Albumin dialysis using the molecular adsorbent recirculating system (MARS) is a new therapeutic approach for liver diseases. To gain insight into the mechanisms involved in albumin dialysis, we analyzed the peptides and proteins absorbed into the MARS strong anion exchange (SAX) cartridges as a result of the treatment of patients with cholestasis and resistant pruritus. Proteins extracted from the SAX MARS cartridges after patient treatment were digested with two enzymes. The resulting peptides were analyzed by multidimensional liquid chromatography coupled to tandem mass spectrometry. We identified over 1,500 peptide sequences corresponding to 144 proteins. In addition to the proteins that are present in control albumin-derived samples, this collection includes 60 proteins that were specific to samples obtained after patient treatment. Five of these proteins  were confirmed to be removed by the dialysis procedure using targeted selected-reaction monitoring MS/MS. Furthermore, capture of HNP-1 and SLURP1 was also validated by Western blot. Interestingly, further analyses of SLURP1 in serum indicated that this protein was 3-fold higher in cholestatic patients than in controls. Proteins captured by MARS share certain structural and biological characteristics, and some of them have important biological functions. Therefore, their removal could be related either to therapeutic or possible adverse effects associated with albumin dialysis. Extracorporeal albumin dialysis (ECAD) using the molecular adsorbent recirculating system (MARS) has been used in patients with acute-on-chronic liver failure or acute liver failure, and in the treatment of primary graft dysfunction after liver transplantation with favorable, although indefinite, results in terms of survival The clinical effects of MARS are presumably due to the removal of albumin-bound substances from the patient's blood In the present study, we used a shotgun proteomic approach combined with targeted MS/MS procedures to describe the set of peptides and proteins that were captured by the MARS system from patients' blood and absorbed in the SAX cartridges in patients treated for resistant pruritus . Our aimParallel analysis by MDLC-ESIMS/MS of tryptic and GluC (Staphylococcus aureus protease V8) digests of the MARS SAX extracts led to the identification of a total of 1,726 distinct peptides (FDR<1%). Proteins identified from only one peptide were re-evaluated with PEAKS to avoid false positives. Only peptide identifications that coincided in PEAKS and SEQUEST analyses were considered. Using this approach, 1,538 peptides corresponding to 144 proteins were identified with high confidence . From thTwelve of the 60 proteins that were putatively derived from patients were selected for targeted MS/MS analysis in control- and patient-derived extracts. In addition, 5 proteins that were common to both samples were taken as references .Targeted proteins were selected on the basis of their molecular weight and the sequence coverage observed in previous experiments. Thus, we selected proteins of low molecular mass (<15 kDa) that were identified in both replicates with high sequence coverage (>25%). An exception was made for the fibrinogen alpha chain (98 kDa). Although this chain was larger than the MARS membrane cutoff (50 KDa), we selected it as it was identified from a high number of different peptides (58 peptides). This indicates that high amounts of this protein or, more probably, fragments of the protein, are removed during the MARS dialysis process.Each targeted protein was monitored through up to four of its previously detected tryptic peptides. These peptides were selected to provide a collection of 34 peptide markers evenly distributed throughout the chromatographic separation. Seventeen of the targeted peptides that represent 10 different proteins were effectively detected and measured by SRM analysis . These iThe presence of HNP-1 and SLURP1 was assessed by Western blot on SAX extracts from the five patients treated with MARS. Alpha-1B-glycoprotein (A1BG), which was identified in control and treated samples by MDLC-ESIMS/MS and SRM analysis, was used as a control for the Western blot validation. In agreement with the above-described mass spectrometry results, the presence of HNP-1 was confirmed in all patient samples, but was not detected in the control albumin sample. SLURP1 was not detected in the control sample either. However, it was present in the SAX extract from the four patients with resistant pruritus, but not in the patient with Wilson's disease .One of the SAX cartridge extracts analyzed in the previous experiments was used as a SLURP1 positive control, and the intensities of SLURP1 signals were normalized to this control sample . SLURP1 There is still little knowledge of the proteins and other biological compounds that are removed from blood during albumin dialysis. Most of the substances that are known to be removed are small molecules, such as ammonia, bile acids, bilirubin or tryptophan. Several proteins have also been reported to be removed by MARS, including regulators of the inflammatory cascade (tumor necrosis factor-α or interleukin-6) and hepatic growth factors . These molecules were identified in studies that analyzed their presence in blood from patients and in the MARS albumin circuit before and after perfusion through the SAX and charcoal cartridges and measured their relative concentrations before and after dialysis In preliminary experiments, the analysis of proteins absorbed in the MARS cartridges during patient treatment was hampered by the presence of many different proteins that were originally present in the commercial albumin used for the MARS internal circuit. These albumin solutions are industrially prepared from human plasma by the Cohn process To identify the proteins derived from patients after MARS treatment in these complex albumin extracts, we followed an approach consisting of a shotgun analysis followed by a targeted MS/MS analysis. Molecules identified in the shotgun analysis that had not been identified in control albumin-derived samples were selected for the semi-quantitative targeted MS/MS analysis, in which their presence/absence in control and patient-derived samples was confirmed. For this purpose, we applied an SRM method that provided a high-sensitivity procedure for monitoring the target peptides .Overall, the number of peptides and proteins identified in the analysis of patient-derived samples was similar to that reported in the analysis of control albumin Our search focused on proteins removed from the patients that could not be detected in the albumin control extracts. Other proteins that are originally in the albumin solution may also be removed from blood during the treatment. Identification of these proteins would require the use of isobaric tag for relative and absolute quantitation (iTRAQ) http://plasmaproteomedatabase.org)  . A largeIn addition, many of these high-molecular-weight proteins  were identified with low coverage, although the sequences that justify the assignation were detected with a high number of counts. The number of counts for a peptide (the times it was detected by MS) is related to its abundance in the sample Overall, and although it is possible for proteins of non-globular/irregular shapes to pass through membranes with a pore size smaller than its specified MWCO, our data suggest that most of these identifications correspond to sequences derived from protein fragments and not from the full-length protein sequence.http://plasmaproteomedatabase.org). It is interesting to note that a similar enrichment in proteins with disulfide bonds  is found in the components of the albuminome described in previous work The set of proteins identified only in albumin after dialysis was significantly enriched in proteins with disulfide bonds  and one patient with a neurologic form of Wilson's disease with no liver involvement who was treated to remove circulating copper. Serum samples from 27 patients with PBC and 10 healthy subjects were also taken and stored at −80°C for subsequent Western blot analysis to validate some of the results of the proteomic analysis of the compounds adsorbed in the MARS SAX cartridge.ECAD was performed with a MARS™ system using a constant flow of 20% human serum albumin  in the extracapillary compartment, as described before Control samples were prepared by recirculating control albumin through a SAX column. For this purpose, a 14-cm long×2.6-cm-i.d. Econo-Pac polypropylene column (20 mL) provided with a frit was filled with 20 mL of resin obtained from a MARS SAX cartridge . This support is a strong anion exchange (SAX) resin based on cholestyramine. Then, 50 mL of commercially available HUMAN ALBUMIN GRIFOLS® 20% solution w/v were recirculated for 6–7 hours at ca. 30 mL/h. The column was stored for a maximum of 16 h at 4°C before the peptides and proteins bound to the support were extracted.The overall procedure used for peptide and protein identification is depicted in Staphylococcus aureus protease V8) , respectively. The resulting peptide samples were analyzed by multidimensional liquid chromatography electrospray tandem mass spectrometry (MDLC-ESIMS/MS), using an Agilent 1200 HPLC  system coupled to a linear LTQ ion trap equipped with a microESI ion source . Peptides and proteins were identified as described elsewhere The protein extract derived from one patient with pruritus and the control sample were digested and analyzed as described before The same MDLC-ESIMS/MS setup as described above was used for SRM analyses. For each of the targeted peptides, the two most intense fragment ions observed in the previous MS/MS analyses were selected for monitoring in the SRM analysis. Ions corresponding to the loss of water from the precursor ion were not taken into account in the selection. The acquisition time for each transition was restricted to a 5–10 min window. Other parameters were: scan time for each SRM event, 3 microscans; maximum injection time, 10 ms; parent ion isolation window, 3 m/z; collision energy, 45%. Collected data on SRM-transitions were evaluated by QualBrowser in Xcalibur 2.0 .Protein extracts obtained from the SAX MARS cartridges from the four patients with pruritus and the one with Wilson's disease were analyzed by Western blot to confirm the presence of secreted Ly-6/uPAR-related protein 1 (SLURP1) and neutrophil defensin 1 (HNP-1), two of the relevant peptides identified by the proteomic analysis. Moreover, SLURP1 was also assessed in serum samples of patients and healthy controls as follows. To preconcentrate the protein and eliminate interferences in the Western blot analysis, 200 µL of serum were incubated with SAX resin for 4 hours and then extracted with 500 mM TFAM 20%ACN. Equal amounts of SAX-eluted protein were loaded onto a 15% acrylamide∶bisacrylamide gel , and blotted onto polyvinylidene difluoride membranes . The proteins were transferred to the membrane at 100 V for 30 min.The PVDF blots were incubated for 1 hour with blocking solution . Then, three parallel overnight incubations were carried out with primary antibodies for HNP-1 , SLURP1  and A1BG . The three PVDF membranes were washed three times in the blocking solution, incubated with the diluted secondary antibodies for 2 h  and washed twice in PBS. Staining was then visualized using the SuperSignal West Pico Chemiluminescent Substrate 1∶1 .Figure S1Interaction network for 51 of the 60 proteins unique to the MARS extracts after patient treatment that were available in the STRING database. Thirty proteins were found linked at the medium confidence level (score 0.4). Albumin is indicated as ALB .(TIF)Click here for additional data file.Table S1Complete peptide data set identified by Bioworks and PEAKS.(XLS)Click here for additional data file.Table S2Data set of proteins mapped by the identified peptides.(XLS)Click here for additional data file.Table S3Peptides and proteins monitored by SRM. Retention times and SRM transitions.(XLS)Click here for additional data file.Table S4Gene Ontology analysis of the protein set unique to the MARS-derived extracts DAVID .(XLS)Click here for additional data file.


