Three experiments provide evidence of an incipient sense of fairness in preverbal infants. Ten-month-old infants were shown cartoon videos with two agents, the ‘donors’, who distributed resources to two identical recipients. One donor always distributed the goods equally, while the other performed unequal distributions by giving everything to one recipient. In the test phase, a third agent hit or took resources away from either the fair or the unfair donor. We found that infants looked longer when the antisocial actions were directed towards the unfair rather than the fair donor. These findings support the view that infants are able to evaluate agents based on their distributive actions and suggest that the foundations of human socio-moral competence are acquired independently of parental feedback and linguistic experience. How do people acquire the ability to judge the fairness of resource distributions? What is the nature of the learning mechanisms involved in the development of this fundamental aspect of the human social brain? Several theories in moral philosophy and psychology posit that we rely on domain general learning mechanisms, either associationist e.g.  or constFor many decades, developmental research has been guided by theories in the first camp and most of the results appeared to provide strong support for the constructivist models. Several studies have reported a slow development in children's judgments, showing that preschoolers' distributions are mainly guided by self-interest and that they are equitable only towards friends kin selection) and who have acted pro-socially towards them (direct reciprocity) or other people (indirect reciprocity). Therefore, preschoolers' judgments and distributions are not solely guided by self-interest  is not compelling evidence that they have a sense of fairness unless it is also shown that infants assign different values to different types of distributions.One way to test whether such attribution of value takes place early in development is to look at infants' reactions to pro-social and antisocial actions, such as giving or taking away resources, directed towards agents who have previously performed a fair or an unfair distribution of resources. For example, in Meristo and Surian In the present study, infants saw two agents, the donors or distributors, performing either an equal or an unequal distribution of goods. Then, a third party either repeatedly hit and pushed away one of the two donors (Experiment 1), or took resources away from him (Experiment 2). If infants spontaneously link such anti-social actions to the previous distributive actions performed by the donors, they should look differently at the two test events.M = 303 days; SD = 8 days; 10 female, 6 male). Infants were recruited by contacting their parents who were randomly chosen from the birth register of the Swedish Tax Agency among families living in Gothenburg (Sweden). The parents were informed about the purpose and procedure of the study and gave signed consent. An additional 8 infants were tested, but excluded from the sample due to fussiness (3), technical problems (2) and because they did not look for at least 2.5 seconds to the test event (3). The Regional Gothenburg Ethical Review Board approved the study.Sixteen full-term healthy 10-month old infants participated  identity of the fair donor (yellow triangle vs. blue square), (2) order of donor familiarisation events , (3) side of the fair donor in the test event (Left vs. Right), and (4) type of test event (hitting the fair vs. hitting the unfair donor), resulting in 16 different testing sessions. The side of delivery of the first strawberry in both the equal and the unequal distribution events (Left vs. Right) co-varied with the identity of the fair donor . All infants followed at least three donor familiarisation events.The dependent measure was the time the infant spent looking at the still picture at the end of the test movie, from the moment when the anti-social agent finished the hitting see , until hM = 11.85 sec, SD = 3.17 sec) than at the event involving the hitting of the fair donor , t(14) = 3.25, p  = .006, two-tailed, partial η2  = .43, see et al.Infants looked reliably longer at the test event involving the hitting of the unfair donor . The recruitment procedure was the same as in Experiment 1. Seven additional infants were tested but excluded because they did not look for at least for 2.5 seconds at the test event (6), or because of fussiness (1).A new group of 16 full-term healthy 10-month-old infants participated  than at the test event involving taking the strawberry away from the fair donor , t(14) = 2.22, p  = .044, two-tailed, partial η2  = .26 , and when it involved taking away an attractive object (Experiment 2). One possible explanation of these findings is that infants looked longer at events that they perceived as more coherent with the sequences of events seen in the previous familiarisation phase. However, in Meristo and Surian In Experiment 3, the procedure was similar to that of Experiments 1 and 2, except that the anti-social test event was replaced with a neutral action performed with an object that was very close to one of the donors. That object, instead of being taken away as in Experiment 2, was simply moved horizontally.M = 305; SD = 3 days; 7 female, 9 male). The recruitment procedure was the same as in Experiments 1 and 2. Seven additional infants were tested but excluded because they did not look for at least for 2.5 seconds at the test event (1) or because of fussiness (6).A new group of 16 full-term healthy 10-month-old infants participated  were visible on the lower part of the screen, one on the right side and the other on the left side . Above eM = 8.81 sec, SD = 3.23 sec) and the actions directed towards the fair donor , t(14)  = .59, p  = .57, two-tailed, partial η2  = .03. An additional two 2 X 2 ANOVAs with Experiment as a one of the between-subject factors and Condition (fair vs. unfair donor targeted) as the second between-subject factor, yielded interaction effects that approach significance  = 3.36, p  = .077 two-tailed, partial η2  = .11; Experiments 2 and 3: F = 3.24, p  = .082 two-tailed, partial η2  = .10. This outcome provides no support for the claim that infants have a bias for looking at actions performed towards unfair donors and that such a bias can account for the results of Experiments 1 and 2 of the present study, as well as for the results reported by Meristo and Surian In contrast to the results in Experiments 1 and 2, infants were equally attentive to the test events involving actions directed towards the unfair donor . This suggests that they preferred to look at the test events that were a coherent completion of the familiarisation events, displaying the donors' distributive actions, assuming that infants valued both the unfair distributions and the antisocial actions negatively. This conclusion is supported by Experiment 3, where the antisocial actions were replaced by a neutral object displacement action and where the infants looked equally long when such an action was performed with the object close to the fair or the unfair donor.An alternative interpretation of our results is that, in our test situations, infants noticed that the third agent acting anti-socially in the test event was absent when the distributions happened previously, and therefore inferred that he did not know anything about the donors' fairness. Infants then looked longer at interactions with the unfair donor because of a general negativity bias; that is, a tendency to attend to potentially anti-social characters, demonstrated by Hamlin, Wynn and Bloom However, we found in Experiment 3 that infants did not look longer at the interactions with the unfair donor when the action directed towards it was neutral. Yet, the purpose of the action in Experiment 3 might not be easily encoded, which might have hindered the infants to generate any expectations, or their expectations might have been masked by their effort to understand the ambiguous action. On the other hand, the findings reported by Meristo and Surian Another possible explanation that needs to be discussed centres on infants' expectations about affiliative biases. When infants are facing a context in which a new third party is entering the scene, they generate expectations about its affiliative biases and look longer when the third party approaches an unfair rather than a fair distributor, provided that the purpose of the approach is easily encoded. When instead the purpose of the approach is not easily encoded, as it might have been the case in Experiment 3, infants do not generate any expectation, or their expectations are masked by their effort to understand the ambiguous action. Infants' affiliative biases towards one of the distributors are well documented in previous studies e.g.  and thisSeveral studies on infants' socio-moral competence have found evidence consistent with the predictions generated following the violation of expectation paradigm. For example, Hamlin et al. Infants' preference for events showing anti-social actions directed towards unfair agents may be related to later biases of punishments directed towards deserving agents found in children and adults. Children and adults endorse the punishment of agents that are causally responsible for harm Free riders are also punished in other well-known tasks used to investigate economic reasoning, such as the Common Good Game Piaget Critics of the nativist perspectives on infants' sense of fairness emphasise the effect of experience e.g. . The cru


