It is also possible to obtain the inverted coefficients matrix using a bit number of simple arithmetic operations. In this regard, peers sustain very low computational complexities. As a result, the MATIN permits random network coding to be more efficient in P2P video streaming systems. The results obtained from simulation using OMNET++ show that it substantially outperforms the RNC which uses the Gauss-Jordan elimination method by providing better video quality on peers in terms of the four important performance metrics including video distortion, dependency distortion, End-to-End delay and Initial Startup delay.In recent years, Random Network Coding (RNC) has emerged as a promising solution for efficient Peer-to-Peer (P2P) video multicasting over the Internet. This probably refers to this fact that RNC noticeably increases the error resiliency and throughput of the network. However, high transmission overhead arising from sending large coefficients vector as header has been the most important challenge of the RNC. Moreover, due to employing the Gauss-Jordan elimination method, considerable computational complexity can be imposed on peers in decoding the encoded blocks and checking linear dependency among the coefficients vectors. In order to address these challenges, this study introduces MATIN which is a random network coding based framework for efficient P2P video streaming. The MATIN includes a novel coefficients matrix generation method so that there is no linear dependency in the generated coefficients matrix. Using the proposed framework, each peer encapsulates one instead of  Nowadays, live video multicasting has been of great interest among the users. More than 1.4 billion Internet users, near three billion gadgets, annual global IP traffic about 667 Exabyte in 2013 RNC has established this fact that it can be a suitable technique in multicasting video streams so that the probability of playback skip can considerably be decreased The rest of this paper is organized as follows. Next sections explain random network coding, the problem statement, the MATIN framework and simulation results, respectively. Finally, the paper is concluded in the last section.T performs one less transmission using XoR-based network coding 20] which results in higher network throughput. Moreover, peers 3 and 4 have their requested packets b and a in lower end-to-end delay, respectively. Although this method has shown that it can increase video quality in P2P systems 21,22], it is topology dependent and needs intelligent algorithms in encoding process for increasing the decoding probability among receivers as much as possible In recent years, network coding promises an efficient solution for high quality video multicasting, especially in P2P systems Ho et al.n k-byte blocks i = ,i = 1,2,...,nB. All blocks iB related to a segment can be arranged in a matrix, named n×kB. Then, the video source selects 2n random values over finite field GF(2m)  29,30]  and arranges them in the coefficients matrix n×nC. i = ,i = 1,2,...,nC to the encoded block i = ,i = 1,2,...,nX and sends them to the next hop as an encapsulated packet. A receiver can decode the encode segment if it can receive n linearly independent encoded blocks iX. All arithmetic operations are performed in GF(2m). For example, suppose that 1g∈GF(2m), then, 1g+1g = 0 and 1g×0 = 0. In T selects four random coefficients c1 to c4 and generates two encoded packets before sending them to peers 3 and 4.RNC, which is originally proposed by  = bi1,bi,…,bik,i m, because the number of innovative packets can be affected by the amount of this parameter. All previous studies used GF(28) which is sufficient for data dissemination in a network with hundreds of links.The decoding probability is an important parameter in selecting suitable values for All in all, RNC improves network throughput and video frame localization. It also decreases playback skips, video distortion and delivery delay in the network. We refer interested researchers to the mentioned references for more information about the benefits of using network coding, especially RNC, in P2P streaming. Actually, the main goal of this study is not to prove the efficiency of RNC in P2P live video streaming. We aim to address existing challenges in RNC which are mentioned in previous studies and are categorized in the next section. Consequently, RNC can be more efficient in a P2P video streaming system using the MATIN framework.What can be inferred from previous studies is that RNC is an efficient method for video multicasting over P2P networks and can be one of the most attractive research areas in the computer communication field in near future. However, according to these studies, RNC introduces new challenges to the system which are categorized as follows:n-byte coefficients vector iC to each k-byte encoded block as its header before sending it to the next hop in the network. This results in high transmission overhead, especially when the values of k and n are small and large, respectively iC as header to other peers. Obviously, the more number of video segments is transferred in each transmission, the better video quality can be provided on receivers. This can be achieved if the header size