Human sperm assay (HSA) is a preferred in house quality control and proficiency test (PT) practiced in fertility laboratories. HSA is performed over varying durations, apparently without following set criteria. To better understand the assay time required for reprotoxicity testing in embryo culture media, we compared American-Association-of-Bioanalysts-(AAB-) administered HSA data to our own assay performed using PT samples obtained from AAB. Participating laboratories were required to culture sperm for 48 hours to determine media acceptability. Conclusions drawn from 48- and 24-hour observations were the same, suggesting that HSA could identify reprotoxic media in less time than required by AAB. Our assay revealed that changes in motility grade in adulterated media are significantly different from those in control media. Furthermore, grade changes can be identified earlier than differences in motility loss between samples. Analyzing motility and motility quality together provides a method for establishing an optimal time for HSA. Quality control (QC) is an essential component of a successful human in vitro fertilization (IVF) program. The success of IVF depends critically on the quality of the products used in the laboratory procedures. The reprotoxicity testing of reagents, media, and consumables utilized in the fertility laboratory is therefore essential –4. AccorAmong these bioassays, the human sperm assay (HSA) has been an integral part of IVF since its inception. Edwards and Steptoe, the 2 pioneers of human IVF, utilized HSA for testing the suitability of the solutions and materials used in the first successful IVF pregnancy in the world , 9. The Review of the HSA literature indicates that human sperm bioassays are being performed over varying durations of time , 12–14. In this study, we explored AAB-administered PT to develop a better understanding of the time required for HSA in AAB-set culture conditions. The AAB provides participating laboratories with 2 media samples in each PT event. The quality of one media is intentionally made poorer than that of the other. The laboratories are required to perform HSA for 48 hours to identify the media as being of acceptable (MAQ) or unacceptable (MUQ) quality. The purpose of this PT is for fertility laboratories to develop the skills needed for evaluating the quality of their own media to be used in IVF procedures. The AAB has been administering this PT for more than 10 years. We evaluated the AAB-compiled HSA data for the past 2 years and also performed our own assay using a set of AAB-provided PT samples. We are able to demonstrate that the assay time for HSA utilized in AAB PTs is lengthy, and that evaluation of the motility grade, along with the motility, enhances the efficiency of the assay, therefore helping to optimize the assay time.http://www.aab-pts.org/) [E1, 08 − E2, 09 − E1, and 09 − E2), in each of which EC1 and EC2 were identified either as MAQ or MUQ by performing HSA.The data of AAB-conducted PT for determining the suitability of culture media utilizing HSA was used (data from AAB: ts.org/) , 21. TheMore than 130 fertility laboratories across the United States participated in the AAB-administered PT. The participating laboratories recorded sperm motility as an indicator of sperm survival in EC1 and EC2 at 0, 24, and 48 hours in order to reveal the differences in their quality. The methods used in AAB-administered HSA fall under 2 categories: sperm culture with and without oil overlay. The laboratories were also required to culture sperm with and without protein supplementation.n ≥ 130) was performed to see if the motility difference between EC1 and EC2 was significant at 24 and 48 hours. The 95% confidence interval for the difference in means between the 2 media was calculated (EC1 versus EC2) in each PT event . The level of significance  was adjusted using the Bonferroni correction to account for multiple comparisons, and a t-test utilizing pooled variance was used.Statistical analysis of the AAB-compiled sperm motility data (average ± SD) in EC1 and EC2 reported by the participating laboratories , exhibiting ≥90% motility and a motility grade predominantly of 4, were used for the convenience of experiment. The cultures, composed of EC1 and EC2 media of 1.0 mL volume containing 4-5 × 106/mL sperm, were established in 5 mL Falcon culture tubes (Becton and Dickinson). All culture conditions were maintained in duplicates in a gas (6% CO2) and temperature (37°C) controlled incubator. Sperm motility and motility grade in the culture were evaluated at the indicated time points by assessing aliquots of samples in the Makler chamber following WHO criteria but with a modified grading system [The gradient-washed sperm samples  at 24 hours as well as at 48-hour observations in all PT events  of years 2008 and 2009 . Further, the significant difference between EC1 and EC2 at 24 hours was revealed by both assay methods  and independently of protein supplementation .The motility difference between EC1 and EC2 (categorized either as MAQ or MUQ) was statistically significant  and hamster sperm motility assay (HSMA), the alternatives to HSA, may be commercially available but are expensive and labor intensive. Most importantly, when human sperm is used, no species differences have to be taken into account in interpreting and validating the outcome of the test. Since the first report of successful human IVF, various modifications have been introduced to improve the IVF techniques, and HSA has played a role in such improvements , 9, 15. The AAB, being an authorized PT provider, has been collecting HSA data from fertility laboratories for more than 10 years. The participating laboratories are required to perform the assay for 48 hours to fulfill AAB requirements. The cost and labor required for an assay are directly related to the assay time. The longer the assay duration, the more expensive the assay becomes. We are able to show that HSA values in EC1 and EC2 are significantly different at 24, as well as at 48, hours in all 4 PT events conducted in the years 2008 and 2009. We argue that when the difference between EC1 and EC2 can be confidently confirmed by 24 hrs, then prolonging the assay up to 48 hours is not necessary. We further argue that the loss of motility in any culture, even one completely free of any harmful elements (toxicants), is expected to occur as time progresses. This natural phenomenon of sperm motility loss in culture may overshadow the real toxicant-induced motility loss, producing erroneous results if the assay time is extended beyond that actually required. It thus appears that there is no gain in prolonging the culture for an additional 24 hours in AAB-administered PTs.The participating laboratories did not have an option for evaluating motility in the culture before 24 hours in AAB-administered PTs. It is possible that a significant motility difference between EC1 and EC2 may have emerged prior to that time (24 hours) but was missed because of lack of investigation. The preliminary results of our own bioassay support this notion. We documented increased motility loss in EC1 compared to EC2 at 6-hour and 12-hour observations but were unable to validate its statistical significance because of our small sample size. It will be worth investigating our observation further with a larger sample size. The evaluation of the quality of the sperm motility (motility grade) is now a routine practice in semen analysis . We realWe must admit that motility grading in its current state is subjective, and it is difficult to give it a true quantitative face. There may be concern about the concept of sperm motility grade being used as a tool in sperm bioassay due to the subjective nature of distinguishing between the various grades. Identifying motile and nonmotile sperm is a much more straightforward issue than distinguishing between the various grades of sperm motility. However, difficulty in grading motility should not hinder the benefit we can achieve by incorporating motility grade in the assay. We used a grading scheme of our own—which may not be a perfect one—in placing sperm under different motility grades. A lot is now known about the motility characteristics of mammalian sperm; therefore, developing a consensus on grading motility will be easier than before. In our view, overcoming the problem of grading motility involves emphasizing the importance of the issue, understanding the obstacles in quantifying the grades, and then developing consensus on grading. It is our expectation that in the near future, consensus will be developed toward a unified motility grading method by utilizing the technologist's skill in assessing motility grade both in one's own laboratory and between laboratories. The existing difficulty in grading motility should be conquered to obtain the benefit it can provide.Uniquely, human sperm can remain in culture for a lengthy time , 24, 30.Our study found that in AAB-administered PTs, the collection of data at 48 hours was not necessary to identify EC1 and EC2 since the conclusion drawn from 48 hours observation was the same as that of 24 hours. In other words, sperm culture for 24 hours and 48 hours revealed the same conclusion about the quality of EC1 and EC2 media in each of the 4 consecutive PT events investigated. Further, it was revealed that the mode of change in motility quality is different in adulterated media compared to the control media, and that change can be identified earlier than the difference in motility loss between the 2 samples. Thus, it appears that motility and motility quality combined can sharpen the sensitivity of the assay and, thus, can help in determining the minimum time required for the assay. Evaluation of motility grade along with motility seems to strengthen the power of the human sperm bioassay. This technique holds promise in our center and now needs to be validated at additional sites, with the hope that it may shorten the time used by the AAB method.