Trachycephalus resinifictrix (Hylidae) on a wooden stick that was used to mimic a small tree branch. We observed two different landing behaviours: (1) landing on the abdomen and (2) attachment with the toes of either the forelimb or the hindlimb. In the latter case, the frogs performed a cartwheel around the stick, while they were only attached by their adhesive toe pads. We estimated the forces that act on the toes during this behaviour to be up to fourteen times the body weight of the animals. This behaviour demonstrates the remarkable adhesive capabilities of the toe pads and the body control of the frogs.Frogs (Lissamphibia: Anura) are famous for their saltatory or hopping locomotion, which is related to numerous anatomical specialisations that are characteristic for the group. However, while the biomechanics of take-off in frogs have been studied in detail, much less is known on how frogs land after a jump. Besides terrestrial and aquatic species, several lineages of frogs adopted an arboreal lifestyle and especially the biomechanics of landing on challenging, small, and unpredictable substrates, such as leaves or branches, are virtually unknown. Here we studied the landing kinematics of the arboreal frog The online version of this article (doi:10.1007/s00359-016-1069-0) contains supplementary material, which is available to authorized users. Prosalirus bitis that dates back to the early Jurassic (Shubin and Jenkins Frogs (Anura) are well known for their saltatory locomotion, which is one of the key characteristics of the group. Anatomical specialisations for jumping have even been identified in the representative of the stem-group anuran Bufo marinus was suggested to be facilitated by an elastic recoil mechanism  on a thin substrate. T. resinifictrix is an arboreal species that is native to South America  to estimate the velocities of the frogs before landing, and (3) to estimate the effectiveness of adhesive pads under the typical behavioural situation and at the natural range of forces that act on the adhesive toe pads during landing in vivo.Here we used high-speed videography and kinematic analysis to observe the landing behaviour of Trachycephalus resinifictrix (Goeldi 1907) were available for this experiment. This species of frog is often kept as a pet and traded under the name Amazon milk frog, but it is also commonly referred to as Mission golden-eyed treefrog (Frank and Ramus Gryllus bimaculatus).Four specimens of x Goeldi 907 were We performed two sets of experiments for which we recorded the movements of the frogs with a high-speed video camera  at 1000 frames per second: (1) free hanging, and (2) landing after a jump. For scaling, we further recorded a ruler that was placed at the same position as the frogs during the experiment with the identical camera settings as during experimental trials. In both experiments, we used a wooden cylindrical stick with a diameter of 1.0 cm as target surface for the frogs. By using a white-light interferometer  we determined the root mean square roughness of this stick to vary between 1.3 and 4.3 µm.We placed the frogs by hand underneath a horizontally oriented wooden stick and allowed them to place one hand on the side face of the stick. We then removed our hands and let the frogs hang free from the stick. Immediately after we removed the hand, the frogs pulled themselves up towards the stick, while we captured the movements of the frog in dorsal view from a perspective perpendicular to the stick.Figure http://www.imagej.nih.gov/ij/). For each experiment, we adjusted the pixel to cm ratio based on the video recordings of the ruler. Each landing video sequence was then analysed frame by frame, which at a recording frequency of 1000 frames per second resulted in a temporal resolution of 1 ms. For the experimental trials in which the frogs were free hanging at the stick, we tracked the position of the toe pads attached to the stick, to identify sliding movements. However, it turned out that in all experimental trials, the toe pads remained stationary once they are attached and sliding was never observed. For landing trials we traced the position of the rostral tip of the nasal capsule and the position of the toes in lateral view. MTrackJ outputs the x- and y-coordinates of each track of landmarks, as well as the travelled distances, and velocities over time. The velocity data in MTrackJ corresponds to the travelled distance per frame, i.e. per ms. For further analysis, we imported these data into the statistical computation software R 3.1.1 (available at http://www.r-project.org). For each trial we calculated a linear regression of distance over time for times ranging from the onset of the recorded videos until the frogs came in contact with the target. The slope of this regression is a measure of the average velocity of the frogs during the approach, which is different from the frame-by-frame velocities that MTrackJ calculated  were able to carry their own body weight on only two digits on either forelimb, if the contact was made by digits three and four. In one case, the frog used only one toe pad (digit three) for free hanging. Once attached to the stick, the toes were not moving or slipping down. However, usually the frogs started to pull themselves up or brought the second forelimb into contact immediately after the onset of the experiment (Supplementary Video 1). Thus, the time of free hanging was limited and ranged from 51 to 277 ms . In one case, the frog hung for approximately 5 s, which exceeded the time we were able to record with our experimental setup (1.54 s) and thus this measurement had to be excluded from the calculation of the average free hanging time.To estimate the performance of the toe pads in T. resinifictrix jump onto a horizontally oriented wooden stick with 1 cm diameter and filmed a total of 40 approaches in lateral view with a high-speed camera at 1000 frames per second. We never observed frogs missing the wooden stick in our experiment nor did we see trials where the animals failed to arrest the jumps. Kinematic analysis of the video sequences revealed that the frogs approached the stick with an average velocity of 1.34 ± 0.19 m/s . We found that the frog ID1 was significantly slower during the approach than the animal ID2 but we did not observe further statistically significant differences between the animals . While the frogs are in the air, their limbs are widespread to increase the reach between their arms and legs, respectively, and also to stabilise their flight.To study the kinematics of landing on a thin substrate, we had N = 20; Fig. N = 20; Fig. N = 16; Fig. N = 4; Fig. N = 8; Fig. N = 8; Fig. N = 4; Fig. After the aerial phase, we observed two different landing strategies Fig. : (1) lanIf the frogs used the limbs for attachment, they always performed a yaw movement before touchdown to orient their limbs towards the target; in some cases this behaviour was also observed during abdominal landing and helped the frogs to orient themselves parallel to the target. Alternatively, if landing on the abdomen, the frogs did also touchdown in perpendicular orientation to the target in which case the body folded around the stick, which decelerated the frog immediately Fig. a. After In experimental trials in which the frogs touched the target stick with one of their limbs to initiate landing, the toe pads in contact did immediately stick to the target without sliding along the substrate. During these trials, the frogs first proceeded on their parabolic jump trajectory after one limb was in contact with the target until this limb was fully stretched. Then, due to the fixation of the animal at the toe pads in contact, the frogs were redirected on a circular path and performed a cartwheel movement  or digits two, three, four, and five simultaneously (N = 3)  and these were excluded from the analysis. In the remainder of the trials, deceleration ranged from 6.1 m/s2 (specimen ID2 leaping over the target and reaching backwards with digit D4 of the forelimb) to 141.6 m/s2 (specimen ID3 reaching forward with the forelimb and making contact with digit D4) and was on average 47.3 ± 42.4 m/s2 , are important to secure the attachment of tree frog toe pads , F0 as the holding force (i.e. the force acting on the toe), µ as the frictional coefficient between two contacting solids, and θ as the angle that is swept by the holding limb. Thus, wrapping the limb around the stick (i.e. enlarging the angle θ) has a similar effect as an increase in the frictional coefficient and allows for higher loading forces with the same amount of holding force  more often performed abdominal landings compared to the smaller animals (Supplementary Table 1). Ontogenetic and scaling effects on the choice of landing strategies in frogs were not in focus here and will require further research. Further, other than scaling, experience, training level, and individual preference might all influence the choice of the landing method.T. resinifictrix further increased the chances for a successful contact by exhibiting a diversity of grip types that can be applied. In our experiment, we observed eight different combinations of digits that were brought into contact with the target. For comparison, in a previous study on arboreal locomotion in frogs, Herrel et al. Supplementary Video 2: Exemplary trial of a landing experiment at which the frog lands on the abdomen (MOV 7496 kb)Supplementary Video 3: Exemplary trial of a landing experiment at which the frog leaps over the target and reaches backward (MOV 10366 kb)Supplementary Video 4: Exemplary trial of a landing experiment at which the frog attaches a hindlimb first (MOV 12473 kb)Supplementary Video 5: Exemplary trial of a landing experiment at which the frog descends before the target and reaches forward (MOV 8694 kb)Supplementary Table 1: Raw data for observed landing movements and calculation of velocities during the approach. Supplementary Table 2: Results of regression analysis to calculate negative accelerations after the toe pads attached to the target (PDF 145 kb)Below is the link to the electronic supplementary material.


Neuromyelitis optica (NMO) is a severe demyelinating disorder of the central nervous system (CNS) associated with the presence of an autoimmune antibody response (AQP4-IgG) against the water channel aquaporin-4 (AQP4). It remains unclear whether pathologic AQP4-IgG in the CNS is produced entirely by peripheral plasma cells or is generated in part by infiltrating B cells. To determine the overlap of AQP4-IgG idiotypes between the CNS and periphery, we compared the immunoglobulin G (IgG) transcriptome of cerebrospinal fluid (CSF) plasmablasts with the CSF and serum IgG proteomes in 7 AQP4-seropositive NMO patients following exacerbation.CSF variable region Ig heavy- (VH) and light-chain (VL) transcriptome libraries were generated for each patient from CSF plasmablasts by single cell sorting, reverse transcriptase polymerase chain reaction (RT-PCR), and DNA sequencing. Recombinant antibodies were generated from clonally expanded, paired VH and VL sequences and tested for AQP4-reactivity by cell-binding assay. CSF and serum IgG fractions were searched for sequences that matched their respective CSF IgG transcriptome. Matching peptides within the same patient’s CSF and serum IgG proteomes were also identified.In each NMO patient, we recovered CSF IgG VH and VL sequences that matched germline-mutated IgG protein sequences from the patient’s CSF and serum IgG proteomes. Although a modest variation was observed between patients, the overlap between the transcriptome and proteome sequences was found primarily, but not exclusively, within the CSF. More than 50% of the CSF IgG transcriptome sequences were exclusively found in the CSF IgG proteome, whereas 28% were found in both the CSF and blood IgG proteome, and 18% were found exclusively in the blood proteome. A comparable distribution was noted when only AQP4-specific IgG clones were considered. Similarly, on average, only 50% of the CSF IgG proteome matched corresponding peptide sequences in the serum.During NMO exacerbations, a substantial fraction of the intrathecal Ig proteome is generated by an intrathecal B cell population composed of both novel and peripherally-derived