of an encoded blocks decreases using an efficient coefficients matrix generation method, because the peer can transfer next video blocks instead of coefficients vectors related to the previous video blocks.As it was mentioned before, by using RNC, each peer attaches n×nC and checking any linear dependency among its vectors. If there is any linear dependency between two vectors, the decoder fails to decode the whole segment S, suppose that thi coefficients vector , which is related to the thi block (Xi), has linear dependency with another one  in this segment. The peer needs to wait for the same block number i but with different coefficients vector from another or the same sender. As it was mentioned earlier, this not only decreases the video quality on peer, but also wastes its computation processing power, because the peer needs to perform additional processing for decoding the new received encoded block. In summary, the Gauss-Jordan elimination method consumes high CPU  time and imposes very high computational complexity on the system as they are mentioned in As soon as an encoded block receives, in the decoding step, peers use the Gauss-Jordan elimination method The Gauss-Jordan elimination method needs to perform many arithmetic operations in decoding step. Therefore, the imposed computational complexity on the system considerably increases.m. However, the first solution considerably degrades the performance of the network and video quality on peers m (e.g. m = 16), the computational complexity in encoding/decoding process and the required bandwidth for segment transmission sharply increases n×nC irrespective of its assigned values.As two solutions for overcoming these challenges, some previous studies suggested to decrease the packet batch size or increase the size of the finite field Therefore, the big question of this study puts forward as follows: Is there any efficient solution which not only addresses these problems, but also improves the performance of the RNC in a P2P live video streaming system? Next section will propose the MATIN to answer this question and efficiently address the mentioned challenges in this section. Moreover, there is no need to use parallel decoding algorithms which both imposes high energy cost on the system and needs considerable CPU resources in all peers This section introduces MATIN, which includes a novel coefficients matrix generation method, in figure of a tailored framework so that it impressively addresses existing challenges in RNC. In summary, it:sharply decreases the imposed transmission overhead by RNC,completely removes the necessity of checking any linear dependency among the coefficients vectors. This increases the number of innovative packets in the network,considerably reduces the imposed decoding computational complexity,As illustrated in RNC-Encoder: it receives original video segments from the buffer and applies RNC on them. Then, it encapsulates each coefficient entry ia and encoded block iX into a packet iP before sending them to the upload scheduler. Section A.2 explains this process in more details.RNC-Decoder: this component progressively decodes the received encoded blocks iX  in order to regenerate the original video blocks iB. Section A.4 particularly discusses on this process.Buffer: all decoded video segments are stored in the buffer. These video segments generate the sequence of video frames for playing in the media player.Peer Manager: joining, leaving and other related managerial processes to the peer management are performed by this component.Upload/Download Schedulers: these components are responsible for transferring/receiving the encoded blocks to/from other neighbors in the system.Coefficients Matrix Generator: this component is the most important component of the MATIN which includes the contribution of this study. Actually, it efficiently generates the required coefficients matrix and its inversion as it is discussed in section A.2.2.In this framework, solid and dashed lines refer to the data and the control messages, respectively.n×kB, it performs the following steps respectively:As soon as the RNC-Encoder intends to encode segment n k-byte blocks.It first divides the segment into n) to the coefficients matrix generator component.Then, this component sends a request message (REQ_MSG) containing the number of blocks .After receiving the coefficients matrix ia1×nεA to the encoded blocks iX in figure of packet iP before sending it to the upload scheduler for transferring to receivers.Finally, it attaches coefficient entry This process can be performed for many times until the peer either leaves the network or the video streaming finishes. Algorithms 1 and 2 show the encoding process when the most recently used method, the Gauss-Jordan elimination, and the MATIN framework are employed in the system, respectively.Algorithm 1. Generating encoded blocks in the current RNC RNC in usedi, i = 1,2,...,nXTo generate the encoded blocks 2n random values over field GF(2m)1.1. Select n×nC based on 2n values1.2. Generate iX using the 1.3. Generate all encoded blocks Xi, i = 1,2,...,nFor all i  = C εn×n,i = 1,2,...,nC2.1. For all iC and iX into Packet iP2.1.1. Encapsulate iP to the next hop // next peer(s) in