We looked for herpes simplex virus types 1 and 2 , varicella zoster virus (VZV), Epstein–Barr virus (EBV) and cytomegalovirus (CMV) DNA in Malawian adults with clinically suspected meningitis.We collected cerebrospinal fluid (CSF) from consecutive adults admitted with clinically suspected meningitis to Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi, for a period of 3 months. Those with proven bacterial or fungal meningitis were excluded. Real-time polymerase chain reaction (PCR) was performed on the CSF for HSV-1 and HSV-2, VZV, EBV and CMV DNA.A total of 183 patients presented with clinically suspected meningitis. Of these, 59 (32 %) had proven meningitis , 39 (21 %) had normal CSF and 14 (8 %) had aseptic meningitis. For the latter group, a herpes virus was detected in 9 (64 %): 7 (50 %) had EBV and 2 (14 %) had CMV, all were human immunodeficiency virus (HIV)-positive. HSV-2 and VZV were not detected. Amongst those with a normal CSF, 8 (21 %) had a detectable herpes virus, of which 7 (88 %) were HIV-positive.The spectrum of causes of herpes viral meningitis in this African population is different to that in Western industrialised settings, with EBV being frequently detected in the CSF. The significance of this needs further investigation. In African settings, aseptic meningitis is thought to be common, but the aetiology is rarely identified and there have been few studies examining this question , 2. In iReactivation of herpes simplex virus (HSV) types 1 and 2, varicella zoster virus (VZV), Epstein–Barr virus (EBV) and cytomegalovirus (CMV) are common in the context of human immunodeficiency virus (HIV) infection and HIV–herpes virus co-infection has been linked with a more severe disease course , 6. WhilIn sub-Saharan Africa, very little is known about the prevalence of HSV, VZV, EBV and CMV as causes of aseptic meningitis in the context of HIV. We, therefore, looked for HSV, VZV, EBV and CMV infections in Malawian adults with clinically suspected meningitis, but without evidence of bacterial or fungal infection.3). Patients with a diagnosis of a CNS infection within the last month were also excluded; this was primarily done to ensure that our cohort of patients had new clinical events and to avoid selecting those with a relapse of past CNS infection. Patients with ≥5 white cells per mm3 of CSF were defined as having an aseptic meningitis, whilst those with <5 white cells per mm3 were defined as not having meningitis using a laboratory diagnostic criteria [This study was a sub-analysis of cerebrospinal fluid (CSF) from a cohort of adults (≥16 years of age) with clinically suspected meningitis admitted to Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi, from September through to December 2007 . Demogracriteria . A CD4 cThe study was approved by the local ethics committee at the College of Medicine, University of Malawi, Blantyre, Malawi (P05/06/471) and the ethics committee medical faculty, University Heidelberg, Heidelberg, Germany (S 026/2007).CSF samples were aliquoted and stored at −70 °C prior to molecular diagnostics for HSV, VZV, EBV and CMV. The samples were tested as a batch at the time of this study.DNA was extracted from 200 µl of CSF using a QIAamp DNA kit as per the manufacturer’s instructions. DNA recovery was confirmed by spectroscopic analysis. We used 5 µl of DNA for each polymerase chain reaction (PCR) for the HSV, VZV and EBV assays, and 10 µl for the CMV assay.Primers and probes targeting conserved regions of the DNA polymerase gene were used for HSV-1 ; HSV-2  and VZV . Similarly, the tegument protein (BNFR1 gene) for EBV and UL123/UL 55 genes for the CMV genome were targeted .Applied Biosystems  AmpliTaq Gold (2×) Master Mix was used for all the real-time PCR assays. All reactions were carried out in the ABI 7300 real-time PCR machine using 96-optical-well plates, with a total volume of 25 µl per well. The PCR thermocycler conditions for all assays included three stages; activation of UNG at 50 °C for 2 min, activation of Taq at 95 °C for 15 min, followed by amplification using 45 cycles at 95 °C for 15 s and 60 °C for 1 min.n = 3) and the sample was then considered to be positive if the test result was positive (n = 1). Quantification was only performed with EBV and shown as genome copies per millilitre of CSF. Serial dilution of 100–108 copies/ml of virus stock was used to obtain a standard curve with an efficiency performance of 92 %. The concentration of EBV genome in the samples was interpolated from this standard curve.A positive control  for each herpes virus was run with unknown specimens to ensure DNA recovery. Similarly, specimens were processed in parallel with aliquots of molecular-grade water to monitor for false-positive results; the run was discarded if this was the case. Each individual sample was processed in triplicate and the sample was considered to be positive if two or more of the three test results were positive. When only one out of the three test results was positive, the PCR test was repeated . We found the limit of detection to be 10http://www.spss.com). Quantitative variables were expressed as medians with interquartile ranges and qualitative variables as frequencies and percentages. Fisher’s exact test was used to compare categorical data and the Mann–Whitney U test for continuous data; a p value of <0.05 was taken to be statistically significant.The data were analysed using PASW Statistics 18, Release Version 18.0.0 .A total of 183 patients presenting with clinically suspected meningitis were identified over the 3-month study period. Of these, 59 (32 %) had proven meningitis , 39 (21 %) had normal CSF and 14 (8 %) had an aseptic meningitis. In 48 (26 %) patients, there was a relapse of a previous CNS infection ; all of them were HIV-positive. Amongst those with a normal CSF, 8/39 (21 %) had a detectable herpes virus; of these, 7 (8 %) were HIV-positive.The prevalence of the individual herpes virus in those with aseptic meningitis 14) was 7 (50 %) EBV and 2 (14 %) CMV. In those with a normal CSF (39), 6 (15 %) had EBV and 2 (5 %) had HSV-1 detected. No patient had HSV-2 or VZV detected , compared with 16 HIV-positive patients. Thirteen (36 %) patients with HIV infection had EBV detected in the CSF.3) was 1,202 copies/ml, compared to 6,680 copies/ml in those with an abnormal CSF (≥5 cells/mm3) (p = 0.048). The median CSF EBV titre in patients with advanced HIV infection was 2,223 copies/ml, compared with 1,239 copies/ml in those with less advanced disease (p = 0.52).To investigate the relevance of the high EBV prevalence found exclusively in the HIV-positive group, we explored the relationship between CSF EBV titre, CSF white cell count (WCC) and CD4 count. The median titre of EBV in those with a normal CSF (<5 cells/mmWe have found that aseptic meningitis constituted 8 % of the patients with clinically suspected meningitis in our setting. Surprisingly, no HSV-2 or VZV DNA was detected in the aseptic CSF; instead, we detected a high prevalence of EBV and all of these patients were HIV-positive.The prevalence of aseptic meningitis in our cohort was lower than in Western industrialised countries, where aseptic meningitis accounts for about 50 % of acute meningitis . As far It is conceivable that the low frequency could be due to an ascertainment bias, as patients with aseptic meningitis have milder self-limiting disease and, therefore, do not present to hospital. Alternatively, advanced HIV infection might modulate the inflammatory response to viral infections in the CNS and, so, there is no pleocytosis. This might be relevant to the 6 of 39 patients with normal CSF and detectable EBV.Our failure to detect HSV-2 and VZV in this cohort was unexpected, since both are common identifiable causes of viral meningitis in Western industrialised countries , 17. It A striking finding was the high incidence of EBV in the CSF of HIV-infected individuals, even in those with a normal CSF. The presence of EBV in the CSF of people with HIV is usually associated with primary CNS lymphoma rather than meningitis , 18. It The high detection of EBV in the CSF could be relevant to the clinical presentation or it could reflect activation in immunocompromised patients that is of no pathogenic significance. However, the detection of EBV was significantly higher in those with a raised CSF WCC. Ideally, we would have liked to explore the association of EBV with clinical outcome, but the retrospective nature of our study limited this. We recently described an association with high EBV titres in the CSF and poor outcome in patients with bacterial meningitis, which suggests that there might be more to this story .In conclusion, we saw a different spectrum of herpes virus infections in this population than that seen in Western industrialised settings. EBV was frequently detected in the CSF, regardless of whether there was a pleocytosis or not, though higher viral titres were seen in patients with a pleocytosis and in HIV-positive patients. The significance of these findings merits further investigation.


