: (200/day)). This patient was also managed conservatively with dietary management. Leak gradually subsided spontaneously in 7 days .Both patients remained symptom-free on followup. Incidentally, the recipients of both kidneys did well. After reviewing the literature for this complication, we found that we are first to report two cases of chyle leak following open donor nephrectomy. However, there are few reports of chylous ascites following laparoscopic donor nephrectomy  1]..1].Most reports listed the complication occurring after left laparoscopic donor nephrectomy. In our study also, both the cases had undergone left donor nephrectomy. The cause for this finding is not known. It may be incidental as the frequency of left donor nephrectomy is much greater than right donor nephrectomy or this complication is unique to left side. Further inquiries and investigative efforts are needed in this regard.Chyle leak has been reported after other abdominal surgeries also. Abdominal surgical procedures usually causing chyle leak include Abdominal aortic aneurysm repair , Retro-peritoneal lymph node dissection, Inferior vena caval resection & Liver transplant .The cause of chyle leak following donor nephrectomy is not exactly known. Chyle leak has been increasingly reported in the recent past, indicating the possible role of inadequate control of perivascular lymphatics during nephrectomy performed by minimally invasive techniques where energy sources like harmonic, LigaSure, and electrocautery are used. These modalities may not be as effective in control of lymphatics as suture ligation or clipping as suggested by Nishizawa et al. with respect to chylous ascites following laparoscopic radical nephrectomy .Treatment options in patients with postoperative chylous ascites primarily involve conservative management in the form of dietary modifications. Constant loss of protein and lymphocytes may cause nutritional and immunological disturbances. The major bulk of fat in diet should comprise of free fatty acids and medium chain triglycerides as these are absorbed directly from the gut into the portal venous circulation. Total parenteral nutrition (TPN) is required in cases who do not respond to dietary modifications. Essential fatty acids and fat soluble vitamins need to be supplemented. Somatostatin has been shown to be effective in one study . Other oChyle leak following donor nephrectomy may be prevented by meticulous surgical dissection and clipping/ligating all the lymphatic tissue around the renal vessels. It can be managed with conservative treatment in most of the cases.


Clerodendrum inerme (L.) Gaertn is a neuroprotective agent was investigated. The effect of acacetin on endogenous glutamate release in rat hippocampal nerve terminals (synaptosomes) was also investigated. The results indicated that acacetin inhibited depolarization-evoked glutamate release and cytosolic free Ca2+ concentration ([Ca2+]C) in the hippocampal nerve terminals. However, acacetin did not alter synaptosomal membrane potential. Furthermore, the inhibitory effect of acacetin on evoked glutamate release was prevented by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker known as ω-conotoxin MVIIC. In a kainic acid (KA) rat model, an animal model used for excitotoxic neurodegeneration experiments, acacetin (10 or 50 mg/kg) was administrated intraperitoneally to the rats 30 min before the KA (15 mg/kg) intraperitoneal injection, and subsequently induced the attenuation of KA-induced neuronal cell death and microglia activation in the CA3 region of the hippocampus. The present study demonstrates that the natural compound, acacetin, inhibits glutamate release from hippocampal synaptosomes by attenuating voltage-dependent Ca2+ entry and effectively prevents KA-induced in vivo excitotoxicity. Collectively, these data suggest that acacetin has the therapeutic potential for treating neurological diseases associated with excitotoxicity.An excessive release of glutamate is considered to be a molecular mechanism associated with several neurological diseases that causes neuronal damage. Therefore, searching for compounds that reduce glutamate neurotoxicity is necessary. In this study, the possibility that the natural flavone acacetin derived from the traditional Chinese medicine  For example, berberine (an active component of berberis), curcumin (an active component of turmeric), honokiol , and tanshinone IIA (an active component of danshen), were able