the network2.1.2. Send Algorithm 2. Generating encoded blocks using MATIN frameworki, i = 1,2,...,nXTo generate the encoded blocks n random values over field GF(2m)-{zero} and arrange them in 1×nA1.1. Select n×nC from the given matrix 1×nA1.2. Generate iX using the 1.3. Generate all encoded blocks i, i = 1,2,...,nX2. For all iaε1×n, i = 1,2,...,nA2.1. For all ia and iX into Packet iP2.1.1. Encapsulate iP to the next hop // next peer(s) in the network.2.1.2. Send n random values over field GF(2m)-{zero} and arranges them in matrix 1×n = A. Recall that, current RNC in use needs 2n random values for generating the matrix n×nC. Based upon 1An×, the diagonal matrix n×nA can be generated as follows. Definitely, the determinant value of the matrix n×nA is not equal to zero (det(n×nA)≠0); because all entries ia≠0. Therefore, the matrix n×nA is undoubtedly invertible. MATIN does not consider n×nA as the final coefficients matrix for sending to the RNC-Encoder, because having many zero values in the coefficients matrix can decrease the decoding probability. In order to resolve this problem, it is possible to add some multiplication of one row/column of n×nA to another row/column. Certainly, the value of the det(n×nA) will not change By receiving a request from the RNC-Encoder, this component first selects n×nC, this component follows the following rules:In this regard, for generating the coefficients matrix Rule i: It replaces thj column of n×nA by column j plus column j-1, for j = 2,3,…,n, respectively.Rule ii: Then, it replaces thj column of n×nA by multiplying 3 by column j plus column n, for j = 1,2,..,n-1, respectively.n×nC can be sent to RNC-Encoder as follows ).Now, the coefficients matrix n×n)C = det(n×nA)×3n−1≠0. Thus, matrix n×nC, similar to the n×nA, will be undoubtedly invertible and no linear dependency exists among its vectors. It is necessary to be pointed out that a received packet in a peer can be innovative if it does not have any linear dependency with the current received packets in its buffer 1×nA will alter n×nC uniquely, because the thn column of n×nC is the transposed of 1×nA. It is necessary to emphasize that each peer directly obtains n×nC from the given 1An×. Consequently, there is no additional computational complexity in coefficients matrix generation process.After applying the second rule, det. Therefore, the data transmission overhead in Algorithms 1 and 2 can be written as Equations 3 and 4, respectively (n and k are in byte).Transmission overhead in the MATIN is very low, because the encoder attaches each entry of the matrix Mirshokraie et. al. n) increases. However, as depicted in Equation 4, transmission overhead of the MATIN is completely independent of the number of blocks. It means that the MATIN lets us ignore both the imposed computational complexity and the transmission overhead due to employing and sending the coefficients matrix n×nC, respectively. As a result, better video quality can be provided on peers.According to -1n×nC using small numbers of arithmetic operations when the first entry 1a is received. Suppose −1n×nC is written as follows and node P1 is receiving segment n×kX. Obviously, each encoded block iX is anchored by a coefficient entry ia and the value of i is determined in the header. The decoding process in P1 can be performed in the following order:Similar to the Gauss-Jordan elimination method, the decoding process can be performed progressively. The MATIN progressively starts to generate the matrix irbεn×kB  to zero before starting the decoding process.i. It first sets all values ia is received, P1 generates two related entries ijzε−1n×n C. For example, suppose that P1 receives packet 1P = . In this case, P1 can easily generate 11z and n1z from the given 1a as depicted in the first row of ii. As soon as a coefficient entry ijz to the Function 1 for progressively obtaining entries irbεn×k.B This function will not be called for zero entries and ijz = 0 in matrix −1n×nC, because the value of irb will not be changed in this case. This considerably decreases the imposed computational complexity in decoding.iii. Finally, P1 sends the calculated irbεn×kB are calculated. This process can be performed for other received encoded segments. In contrary to n×nC, having many zero values in the matrix -1n×nC leads to very low computational complexity in decoding. This is another reason for this fact that the MATIN sharply decreases the computational complexity of RNC.P1 will repeat steps ii and iii until all entries Function 1. Progressively Decoding Process in MATINijz, i, j)Function MATIN_Decoder (r = 1 to k DoFor irb = irb+(ijz×jrx)EndFori = j = 1Then n1 = z11// because zIf r = 1 to k DoFor nrb = nrb+(ijz×jrx)EndForEndIfEndiX in iteration i. This Function can be called two times in each iteration . 