clones. Intrathecal CSF B cell clones may contribute to NMO disease exacerbation and lesion formation and may be an important target for preventative therapies. Neuromyelitis optica (NMO) is a severe central nervous system (CNS) autoimmune disorder with predilection for the optic nerves and spinal cord . SpecifiThe relationship between serum and CSF titers of AQP4-IgG and the presence of intrathecal AQP4-IgG production, however, remains unclear. Several studies have suggested that AQP4 autoantibodies are passively transferred from serum to CSF ,8-10. InThe identification of clonally expanded CSF plasmablasts expressing AQP4-IgG in an NMO patient  suggestsAQP4-seropositive NMO or NMO spectrum disease (NMOSD) patients experiencing an acute exacerbation were recruited at the University of Colorado  and the Technische Universität München (TUM09-527). CSF analysis was performed within 84 days of symptom onset . Diagnoses were made according to published criteria ,15. PatiCSF IgG variable region heavy- (VH) and light-chain (VL) sequences were recovered from CD19-CD138+ plasmablasts by single cell fluorescent-activated cell sorting (FACS), reverse transcriptase PCR (RT-PCR), and DNA sequencing as described previously . RecombiS. aureus V8 protease (Glu-C). Following digestion, the tryptic mixtures were extracted in 1% formic acid/50% acetonitrile). Samples were analyzed on a linear trap quadropole (LTQ) Orbitrap Velos mass spectrometer  coupled to an Eksigent nanoLC-2D system  through a nanoelectrospray LC-MS interface using a 90-minute gradient from 6 to 40% ACN. Peptide fragmentation was performed in a higher energy collisional dissociation cell with normalized collision energy of 40%, and tandem mass spectra were acquired in the Orbitrap mass analyzer. Data acquisition was performed using Xcalibur software .CSF and serum IgG (0.5 to 2.0 ml) was applied to protein A sepharose columns  and purified according to the manufacturer’s instructions. Protein concentrations were determined by bicinchoninic acid (BCA) Protein-Assay , and the purity analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis. Excised heavy- and light-chain gel pieces were destained in ammonium bicarbonate/50% acetonitrile (ACN) and dehydrated in 100% acetonitrile. Disulfide bonds were reduced by dithiothreitol, and cysteine residues were alkylated with iodoacetamide. Heavy-chain proteins were digested with trypsin, light chains with trypsin and Tandem mass (MS/MS) spectra were converted into mgf files using an in-house script. Mascot  was used to perform database searches against the Swiss-Prot database and a database containing translated CSF B cell transcriptome repertoires. Peptide tolerance was set at ± 15 ppm with an MS/MS tolerance of ± 0.1 Da from spectra. Full trypsin specificity was required and one missed cleavage was allowed; carbamidomethylation on cysteine was defined as a fixed modification; methionine oxidation, N-terminal pyroglutamic acid formation and N-terminal (protein) acetylation were defined as variable modifications in the database search. Scaffold  was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted at a > 95.0% probability, protein identifications at a > 99.0% probability.http://www.vbase2.org/).To search for an overlap between IgG transcriptome sequences and IgG proteome peptides, recovered peptides were aligned to the same patient’s transcriptome sequences using Scaffold software . Peptides were considered unique when they were identical to the somatically mutated cDNA sequence of the VH or VL sequence Figure . To deteWe generated subject-specific variable region VH and VL repertoires from seven NMO and NMOSD patients and in conjunction assessed IgG VH and VL peptide libraries from the patients’ CSF and serum. Using the CSF VH and VL transcriptome repertoires as databases, we examined the serum and CSF IgG proteomes for matching unique sequences defined as peptides perfectly matching somatically-mutated CSF transcriptome sequences Figure . No uniqIgG peptides that uniquely matched CSF transcriptome sequences showed a common distribution among NMO patients Table . SurprisIn NMO, pathogenic AQP4-IgG is presumed to passively transit into the CNS where it initiates astrocyte injury and secondary demyelination. Surprisingly, on average, only 28% of the IgG peptides matching CSF transcriptome sequences were recovered from both the CSF and serum of the same individual, which may be the result of the limited number of reference CSF transcripts. To complete a more extensive analysis of the overlap between the CSF and serum IgG proteomes, we searched the CSF and serum IgG proteomes for identical, unique VH and VL peptides independent of the transcriptome repertoires. By aligning the peptides from patients’ serum and CSF to a random human protein database (Swiss-Prot), we recovered on average 995 VH  and 584 VL  CSF and serum Ig peptides per patient. Further evaluation of these peptides for mutations from germline identified 59 unique (non-germline) IgG peptides in 5 out of 7 patients. No common unique IgG peptides were identified in different patients. In contrast to expectations, only 45% (range 0 to 77.8%) of the unique VH or VL peptides were found in both the CSF and serum compartments. Twenty-nine percent (range 11.1 to 52.9%) of the unique IgG peptides were found exclusively in the CNS compartment, 26% (range 0 to 75%) in the peripheral compartment only.The detection of AQP4 autoantibodies has been shown to be a highly specific marker for NMO and AQP4-IgG plays a direct role in disease pathology. However, it is still unclear, whether CNS AQP4-IgG is primarily produced by peripheral plasma cells and passively leaks into the CNS through an open blood-brain barrier (BBB) or is generated in part by local CNS B cells. To date, serologic studies have favored a passive influx of AQP4-IgG from serum to CNS: the CSF:serum ratio of AQP4-IgG is approximately 1:500  and stanIn this study, we show that the overlap between the CSF IgG transcriptome and proteome in NMO patients was found primarily, but not exclusively, within the CSF IgG fraction following acute exacerbations. These data indicate that CSF plasma cells in NMO contribute to the production of CSF AQP4-IgG and a significant fraction of the IgG idiotypes are unique to or highly enriched within the CNS compartment. The concurrent presence of many matching peptides in the CSF and serum proteomes indicates that some of the intrathecal plasma cells originate from memory B cells that had previously differentiated and populated peripheral plasma cell niches. Analyses of the overlap between the CSF and serum IgG peptides, independent of the CSF B cell transcriptomes, also reveals that a significant fraction of intrathecal IgG is unique to the CSF. Similar distributions were observed for AQP4-specific VH and VL peptide sequences. The data contrast with the expected results of a passive influx of serum AQP4-IgG, where a near complete overlap of serum and CSF IgG would be anticipated. Indeed, in our analysis, CSF metrics indicated a dysfunction of the blood-CSF barrier in only two of seven patients Table .Interestingly, approximately 20% of CSF VH transcripts matched only serum Ig peptides. The absence of overlap between these CSF B cell transcripts and the CSF Ig proteome could result from methodological limitations. While there is no data on the sensitivity and specificity of similar analyses , the higAlbumin. These results suggest that the extent of BBB disruption in active NMO may not be as global as anticipated and, dependent on the lesion localization and volume, QAlbumin might not be a sensitive enough marker in NMO [Prior studies have generated disparate results on the relationship between BBB disruption and CSF AQP4-IgG. Jarius and colleagues  noted anr in NMO . Additior in NMO ,21, therIgG index and oligoclonal banding. These measures may also be skewed by aspects of NMO pathology  that alter biophysical parameters such as blood-CSF diffusion and CSF flow. Furthermore, the passive transfer of serum IgG into the CSF may obscure faint bands of intrathecally produced IgG on isoelectric focusing. Finally, a significant fraction of the AQP4-IgG produced intrathecally may remain bound to glial tissue [The combined proteomic and transcriptome analysis of CSF IgG in NMO patients suggests that CNS AQP4-IgG is a product of both passive transit from circulating serum IgG and intrathecal plasmablasts and plasma cells Figure . Approxil tissue . TherefoAlbumin [How might AQP4-IgG producing B cells and serum AQP4-IgG combine to initiate an NMO relapse? The migration of AQP4-IgG producing B cells into the CSF could be the initial step. Although AQP4 is highly expressed on perivascular astrocyte foot processes, the possibility that the limited fraction of circulating serum AQP4-IgG might initiate CNS injury is confronted by several quandaries. AQP4-specific autoantibodies are only a minor component of the total serum IgG fraction , CSF AQPAlbumin , CSF AQPAlbumin , and onlAlbumin .In situ production of AQP4-IgG by these transiting cells may be sufficient to produce local disruption of the BBB and facilitate passive entry of serum AQP4-IgG, lesion propagation, and clinical symptomatology.While local physiologic disturbance of the BBB due to stress or infection may precipitate an influx of a sufficient amount of circulating AQP4-IgG to induce astrocytic injury, an alternative explanation, based on our transcriptome and proteome data, is that NMO lesion formation is initiated following the transit of pathologic AQP4-IgG producing B cells to the CNS. The exclusive identification of the majority of the CSF Ig transcriptome in the CSF Ig proteome indicates that CSF plasmablasts are composed, in part, of a novel B cell population that might emerge during an acute exacerbation. In NMO CSF, plasmablasts show a remarkable degree of intraclonal diversity (reference  and unpuIn this study, the qualitative picture of the overlap between the CSF IgG transcriptome and the CSF and serum proteomes may have been limited by differences in the concentration and diversity of CSF and serum IgG, the small number of patients analyzed, and the timing of CSF analysis relative to relapse onset. In addition, the proportion of sequences and peptides recovered may not reflect their absolute number. Despite these caveats, the overlap of the IgG proteome and transcriptome sequences remained consistent among patients and showed evidence of both intrathecal AQP4-IgG production by CNS B cell clones and passive influx of serum IgG.In this study we show, that a fraction of intrathecal IgG including AQP4-IgG is generated by an intrathecal B cell population composed of both novel and peripherally-derived B cell clones. The production of intrathecal AQP4-IgG by CSF B cell clones indicates that future treatment in NMO may need to address both peripheral and CNS B cell populations for acute and prophylactic strategies -24.