Objective. To assess the efficacy and the effect on Qol of solifenacin for the treatment of OAB in MS patients. Patients and Methods. Thirty MS patients suffering from OAB were treated with solifenacin 5/10 mg for 8 weeks. The first 4 weeks patients received solifenacin 5 mg. At week 4 patients could request a dose increase to 10 mg. The efficacy was evaluated at 8 weeks. Results. After 4 weeks of treatment, 28 patients reported acceptable or no side effects. 17 continued the study with the 10 mg dosage, and 11 stayed on 5 mg solifenacin. Two patients withdrew from the study due to side effects. Solifenacin 5/10 mg for 8 weeks resulted in a significant decrease in number of micturitions and number of pads used per day compared to baseline. Also the severity of urgency prior to voiding decreased significantly, and an increase was seen in the volume per void. Twenty out of 30 patients chose to continue solifenacin therapy after termination of the study. The majority of patients reported global QoL improvement. Conclusions. Solifenacin is effective in the treatment of MS patients with OAB symptoms. This is the first study with solifenacin in a specific neurogenic patient group with a neurogenic disease-specific QoL outcome measure (MS-QoL 54). Solifenacin is a once-daily oral antimuscarinic agent that has been available in The Netherlands since September 2004 for the treatment of overactive bladder (OAB). The efficacy and safety of solifenacin has already been demonstrated in randomised, double-blind placebo controlled studies , 2. HoweThis is a prospective, open-label study to assess the efficacy and effect on quality of life of solifenacin 5/10 mg for 8 weeks in the treatment of MS patients with symptoms of OAB. The study protocol was approved by the local ethics committee of the University Medical Centre Nijmegen (CMO no. 2004/194). Patients provided written informed consent before enrolment. Men and women with a classified MS diagnosis according to the criteria of McDonald et al.  and sympAll medication that could influence bladder function was stopped at least 2 weeks prior to treatment or continued with no dose changes during the study. Patients were not allowed to use other antimuscarinic drugs prescribed for bladder dysfunction 2 weeks prior to or during the study. All methods, units, and definitions used in this study were done according to ICS standards .At baseline patients were evaluated by 72-hour voiding diaries, as well as an MS-specific quality of life questionnaire MS-QoL 54). The MS-QoL 54 is a multidimensional health-related quality of life measure that combines both generic and MS-specific items to a single instrument [4. The MSIn the voiding diary the voiding frequency, voided volume per void, severity (degree) of urgency prior to any void, number of incontinence periods, severity of incontinence periods, and number of pads used were recorded. The degree of urgency was described on a scale of 0–3 . The severity of urine loss was described on a scale of 0–3 .Patients that met all inclusion criteria, and none of the exclusion criteria received solifenacin 5/10 mg for 8 weeks. All patients received solifenacin 5 mg once daily in the the first 4 weeks. At week 4, patients could continue 5 mg solifenacin treatment for another 4 weeks or request a dose escalation to 10 mg once daily in case of subjective insufficient efficacy and no or acceptable side effects. Both solifenacin 5 and 10 mg once daily have been shown to be effective and well tolerated for treating symptomatic OAB . A startAfter a total treatment period of 8 weeks the efficacy of solifenacin was evaluated by 72-hour voiding diary and MS-QoL 54. Global patient perception was assessed by enquiring patient satisfaction and the patients' desire to continue the treatment. Primary endpoints were defined as change from baseline in mean number of micturition per 24 hours, change from baseline in mean voided volume per void, and change from baseline in number of incontinence episodes per 24 hours and number of pads used per 24 hours. Change in quality of life (QoL), as measured with the MS-QoL 54, was a secondary endpoint.Changes from baseline to endpoint were subjected to the Wilcoxon signed-ranks test.t-test).The quality of life questionnaires were compared using the test for paired samples correlations  were diagnosed with OAB. Nine patients suffered from “OAB-dry” and 21 of “OAB-wet” Seven patients had received antimuscarinic therapy in the past without the desired effect. Twenty-three patients have not been treated with antimuscarinic agents prior to inclusion in the protocol. No patients had received other therapy than oral antimuscarinics for the OAB symptoms prior to the study.After 4 weeks of treatment with solifenacin 5 mg, 28 patients reported acceptable or no side effects. Two patients withdrew from the study due to adverse events  Both adverse events fully disappeared within days from discontinuation of the therapy. Of the remaining 28 patients, 11 patients (39%) chose to continue treatment with the 5 mg dosage. Seventeen (61%) patients requested a dose escalation to 10 mg solifenacin due to subjective insufficient efficacy. P < .0001) and number of pads used per 24 hours . Also the degree of urgency prior to voiding decreased significantly . Additionally a significant increase with 33 mL (P < .0001) was seen in the median voided volume per void. Whilst the median number of incontinence episodes per 24 hours and the median severity of urine loss improved numerically, these changes were not statistically significant . All other MS-QoL 54 subscales detected no significant differences from baseline to end of study  chose to continue solifenacin therapy after the study, due to beneficial effects. Six patients (20%) chose to terminate the use of the drug because of lack of effect. Only in 2 out of 30 (7%) patients the reason for discontinuation of therapy was side effects.Lower urinary tract symptoms are common in MS. Up to 90% of patients have voiding complaints at some time during the course of the disease, especially with a disease duration longer than 10 years . The mosUnlike the bladder dysfunction that follows spinal cord injury and causes life-threatening upper urinary tract conditions, MS very rarely causes severe upper urinary tract involvement but rather results in morbidity that influences the quality of life .Anticholinergic agents are commonly used in the management of the OAB. For more than 30 years oxybutynin has been the drug of choice in patients with a neurogenic cause of OAB. Randomised controlled studies comparing oxybutynin to other antimuscarinic agents in neurogenic patients date from the late 1980s and early 1990s , 11. A lMost of the time neurogenic patients are excluded from registration studies because they respond differently to medical treatments or they deal differently with common primary endpoints in OAB studies such as micturition frequency. When a wheelchair-bound patient has to tell you how many times he or she goes to the toilet per day, the answer will be highly dependent, amongst others, on environmental factors such as the availability of proper wheelchair toilets. Moreover MS is a progressive disease, so the clinical endpoint may differ from baseline to end of study even without intervention. The issue is that we do not have objective parameters to define a stable patient, apart from that MS patients are not a homogeneous group and that they may have all kinds of disabilities. This study shows that solifenacin is effective in the treatment of MS patients with OAB symptoms when assessed by means of 72-hour voiding diaries. Significant improvements were observed on severity of urgency, frequency and urge incontinence and number of pads used in 24 hours. Also subjective improvements evaluated by means of the global patient perception question suggested that patients were satisfied with the efficacy of solifenacin on their symptoms. 73% of patients wanted to continue therapy due to favourable results. We recognize that the study design is limited by the absence of a control group. It would be useful to do a head to head comparison in a randomized controlled trial in the future.The effects on QoL, measured with the MS-QoL 54, were not apparent. The developers of the MS-QoL 54 utilized the Short Form 36 (SF-36) as the generic component to which 18 items were added to tap MS-specific issues . TherefoSolifenacin is efficacious in treating OAB symptoms in MS patients. Solifenacin significantly improved frequency, severity of urgency, volume voided, and number of pads used per 24 hours. The MS-QoL 54 showed no significant changes, other than a modest improvement in one of the subscales. Possibly, MS-QoL 54 is not specific enough to detect changes in OAB symptoms in an MS patient population. Further randomised clinical studies with antimuscarinics in this specific patient group are warranted.


Human immunodeficiency virus (HIV) and mental illness are interlinked health problems; mental illness may pose a risk for contracting HIV and HIV-positive individuals are at higher risk of mental illness. However, in countries with high HIV prevalence, the main focus of HIV-related health programmes is usually on prevention and treatment of somatic complications of HIV, and mental illness is not given high priority. We examined HIV prevalence, uptake of HIV services, and HIV-related risk behaviour among people attending a mental health clinic in rural Malawi.Semi-structured interviews were performed with patients capable to consent (94%), and with those accompanied by a capable caregiver who consented. HIV counselling and testing was offered to participants.Among 174 participants, we collected 162 HIV test results (91%). HIV prevalence was 14.8%. Women were three times as likely to be HIV-positive compared to men. Two-thirds of participants reported having been tested for HIV prior to this study. The uptake of HIV-services among HIV-positive patients was low: 35% did not use recommended prophylactic therapy and 44% of patients not receiving antiretroviral treatment (ART) had never been assessed for ART eligibility. The reported rate of sexual activity was 61%, and 9% of sexually active participants had multiple partners. Inconsistent condom use with stable (89%) and occasional (79%) sexual partners, and absence of knowledge of the HIV status of those partners  indicate high levels of sexual risk behaviour.HIV-prevalence among persons attending the clinic, particularly men, was lower than among the general population in a population survey. The rate of HIV testing was high, but there was low uptake of preventive measures and ART. This illustrates that HIV-positive individuals with mental illness or epilepsy constitute a vulnerable population. HIV programmes should include those with neuropsychiatric illness. Globally, the burden of disease attributable to mental, behavioural and neurological disorders is rising and calculated at 10.4% of all disability-adjusted life years Resource limitations have become more apparent in the context of the HIV pandemic. The overwhelming demand for HIV care has drawn heavily upon available financial and human resources. In countries with a high HIV prevalence, the focus has been on the prevention and treatment of somatic complications, although people infected with HIV are also at a higher risk of developing mental illness Several studies from developed countries have reported that adults with severe mental illness have elevated rates of HIV-infection In high-income countries this increased HIV prevalence is associated with high-risk behaviour such as drug injection use and high-risk sexual behaviour Councelling and testing for HIV is an important entry point for HIV care, especially since people with mental disorders may delay seeking health care and are less likely diagnosed with other health conditions In Malawi, a country with one of the highest HIV prevalence rates in the world, there is a lack of documentation of HIV prevalence among people suffering from neuropsychiatric disorders. Extrapolating data from other countries, a substantial part of the Malawian population is likely to suffer from neuropsychiatric disorders. As in many low- and middle-income countries, mental health services in Malawi are concerned with neuropsychiatric disorders: mental, behavioural, and neurological conditions, such as epilepsy.The aim of this study was to systematically examine HIV prevalence, HIV service uptake and HIV-related risk behaviour among patients with neuropsychiatric disorders attending a mental health clinic in rural Malawi.This was a descriptive cross-sectional study of patients attending a mental health and epilepsy clinic.The study was conducted from August 1st, 2009, to May 1st, 2010, in Thyolo District, a predominantly rural area in Southern Malawi, a very low-income country in sub-Saharan Africa. The district population was estimated at 617,000 in 2010 and had an HIV-prevalence rate of 21% in 2004 Inpatient mental health care is delivered at the district hospital wards, and irregular outreach clinics take place throughout the district. Most mental health care in the district is provided by nurses trained in basic mental health care. There is a central referral hospital for mental health, Zomba Mental Hospital, located one hundred kilometres away. Referral from Thyolo to Zomba occurs in case of severe psychiatric symptoms, and can be seen as an indicator for severity. Diagnoses are usually given by the psychiatric nurses and documented in the patient’s ‘health passport’. The number of diagnoses applied is limited. However, in absence of specialist care, this basic list is practical in the local setting. Medication from the essential health package for common neuropsychiatric conditions is available.In Thyolo District, ART became available in 2003 and district-wide coverage was achieved in 2007 A study sample of 156 was calculated to assess a difference in the HIV-prevalence among the clinic population compared to the general population, with a 95% confidence interval and 5% precision. The general population prevalence in Thyolo District of 21% was taken from the District Health Survey (DHS) Patients attending the clinic who were age eighteen years or above were included. Excluded were: (A) those under the acute influence of alcohol or drugs, (B) those with an acute disturbance of the mental state, (C) those that suffered from a physical condition for which they required admission or (D) those with a severely impaired mental capacity without an accompanying caregiver.Participants were randomly included. Persons in a strictly kept queue were assigned to either the room for routine check-up or the study room, depending on which was available for the next consultation. This procedure continued until all persons in the queue had been seen in one of the rooms. The consultation started with an interview in which the primary investigator (KL), who is a general physician with mental health experience, judged eligibility and capacity to provide informed consent. When a patient lacked capacity to provide consent, a committed caregiver was asked to give consent on behalf of the patient. The purpose of the research was explained, and the patient or caregiver was invited to read the information sheet and consent form in Chichewa or this was read out to illiterate patients. Written consent was obtained from the patient or caregiver or their thumbprint was used.After obtaining informed consent, semi-structured interviews were performed. Information collected and categorised included: neuropsychiatric diagnosis and treatment , socio-demographic factors , variables for the neuropsychiatric condition , general health perception and