Since September 2018, nearly 900 notifications of classical swine fever (CSF) have been reported in Gifu Prefecture (Japan) affecting domestic pig and wild boar by the end of August 2019. To determine the epidemiological characteristics of its spread, a spatio-temporal analysis was performed using actual field data on the current epidemic. The spatial study, based on standard deviational ellipses of official CSF notifications, showed that the disease likely spread to the northeast part of the prefecture. A maximum significant spatial association estimated between CSF notifications was 23 km by the multi-distance spatial cluster analysis. A space-time permutation analysis identified two significant clusters with an approximate radius of 12 and 20 km and 124 and 98 days of duration, respectively. When the area of the identified clusters was overlaid on a map of habitat quality, approximately 82% and 75% of CSF notifications, respectively, were found in areas with potential contact between pigs and wild boar. The obtained results provide information on the current CSF epidemic, which is mainly driven by wild boar cases with sporadic outbreaks on domestic pig farms. These findings will help implement control measures in Gifu Prefecture. Pestivirus, family Flaviviridae. CSF is described by the World Organisation for Animal Health as a highly contagious febrile disease with potential for high mortality that causes enormous economic loss in the pig industry worldwide [Classical swine fever (CSF) is caused by infection with the CSF virus (CSFV), which belongs to the genus orldwide . CSFV isorldwide . During orldwide . Outbreaorldwide . Based oorldwide ,5. The vorldwide ,7. Althoorldwide ,9. Moreoorldwide . No notifications of CSF were reported in Japan since 1992, and the country had an 11-year stretch of CSF-free status defined by the OIE Terrestrial Animal Health Code since 2007. However, CSF reemerged in Japan in September 2018 in Gifu Prefecture, which is located in the central part of the main island of Japan. Phylogenetic analysis revealed that the CSFV strain isolated in Japan in 2018 showed the highest identity in the complete E2 gene sequence with Chinese strains isolated between 2011 and 2015 and in the partial 5’-UTR sequence with strains isolated in China and Mongolia in 2014 and 2015 [By the end of August 2019, a total of 39 CSFV outbreaks on pig farms in four prefectures and 1,071 cases in wild boar in seven prefectures have been reported . DespiteThe present study conducted a spatio-temporal analysis to obtain epidemiological information on current epidemics of CSF in Japan. Based on the official CSF reports on domestic pig farms and wild boar, notified in Gifu Prefecture from September 2018 to June 2019, we assessed the direction of the spread of the disease and identified areas with high densities of notifications. In addition, to identify spatio-temporal aggregation of notifications and to characterize land cover vegetation in areas of disease aggregation, a clustering analysis was conducted, and obtained clusters were then overlapped with quality habitat map. The obtained information can be used to develop more effective disease control measures for application in both domestic pigs and wild boar. A standard deviational ellipse analysis was applied to describe the directional trend and dispersion of CSF notifications in the study area throughout the study period. The study covered the period between September 2018 and June 2019, which was divided into three stages . The multi-distance spatial cluster analysis was applied to explore the maximum distance between cases of CSF notifications. The results indicated that 23 km was the maximum distance of the significant spatial association between CSF notifications in Gifu Prefecture. The obtained maximum distance was used in the subsequent analyses.The kernel density estimation analysis was applied to describe the spatial distribution of the CSF notifications. The analysis showed that the highest density of CSF notifications was located in the southern part of Gifu Prefecture  with furP < 0.05) in Gifu Prefecture during the study period. Cluster 1, which had a radius of 12.12 km, covered 9 September 2018 to 13 January 2019, and contained 83 notifications, including 4 outbreaks on domestic pig farms. Cluster 2 had a radius of 19.79 km, spanning the period from 11 February 2019 to 19 May 2019, and contained 198 notifications, including three outbreaks in domestic pigs , a closed (>40%) needle-leaved evergreen forest (>5 m) (QAH 1.5), a mosaic of cropland (50%–70%) and vegetation (grassland/shrubland/forest) (20%–50%) (QAH 1.75), a mosaic of vegetation (grassland/shrubland/forest) (50%–70%) and cropland (20%–50%) (QAH 2), closed (>40%) broadleaved deciduous forest (>5 m) (QAH 2), and closed to open (>15%) mixed broadleaved and needle-leaved forest (>5 m) (QAH 2).Although different patterns of land cover vegetation were observed between clusters 1 and 2, nearly 50% of CSF notifications within cluster 1 and more than 75% within cluster 2 were notified in QAH 1.5–2, which provides the greatest opportunities for food and shelter for wild boar. From 2018 until August 2019, all notifications of CSF outbreaks in Japan have been made in Gifu Prefecture as well as in the surrounding four prefectures. A total of 1110 notifications had been reported so far, with 1071 affecting wild boar and 39 affecting domestic pig farms. The continuous notification of CSF in the area might have been attributed to wide spread of the virus within wild boar populations favored by free animal movements, as well as to the emergence of epidemiologically related domestic pig farms. To prevent the disease spreading in wild boar, control measures including (i) fencing to restrict animal movements, (ii) hunting activities for active monitoring and to reduce susceptible populations, and (iii) disseminating baits for oral immunization, were implemented. However, the efficacy of these strategies has not been confirmed. Therefore, we conducted a spatio-temporal analysis to obtain epidemiological information of the spread of CSF in Gifu Prefecture. Results from this analysis could help to increase our understanding of the current CSF epidemic and to contribute strategies for the containment of the disease in domestic pigs and wild boar. Japan is an island country that has achieved the status of freedom from several contagious animal diseases by implementing adequate control measures that take advantage of the country’s geography. Nevertheless, Japan has imported outbreaks of contagious animal diseases from neighboring countries. In 2010, there was an outbreak of foot-and-mouth disease (FMD) in Miyazaki Prefecture in the southern part of Japan, which caused extensive losses in animal husbandry. According to the high degree of sequence homology between an original virus isolated in Japan and viruses that were circulating widely in East Asia, it was suspected that the FMD virus might have been introduced via movement of people or commodities from East Asia . The higIn the present study, standard deviational ellipse analysis was conducted to measure the standard distance of CSF notifications. Shifting the centroids of identified ellipses indicated that the disease notification has spread in a northeast direction. Overlaying the three identified ellipses with a map of snowfall area in Gifu Prefecture revealed that the disease spread along the border of the snowfall area. In the south of Gifu Prefecture, there is a widespread area of flat land with field crops or animal farms, residential areas, and forests surrounded by mountains to the north. As suggested by other authors ,30, wildAnother concern regarding the spread of the disease is the potential for it to jump to remote areas. During the epidemic, CSFV infections were confirmed on seven farms that were geographically distant from, but epidemiologically linked, to the farms affected by CSFV  . Given tIn the present study, the results of the multi-distance spatial cluster analysis revealed that the maximum distance of relationship between CSF notifications was 23 km. Because of the small number of CSF outbreaks on domestic pig farms, we estimated the maximum distance of the relationship between notifications of domestic pigs and wild boar. This assumption could have influenced our estimated distance resulting in overestimation due to long distance spread observed on domestic pig farms. Nevertheless, similar approaches have studied another transboundary animal disease, African swine fever (ASF), which shares hosts and most of the transmission mechanisms with CSF ,32,33. WThe application of cluster analysis to identify areas with significant spatio-temporal aggregation of the ASF outbreaks in Sardinia from 2004 to 2013 indicated four clusters, the largest of which had a radius of 30 km . This doData on wild boar cases consisted of animals found dead and/or captured during surveillance activities. Many of wild boar were captured during active surveillance activities by setting traps and conducting hunting activities. Considering that most of the reported wild boar cases were located close to human habitats, the wild boar capture area may have been biased. Therefore, the disease could be wider spread in the area than what has been reported in official notifications, and the identified clusters could have had a shorter radius. Ideally, active virologic surveys should be intensively implemented to decrease the reporting biases by providing more samples to detect low levels of prevalence ,35. The According to the investigative report of the affected farms, there were some factors that might have increased the risk of CSFV introduction into affected farms, including (i) improper preparedness against invasion of wild or small animals into farms; (ii) imperfect clothing and boot changes in farms and pig pens, or disinfection of those materials; and (iii) inadequate vehicle disinfection . To prevFinally, we analyzed the QAH level of areas within the two identified clusters to characterize land cover vegetation in areas of disease aggregation. According to Bosch et al., a QAH 1 level corresponded to suitable areas for food or shelter for wild boar  . In clusInterestingly, the QAH map could also identify routes of CSF introduction or spread, mediated by wild boar, through vegetation or travel corridors. Travel corridors are either unbroken vegetation corridors or patches of habitat that enable animals to travel securely from one habitat to another . These pIt should be noted that vegetation types and wild boar behavior could vary among geographical features. For example, mountains usually have gentle slopes in Germany, whereas Japanese mountains tend to have precipitous slopes . These tAlmost one year has passed since the first notification of the CSF outbreak in Japan, and the spread of the disease has been confirmed mainly in wild boar. Fortunately, CSF outbreaks on domestic pig farms have been limited. Nevertheless, the potential risk of CSF introduction on farms could be high due to limited biosecurity, high number of wild boar cases in the area, and difficulties in implementing disease control measures in wildlife . The resGiven the potential risk of the ASF introduction from neighboring countries, we should summarize and disseminate the lessons learned from the current CSF outbreak to achieve the protection of ASF invasion or rapid containment of its occurrence even if it occurred.Epidemiological data for the periods from 9 September 2018 to 25 June 2019 were provided by the Gifu Prefectural Government, which provided the dates and coordinates (latitude and longitude) of the notifications of CSF in domestic pigs and wild boar. A total of 743 CSF notifications, 16 