Taenia solium is the postoncospheral (PO) form, which has not yet been completely characterized. The aim of this work was to standardize a method to obtain T. solium PO forms by in vitro cultivation. We studied the morphology of the PO form and compared the expression of antigenic proteins among the PO form, oncosphere, and cysticerci stages.The transitional period between the oncosphere and the cysticercus of T. solium activated oncospheres were co-cultured with ten cell lines to obtain PO forms, which we studied at three stages of development–days 15, 30, and 60. A high percentage (32%) of PO forms was obtained using HCT-8 cells in comparison to the other cell lines. The morphology was observed by bright field, scanning, and transmission electron microscopy. Morphology of the PO form changed over time, with the six hooks commonly seen in the oncosphere stage disappearing in the PO forms, and vesicles and microtriches observed in the tegument. The PO forms grew as they aged, reaching a diameter of 2.5 mm at 60 days of culture. 15–30 day PO forms developed into mature cysticerci when inoculated into rats. Antigenic proteins expressed in the PO forms are also expressed by the oncosphere and cysticerci stages, with more cysticerci antigenic proteins expressed as the PO forms ages.in vitro production method of T. solium PO forms. The changes observed in protein expression may be useful in identifying new targets for vaccine development. In vitro culture of PO form will aid in understanding the host-parasite relationship, since the structural changes of the developing PO forms may reflect the parasite’s immunoprotective mechanisms. A wider application of this method could significantly reduce the use of animals, and thus the costs and time required for further experimental investigations.This is the first report of an  T. solium, which is a neglected disease. The postoncospheral (PO) form is an intermediate form between the oncosphere, which is the larva, and the fully developed cysticercus, which is a cyst with a scolex. The morphology, development, and protein and antigen expression of the PO form have not previously been characterized. Here, we report the novel in vitro cultivation of T. solium PO forms and characterize the morphology, development, and expression of antigenic proteins. This new method will allow for better study of this transitional form, which is very difficult to study in the intermediate host. With the increased availability of secreted proteins and antigens, in vitro cultivation will help improve diagnostic assays and provide new targets for vaccine development to block transmission.Neurocysticercosis is caused by  Taenia solium which forms cysts in the muscles or the central nervous system of its intermediate host. Both humans and pigs can acquire cysticercosis through fecal-oral transmission by ingesting T. solium eggs released by the adult tapeworm, which lives exclusively in the small intestines of humans. This parasitic disease is a major public health problem in developing countries where pigs are raised as a food source and causes great economic loss to farmers. It is strongly correlated with poor sanitary conditions and deficient sanitation infrastructure in regions where pig rearing is common , ..5].T. solium contain a six-hooked larva (hexacanth) called the oncosphere  or orin viteformis) , 12]..in vitroT. solium PO forms have only been done in vivo using pigs, focusing on the morphology and distribution of the PO forms . I. IT. solIn order to determine which cell line was the most successful in producing PO forms, we evaluated different cell lines: human intestinal cells , human neuroblastoma cells (SH-SY5Y), human choriocarcinoma cells (BeWo), rat myocardium cells (H9C2), human lung cells (MRC-5), Chinese hamster ovary cells (CHO-K1), African green monkey kidney cells (VERO), and Rhesus monkey kidney cells (LLC-MK2). All of these cell lines were obtained from American Tissue Culture Collection .2 and grown in specific media as recommended by ATCC . All media were supplemented with 10% fetal bovine serum and changed every 2 days. Once cell confluency was obtained, cells were harvested using trypsin-EDTA . Cells were placed into 24-well plates (1x105 cells per well), and the maturation assay described below was performed when cells formed a monolayer.Cells were incubated at 37°C in 5% CO2CO3, and 1 ml of real pig bile, with enough RPMI 1640 medium [pH 8.04] to make 100mL). After activation, the oncospheres were washed three times with RPMI medium and counted using a Neubauer chamber.Tapeworms were collected after medical treatment of newly diagnosed patients as described by Jeri et al . HatchinWe performed three experiments to define the best conditions to obtain PO forms. A parasite was considered to have developed to PO form if we observed the following characteristics: increased number of cells, increased size, loss of hooks, formation of inner cavity with no scolex, and change of morphology.The first experiment was aimed at identifying the best cell line to obtain PO forms. Ten thousand activated oncospheres were placed in each well of 24-well plates containing one of the different confluent cell lines listed above; they were incubated in 5% CO2 at 37°C, and the cell medium was changed every 3 days for a period of two weeks. This experiment was repeated three times.The free-floating PO forms were collected and rinsed three times with sterile PBS buffer. Washes consisted of transferring the PO forms into a 1.5 mL tube, allowing it to settle for 5 minutes, then the supernatant was removed and 1 mL of sterile PBS was added before the process was repeated. Finally, the PO forms were resuspended in 1 mL of PBS, mixed gently, and counted using an inverted microscope.The second experiment aimed to evaluate if the oncosphere can develop to PO form in the absence of a cell monolayer. Based on the results of the first experiment, activated oncospheres  were cultured for two weeks in: A) HCT-8 cell monolayer (positive control), B) culture media alone, C) supernatant from HCT-8 cell monolayer and D) supernatants from HCT-8 cell monolayer that had been incubated with 2-week old PO forms.The third experiment was done to evaluate if the oncosphere could develop into PO form in the absence of direct contact with the selected feeder cell (HCT-8). We co-cultured the feeder cells and activated oncospheres  in two types of transwell systems; one with a collagen-coated membrane (Transwell-PTFE/COL) and the other one with a polyester membrane (Transwell-PET). The activated oncospheres were added to the top of each transwell insert . One milliliter of medium was placed in the bottom well, which contains the HCT-8 monolayer cells. Two types of controls were included in this experiment; one with activated oncospheres in a transwell system (collagen or polyester membrane) in the absence of monolayer cells, and the second one with activated oncospheres in direct contact with the monolayer cells. The 24-well plate was incubated for two weeks in 5% CO2 at 37° and the medium in the bottom well was replaced every three days.The HCT-8 intestinal cell line was selected to study the morphological changes of the parasite because it yielded the highest percentage of PO forms. Ten thousand activated oncospheres were cultured in confluent HCT-8 cell monolayer and cell medium was changed every three days. The free-floating PO forms were collected and rinsed twice with fresh medium, then transferred to another well containing confluent HCT-8 cell monolayers. This process was repeated every three days for up to two months to allow the PO form to continue development. Cultures were inspected daily using an inverted microscope (Leitz labovert FS). PO forms were collected at 15, 30, and 60 days of incubation. After 2 months of incubation, the PO forms began to die off.The PO forms collected were divided into 3 aliquots. The first aliquot was dried on a slide coated with poly-L-Lysine 0.1% solution  followed by fixation with methanol-acetone (1:1) for 10 min at -20°C, and the slides were then washed three times with PBS, mounted with Prolong gold antifade reagent with DAPI , and examined by UV microscopy (Leica DM 500). The second aliquot was fixed with 2% of glutaraldehyde (Grade I: 70% solution) in 1% sucrose in PBS to be examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The third aliquot was fixed with 4% of paraformaldehyde in PBS and embedded in paraffin. Two 4-μm sections from all samples were stained with H&E.2 . Using an inverted microscope, samples were positioned in stubs and gold coated (SPI-Module Sputter coater). Specimens were observed using a Zeiss 1450EP Scanning Electron Microscope.The fixed PO forms were then fixed in 1% osmium tetroxide followed by sequential dehydration steps from 25% to 100% ethanol. Ethanol was exchanged for liquid COThe fixed PO forms were then fixed in 1.5% osmium tetroxide, dehydrated in ethanol, and embedded in epoxy, and 50-nm sections were cut. Sections were then stained with lead hydroxide and uranyl acetate and were examined using a Phillips electron microscope  operating at 75 kV.To determine the viability and infectivity of the PO forms, 15 day old Hotzman rats, purchased from Universidad Peruana Cayetano Heredia (UPCH), Lima, Peru, were infected intracranially (in the bregma) as described by Verastegui  with 15 Necropsy was done four months later. Briefly, the rats were anaesthetized with ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight). Anaesthetized rats were perfused with 200 ml of PBS and then with 100 ml of 4% paraformaldehyde in PBS. Brains were carefully removed, post-fixed for 24 hours at 4°C with 4% paraformaldehyde in PBS, and stored in 70% ethanol. Brains were observed macroscopically to identify extraparenchymal cysticerci; coronal sections of the brain (5 mm) were done until the intraparenchymal cysticerci were observed. Coronal sections of the brain were paraffin embedded, cut in 3 μm thick sections and Masson’s trichrome stained.All animal procedures were performed in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal of the Universidad Peruana Cayetano Heredia, Lima, Peru (Permit Number: 61242).The total proteins present in PO forms, activated oncospheres, and cysticerci were incubated with anti-oncosphere and anti-cysticercus rabbit sera in order to evaluate the change in the expression of antigenic proteins during PO form development. Total proteins of the PO form were obtained from PO forms cultured in HCT-8 cells as described previously. PO forms were collected at 15, 30, and 60 days of incubation, rinsed three times with PBS buffer, sonicated, and centrifuged. Hatched and activated oncospheres were obtained as described above; cysticerci removed from an infected pig were also rinsed, sonicated and centrifuged. The supernatants were used as total protein, and examined by western blot.Protease inhibitors of 1mg/ml of Leupeptin (1/1000) , 0.1 M Pefabloc SC PLUS (1/100)  and 1mg/ml of Pepstatin (1/1000)  were added to the total protein prior to storing at -70°C. Three μg of total proteins were resolved in 15% SDS-PAGE non-denaturing gels and transferred to nitrocellulose (NC) membranes . NC membranes were incubated overnight at 4°C with rabbit sera immunized with total proteins of activated oncosphere and cysticerci (1:200) ). T). TEchinparasite .Interestingly, the PO forms did not grow in LL-CMK2 monolayer cells which may be due to lack of essential nutrients, growth factors, cytokines or other possible growth stimuli, and the fact that different cell lines likely express different surface molecules. The transformed cell lines that are used in the present study usually express a variety of growth factors that normal cells would not produce in their tissues of origin. These factors may also explain the different growth rate of the parasites among the cell lines.Echinococcus granulosus . H. H22]. HT. solium PO form has the general pattern of postoncosphere development reported in Taenia serialis, Taenia ovis, Echinoccoccus granulosus, Taenia hydatigena and Taenia pisiformes when these are grown in medium supplemented with serum [T. solium postoncospheral development was 1) cell multiplication as seen with DAPI stain, 2) cavity formation, which was observed from 15 to 60 days PO forms, and 3) further growth, and where applicable movement which is observed in all three age points. Additionally, when the 15 and 30 day PO forms were injected into rat brains they continued to mature and formed cysticerci, proving their viability; this is a highly efficient method of infecting an animal model. Finally, the PO forms started to die off at 60 days of culture.The th serum . In our in vitro study. First, it is likely that the PO forms were not viable long enough to complete its development as it was reported for T. saginata [No scolex formation was observed in this saginata . Secondlsaginata . Howeversaginata .T. solium oncosphere antigens [We observed the transition of immunogenic proteins from oncosphere to cysticerci. The 15 day PO forms stopped producing some oncosphere proteins, such as those of the size of 22.5 and 31.3 kDa, and, as the PO forms aged, started the expression of a majority of the cysticerci antigenic proteins. Bands of 22.5 and 31.3 kDa were reported as specific antigens ; howeverantigens . The chaT. solium oncospheres to obtain PO forms for up to 60 days in the presence of an HCT-8 feeder cell monolayer. These PO forms can complete their development to mature cysticerci when injected in rat brains, and changes in the expression of antigenic proteins during their development are related to the changes observed in their morphology.In conclusion, we have developed and standardized a method for culturing S1 Multimedia File(MOV)Click here for additional data file.S2 Multimedia File(MOV)Click here for additional data file.S3 Multimedia File(MOV)Click here for additional data file.