problems  treatment, history of tuberculosis (TB) treatment), HIV/AIDS services utilization  and HIV-related risk behaviour. With regard to risk behaviour we asessed alcohol use, binge drinking, cigarette and drug use in the previous month. In addition, we interviewed participants about sexual risk behaviour in the previous year. Questions included the type of relationship , number of other sexual partners, condom use with all sexual partners  and knowledge about HIV status of sexual partners. ‘Sex’ was not specified to the participants. When participants lacked capacity to consent and were accompanied by a caregiver, sexual behaviour was discussed without the caregiver’s presence.HCT was offered. Results were de-identified and documented in a patient record. HTC was done in a dedicated room that ensured privacy. Counsellors received an additional one-day training about HIV counselling for mental health patients from the first author (KL) and an experienced psychiatrist (RS). Counselling and testing were provided according the National HIV/AIDS counselling and testing Guidelines positive HIV-test result (3) a test result obtained after referral for HTC as part of the study. For patients who refused referral for HTC we documented the reason given.We accepted three types of HIV test results: (1) a documented test result obtained in a recognized HTC clinic within three months prior to inclusion, and (2) a previously properly documented For HIV testing, the local HIV-testing algorithm and assays were used. The district used an algorithm of three simple rapid HIV assays, which is based on the use of Determine™ HIV-1/2 assay for screening, followed by confirmatory testing of reactive samples by Uni-Gold™ HIV-1/2, with SD Biolin as the tiebreaker for discordant results.All relevant data were entered in a Microsoft Excel database. Missing data were treated as missing in the analysis. The data were analyzed using the statistical programs: Openepi, Version 2.3 Two-by-two tables were used to evaluate the association between a possible risk factor and HIV infection. Odds ratios and their 95% confidence limits were calculated with Fisher Exact method.The study was performed according to Malawian national guidelines. The Thyolo District Health Office of the Ministry of Health and the Department of Mental Health of the College of Medicine in Blantyre took part in the study design. We received ethical approval from the College of Medicine Research Ethics Committee in Malawi and the the Human Research Ethics Committee of the Faculty of Health Sciences of the University of Cape Town was informed about the study.185 patients were assessed for eligibility. Of this group, six patients were found to be under the age limit, two had an acute disturbance of their mental state and three had severely impaired mental capacity without an accompanying caregiver. All of the remaining 174 patients or their caregiver consented to participating in the study.More than half of the participants had epilepsy . One person in this group was also diagnosed with a developmental disorder. Almost half of the patients with epilepsy (48%) reported no convulsions in the previous month, and 47% reported between one and four convulsions.Of 75 participants with a psychiatric diagnosis (43.1%), 56 had schizophrenia spectrum disorders (74.7%). Within this group, one patient had an additional diagnosis of alcohol misuse and one of drug misuse (cannabis). Other diagnoses (n = 20) included affective disorder , and organic and acute psychosis .Most participants  were assessed to have the capacity to provide informed consent. Patients who themselves lacked the capacity to consent and who needed caregivers to provide consent on their behalf were more often found in the group with psychiatric disorders than in the group with epilepsy (12% vs. 2%) (p = 0.018). Of these patients (n = 11) only one patient was not able to discuss sexual behaviour. In the group of participants with a psychiatric diagnosis, 39 (52%) had had a previous admission to the central psychiatric referral hospital. No participants with a diagnosis of epilepsy had been referred to central level. Regarding adherence to medication, most of the participants  had not missed any of the five previous appointments for drug refill.Sociodemographic characteristics are shown in Of participants, 110 (63.2%) reported to have been tested for HIV prior to the study, and 65 (37.3%) had documented evidence of a test result. Of those with documented evidence 17 (26%) had tested HIV-positive. Based on this, the pre-study recorded HIV prevalence was calculated at 9.8%.Of the 17 individuals who tested positive prior to the study, six (35%) did not use co-trimoxazole prophylaxis according to national guidelines. Nine (53%) did not receive ART: five (56%) had their CD4 count assessed in the previous year and were found not eligible for ART, four (44%) had never been staged according to WHO staging criteria.Patients with a psychiatric disorder and a history of severe symptoms  showed a trend towards being more likely to have been tested for HIV compared to patients without referral . There were no statistical differences between the patients with epilepsy and psychiatric disorders for uptake of HIV services and the pre-study prevalence of HIV.Among 174 participants, 162 test results were collected. From twelve participants (6.9%) an HIV test result was not obtained: five did not return after going for HTC and seven refused testing. Reasons for refusal were: ‘no interest’, ‘want to discuss with partner or caregiver’, ‘presupposed knowledge about a negative HIV-status’ or, ‘too old to be tested’.Of the 162, 24 were found to be HIV positive , 13 (54%) of whom were in the 25–34 age group. Women were three times more likely to be infected with HIV compared to men . Bivariate analysis showed no association between other demographic factors  and risk of HIV infection.HIV- prevalence among participants with psychiatric conditions was 17.7% (12/68), versus 12.8% (12/94) among those with epilepsy . No association was found between HIV status and the capacity to consent to the study, duration of symptoms or admittance into the central referral hospital.Of all patients, 129 (74.1%) did not report any physical health problems, 16 (9%) reported having undergone treatment for sexually transmitted infections, and two reported having been treated for tuberculosis. Ten participants reported use of alcohol, six binge drinking, four cigarette use and one cannabis use in the previous month.The reported rate of sexual activity within the previous year was 61%. Of all sexually active participants, 15 (9%) had multiple (2 or more) partners. Among 91 participants with a stable relationship , 11 (12%) had one or more other partners, 76 (89%) never or inconsistently used condoms with their partners and 46 (53%) reported not knowing the HIV status of their partner.Among those with a stable relationship and additional sexual contact(s) 10 (91%) did not use condoms or used condoms inconsistently with their stable partner, and nine out of these 10 did not use condoms with additional sexual contact(s) either. Six (54%) reported not knowing the HIV status of their stable partner and seven (64%) that of other sexual partner(s).Of 15 participants without a stable relationship with occasional sexual partners, ten reported never or inconsistently using a condom, seven of whom reported not knowing the HIV-status of their occasional sexual partners.With regard to possible risk factors and HIV-infection, bivariate analysis showed that participants who reported physical health problems were more likely to be found HIV positive . There were no associations between HIV-status and being sexually active, history of STI treatment, history of TB treatment, multiple sexual partners, condom use with spouse, condom use with non-spousal partner(s), alcohol use, binge drinking, cigarette and cannabis use and history of HIV testing. Also, no differences were found between those with epilepsy or psychiatric disorders with regard to the HIV risk factor variables.Compared to female participants with a spouse, male participants with a spouse more often had one or more additional sexual partners. Alcohol use in the month prior to inclusion was higher among male than among female participants. No other differences between males and females were found with regard to possible risk factors for HIV acquisition see .Those attending the mental health clinic population were less likely to be infected with HIV compared to the general population. Differences in HIV-prevalence between the clinic population  and the general population  Men in the clinic population were half as likely to be HIV-positive compared to men in the general population. The prevalence among women attending the clinic and women in the general population was comparable.Of the clinic population, 97 persons (56%) completed standard 4 or more, compared to 243 persons (39%) in the sample of the general population (p<0.05); the clinic population had better access to electricity (n = 15 (9%) versus n = 250 (2%), p<0.05) Sexual activity in the past year was significantly lower in the clinic population compared to the general population (n = 106 (61%) versus n = 660 (84%), p<0.05), both for male participants (n = 59 (63%) versus n = 140 (83%), p<0,05), and female participants n = 47 (59%) versus 520 (84%), p<0.05). However, the percentage of men and women in the clinic population having multiple partners (n = 12 (13%) and n = 3 (4%)) were higher than the percentages of men (n = 15 (11%), p = 0.76) and women (n = 2 (0.4%), p<0.05) in the general population having multiple partners The uptake of HIV testing among the clinic population was high (n = 110 (63%)) compared to the 25% uptake of HIV testing among the adult population in Malawi This is the first published study of HIV among people with psychiatric disorders and epilepsy visiting an outpatient clinic in Sub-Saharan Africa. We found a trend towards a lower HIV-prevalence among this population, particularly men attending the clinic were significantly less likely to be infected than their male counterparts in the general population.These findings differ from studies in high income countries that suggest that mental illness is a risk factor for developing HIV infection We did not find an explanation for the finding that men in the study population had a lower HIV prevalence rate and women a similar prevalence rate compared to the general population: the rates of sexual activity of both men and women were comparable, and risk factors for HIV acquisition were either similar for men and women, or more prevalent among men. Both men and women were exposed to health promotion and other preventive measures during the monthly clinic visits.Possible reasons for the relatively low number of HIV-positive men in our sample are that HIV-positive men in Malawi are less likely to seek health care In addition, we found a lower rate of sexual activity among both men and women compared to the general population, which can be explained by stigmatization and sexual disinterest or dysfuntion due to neuropsychiatric symptoms and side effects of medication People with severe mental illness have higher all–cause mortality than those without A number of possible study limitations merit discussion. Participants were recruited from a treatment site and results may not be generalizable to all people with mental illness. Second, a small number chose not to undergo HIV testing or were lost to follow up, so the sample may not be entirely representative. However, we estimate that these limitations had minimal effects on our findings: most people consented, the sample exceeded the calculated sample size and losses-to-follow up and opt-out rates were low.The fact that the main researcher was one of the two doctors in the hospital and known by many of the patients attending the clinic, may have led to socially acceptable answers. Therefore, in reality, rates of high-risk sexual behaviour and substance abuse for instance, may be higher, and history of HIV testing lower than the rates reported. Discussing sexual risk behaviour with participants who lacked capacity to consent may have led to some inaccurate responses. However, we found that most participants in this relatively small group were able to discuss sexual behaviour.The population attending the clinic was doing fairly well and symptoms generally appeared to be under control. The fact that the clinic functioned reasonably well, sustained an adequate drug supply, had a low staff turnover and provided weekly consultations is likely to have contributed. Furthermore, we found in the group of patients with psychiatric disorders very few dual diagnoses of psychiatric disorders and substance abuse (2.6%) compared to literature from high income countries Our sample shows similar rates of sexual activity to studies from around the world Uptake of HIV testing was unexpectedly high. The general testing rate in Thyolo is unknown, and may be higher than the testing rate in Malawi due to a large HIV/AIDS project operating in the area. The high uptake of HTC may be related to high utilization of health services by the clinic population and a strong therapeutic alliance with care providers, similar to reports from developed countries The finding from previous studies that patients with severe psychiatric symptoms were less likely to be tested However, mental health patients did not easily access treatment for their HIV disease. Many did not use co-trimoxazole prophylaxis or had not been assessed for ART eligibility. These findings may be caused by health workers’ reluctance to start ART or co-trimoxazole in mental health patients because of prejudice, fear of poor adherence, inadequate knowledge or high work pressure.Our sample is a mixture of patients with mental illness and epilepsy. Little is known about HIV prevalence among people with epilepsy in low-income countries compared to patients with severe mental illness, or about social exclusion rates and cognitive deficit among this specific population. Those with epilepsy visiting the clinic were generally faring quite well, and had a low rate of monthly seizures. These patients were either able to visit the clinic by themselves or supported by caring guardians. This makes ‘social exclusion’ and ‘cognitive deficit’ probably minor factors in this specific population.We found no specific patient-related factors that justify withholding HIV testing and care from patients with mental disorders. Almost all patients were found to have the capacity to consent to taking part in the study, or were accompanied by a capable caregiver. Most patients came in time for their drug refill, indicating good adherence.In fact, the high rate of high-risk sexual behaviour among this population and the generally high HIV-prevalence in Thyolo District should encourage health staff to offer HTC to mental health patients. Our study suggests that patients with double diagnoses (and related stigma) of mental illness and HIV may miss out on services available within the health system.HIV-prevalence among patients attending a mental health clinic in rural Malawi was lower than in the general population, particularly due to a lower HIV-prevalence in males. The HIV testing rate was high, but uptake of preventive measures and HIV care low. Our findings indicate treatment gaps in: (1) male patients, who may delay seeking health care, and (2) all patients with neuropsychiatric illness, despite the fact that this clinic population was very well able to undergo testing and counselling for HIV. HIV prevention and care programmes should be available to patients with neuropsychiatric illness.