outbreaks on domestic pig farms, and 727 cases in wild boar were confirmed by RT-PCR and/or ELISA tests in the laboratory . As we fStandard deviational ellipse (SDE) analysis is a tool that provides the orientation and shape of a distribution, as well as its location, and dispersion or concentration of the data . It requA is the area, N is the number of events, d is the distance, and k is the weight, in which it is 1 when the distance between i and j is less than or equal to d and it is 0 when the distance between i and j is greater than d. To analyze the spatial pattern of CSF notifications, Observed K values were compared to the Expected K values of a completely random spatial distribution of CSF notifications with 999 simulations, which is equal to confidence levels of 99.9%. A multi-distance spatial cluster analysis tool in ArcGIS software version 10.6.1 was used to identify the maximum distance of the relationships between CSF notifications according to the guide on the manufacture’s website . In brieThe Diff K values contain the Observed K values minus the Expected K values. In the present analysis, the Expected K values that yield the highest Diff K values were applied as the maximum distance for relationships between notifications of CSF outbreaks in Gifu Prefecture.Kernel density estimation is a non-parametric estimator for describing the spatial extent of a series of events . In the P < 0.05). Analyses were conducted in SaTScan software v9.6  [A space-time permutation technique was applied to examine the presence of space-time clusters in Gifu Prefecture. The upper limit on the geographical size of the cluster was set as 23 km, the minimum time aggregation as seven days, and the maximum temporal cluster size as 50% of the total study period (default setting) . A MonteMA, USA) .CSF notifications within significant space-time clusters were overlaid on a QAH map to characterize land cover vegetation in areas of disease aggregation. The QAH map developed by Bosch et al.  is a car


The abnormal alterations of proteins 14-3-3 and tau in cerebrospinal fluid (CSF) are widely used for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD), while the situations of CSF biomarkers in genetic prion diseases (gPrDs), particularly in Chinese gPrDs patients, have not been well documented.Here, with the help of commercial 14-3-3 and total tau ELISA kits, we evaluated the levels of proteins 14-3-3 and tau in the CSF samples of 140 Chinese patients of 14 different types of gPrDs.We found that CSF 14-3-3 ELISA values in the patients with P102L GSS and D178N FFI were remarkably low, while those in the patients with T188K, E196A, and E200K gCJD were relatively high. Linear correlation assays identified a positive correlation between positive rate in Western blot (WB) and ELISA values of CSF 14-3-3. ELISA assays for total tau in CSF samples identified relatively high levels in the cases of T188K, E196A, and E200K gCJD , but relatively low levels in those of P102L GSS and D178N FFI (median: 64397.77 and 43856.79 AU/ml).PRNP.These data illustrate heterogeneous profiles of CSF 14-3-3 and tau in various types of gPrDs, depending on the differences in the mutations in  PRNP) have been noted to be directly associated with gPrDs (Human genetic prion diseases (gPrDs) account for approximately 10–15% in all human prion diseases worldwide  and consth gPrDs . Largelyth gPrDs .In the past two decades, screening biomarker in CSF for diagnosis of human prion disease has constantly attracted great attention. Several surrogate biomarkers in CSF have been proposed . HoweverPRNP. Among them, the case numbers of D178N FFI, T188K gCJD, E200K gCJD, P105L GSS, and E196A gCJD were top five. Besides routine 14-3-3 specific WBs, all CSF samples were subjected to the ELISA tests for 14-3-3 and tau with commercial kits. In addition, the possible associations of the elevations of the ELISA values of CSF 14-3-3 and tau with some main clinical and laboratory indexes were statistically analyzed.In the present study, we tested 140 CSF specimens from the Chinese patients of gPrDs covering 14 different subtypes of mutations in PRNP sequencing were collected from the database of the Center of Chinese CJD Surveillance System.A total of 140 CSF samples from the patients with various gPrDs were obtained from the CSF bank in the Center of Chinese CJD Surveillance System . The demAll enrolled CSF samples were obtained by standard clinical procedures and were free of blood contamination. Routine CSF biochemistry assays of those specimens, including cell count, glucose and total protein were all in the normal ranges.Besides the WB for CSF 14-3-3, the protein 14-3-3 in CSF of each patient was measured by a commercial double-antibody sandwich ELISA and CSF samples were diluted by 40-fold dilution with dilution buffer according to the manufacturer’s instructions . Briefly, 2.5 μl of CSF sample together with 97.5 μl of sample dilutions were transferred into a 96-well 14-3-3 Gamma ELISA kit and incubated at room temperature (RT) for 60 min. After being thoroughly washed with washing buffer four times, the solution of 14-3-3 Gamma detection antibody was added and incubated at RT for 60 min. After being washed, the solution containing HRP-conjugated antibody was added and incubated at RT for 60 min. The reactions were developed with addition of the solution of substrate for 15 min and terminated with the stop solution. Each reaction was measured automatically at 450 nm in an ELISA reader . The values of CSF 14-3-3 were correlated to the external standard curve supplied by the manufacturer and measured in arbitrary unit (AU) per ml. Three CSF samples  showed the negative reaction, whose data were under the threshold of the standard curve range and were set as 0 in the following calculation.2SO4. CSF tau concentrations were calculated based on a tau standard curve.The values of total tau protein in CSF were quantitatively measured with a commercial ELISA kit . Briefly, 25 μl of CSF sample was diluted together with dilution buffer supplied by the manufacturer and added to duplicate wells of the antibody-coated plate. Subsequently, the plate was incubated overnight at RT. After being washed well five times, 100 μl of HRP-conjugated detection antibodies were added into each well and incubated at RT for 30 min. The reactions were developed with an addition of 100 μl substrate working solution for 30 min in the dark, and terminated with the stop solution. Absorbance at 450 nm was measured by a microplate reader  after terminating the reaction by addition of 2 M HU test was performed for statistical analysis among the groups of positive and negative data in 14-3-3 WB and the categorical variables were compared using the Chi-square test. Multivariate logistic regression was used to analyze associations of CSF 14-3-3 or tau with relational influence factors. The p-values were corrected by the Bonferroni’s correction method and presented as ∗∗∗ (p < 0.001), ∗∗ (p < 0.01), ∗ (p < 0.05) and ns (non-significant) on the graphs.The data were processed with SPSS 17.0 statistics software, and descriptive data were expressed as median (range) for continuous variables and as percent (%) for categorical variables. The Mann–Whitney PRNP by sequencing. As shown in In total, 140 Chinese patients of various gPrDs were enrolled in this study. All cases were verified to contain a special disease-associated mutation in p < 0.01). It implies that P102L GSS and D178N FFI cases have different homeostasis of CSF 14-3-3 compared with the other genetic prion mutants.Using a commercial 14-3-3 gamma detection ELISA kit, the values of CSF 14-3-3 in 140 patients of different gPrDs were measured. The ELISA values of CSF 14-3-3 in 140 CSF samples ranged widely from 0 to 1944890.83 AU/ml . The medp < 0.0001). Only the 20.7% of WB-negative samples were in the groups of high ELISA values . Subsequently, the WB-positive rate and ELISA values of CSF 14-3-3 in each group that contained more than 10 cases were separately analyzed. The WB-positive rate of D178N FFI, P102L GSS, E196A, T188K, and E200K gCJD were 36.59, 45.45, 70, 71.79, and 80%, while the corresponding ELISA median values were 43856.79, 64397.77, 159992.80, 133840.81, and 153342.92 AU/ml, respectively. Linear correlation assays identified a positive correlation between WB-positive rate and ELISA values of CSF 14-3-3 . Additionally, one parameter of clinical auxiliary examinations, sCJD-associated abnormal changes in MRI, showed remarkable correlations with the elevation of CSF 14-3-3 (p < 0.05). Meanwhile, the patients with relative short disease duration showed association with a high level of CSF tau , the concentrations vary dramatically, even in the same types of gPrD. Generally, the cases with the mutations in the N-terminal part of PrP (before aa 183) seem to contain lower CSF 14-3-3 levels than those having mutations in the C-terminal part of PrP, despite that many mutants enrolled in the present study contain only a few cases. Among 14 different types of gPrDs in this study, 5 have 10 or more cases. As expected, the median ELISA values of CSF 14-3-3 in the groups of T188K, E196A, and E200K gCJD cases are higher than that of P102L GSS and D178N FFI cases. From the aspect of clinical manifestations, T188K, E196A, and E200K gCJD are more like sCJD . In CSF Detection of CSF 14-3-3 protein by 14-3-3-specific WB is widely used as a diagnostic criteria for probable sCJD . Some stThe increase of CSF total tau has been repeatedly observed in the patients affected by sCJD, and it is considered to be another good diagnostic biomarker for sCJD . The altPRNP influences the levels of CSF 14-3-3 and tau in gPrD cases (We have analyzed the potential factors that may contribute to the increases of CSF 14-3-3 and tau measured by ELISA in gPrDs. Similar as the positive CSF 14-3-3 in WB, no special clinical element is closely associated with the elevation of 14-3-3 in CSF, besides MRI abnormality and PSWC in EEG. CSF 14-3-3 positive in WB is the most significant factor responsible for elevation of CSF tau in all gPrDs, which may somehow reflect the abnormality in CSF homeostasis . AnalysirD cases . HoweverBecause the main aim of this study is evaluation of the possible different levels of CSF 14-3-3 and tau in various gPrDs via ELISA method, the diagnostic potential for gPrD is not addressed. Although different gPrDs appear in the distinguishable ELISA profiles of CSF 14-3-3 and tau, the individual concentrations of CSF 14-3-3 and tau vary largely even in the same subtype of gPrD, which may hinder the usage for diagnosis of gPrDs. Certainly, the study here does not contain controls, such as the CSF samples of sCJD, non-CJD and other diagnosed neurodegenerative diseases. It is impossible to figure out the cutoff values in those two ELISA tests for gPrDs. Therefore, the clinical significances of ELISA 14-3-3 and tau in gPrDs need further study.PRNP.These data illustrate heterogeneous profiles of CSF 14-3-3 and tau in various types of gPrDs, highly depending on the differences in the mutations in All datasets generated for this study are included in the manuscript and/or the Usages of stored CSF samples and relevant clinical information of the patients with gPrDs in the Center of Chinese CJD Surveillance System has been approved by the Ethics Committee of the National Institute for Viral Disease Control and Prevention, China CDC.CC and CH designed the study and drafted the manuscript. WZ provided the patients data. CH, JC, and QS carried out the ELISA. KX, YW, LL, and YX performed the statistical analysis. X-PD conceived of the study, participated in the study design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.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.