Human herpesviruses 6 and 7 were infrequently found in cerebrospinal fluid of children with central nervous system infection. The role and frequency of human herpesviruses (HHV)-6 and -7 in central nervous system (CNS) diseases of children are unclear. Cerebrospinal fluid samples from 245 pediatric patients (median age 43 days), submitted for evaluations of possible sepsis or of neurologic symptoms, were tested for HHV-6 and HHV-7 DNA by polymerase chain reaction. HHV-6 DNA was found in 3 of 245 samples, and HHV-7 was found in 0 of 245 samples. The three patients with HHV-6 DNA were <2 months of age. HHV-6 was likely pathogenic in two patients with meningitis who lacked evidence of another microbiologic cause. HHV-6 and HHV-7 are uncommon causes of CNS infection in children. HHV-6 may occasionally cause meningitis in young infants. Human herpesvirus (HHV)-6 and HHV-7 are ubiquitous T-lymphotropic viruses that infect most humans. Infections with either agent occur primarily during childhood. Seroprevalence of HHV-6 reaches >80% in children >2 years –3. AntibThe role of HHV-6 and 7 in central nervous system (CNS) disease is an area of ongoing investigation. The range of CNS manifestations ascribed to these viruses includes asymptomatic infection, febrile convulsions, seizure disorders, meningitis, meningoencephalitis, facial palsy, vestibular neuritis, demyelinating disorders, hemiplegia, and, rarely, fatal encephalitis –18. InveBecause of the conflicting results in the medical literature, the frequency at which HHV-6 and HHV-7 are associated with neurologic disease is unclear. The goal of this study was to further define the role of HHV-6 and HHV-7 as causes of CNS disease in children.The study, approved by the University of Colorado Multiple Institutional Review Board, was conducted with all CSF clinical samples from pediatric patients submitted for herpes simplex virus (HSV) PCR to the Clinical Virology Laboratory at the University of Colorado from December 1998 through February 2000. When multiple specimens were submitted for one patient, only the first one was tested. Specimens positive for other microorganisms were not excluded. Peripheral blood specimens from these patients were not available to study. Information regarding demographics, clinical manifestations, diagnostic studies, management, discharge diagnosis, and outcome was gathered by retrospective chart review for patients seen at The Children's Hospital, Denver. CNS diagnoses and classification of seizures were based on the assessments of the primary treating physicians.6/L for preterm neonates, >22 leukocytes x 106/L for term neonates, and >7 leukocytes x 106/L for all other patients. Infections not involving the CNS were classified as other infections.Infectious and postinfectious encephalitis were defined as the presence of encephalopathy or focal neurologic abnormalities, an abnormal CSF profile but negative CSF microbiologic studies, and a history or serologic result consistent with a current or preceding acute infectious illness. CSF pleocytosis was defined as >25 leukocytes x 10HHV-6 PCR was performed  with theThe analytical sensitivity of HHV-6 PCR was 8–10 copies of genomic DNA per reaction tube, as determined by serial dilutions of a sample with known DNA copy number. To rule out the presence of inhibitors in the extracted DNA, 20 HSV-negative CSF samples were spiked with the equivalent of 2 HSV DNA copies per PCR reaction tube. All samples yielded a positive result for HSV, whereas the unspiked specimens remained HSV-negative. The ability to detect HHV-6 strains from different geographic regions was confirmed by using 14 HHV-6–containing specimens obtained from diverse laboratories with recognized expertise in diagnosing HHV-6 infections as well as our laboratory. No cross-reactivity occurred with other DNA viruses, including other herpesviruses, parvovirus, adenovirus, and polyomavirus, thereby establishing the specificity of the HHV-6 PCR.HHV-7 PCR was performed by using the methods described for HHV-6 with modified sample preparation. The primers were 5´ TAT CCC AGC TGT TTT CAT ATA GTA AC and 5´ GCC TTG CGG TAG CAC TAG ATT TTT TG, and the probe was 5´ AGA ATT CTG TAC CCA TGG GCA CAT TTG TAC . Testing with control viral DNA indicated that boiling did not affect the sensitivity of the PCR. The presence of inhibitors was ruled out by spiking 20 HSV-negative CSF samples with the equivalent of two HSV DNA copies per reaction tube and showing that all spiked samples became HSV-positive, whereas unspiked samples remained negative. The ability to detect HHV-7 DNA after at least three freeze-thaw cycles remained intact.CSF samples obtained from 245 patients were tested for HHV-6 and HHV-7 DNA by PCR. Clinical data were available for the 218 patients hospitalized at The Children's Hospital, Denver . The med6/L (46% neutrophils and 21% bands) and a platelet count of 40,000 x 106/L. His CSF showed 40 leukocytes x 106/L , 4,110 erythrocytes x 106/L, protein 169 g x 10-2/L, and glucose 2.3 mmol/L. CSF HSV PCR, viral culture, and bacterial cultures of CSF and blood and urine were negative. Diagnosis at discharge was aseptic meningitis. The patient recovered spontaneously without sequelae.PCR identified HHV-6 DNA in CSF of 3 of 245 patients . Patient6/L , 1 erythrocyte x 106/L, protein 48 g x 10-2/L, and glucose 2.3 mmol/L. PCRs of CSF for HSV and enterovirus and bacterial cultures of CSF, blood, and urine were negative. She recovered spontaneously without sequalae. Her discharge diagnosis was aseptic meningitis.Patient 2 was a 38-day-old full-term female infant who had a fever of 39.2ºC and irritability. The physical examination showed a few vesicular lesions on the tonsillar pillar as well as a diffuse, erythematous, macular blanching rash. CSF studies found 19 leukocytes x 10Candida. A lumbar puncture was performed when the blood grew Candida. The patient did not have any neurologic symptoms or signs. CSF had 1 leukocyte x 106/L, 1,780 erthrocytes x 106/L, protein 105 g x 10-2/L, and glucose 8.1 mmol/L. CSF studies, including PCRs for HSV and enterovirus, as well as bacterial, viral, and fungal cultures, were negative. He subsequently died of the complications of prematurity and candidal sepsis, which were unrelated to the CNS.Patient 3 was a male infant of 24 weeks' gestation with respiratory distress syndrome. A blood culture was taken on day 10 of life because of worsening respiratory distress; results were positive for All 3 were <2 months of age, and 2 had CSF pleocytosis. Overall, HHV-6 DNA was detected in 2 (2.2%) of 90 specimens from patients with pleocytosis and in 2 (3.3%) of 61 patients with a discharge diagnosis of meningitis. HHV-6 DNA was not found in any patients who had encephalitis, febrile seizures, or a seizure disorder. None of the 19 immunocompromised patients had HHV-6 DNA in CSF.HHV-7 DNA was not found in any of the 245 CSF samples tested by PCR. Spiking random samples with HHV-7 DNA generated positive PCR results.HHV-6 DNA was uncommon in CSF of children (1.2%), and HHV-7 DNA was not detected in any CSF specimen in this study. HHV-6 DNA was found in a small percentage of meningitis patients (3.3%), but neither HHV-6 nor HHV-7 DNA was detected in samples from any patients with encephalitis, febrile seizures, or seizure disorders. Our limited detection of HHV-6 and HHV-7 contrasts with results of other studies, such as the report of Caserta et al. , in whicTechnical laboratory problems were unlikely. Our assays are as sensitive or more sensitive than those previously reported that use plasmid-encoded DNA, and we detected a panel of samples identified by other laboratories, which use different primers or probe sets.. However, these latter experiments were limited in number and could not completely rule out sensitivity differences related to the primers and probe sets. The presence of inhibitors was ruled out by spiking experiments. We tested the effect of freezing and thawing by subjecting several samples to three to four freeze-thaw cycles and found good preservation of both HHV-6 and HHV-7 DNA. Also, the PCR method used in this study detected cytomegalovirus DNA in CSF specimens stored at –70°C for up to 6 years . YoshikaOur results are not likely to be biased by the study population. Although lower seroprevalence rates have been found in some regions of the world, for example, Morocco (20% for HHV-6) and northern Japan (44% for HHV-7) ,26, no eThe clinical features of patients 1 and 2 and the absence of another identified infectious agent suggest that HHV-6 may have been the cause of fever, pleocytosis, and the clinical aseptic meningitis diagnosed in these two infants. Other studies have not found an association between HHV-6 DNA in CSF and CSF pleocytosis. However, we note that the CSF of patient 1 was contaminated by blood. Therefore, the presence and magnitude of pleocytosis are difficult to state with certainty; moreover, we cannot be certain whether HHV-6 DNA was truly present in CSF or derived from contamination with peripheral blood.In patient 3, the identification of a well-established pathogen and the normal results of neurologic and CSF examinations suggest that HHV-6 DNA in the CSF may have represented an asymptomatic infection or contamination with peripheral blood. The cases of HHV-6 infection of CNS occurred in infants <2 months of age, and two occurred in the early neonatal period. Infections in newborns have been previously described. Although horizontal transmission through saliva is postulated to be the most common method of transmission of HHV-6 and HHV-7 –29, infeOur study included 29 patients with CNS disorders caused by a known pathogen other than HHV-6 and HHV-7 ; none of these patients had HHV-6 or HHV-7 DNA in CSF. This finding suggests that HHV-6 or HHV-7 DNA is not frequently detected in CSF in association with other CNS infections, as might be expected if these viruses established latency in CNS and reactivated nonspecifically with other pathogens. Conversely, this finding supports the idea that detecting HHV-6 or HHV-7 DNA in CSF of patients with neurologic disorders is consistent with a pathogenic role. Our study population included a modest number of immunocompromised patients of whom a small subset had neurologic diagnoses. These numbers are insufficient to ascertain the roles of HHV-6 and HHV-7 in neurologic disease in immunodeficient patients.In conclusion, our data indicate that HHV-6 and HHV-7 are uncommon causes of CNS infection in children. HHV-6 may occasionally cause meningitis in young infants. Further studies are required to define the role of HHV-6 and HHV-7 in neurologic disorders in immunocompromised hosts.