There was a positive correlation between radiocesium concentrations in the soil samples and frogs, and the highest concentration in frogs was 47,278.53 Bq/kg-wet. Although we conducted a histological examination of frog ovaries and testes by light microscopy to detect possible effects of radionuclides on the morphology of germ cells, there were no clear abnormalities in the gonadal tissues of frogs collected from sites with different contamination levels.We investigated the accumulation of radionuclides in frogs inhabiting radioactively contaminated areas around Fukushima Daiichi Nuclear Power Plant (FDNPP) to search for possible adverse effects due to radionuclides. We collected 5 frog species and soil samples in areas within and outside a 20-km radius from FDNPP in August and September 2012 and determined their radiocesium concentrations ( The Fukushima Daiichi Nuclear Power Plant (FDNPP) was severely damaged by the Great East Japan Earthquake and Tsunami of 11 March 2011, releasing large amounts of radionuclides over a wide area1Reports about the radioactive contamination and radiation effects on animals and plants in Fukushima are gradually increasing. The Fukushima prefectural government and the Ministry of the Environment are monitoring the accumulation of radionuclides in the wildlife after the FDNPP accident, and most of the examined species are popular for hunting and fishing3567After severe nuclear accidents such as the Chernobyl accident, the biological impact of radioactive exposure has been investigated, and several studies have reported on the radiation damage to wildlife911In this study, we investigated the accumulation of radioactive contamination in frogs and its possible adverse effects. In Japan, most frog species inhabit rice paddy fields, and they are important organisms in paddy ecosystems. The areas around the FDNPP include many rice paddy fields. Frogs live close to the ground and they have a small home range. They are thin skinned and they absorb water through their skin. Therefore, they are constantly exposed to radionuclides in the environment both internally and externallyWe collected frogs and soil samples at survey sites in the highly contaminated area within a 20-km radius from the FDNPP and a neighbouring area in August and September 2012, and determined radiocesium concentrations in the frogs and soil samples. We conducted histological examinations of the ovaries and testes of the frogs caught at the survey sites by using light microscopy to search for aberrations in the morphology of germ cells, because we were more concerned about the impact of radioactivity on the individual frog's reproductive ability rather than on its survival ability. This is a preliminary analysis of the effects of radionuclides on the reproduction of frogs in wild populations.Rana japonica, R. ornativentris, R. tagoi tagoi, Pelophylax porosus porosus, and Hyla japonica; P. porosus porosus, although we found it at Naraha. We heard calls of R. catesbeiana at Futaba B and found a larva of Rhacophorus arboreus at Namie C. In the September survey, we heard calls of H. japonica at Namie A, Namie B, Futaba A, Futaba B, and Tamura.We collected 135 live individuals of 5 species at 9 sites . We detected radiocesium in 21 composite samples of oi tagoi . Ten aniR. japonica at Minamisoma showed the lowest concentration of radiocesium (the sum of 134Cs and 137Cs)  = 62.68 mm), and a composite sample of 5 males and 3 juveniles (sex unknown) of R. tagoi tagoi at Namie C showed the highest concentration of radiocesium .A female r = 0.591, P = 0.001, n = 27, R. japonica, for which we could get animals at most survey sites, the radiocesium concentrations in frogs were correlated with those in the soil .The radiocesium concentrations in all species of frogs were positively correlated with those in soil samples , but not with the distance from the FDNPP to each site  were 1.062 ± 0.074 SD for frogs and 0.997 ± 0.029 SD for soil samples, and these 134Cs/137Cs ratios were nearly equivalent to that of the FDNPP falloutRadiocesium concentrations in soil samples were correlated with the air radiation dose rates at sites , which looked like degenerative oocytes, in the ovary. It is difficult to statistically confirm the difference in frequencies of females with DSCs between sites with different levels of radioactive contamination owing to the small numbers of animals. Of 28 females caught in the August survey and 12 females caught in the September survey, 6 and 7 females, respectively, showed DSCs. The frequency of females with DSCs in the September survey was significantly greater than that in the August survey . Females with DSCs were likely mature according to their body sizeR. japonica. Of 7 females of H. japonica, 6 females showed DSCs, and these were caught in the less contaminated sites of Minamisoma and Naraha.Of 29 males, 3 males of Buergeria buergeri 160,000 Bq/kg-wet at 11.3 μSv/h) caught at a certain location in Minamisoma. B. buergeri inhabits forests. Similarly, Rana tagoi tagoi, which showed the highest concentration of radiocesium in our surveys, inhabit forests although the lives of both species are ecologically different. However, it is difficult to indicate the relationship of contamination levels among frog species and the environment of habitats because only a small number of samples were investigated in the present study.In this study, we found that the radiocesium concentrations were rather high in frogs caught within a 20-km radius of the FDNPP. The concentrations found these frogs were positively correlated to radiocesium concentrations of the soil at the survey sites. Since there are very few studies of radioactive contamination of amphibians in Fukushima, our study is an important report about high radioactive contamination in frogs captured within a short period after the accident. The Ministry of the Environment reported radiocesium concentrations in some frog species within a 20-km radius of the FDNPP in May 2012, and high concentrations of radiocesium in four frog species  were foundR. japonica was independent of contamination levels. In wild populations in several sites of Japan, however, male frogs with testis-ova were found frequently before the FDNPP accident. Male frogs of six species with testis-ova were observed at the ratio of 0% to 62.7% in Japan20R. japonica with testis-ova was 11.1%R. japonica, nor were there clear differences in the morphologies (i.e. size and colour) and germ cells of the ovaries of females among survey sites. There is very little information about the radiation effects on frogs in wild populations. For example, in the investigations that were carried out both in the year when the Chernobyl accident happened and 3–8 years after the accident, chromosome aberrations and aberrant cells in bone marrow were observed in R. temporaria L. in contaminated regions in BelarusIn the present study, we did not observe clear aberrations in the morphology of germ cells in the testes and ovaries of frogs caught in the contaminated sites; however, not all aberrations may have been visible to us. The presence of testis-ova in male R. japonica, appeared to have been born in the spring of 2012 based on their body size. In April 2013, we found egg masses of R. japonica and R. ornativentris in survey sites within the contaminated area. These observations indicate that frogs could oviposit their eggs there in 2012 and 2013. Therefore, internal and external exposure to radioactive materials does not seem to have severely affected the reproductive behaviour or the maturation of eggs and sperm in frogs. As R. japonica has a lifespan of several yearsMost of these frogs in Namie A, where we caught the most −1 from 60Cobalt and 0.05–0.26 Gy h−1 from 137Cs−1 and 490 μGy h−1 (corresponding to 11.76 mGy d−1), respectively). However, these studies did not examine the effects of internal exposure to radioactive materials. Both tadpoles and adults in contaminated areas always assimilate radiocesium in the natural environmentOn the other hand, the effects of radiation  on the early developmental stages of frogs (e.g. eggs and tadpoles) were unknown in this study. In some laboratory experiments that were performed with high dose irradiation, sensitivities of zygotes and hatchings to radiation were higher than that in juvenile and adult frogs22R. japonica, H. japonica, P. porosus porosus, Glandirana rugosa, and Rhacophorus schlegelii)R. schlegelii uses paddy fields as reproductive sites in early summer and live in forests in other seasons. G. rugosa is an endangered species in the Fukushima prefectureR. japonica, H. japonica, and P. porosus porosus). Unexpectedly, however, we found only a small number of frogs at most survey sites. One possible reason is that it rained less than usual in the summer of 2012. Therefore, most of the frogs might have left for moist environments since the complete evacuation of residents from the area meant that there was no water body in the paddy fields. However, we caught many frogs at Minamisoma, where water was supplied from a spring and grooves. Therefore, this site was a suitable wet environment for inhabitation and breeding of frogs.We caught common species of frogs in our survey area and found the species on which our surveys about biota and the environment in the eastern area of Fukushima prefecture would be based2829Another reason is that most paddy fields had changed into grasslands without pools of water. There were many paddy fields in these areas. Every year, rice producers irrigated rice fields to cultivate rice from the spring through the summer. Therefore, paddy fields had been used by amphibians as breeding pools in place of wetlands. Since all residents in the radioactively contaminated area were immediately evacuated after the FDNPP accident, no agricultural activity had been conducted in the area and neighbouring areas since the accident, and consequently, most paddy fields have dried and changed into grasslands. For many amphibians, the aridification of paddy fields means a rapid decrease in suitable environments. The same is also true for many other organisms of paddy ecosystems, such as aquatic insects and fishes. We should pay attention to rapid changes in environments caused by no human activity due to exhaustive escape from the accident and consider the biodiversity conservation in radioactively contaminated areas as well as radiation impacts on wildlife health.We surveyed and caught frogs at 5 sites within the 20-km radius area and at 4 sites surrounding this area , Table 1The surveys in restricted area were always performed by 3 people. Two people walked around a site, searching for and catching frogs for thirty minutes, while the other was in charge of safety management and helping the researchers take off protective clothing. When frogs were not caught but they were observed or their calls heard, we recorded their species. These surveys were conducted twice in 2012 (27–28 August and 20–21 September). In August, we did not conduct the survey at Tamura, Katsurao, or Namie C.Except for Katsurao and Namie C, paddy fields were being used as survey sites before the accident. In Namie C and Katsurao, there were pools surrounded by forests and there were paddy fields used before the accident within 500 m from these sites. When we surveyed, in Naraha and Tamura sites, there were experimental paddy fields created to check for radioactive contamination in rice. Other paddy fields had pools or marsh in grasslands, although most paddy fields there dried to change into grasslands owing to lack of agricultural activity in the evacuated area. Thus, these survey sites were selected as the place where frogs may be able to inhabit.R. ornativentris in all pools in March 2012. Therefore, although we collected surface soil from beside each pool to take account of the differences in contamination between pools, all frogs collected around pools were combined because we could collect only a few frogs there  and CsI scintillator (HORIBA PA-1000 Radi). For the determination of radionuclides in soil, soil samples were collected from the surface where there was very less vegetation . At Namie C, there were 3 small pools within 840 m and we observed eggs of here see .We collected frogs irrespective of the body size and age. No tadpoles were collected, neither for histological examination nor for determination of radiocesium levels. All live frogs caught were transferred to a laboratory in Tsukuba and kept alive in a room maintained at 20°C until dissection of the gonads. Under deep anaesthesia with diethyl ether, live frogs were measured (snout-vent length) and weighed, and the gonads were immediately dissected for histological examination. All tissues removed for dissection were preserved in 70% ethanol after fixation in Bouin's solution for 48 h and then embedded in paraffin. They were cut into 5–6 μm sections using a microtome and subsequently stained with hematoxylin and eosin. The experiments were carried out in accordance with the approved guidelines prepared by National Institute for Environmental Studies, Japan.134Cs and 137Cs) concentrations of frogs and soil samples were analysed using a gamma spectrometer with a germanium semiconductor detector . Gamma-ray emissions were measured at energies of 604.7 keV (134Cs) and 661.6 keV (137Cs). Detection time was 203–40,000 s and 26–995 s for frogs and soil samples, respectively. All soil samples and the tissues of frogs were maintained at 4°C and −20°C, respectively, until measurement. Whole bodies of individuals whose gonads maintained removed and individuals that died before dissection were used for the determination of radiocesium. Frogs were separated according to body size and sex for determination of radiocesium. Large frogs were measured alone. To meet the minimum requirements for determining radiocesium contents, small frogs were collected and measured as composite samples .N.M., S.I. and T.H. designed the study and collected the samples. M.T. analysed all sections of gonadal tissues of frogs. N.M. analysed all of the data and wrote the manuscript text with information from all the authors.