We quantified chemotaxis, oxidative burst, and phagocytosis before and after treatment and detected significantly reduced chemotactic activity upon G-CSF treatment. Similarly, in vitro treatment of previously untreated neutrophils with G-CSF led to reduced chemotactic activity. In addition, we revealed that this effect persists in the allogeneic SC recipients up to 4 weeks after neutrophil engraftment. Our data indicates that neutrophil quantity, as a sole measure of immunocompetence in high-risk patients should be considered cautiously as neutrophil functionality might be affected by the primary treatment.Neutrophils are crucial for the human innate immunity and constitute the majority of leukocytes in circulation. Thus, blood neutrophil counts serve as a measure for the immune system's functionality. Hematological patients often have low neutrophil counts due to disease or chemotherapy. To increase neutrophil counts and thereby preventing infections in high-risk patients, recombinant G-CSF is widely used as adjunct therapy to stimulate the maturation of neutrophils. In addition, G-CSF is utilized to recruit stem cells (SCs) into the peripheral blood of SC donors. Still, the actual functionality of neutrophils resulting from G-CSF treatment remains insufficiently understood. We tested the  All parts of the study were performed according to the Declaration of Helsinki. The patients were included after informed written consent.Blood samples were collected between 2010 and 2017 at Umeå University Hospital, Sweden. The first cohort consisted of healthy individuals selected by Hematology Unit clinicians to donate for bone marrow transplantation Table . Before 9/l  and incubation occurred under standard cell culture conditions: 37°C, 5% CO6 neutrophils/ml were stained with 3.3 μM bis-2-carboxyethyl-5-[and-6]-carboxyfluorescein-AM (Sigma-Aldrich) for 20 min at room temperature, washed, and resuspended in medium with 0.05% human serum albumin. Fluorescence-impermeable transwell inserts  were loaded with 5 × 105 cells each and placed into wells of 24-well plates containing 600 μl RPMI, with or without 10 nM formyl-methionyl-leucyl-phenylalanine (fMLF). Fluorescence intensity (FI) with excitation 485 nm and emission 520 nm was detected in a plate reader  with 5% CO2 and 37°C over the course of 30 min. Medium only served as blank and 5 × 105 stained cells seeded directly into the lower compartment served as 100% FI reference. If indicated, we added 10 ng/ml or 50 ng/ml G-CSF  to the respective neutrophil sample. Subsequently, cells were washed in medium and used as described. Thus, if not stated otherwise, neutrophils are unstimulated prior to the analysis. Migration was calculated as percent of 100% reference Migration of unstimulated cells was subtracted from the fMLF-induced migration. Half migration time was determined as time required for 50% of maximum migration. Velocity at half migration time was calculated as Δ%/min . Neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) or left untreated. Chemiluminescence as a measure of hydrogen peroxide production was detected every 2 min for 3 h in an Infinite 200 luminometer (Tecan). ROS were quantified by the area under the curve (AUC) calculated for the time given (3 h) and reduced by background of unstimulated cells.Oxidative burst was quantified by luminol-based chemiluminescence . BrieflyS. aureus bioparticles conjugated for phagocytosis, Lifetechnologies) that change their FI pH-dependently. Briefly, 5 × 104 neutrophils per well were added into a black 96-well plate with 100 μg beads/well. Beads put directly into a buffer with pH 4 served as 100% FI control. Beads in medium with pH 7.4 served as blank. Neutrophils pre-treated with 10 μM cytochalasin D for 15 min to block phagocytosis served as negative control. FI (540/580 nm) was measured every 60 min for 3 h .Phagocytosis was assessed using pH rodo beads  using Luminex ELISA assays . Assays were performed according to manufacturer's protocol using a magnetic beads washer. For G-CSF quantification, the samples  were diluted 1:4 in sample diluent. For IL-8 quantification in plasma from HSC donors, a high-sensitive Luminex assay (BioTech) was used with standard range 0.78–3200 pg/ml. Samples were diluted 1:2 in calibrator diluent RD6-40 and added to plates for subsequent analysis with a Bio-Plex reader . Plasma samples were used for the analysis of C-reactive protein (CRP) which was quantified using the CRPL3 cobas® assay (Roche Diagnostics Scandinavia) performed by the Clinical Chemistry Laboratory at Umeå University Hospital according to the manufacturer's recommendations and the standard ISO/IEC 17025:2005 with continuous evaluations by the Swedish Board for Accreditation and Conformity Assessment.5 neutrophils were treated with 50 ng/ml G-CSF. After 30 min, 1 μg/ml LPS was added as a co-stimulant for 6 h. As control, cells were treated with medium only. After 6 h, supernatants were collected and directly frozen at −80°C. The IL-8 concentration was quantified according to manufacturer's protocol (BioLegend Human IL-8 ELISA Max standard set).To analyze IL-8 release upon G-CSF stimulation, neutrophils were isolated from healthy volunteers, and 5 × 104 cells were analyzed per sample and all samples were run in duplicates.G-CSF treated as well as untreated neutrophils were stained with labeled monoclonal anti-fMLP receptor antibody coupled to PE  in PBS supplemented with 3% FCS in a final dilution of 1:10 and incubated for 30 min at 8°C. Cells were washed 3 times in PBS with 3% FCS followed by flow cytometry analysis (BD Biosciences LSR II). The population of neutrophils were identified using FSC-A and SSC-A. At least 10t-test, and one-way ANOVA with Tukey's multiple comparison tests. Mann-Whitney U-, Wilcoxon signed-rank and Kruskal-Wallis tests were used when data was not normally distributed. A p-value below 0.05 was considered significant.We used Graph-Pad Prism V 5.04 for statistical analysis. Data was analyzed using Students ex vivo, we detected that neutrophils isolated from HSC donors after G-CSF-treatment displayed significantly increased half migration time and a reduced velocity at half migration time compared to neutrophils before treatment  is a central chemokine for neutrophil recruitment contributing to the extravasation of neutrophils to the site of infection . C-reactin vitro . We therin vitro . Notablyin vitro . After ti Figure . In conti Figure .We compared neutrophil chemotaxis from controls and allogeneic transplant recipients 2 weeks after neutrophil engraftment Figure  and obseAnalysis of neutrophils sampled 4 weeks after neutrophil engraftment and beyond Figure  showed tROS, produced by the NADPH oxidase complex in phagocytes, are important mediators of cell signaling and very powerful effectors of antimicrobial killing. Thus, we investigated whether G-CSF treatment alters total ROS production in neutrophils stimulated with PMA for 3 h. We could not detect a difference in ROS production by neutrophils from HSC donors before and after G-CSF-treatment Figure . SimilarStaphylococcus aureus. Upon engulfment, these beads increase fluorescence intensity in the acidic milieu of the mature phagosome. To confirm actual phagocytosis, we included neutrophils treated with cytochalasin D as a negative control. Cytochalasin D inhibits cytoskeleton rearrangements and thus allows discrimination between adherence and phagocytosis. When comparing the phagocytic uptake by neutrophils from HSC donors before and after G-CSF treatment as well as the neutrophils from the allogeneic transplant recipients at different time points of harvest we could not observe any significant difference  and the resulting plasma concentrations were in the expected ranges (ex vivo chemotaxis toward fMLF. Other key functions tested, i.e., ROS production and phagocytosis, seemed to remain unaltered. Strikingly, the chemotactic impairment observed in neutrophils from HSC donors persisted in the group of allogeneic transplant recipients. Even after transplantation and novel granulopoiesis within the transplant recipient, newly emerging neutrophils showed the same migration defect as the neutrophils from the HSC donors. Of note, the recipients themselves received no prophylactic G-CSF. According to our data, it took several weeks until normal neutrophil activity was regained. Notably, we observed the chemotactic impairment even though the allogeneic transplant recipient group was heterogeneously composed with diversity regarding the underlying disease. Nevertheless, these patients frequently receive immunosuppressive therapy, such as steroids or calcineurin inhibitors, which in turn were shown to impair neutrophil functions. However, the effects of these type of drugs are usually pleiotropic and do not specifically target neutrophil chemotaxis. For instance, calcineurin inhibitors can impair chemokinesis of neutrophils on vitronectin (Using G-CSF to mobilize HSCs has revolutionized the treatment of blood malignancies as it often replaces the classical bone marrow transplant . Insteadd ranges , 35. As ronectin , reduce ronectin  and dimironectin . Furtherin vitro effects as discussed in more detail below. Interestingly, in vitro studies triggering neutrophils directly with G-CSF observed a rapid stimulation of random mobility that declined within about half an hour (in vitro chemotaxis did unlikely result from decreased surface exposure of the fMLF receptor.Our results confirm previous studies describing impaired neutrophil functionality in allogeneic transplant recipients. Sosa et al. reported that neutrophil chemotaxis is impaired up to 4 months after HSC transplantation  and Kato an hour . In our More strikingly, we demonstrate that reduced neutrophil chemotaxis persisted within the allogeneic transplant recipients at 2 weeks post HSC engraftment. The average life span of neutrophils in circulation is relatively short; it is estimated to be not longer than a few days –48. So, + cells that are used as a measure for HSC mobilization. Again, this is in contrast with our findings: We could not detect any correlation between IL-8 levels and amount of harvested CD34+ cells or the neutrophil count in circulation. Since IL-8 is pivotal for neutrophil chemotaxis, we conclude that the significant reduction of IL-8 plasma levels might contribute also to reduced chemotaxis of neutrophils. However, it remains unclear, which cells are responsible for IL-8 production under these conditions.In addition to reduced chemotaxis, we show that IL-8 plasma levels of HSC donors are reduced compared to untreated controls after G-CSF treatment. That decrease is contradictory to a study by Watanabe et al. where the authors reported elevated levels of IL-8 after G-CSF-treatment even though the G-CSF treatment regimen was virtually identical to our donors . Moreovein vivo by chemotaxis, others described an unchanged infection risk in G-CSF-treated HSC donors (Neutrophils are crucial for the innate immunity. G-CSF is therefore used in neutropenic patients as a preventive treatment to avoid infections. Still, a study with transplant recipients reported that preventive G-CSF treatment did not alter infection-related mortality in these patients . While tC donors . Taken tin vitro (Previously, it was shown that CRP levels increased upon G-CSF treatment . Our datin vitro .in vitro experiments with purified neutrophils, 10 ng/ml G-CSF had no effect on neutrophil chemotaxis, only 50 ng/ml G-CSF impacted neutrophil chemotaxis. Taking this into consideration, our findings indicate that lower IL-8 and higher CRP concentrations in combination with a direct effect of G-CSF might render neutrophils less motile in G-CSF-treated individuals. Reduced IL-8 levels in plasma of G-CSF treated individuals could stem from non-neutrophil IL-8-sequestering cells.To judge whether reduced chemotaxis in neutrophils is a direct effect of the G-CSF present in blood, we have detected G-CSF plasma levels. Even in treated HSC donors, G-CSF plasma levels ranged around 1 ng/ml. Other studies have described slightly higher plasma concentrations ranging from 4 to > 15 ng/ml . In our E. coli, but not when stimulating with PMA, confirming our findings (In our cohorts, ROS production and phagocytosis were not affected by systemic G-CSF treatment in healthy HSC donors or in allogeneic recipients. Oxidative burst and phagocytosis remained unchanged in all cohorts and at all time points of sampling. This specificity suggests that the reduced chemotaxis in our cohorts is not a result of general immaturity of neutrophils mediated by G-CSF as immaturity of neutrophils is usually accompanied by the impairment of other functions as well , 55. It findings . Macey afindings .Allogeneic transplant recipients are susceptible to infections . With a DE, AW, CA, MN, and CU designed the research study. AW, FÅ, and AT recruited and took care of the donors and patients. AT, MN, AH, DE, MS, and MR performed the experiments. AT, MN, AH, MS, and CU analyzed the data. AT, MN, DE, and CU wrote the paper.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.