Midazolam concentration is applied as a probe to determine the CYP3A enzyme activity. This study was done to determine piperine pretreatment role on midazolam plasma concentration.Some studies showed that piperine  received oral dose of piperine (15 mg) or placebo for three days as pretreatment and midazolam (10 mg) on fourth day of study and the blood samples were taken at 0.5, 2.5 and 5 h after midazolam administration. The midazolam plasma levels were assayed using HPLC method . Data were fit in a “one-compartment PK model” using vs. 102±43 min, p<0.0001). Half-life and clearance of midazolam were higher in piperine pretreatment group compared to placebo .The mean ±SD of the age and body mass index were 24.3 ± 1.83 years (range: 21–28 years) and 23.46± 2.85, respectively. The duration of sedation in piperine receiving group was greater that the placebo group (188±59 According to the results, piperine can significantly increases half-life and decreases clearance of midazolam compared to placebo. It is suggested that piperine can demonstrate those effects by inhibition CYP3A4 enzyme activity in liver microsomal system. Some specific substances of food products and additives can manipulate drugs disposition and fate in body through mechanisms that make changes their plasma concentration -3. TheseGrilled meat using wood charcoal, smoked meats, foods containing cruciferous vegetables, and some medicinal plants can induce several microsomal and transporter enzymes and decrease plasma concentration of xenobiotics, such as cyclosporine A and anti-HIV protease inhibitors . Other dCYP3A enzymes are the most important enzymatic group that involve in drug metabolism. More than 50% of microsomal enzymes are made up of CYP3A and about 60% of drugs used in treatment  are metabolized by CYP3A . Plasma Based on the mentioned effect of piperine and the important role of CYP3A in drug metabolism, this study was designed to investigate the effect of three days pretreatment of low dose piperine on midazolam plasma concentration in healthy volunteers.This study has been approved by ethics committee of Babol University of Medical Sciences  and recorded in IRCT  data bank with registration number:IRCT201203129271N1. All healthy volunteers were given information of the aims of the study, methods and possible outcomes of the treatment so that they could understand the main objective of the investigation. All selected participants signed a written informative consent form and then they were entered into the study. Although, neither midazolam nor piperine have serious side effects at lower doses, all participants were allowed to leave the study at any time of the follow up when they developed considerable complications.This was a cross-over controlled study on two groups (n=10) of healthy volunteers. A one-month period was considered as wash out time between two trials. Based on previous reports in 100 healthy volunteers of Mazandaran province (Northern Iran) to find out the CYP3A activity and recorded moderate activity in this sample size of the population ,18, all Midazolam , EDTA , piperine , n-hexan, HPLC grade methanol, isoamyl alcohol and HCl  and deionized water were used. Standard form of midazolam and diazepam as base  were prepared from Dr. Abidi Pharmaceuitical Company .Centrifuge  and HPLC  were used.All participants received adequate description about the setting, treatment schedule and outcome of drug administration. Then they recorded their personal information including age, sex, height, weight, general health situation, and history of drug use, food sensitivity, smoking, occupation, education and consent to the study in a special form.Healthy volunteers regardless of gender were randomly divided into two control and piperine receiving groups (n=20 per group). The subject received placebo or piperine (1 capsule 15 mg of piperine daily for 3 days before midazolam) and midazolam  on fourth day of the study. Total period of the study is 4 days. Blood samples (maximum 10 ml) were taken at 0.5, 2.5 and 5 hour after midazolam administration. After a month as a wash out period, the treatment program was exchanged between placebo and piperine groups and all steps were done as same as the previous.Blood samples were transferred into labeled tubes containing Ca-EDTA and shake gently to mix Ca-EDTA with blood for preventing of coagulation.The samples were centrifuged at 3500 ×g for 15 min and supernatant plasma layer transferred into a blank tube. Until the assay of midazolam or diazepam concentration, the plasma samples were kept at −20°C.For measurement of the drugs concentration, plasma samples were removed from the freezer condition and left in the laboratory to convert from frozen into a liquid sample. Then 0.5 ml of thawed plasma sample was transferred into a capped falcon tube. In the next step, 100 micl of 1 M NaOH and 100 micl isoamyl alcohol were added. After a gently shaking, 4.5 ml of n-hexan was added and shake for 1 minute. The final solutions were centrifuged at 300 ×g for 5 min. After this step, 4.2 ml of supernatant was drawn and transferred into a plastic tube and 100 miclof 0.05 M HCl was added to the solution and centrifuged similar as the previous. Then the supernatant (organic layer) was discarded and the remaining was directly injected into the HPLC.The separation was carried out at ambient temperature using a single-column isocratic reverse-phase method. The mobile phase consisted of 75% methanol and 25% water. Flow rate was set at 0.8 ml/min. The extracts were detected by UV detector at 242 nm wavelength. Twenty micl of the aqueous layer of final extract was injected into the HPLC using a Hamilton’s syringe. The injection was repeated 3 times for each sample. Peaks area and height were considered for midazolam and diazepam detection and used for calculation of the drugs concentration in the final plasma extract.A working solution containing 10 micg of midazolam in 1 mL of mobile phase was prepared. Six standard solutions of different concentrations of midazolam  were prepared. Then 20 micl of 25 ng/ml of diazepam solution as internal standard as added to each standard concentration of midazolam.Data were analyzed in two steps. The first; personal information of the participants and data of drugs assay were handled using Excel software. Calibration curves were plotted based on peak area and/or height ratio of midazolam to diazepam.2coefficient were calculated. Coefficient variation (CV) for this analysis was 3.8%.The slope, intercept and RP-Pharm software.The second; pharmacokinetic (PK) parameters such as absorption rate constant (Ka), clearance, half-life and volume of distribution of midazolam in the placebo and piperine groups were calculated based on assumed one-compartment kinetics using Based on non-parametric distribution, data of three concentrations by time were analyzed using Friedman test. Wilcoxon U-test was used for the results of clearance, volume of distribution and half-life of midazolam in piperine group compared to placebo. The difference of the PK parameters between two groups was considered statistically significant at p<0.05.The age range of the participants was from 21 to 28 year (24.3±1.83). Six subjects were female and 14 were male . All participants were healthy volunteer and selected from Mazandaran province. The subjects’ weight ranged from 49 to 101 kg (68.05±2.85). Their average (±SD) BMI was 23.46 (±2.85).vs. 102±43 min, p<0.0001) . The mean duration of the sedation in piperine receiving group was greater than placebo   constant and the line equation are shown in the Figure Six concentrations  as standard solution of midazolam were prepared. Equal volume of diazepam solution (25 ng/ml) as internal standard was spiked on each concentration of midazolam. Based on peak area ratios of different concentration of midazolam on diazepam, the standard curve was obtained  and diazepam (25 ng/ml) are shown in Figure 0-5 of placebo compared to piperine pretreatment group .Pharmacokinetics parameters were estimated in an assumed one-compartment model using P-Pharm software. For comparing final estimation, non-parametric paired Wilcoxon U-test was applied.The comparison of primary data of drug concentrations showed a significant different between placebo and piperine pretreatment groups at 2.5 and 5 hours after midazolam . Analysis of intra-group variation of concentration by time showed a significant profile (p=0.03). There was no significant difference in AUCAll PK parameters except of Vd, in piperine pretreated subjects has demonstrated a significant difference compared to placebo (p<000.1). Half-life of midazolam in piperine pretreatment group is considerably greater than in placebo Table . For thiAfter modeling with input of initial data assumption , concentration-time curves were plotted. Individual Bayesian fits of concentration (ng/ml) of midazolam by time (h) profile placebo and piperine pretreatment are shown in Figure The population plasma midazolam concentrations time curves in placebo (A) and piperine (B) treatment groups are shown in Figure This investigation showed 3 days piperine pretreatment can significant increase elimination half-life (t1/2) and decrease clearance of midazolam when compared to control. Our results can demonstrate a possible relation between midazolam concentration and CYP3A activity. This enzyme has an important role in microsomal drug metabolisms. Thirty percent of cytochrome P450 enzymes of liver are made of CY3A4 . It has In the present study piperine the main alkaloid of black pepper , could sThe genes of CYP3A4 and p-glycoprotein are expressed in enterocytes and the bioavailability of many drugs such as cyclosporine A, midazolam, verapamil, HIV protease inhibitors and digoxin could be affected by the enzyme and transporter ,36,38-40Individual PK variations can significantly determine CYP3A activity in general population. This may predispose patients to demonstrate unexpected drug manifestations compared to normal subjects. According to the present investigation, piperine may inhibit CYP3A4 activity and increase the level of midazolam as a substrate of the enzyme.The authors declared that they have no competing interests.MMR: data collection and executive affairs; SK: laboratory works and review literature searching; MTK: lab tests and data collection; SGh: HPLC model setting; EY and HGh: data collection; MRS: study design and modeling; AAM: study design, to register study in IRCT, data handling and statistical analysis, manuscript writing and correction. All authors read and approved the final manuscript.


Bacteria persist within biofilms on the middle ear mucosa of children with recurrent and chronic otitis media however the mechanisms by which these develop remain to be elucidated. Biopsies can be difficult to obtain from children and their small size limits analysis.in situ hybridisation (FISH) to determine bacterial species present. Most MEEs had live bacteria present using viability staining (32/36) and all effusions had bacteria present using the universal FISH probe (26/26). Of these, 70% contained 2 or more otopathogenic species. Extensive DNA stranding was also present. This DNA was largely host derived, representing neutrophil extracellular traps (NETs) within which live bacteria in biofilm formations were present. When treated with the recombinant human deoxyribonuclease 1, Dornase alfa, these strands were observed to fragment.In this study we aimed to investigate biofilm presence in middle ear effusion (MEE) from children with recurrent acute otitis media (rAOM) and to determine if these may represent infectious reservoirs similarly to those on the mucosa. We examined this through culture, viability staining and fluorescent Bacterial biofilms, composed of multiple live otopathogenic species can be demonstrated in the MEEs of children with rAOM and that these contain extensive DNA stranding from NETs. The NETs contribute to the viscosity of the effusion, potentially contributing to its failure to clear as well as biofilm development. Our data indicates that Dornase alfa can fragment these strands and may play a role in future chronic OM treatment. Recurrent acute otitis media (rAOM) affects between 10–20% of children Haemophilus influenzae (NTHi) and Streptococcus pneumoniae in vitro and in the chinchilla model of otitis media (OM) S. pneumoniae and NTHi can resist NET killing Biofilms on mucosal surfaces are a result of interactions between bacteria and the host and these differ physiologically from biofilms formed on inert surfaces Staphylococcus aureus biofilm has been demonstrated in otorrhoea smears from children with CSOM in situ hybridisation treatments, reducing bias in selecting probes and determining non-specific interactions between the probes and the sample. The presence of DNA in MEE from children with chronic and/or recurrent OM has not been previously demonstrated. In this study, we used FISH and viability staining to investigate whether bacterial biofilm, intracellular pathogens and extracellular DNA are present in MEE samples from children undergoing VTI for rAOM.While middle ear mucosal biopsies have been used for demonstrating biofilm or intracellular presence of bacteria MEE samples were obtained from children aged between 9 and 36 months with a history of at least 3 episodes of AOM and undergoing VTI as previously reported Samples were collected as previously described 2 for 48 h and all predominant bacteria recorded.MEE (100uL) and nasopharyngeal samples (10uL) were assayed with standard culture techniques Viability of bacteria in the MEEs was assessed using the generic nucleic acid labelling of cells with the BacLight kit (Invitrogen Technologies). Briefly, 50ul of fresh MEE in saline was air dried for 30 min at room temperature on SuperFrost plus slides . Samples were incubated with 50 µl of the BacLight working solution (consisting of propidium iodide and SYTO9)  for 30 min at room temperature before three 5 min washes with PBS. Slides were mounted using an in-house low fade mounting media and stored at −20°C until CLSM imaging using the Leica TCS SP2 AOBS multiphoton confocal.et al