to penetrate the brain and protect it against brain damage in various animal models with neurological disorders + influx, and the activation of the voltage-dependent Ca2+ channels (VDCCs). The isolated presynaptic terminals (synaptosomes) also represent a model for directly investigating the underlying molecular mechanisms of presynaptic phenomena. Specifically, synaptosomes are capable of accumulating, storing, and releasing neurotransmitters, and is devoid of functional glial and nerve cell body elements that might cause the findings to be misinterpreted because of modulatory loci at the non-neuronal, postsynaptic, or network levels Considering that the excitotoxicity caused by excessive glutamate is believed to play pivotal roles under numerous neuropathological conditions Clerodendrum inerme, as described previously Clerodendrum inerme (3.0 kg) were grinded and repeatedly extracted with 95% EtOH (10 L) for three times. The combined EtOH layers were concentrated in vacuo to give a residue (246 g). The residue was triturated with n-hexane, CH2Cl2, n-BuOH and H2O, respectively. CH2Cl2 layer was concentrated in vacuo to provide a residue (16 g) and then subjected to silica gel chromatography to yield fractions 1–9. One compound that was crystallized from fraction 7 using EtOH as a solvent was identified as acacetin based on NMR spectrum comparable to literature reported. The purity of the compound was more than 98% on high-performance liquid chromatography (HPLC). 3′, 3′, 3′-dipropylthiadicarbocyanine iodide [DiSC3(5)] and fura-2-acetoxymethyl ester (Fura-2-AM) were purchased from Invitrogen . 4-aminopyridine (4-AP), ω-conotoxin MVIIC (ω-CgTX MVIIC), dantrolene, 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157), kainic acid (KA), and all other reagents were purchased from Sigma-Aldrich . Acacetin, DiSC3(5), SBFI, and Fura-2 were dissolved in 0.1% dimethylsulfoxide (DMSO). 4-AP, ω-CgTX MVIIC, dantrolene, and CGP37157 were dissolved in normal saline.Acacetin was isolated from Adult male Sprague-Dawley rats (150-200 g) (n = 45) were used in this study. All of the animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals , and were approved by the Fu Jen Institutional Animal Care and Utilization Committee . All efforts were made to minimize the number of animals used and their sufferings.3, 1 mM MgCl2⋅6H2O, 1.2 mM Na2HPO4, 10 mM glucose, and 10 mM HEPES (pH 7.4). Protein concentration was determined using the Bradford assay. Synaptosomes were centrifuged in the final wash to obtain synaptosomal pellets with 0.5 mg protein. The synaptosomal pellets were stored on ice and used within 4–6 h.Percoll-purified synaptosomes were prepared using the hippocampus of rats, as described previously + (2 mM), glutamate dehydrogenase  and CaCl2 (1.2 mM) were added after 3 min. After an additional 10 min of incubation, 4-AP (1 mM) or high external KCl (15 mM) was added to stimulate glutamate release. Glutamate release was monitored by measuring the increase in fluorescence  caused by NADPH being produced by the oxidative deamination of released glutamate by GDH. Data were accumulated at 2-s intervals. A standard of exogenous glutamate (5 nmol) was added at the end of each experiment, and the fluorescence response used to calculate released glutamate was expressed as nanomoles of glutamate per milligram of synaptosomal protein (nmol/mg). Values quoted in the text and depicted in bar graphs represent the levels of glutamate cumulatively released after 5 min of depolarization, and are expressed as nmol/mg/5 min. Estimation of the IC50 was based on a one-site model [Inhibition  =  (Inhibition MAX × [acacetin] / (IC50 + [acacetin])], and calculated using the nonlinear curve-fitting function provided in MicroCal Origin. Cumulative data were analyzed using Lotus 1-2-3.The glutamate release was assayed by using on-line fluorimetry, as described previously 3(5)3(5) were added and allowed to equilibrate before the addition of CaCl2 (1 mM) after 4 min of incubation. 