AimTo study the tibial tuberosity-trochlear groove distance (TT-TG) in normal Indian population and the variation of the same in relation to tibial size using computed tomography (CT) of knee.MethodsCT of 100 knees  were assessed. TT-TG distance and maximal medio-lateral (MML) distance of tibia was measured on axial CT scans. The modified TT-TG (mTT-TG) was calculated as the ratio of TT-TG and MML.ResultsThe average TT-TG distance was 13.01 (±2.84) mm for the entire group with males and females having 12.82 (±2.95) and 13.32 (±2.66) mm, respectively (p > 0.05). The MML distance was 75.99 (±3.78) and 66.77 (±4.33) mm for males and females, respectively (p < 0.05). The average modified TT-TG was 0.18 ± 0.04. The TT-TG distance of Indian knees was similar to values obtained in Caucasian knees and higher than other Asian knees (p < 0.05).ConclusionThe average TT-TG distance in Indian population is 13.01 mm, with no difference between males and females. The ML/TT-TG ratio was 0.18. The TT-TG distance in Indian population is found to be similar to the Western population and significantly higher than other Asian population. Tibial tuberosity-trochlear groove distance (TT-TG) relates to the relationship of the tibial tuberosity with the femoral trochlea and is used to assess the degree of external torsion and lateralisation of the tibial tuberosity in patellar instability . UnderstWe here present a CT-based assessment of TT-TG distance in normal Indian population and the variation of the same in relation to tibial size. The null hypothesis kept at the start of the study was that TT-TG distance in Indian population was different from Western population.This was a CT scan-based observational study of 100 knees. CT scans of patients who had undergone CT angiography of lower limbs for various reasons with normal knee CT, were used for the study. The sample size of 100 knees was calculated on the basis of study by Mullaji et al. . CT scanThe TT-TG distance was measured between the most anterior point of tibial tuberosity with the deepest bony point of trochlear groove which is perpendicular to the tangent to bony borders of the posterior condyles of distal femur on axial CT scans . First, Measurements were made using Horos Dicom viewer. The data obtained was assessed using Microsoft Excel 2010 and XL-STAT software. The values were expressed as mean ± standard deviation. The normality of test was assessed with Shapiro-Wilk test. Student’s t test or Wilcoxon-Mann-Whitney test was used to assess the statistical significance and p-value of <0.05 was considered significant. The normal range of MML-TT-TG was determined with 95% confidence interval. The TT-TG values obtained in our study were compared with similar studies in literature on Caucasian and other Asian population ,9. T-tesThe mean TT-TG distance for the entire study group was 13.01 mm (±2.84). There was no significant difference in TT-TG distance between male and female population (p > 0.05). The MML distance was 75.99 mm (±3.78) and 66.77 mm (±4.33) for the male and female group, respectively. This was found to be statistically significant (p < 0.05). The average ML/TT-TG ratio (mTT-TG) was 0.18 (±0.04). There was significant difference in this ratio between male and female subjects. The ML/TT-TG ratio averaged 0.18 (±0.04) with males and females having 0.17 (±0.04) and 0.2 (±0.05), respectively. This difference was found to be statistically significant (p < 0.05)  mm for Korean population . There wPatellar instability is a relatively common orthopaedic problem for which the TT-TG distance is a predominant factor in evaluation. An increased tibial tuberosity-trochlear groove distance is considered as an isolated and independent predisposing factor leading to patellofemoral instability and even more as an indication for performing distal realignment procedure around knee like medialisation of the tibial tuberosity . It is oThe tibial tuberosity position relative to the femoral trochlea is pertinent for the quadriceps mechanism to function normally as it decides the direction of the inferolateral force vector on the patella and pull of the quadriceps mechanism . In a noA TT-TG distance in the range 10-15 mm is usually considered as normal, and an increased distance is an indication for surgical intervention in the form of medial tibial tuberosity transfer to correct patellofemoral malalignment . HoweverOur study revealed the mean TT-TG distance to be 13.01 ±2.84) mm with no significant difference between gender. Twenty-four percent of the subjects had a value of more than 15 mm. Dejour et al. used CT scans with improved accuracy and reliability to calculate the TT-TG distance [4 mm withPennock et al. found an association of TT-TG distance with height and observed a variation of TT-TG distance by 0.12 with each centimetre increment . BalcareThe study has its limitations. Firstly, the sample size we had in our study was small. But, this is comparable to the rest of the literature at present. Secondly, MRI-based assessment of the TT-TG distance is being considered by many as a single investigation that would identify the underlying pathology and assess the status of the cartilage. But, there is a lack of clarity in respect to the validity of the techniques as Aarvold et al. have demonstrated that the MRI done with dedicated knee coils often gives altered distance . This stThe present study reveals the average TT-TG distance to be 13.01 mm in Indian population with males having average distance of 12.82 mm and females 13.32 mm. The ML/TT-TG ratio was 0.18. The TT-TG distance in Indian population is found to be similar to the Western population and significantly higher than other Asian population.


Handwashing prevalence in schools in Kenya is low due to lack of access to water and soap and lack of drive for handwashing. Soapy water made from detergent powder is an inexpensive alternative to bar soap and disgust and social norms change can be powerful drivers of handwashing, but their effectiveness has not been assessed in school setting. In Kenyan public schools, we evaluated an equipment-behavior change intervention’s effect on handwashing outcomes. We also monitored functionality of the Povu Poa prototypes to identify design improvements necessary for continued high usage in institutional settings.The intervention included the “Povu Poa”, a new type of handwashing station that dispensed foaming soap and rinse water, combined with school-wide behavior change promotion based on disgust and social norms. In this stepped-wedge cluster-randomized trial, we randomly selected 30 schools and divided them into 3 groups of 10. Following baseline data collection, we delivered the intervention sequentially . We observed outcomes [1] availability of handwashing materials at handwashing places, and; 2) observed handwashing behavior after toilet use among schoolchildren) at baseline and in three follow-up rounds. We compared the outcomes between schools that had received the intervention and schools that had not yet received the intervention.Water and soap/soapy water were available at 2% of school visits before intervention, and at 42% of school visits after intervention.. Before intervention, we observed handwashing with water after 11% of 461 toilet use events; no one was observed to wash hands with soap/soapy water. After intervention, we observed handwashing after 62% of 383 toilet use events  and handwashing with soap/soapy water after 26% of events . Foaming soap dispenser caps were cracked in 31% of all observations, but were typically still functional.Our combined equipment-behavior intervention increased availability of handwashing materials and improved the compliance with handwashing after using the toilet, but handwashing with soap was still rare. Equipment durability must be improved for deployment in schools at scale.American Economic Association’s Registry for Randomized Controlled Trials; Trial Registry Number (TRN): AEARCTR-0000662; Date of Registry: April 14, 2015.The online version of this article (10.1186/s12889-019-6902-2) contains supplementary material, which is available to authorized users. Diarrhea and respiratory infections are among the most common causes of death in children in low-income countries , 2. HandApproximately 15% of the population in low and middle-income countries wash their hands with water and soap after fecal contact . In schoPrevious interventions to increase handwashing in schools include efforts to overcome the lack of soap by budgeting for soap purchases , 12, hirSchools in Kenya often lack funds for purchasing soap, and bar soap tends to be stolen or lost , thus maThere is a well-recognized gap between knowledge about handwashing and actual handwashing practice, even following exposure to handwashing promotion –22. PrioThis study assessed the effect of providing Povu Poa handwashing stations with foaming soap dispensers combined with behavior promotion utilizing disgust and social norms in public primary schools in Western Kenya on two primary outcomes: 1) availability of handwashing materials at handwashing places; 2) observed handwashing behavior after toilet use among schoolchildren. We also used the school pilot as an opportunity to monitor the durability of the product equipment over a 1-year period in a high-usage setting to identify design improvements that would increase robustness and extend the product lifespan in school settings.Povu Poa (“Cool Foam”) handwashing stations combined with a behavior intervention designed to change social norms and use disgust as a behavioral trigger. The Povu Poa handwashing stations are portable handwashing systems consisting of a water container in the form of a bucket or pipe, a wooden or plastic stand, a soap-frugal foaming dispenser (that converts soapy water into foam), and a water-conserving tap [We conducted a cluster randomized trial using a stepped wedge design to evaluate the use of ving tap .We chose the stepped-wedge design for several reasons. First, as the intervention posed no harm and brought benefit to the participating schools by creating places to wash hands with soap, it would be unethical to withhold the intervention in some participating schools. Second, the sequential roll-out of the intervention enabled the enumerators to deliver the intervention to the 30 participating schools while minimizing logistical constraints.Consistent with the stepped-wedge design, we first collected baseline data at all schools, then randomly allocated the schools into 3 groups of 10, and rolled out the intervention sequentially by group. The order in which each group of schools received the intervention was randomly assigned . All datWe approached the intervention based on existing theories of behavior change . We hypoWe conducted in-depth interviews and focus-group discussions to elucidate factors influencing handwashing behavior with household members, students, and teachers in the study area. The non-profit Catapult Design developed the soapy water handwashing station (SW-HWS) and pilot-tested the preliminary designs in the study region  schools in Kisumu County were randomly selected using a computerized random number generator.We obtained a list of public primary schools in Kisumu County from the local Kenyan Ministry of Education office and used the list as the sampling frame. Thirty  be public; 2) be a day school, without boarding students; 3) have an existing handwashing system with soap and water; 4) have at least 200 students, and 5) lack access to a piped connection providing continuous water supply (schools with intermittent water supply were eligible).We recruited enumerators from Innovations for Poverty Action - Kenya’s roster of local staff. Enumerator training lasted approximately 1 week and included overview of the