and Thornton et alGCT GCC TCC CGT AGG AGT) GTG ATG CAA GTG CAC CTT) for S. pneumoniaeCCG CAC TTT CAT CTT CCG) for H. influenzaeA GCA AGC TTC TCG TCC G) for S. aureusMoraxella catarrhalisTCT CGG CCT TGA AAC CCC) for Pseudomonas aeruginosaFISH was conducted as described by Hall-Stoodley 2 EDTA) for 2 h at 37°C. Slides were then washed with PBS. Samples were treated with 40 µl of hybridisation buffer and 50 ng/ml of each probe at 46°C inside a hybridisation chamber for 2 h. Slides were then washed for 15 min at 48°C and incubated overnight with 1.2 µg/ml Hoechst 33342  to stain the nuclei of the host cells. Slides were mounted in low fade mounting media and imaged using four colour CLSM using the Leica TCS SP2 AOBS multiphoton confocal. Images were analysed for presence of bacteria, specific bacterial species, biofilm structure and composition.MEE samples stored in 50% PBS/Ethanol at −20°C were used for FISH. A 50 µl aliquot from each MEE was air dried as above, incubated for 30 min at room temperature in 80% and then 100% ethanol. Samples were transferred to hybridisation chambers and incubated with lysozyme (10 mg/ml lysozyme in 0.1 M Tris (hydroxymethyl) aminomethane hydrochloride and 0.05 M Naet alSamples were scored positive for biofilm presence when CLSM images showed bacterial aggregates resembling microcolonies or more extensive morphology, bacterial morphology based on size (0.5–2 µm) and shape (cocci or coccobacilli) was correct, biofilm ultrastructure was present and fluorescent signal was only present in the appropriate fluorescent channel. Host cells were identified through the use of the Hoechst 33342 nucleic acid stain. Bacteria were marked as being intracellular when discrete clusters of bacteria were in close proximity to intact host cell nuclei as per Thornton A subset of 10 MEEs was evaluated for neutrophil elastase by staining with anti-human neutrophil elastase antibodies . Fifty µl of MEE in saline was air dried for 30 min at room temperature on SuperFrost plus slides . MEE was blocked with Zuk's blocking buffer and incubated at room temperature for 30 min. Block buffer was removed and MEE was incubated with a 1/100 dilution of rabbit anti-neutrophil elastase  in Zuk's buffer with 1% saponin  for 60 min at room temperature. Antibody was aspirated and the MEE was washed 3×10 min in PBS. MEE was then incubated with 1/100 AlexaFluor 647 labelled goat anti-rabbit antibody  in Zuk's buffer with 1% saponin for 60 min at room temperature in the dark. Antibody was aspirated and the MEE was washed 3×10 min in PBS at room temperature before being counterstained with propidium iodide , mounted in low fade mounting media and stored at −20°C until imaging using the Leica TCS SP2 AOBS multiphoton confocal.Seven MEEs were evaluated for actin presence by staining with Phalloidin. Briefly, 50ul of MEE in saline was air dried for 30 min at room temperature on SuperFrost plus slides. Samples were incubated with 50 µl of TRITC labeled Phalloidin  at room temperature for 60 min, washed 3 times with PBS, stained for 30 min at room temperature with 50 µl of SYTO9 and washed 3×5 min in PBS. Slides were mounted using a low fade mounting media and stored at −20°C until imaging using the Leica TCS SP2 AOBS multiphoton confocal.A subset of 10 MEEs was stained for lectins by staining with Concanavalin A (ConA) and Wheat Germ Agglutinin (WGA). Fifty µl of MEE in saline was air dried for 30 min at room temperature on SuperFrost plus slides. MEE were fixed in 4% formaldehyde for 15 min at 37°C. Samples were then washed 3×10 min with PBS at room temperature. Samples were incubated at room temperature in the dark with 50 µl of a 5 µg/ml of WGA conjugated with Oregon Green 488  in PBS for 10 min. Samples were washed twice in PBS. MEEs were then stained with 50 µl of 50 µg/ml FITC labelled ConA  for 30 min at room temperature. Samples were then washed 3×5 min in PBS before being counterstained with propidium iodide and mounted in low fade mounting media and stored at −20°C until imaging.Seven MEEs were treated with two concentrations of a highly purified solution of recombinant human deoxyribonuclease I  which is an enzyme that selectively cleaves DNA. Briefly, 250 µl MEE was incubated with 250 µl of 5 ug/ml or 1 mg/ml Dornase alfa for 15 min at room temperature. Samples were centrifuged using a serofuge and rinsed three times using PBS and stained as previously described using the BacLight kit (Invitrogen Technologies) followed by CLSM imaging.Thirty eight MEEs obtained from 24 children undergoing VTI for rAOM were assessed for the presence of extracellular DNA and live bacteria using generic nucleic acid staining and CLSM. Five children were female (20%) and the median age was 17.9 months , 7 S. pneumoniae (29%) and 1 S. aureus. Nine children had 2 or more otopathogens isolated from the nasopharynx. Eleven MEEs (29%) were culture positive for at least one bacterial species, with 3 being positive for S. pneumoniae and 6 for NTHi , 6 for NTHi . Three Mccasions .To investigate whether bacteria within the MEE were viable despite low culture rates we used BacLight viability staining. Live bacteria were identified in 32 of the 36 MEE samples tested. In 20 of these MEE only live bacteria were present, while in 12 both live and dead bacteria were identified. Bacteria were present in extensive biofilms, microcolonies and in the planktonic state . NucleicS. pneumoniae, 17 (65%) for H. influenzae, 7 (27%) for M. catarrhalis, 7 (27%) for S. aureus and 1 (4%) was positive for P. aeruginosa. Using the NONEUB338 probe non-specific hybridisation was not observed for any of the MEE samples (data not shown). Bacteria were evident within epithelial cells  and at 5µg/ml, complete fragmentation of the DNA matrix was observed in all samples within 15 minutes .Bacterial biofilm is increasingly recognised to play a role in the recurrence and persistence of infections such as OM. Demonstrating biofilm in very small tissue biopsies from the middle ear of children is technically difficult and may be biased by sampling and probe selection In this study, bacterial biofilms were demonstrated in 84% of MEE from children undergoing VTI for rAOM. This is similar to rates of biofilm presence on the middle ear mucosa of children with chronic OME and rAOM (64–92%) Using FISH, biofilms within MEE were shown to be polymicrobial, consistent with those observed in the environment in vitro studies S. pneumoniae and H. influenzae the most commonly identified pathogens in the MEE of children with rAOM. These species can resist NET killing despite high levels of antimicrobial proteins We demonstrated extensive extracellular DNA stranding in MEE, predominantly neutrophil derived through NET formation, and live bacteria within these. Both animal In the MEE, it is important to use both the nucleic acid staining and the specific FISH methodology to assess biofilms. Generic staining protocols such as the BacLight kit do not involve enzyme treatments or stringent wash steps, which are necessary to conduct FISH staining, which means that the biofilm morphology within the MEE is retained more reliably. Indeed biofilm presence was more often scored using this technique compared with using FISH. FISH however allows identification of specific species and their relationship within the sample. Intracellular pathogens were detected in 45% of effusions, and 36% of children. These rates are lower than those detected in biopsies from children in previous studies (71%) H. influenzae followed by S. pneumoniae. Discrepancies were present comparing FISH and culture, with bacteria observed more often using direct microscopic examination. These differences have been observed previously when comparing standard culture and a direct detection method such as FISH The presence of non-cultureable but metabolically active bacteria in the MEE provides further evidence of biofilm involvement in OM. In this study 21% of children were culture positive for any of the main OM pathogens which is comparable with other published studies H. influenzae and M. catarrhalis OM, where M. catarrhalis is seen to be exclusively associated with the epithelial surface while H. influenzae is detected in both the epithelium and the MEE FISH represents a specific and sensitive tool for assessing bacteria in the MEEs and in addition provides spatial and morphological data. This could be important when considering larger scale studies, particularly monitoring clinical interventions to treat biofilm, where it is easier to collect and examine MEEs than biopsies. Using MEE to monitor intracellular persistence may not be as reliable as much lower rates of intracellular infection were observed in the MEE when compared to mucosal biopsies. Direct comparisons of MEE and middle ear biopsies from the same child are required to confirm this. Data from animal models have also demonstrated a difference in the niches occupied by bacterial species in polymicrobial infections. Specifically, in the chinchilla model of Studies often use nasopharyngeal carriage of otopathogens as a surrogate for the infecting pathogens of the middle ear. This may be indicative with acute infection but less reliable in children with recurrent AOM. In this small study, we did not see a direct correlation between the pathogens in the middle ear with any of the detection methodologies with those cultured from the nasopharynx. This suggests that different bacteria may be found only in the middle ear space reflecting the presence of biofilm as a separate source of microbes causing the recurrent acute infections. Investigation of MEE and NPS samples using molecular detection techniques rather than relying solely on culture methodology is required as previous studies have demonstrated that non-cultureable organisms can be present in up to 36% of NPS using molecular methods in vitro systems of neutrophil induced biofilms by P. aeruginosa, DNases had decreased activity in treating thicker, more mature biofilms or those containing mucoid strains of bacteria, possibly due to increased polysaccharide masking DNA cleavage sites Dornase alfa is the only licensed DNAse with efficacy in the treatment of CF This study includes a relatively small number of children with a specific clinical presentation of OM, being persistent effusion following rAOM episodes. Investigation of the role of biofilm in MEE and the middle ear mucosa in a population of children with a broader OM spectrum, including children with chronic OME and CSOM, and the potential of Dornase alfa are required.These data provide an insight into the role of biofilm in the middle ear which is not only surface associated but present in the entire middle ear cavity. Live bacteria in microcolonies are present in extensive DNA stranding from NETs in the MEE of children with rAOM, contributing to effusion viscosity and may affect its failure to clear. The involvement of these biofilms with NET associated DNA indicates a potential for the novel use of Dornase alfa combined with currently used topical antibiotics at the time of surgery. We are currently conducting a clinical trial to assess the use of Dornase alfa as an adjunct therapy to VTI to prevent recurrent episodes of OM and possibly the need for repeat surgery. The potential use of Dornase alfa as a treatment for recurrent or chronic OM prior to surgery requires development of suitable methods of delivery to the middle ear and this warrants further investigation.


More than 160 Active Pharmaceutical Ingredients (APIs) have been detected in surface waters, although generally at low concentrations (ng-ug/l). Most available ecotoxicological information on these pharmaceuticals originates from acute testing using algae, Daphnia and fish; results from long-term exposures are still very scarce, particularly in vertebrates. There is, however, emerging evidence to suggest that several human and veterinary pharmaceuticals may pose a serious threat to aquatic wildlife. Still, there are only two examples where exposure to pharmaceuticals has been indisputably linked to adverse effects in wild vertebrates, i.e. the feminizing effects of the synthetic estrogen ethinylestradiol , frogs and fish (exposure via ambient water). The results show that the developing Müllerian duct (absent in fish) is a highly sensitive target for endocrine disruption and developmental toxicity in amphibians and birds.2, injected into the embryonated egg, cause a disrupted differentiation and malformations in different parts of the oviduct, including the shell gland. Egg injection of EE2 also resulted in eggshell thinning in the next generation of eggs, and a dramatically altered pattern of expression of the enzyme carbonic anhydrase in the shell gland. Eggshell thinning is probably the most serious ecotoxic effect that has afflicted avian wildlife. Based on these and other data, we propose that eggshell thinning could represent a developmental effect in the Müllerian duct rather than a direct effect of pollutants in the oviduct of the adult egg-laying bird.In birds, both diethylstilbestrol (DES) and EE2 resulted in a skewed sex ratio with almost complete feminization at an ecologically relevant concentration. A partial or complete lack of oviducts was observed in these frogs, resulting in ovulation directly into the abdominal cavity. Also the gestogenic pharmaceutical levonorgestrel (LNG), used in contraceptives and other hormone therapies, has recently been demonstrated to give rise to oviductal agenesis following developmental exposure. Unlike the impact in EE2-exposed frogs, the adult ovaries of the LNG-exposed frogs also displayed an increased fraction of immature oocytes, arrested in early meiotic prophase. We conclude that both EE2 and LNG are potent developmental toxicants at environmentally relevant exposures in frogs, giving rise to dysfunctional female sex organs with subsequent sterility.The Müllerian duct also proved to be a sensitive target of toxicity in frogs. Exposure of tadpoles to EEIn conclusion, exposure to pharmaceutical steroids during early life stages can permanently impair female reproductive organ development and fertility in lower vertebrates.