Background: Given the paucity of data on possible testis changes in opioid dependents, we sought to compare the testis volumes between a group of opium dependents and a group of healthy controls.Objective: Comparison of testis volume between opium dependents and healthy controls.Materials and Methods: This case-control study recruited 100 men with opium dependency (cases) and 100 healthy men (controls) in Iran, in 2008. A checklist containing questions about age, height, weight, daily amount of cigarette use, and duration of cigarette use for all the participants as well as daily amount of opium use (grams) and duration of opium use (years) for the case group was completed. Additionally, the dimensions of each testis were measured by a single person using calipers, and the mean of the left and right testes volume was compared between these two groups.Results: The mean of the testis volumes in the case group was significantly lower than that of the case group . The results of the ANCOVA test showed that even after the omission of the cigarette smoking effect (p=0.454), the testis volume remained lower in the opium dependents . In the case group, there were significant reverse correlations between testis volume and age , daily amount of opium use  and duration of opium use .Conclusion: We found that the testis volume in the male opium dependents was lower than that of the healthy controls. We would recommend that future studies into the impact of drugs on the testis dimensions pay heed to possible histological changes in the testes owing to opium dependency. According to the World Drug Report in 2007 an approximate 200 million people in the world  aged 15 and above had used substances in the year before . The American Attitudes on Substance Abuse XIII underscored the availability of prescription opioids for abuse as reported by teens and parents alike . Indeed,What is more, maintenance treatment by opioids for opioid dependency was once reported to have accounted for a minimum of 460,000 cases in Europe and 241,000 cases in the USA , 6. UndeA case in point is endocrine gland dysfunction and its concomitant sexual dysfunction, which has been the subject of much research . About tAn estimated 5 million people in the US and Canada are believed to be inflicted with this dysfunction , 10. TheTo the best of our knowledge, there is a dearth of data in the existing literature on changes in the testes, as one of the major endocrine glands, in opioid dependents and there is also a lack of comparative studies into possible differences in the testis volume between opioid dependents and control groups , 15. TheDesign and settingThis case-control study was conducted by opioid dependency treatment clinics in the Iranian city of Arak in 2008. Informed written consent was obtained from all the participants, and the study protocol was approved by ethical committee of Arak University of Medical Sciences.ParticipantsThere were 100 men in each study group. The case group was comprised of opium dependents selected from opioid dependency treatment clinics in Arak, and the control group comprised male inpatients without a history of any drug use in orthopedic wards of Vali-Asr Hospital of Arak. The inclusion criteria for the case group were in accordance with the DSM-IV criteria for opioid dependency, with the minimum fulfillment requirement being three of the following conditions within the previous year: withdrawal, unsuccessful withdrawal attempts, opioid use more than or longer than the prescribed amount or duration by a physician, lengthy period of recovery from the side effects of opioids, and social or occupational or recreational dysfunction . The excMeasurements and outcomesThe research assistants introduced themselves and fully explained the aims to the participants before completing a checklist of basic characteristics such as age, height, and weight in addition to variables pertaining to cigarette use, for instance daily amount and duration of use. Information on opium use such as daily amount (grams) and duration of use (years) was also recorded for the case group. The main outcome was the mean of the volumes of the right and left testes as measured by a single examiner in both groups. The examiner measured the testes by tensioning the scrotum skin completely by his left hand and measuring the dimensions of each testis with calipers . The meaStatistical analysisStatistical analysis was conducted using SPSS for Windows 13 software. The quantitative variables were described in range and mean (SD). The independent t-test was utilized to make comparisons between the two groups with respect to age, body mass index, cigarette use, and testis volume. The ANCOVA  test was employed to assess the independent effect of cigarette use by considering the case and control groups as fixed factors, cigarette use as a simultaneous variable, and testis volume as a dependent variable. Furthermore, the bivariate correlation test helped assess the association between testis volume and independent quantitative variables in the case group. P-value<0.05 was considered significant.Basic characteristicsThe age ranges of the case and control groups were 20-49 and 21-48 years, respectively. The respective body mass index ranges of the case and control group were 16.8-31.1 and 18.3-29.3 kg/m². The ranges of the amount of cigarette use were 0-5 and 0-8 packs/year  in  the case and control groups, respectively. The range and mean (SD) of the amount of opium use in the case group were 1-12 and 2.7±1.8 gr/day, and the range and mean (SD) of the duration of opium use were 1-28 and 8.7±6.2 years. No significant difference was seen between the case and control groups in terms of age and body mass index (p<0.001) .Dimensions and volumes of testes:The mean (SD) of the right and left testis volumes in all participants were 18.2±7.4 and 18.1±7.4cm³, respectively. The volumes of the right (p<0.001) and left (p<0.001) testes as well as the mean of the volumes of both testes (p<0.001) in the case group were significantly lower than those of the control group .Independent effect of cigarettes on testis volumeThe results of the ANCOVA test for an assessment of the simultaneous effect of cigarette and opium use on the testis volume revealed that the testis volume in the patients with opium dependency was significantly lower than that of the control group , despite the deletion of the effect of cigarette use (p=0.454).Factors relating to testis volume in opium dependencyIn the opium dependents, there was a significant reverse correlation between age , daily amount of opium use , and duration of opium use  and testis volume. The values of body mass index (p=0.501) and amount of cigarette use (p=0.501) showed no significant relation with the testis volume.The testis volume in the opium dependent group was significantly lower than that of the healthy controls in our study. Cigarette use was more common among our case group, but an assessment of the independent effect of cigarette and opium use on the testis volume revealed that not only did the amount of cigarette use did not affect the testis volume but also after the omission of the effect of cigarette use, opium use still exerted a significant effect on the testis volume. Furthermore, an evaluation of the basic characteristics and variables pertaining to cigarette and opium use showed that although increasing age and amount and duration of opium use could lead to a reduced testis volume, body mass index and cigarette use had no impact on the testis volume. There is currently no disagreement in the scientific community about the negative impact of all opioids on the function of endocrine glands and sexual health , 20.The testis, an endocrine gland secreting sexual hormones and producing sperms, is certainly no exception. For all the evidence in the existing literature on the reduction in the volume and morphological changes in the testes in opioid dependents , 15, 21.Opioids, whether produced endogenously or exogenously, are capable of banding to opioid receptors in the hypothalamus, pituitary, and even testis and influence the function of the sexual glands . A decreMoreover, these substances can directly affect the testis tissue and lessen the production and secretion of sexual hormones as well as the interstitial fluid of testes . PreviouResearch also shows that an increase in the duration of opium use precedes a decrease in sexual hormone levels , 27. MucTo the best of our knowledge, the present study is the first of its kind to assess the testis volume in opioid dependents with a sufficient sample volume. Be that as it may, it suffers from some limitations such as non-measurement of the values of different hormones secreted by the endocrine glands  and absence of testis-function assessor tests  in opioid dependents. The findings of this study showed that the testis volume in the men with opium dependency was significantly lower than that of those without opium dependency. In addition, whereas an increase in age and amount and duration of opium use was in tandem with a continuous reduction in the testis volume in the opium dependents, body mass index and amount and duration of cigarette use had no effect on the testis dimensions. We would, therefore, recommend that people under treatment for problems created by opium during the withdrawal process receive special attention to testicular atrophy through consultation with an urologist alongside treatment for other mental and physical side effects.