study, codes of conduct at the target schools, detailed training on intervention delivery, detailed training on the study instrument, and protocols for data collection and quality control.Trained enumerators approached the randomly selected schools, asked to meet with the head teacher to explain the study and sought consent for the school to enroll in the study. The consent form included information on purpose of the study, intervention delivery, interviews and observations to be made at the school, follow-up visits, benefits and risk from the study, confidentiality, voluntary nature of participation, and contact details of research staff. The Maseno Universty Ethics Review Committee approved of the procedures for this study. MUERC No.: MSU/DRPC/MUERC/000099/14.After obtaining consent, the enumerators collected baseline data by interviewing the head teachers and the teachers responsible for school water, sanitation and hygiene. The content of the interview included characteristics of the school, availability of water and soap, availability of budget for soap purchase, and activities related to water, sanitation and hygiene at the school. Schools that met the eligibility criteria were included in the study. One sampled school was ineligible because it had fewer than 200 students, and we replaced the school with another randomly selected school.In addition to the interviews, the enumerators also conducted rapid observation of handwashing place, where the enumerators observed the presence of soap and water at the school’s handwashing place. After observation of handwashing place, the enumerators also conducted structured observation of handwashing behavior among schoolchildren. We trained the enumerator to position themselves in a discreet location that was adequately far away from the handwashing palce to not be immediately noticed, but still had a direct line of sight to the handwashing place. Enumerators then followed the instructions in the data collection instrument, and observed handwashing behavior after toilet use among students for 1 h or until there were 10 observed events, whichever came first. These observations of handwashing place for presence of soap and water and observations of handwashing behavior after toilet use were repeated during each of the additional visits to the school.We delivered the intervention in sequence: to the first group of 10 randomly-selected schools (Group 1) in early-June 2015, the second group of 10 schools (Group 2) in early-July 2015, and the third group of 10 schools (Group 3) in mid-October 2015. Group 1 received the intervention 3–5 weeks after the baseline period. Group 2 received the intervention 6–8 weeks after baseline. Group 3 received the intervention 19–24 weeks after baseline.On the day that the intervention was implemented, enumerators provided the teacher and student leaders responsible for water, sanitation and hygiene activities with 2 soapy water handwashing stations and briefed them on use and maintenance. Enumerators also delivered the handwashing behavioral intervention to the entire school via school-wide assembly Table . The behStudy team members made 3 rounds of follow-up visits to each participating school after baseline Fig. . The avaWe used descriptive statistics and log-binomial regression analyses to assess the effect of the intervention on two outcomes: 1) availability of soap and water at the school’s handwashing place; and 2) handwashing with water (and soap) after toilet use during structured observation. We used descriptive statistics to compare outcomes in participating schools after receiving the intervention (post-intervention schools) to outcomes in the same participating schools before receiving the intervention (pre-intervention schools). We then used log-binomial regression to calculate prevalence ratios (PR) and 95% confidence interval. We used the school ID number as the repeated variable to account for the effect of clustering at the school level. We stratified the analyses by levels of co-variables to assess for effect modification. All analyses were conducted using SAS version 9.4.The characteristics of the participating schools are summarized in Table Post-intervention schools had significantly higher probability of having water and soap at the handwashing place compared to pre-intervention schools Table . HoweverStructured observation included observation of 461 toilet use events in pre-intervention schools, and 383 toilet use events in post-intervention schools. There was a similar proportion of female and male students at both pre-intervention and post-intervention schools, and a similar proportion of younger students (age ≤ 10 years) and older students (age > 10 years) Table . The preOur study assessed the effectiveness of two major innovations to motivate handwashing: a cost-saving soap foam dispenser with a water-frugal tap, and a behavior change intervention aimed at triggering disgust and promoting handwashing as a social norm. This study is the first to assess the effect of both interventions delivered in combination at schools in a low-income country setting. Availability of water and soap at handwashing place and observed handwashing after toilet use were significantly higher after receiving the intervention than before the intervention. Observed handwashing with water and soap increased from non-existent to 26% of toilet use events. In the study area, WASH  clubs are clubs of students with one supervising teacher each with the purpose to engage schoolchildren in activities related to water, sanitation and hygiene. Schools that had an active WASH club might have a more rapid adaptation of the intervention than schools that did not have a WASH club because the club’s purpose aligned with implementation of the intervention.The behavior change intervention activities in our study aimed to address multiple drivers of behavior, including injunctive norm, i.e., the perception of what is commonly approved and disapproved among relevant others . The “ToWith regards to disgust and social norms, after the “Toilet and Shake” Skit, there was a session to teach handwashing song and a handwashing pledge. The aim of the song and the public pledge was to create a sense of awareness that each student’s peers also had disgust for unclean hands and a commitment for handwashing, which had the potential to lead to formation of injunctive norm in each participating student. Experimental data from a previous study suggested that the use of strong injunctive normative elements as focal point of a behavioral decision can be effective in creating desirable conduct . HoweverOur study only measured handwashing-related outcomes and did not measure changes in any of the potential psychosocial determinants of handwashing. We do not know which psychosocial behavioral determinants, or a combination thereof, were affected by the intervention and were associated with changes in handwashing behavior. Limitations with regards to lack of measurement of psychosocial drivers of handwashing behavior and negative consequences of the interventions should be taken into consideration when assessing suitability of this type of intervention.Post-intervention availability of handwashing materials was higher in this study than in the 2012 evaluation of a school and household handwashing promotion program implemented in the same region of Kenya, which used health education based on a soap mascot, and found that only 13% of schools had a handwashing place with soap and water at post-intervention . The useHowever, at post-intervention, approximately half of all observed handwashing systems still did not have water and soapy water available for handwashing. Anecdotally, field staff reported that the caps and water taps were not resistant to ultraviolet radiation from the sun and became brittle and cracked, which may have affected functionality. This cracking might have been prevented by using UV resistant plastic to manufacture the soap dispenser caps and water dispenser taps. There were also more than 400 students in three-fourths of the participating schools, thus the student-per-handwashing-station ratio was at least 200:1 contributing to crowding and frequent usage. A general guideline for the student-per-handwashing-station ratio does not exist, but the guideline for sanitation recommends 20–40 students per latrine . ProvidiProbability of handwashing with soap after toileting at the first, second and third post-intervention visits were all significantly higher than the probability of handwashing with soap pre-intervention, suggesting that the effect could last beyond the study period. In that regard, stepped-wedge trials are not free from potential biases, particularly when there are secular trends associated with the study outcome . HoweverEven at post-intervention, only one-third (32%) of observed toilet use events was followed by handwashing with soap. Although the change in probability of handwashing with soap after toilet use was significant, it still was less than ideal. As mentioned earlier, the theoretical framework for the intervention posits that the intervention changes physical and psychosocial factors for handwashing, which then drives compliance to handwashing after toilet use. The data showed that the physical factors, i.e., the availability of water and soap for handwashing, improved after the intervention. According to the theoretical framework, it is possible that the intervention might not have been ideal in changing psychosocial factors, or the psychosocial factors that were targeted by the intervention were not strong determinants of handwashing in our study setting.Future studies should consider other interventions that yield high probability of handwashing with soap at post-intervention. A 6-week pilot intervention study in Bangladesh that employed only visual cues (nudges) increased the probability of handwashing with soap from 4% at baseline to 68% post-intervention . IncorpoThere are five major limitations in our study. First, it was not possible to blind the enumerator from the intervention status of the school because our study involved installation of handwashing equipment and the soapy water handwashing stations were the focus location of the structured observation. The enumerators could have introduced observer bias into the study. However, we trained the enumerators to adhere strictly to established observation protocols and there was no incentive for the enumerator to over-report handwashing behavior in the post-intervention period. Second, we could not observe handwashing behavior following 12% of toileting events in pre-intervention schools and 21% of toileting events in post-intervention schools  because there were multiple children or events to observe at the same time, and the children might crowd around the handwashing station, making detailed observation difficult. These non-observations were classified as missing data. If the non-observations did not occur at random, then it could have biased our estimate of the effect of the intervention either away from the null or toward the null, depending on what was not observed during the pre-intervention period and the post-intervention period. However, non-observation is unlikely to be non-random because the interviewers were trained to follow established observation procedure that was identical for pre-intervention and post-intervention schools. Third, our study did not include measurement of psychosocial drivers . We could not assess the effect of the intervention according to the theory of change, thus we could not conclude whether the observed effect of the intervention happened through changes in disgust, social norms, other psychosocial drivers of handwashing, or a combination thereof.Fourth, our observation of handwashing behaviors did not include non-toileting events, such as handwashing before eating and after touching respiratory fluids. Therefore, we cannot comment on the efficacy of the intervention for handwashing behavior change at other times of possible pathogen transmission. Lastly, we conducted this study in public primary schools in one county in the western part of Kenya, and thus, the results may not be generalizable to other settings with different infrastructural and social contexts. Future studies should consider: 1) interventions that can further improve handwashing behavior, aiming for > 90% handwashing compliance after toileting; 2) replication of the study to include all pathogen hand transmission events on a larger scale in other low-income country settings, and; 3) measurement of pre-intervention and post-intervention social norms if the intervention includes a component that aims to change social norms.A stepped wedge cluster randomized trial at public primary schools in western Kenya had an intervention that included providing water-frugal handwashing system with foaming soap and a behavior change program employing disgust, norms, and pledging. Comparing pre-intervention outcomes to post-intervention outcomes, the intervention improved availability of soap and water at handwashing places, and enhanced the probability of handwashing with water and soap after toilet use among schoolchildren.Additional file 1:Table S1. Observed handwashing behaviors at toileting events according to whether there was at least one handwashing place with water and soap during rapid observation (n = 844 toilet use events in 30 schools). (DOCX 35 kb)