ACE, PAI-1 and eNOS gene polymorphisms on endothelial function. The genes are related to atherosclerosis and endothelial dysfunction in coronary slow flow (CSF).We examined the effects of PAI-1 4G/5G, eNOS T-786C and ACE I/D polymorphisms were determined by polymerase chain reaction (PCR) amplification.Thirty-three patients with angiographically proven CSF and 48 subjects with normal coronary flow were enrolled in this study. Coronary flow patterns were determined by the thrombolysis in myocardial infarction (TIMI) frame count method. Endothelial function was assessed in the brachial artery by endothelium-dependent flow-mediated dilatation (FMD). p = 0.009). Mean TIMI frame counts were significantly higher in CSF patients . FMD was significantly lower in CSF patients than in the controls . TIMI frame count and FMD were found to be negatively correlated in a correlation analysis . PAI-1 4G/5G, eNOS T-786C and ACE I/D polymorphisms were similar in the two groups.No difference was found between the groups regarding age, heart rate and blood pressure. Males were more prevalent among patients with CSF than control subjects (58.8 vs 29.8%, This study showed that endothelial function was impaired in patients with CSF. PAI-1, ACE and eNOS polymorphisms were not related to CSF in our study population. Coronary slow flow (CSF) was first reported in 1972 as an angiographic phenomenon, described as delayed passage of angiographic contrast agent along the coronary arteries in the absence of stenosis in the epicardial vessels.6ACE gene located on chromosome 17q23. The ACE gene has an insertion/deletion (I/D) polymorphism in the non-coding region of the gene.11The angiotensin converting enzyme (ACE) is part of the renin–angiotensin system and plays an important role in haemostasis of the vascular wall.et al. originally reported a mutation of thymidine, being replaced by cytosine at the nucleotide -786 (T-786C) gene.19Nitric oxide (NO) is synthesised from L-arginine by nitric oxide synthase and has an effect on endothelial relaxation.PAI-1 gene is located on 7q21.3-22 and polymorphism of the 4G/5G gene is located in the PAI-1 gene promoter region. The fifth guanine (G) base is inserted or deleted in the 4G sequence in the 675th base of the initial transcription point upstream. The PAI-1 gene has three genotypes, namely, 4G/4G, 4G/5G and 5G/5G. 4G/4G allele carriers always have higher plasma PAI-1 activity than 4G/5G and 5G/5G carriers.22Tissue plasminogen activator inhibitor 1 (PAI-1) plays an important role in endogenous fibrinolytic activity. Recent studies demonstrated that elevated PAI-1 activity was related to atherosclerosis, and was an independent predictor of coronary artery disease and myocardial infarction.The aim of this study was to investigate the association between ACE I/D, eNOS and PAI-1 gene polymorphisms and endothelial function, evaluated by FMD, in patients with CSF.A total of 33 patients with CSF (mean age 55.8 ± 10.3 years) and 48 controls (mean age 53.9 ± 11.8 years) with normal coronary arteries were enrolled in this study. Coronary angiography was performed in the cardiology clinic between January 2010 and June 2012 on patients who had an indication for elective coronary angiography due to ischaemia detected on a treadmill test and/or myocardial perfusion scintigraphy.A complete history, findings of the physical examination, risk factors for atherosclerotic heart disease and medications were recorded. Patients who had been treated with antihypertensive drugs or those whose baseline blood pressure exceeded 140/90 mmHg were diagnosed with hypertension (HT). Diabetes mellitus (DM) was defined as fasting blood glucose levels > 126 mg/dl or the use of anti-diabetic medication. Hyperlipidaemia was defined as a total cholesterol level > 200 mg/dl and/or low-density cholesterol level > 160 mg/dl. Patients with known atherosclerotic disease, visualised coronary artery plaque in coronary angiography, peripheral artery disease, malignancy, renal and hepatic insufficiency, and chronic inflammatory disease were excluded from the study. All subjects agreed to participate in the research and the consent of the local ethics committee was obtained.et al.23Coronary angiography was performed with a femoral approach using Judkins catheters and the contrast agent iopramide  with angiographic equipment . The thrombolysis and myocardial infarction (TIMI) frame rate was 30 frames per second (fps) and angiograms were recorded on a compact disc in DICOM format. Coronary blood flow was measured quantitatively using TIMI frame count, which was determined for each major coronary artery of each subject included in the study, according to the method first described by Gibson The left anterior descending coronary artery (LAD) is usually longer than the other major coronary arteries and for that reason the TIMI frame count of this vessel is often higher. Therefore, to obtain the corrected TIMI frame count of the LAD, the TIMI frame count was divided by 1.7.23TIMI frame counts in the LAD and left circumflex (LCx) arteries were assessed in the right anterior oblique projection, and the right coronary artery (RCA) in the left anterior oblique projection. The mean TIMI frame count for each subject was calculated by adding the TIMI frame counts for the LAD/1.7, LCx and RCA and then dividing the value obtained by 3. The corrected cut-off values due to the length of normal visualisation of the coronary arteries were 36.2 ± 2.6 frames for the LAD, 22.2 ± 4.1 frames for the LCx, and 20.4 ± 3 frames for the RCA. Any values obtained above these thresholds were considered CSF.PAI-1, MTHFR and ACE genes and are compared in the results. Three thrombophilic marker genes, plasminogen activator inhibitor-1 ; two polymorphic regions for MTHRF , and ACE I/D (rs1799983) genes were analysed in the results.Peripheral blood samples from CSF patients and healthy controls were used for genotyping for point mutations of Peripheral blood samples containing EDTA were collected from the patients and volunteer controls after a 12-hour overnight fast. All routine biochemical tests were carried out on an autoanalyser with the Cobas 6 000 Integra  auto-analyser device using the chemiluminescence method. Venous blood was collected in 6-ml EDTA tubes for isolation of the genomic DNA and stored at –20°C. A total of 81 DNA samples from patients with CSF and controls were genotyped by real-time polymerase chain reaction (PCR) analysis.The total genomic DNA was extracted by the MagnaPure Compact (Roche) and Invitek kit extraction techniques . Target genes were amplified by real-time PCR, LightCycler 2.0 methods (Roche) for the CSF cohort and healthy controls. Briefly, LightCycler FastStart DNA Master HybProbes, master mix  and template DNA from patients and controls were used for real-time amplification for each target gene.MTHFR 677C>T consisted of a denaturation step of 10 minutes at 95°C. The amplification conditions for 45 cycles were: denaturation at 95°C for five seconds, annealing at 55°C for 10 seconds, extension at 72°C for 15 seconds, melting curve step with denaturation at 95°C for 20 seconds, annealing at 40°C for 20 seconds, melting at 85°C for two seconds and the cooling step at 40°C for 30 seconds. A software program  was used for detection of the mutated (channel 640 at 54.5°C) and wild genotype (channel 640 at 63°C) profiles for target 677 C>T SNP analysis.The amplification protocol for MTHFR 1298A>C consisted of a denaturation step of 10 minutes at 95°C. The amplification conditions for 40 cycles were: denaturation at 95°C for five seconds, annealing at 62°C for 10 seconds, extension at 72°C for six seconds, melting curve step with denaturation at 72°C for 30 seconds, annealing at 95°C for 20 seconds, melting at 40°C for one second and the cooling step at 40°C for 30 seconds. A software program  was used for detection of the mutated (channel 640 at 59°C) and wild genotype (channel 640 at 65°C) profiles for target 1298A>C SNP analysis.The amplification protocol for PAI-1 5G/4G consisted of a denaturation step of 10 minutes at 95°C. The amplification conditions for 40 cycles were: denaturation at 95°C for three seconds, annealing at 60°C for 10 seconds, extension at 72°C for 13 seconds, melting curve step with denaturation at 95°C for 30 seconds, annealing at 40°C for one minute, melting at 85°C for two seconds and the cooling step at 40°C for 30 seconds. A software program  was used for detection of the mutated (4G) (channel 640 at 54°C) and wild genotype (5G) (channel 640 at 61°C) profiles for target PAI-1 5G/4G analysis.The amplification protocol for ACE I/D consisted of a denaturation step of 10 minutes at 95°C. The amplification conditions for 45 cycles were: denaturation at 95°C for three seconds, annealing at 60°C for 10 seconds, extension at 72°C for 10 seconds, melting curve step with denaturation at 95°C for 30 seconds, annealing at 40°C for one minute, melting at 85°C for 10 seconds and the cooling step at 40°C for 30 seconds. A software program  was used for detection of the mutated  (channel 640 at 85°C) and wild genotype  (channel 640 at 93°C) profiles for target ACE I/D analysis.The amplification protocol for Two-dimensional, M-mode, pulsed and colour-flow Doppler echocardiographic examinations were performed on all patients by one cardiologist with a Vivid 7 Pro echocardiography system . During echocardiography, a single-lead electrocardiogram was recorded simultaneously. Data were recorded from the average of three cardiac cycles. M-mode and Doppler measurements were performed, adhering to the American Society of Echocardiography guidelines.Endothelial function of all subjects was assessed by a single ultrasonographer blinded to the coronary flow groups. Measurements were performed in a temperature-controlled room (22°C) in the morning and after eight to 12 hours of a fasting period. Ingestion of substances that might have affected measurements, such as caffeine, high-fat foods and vitamin C was not allowed for 12 hours before the study. Any vasoactive medication was discontinued at least five serum half-lives before the brachial studies.The right brachial artery was imaged above the antecubital fossa in the longitudinal plane. Upon acquiring an appropriate image, the surface of the skin was marked. The arm and the ultrasound probe were kept at the same position by the ultrasonographer during the entire study. The diameter of the brachial artery was measured from longitudinal images in which the lumen–intima interface was visualised on the anterior and posterior walls at end-diastole (onset of the R wave on the electrocardiogram), and the mean of the three highest measurements from five consecutive cardiac cycles was taken.After the basal lumen diameter and blood flow were noted at rest, a sphygmomanometer cuff was placed on the forearm and the cuff was inflated to 250 mmHg for arterial occlusion. After five minutes, the cuff was deflated and the lumen diameter was recorded one minute later, to assess endothelium-dependent flow-mediated dilatation (FMD) This was defined as both the maximum absolute change and maximum percentage change in vessel diameter during reactive hyperaemia:FMD= (diameter of reactive hyperaemia-diameter of baseline)diameter of baseline ×100U- or student t-tests. Categorical variables (risk factors and polymorphisms) were analysed using the chi-square test. Spearman’s correlation test was used for correlation between TIMI frame count and endothelial function.All continuous variables were expressed as mean ± standard deviation and median (interquartile range). All measurements were evaluated with the Kolmogorov–Smirnov test, and the Shapiro–Wilk test was used to determine normal distribution. Comparisons of parametric and non-parametric values between the two groups were performed by means of Mann–Whitney p-values < 0.05 were accepted as statistically significant. Risk estimations for the association of SCF with the polymorphisms were calculated using odds ratios (OR) and 95% confidence intervals (CI) by comparing the genotypic combinations.All statistical studies were carried out with the program SPPS ; p = 0.456 and 126.4 ± 127.4 vs 127.4 ± 127.4 mmHg, p = 0.712, respectively). The TIMI frame counts for each