Avulsion of spinal nerve roots in the brachial plexus (BP) can be repaired by crossing nerve transfer via a nerve graft to connect injured nerve ends to the BP contralateral to the lesioned side. Sensory recovery in these patients suggests that the contralateral primary somatosensory cortex (S1) is activated by afferent inputs that bypassed to the contralateral BP. To confirm this hypothesis, the present study visualized cortical activity after crossing nerve transfer in mice through the use of transcranial flavoprotein fluorescence imaging. In naïve mice, vibratory stimuli applied to the forepaw elicited localized fluorescence responses in the S1 contralateral to the stimulated side, with almost no activity in the ipsilateral S1. Four weeks after crossing nerve transfer, forepaw stimulation in the injured and repaired side resulted in cortical responses only in the S1 ipsilateral to the stimulated side. At eight weeks after crossing nerve transfer, forepaw stimulation resulted in S1 cortical responses of both hemispheres. These cortical responses were abolished by cutting the nerve graft used for repair. Exposure of the ipsilateral S1 to blue laser light suppressed cortical responses in the ipsilateral S1, as well as in the contralateral S1, suggesting that ipsilateral responses propagated to the contralateral S1 via cortico-cortical pathways. Direct high-frequency stimulation of the ipsilateral S1 in combination with forepaw stimulation acutely induced S1 bilateral cortical representation of the forepaw area in naïve mice. Cortical responses in the contralateral S1 after crossing nerve transfer were reduced in cortex-restricted heterotypic GluN1 (NMDAR1) knockout mice. Functional bilateral cortical representation was not clearly observed in genetically manipulated mice with impaired cortico-cortical pathways between S1 of both hemispheres. Taken together, these findings strongly suggest that activity-dependent potentiation of cortico-cortical pathways has a critical role for sensory recovery in patients after crossing nerve transfer. Amelioration of functional disabilities after peripheral nerve injury remains extremely challenging Genetically manipulated strains of mice are helpful for investigating molecular mechanisms that underlie cortical changes after crossing nerve transfer. Neural plasticity is expected to played a critical role in functional recovery from BP injury Another merit in the use of mice is that cortical activity can be investigated using transcranial imaging, with no surgical damage to the cortex In naïve mice, vibratory stimulation applied to the palm of the forepaw resulted in localized cortical responses in the S1 contralateral to the stimulated side, while the ipsilateral S1 was only weakly activated . The senSince sensory information from the injured forepaw area is lost after crossing nerve transfer, the contralateral S1 forepaw area could be occupied by afferent inputs originating outside the forepaw area One of the drawbacks to crossing nerve transfer is that the healthy BP must be cut during surgery. To induce as little damage as possible to the healthy BP, the crossing nerve transfer was also performed in an end-to-side fashion: the nerve graft was sutured to a window of the epineural sheath on the right healthy BP Cortical responses elicited by left forepaw stimulation were observed in the ipsilateral S1 at 4 weeks and the contralateral S1 at 8 weeks after crossing nerve transfer, respectively . This orIn our previous study, cortico-cortical pathways between S1 in both hemispheres are temporarily potentiated by high-frequency cortical stimulation applied to the ipsilateral S1 of anesthetized rats Since NMDA receptors play important roles in various types of synaptic potentiation Protocadherins are neuron-specific cell adhesion molecules that are thought be involved in synaptic formation Results from the present study demonstrated that cortical activity elicited by forepaw stimulation in mice was restored in the contralateral S1 after crossing nerve transfer. However, peripheral nerve injury is sufficient to induce reorganization of somatosensory cortical maps within a hemisphere The appearance of cortical responses in the ipsilateral S1 after crossing nerve transfer was easily expected, because primary sensory neurons projecting to the ipsilateral S1 have an inherent capacity to regenerate of their lesioned axons, and the regenerated axons are led to the lesioned forepaw via the nerve graft used for crossing nerve transfer NMDA receptors play an essential role in induction of synaptic potentiation that is dominated by the Hebbian rule When BP injuries are repaired by nerve transfer between the injured nerve and accessory nerve or intercostal nerve Restoration of contralateral cortical activity was more efficient in mice operated in an end-to-side fashion. Our research group examined a patient case operated in an end-to-side fashion. The patient exhibited avulsion injury to the right C7, C8, and Th1 roots, as well as laceration of the right upper trunk of BP (C5 and C6 roots), as a result of a motorcycle accident. Surgery was performed in an end-to-side fashion at 43 days after the accident. At six months after surgery, tapping of the nerve graft in the cervical subcutaneous tunnel resulted in a radiating pain sensation in the right thumb, with no tingling sensation on the contralateral left side. At nine years after surgery, tapping of the graft resulted in pain sensation in the right thumb, index finger, and palm with no tingling sensation in the contralateral left side. Subcutaneous injection of mepivacane, a local anesthetic, at the nerve graft locating in the anterior neck resulted in a transient sensory loss in the right forearm and hand, with no apparent effect on the contralateral left side. However, it is unknown why the unreversed sensation, rather than a synchronized sensation, was achieved in this patient by crossing nerve transfer in an end-to-side fashion. Somatosensory information processing in S1 is followed by processing in the secondary somatosensory cortex The present study was performed according to the guidelines for animal experiments of Niigata University and had the approval of the ethics committee of Niigata University. Male C57BL/6 mice were used. Cortex-restricted heterozygous GluN1 knockout mice Mice were anesthetized at 8 weeks of age with pentobarbital , and fastened in a supine position on a flat surface. BPs of both sides were exposed after an infraclavicular hockey stick incision of the skin under sterile conditions. The pectoralis major muscle was split along the arcuate line, and the pectoralis minor muscle was laterally retracted. The medial cord, median nerve, and ulnar nerve were identified under a binocular microscope. On the recipient side (left), the medial cord was cut at the level just proximal to the point where the medial cord diverges into the median nerve and ulnar nerve. When nerve transfer surgery was performed in an end-to-end fashion , the med0), which were obtained by dividing increased in fluorescence intensity (ΔF) in each pixel by averaged intensity in 5 frames immediately prior to stimulation (F0). Response amplitude was evaluated as ΔF/F0 in a square window of 10 by 10 pixels (0.36 by 0.36 mm) including the response peak, which was found by visual inspection in serial pseudocolor images. The window location was adjusted, so that the response amplitude in the window was maximal. When ΔF/F0 was measured in a hemisphere with almost no response, the window was placed at the location symmetrical to the response area in the contralateral hemisphere. To estimate the extent of bilateral cortical representation in S1, bilaterality index was calculated as the ratio of response amplitudes in both hemispheres. In naïve mice, it was defined as ipsilateral response amplitudes normalized by contralateral amplitudes. In mice with crossing nerve transfer, the bilaterality index was defined as contralateral response amplitudes normalized by ipsilateral amplitudes. After the imaging experiments, the mice were sacrificed with an overdose of pentobarbital (i.p.). Using a binocular microscope, it was confirmed that the left forepaw of each mouse with crossing nerve transfer was connected to the brain via the nerve graft only.Imaging experiments were performed as previously described 2) was approximately 1 mm or less. The tip of the light guide attached to the laser was placed approximately 1 cm away from the skull surface, so that an area with a diameter of approximately 1 mm was illuminated. Irradiation was maintained for 90 min, and neural activity was compared before and immediately after light exposure.Cortical activities in S1 were transcranially suppressed by photobleaching of flavoproteins and resulting suppression of aerobic energy metabolism, as previously described To produce S1 bilateral cortical representation in naïve mice, direct cortical stimulation at 100 Hz was applied for 1 s to the left S1 around the forepaw area When functional cortico-cortical connection between S1 in both hemispheres was estimated, the forepaw area was transcranially stimulated, as previously described Statistical significance was tested using StatView software . Differences between unpaired data were evaluated using the Mann-Whitney U test, and differences in paired data were evaluated using the Wilcoxon signed rank test. Only significant differences (P<0.05) are shown in the figures.