Hemiechinus auritus). Our results show that live, non-apoptotic, germ cells are massively depleted by desquamation during the testis regression process. This is concomitant with both decreased levels of serum testosterone and irregular distribution of the cell-adhesion molecules in the seminiferous epithelium. The inactive testes maintain some meiotic activity as meiosis onset is not halted and spermatocytes die by apoptosis at the pachytene stage. Our data support the notion that apoptosis is not the major testis regression effector in mammals. Instead, desquamation appears to be a common mechanism in this class.Testes of seasonally breeding species experience a severe functional regression before the non-breeding period, which implies a substantial mass reduction due to massive germ-cell depletion. Two alternative mechanisms of seasonal germ-cell depletion have been described in mammals, apoptosis and desquamation (sloughing), but their prevalence has not been determined yet due to reduced number of species studied. We performed a morphological, hormonal, and molecular study of the mechanism of seasonal testicular regression in males of the Egyptian long eared-hedgehog ( In the north, reproduction occurs in summer and males undergo complete testis regression in winter. On the other hand, in the south reproduction takes place in winter and no testis regression occurs during the non-breeding season . The authors proposed that this isa case of adaptive lack of seasonal involution.In temperate areas of the Earth, animals reproduce when the environmental conditions are optimal to maximize growth rate and survival of newborns. In most species living at these latitudes, photoperiod is the cue controlling their reproductive rhythm. However, other factors, such as food availability, stress, and weather, can either modify or even overcome the influence of photoperiod. Environmental cues act by modulating the expression of the hormones released by the hypothalamic–pituitary–gonadal (HPG) axis. The levels of serum gonadotropins are lower in the non-breeding period, a fact that in males leads to a reduction of circulating testosterone, which is associated to spermatogenesis inhibition and, as a consequence, to the loss of the germinative epithelium and the subsequent reduction in testicular size and mass . Interspet al.,  reportedIn addition to germ cells, Leydig cells also show species-specific differences in the regressed testis .Talpa occidentalis [Chaetophractus villosus [et al. [et al. [Lagostumus maximus.The mechanisms of testis regression are also variable among species. Most studies have pointed to apoptosis as the main cellular event responsible for testis regression , 9. Howedentalis  and the villosus . Dadhich [et al.  proposed [et al.  proposedHemiechinus auritus, is one of the six African species of spiny hedgehogs belonging to the family Erinaceidae. It is distributed in the coastal semi-desert areas of Libya and Egypt, in the Cypriot island, the Middle East, and Central Asia. H. auritus is a terrestrial nocturnal mammal that lies in burrows and prefers mesic habitats including gardens, olive groves, and cultivated areas [H. auritus is a seasonal breeder. Available data indicate that, independently of the geographical location, hedgehogs of this species breed in late spring-summer and are inactive in late autumn and winter [Thus, analysing the circannual testicular dynamics in new mammalian species would help 1) to elucidate which mechanisms of testis regression are evolutionary conserved and 2) to estimate the inter-species variation existing in the process. The long eared-hedgehog, ed areas . This sped areas . Consistd winter –18.Here we analysed the circannual testicular changes occurring in the long-eared hedgehog from northwestern Egypt during its seasonal breeding cycle. Testicular histology was studied and the spatial gene-expression patterns of several somatic and germ cell types were determined. The incidence of apoptosis and the serum testosterone levels were also quantified in both sexually active and inactive hedgehogs.2 inhalation.Twelve adult males of long-eared hedgehog were captured alive in Matrouh governorate  in the northwestern part of Egypt, 400 km from Cairo. Two study groups were established: animals captured in summer  and those captured in winter . We also collected two individuals in the period of testis regression, at the end of September. Juvenile males (N = 2), identified on the basis of extremely low testis mass (<200 mg), reduced seminiferous tubule diameter (<70 μm), and low body mass (<140 g), were excluded from the study. Testes and epididymides were collected, weighed, and fixed overnight in a 50× volume of Serra's fixative . This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The capture and experimental protocols were approved by the Ethics Committee for Animal Experimentation of the Zoology Department, Faculty of Sciences, Fayoum University. The long-eared Hedgehog is categorized by the Egyptian Wildlife Protection System as (LR/lc), (Lower risk/ least concern), thus, it is not an endangered or protected species. Animals were captured by a professional hunter with permission from the Egyptian Federation of Wild Hunting. To minimize animal suffering, hedgehogs were captured by hand in their burrows and immediately transported to the laboratory where they were euthanised by C0Table 1 summarizes the antibodies and working concentrations used in this study.Testes were embedded in paraffin, sectioned (5 μm), mounted on polylysine-coated slides , and stained with hematoxylin and eosin according to standard procedures for morphological analysis. Single and double immunofluorescences were performed as previously described . Table 1+ cells were counted in a total of 100 seminiferous tubule sections of each animal.Apoptosis was assessed with the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) assay, using the Fluorescent In Situ Cell Death Detection Kit (Roche ref. 11684795910), according to the manufacturer's instructions. In order to quantify the incidence of apoptosis, the total number of TUNELt‐test to compare the respective means.The diameters of 30 transversely sectioned seminiferous tubules of different testis sections were measured as previously described . SeminifH. auritus males included in this study were stored at 4°C overnight and centrifuged next morning at 6,000 rpm for 20 min at 4°C. The supernatant (serum) was then stored at -80°C until further use. Testosterone levels were measured using the chemiluminescent enzyme immunoassay method in an Immulite 2000 analyzer .Fresh blood samples obtained from the t-test, P = 0.019; Fig 1A). In the summer group, the mean testis mass was three times higher than that in the winter group . At the histological level, testes in the summer group presented all features of an active and fertile testis, with seminiferous tubules in all stages of the spermatogenic cycle and spermiogenesis completed . Accordingly, we found abundant sperm in the epididymides . In contrast, winter testes showed marked reduction in seminiferous tubules . In these tubules, no stage of the spermatogenic cycle could be identified and most of them were filled with primary spermatocytes, lacking secondary spermatocytes, spermatids, and sperm . Consistently, the diameter of the epididymal tubule was reduced and empty .Long-eared hedgehogs captured in summer were significantly larger than those from winter . In the inactive testes, SOX9+ cells were also localized at the periphery of testis tubules; however, the distance between neighbouring SOX9+ cells was notably reduced when compared to that of active tubules, confirming that Sertoli cells in the regressed testes undergo a severe shrinkage . We also analysed α-smooth muscle actin (ACTA2), which in the testis is a marker for peritubular myoid cells and arterial muscle fibres [Fig 3Ab and 3Ac). In the inactive testes, the staining for both ACTA2 and LAM, was weaker and irregular in shape, again, as a consequence of the involution of the seminiferous tubules . Finally, we also studied the expression pattern the cholesterol side-chain cleavage enzyme, P450scc, a protein involved in the synthesis of testosterone which is produced by Leydig cells. In both active and inactive testes, P450scc expression was clearly visible in the interstitial cells, indicating that Leydig cells of regressed testes maintain the steroidogenic function in some extent .Next, we studied the expression of somatic-cell-specific molecular markers by means of immunofluorescence in both summer (hereafter referred to as active) and winter (hereafter referred to as inactive) testes of function . In the e fibres , and lame fibres . In the Fig 3Ba–3Bc), which were located at the periphery of the seminiferous tubules showing an expression pattern that varied depending on the spermatogenic cycle stage, as described previously [+ cells were only observed in tubules of the spermatogenic stages VII-IX , whereas PCNA was expressed in proliferating spermatogonia as well as in a variable number of spermatocytes depending on the stage of the spermatogenic cycle . Both proteins co-localized in zygotene and early pachytene spermatocytes of seminiferous tubules at stages VII-IX , but not in seminiferous tubules containing later spermatocytes . The inactive testis also showed a dynamic pattern of expression for both markers, although immunoreactive cells did not exhibit a ring-like organization and clustered together . At a higher magnification, we could observe that the centres of the testis tubules were completely filled with either DMC1+ PCNA+ (zygotene and early pachytene) or DMC1- PCNA+ (later pachytene) spermatocytes . Altogether, our results show that in the inactive testes of H. auritus meiosis onset is not halted but meiotic arrest occurs at the pachytene stage.We also studied the expression of germ cell-specific markers. DMC1 is present in leptotene-to-early pachytene primary spermatocytes  and the eviously , 24. DMCH. auritus. Active testes only contained some few positive cells in a reduced number of seminiferous tubules, whereas the number of apoptotic cells was