epicardial artery were higher in patients with CSF than control subjects. Mean TIMI frame count was also significantly higher in CSF patients .Clinical and laboratory findings of the subjects are shown in p = 0.029). TIMI frame count and FMD were negatively correlated in the correlation analysis .Echocardiographic and FMD measurements of the subjects are summarised in PAI-1 5G allele was found to be marginally associated with the possibility of CSF, however it was not statistically significant  Genotype properties and allele frequencies were similar in the two groups. The ACE, PAI and eNOS gene polymorphisms were not related to CSF in our population. Brachial artery FMD was impaired in patients with CSF, and the TIMI frame count was negatively correlated with FMD.This study showed that ACE D/D allele genotype was associated with higher serum ACE activity. Several studies have reported a relationship between the D allele and cardiovascular disease,et al. reported that concentrations of plasma endothelin-1 were increased and NOS were decreased in patients with CSF, as a result of microvascular vasomotor dysfunction, which may be important in this phenomenon.31ACE plays an important role in vascular wall haemostasis and endothelial function. The et al. demonstrated, with intravascular ultrasonography, decreasing fractional flow reserve in the coronary arteries in patients with CSF due to diffuse atherosclerosis.et al. reported that the ACE I/D polymorphism correlated with carotid intima–media thickness, which is a sign of subclinical atherosclerosis.et al. reported that the frequency of the DD genotype and D allele were higher in patients with CSF, and DD genotypes were related to a possibility of CSF.Pekdemir PAI-1 is a key regulator of the fibrinolytic process and is related to the PAI-1 promoter 4G/5G polymorphism, although it is regulated by several factors, including cytokines, growth factor and insulin.37et al. reported that plasma PAI-1 activity was higher in carriers of the 4G/4G genotype and this was correlated with atherosclerotic heart disease, as determined by coronary angiography.39There are few and conflicting results regarding the association of 4G allele carriers and coronary events. Some studies suggested that PAI-1 may play a role in atherogenesis due to increased PAI-1 expression, which has been demonstrated in atherosclerotic plaques.et al. reported that the PAI-1 4G/4G genotype was related to a lower risk of the development of stable coronary artery disease because of the inhibitory effects of PAI on cellular migration.PAI-1 polymorphism in the literature. Our study, surprisingly, showed that PAI-1 4G allele carriers had a protective effect on CSF and the 5G allele was related to increasing risk for CSF.By contrast, Onalan eNOS gene is associated with reduction in gene promoter activity and the resulting reduction in NO levels, increasing the risk for coronary spasm.45eNOS is a regulator enzyme in the cardiovascular system for functions such as vasodilatation, inhibition of leucocyte adhesion to the endothelium, vascular small muscle cell migration and proliferation, and platelet aggregation. Reduced endothelial NO concentration is an important cause of endothelial dysfunction.et al. reported that FMD of the brachial artery was impaired and decreased plasma NO levels in patients with CSF. They concluded that endothelial dysfunction might be a cause of CSF.et al. reported an association between CSF and T-786C polymorphism of the eNOS gene, and a positive correlation between TIMI frame count and the C allele.Sezgin et al. reported impaired FMD of the brachial artery in patients with CSF and a negative correlation between TIMI frame count and FMD.6 In our study, FMD was impaired in patients with CSF, and negatively correlated with TIMI frame count. We found no correlation between genotyping and FMD in this study. These results suggest that endothelial dysfunction is an important process in CSF.Several mechanisms, including endothelial dysfunction, diffuse atherosclerosis and small-vessel disease have been proposed as a cause of CSF.The most significant limitations of the present study include the small sample size; the control group was not a normal population of subjects, for ethical reasons; we could not measure inflammatory markers such as C-reactive protein, interleukins, NO and PAI-1 levels; and we could not perform intravascular ultrasonography on the patients for the determination of intimal thickening and calcification.PAI-1 4G/5G polymorphism was less prevalent among CSF patients. However, large-scale genetic studies need to be undertaken in CSF populations in order to understand the underlying mechanisms of aetiopathogenesis.This study shows that brachial artery FMD was impaired and the 4G allele of the


Current approaches for clinical use are based on the infusion of freshly isolated or ex vivo polyclonally expanded Tregs into graft recipients with an aim to redress the in vivo balance of T effector cells to Tregs. However mounting evidence suggests that regulation of donor-specific immunity may be central to achieving immunological tolerance. Therefore, the next stages in optimizing translation of Tregs to organ transplantation will be through the refinement and development of donor alloantigen-specific Treg therapy. The altering kinetics and intensity of alloantigen presentation pathways and alloimmune priming following transplantation may indeed influence the specificity of the Treg required and the timing or frequency at which it needs to be administered. Here we review and discuss the relevance of antigen-specific regulation of alloreactivity by Tregs in experimental and clinical studies of tolerance and explore the concept of delivering an optimal Treg for the induction and maintenance phases of achieving transplantation tolerance.Current clinical strategies to control the alloimmune response after transplantation do not fully prevent induction of the immunological processes which lead to acute and chronic immune-mediated graft rejection, and as such the survival of a solid organ allograft is limited. Experimental research on naturally occurring CD4 Transplantation presents a life-saving treatment for patients with end stage organ failure, however the success of this procedure is restricted by the recipient immune response directed against donor graft alloantigens and the clinical caveats associated with immunosuppressive drugs aimed at controlling this immune response. Current strategies for clinical management of transplant recipients using sustained immunosuppression do not fully prevent induction of the immunological processes which lead to graft rejection, namely chronic allograft failure, and as such the survival of a solid organ allograft is limited  have been recognized for many years and substantial research efforts have sought to exploit their suppressive functions to deliver a tolerogenic cell therapy for transplantation  molecules expressed by donor allograft derived cells are directly presented to recipient T cells. The most potent driver of CD4+ and CD8+ T cell responses with specificity for alloantigen presented by the direct pathway is through the migration of donor antigen-presenting cells (APCs) from the allograft to the secondary lymphoid organs , Brown et al. were able to demonstrate activity of the semi-direct pathway as early as 8 days post-transplantation by counter-staining lymphoid tissues for both recipient and donor MHC class II expression  and I-Ab (C57BL/6 MHC Class II) expression (~30%), which remained detectable for extended periods of over 80 days post-transplantation. In the study by Tokoyawa et al., the semi-direct pathway is, therefore, a more likely explanation for prolonged direct pathway donor alloantigen presentation, particularly as in this same study they also detected upregulation of MHC class II expression by donor allograft epithelial and endothelial cells during inflammation, which could have provided a continuous source of donor alloantigen. This would however need to be confirmed by staining for both donor and recipient MHC expression.Recent data supports this view by providing evidence of the presentation of donor alloantigen by the semi-direct pathway for prolonged periods post-transplantation. In a rat model of complete MHC mismatched (LEW→BN) liver transplantation, Toyokawa et al. were able to detect donor MHC Class II L21-6+ T cells with direct allospecificity are well placed to provide help to alloreactive CD8+ T cells, through the likely 3-cell clusters formed between direct alloreactive CD4+ and CD8+ T cells interacting with a donor-derived APC expressing both allogeneic MHC class I and II, there must be an alternative provision of help for CD8+ T cells, as otherwise the clearance of donor passenger APCs would result in a parallel reduction of alloreactive CD8+ CTL responses. This long-standing conundrum is partly resolved by demonstration of a helper T cell-independent mechanism of CD8+ T cell alloactivation  and MHC class II I-Ad double positive APCs within lymphoid tissue of C57BL/6 (H2-Kb) mice that have received a BALB/c (H2-Kd) heart graft, demonstrating the capacity of recipient APCs to simultaneously prime both direct and indirect T cell alloresponses , or the indirect pathway (H2-Kd:I-Ab complex) into C57BL/6 mice (I-Ab) which were then challenged with BALB/c (I-Ad) heart or skin grafts. On adoptive transfer, T cells with indirect allospecificity proliferated with much more rapid kinetics compared to T cells with direct specificity, which was also reflected by endogenous alloreactive T cell populations. Using a murine transplant model Gupta et al. were able to measure the kinetics of alloantigen presentation though the direct or indirect pathways by grafting skin from a BALB/c or CB6F1 (C57BL/6 × BALB/c F1) mouse onto a C57BL/6.TEa.Rag2−/− recipient mouse, in which the T cells have indirect specificity for I-E alpha peptide presented by C57BL/6 MHC class II I-Ab , suggesting this short delay was caused by the time lag required for antigen processing and presentation by the recipient DCs before subsequent T cell proliferation. Although these findings concur with the concept that late graft rejection is associated with a gradually priming and maintenance of the indirect pathway, they fundamentally differ from the findings by Brennan et al., however these differences may be attributed to the allograft model or TCR transgenic T cells used, suggesting although these models are ideal for dissecting mechanistic basis of alloimmune priming, examining clinical data may be more informative for developing practical therapeutic strategies for intervention.In this respect, the contribution of the indirect pathway toward allograft rejection has been firmly established by studies using donor grafts from MHC class IIThe experimental findings described above underpin two main features of alloimmunity, firstly that the indirect pathway is active immediately following transplantation and secondly that the direct pathway can contribute toward the maintenance of the indirect pathway and may be active later in the post-transplantation period through the semi-direct pathway. It may, therefore, be tempting to speculate that Treg therapy would be best applied either pre-transplantation or at the time of transplantation to prevent any initial priming of memory alloreactive T cell responses, perhaps through modulation of APC activities  is associated with an expansion of splenic Tregs. In parallel, the study detected the persistence and even proliferation  of donor DCs for over 100 days post-transplantation, which were restricted to the allografted tissue, achieving a localized tissue chimerism  was lost when second donor-matched hearts were transplanted 200 days after the removal of the primary hearts, that is, once all donor-alloantigen had been cleared from the recipient mouse  bearing donor cells. These data allow speculation that donor-specific Treg mediated suppression occurs primarily at sites of alloantigen expression and effector target tissue, and possibly acts through early interaction and modulation of APC function and effector cell recruitment, as implicated by other in vitro and in vivo studies  and kidney allograft patients whom