Bacterial and fungal infections continue to pose a major clinical challenge in patients with prolonged severe neutropenia after chemotherapy or hematopoietic stem cell transplantation (HSCT). With the advent of granulocyte colony-stimulating factor (G-CSF) to mobilize neutrophils in healthy donors, granulocyte transfusions have been broadly used to prevent and/or treat life-threatening infections in patients with severe febrile neutropenia and/or neutrophil dysfunction. Although the results of randomized controlled trials are inconclusive, there are suggestions from pilot and retrospective studies that granulocyte transfusions may benefit selected categories of patients. We will critically appraise the evidence related to the use of therapeutic granulocyte transfusions in children and adults, highlighting current controversies in the field and discussing complementary approaches to modulate phagocyte function in the host. Bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological malignancies receiving dose-intensive chemotherapy and hematopoietic stem cell transplantation (HSCT). Once in vogue in the 1980s, granulocyte transfusions were later relegated to a marginal role, due to the inconveniences of harvesting cells, infusion-associated toxicities and limited clinical efficacy . The imphttp://www.ClinicalTrials.gov) and the International Clinical Trials Registry Platform .The results of new randomized trials of granulocyte transfusions for life-threatening infections have now been published. Also, new-generation leukapheresis devices have been optimized to further improve the yield and purity of granulocyte collections. It thus is an appropriate time to review existing data on granulocyte transfusions in neutropenic hosts, and to critically appraise the immune-biological effects and safety issues related to G-CSF administration to healthy donors. The following PubMed query was used to retrieve relevant clinical trials: (infection OR sepsis) AND (neutropenia OR granulocytopenia) AND (“granulocyte transfusion” OR “granulocyte transfusions”). We also searched both the U.S. National Institutes of Health registry of clinical studies (The effects of single-dose G-CSF (5–10 µg/kg) on peripheral blood counts have been explored in 261 normal subjects donating peripheral blood progenitor cells (PBPC) for research. Following G-CSF administration, the neutrophil count increased 6.2- to 7.4-fold over baseline values, depending on G-CSF dose. Sixty-nine percent of the donors experienced one or more dose-related side effects 24 h after G-CSF administration, most commonly muscle and bone pain, headache, fatigue and nausea .The combination of G-CSF and dexamethasone has been extensively characterized as a granulocyte mobilization regimen. Neutrophilia after single-dose G-CSF and dexamethasone is attributed to shifts of neutrophils from the bone marrow storage pool into the peripheral blood . The anaIn a prospective study, 52 healthy unrelated volunteers were treated with a single subcutaneous injection of glycosylated G-CSF, lenograstim, at a median dose of 3.1 μg/kg plus dexamethasone  or with a median dose of 11.8 μg/kg of lenograstim without dexamethasone (n = 23). Mobilization kinetics and leukapheresis yields were similar in the low-dose compared with the high-dose G-CSF group. Donor adverse reactions were of greater clinical significance in donors given high-dose G-CSF alone. The combination of glycosylated G-CSF and dexamethasone allowed a significant reduction of G-CSF dose and enhanced the tolerability of the mobilization regimen to the donors .Another study evaluated the efficacy of four different granulocyte mobilization regimens. Donors received G-CSF, either intravenously or subcutaneously, with or without dexamethasone (8 mg) 18 h before apheresis. Whereas G-CSF administration route had no impact on neutrophil counts at hours 2 through 8, subcutaneous G-CSF and dexamethasone sustained 24-h neutrophil counts more effectively than intravenous G-CSF and dexamethasone [A possible strategy to minimize the number of donors for each patient, thus limiting the risk of alloimmunization, would be to use sequential granulocyte collections from a single donor given G-CSF daily. This approach has been prospectively evaluated in 76 healthy donors, who were allowed a maximum of five consecutive donations . This moG-CSF has long been recognized as a potent immediate activator of neutrophils in vivo . An initl-selectin (CD62L), Fcγ receptor (FcγR) III , and the leukocyte function antigen (CD11a) decreased during the course of G-CSF administration. By contrast, FcγRI (CD64) and lipopolysaccharide-binding protein receptor (CD14) levels increased. The expression of amino-peptidase N (CD13), C3bi receptor (CD11b), and the neutrophil β2-integrin (CD18) did not change during the administration of G-CSF, but levels of both CD13 and CD18 increased 3 days after the last dose. Expression levels of neutrophil-specific antigen NB1 (CD177) initially increased, returned to pre-G-CSF levels after 4 days, and increased again after 10 days of G-CSF administration. Overall, this study indicated that most changes in neutrophil phenotype, potentially affecting the function of mobilized granulocytes, occur after one dose of G-CSF. Importantly, the down-regulation of CD62L after G-CSF treatment may result from shedding and likely limits neutrophil-endothelial cell interactions, thus preventing the pulmonary sequestration of granulocytes [The expression of neutrophil antigens has been thoroughly evaluated in seven healthy donors receiving 5 μg/kg of G-CSF for 10 days . The expulocytes , 22.CAST gene, which encodes calpastatin, was induced after G-CSF/dexamethasone treatment both in vivo and in vitro. This observation may account for the prolonged survival ability imparted on granulocytes by the combined treatment with G-CSF and dexamethasone. Irrespective of phenotypic changes, granulocytes collected after G-CSF and dexamethasone benefited 11 out of the 16 children (70 %) who were treated with granulocyte transfusions for febrile neutropenia [Oligonucleotide microarrays were also used to identify genes that are differentially expressed before and after mobilization with single-dose G-CSF and dexamethasone . More thtropenia .http://www.ncbi.nlm.nih.gov/gds). We identified a priori a panel of genes regulating neutrophil effector functions , CD64, CD16b, and CD62L increased after donor treatment with G-CSF [SOD1 mRNA expression declined after neutrophil mobilization. Compared with G-CSF alone, G-CSF and dexamethasone induced a decrease of CD177 and CD64 mRNA expression  or G-CSF and dexamethasone  to those of the COBE Spectra apheresis system (Terumo-BCT) in 32 healthy donors, who received G-CSF and dexamethasone. The collection efficiency of granulocyte procedures using the Spectra Optia® system was approximately 23 % higher than that of the COBE Spectra system. In particular, Spectra Optia® collections generated a higher total neutrophil yield per liter of blood processed compared with the COBE Spectra. No differences in granulocyte viability, chemotaxis and bacterial killing were evident between the two devices. Not unexpectedly, fewer operator adjustments were required when using the Spectra Optia®. Importantly, there was no difference in the number or intensity of adverse donor events between instruments [A recent prospective, multicenter, randomized trial compared the performance and safety of a novel granulocyte collection protocol with the Spectra Optiatruments .® device is not inferior to the COBE Spectra forerunner and produces high-quality granulocyte concentrates with greater absolute neutrophil yields. In another study, target doses of 1 × 1010 neutrophils were achieved in all but one collection with the Spectra Optia® device [® collections were 20 % more efficient compared with those performed with the predecessor device.The Optia device has also been used to collect granulocytes from healthy blood donors and family donors mobilized with prednisolone or G-CSF. . One stu® device . Again, Since the dose of neutrophils transfused is important in determining clinical response, the ability to collect higher numbers of granulocytes will favorably impact the efficacy of therapeutic granulocyte transfusions.Although G-CSF and dexamethasone delay granulocyte apoptosis, the high cell counts achieved in granulocyte concentrates may reduce nutrients and lower pH, resulting into neutrophil death. The production of pyrogenic cytokines may also be increased. According to current standards, granulocyte storage should be limited to 24 h. After 2 days of storage in RPMI-1640 medium at 4 °C, only 2–7 % of the granulocytes remain viable . InfusibThe storage characteristics of granulocyte concentrates were compared after mobilization of donors with G-CSF and dexamethasone, either alone or in combination . Ten graNeutrophil chemotaxis and NADPH oxidase activity were also examined after apheresis collection and neutrophil storage to 48 h . After iOverall, commercial leukocyte storage solutions can prolong granulocyte survival up to 7 days. However, testing of granulocytes with monoclonal antibodies or alloantibodies is problematic after 4 days due to nonspecific staining.9/L [9/L [9/L [Infections in neutropenic patients are associated with a severe prognosis, despite treatment with broad-spectrum antibiotics. In children with malignancy and septic shock, mortality can be greater than 40 %  or even 9/L –41, 0.2 9/L [9/L  or 0.1 ×9/L [9/L , associaTo date, there have been no randomized controlled GTX trials in children, and most available data are derived from observational studies . Sachs e9. Infection-related survival and overall survival rates were 82 and 77 %, respectively, at day 30 [A retrospective analysis including 13 children with neutropenia and proven or suspected infection also supports the efficacy of granulocyte transfusions. Although short-term survival was promising, eight of the 13 patients ultimately died of their infection . In anott day 30 . Anothert day 30 , who werA 59 % overall survival rate was obtained in a cohort of 32 children, with particularly favorable results in bacterial infections  and fungal infection  . In anot8 granulocytes/kg likely contributed better clinical outcomes. This minimum recommended dose was derived from a Cochrane meta-analysis [8/kg neutrophils per concentrate was able to generate a stable ANC increment, to shorten the duration of neutropenia, and to support the control of infections in neutropenic patients with high-risk infections.Seidel et al.  showed tanalysis . They al9/kg granulocytes. Two out of three patients survived the infectious episode [Granulocyte transfusion therapy has been used in three patients with chronic granulomatous disease (CGD) and disseminated invasive aspergillosis. Healthy donors were mobilized with 450 µg G-CSF and dexamethasone approximately 12 h before collection. Patients received between 0.4 and 3.0 × 10 episode .Some studies also suggest a role for granulocyte transfusions in preventing infections or progression of infections in children who are expected to experience prolonged neutropenia after HSCT or chemotherapy , 50. GraConcern for potentially serious pulmonary complications is one of the major limiting factors for the routine use of GTX. Some studies of GTX recipients have documented acute pulmonary transfusion reactions with shortness of breath, dyspnea, hypoxemia, and lung edema , 45, 51.Although randomized controlled trials are not available in children yet, the current evidence supports the early use of GTX, especially for patients with bacterial infections. However, patients should be closely monitored for adverse pulmonary transfusion reactions.Published and ongoing clinical trials in adults are summarized in Table A community blood bank GTX program was developed at the Fred Hutchinson Cancer Research Center. Donors received G-CSF and dexamethasone. This program treated 19 patients with documented fungal or antibiotic resistant bacterial infections who were either waiting for or recently given HSCT . AdverseIn another study G-CSF-mobilized GTX collected from related donors were administered to 15 neutropenic patients with hematologic malignancies and fungal infections . Eleven In a similar study, thirty patients with hematological malignancies received granulocyte transfusions for neutropenia and severe infections during a 12-year period . The donA prospective, non-randomized study evaluated the efficacy of granulocyte transfusions for controlling and preventing recurrence of severe infections in patients with hematological malignancies . Fifty-t9. The composite primary endpoint was survival plus a microbial response evaluated 42 days after randomization. The median number of transfusions in subjects randomized to the GTX arm was five. Success rates were 42 % (20/48) and 43 % (21/49) for the granulocyte and control groups, respectively, on intention-to-treat analysis, and 49 % (17/35) and 