notably higher in inactive testes . To identify the cell types dying in the inactive testes, we performed TUNEL assay together with immunofluorescence for cell type-specific markers. We observed that SOX9+ cells were never TUNEL+ , suggesting that Sertoli cells were not affected. In contrast, all TUNEL+ cells showed immunoreactivity for PCNA , showing that an apoptosis-mediated massive loss of germ cells occurs in the inactive testes of H. auritus.Since apoptosis is a process normally occurring in the regressed of testes of seasonal breeding mammals , we perfFig 5Aa), showing that they were sloughed from their usual location in more basal regions. We also detected this type of germ cells within the lumen of the epididymal tubule . We performed TUNEL assay to check whether apoptosis was responsible for the massive loss of germ cells that takes place in the regressing testes of the long-eared hedgehog. Dying cells were observed within testis cords of inactivating testes, although few of them were located at, or nearby, the lumen , thus indicating that desquamation mainly affects living cells. These results suggest that, although apoptosis may contribute in some extent to the massive germ cell depletion affecting inactivating testes, additional mechanisms must be involved in the process. Two recent studies have shown that in the testes of two seasonal breeders, the Iberian mole T. occidentalis [C. villosus [H. auritus, we performed immunofluorescence for several important adhesion molecules on histological sections of active, inactivating, and inactive testes. N-cadherin (NCAD) and β-catenin (β -CAT) are structural components of the adherens junctions as well as of the ectoplasmic specialization existing between Sertoli-Sertoli and Sertoli-germ cells [Fig 5Ca and 5Cd). Claudin 11 (CLDN11) is a transmembrane protein of the tight junctions forming the blood-testis barrier established between adjacent Sertoli cells [Fig 5Cg and 5Cj). In contrast, in both inactivating and inactive testes, a homogeneous disorganized staining was observed for the four molecules throughout the entire germinative epithelium . These results suggest that the adhesion between Sertoli cells and between Sertoli and germ cells is compromised in the regressing testes, thus explaining the germ cell sloughing taking place during this period.Two individuals captured at the beginning of Autumn (end of September) presented testes in which the process of regression was taking place (hereafter referred to as inactivating). In these testes, the mean diameter of the seminiferous tubules was in between those of the summer and the winter group (120 ± 12 μm). The germinative epithelium looked disorganised in many tubules and primary and secondary spermatocyte were anomalously located in the lumen .Reduction of the levels of serum testosterone in males during the non-reproductive season is a common feature of all seasonal breeding mammals studied to date. In the case of H. auritus. Our results show that in this species the main testis regression effector is germ-cell desquamation, as described previously in both the Iberian mole T. occidentalis [C. villosus [H. auritus.Testis regression is the process by which males of seasonally breeding species inactivate their gonads before they enter the resting period of the circannual reproductive cycle. This is necessary if the non-breeding period is long enough to make testis regression efficient in terms of energy saving, as spermatogenesis is too expensive to be maintained when it is not needed . After tdentalis  and the villosus , 29. TheCapreolus capreolus [T. occidentalis [C. villosus [H. auritus. The low abundance of apoptotic cells in regressing testes supports this finding. Nevertheless, as in the mole, apoptosis plays an important role in the inactive testes of H. auritus, by eliminating all primary spermatocytes that reach the pachytene stage.Apoptosis is an essential process occurring normally in functional fertile testes. It facilitates the regulation of the spermatogonial population density and safeguards the genetic integrity of the male gamete by eliminating cells unable to pass checkpoint transitions derived from improper meiotic chromosome synapsis , 30–33. apreolus , the Ibedentalis , and thevillosus , we foun+ cells and others lacking them in inactive testes (like in active ones), a finding also described in the Iberian mole [Cynops pyrrhogaster [Vulpes vulpes [Mesocricetus auratus [Pelodiscus sinensis [Corvus macrorhynchos [Microtus duodecimcostatus (our unpublished results), and the long-eared hedgehog H. auritus (present paper), amongst others. On the other hand, complete inhibition of meiosis entry occurs in the inactive males of the white-footed mouse Peromyscus leucopus [Sturnus vulgaris [Chaetophractus villosus [This process of apoptosis-mediated spermatocyte depletion is needed because meiosis onset is not halted in the inactive testis of this hedgehog species. The presence of both seminiferous tubules containing DMC1ian mole , suggesthogaster , the sils vulpes , the Syr auratus , 37, thesinensis , and therhynchos , as wellleucopus , the Eurvulgaris , and thevillosus , 29. Thevillosus , speciesMustela vison [Phodopus sungorus [T. occidentalis [in vivo molecular tracing experiments with the hedgehogs included in this study, but the disorganised expression pattern observed for CLDN11 in the testes of inactive males of this species clearly resemble what we previously described in the mole, and suggests that the BTB of the H. auritus testis also becomes permeable during seasonal regression.A common feature of seasonal testis regression is the permeation of the blood-testis barrier (BTB), a specialised junctional complex that defines an adluminal and a basal compartment in the germinative epithelium and preserves meiotic and post-meiotic germ cells  from the action of the immune system , 47. Seala vison , the Djusungorus , and thedentalis . In all T. occidentalis [C. villosus [H. auritus, thus suggesting that it could be relatively common in mammals. The testis regression process of this hedgehog species is similar to that described for the mole, as it includes 1) massive desquamation of meiotic and post-meiotic germ cells, 2) irrelevance of apoptosis during regression, 3) disorganised expression of cell-adhesion molecules, 4) persistent meiosis onset, 5) apoptosis of pachytene spermatocytes, and 6) probable BTB permeation. The only noticeable difference between these two species concerns the re-organization of the interstitial tissue during testis regression. In the mole, Leydig cells form a dense, continuous cell matrix that occupies most of the inactive testis volume [M. auratus [C. capreolus [T. occidentalis [Subsequent to the identification of germ cell desquamation as the principal testis regression effector in the Iberian mole dentalis , this mevillosus , 29. Hers volume , a pheno auratus ) belong apreolus  and the dentalis . Furtherdentalis  and the dentalis , present


To evaluate the interest of cerebrospinal fluid (CSF) lactate assay for the diagnosis of post-neurosurgical bacterial meningitis (PBM).We conducted at our neurosurgical resuscitation unit a prospective study of patients who underwent elective or emergency craniotomy. Lumbar puncture was performed in all patients who had clinical suspicion of PBM for CSF culture and cytological and chemical analysis . The diagnosis of PBM is made according to the criteria proposed by the Center for Disease Control and Prevention (CDC). Receiver Operating Characteristic (ROC) was used to determine the diagnostic accuracy of CSF lactate.72 patients were studied and only 32 of them had the clinical and biological criteria of the diagnosis of PBM. Median CSF lactate was 6.18 mmol/L for PBM vs 2.63 mmol/L for no PBM (p < 0.001). CSF lactate may predict the presence PBM, with a AUC of 0.98 and NPV of 99.1. The analysis of Youden's index also confirms the good diagnostic power of CSF lactate with a value of 83 at a cut-off value of 4 mmol/L and a sensitivity of 92.3% and specificity of 91.6%.Our study shows that the CSF lactate as an indicator for PBM. It is a fast and simple test that can help the clinician to optimize the management of PBM and decrease premature cessation of antibiotics.Lotfi R, Ines B, et al. Cerebrospinal Fluid Lactate as an Indicator for Post-neurosurgical Bacterial Meningitis. Indian J Crit Care Med 2019;23(3):127-130. The diagnosis is difficult, the symptoms being frustrating and nonspecific in the postoperative period3. Contamination of the subarachnoid space with blood or degradation products resulting in a secondary inflammatory response, combined with antibiotic prophylaxis may make it difficult to interpret routine CSF analyses4. In addition, direct bacteriological examination results are often negative5. Several studies have shown that CSF lactate is a potential marker for identifying patients who develop bacterial meningitis6. The CSF lactate is a simple test and sensitive even in the case of blood contamination of the CSF2. The purpose of this study was to evaluate the efficacy of CSF lactate concentration in diagnosing PBM.Bacterial meningitis is relatively uncommon in post-neurosurgicalWe conducted a prospective study of patients admitted to Burns and Trauma Center, Ben Arous who underwent elective or emergency craniotomy between June 2015 and December 2016. Patients with external or internal CSF shunts were excluded. The patients were prospectively followed for the development of meningitis during the first 30 postoperative days. Lumbar puncture was performed with CSF sampling in all patients with clinical symptoms of bacterial meningitis such as fever, headache, and deterioration of neurological status within two to three days of craniotomy. Responsible parents of patients have been informed and received the signed consent from then.Demographic data obtained included age, sex, Glasgow Coma Scale (GCS), indication for surgery, steroid use, antibiotic prophylaxis, operation duration, mechanical ventilation and length of UCI stay.7. Patients were divided into PBM group (Proven PBM and Presumed PBM) and no PBM group.The diagnosis of PBM is made according to the criteria proposed by the Center for Disease Control and Prevention (CDC)Patient status was categorized following predefined criteria:PBM: positive bacterial CSF culture or Gram stainor Patient has at least 1 of the following symptoms with no other recognized cause: fever (>38 °C), headache, stiff neck, meningeal signs, cranial nerve signs, or irritability And positive CSF examination: CSF WBC count ≥100/ mm3 and C SF glucose <2.5 mmol/L or ratio of CSF glucose to blood glucose <0.4.No PBM: neither proven nor presumed criteria.CSF samples were obtained by lumbar puncture in a sterile manner. The CSF lactate and other biomarkers, including WBC count, CSF protein and CSF glucose were assessed quantitatively.The statistical analyses were performed using SPSS software version 24. MedCalc statistical software was used to analyze the ROC curves.The Kolmogorov-Smirnov test was used to determine the distribution of continuous variables. The medians of the CSF markers were compared using the non-parametric Mann-Whitney test. p value <0.05 was considered to be significant. The receiver operator curve (ROC) analysis was used to calculate the optimal cut-off value for CSF lactate. Youden index was used to define the diagnostic cut-off level for PBM.A total of 442 patients who underwent craniotomy in our center between June 2015 and December 2016 were identified. Among the 442 patients studied, 72 had a clinical suspicion of PBM and were divided into 32 patients to the PBM and 40 to the no PBM according to our preset criteria.Demographic data are presented in CSF markers data are presented in The difference in the CSF