develop operational tolerance , recipient mice received an autologous BMT (I-Ak+) which had been retrovirally transduced to express a single donor MHC Class II donor allele (I-Ab), followed by a donor or 3rdParty heart allograft. This protocol resulted in the induction of donor-specific tolerance, in the complete absence of sustained immunosuppression, which was associated with immune deviation from a Th1 to Th2 predominate cytokine response and with no indications of chronic rejection associated vasculopathy  in experimental and clinical transplantation  or T cells with direct allospecificity (CD4+ T cells with direct specificity for I-Abm12), Conlon et al., were able demonstrate that only T cells with indirect allospecificity had the capacity to provide B cell help and induce an alloantibody response to a heart allograft (BALB/c→C57BL/6), whereas T cells with direct allospecificity alone were incapable  results in aberrant B cell IgM, IgA, IgG1, and IgG2b antibody production  and Tregs. Bregs have recently been identified to play roles in regulating autoimmunity in experimental models of collagen-induced arthritis, experimental autoimmune encephalitis (EAE) and colitis, which is associated with B cell production of IL-10 (BR1 or B10 cells in human or mouse respectively) or TGFβ production  effect in vivo, with no adverse or aggressive GvHD effects reported as a direct consequence (Edinger and Hoffmann, in vivo studies are required to understand the real risk of donor-specific Treg infusion in cell therapy.A potentially serious caveat to using donor alloantigen-specific Tregs Another potential limitation of Treg therapy is assessing its capacity to control alloreactive memory. Memory T cell responses naturally provide rapid and potent T cell immunity and are a barrier to most transplantation tolerance induction strategies (Lakkis and Sayegh, In summary, review of experimental and clinical data on transplantation tolerance support the use of donor-specific Treg therapy for establishing immunological tolerance in the clinc, in particular, Tregs with indirect allospecificity. Although current studies lack a clear demonstration of the comparative efficacy of Tregs with either direct or indirect allospecificity, there is strong evidence for an integral role of Tregs in establishing tolerance, although their contribution toward maintaining the stable tolerant state is unclear, and requires further investigation. Treg cell therapy may, therefore, be envisaged as being administered early during the post-transplantation period to accelerate the generation of other associated immunoregulatory processes that act toward maintaining stable immunological graft acceptance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tree frogs climb smooth surfaces utilising capillary forces arising from an air-fluid interface around their toe pads, whereas torrent frogs are able to climb in wet environments near waterfalls where the integrity of the meniscus is at risk. This study compares the adhesive capabilities of a torrent frog to a tree frog, investigating possible adaptations for adhesion under wet conditions. We challenged both frog species to cling to a platform which could be tilted from the horizontal to an upside-down orientation, testing the frogs on different levels of roughness and water flow. On dry, smooth surfaces, both frog species stayed attached to overhanging slopes equally well. In contrast, under both low and high flow rate conditions, the torrent frogs performed significantly better, even adhering under conditions where their toe pads were submerged in water, abolishing the meniscus that underlies capillarity. Using a transparent platform where areas of contact are illuminated, we measured the contact area of frogs during platform rotation under dry conditions. Both frog species not only used the contact area of their pads to adhere, but also large parts of their belly and thigh skin. In the tree frogs, the belly and thighs often detached on steeper slopes, whereas the torrent frogs increased the use of these areas as the slope angle increased. Probing small areas of the different skin parts with a force transducer revealed that forces declined significantly in wet conditions, with only minor differences between the frog species. The superior abilities of the torrent frogs were thus due to the large contact area they used on steep, overhanging surfaces. SEM images revealed slightly elongated cells in the periphery of the toe pads in the torrent frogs, with straightened channels in between them which could facilitate drainage of excess fluid underneath the pad. Tree frogs stick by wet adhesion in that a fluid film fills the contact zone between the adhesive pad and the substrate Amolops had distinct anatomical differences from the typical pattern seen in tree frogs. The only relevant biomechanical work is a preliminary study of adhesion in the Trinidadian stream frog, Mannophryne trinitatisMuch less is known about adhesion and toe pad morphology in rock or torrent frogs. Ohler Brunei, with several species of torrent frog capable of adhering to vertical rocks in waterfalls, and rock frogs capable of fast movement over wet rocks This was a study of free-ranging animals temporarily brought into a field-based laboratory and later released at their site of capture. Individuals used for electron microscopy where given a lethal dose of Benzocaine. The experimental protocol adhered to the Animal Behaviour Society guidelines for the use of animals in research and was approved by the University of Brunei Darussalam Research Committee (UBD/DVC/32.10) and an export permit JPH/TAD/30 PT 10. We confirm that the species investigated are not endangered or protected.Rhacophorus pardalis) and male individuals of the Black-spotted Rock frog (Staurois guttatus). For an easier distinction between the two, we will refer to R. pardalis as the tree frog and to S. guttatus as the torrent frog. Both species were abundant around the Kuala Belalong Field Studies Centre, Ulu Temburong National Park , where the experiments were carried out during two six-week visits (May/ June 2010 and 2012). The torrent frogs were found near waterfalls on fast flowing streams, where they could be captured on rocks (day) or surrounding vegetation (night). The tree frogs were caught at night on vegetation near small ponds in the forest. Although not identical in either body mass or snout-vent length (SVL), they were the best species match that was obtainable in sufficient numbers in the local area. Body mass was measured using an electronic balance (Mettler), while SVL was measured using callipers. Values are given in Our study focused on the comparison of a tree frog and a torrent frog species . Tree fr−1. The platform consisted of a wooden base  which wets the platform, and a fast flow rate , both within the range of flow rates found in the torrent frog's natural habitat. The water formed a more or less uniform layer across the surfaces, and stayed attached to it until the platform was close to the upside down orientation (ca. 170°).We also equipped the platform with a running water system to flood the surfaces in a controlled manner . The watFrogs were placed onto the platform, starting from a horizontal position (0°). In the majority of cases, frogs that initially faced downhill would turn so that they faced uphill as platform rotation progressed. We recorded the angle at which the frogs showed an initial slip and when they were completely dislodged from the platform. From those angles we calculated the maximum friction and adhesion forces using trigonometry. In cases where the frog slipped completely off the platform below 90°, the values for the fall angles were set equal to the slip values. In cases where the frogs showed an initial slip beyond 90°, the slip angles were set to 90°, since this would be the orientation where maximum friction would occur. The angles were measured by reading the position of a needle mounted on the platform against a 360° protractor (estimated measurement error was below 2°).Each individual frog was tested ten times on all three surfaces and all three flow regimes. The order of each combination of surface roughness and flow regime was randomised to avoid order effects. The frogs were given time to recover between trials. In a few cases, where the performance of the frog deteriorated significantly, we stopped the experiment and replaced the frog with a fresh individual.−1. A digital video camera  was mounted onto the platform and rotated with it in order to film the ventral body area of the frogs in contact with the glass surface and the protractor that monitored the angle of tilt of the platform. The video recordings (25 frames−1) were captured using Streampix  and analysed frame by frame using custom-built Matlab routines In order to see which body parts of the frogs were in contact with the substrate, we used a special illumination technique on a transparent glass surface  in addition to the adhesive toe pads. They were tested under both dry and wet conditions on surfaces of different roughness . Pads were tested under ‘wet’ conditions by pipetting 10 −1).The experimental set-up was similar to that used in previous single toe pad studies on tree frogs −1. A custom-built LabVIEW interface  was used to display and save force recordings, which were retrieved from the transducer via a portable data acquisition device . The video recordings and the force data acquisition were synchronised using a stop trigger pulse from a manual switch.Forces were measured in two dimensions, lateral (which measured friction) and normal (which measured adhesion). The applied vertical or horizontal movements were made manually by turning the knob of a micromanipulator and were recorded by means of a potentiometer. These inevitably led to some variation between different movements, means and standard deviations being as follows: amplitude 2.9For a closer inspection of the adhesive toe pads and other areas of ventral skin used by the frogs to aid their adhesion on steep and overhanging surfaces (belly and thigh skin), a few individual frogs were sacrificed using a lethal dose of Benzocaine. Fore- and hind limbs were severed at the base of the wrist or ankle, respectively, while belly and thigh skin samples were obtained by carefully cutting out rectangular pieces of skin using a scalpel. All specimens were fixed in a solution of 2.5% gluteraldehyde buffered at pH 7.4 for 24 h. After washing in distilled water, specimens were dehydrated in an alcohol series and critical point dried. Samples were mounted and gold-coated before viewing with a JSM-7500F scanning electron microscope (JEOL UK Ltd.).All statistical tests were carried out using the statistical toolbox in Matlab . Data for contact area was extracted at angles of 45° intervals from 0° to 180°, which allowed us to draw statistical comparisons between these categories.p). Test results are either highlighted in the text or summarised in tables listed in For comparisons between two samples we used the Mann-Whitney U-test. For each test we provided the sample size  and coarse rough (1125 μm diameter particles) surfaces attached to a rotating platform. Each surface was used under three flow regimes: ‘dry’ ‘low flow rate’and ‘high flow rate’ These data allowed us to examine both the effects of changing flow rate on each surface and R. pardalis) slipped at angles below 90°, regardless of surface roughness. Only on the flooded surfaces (high flow rates) did performance decrease dramatically . Although, as S. guttatus , in contrast to the tree frog , is extremely well adapted to adhering to rough surfaces under flooded conditions.To summarise, there was no significant difference in performance between the species on all the dry surfaces. However, differences appeared on some of the wet surfaces (low flow rate) and were most dramatic under the high flow rates on the two rougher surfaces. The results clearly indicate that the torrent frog  illuminated glass surface so that areas of contact were highlighted  used a similar amount of total ventral body area when they were resting against a horizontal surface (0 rotation of the platform). Pad contact area was, however, much smaller (approximately 