This study describes the single center experience and long-term results of ABOi kidney transplantation using a pretransplantation protocol involving immunoadsorption combined with rituximab, intravenous immunoglobulins, and triple immune suppression. Fifty patients received an ABOi kidney transplant in the period from 2006 to 2012 with a follow-up of at least one year. Eleven antibody mediated rejections were noted of which 5 were mixed antibody and cellular mediated rejections. Nine cellular mediated rejections were recorded. Two grafts were lost due to rejection in the first year. One-year graft survival of the ABOi grafts was comparable to 100 matched ABO compatible renal grafts, 96% versus 99%. At 5-year follow-up, the graft survival was 90% in the ABOi versus 97% in the control group. Posttransplantation immunoadsorption was not an essential part of the protocol and no association was found between antibody titers and subsequent graft rejection. Steroids could be withdrawn safely 3 months after transplantation. Adverse events specifically related to the ABOi protocol were not observed. The currently used ABOi protocol shows good short and midterm results despite a high rate of antibody mediated rejections in the first years after the start of the program. Matching for the antigens of the human ABO blood group system is necessary when foreign cells or organs are considered for donation. If not matched properly, the circulating anti-A and/or anti-B blood group antibodies of the recipient will bind to the antigenic moieties of the cell surface-bound A and B blood group molecules within the kidney transplant . The antPretreatment of the recipient is aimed at substantial lowering of the concentration of circulation antibodies before transplantation and reducing the subsequent production of these antibodies. To this end, a number of protocols have been developed that originally included plasmapheresis for antibody removal and splenectomy for permanent reduction of antibody production. In most protocols, high dose intravenous immunoglobulins were also perioperatively given as they exert a pleiotropic immune suppressive effect, particularly in the case of antibody mediated immune diseases. However, in recent years new protocols have been developed based on the use of the B cell depleting antibody rituximab and the availability of an immunoadsorption column that specifically binds anti-A or anti-B antibodies. This column is able to clear efficiently these antibodies from the plasma, thereby obviating the need for plasma exchange. The Swedish ABOi kidney transplantation protocol was among the first that successfully combined these new treatment modalities into a highly effective pretreatment protocol . PublishIn our transplantation center in The Netherlands, we adopted the Swedish protocol and started the ABOi transplantation program in 2006. Over the years, we have left out parts of this protocol and the use of steroids was stopped after 3 months in the posttransplantation period, similar to our standard immune suppressive protocol for ABOc patients.The long-term results of the first 50 ABOi kidney transplants in a period of 5 years are now reported in detail and safety and long-term results are in accordance with other reports.However, early antibody mediated rejections were observed more frequently than previously described although in the majority timely treatment was effective. Postoperative removal of antibodies and continuation of prednisone beyond three months after transplantation did not appear to be essential for the success of the program.2) one month prior to kidney transplantation. Two weeks before transplantation, mycophenolic acid (1000 mg bid), tacrolimus twice daily (target through level 10–15 mg/L), and prednisone 20 mg once daily were given. The immunoadsorption procedure was performed daily before the transplantation. The number of sessions was dependent on the height of the titer of antidonor blood group antibodies and the rebound after every session. Therefore, the number of pretransplantation immunoadsorptions varied with a median of 4 sessions (range from 0 to 7). During the procedure, 6 liters of plasma was passed over the column and antibody titers were assessed before and after the procedure. The kidney transplantation was performed the day after the last immunoadsorption but only if the post-adsorption IgM and IgG antibody titers were below 1 : 8. In 4 patients no immunoadsorption was performed as the anti-ABO titers were already <1 : 8. Following the original protocol, we performed 3 immunoadsorptions at days 1, 4, and 7 postoperatively in the first 25 patients. The day before kidney transplantation, after the last immunoadsorption, 0.5 gram/kg IVIG was given. In all patients, prednisone was stopped 3 months after transplantation and tacrolimus and mycophenolic acid continued, following our standard posttransplantation treatment protocol. No T cell depleting agent was used for induction therapy.Living ABOi kidney donor-recipient combinations were evaluated for the ABOi procedure after routine pretransplantation screening. Patients with titers of IgM and IgG antibodies against blood group A or B below or equal to 1 : 128 were considered eligible for ABOi kidney transplantation. At first, only O recipients and AB donors were included. The remaining ABOi couples participated in the Dutch national kidney exchange program, because of their good chance to find suitable donors. The protocol described by Tydén et al.  was follThe percentage of CMV seropositive patients was 66% and routinely all our transplantation patients, except for CMV−/− combinations, received prophylaxis with valganciclovir during the first 6 months after kidney transplantation. Routine kidney biopsies were performed in the first years of our ABOi kidney transplantation program but this policy was abandoned, as it did not contribute to clinical decision-making see .A kidney biopsy by indication was performed if rejection was suspected on the grounds of an unexpected halt in improvement or worsening of kidney function. Histological confirmation of rejection was obtained by following the Banff criteria 07 for acute cellular and antibody mediated rejection (AMR). Acute AMR was treated with extra immunoadsorptions, high dose steroids (1000 mg prednisolone/day for 3 days), and IVIG (1 gram/kg). In case of unresponsiveness, T cell depleting therapy was given according to our local protocol. The first 25 patients received postoperative immunoadsorptions per protocol and were routinely monitored for isoagglutinin titers after transplantation. As the isoagglutinin titers remained low and no rebound occurred, we abandoned the policy of routine posttransplantation immunoadsorptions.For the purpose of comparison, 2 cases of ABO compatible kidney transplantation were selected from the same period and matched for age and number of HLA mismatches for every case of ABOi kidney transplantation. The posttransplantation immune suppressive medication protocol was similar to the ABOi protocol except for induction therapy with basiliximab in all patients from 2007 onwards.Patient serum was tested for the presence of IgM and IgG antibodies against the ABO-blood group antigens of the donor using the red blood cells (RBC) of the donor. Hemagglutination was assessed fully automated using the ORTHO BioVue system column agglutination technology. Test RBC, with or without serum, were placed in the chamber above the 6-microcolumn cassette preloaded with diluent and/or reagent and glass beads. Upon centrifugation, RBC are forced through the bead column where agglutinated cells are trapped, while unagglutinated RBC travel to the bottom of the column forming a discrete pellet. The isohemagglutinin titer is determined by the highest dilution of patient serum that still results in donor RBC agglutination.t-tests. Skewed distributed continuous variables are presented as medians and compared using the nonparametric Mann-Whitney U test. Categorical variables are presented as numbers and/or percentages. For selected comparison between two group proportions, the chi-square test was used. Survival percentages were analyzed with the Kaplan-Meier method. A P value of less than 0.05 was considered significant; all probabilities were 2-tailed.The SPSS software version 18.0 was used for all statistical tests. Descriptive statistics were used to summarize baseline characteristics. Distribution of data was tested using the Kolmogorov-Smirnov test. Continuous variables with a normal distribution are presented as means (SD) and compared using parametric From the start of the ABOi program in 2006 until March 2012, a total of 50 patients received an ABOi kidney transplant from a living donor and clinical follow-up was at least one year with a median follow-up of 38 months. The clinical and demographical patient characteristics are shown in P = 0.43). Two patients in the ABOi group died during follow-up; one committed suicide and one patient died because of abdominal sepsis due to an incarcerated hernia cicatricalis. Serum creatinine concentrations at 1 and 3 years follow-up were significantly higher (P < 0.05) in the ABOi group (median 137 μmol/L and 154 μmol/L) compared to the ABOc group (median 114 μmol/L and 123 μmol/L). This difference in graft function was largely attributable to the decreased graft function in ABOi patients after their AMR had resolved. The median serum creatinine concentration in the ABOi patient group without AMR was 135 μmol/L at 1 year and 124 μmol/L at 3 years follow-up. For ABOi patients with early AMR, these values were 172 μmol/L and 172 μmol/L, respectively. Two patients had an exceptionally poor renal function at 3 months after transplantation. One case was caused by a surgical problem leading to severe problems with renal blood supply and subsequent partial renal infarction and the other case was due to an AMR resistant to treatment. Of note is one patient that presented 5 months after transplantation with an urosepsis with Serratia species and a very severe acute AMR necessitating transplantectomy. Anti-A titers were sharply risen (IgM > 5000 and IgG 512).The majority of ABOi patients showed an uncomplicated clinical course and graft survival censored for death at 1 year, 3 years, and 5 years was 96%, 90%, and 90% compared to, respectively, 98%, 96%, and 96% in the ABOc group . ABOi grIn eleven patients, acute AMR was observed, all within the first week after transplantation except for the case of late acute AMR described above. Donor specific antibodies against HLA were only found in 2 patients, both having anti-HLA-DQ7 antibodies with a MFI of 20.000 in the single antigen beads Luminex assay. The C4d staining was positive in 8 biopsies, weak to focally positive in 2, and negative in 1 kidney biopsy. Five of these early rejections were categorized as AMR at the time of biopsy and responded to treatment with IVIG and steroids. In 5 cases, the renal biopsy showed a mixed type rejection with evidence for the coexistence of both cellular and AMR. T cell depleting therapy was used in case of an inadequate response to IVIG and steroids. In one of these patients, graft loss occurred 6 months after transplantation because of treatment resistant AMR. One patient had an acute cellular rejection at 7 months when his tacrolimus through level was inadvertently low.Routine allograft kidney biopsies at day 7 after kidney transplantation showed C4d positive staining in 8 out of 19 (42%) patients but without other positive criteria to support the diagnosis of AMR.We could not find any significant relation between