lactate levels between the PBM and the no PBM groups was statistically significant (p < 0.001).Median values of CSF leukocytes, proteins, glucose and CSF/ blood glucose ratio were also significantly higher in PBM than no PBM patients.The ROC curve analysis of the CSF lactate data showed that the cut-off value was 4 mmol/L with AUC = 0.98, p < 0.001 . The anaFive cultures of CSF samples were positive . Gram-positive organisms predominated. Coagulase- negative Staphylococci were isolated from three patients, Pseudomonas aeruginosa from one patient and Klebsiella pneumonia from one patient.9. The use of only bacterial culture may misidentify many patients with postoperative infections. Routine CSF analysis, such as glucose levels, cell counts and protein concentration are retained by most authors as criteria to diagnose PBM12. In many diagnostic studies in patients with PBM, CSF routine examinations, including WBC and neutrophil counts, protein levels and hypoglycorrhachia, were neither specific nor associated with high positive and negative predictive values13. Tamune et al. demonstrated that the CSF/blood glucose ratio may predict the presence of bacterial meningitis more precisely than other routinely measured markers in CSF14. The cell index (a ratio of WBC to RBC in the CSF divided by the ratio of WBC to RBC in blood) has been suggested to account for erythrocytes in the CSF because of hemorrhage. A cutoff value of 5 has been proposed to be indicative of infection but this has not been validated15.Our study confirms the value of CSF lactate assays for the diagnosis of PBM. An early and precise diagnosis of PBM is essential to start appropriate empiric antibiotic therapy. Since the CSF bacterial culture is routinely used as gold standard, the percentage of positive cultures does not exceed 10%, and this is due to several factors such as injection of antibiotics, contamination of the CSF sample16. In a recent meta-analysis, Xiao et al. have included 404 post-neurosurgical patients from 5 studies to evaluate the effectiveness of CSF lactate in the diagnosis of PBM17. It has shown that CSF lactate concentration has a good efficacy in the diagnosis of post-neurosurgical bacterial meneingitis with sensitivity at 0.92 and a specificity at 0.8817. In this meta-analysis, the cutoff values for CSF lactate ranged from 3.45 mmol/L to mmol/L (4.41 ± 0.85 mmol/L). Maskin et al.11. confirms, in 79 patients with signs of PBM that CSF lactate is relatively a good marker to distinguish between PBM and aseptic meningitis, even after antibiotic but the CSF lactate cutoff (4 mmol/L) value was defined before the analysis. Li et al12. retrospectively studied 178 patients and shows that the CSF lactate assay is very useful for the diagnosis of PBM. In their study, the cut-off value of CSF lactate was 3.45 mmol/L with higher sensitivity (90.0%) and specificity (84.6%), however, the author relied for the diagnosis of PBM on the old criteria cited in the study by Gray and Fedorko18. Our study shows the usefulness of CSF lactate for the diagnosis of PBM, with a AUC of 0.98 and NPV of 99.1. Youden Index for CSF lactate level was 0.83 at a cut-off value of 4 mmol/L, with a sensitivity of 92.3 and specificity of 91.6% confirming that CSF lactate concentration is a good indicator of PBM (Several studies have investigated the value of the CSF lactate assay to diagnose bacterial meningitis. Currently, it has been well established that astrocytes are able to produce lactate in response to a bacterial infection and that CSF lactate is little affected by serum lactate and therefore can be considered a good marker of intracranial infectionr of PBM .Previous recommendations proposed, if CSF Gram-stain and culture are negative, stopping of antimicrobial therapy; but this approach is not applicable with patients who have treated with antibiotics.Our results show that if CSF lactate is lower 4 mmol/l, CSF glucose is normal and the Gram stain is negative, bacterial meningitis is unlikely. This algorithm can be used to decrease premature cessation of antibiotics.19.Recently several studies have tested other markers such as procalcitonin, sCD163 and C-reactive protein but most of them were retrospective or with small sizes20.In our study, coagulase-negative Staphylococci were the most commonly pathogens isolated in PBM group, which joins the literature data with a predominance of Gram-positive cocci pathogens in postneurosurgical infectionOur study presents some limitations. First it is a monocentric study with a small number of patients presenting a PBM. Second, the diagnosis of PBM according to the CDC criteria remains not applicable for patients with pre-existing neurologic deficits or who are sedated.Our study shows that the CSF lactate as an indicator for PBM. It is a fast and simple test that can help the clinician to optimize the management of PBM and decrease premature cessation of antibiotics. More investigation is needed to confirm our results.


Pegaptanib might be a promising anti‐tumour drug targeting VEGF to inhibit tumour vascular endothelial cell proliferation. However, the poor biostability limited its application. In this study, we took tetrahedron DNA nanostructures (TDNs) as drug nanocarrier for pegaptanib to explore the potent anti‐angiogenesis and anti‐tumour activity of this drug delivery system.The successful synthesis of TDNs and pegaptanib‐TDNs was determined by 8% polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis and dynamic light scattering (DLS). The cytotoxicity of pegaptanib alone and pegaptanib‐TDNs on HUVECs and Cal27 was evaluated by the cell count kit‐8 (CCK‐8) assay. The effect of pegaptanib and pegaptanib‐TDNs on proliferation, migration and tube formation of HUVECs induced by VEGF was examined by CCK‐8 assay, wound healing assay and tubule formation experiment. The cell binding capacity and serum stability were detected by flow cytometry and PAGE, respectively.Pegaptanib‐TDNs had stronger killing ability than pegaptanib alone, and the inhibiting effect was in a concentration‐dependent manner. What's more, pegaptanib‐loaded TDNs could effectively enhance the ability of pegaptanib to inhibit proliferation, migration and tube formation of HUVECs induced by VEGF. These might attribute to the stronger binding affinity to the cell membrane and greater serum stability of pegaptanib‐TDNs.These results suggested that pegaptanib‐TDNs might be a novel strategy to improve anti‐angiogenesis and anti‐tumour ability of pegaptanib. The hydrodynamic sizes of TDNs and pegaptanib‐TDNs were measured by A Zetasizer Nano‐ZS .The successful synthesis of TDNs and pegaptanib‐TDNs was examined by 8% polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis.2.3v/v) foetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin antibiotics and maintained at 37°C in 5% CO2.HUVECs and Cal27 were purchased from the American Type Culture Collection . They were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% , and the cell viability was measured by CCK‐8 assays. To detect the effect of these drugs on proliferation of HUVECs induced by VEGF, HUVECs were cultured in VEGF (25 ng/mL), VEGF (25 ng/mL) +TDNs (250 nmol/L or 375 nmol/L), VEGF (25 ng/mL) +pegaptanib (250 nmol/L or 375 nmol/L) and VEGF (25 ng/mL) +pegaptanib‐TDNs (250 nmol/L or 375 nmol/L). After 48 hours, the cell viability was monitored by CCK‐8.HUVECs and Cal27 were cultured in 96‐well microtitre plates at a density of 8 × 102.5This section was prepared on the basis of the previously reported methods.2.65 cells/mL. 100 μL of the resuspended HUVECs was added to the Matrigel‐coated wells and incubated for 30 minutes to allow cell attachment. Subsequently, the media were replaced with high glucose DMEM containing VEGF (25 ng/mL), VEGF (25 ng/mL) +TDNs (375 nmol/L), VEGF (25 ng/mL) +pegaptanib (375 nmol/L) and VEGF (25 ng/mL) +pegaptanib‐TDNs (375 nmol/L). The cells were incubated at 37°C in 5% CO2 for 10 hours. After incubation, HUVECs were imaged by an inverted fluorescence microscope (Olympus IX73). The mean tube length was analysed by ImageJ.50 μL Matrigel solution was added to each well of a 96‐well plate and incubated for 1 hour at 37°C. After being serum‐starved overnight, HUVECs were trypsinized and resuspended in high glucose DMEM with 0.5% FBS at 1 × 102.7g for 5 minutes. The cell pellets were resuspended into 500 μL PBS. Subsequently, cell suspensions were measured by flow cytometry.HUVECs were seeded in 6‐well plates. After 24 hours, pegaptanib‐cy5  and pegaptanib‐TDNs‐cy5  were added to the culture media. Cells were cultured for another 2 hours. Then, the cells were rinsed three times with PBS and digested with trypsin. The detached cells were collected into centrifuge tubes and centrifuged at 350 2.8v/v) FBS and incubate at 37°C in 5% CO2 for 0, 2, 6, 8, 10, 12, 24, 36 hours, respectively. 8% PAGE was used to detect the degradation of pegaptanib and pegaptanib‐TDNs at different time points.Pegaptanib and pegaptanib‐TDNs were suspended in the high glucose DMEM with 10% (2.9P < 0.05 indicated that group means were significantly different. All quantitative results were presented as mean ± standard deviation (SD).One‐way ANOVA  or Student‐Newman‐Keuls test was used to perform statistical analysis of data and 33.1Pegaptanib was linked to the 5’ terminal of S4 to form pegaptanib‐S4. The ssDNA sequences were listed in Table 3.2The cell proliferation was evaluated by CCK‐8 assay. As shown in Figure 3.3VEGF, an angiogenic factor, is crucial to promote the proliferation of HUVECs. HUVECs were incubated with VEGF (25 ng/mL), VEGF (25 ng/mL) +TDNs (250 nmol/L or 375 nmol/L), VEGF (25 ng/mL) +pegaptanib (250 nmol/L or 375 nmol/L) and VEGF (25 ng/mL) +pegaptanib‐TDNs (250 nmol/L or 375 nmol/L) for 48 hours. In Figure HUVECs migration is a necessary process of angiogenesis. VEGF is a chemokine of HUVECs, which can promote HUVECs migration by activating cytoskeleton remodelling signalling pathways.P < 0.01; VEGF + pegaptanib‐TDNs vs VEGF + pegaptanib: P < 0.05).Tube formation assay was carried out in order to test the influences of these materials on angiogenesis. Capillary tube formation induced by VEGF was reduced when HUVECs incubated with 375 nmol/L pegaptanib‐TDNs, but the inhibition effect was not obvious in the 375 nmol/L pegaptanib group Figure A. QuantiThese results suggested that pegaptanib loaded onto TDNs could effectively enhance the ability of pegaptanib to inhibit proliferation, migration and tube formation of HUVECs.3.4Pegaptanib and pegaptanib‐TDNs were labelled with cy5. Fluorescent signal was detected by flow cytometry to measure pegaptanib binding to HUVECs. When the concentration was below 50 nmol/L, the fluorescence signal of pegaptanib‐TDNs slowly increased, but with the increase of the concentration, especially at 250 nmol/L, the fluorescence signal obviously increased whereas the fluorescent signal was not observed any change in pegaptanib group  FBS at 37°C in 5% CO2 for 24 hours, but pegaptanib occurred obvious degradation at 2 hours and complete degradation <6 hours (Figure Subsequently, the serum stability of pegaptanib and pegaptanib‐TDNs was evaluated. The result of gel electrophoresis demonstrated that pegaptanib‐TDNs was almost completely degraded after incubated with 10% (4In this study, we synthesized pegaptanib‐TDNs and investigated the effect of anti‐tumour and anti‐angiogenesis. Pegaptanib, an RNA aptamer, was successfully linked to a vertex of TDN Figure . Pegapta5Taken together, we put forward a novel drug carrier for pegaptanib. TDNs could help pegaptanib overcome the limitations of aptamer and broaden its application in VEGF‐targeting cancer therapy. Our results demonstrated again that TDNs might be a vehicle of potential value for disease therapy.All authors declare no conflict of interest.