4-AP was then added to depolarize the synaptosomes for 10 min, and DiSC3(5) fluorescence was monitored at excitation and emission wavelengths of 646 and 674 nm, respectively. Cumulative data were analyzed using Lotus 1-2-3 and expressed in fluorescence units.The plasma membrane potential was determined using a membrane-potential-sensitive dye, DiSC2+]C was measured using the Ca2+ indicator fura-2. Synaptosomes (0.5 mg/mL) were resuspended in HBM containing 0.1 mM CaCl2 and loaded with 5 µM Fura-2-AM for 30 min at 37°C. The synaptosomes were washed with HBM by being centrifuged, resuspended in 2 mL of HBM containing BSA, and placed in a Perkin-Elmer LS-55 spectrofluorometer at 37°C with stirring in the presence of 1.2 mM CaCl2. The synaptosomes were incubated for 10 min in the presence of acacetin (30 µM) prior to being depolarized with 4-AP (1 mM). Fura-2-Ca fluorescence was determined at excitation wavelengths of 340 and 380 nm , and data were accumulated at 2 s intervals. [Ca2+]C (nM) was calculated by using calibration procedures The [Ca+ measurements were performed in essentially the same manner as those performed in the [Ca2+]C determinations, except the synaptosomes were preincubated with 5 µM SBFI-AM instead of fura-2 Na2.Rats (n = 24) were randomly divided into four experimental groups: (1) control group; (2) KA-treated group; (3) KA and acacetin 10 mg/kg-treated group; and (4) KA and acacetin 50 mg/kg-treated group. Acacetin was injected intraperitoneally (i.p.) 30 min before KA  injection. Rats were sacrificed 3 days after KA (15 mg/kg) injection by using an overdose of anesthetics . The animals were then perfused transcardially with saline (room temperature) followed by cold 4% paraformaldehyde in 0.1 M PBS. The brains were removed immediately and post-fixed in the same fixative overnight at 4°C, and then cryoprotected in 30% sucrose for 24–48 h. To perform neutral red staining, the brains were sectioned coronally into 30-µm-thick sections in a cryostat. The sections were then mounted on gelatin-coated slides, air dried and then stained with neutral red solution. Fluoro-Jade B  staining was performed as described previously Fixed brains were cut into 40-µm-thick coronal sections in a cryostat and then free-floating staining was performed using an immunohistochemical ABC method. In summary, after rinsing the sections 3 times with PBS, the sections were blocked with 2% normal goat serum containing 0.3% Triton X-100 for 1 h at room temperature. The sections were then incubated overnight at 4°C with a mouse monoclonal anti-OX-42 antibody . The sections were then incubated with a goat biotinylated anti-mouse secondary antibody  for 2 h, and subsequently incubated with ExtrAvidin peroxidase  for 1 h at room temperature. After rinsing the sections in 0.1 M PBS for 20 min, the sections were reacted with 0.025% 3,3′-diaminobenzidine tetrahydrochloride (DAB) solution in PBS containing 0.0025% hydrogen peroxide for 6 min. The sections were then mounted on gelatin-coated glass slides, air-dried, dehydrated, cleared with xylene, and coverslipped with Entellan mounting medium .t test or by using one-way ANOVA accompanied by post-hoc LSD comparison tests for multiple comparisons. The analysis was completed using SPSS software . P<0.05 was considered to represent a significant difference.Data were expressed as mean ± SEM. The data reported were analyzed by using the unpaired Student's +] to investigate the effect of acacetin on glutamate release. In synaptosomes incubated in the presence of 1 mM CaCl2, 4-AP (1 mM) evoked a glutamate release of 7.2±0.1 nmol/mg/5 min. The application of acacetin (30 µM) decreased the amount of 4-AP-evoked glutamate release to 4.0±0.1 nmol/mg/5 min (P<0.001) without altering the basal release of glutamate  from 127.1±1.2 nM to a plateau level of 196.8±5.5 nM (P<0.001). Applying acacetin (30 µM) did not affect basal Ca2+ levels, but caused an approximately 18% decrease in the 4-AP-evoked rise in [Ca2+]c . 3(5) fluorescence of 16.3±0.8 fluorescence units/5 min. Preincubation of the synaptosomes using acacetin (30 µM) for 10 min before adding 4-AP did not alter the resting membrane potential, and produced no substantial change in the 4-AP-mediated increase in DiSC3(5) fluorescence . In addition, the Na+-sensitive probe SBFI was used to measure cytosolic Na+ levels. + influx, but acacetin (30 µM) failed to affect this increase (P = 0.792). The failure of acacetin to produce an effect on this increase was not caused by an insufficient level of sensitivity of the SBFI probe to alterations in Na+ channel activity because in parallel experiments, the Na+ channel blocker tetrodotoxin  caused an 82% inhibition of 4-AP-evoked Na+ influx (P<0.001). These results indicate that the observed effect of acacetin on [Ca2+]C is most likely caused by a direct modulation of VDCCs activity.Transmitter release can be modulated by regulating the plasma membrane potential, and