antibody titers postoperatively and subsequent AMR, as all titers remained low after transplantation . Because of this observation, we stopped the posttransplantation immunoadsorptions after the first 25 patients. This change in protocol did not alter the frequency of AMR and, in fact, most AMR episodes were observed in the patients that received posttransplantation immunoadsorptions (9 out of 11 patients with AMR). In the group of ABOi patients with an early AMR episode, only one patient was diagnosed with transplant glomerulopathy seven years after transplantation.n = 2), BK virus (n = 3), and herpes zoster virus (n = 3). In addition, 1 case of Pneumocystis carinii pneumonia was recorded. The frequency of these infections is in the range of our ABO compatible program and cannot be specifically related to the use of rituximab or IVIG.All adverse events, possibly related to a change in immune suppression, notably the use of rituximab and IVIG, were recorded. The posttransplantation viral infections in the ABOi group were caused by cytomegalovirus (Newly developed desensitization protocols have greatly facilitated the development of ABOi kidney transplantation programs in many countries. Similar to other groups, we have successfully applied the Swedish desensitization protocol for more than 5 years and did not observe any significant side effects. By combining the immunoadsorption procedure with hemodialysis, it was also possible to reduce the total number of procedures. This is not only time-effective but also less burdensome for dialysis patients.In accordance with a number of other studies –11, the In agreement with the experience of others , 18, we We did not observe a relation between posttransplantation anti-A and B titers and rejection and all posttransplantation titers remained low. Therefore, we removed the posttransplantation immunoadsorptions from the protocol. As stated before, the frequency of AMR actually was lower in the 25 patients who did not receive the postoperative immunoadsorptions. Other groups have also reported on the results of ABO-incompatible transplantation without posttransplantation plasmapheresis or immunoadsorption, confirming that such a strategy can indeed be followed without a significant increase in the incidence of AMR , 15, 20.The combined elements of the current ABOi protocol have been chosen on a rationale based on the proposed mechanism of action of each drug. For instance, pretreatment with rituximab depletes the circulating B cell population. This may be important in the prevention of increased anti-A and anti-B antibody synthesis after ABOi kidney transplantation as rituximab treatment diminishes de novo humoral immune responses. However, a recent report showed that a rituximab-free ABOi protocol yields similar excellent short- and long-term results after kidney transplantation . This isGiven these results, it seems that a simplified protocol with immunoadsorption and immune suppressive drugs in the pretransplantation period is equally effective. The need for IVIG in the ABOi protocol remains to be established as this is not a standard procedure in some other ABO-incompatible kidney transplantation protocols , 20, 24.After transplantation, we routinely stopped prednisone and continued with tacrolimus and MMF. Others found that this policy may lead to an increase in the incidence of acute rejection and is associated with an decreased graft survival . HoweverIn conclusion, the “Swedish protocol” with immunoadsorption, IVIG, and rituximab, added to a standard triple immune suppressive regimen, has been used successfully for more than 5 years in our transplantation center despite an initial high frequency of AMR. It allows for ABOi kidney transplantation with good long-term graft survival comparable to ABO compatible kidney transplantation. Important questions, such as the upper level of anti-A and anti-B that can be accepted before kidney transplantation and the optimal preconditioning treatment, remain to be answered. The ABOi treatment protocol is of value for those patients that cannot be matched to a donor kidney because of blood group incompatibility. Although our center is involved in an effective national donor kidney exchange program, the O positive recipients benefit in particular from the ABOi protocol as they have the lowest chance of success in the kidney exchange program , 27.


We have used cultured human synovial fibroblasts that respond to adiponectin by increasing the expression of IL-6, TNF-alpha and IL-1beta via ADIPOR1. The biological effect on fibroblasts of osmotin and its peptide derivative has been found similar to that of adiponectin confirming the results found in silico.Osmotin, a plant protein, specifically binds a seven transmembrane domain receptor-like protein to exert its biological activity via a RAS2/cAMP signaling pathway. The receptor protein is encoded in the gene ORE20/PHO36 and the mammalian homolog of PHO36 is a receptor for the human hormone adiponectin (ADIPOR1). Moreover it is known that the osmotin domain I can be overlapped to the β-barrel domain of adiponectin. Therefore, these observations and some already existing structural and biological data open a window on a possible use of the osmotin or of its derivative as adiponectin agonist. We have modelled the three-dimensional structure of the adiponectin trimer (ADIPOQ), and two ADIPOR1 and PHO36 receptors. Moreover, we have also modelled the following complexes: ADIPOQ/ADIPOR1, osmotin/PHO36 and osmotin/ADIPOR1. We have then shown the structural determinants of these interactions and their physico-chemical features and analyzed the related interaction residues involved in the formation of the complexes. The stability of the modelled structures and their complexes was always evaluated and controlled by molecular dynamics. On the basis of these results a 9 residues osmotin peptide was selected and its interaction with ADIPOR1 and PHO36 was modelled and analysed in term of energetic stability by molecular dynamics. To confirm  These experimental observations suggest that osmotin binds the adiponectin receptor (ADIPOR1) in a cellular environment, made up of human cells, by activating the same signaling path of adiponectin OSM) and evaluated its ability to interact with ADIPOR1 and PHO36. The peptideOSM/ADIPOR1 complex shows that the peptide is firmly positioned in the same area of receptor with which both the adiponectin and the osmotin interact. Finally, experiments on synovial fibroblasts have shown that this peptide, as well as the whole osmotin, interacts with ADIPOR1 by activating the same signaling pathway activated by the adiponectin. The results, even if at an initial stage, show that the peptideOSM can be taken into account as adiponectin-receptor agonist, once suitably amended to make it enzymes resistant.On the basis of this hypothesis we have searched answers to the following questions: a) What are the structural similarities in the interaction of osmotin and adiponectin with the adiponectin receptor (ADIPOR1)? b) Knowing the structural basis of their interaction with the receptor, is it possible to isolate a biologically active peptide which mimes the adiponectin binding to ADIPOR1? We have used some methods of molecular modeling for searching and having those structural answers able to address functionally our questions. Therefore, in this paper we report the 3D modeling of the globular domain of the human adiponectin trimer (ADIPOQ), that of its receptor, ADIPOR1, that binds the globular domain, and that of PHO36. Moreover, we also modeled the ADIPOQ/ADIPOR1, osmotin/PHO36 and osmotin/ADIPOR1 complexes. Then, all the amino acids at the interface in these complexes were evaluated in order to know the structural and chemical features of these interaction residues that might be useful in the drug design involved in the treatment of obesity-related insulin resistance. Therefore, we have selected a small osmotin peptide  was performed by a comparative modelling strategy using as template structures the related mouse adiponectin chains (PDB code: 1C3H). Since the template structure contained two trimers having different loop conformations among beta-strands, we have used: i) the murine A and D chains to model the human monomer A, ii) the murine B and E chains as template for the human monomer B and iii) the murine C and F chains as template for the human monomer C. The sequence alignment was made by using CLUSTALW program http://opm.phar.umich.edu/) as well as a fold recognition program LOOPP Natronomonas pharaonis) phototaxis protein rhodopsin II (PDB code: 1GU8) presented similar lengths of helices and loops between the helices respect to ADIPOR1 and, thus, suggested that this protein was the correct template for predicting the three-dimensional structure of ADIPOR1 by comparative modelling strategy. Moreover, in the obtained model the loop regions were refined using the LOOPY module of Jackal package, that appeared to yield the best results in loop modelling, with models that are on average of 2–8% better than those generated by other programs Natronomonas pharaonis) phototaxis protein rhodopsin II (PDB code: 1GU8).Modeling of the human adiponectin receptor 1 (ADIPOR1),  was obtained using an integrated protocol based on the method of fold-recognition and comparative modeling and already used in our recent paper Firstly PatchDock web server OSM. The peptide shows a disulphide bond between Cys 157 and Cys 162 that stabilizes it during the simulations. This structural feature proved to be important in comparison with all other different peptides screened from osmotin as well as the same adiponectin and involved in the interaction surface between osmotin/adiponectin and ADIPOR1. These peptides were poor ligands from an energetic point of view probably because of their high flexibilities. The chosen peptide was subjected to molecular dynamics by GROMACS software package (v3.3.1) OSM and ADIPOR1 was modeled and analyzed using the same procedure reported above.Starting from the osmotin structure we have selected the peptide CTQGPCGPT, (segment 157–165), that hereafter we will name as PeptideOSM/PHO36 complexes were modeled and analyzed using the obtained model for PHO36 by comparative modelling strategy, the crystallographic structure of Osmotin (PDB code: 1PCV) Osmotin/PHO36 and Peptide18 column and its purity was checked by MALDI-TOF mass measurements (Voyager-DE Biospectrometry). Disulfide bond was formed after cleavage and deprotection of the relevant linear peptide with Cys' protecting groups. The two side-chains protecting groups of cysteine were removed at the same time as peptide cleavage and the disulfide bond was formed using 0.95 equiv. of 2,20-dithiodipyridine (PySSPy) and 1 mM peptide in 0.1% (v/v) TFA in water at room temperature. The oxidation was completed after 2 h and the product was purified by preparative RP-HPLC. The product was identified by MALDI-TOF. The peptide was poorly soluble in water at low pH. Its solubility increased at higher pHs and at pH 6.5 was completely soluble. A batch solution 1×10−3 M PeptideOSM, 5×10−3 M K-phosphate buffer at pH 7.4 was used through the experiments diluting it each time to the appropriate concentration. The peptide concentration was determined by using a molar extinction coefficient at 276 nm of 165 M−1 cm−1 for Cistine as suggested by Pace et al. (1995) The solid-phase peptide synthesis by Fmoc chemistry was used to obtain our peptide (having pI = 5.50), combined with the S-acetamidomethyl protection for cysteines. Rink-amide resin was used as a solid-phase carrier, and TBTU was