11b can be obtained at the end of the second iteration, because 1j = 0 z will not be sent to Function 1. This is the same for 12b. In this regard, 21b and 22b can be obtained at the end of the iteration 3 and so on. Suppose that iX is not received or received with error. In this case, the MATIN, just like as the Gauss-Jordan elimination method, can use the received encoded blocks ″iX from another node instead of i,X because both of them are generated based upon the same matrix n×kB but with different coefficients matrices ″n×nC and n×nC, respectively.In order to illuminate the operation of Function 1, suppose that node P1 receives encoded bock n×nC based upon the same given matrix 1×nA.Finally, it is necessary to be pointed out that each peer downloads a small source code in size for just one time when it joins the network. This source code can be used by peers in order to generate the same coefficients matrix As it was mentioned before, the MATIN imposes very low computational complexity on the system. n k-byte blocks for encoding over GF(2m). Based upon sections A.2 to A.4, it is possible to calculate the complexity of MATIN as follows:In order to provide a more comprehensive analysis over MATIN, suppose that there are • Throughput: There is no linear dependency in the generated encoded packets using MATIN. Therefore, all received packets can be as an innovative packet. This removes the necessity of re-transmission due to linear dependency between two received encoded packets. Moreover, because of low transmission overhead, bandwidth utilization considerably increases meaning that MATIN provides very high performance  in terms of the throughput.G. Moreover, addition/multiplication operation imposes constant complexity of A. Generating the matrix 1×nA and applying rules i and ii impose a complexity of O(nG)+2A)OO. Then, the encoder multiplies n×nC by n×kB which leads to complexity of 2k)O. This just leads to decoding complexity of O(nk) in the system.• Decoding Complexity: According to section 4.4, MATIN imposes very low computational complexity on the system in decoding. In fact, just 2×n coefficient entries to each encoded block. Therefore, the imposed packet overhead by it is equal to m bits.Packet Overhead in bit: According to section 4.3, RNC-Encoder attaches one instead of Packet Feedback: Similar to RNC, MATIN needs no feedback.Qureshi et. al.(2012)q, M and B are defined the same as m, n and k in our study, respectively.For better understanding and evaluating the performance of the MATIN in P2P live video streaming, this study carried out a simulation using the INET and the OVERSIM frameworks in OMNET++ n k-byte blocks . The simulation ran for five times based upon the mentioned conditions and parameters in 8).The video source disseminates Star War IV, a single-layer MPEG-4 video trace file available from .In order to measure the performance provided by the MATIN and the RNC-GJE, this study considers four important performance metrics as follows:Video Distortion: The capacity of frames not playback divided by the total capacities of all video frames of the stream.• End-to-End Delay (EED): The time elapsed between transmitting a video frame from the video source and playing it in a peer.• Initial Startup Delay (ISD): The time elapsed between starting to buffer the video frames after joining the network and playing the first buffered video frame. In other words, ISD shows how much time it took in a peer to finish the initial buffer time after joining the network.• Dependency Distortion: According to the existing dependencies among video frames, it is possible that a video frame arrives, however, the decoder cannot decode it due to this fact that it is dependent on a frame which has not received yet. For example, peer P receives frame B3, while frames P1 and P2 are not received. Thus, it is impossible to successfully decode frame B3 (by video decoder not RNC-Decoder) and playback it. This metric shows the capacities of these frames divided by the total capacity of all video frames in percentage.• GP and MP indicate the number of encoded blocks in a packet using the RNC-GJE and MATIN, respectively (MTU shows the maximum transfer units in byte). In this regard, according to Equations 5 and 6, GG and MG depict the number of generated packets for transmitting a segment to a neighbor using the RNC-GJE and MATIN, respectively.This section is divided into three subsections; each of them evaluates the obtained results related to one of the considered churning scenarios. In all scenarios, we use aggregation method so that each packet can contain more than one encoded block. By employing aggregation approach, suppose that Then, for This clearly indicates that RNC-GJE imposes higher traffic on the network which can result in higher video distortion, because using same block and segment size, the capacity of all generated packets by the MATIN is lesser than that of the RNC-GJE.n is large). In this case, the RNC-GJE method needs to attach n bytes as header to each encoded block before encapsulating it in the packet. This increases the transmission overhead which causes high video distortion. The MATIN efficiently overcomes this problem by attaching just one instead of n bytes to each encoded blocks. For instance, suppose that a segment is divided into n = 10 k = 128-byte blocks. The imposed transmission overhead using the RNC-GJE and MATIN will be 4.4 and 0.78 percent, respectively. The difference between the imposed transmission overheads by these methods can be sharply increased when n increases.In an overall view, considering 150 or 300 peers, what can be inferred from these figures is that the MATIN provides