consequently altering the calcium influx. To investigate the potential mechanisms underlying the acacetin-mediated inhibition of glutamate release, the effect of acacetin on intrasynaptosomal Ca+ influx ; P<0.001v2.2 (N-type) and Cav2.1 (P/Q-type) channels 2+ channel activity was involved in the effect of acacetin on 4-AP-evoked glutamate release, we examined the effect of acacetin in the presence of ω-conotoxin MVIIC (ω-CgTX MVIIC), a wide spectrum blocker of Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels. In P<0.001). Although the 4-AP-evoked glutamate release was considerably reduced in the presence of acacetin (30 µM), this effect was prevented by the presence of ω-CgTX MVIIC. The release measured in the presence of both ω-CgTX MVIIC and acacetin was similar to that obtained in the presence of ω-CgTX MVIIC alone   and mitochondria 2+ release in the acacetin-mediated inhibition of glutamate release was tested in the presence of dantrolene, an inhibitor of intracellular Ca2+ release from endoplasmic reticulum, and CGP37157, a membrane-permeant blocker of mitochondrial Na+/Ca2+ exchange. P<0.001), indicating that the Ca2+ release from ER ryanodine receptors contributes substantially to the levels of 4-AP-evoked glutamate release. In the presence of dantrolene, however, acacetin (30 µM) still effectively inhibited 4-AP-evoked glutamate release . Compared to the effects of dantrolene, CGP37157 (100 µM) similarly decreased the levels of 4-AP-evoked glutamate release, but it produced no effect on the acacetin-mediated inhibition of 4-AP-evoked glutamate release   modulation. This finding disagrees with previous electrophysiological studies, which have shown that acacetin inhibits K+ currents in human atrial myocytes and HEK 293 cells In nerve terminals, the inhibition of Na2+ entry through the Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels that are coupled to glutamate exocytosis in the nerve terminals 2+]C. In addition, the inhibitory effect of acacetin on glutamate release was prevented by ω-CgTX MVIIC, a wide spectrum blocker of the Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels. However, neither dantrolene, an inhibitor of intracellular Ca2+ release from the endoplasmic reticulum ryanodine receptors, nor CGP37157, a mitochondrial Na+/Ca2+ exchange blocker, affected the inhibitory effect of acacetin on glutamate release. Based on these results, we suggest that acacetin inhibits evoked glutamate release by reducing presynaptic Ca2+ influx rather than by indirectly affecting neuronal excitability.Therefore, if the effect is not caused by the modulation of synaptosomal excitability, then acacetin possibly inhibits evoked glutamate release by decreasing the levels of CaWe hypothesized that acacetin would have a neuro-protective effect because we discovered that acacetin depressed glutamate release from nerve terminals, and that the excessive release of glutamate is a critical element in the neuropathology of acute and chronic brain disorders. This hypothesis was confirmed in this study by applying the KA model to adult rats. KA is an analogue of glutamate, and the systemic administration of KA to animals causes neuronal cell death or neurodegeneration in specific brain regions, such as the hippocampus, piriform cortex, thalamus, and amygdala Numerous studies have indicated that KA-induced neuronal death is associated with microglia activation in the hippocampus 2+]C, whereas it did not alter 4-AP-mediated depolarization and Na+ influx; (2) acacetin-mediated inhibition of glutamate release was abolished by the N-, P- and Q-type Ca2+ channel blocker, but not by the ryanodine receptor blocker, or the mitochondrial Na+/Ca2+ exchanger blocker.Studies have reported that acacetin, at 10-25 mg/kg, protects neurons against insults induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and ischemia in vivo clinical situations remains to be determined, this investigation enhances the understanding of acacetin action in the brain and demonstrates the therapeutic potential of this natural compound in treating neurological disorders in which excitotoxic neuronal cell death and inflammation processes are involved.In conclusion, this study is the first to demonstrate that acacetin inhibits glutamate release from hippocampal nerve terminals in rats. This may contribute to the substantial neuroprotective effect of acacetin against KA-induced in vivo excitotoxicity. Although, the relevance of our finding to