25% of that in the larger tree frogs). As in the tree frogs, there was little change in behaviour until the platform rotation passed 70°, at which point total contact area began to increase, mainly through use of belly skin. At angles between 90° and 135°, the limbs were spread out sideways as in the tree frogs (though to a lesser degree) and frogs initially facing downhill would reorientate to face head-up . However, under wet conditions, values were significantly reduced. Additionally, preliminary data using rough surfaces (not shown) also produced low levels of adhesion, particularly under wet conditions.Odorrana hosii, also found in the Brunei rainforest in the region of fast flowing rivers . Although usually surrounded by six other cells, the cells are not hexagons, as they mainly have curved rather than straight edges. Their ends are often pointed, especially the ends nearest the edges of the pad (see arrows in S. guttatus), cell shape seems to vary with the region of the pad. In central regions, there is a tendency towards a pattern of roughly regular hexagons, but peripherally they are more elongated, the overall pattern being intermediate between those of the tree frog and O. hosii . However, previous work on torrent frogs g rivers . All thrO. hosii . In suchO. hosii . We haveO. hosii . Like thR. pardalis) and torrent (S. guttatus) frogs adhere equally well to smooth, dry surfaces, the torrent frog species excels on rough surfaces covered by fast-flowing water, i. e. in conditions that replicate their natural habitat of wet rock in the immediate vicinity of waterfalls The first part of this paper describes behavioural experiments which show that, while both representative tree  helped to increase performance significantly. However, at the highest flow rates, friction forces dramatically decreased, presumably because of the lubricating effect of the water flow, with follow-on effects on adhesion. It was here, on the 30 μm and 1125 μm surfaces, that the torrent frog outperformed the tree frog. Indeed, the difference on the roughest surface was dramatic. Almost all of the tree frogs were swept off the platform before the angle reached 90°, while the torrent frogs stayed attached until the platform was almost upside-down. Thus, on these rough, wet surfaces, torrent frogs clearly have better attachment  did not perform well on our tilting apparatus (data not shown).Being small helps: Body mass was only a small factor in this particular comparison, but the much heavier torrent frog S. guttatus. Given the relatively minor role of the toe pads in comparison to thigh and belly skin in our experiments, it is unlikely that they are important here.Use of ventral body skin as well as toe pads: Observations of the torrent frogs on the rough, wet surface indicate that they use the same strategy for adhesion as they show on the dry smooth surface. Thus, as High friction forces: The behavioural data demonstrated that the torrent frogs generated superior friction compared to the tree frogs on rough, wet surfaces. Indeed, their ability not to be swept off by the high flow rate showed that friction was the major factor in their superior performance.Behavioural strategies: Although the few tree frogs that performed well on the rough, wet surface appeared to be using a similar strategy to the torrent frogs , it did appear that not all tree frogs used optimal strategies to remain attached to a smooth surface being rotated from the horizontal to the upside-down position. While adhesion to wet, rough substrates is normal for a torrent frog, the same is not true for tree frogs. Thus, suboptimal behavioural strategies may be expected on such surfaces.Staurois guttatus has abilities not shared by a tree frog of comparable size.In conclusion, we do find anatomical evidence to support the idea that the toe pads of our torrent frog species have some specializations to promote better drainage of fluid from under its pads, but such adaptations seem to be restricted to the edge of the pad. Such features may lend inspiration to man-made adhesive pads which would work under similar (wet) conditions. In contrast, however, neither belly nor thigh skin of either species seems to be specialized for adhesion or friction, but as these body parts have a much greater surface area than the toe pads, they may still play an important role in adhesion and friction. We recognise, as Garland and Adolph Figure S1Reorientation behaviour of the frogs. A & B) Individual frames of a tree- and a torrent frog, respectively, in contact with a transparent surface at two tilt angles (45° and 135°). C & D) Plots of total contact area against rotation angle of the platform. When frogs initially faced downhill, they usually re-orientated themselves during the course of the platform rotation to face uphill again (arrows). While turning around, tree frogs often lost the contact of their belly with the surface; in contrast, torrent frogs managed to increase the contact area of their ventral surface.(TIF)Click here for additional data file.Supplementary Materials S1Supplementary Tables S1 to S8. Table S1, Mann-Whitney U-tests for the comparisons of the slip angles for R. pardalis under different testing regimes. ‘smooth’, ’30 μm’ and ‘1125 μm’ refers to the substrate roughness; ‘dry’, ‘low flow rate’ and ‘high flow rate’ refer to the amount of water on the surface. Table S2, Mann-Whitney U-tests for the comparisons of the fall angles for R. pardalis under different testing regimes. ‘smooth’, ’30 μm’ and ‘1125 μm’ refers to the substrate roughness; ‘dry’, ‘low flow rate’ and ‘high flow rate’ refer to the amount of water on the surface. Table S3, Mann-Whitney U-tests for the comparisons of the slip angles for S. guttatus under different testing regimes. ‘smooth’, ’30 μm’ and ‘1125 μm’ refers to the substrate roughness; ‘dry’, ‘low flow rate’ and ‘high flow rate’ refer to the amount of water on the surface. When the sample size was too small no z-value could be computed; when both samples contained identical values, no p-value could be obtained (‘NA’: not applicable). Table S4, Mann-Whitney U-tests for the comparisons of the slip angles between the two frog species under different testing regimes. ‘smooth’, ’30 μm’ and ‘1125 μm’ refers to the substrate roughness; ‘dry’, ‘low flow rate’ and ‘high flow rate’ refer to the amount of water on the surface.When the sample size was too small no z-value could be computed; when both samples contained identical values, no p-value could be obtained (‘NA’: not applicable). Table S5, Mann-Whitney U-tests for the comparisons of the fall angles for S. guttatus under different testing regimes. ‘smooth’, ’30 μm’ and ‘1125 μm’ refers to the substrate roughness; ‘dry’, ‘low flow rate’ and ‘high flow rate’ refer to the amount of water on the surface. Table S6, Mann-Whitney U-tests for the comparisons of the fall angles between the two frog species under different testing regimes. ‘smooth’, ’30 μm’ and ‘1125 μm’ refers to the substrate roughness; ‘dry’, ‘low flow rate’ and ‘high flow rate’ refer to the amount of water on the surface. Table S7, Mann-Whitney U-tests for the comparison of friction force per contact area for different body parts under different conditions between the two frog species. ‘smooth’, ‘0.3 μm’ and ’16 μm’ refers to the substrate roughness; ‘dry’ and ‘wet’ refer to the absence or presence of water on the surface, respectively. When the sample size was too small no z-value could be computed; when both samples contained identical values, no p-value could be obtained (‘NA’: not applicable). Table S8, Mann-Whitney U-tests for the comparison of adhesive force per contact area for different body parts under different conditions between the two frog species. ‘smooth’, ‘0.3 μm’ and ’16 μm’ refers to the substrate roughness; ‘dry’ and ‘wet’ refer to the absence or presence of water on the surface, respectively. When the sample size was too small no z-value could be computed; when both samples contained identical values, no p-value could be obtained (‘NA’: not applicable).(PDF)Click here for additional data file.Video S1Torrent frog (S. guttatus−1) conditions.) adhering to the rotating platform, on a rough surface (1125 μm particles) under high flow rate  position is reached.(MOV)Click here for additional data file.Video S2Use of the LED-illuminated platform enabled the visualisation and measurement of contact area during adhesion to a rotating glass platform. Here, at near 180 degrees (fully upside-down position), the tree frog  shows dynamic stability, using its toe pads alone. Under the influence of gravity, the limbs slide towards the body and are re-extended at intervals, while the fingers of the fore-limbs slide centripetally towards the hand, again being replaced in an extended position at intervals. Such ‘dancing’ behaviour allows the frog to avoid falling for it keeps the angle between pad and surface as small as possible, avoiding detachment by peeling (see (MPEG)Click here for additional data file.Video S3Comparison of typical turning behaviour of the tree frog  (upper video and animated graph) and the torrent frog (S. guttatus) (lower video and animated graph) on the LED-illuminated platform. Such turning takes place during rotation whenever the frogs find themselves facing downhill. Typically, body turning in the tree frog results in loss of body contact, so that the frog is subsequently attaching by its toe pads alone. However, torrent frogs usually turn without permanently losing body contact. Indeed, body contact area increases with the angle of tilt. The graphs show total contact area against platform rotation angle (a measure of time), the moving vertical line indicating the position in the video.(MPEG)Click here for additional data file.


There were errors in the Author Contributions. The correct contributions are: Conceived and designed the experiments: RE MSR RC HJB ITdA. Performed the experiments: RE MSR MB CF RMK. Analyzed the data: RE MSR TT YMS IR PL PK RC HJB ITdA. Contributed reagents/materials/analysis tools: TT PK RC HJB ITdA. Wrote the paper: RE MSR TT YMS IR PL PK RC HJB ITdA.


To the Editor: We have read with interest many of the articles concerning West Nile virus (WNV) published in the July 2003 issue of Emerging Infectious Diseases. Last summer Ohio was one of the leading states with WNV infection in humans. Consequently, requests for tests for this pathogen have increased. Unfortunately, the turnaround time for testing these specimens may be delayed because of shipping difficulties secondary to associates, the limited number of laboratories that can perform these assays, and an increase in requests at testing facilities.Cytologic examination of cerebrospinal fluid (CSF) from patients with WNV has not been studied. Although cytologic examination of CSF from patients with encephalitis is likely nonspecific, it may provide supportive information of the suspected disease process, and is useful for excluding other conditions, such as neoplasia. Of the 22 patients that were hospitalized at our institution last year with WNV meningoencephalitis, documented by serologic tests and/or reverse transcription-polymerase chain reaction, CSF of 4 of these patients was submitted for cytologic examination. Of these 4, 3 had a sufficient number of cells in the CSF specimen  to afford cytologic examination, whereas one had a paucicellular CSF, with only 2 white blood cells/μl. The cytologic features from the 3 patients >10 cells/μL consistently demonstrated a mixture of lymphocytes at various stages of activation and occasional large monocytic-like cells with cerebriform nuclei reminiscent of the Mollaret cells described in CSF of patients with recurrent meningitis  (1).Mollaret described cells with enlarged nuclei and cerebreform nuclear contours in CSF of patients with recurrent, aseptic meningitis (One of the patients infected with WNV meningoencephalitis who had Mollaret-like cells in CSF died. Postmortem neuropathologic examination showed an extensive perivascular lymphocytic infiltrate which contained mononuclear cells consistent with the Mollaret-like cells in CSF. These mononuclear cells were stained with an immunohistochemical stain directed against the CD68 antigen, which supports a monocytic origin (