used as a coupling reagent. Piperidine in dimethylformamide (20% solution) was used to remove the Fmoc group at every step. The coupling and deprotection reactions were checked by the Kaiser test. Cleavage from resin and side-chain deprotection were achieved using a mixture of 82.5% TFA, 5% thioanisole, 5% m-cresol, 5% water, and 2.5% ethanedithiol). The peptide was analyzed on a reversed-phase high performance liquid chromatography (HPLC) Cicotiana Tabacum seeds. Purification was achieved by using a procedure consisting of an acid treatment step followed by two chromatography steps, already reported for an osmotin-like protein from the seeds of Benincasa hispida−1cm−1 calculated by the known content of Trp (5690 M−1cm−1), Tyr (1280 M−1cm−1 and Cys (120 M−1cm−1) present in the sequence Osmotin was purified to homogeneity from Nin vivo the cellular effects of the osmotin and PeptideOSM in comparison with those exerted by adiponectin, we have examined the IL6, TNF-alpha and IL-1beta profiles from normal Synovial tissues obtained by arthroscopic biopsy To elucidate Human full-length adiponectin  (5 µg/ml), osmotin (5 µg/ml) and peptide (5 µg/ml) administration was followed by 24 h of incubation at 37°C in a humidified incubator and separate plates, the media were then removed and stored at −80°C until assay. IL-6, TNF-alpha and IL-1beta in the medium were assayed using a multiplex biometric ELISA-based immunoassay, containing dyed microspheres conjugated with a monoclonal antibody specific for a target protein and a procedures already used in our previous article The three-dimensional model of the human adiponectin trimer was performed, according to the comparative modelling strategy and using as template structures three murine chains because the sequence identity percentage between human and mouse proteins was equal to 91%. We have modeled ADIPOR1 receptor because it is known to bind the globular part of ADIPOQ (i.e. without the related collagen part). The alignment between ADIPOR1 and the template structure used for the modelling procedure is reported in The ADIPOQ/ADIPOR1 complex was modeled  and the OSM (region 157–165 in the osmotin sequence) is located at the interface between the osmotin and ADIPOR1  and residues to the interface, H-Bonds, salt bridges  and 2. I bridges  and 2.in vivo effect of osmotin and its derivative peptide on the adiponectin receptor ADIPOR1, we have purified osmotin from seeds of Nicotiana Tabacum at homogeneity according to Shih et al. (2001) OSM was synthesized  OSM shows a similar effect activating both the same receptor and the signaling pathway. We have also used two other peptides of nine and eleven residues, respectively, with random sequences but they did not show any biological activity in cultured synovial fibroblasts (data not shown). Even so two unrelated proteins ) were tested with negative results. It is noteworthy that BSA has been already used as a negative control to check the binding on unrelated proteins to osmotin receptor OSM, even if biologically active, should be chemically modified to be used in vivo usefully.It has been recently demonstrated that adiponectin acts as an inducer of inflammatory response by increasing IL-6 production in human synovial fibroblasts. It was found that adiponectin increases the expression of IL-6 and ADIPOR1 but not ADIPOR2 ized see  and its in vivo on cultured synovial cells together with osmotin. The biological response of the adiponectin-receptor was to activate the same signaling pathway enabled by the adiponectin hormone. Experimental data have demonstrated that the suppression of ADIPOR1 with small interfering RNA (siRNA) reduced the increase in fatty-acid oxidation by globular domain of the adiponectin In this article, on the basis of experimental data of several authors Independent results showed that a protein from plants, with no sequence homology with the human adiponectin, interacts with a membrane receptor codified by the same type of gene encoding for the membrane receptor of human adiponectin When the osmotin/ADIPOR1 and ADIPOQ/ADIPOR1 complexes are compared with superimposed ADIPOR1 receptors, the lectin domains of osmotin and of ADIPOQ are not in the same orientation see . Likely,2) and of interaction residues.Comparing the Osmotin/PHO36 and Osmotin/ADIPOR1 complexes one can evidence that ADIPOR1 presents at the interface two positively charged residues (i.e. one Lys and one Arg) whereas PHO36 both positively (i.e. Lys) and negatively (i.e. Glu) charged residues. Moreover, the osmotin exposes a positively charged residue (Lys 169) and two negatively charged residues (Asp 184 and Asp 185) in the Osmotin/PHO36 complex, but at the interface in Osmotin/ADIPOR1 complex there are three positively and three negatively charged residues, as reported above. Even in these complexes interactions are stabilized by many aromatic residues see . This hiAll these results evidence that: i) the interaction regions are conserved in the complexes, ii) the interactions are on electrostatic basis but stabilized also by stacking interactions and iii) these findings can be used for the development of new ADIPORs agonists able to stimulate adiponectin receptors.OSM, is stabilized from a disulphide bond that makes rigid its conformation. In fact, after molecular dynamics simulation, only the C-terminal residues of this peptide have been subjected to conformational changes  evidenced in the other two complexes, i.e., ADIPOQ/ADIPOR1 and Osmotin/ADIPOR1 , of number of interaction residues, of H-bonds, and of salt bridges, too  that was observed at the interface between osmotin and ADIPOR1 as well as between osmotin and PHO36 . This pe changes . In the /ADIPOR1 . We haveHO36 see  with hig too see  and 2.in silico modeling, we have purified osmotin and synthesized the PeptideOSM. Afterward we have compared the in vivo effect of adiponectin, osmotin and PeptideOSM on cellular cultures of synovial fibroblasts which are known to be specifically stimulated through the signaling downstream pathway of the ADIPOR1 receptor each time it is bound to the adiponectin. Our in vivo results demonstrate that both osmotin and PeptideOSM interact with this same receptor by triggering the same specific pathway of adiponectin with the same final products, i.e., IL-6, TNF-alpha and IL-1beta, as already shown by Tang et al. (2007) in silico shows similar cellular effects in vivo, is surprising because, in general, an interaction requires that interacting residues be in clearly complementary sequence positions. This requires that the two structures must also predispose these interacting residues at a suitable distance and well-placed for an efficient interaction. In this case we have two similarly structured proteins with different sequences that interact with the same region of a third part (ADIPOR1) by triggering the same cellular process. Osmotin is not evolutively adapted to interact with ADIPOR1, therefore, we have only two choices: a) the osmotin structure is so flexible to adapt its sequence to the same ADIPOR1 region where adiponectin interacts too; b) the outer ADIPOR1 region is so flexible to adapt itself to different ligands structurally similar. Answer and possible explanation can be given on the basis of some of our recent observations. In fact, we have found that the outer loops and/or segments of numerous cytokine receptors have shown, by the consensus of eight different predictors and charge-hydrophobicity graphs, a consistent and abundant presence of intrinsically disordered structural stretches OSM is structurally stable and structurally rather rigid with a good affinity to the ADIPOR1 receptor and correctly located to mimic the adiponectin binding, at the moment it represents, at best of our knowledge, the only good model of adiponectin agonist in vitro. Another similarly located peptide from adiponectin resulted very flexible and thus with lower affinity (data not shown).To support the conclusion reached by our OSM is quite conserved in the family of the thaumatin-like proteins; in fact, its sequence has an identity percentage of about 73% in respect to thaumatin-like proteins of other species . In the literature no mention was found about an effect on ADIPOR1 exerted by other members of thaumatin-like proteins. However, a comparative analysis should be made on these proteins to study their putative role on the binding with ADIPOR1 but this is outside the aim of this paper.Moreover, the peptideOSM could be used, opportunely modified to overcome difficulties in generating biologically active molecules, as a drug design template for the treatment of obesity-related insulin resistance. However, this study also raises questions about the role of this plant protein for the human health. No studies have been performed to assess if and how the osmotin affects the human health. This protein is enough stable and resistant to acidity and enzymes and thus might circulate through the body as peptides and/or interacting molecule by considering that it is present in many edible fruits and vegetables. In general, we have only a poor knowledge of the biologically active compounds in the foods. Our finding that an osmotin peptide, structurally very stable, is able to mimic the adiponectin, raises the question whether the osmotin could play any role in the health benefits attributed to diets high in fruits and vegetables. However this is another problem that will need different approaches to be studied.In this work, we suggest that the PeptideFigure S13D model of two complexes. (A) Osmotin/PHO36 complex with Osmotin in fuchsia and PHO36 in cyan and (B) PeptideOSM/PHO36 complex with peptide in green and PHO36 in cyan.(DOC)Click here for additional data file.Figure S2Osmotin sequence. Amino acids in the helices are reported in grey but those in beta-strands are underlined. Interaction residues with the ADIPOR1 are evidenced in bold.(DOC)Click here for additional data file.Figure S3RMSD evolution during the molecular dynamics performed on the osmotin peptide (region 157-165).(DOC)Click here for additional data file.Figure S4Pairwise alignment between the sequences of human adiponectin and yeast Pho36 receptors.(DOC)Click here for additional data file.Figure S5Structure of plant osmotin (A) and human adiponectin (B). We have evidenced with a circle the domain I of the osmotin having a similar fold to that of the adiponectin.(DOC)Click here for additional data file.Figure S6Comparison between ADIPOQ/ADIPOR1 and osmotin/ADIPOR1 complexes concerning ADIPOR1 receptors superimposed. 3D model of Osmotin is colored in fuchsia and monomer A, B an C of ADIPOQ in red, blu and green, respectively.(DOC)Click here for additional data file.Figure S7Comparison of the osmotin and adiponectin residues which contact ADIPOR1 obtained superimposing ADIPOR1 receptors in two ADIPOQ/ADIPOR1 and osmotin/ADIPOR1 complexes. In particular, the residues are reported in CPK. Osmotin residues are colored in cyan but those in the adiponectin trimer in yellow.(DOC)Click here for additional data file.Figure S8Binding free energies for five complexes. The bars represent the binding energies .(DOC)Click here for additional data file.Table S1Evaluation of Z-score by ProsaII program (A) and % residues in favored regions (B) for three adiponectin monomers in human and mouse.(DOC)Click here for additional data file.Table S22), number of interchain H-bonds, interaction residues and salt-bridges.Analysis of the interaction among monomers in human and murine adiponectin trimers. The table shows interface accessible surface areas (Å(DOC)Click here for additional data file.