higher video quality than that of the RNC-GJE with slightly lower end-to-end and initial startup delays. According to Xi is anchored by a coefficients vector iC using RNC-GJE method. In this regard, the amounts of Transmitted Data (TD) and the imposed transmission overhead reduce when the amount of k increases. This is why the imposed video distortions on peers slightly decrease for larger values of k. Using the MATIN framework, on the other hand, the amount of video distortion does not sharply increase even if the amount of K increases. This is more visible for 128-byte block size. This means that the MATIN can provide better video quality than that of the RNC-GJE even if the network size and block size increases and decreases, respectively. In order to substantiate this claim, consider 128-byte block size. Using MATIN, the introduced amount of video distortion only increases 0.83 in case of having 300 peers in comparison with the RNC-GJE when there are 150 peers in the network. Moreover, MATIN improves the video distortion about 508 percent in comparison with the RNC-GJE when there are 150 peers in the network and block size is 128 bytes. For the same network size (150 peers), 196 and 150 percent improvement are achieved when block size is equal to 512 and 1024 bytes, respectively. This is approximately the same for 300 peers.Network size can influence the network performance. According to S, the large number of generated packets (GG) due to using blocks size 128 bytes leads to high video distortion, especially when the network size increases. The reason is that the amounts of traffic and end-to-end delay considerably increase. However, using block size 512 bytes results in better video quality than that of block size 1024 bytes when there are 300 peers in the network. The main reason is that the amount of PG increases while the amount of TD decreases. This leads to higher frame diversity in the network, because each packet includes more number of video blocks iX. Although the amount of PG increases more when the block size is 128 bytes, the imposed transmission overhead and the large amount of TD outperform the advantages of having large amount of PG so that the video distortion increases. The obtained results in Considering the RNC-GJE method and segment size S but the MATIN is used in the network. The amounts of TD and transmission overhead on the system remain low and almost constant even if the amount of k increases. This is why approximately the same amounts of video distortion are experienced by 150 peers. In case of large network size (e.g. 300 peers), the amount of the imposed transmission overhead plays more important role than that of other parameters . In fact, low frame diversity due to small number of PM causes higher video distortion using block size 1024 bytes in comparison with the block size 512 bytes.Now, consider the same segment size Dependency distortion is another important metric. This metric evaluates the performance of a method/framework in video streaming in a detailed view. As can be seen in T seconds so that the first peer joins at the beginning of the simulation. The amount of T can be selected randomly between 1 and 3 seconds using normal distribution method. In addition, the averaged lifetime duration of peers in the network is 400 seconds. As can be seen in This behavior is somehow the same for the RNC-GJE in block sizes 128 and 1024 bytes. However, the MATIN provides better video quality than that of the RNC-GJE by introducing lesser amount of video distortion on both 150 and 300 peers. Moreover, according to T is selected randomly between 0.5 and 1 second using normal distribution method and the averaged lifetime duration of peers is 300 seconds. In this regard, peers join rapidly but stay for a shorter time in the network. Although in this scenario the behavior of the network is more dynamic, similar to the scenarios 1 and 2, the MATIN provides higher video quality with lower end-to-end and initial startup delays. This means that MATIN makes the P2P network more robust in high churning scenarios.Finally, it is necessary to be pointed out that the bigger range of the confidence interval in scenarios 2 and 3 is due to churning event. In other words, different runs of the simulation in different seeds cause video distortions with larger differences in their amounts in comparison with the first scenario. Following Although previous studies such as iC for decoding the related encoded block iX. This is why the RNC-GJE imposes considerable transmission overhead on the network which leads to higher video distortion. Moreover, according to In previous sections, we showed that employing the Gauss-Jordan elimination method in decoders causes some problems such as high transmission overhead and computational complexity. The Gauss-Jordan elimination method needs to have the whole coefficients of a coefficients vector n instead of 2n entries in Galois Field so that there is no linear dependency among its vectors. Then, it sends one instead of n coefficient entries as the header of each encoded blocks. Therefore, the transmission overhead sharply decreases.Recent studies have shown that better perceived video quality can be provided on peers using