41 % (16/39), respectively, on per-protocol analysis. Because of low accrual, the power of this study to detect a 20 % difference in the overall success rates was reduced to approximately 40 %. Thus, it cannot be ruled out that a true effect was missed, particularly if the effect is limited to specific patient subsets [The RING study is a recently completed randomized controlled study carried out by the NHLBI Transfusion Medicine/Hemostasis Clinical Trials Network which evaluated the efficacy of high-dose granulocyte transfusion therapy . The des subsets .A retrospective analysis of 74 patients with refractory hematological malignancies, receiving granulocyte transfusions, showed that patients with documented severe infections might have better survival rates compared with those who do not have severe infection . PatientFusarium infections who were treated with GTX showed a 91 % response rate [A recent single-center case series of 11 patients with invasive nse rate . Three pGranulocyte transfusions have been combined with G-CSF administration in 56 patients with severe aplastic anemia and severe infections . The med99mTc-HMPAO- labeled granulocytes, planar imaging at 1 h (early) and 4 h (delayed) after granulocyte infusion allowed the identification of responders and non-responders based on granulocyte uptake, as assessed by the lesion-to-normal lung ratio. By contrast, granulocyte scintiscans of two patients who were non-responders did not show any granulocyte uptake into the infiltrative lung lesions [It has been shown that accumulation of the transfused granulocytes at sites of infection can help predict the clinical response. In four patients given  lesions .The clinical results of the transfusion of granulocytes collected from community donors vs. family donors may be similar . The useCollectively, the available evidence points to the efficacy of GTX as an adjunct treatment modality for severely neutropenic patients who are likely to experience hematopoietic recovery.The Cochrane Library provides high-quality information based on publication types that are crucial in evidence-based medicine . The Coc10 granulocytes were excluded, the relative risk ratio was significantly in favor of the intervention. The authors conclude that the review identified a reduction in mortality due to infection in children and a transfusion with >1 × 1010 granulocytes. However, the studies were published many years ago and supportive care measures have significantly improved over time. This Cochrane review, first published in 2009, has been recently updated [https://clinicaltrials.gov/ct2/show/NCT01204788), leaving a total of 11 trials eligible involving 653 participants. Ten studies included only adults, and two studies included children and adults. Overall, the quality of the evidence was judged to be very low or low across different outcomes according to GRADE methodology. All-cause mortality was reported for nine studies (609 participants) and mortality due to infection was reported for seven studies (398 participants). There was no difference in all-cause mortality measured over 30 days between patients receiving prophylactic granulocyte transfusions and those that did not. Similarly, mortality due to infection over 30 days was not different in patients receiving granulocyte transfusions and in those that did not. In the low-dose granulocyte group (<1.0 × 1010 granulocytes/day), the number of patients with infection was similar in the two patient groups. However, the number of patients with infection was lower among recipients of intermediate doses of granulocytes (1.0–4.0 × 1010/day). Also, the number of patients with bacteremia and fungemia was lower among recipients of prophylactic granulocyte transfusions. This systematic review concluded that there is low-grade evidence that prophylactic granulocyte transfusions decrease the risk of bacteremia or fungemia. Similarly, there is low-grade evidence that the effect of prophylactic granulocyte transfusions is dose-dependent, with doses of at least 1.0 × 1010/day being more effective at decreasing the risk of infection. Collectively, there is insufficient evidence to determine any difference in mortality rates due to infection, all-cause mortality, or serious adverse events.The Cochrane Collaboration has published two reviews that aim to appraise the literature for randomized controlled trials on granulocyte transfusions for preventing and treating infections in patients with neutropenia or neutrophil dysfunction , 62. In  updated . Twelve 10 granulocytes.A third Cochrane review included eight randomized clinical trials, published between 1975 and 1984 . Eight sThe efficacy of granulocyte transfusions may be lower in patients with HLA alloimmunization. A retrospective study of alloimmunization to HLA and neutrophil antigens was performed in 18 patients with chronic granulomatous disease (CGD), who had also received repeated granulocyte transfusions. Sera were tested using lymphocytotoxicity, granulocyte agglutination, granulocyte immunofluorescence, monoclonal antibody immobilization of granulocyte antigen, and immunoprecipitation assays. This study showed that sera from 14 of the 18 transfused patients contained WBC antibodies. Seven serum samples reacted in the lymphocytotoxicity, granulocyte immunofluorescence, and granulocyte agglutination assays; seven reacted in the lymphocytotoxicity and granulocyte immunofluorescence assays, but not the granulocyte agglutination assay, and four did not react. Overall, antibodies to neutrophil antigens other than HLA molecules could be detected in sera from eight patients. When the monoclonal antibody immobilization of granulocyte antigen assay was employed, three sera samples reacted with Fcγ receptor III (CD16), three with the 58- to 64-kDa protein carrying the neutrophil antigen NB1, one with CD11a, and one with CD18. In addition, antibodies from three patients were shown to immunoprecipitate a 60-kDa neutrophil protein. Transfusion reactions, including pulmonary toxicity, were documented in 11 of the 14 patients with WBC antibodies, but in none of the 4 patients without antibodies. The patients with WBC antibodies were given a higher number of granulocyte concentrates. This study shows that recipients of granulocyte transfusions often develop alloimmunization and suggests that screening studies for WBC antibodies are indicated periodically during transfusions, after any adverse reactions, or before subsequent transfusion cycles. If WBC antibodies are present, no further granulocyte transfusions should be given unless the granulocytes are collected from HLA- and/or neutrophil antigen-compatible donors [Dihydrorhodamine-123 (DHR) is a marker for cellular NADPH oxidase activity and has been used to monitor the survival of transfused oxidase-positive granulocytes . This teSeveral studies reported high rates of transfusion reactions after the administration of granulocyte concentrates. Severe pulmonary reactions might be attributed to sequestration of the transfused cells into the pulmonary vascular bed. Most transfusion reactions have been documented in patients who were alloimmunized to leukocyte antigens. Anti-leukocyte antibodies could affect post-transfusion increments of neutrophil counts, alter the kinetics of circulating neutrophils and limit the anti-microbial effects of granulocyte transfusions. Importantly, no significant complications from the transfusions were reported in three RCTs that provided compatible leukocytes –68.G-CSF effects in healthy volunteers, although normally transient and self-limiting, are currently believed to be more complex and heterogeneous than previously appreciated. In addition to its established role in activating neutrophil kinetics and functional status, G-CSF administration can affect monocyte/DC and lymphocyte numbers and/or function, as well as the hemostatic system –71. In vThe available clinical data do not provide unequivocal evidence that G-CSF can transform normal hematopoietic stem cells in the absence of predisposing factors. In patients with acute myeloid leukemia (AML) with altered proportions of G-CSF receptor isoforms, G-CSF may promote the survival of leukemic cells. In severe congenital neutropenia and severe aplastic anemia, G-CSF receptor mutations or alterations in the proportions of specific isoforms in some patients appear to render them susceptible to leukemic transformation in the presence of sustained pharmacologic levels of G-CSF , 75. Nev+ cells from 35 healthy donors after G-CSF administration for 5 days and by spectral karyotyping analysis (SKY) in four individuals to assess chromosomal integrity. The authors also examined 38 granulocyte donors who received up to 42 doses of G-CSF and dexamethasone. No abnormalities in chromosomes 7 and 8 were found in G-CSF-mobilized CD34+ cells and no aneuploidy was detected in G-CSF/dexamethasone-treated donors.A review of data from >50,000 healthy donors given G-CSF was recently published and documented no evidence for an increased incidence of hematological malignancies . Some stRAR-B) gene and Ras association domain family 1A (RASSF1A) gene. Although DNMT activity increased significantly on the day of donation and 1 day after, baseline values were reached by day +7. In addition, differences in the gene methylation of RAR-B and RASSF1A were not detected between both groups, suggesting no long-lasting increase of DNMT activity or enhanced DNA methylation after G-CSF treatment.The influence of G-CSF on DNA methyltransferase (DNMT) activity and on changes in DNA methylation of candidate genes has been analyzed in peripheral blood cells of 20 healthy unrelated stem cell donors within an observation period of 1 year . The autIn vitro immunoglobulin production was increased up to 6 months after mobilization. Although some immunologic parameters may be altered in a more persistent manner than initially believed, most alterations remain transient with restoration of normal values by 1 year. We have shown that G-CSF can perturb mitochondrial function and promote T-cell activation-induced apoptosis through the upregulation of Bax [The immunological alterations induced by G-CSF mobilization were prospectively analyzed in 24 healthy donors . Interesn of Bax . These aA recent report identified 8 longitudinal studies on the incidence of AML among healthy donors mobilized with G-CSF . In aggrFinally, a recent survey on 83 donors who contributed 1120 granulocyte concentrates suggests that G-CSF/dexamethasone stimulation may be safe , 80. Thi®, Filgrastim, Amgen) and does not necessarily apply to other mobilizing agents.Based on of an assessment of a continuing lack of evidence for an increased risk of malignancy in donors receiving G-CSF, the WMDA issued a statement in 2012, which endorses that, ‘Studies following large numbers of unrelated donors have shown that the risk of developing cancer within several years after the use of G-CSF is not increased compared with donors not receiving G-CSF’ . It is r2O2 production index. This study suggests that IL-8-induced neutrophils could be used for transfusion purposes [IL-8 is a pro-inflammatory chemokine that can mobilize hematopoietic stem cells in mice and monkeys. In non-human primates, IL-8 with a dose range of 30–50 μg/kg of body weight induces a 8.7-fold increase of blood neutrophils with peak counts being achieved 45 min after injection. IL-8-mobilized granulocytes were functionally normal, in spite of decreased chemotaxis and adherence abilities, as well as Hpurposes .2 M-CSF from day 0 to 28 after determination of progressive fungal disease, compared with 5 % in 58 similar historical controls [Cytokines such as GM-CSF, M-CSF and IFN-γ have been used to treat specific infections . GM-CSF controls . The surcontrols . In thisIFN-γ1b has been administered concomitantly with granulocyte transfusions to enhance the host defense against fungal pathogens . In thisIt has been shown that the elevation of intracellular phosphatidylinositol -triphosphate signaling with PTEN inhibitor SF1670 can enhance the efficacy of granulocyte transfusions . In miceData regarding efficacy and complications of GTX are still limited in children. A properly designed, randomized controlled trial of GTX in children with clinically relevant endpoints would help resolve the current controversy surrounding the use of GTX in this patient population. The heterogeneity of study populations, types of infection, antimicrobial therapy and dosage of transfused granulocytes, coupled with lack of randomization, power and outcome parameters make it difficult to propose accurate recommendations. In addition, their efficacy may be enhanced by cytokine therapy, including IFN-γ, GM-CSF and G-CSF. Overall, granulocyte transfusions remain an important therapeutic modality in patients with difficult-to-treat opportunistic infections, especially as a bridge towards spontaneous recovery of neutrophil counts in patients who achieve remission of their underlying disease .