RNC. However, this method imposes high transmission overhead and computational complexity on the system as they are mentioned in these studies. The imposed computational complexity is due to large number of arithmetic operations in decoding the encoded blocks and checking linear dependency among the coefficients vectors using the most recently used method, the Gauss-Jordan elimination. In addition, the decoder needs to have all values of a coefficients vector to be able to decode the related encoded block which imposes high transmission overhead. This paper introduced MATIN framework in order to efficiently address these challenges. The MATIN generates the required coefficients matrix using Moreover, it can progressively produce the inverted coefficients matrix using very few numbers of arithmetic operations. As a result, very low computational complexity is imposed on the system. In order to evaluate the performance of the MATIN in P2P live video streaming, we simulated it using a precise simulator in OMNET++ and under different network conditions. Four performance metrics including total video distortion, video distortion due to frame dependency, end-to-end delay (EED) and initial startup delay (ISD) are considered. This study also considered three different scenarios for churning. In the first scenario no churn happened, whereas the second and the third scenarios were based upon two different churning rates and peers' lifetimes.The results showed that the MATIN considerably outperforms the RNC-GJE, which employs the Gauss-Jordan elimination method in decoding, in terms of the four considered performance metrics in all scenarios. In fact, it provides higher video quality by introducing lesser video distortion and delivered video packets in lower end-to-end delay. Finally, peers finished their initial buffer time earlier. In this regard, the MATIN can be a possible answer to the propounded big question in this study and permits RNC to be more efficient in data multicasting, especially video streaming. In future studies, we aim to implement the MATIN framework over the PlanetLab for evaluating its performance in a real-world P2P network.


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Thymic epithelial cells (TECs) are necessary for normal T cell development. Currently, one transcription factor, Foxn1 is known to be necessary for the progression of fetal TEC differentiation. However, some aspects of fetal TEC differentiation occur in Foxn1 mutants, suggesting the existence of additional transcriptional regulators of TEC differentiation. The goal of this study was to identify some of the additional candidate transcription factors that may be involved in the specification and/or differentiation of TECs during fetal development.Foxg1, Isl1, Gata3, Nkx2-5, Nkx2-6 and Sox2 for further studies. Whole mount in situ hybridizations confirmed the expression of these transcription factors within subdomains of the third pharyngeal pouch from E9.5–E10.5. By E11.5 days Foxg1 and Isl1 transcripts were the only mRNAs from this group of genes detected exclusively within the thymus domain of the third pouch. Based on this initial in situ hybridization analysis, we focused on defining the expression of Foxg1 and Isl1 during multiple stages of thymus development and TEC differentiation. We found that Foxg1 and Isl1 are specifically expressed in differentiating TECs during fetal and postnatal stages of thymus development. In addition, we found differential expression of Islet1 and Foxn1 within the fetal and postnatal TEC population.We identified candidate fetal TEC transcriptional regulators via data and text mining. From our data mining we selected the transcription factors Foxg1 and Isl1 may play a role in the regulation of TEC differentiation during fetal and postnatal stages. Our results also demonstrate heterogeneity of TECs marked by the differential expression of transcription factors, potentially providing new insights into the regulation of TEC differentiation.Our studies have identified two developmental transcription factors that are excellent candidate regulators of thymic epithelial cell specification and differentiation during fetal development. Our results suggest that  Thymic epithelial cells (TECs) are a critical component of the thymic microenvironment. TECs are derived from the endoderm of the third pharyngeal pouch. Despite their essential role in thymus function, our current understanding of fetal TEC specification and differentiation is very limited. For example, we do not know which transcriptional regulators are necessary for the earliest specification of the thymus organ domain within the third pharyngeal pouch. In addition we have very limited knowledge about the transcription factors that regulate the differentiation and function of TECs during fetal and postnatal thymus development. Identifying the factors that regulate these key steps in the development of thymic epithelial cells is a key part of understanding the genetic pathways that regulate thymus organogenesis and function.Gcm2 at E9.5 in the parathyroid domain and Foxn1 at E11.25 in the thymus domain of the 3rd pharyngeal pouch marks the patterning of the pouch into the primordium of the two organs Foxn1 which is first detected at E11.25 Foxn1 is first expressed suggests that additional transcription factors are acting within the pouch at times prior to Foxn1 expression. These factors include the transcriptional regulators that activate Foxn1 within the thymus primoridium.Our current knowledge regarding the earliest events in the specification of the parathyroid and thymus suggests that specification occurs early in third pouch development. Localized expression of Hoxa3, Pbx1, Tbx1, Pax1, Pax9, Six1 and Eya1 as necessary for 3rd pouch development. All of these transcription factors, except for Tbx1 are expressed throughout the 3rd pouch at E10.5 prior to detectable Foxn1 expression Tbx1 is initially expressed throughout the 3rd pouch and becomes restricted to the presumptive parathyroid domain at E10.5 in the pouch endoderm Hoxa3, Eya1, Six1, Pax9, Tbx1 and Pbx1 the homozygous mutants either fail to form the 3rd pouch or exhibit very severe early defects in the formation of both the thymus and parathyroid primordia Previous studies have identified the transcription factors in situ hybridization analysis based on previous expression data, our genetic studies have shown that it is necessary for the normal development of the third pharyngeal pouch Gata3 mutants at E12.5 days indicating an early role for this gene in the development of the pouch A major goal of this screen was to identify candidate developmental transcription factors that play an important role in 3rd pharyngeal pouch endoderm development and/or the differentiation of fetal TECs into functional components of the thymus microenvironment. To enable the characterization of the genes identified in our screen we focused our screen on genes for which well-characterized knockout mice are available. In the case of Gata3, which we chose for detailed in situ hybridization data are limited in terms of resolution, they can be used to suggest candidate genes for in depth characterization. We took advantage of this information to focus our in situ hybridization analysis to a short list of candidate transcription factors that were likely to be expressed in localized domains of the 3rd pouch and/or thymic epithelium. For third pharyngeal pouch expression we visually screened data in the MGI/GXD and Emage in situ hybridization databases and examined published reports describing Cre recombinase expression patterns in situ hybridization of para-sagittal sections in the Genepaint database indicated that most of the genes we had identified as being expressed in the third pouch were also expressed within the thymus at E14.5. This suggested that these transcription factors were excellent candidates for genes involved in thymus development and differentiation. After additional literature mining, we selected Nkx2-5, Nkx2-6, Foxg1, Islet1, Gata3 and Sox2 for further analysis in this study. The screen of in situ database information allowed us to focus on performing a detailed characterization of a small group of genes rather than performing a less detailed large-scale screen.A large amount of published and unpublished mouse developmental gene expression data is available in online databases Foxn1 expression within the thymus domain of the third pouch at E11.25 days are not known. One purpose of our study was to search for transcription factors with regionalized expression patterns in the third pharyngeal pouch endoderm between E9.5–E11.25. We analyzed the expression of our candidate genes in E9.5 and E10.5 somite stage matched wild type mouse embryos by in situ hybridization and 3D reconstruction, focusing on their expression in the pharyngeal pouches. By carefully staging embryos by somite counting we found that embryos at the same somite stage had comparable pouch morphologies as determined by 3D reconstruction of paraffin sections. By comparing the expression patterns of the genes we examined to the Gcm2 expression pattern at E10.5 as a landmark for organ specific domains, we found a surprisingly diverse and complex combination of regionalized expression patterns in the third pouch endoderm.Although pouch endoderm as early as E9.5 is capable of developing into a fully functional thymus when transplanted under the kidney capsule Nkx2-5 and Nkx2-6 expression have detected expression of Nkx2-5 in the pharyngeal endoderm and Nkx2-6 in the pharyngeal pouch endoderm. However, these studies have not described the expression pattern of either gene in the 3rd pouch in detail Nkx2-5 is expressed in a localized domain of the 3rd pouch Nkx2-5 at some point in its history Nkx2-5/Nkx2-6 double mutants Previous studies of in situ hybridization analysis revealed that Nkx2-5 and Nkx2-6 are expressed in localized regions of the third pouch. At E9 days (20–23 somites) Nkx2-5 is expressed on the ventral side of the forming third pharyngeal pouch endoderm  indicated that it is expressed in the thymus domain of the E10–E10.5 (30–35 somites) 3rd pouch Gata3 are associated with a syndrome of hypoparathyroidism, sensorineural deafness and renal disease Gata3 in development of 3rd pouch derivatives.Previous studies have detected Isl1 during pharyngeal pouch development. At the 20–23 somite stage, Isl1 is expressed in the cells on the ventral side of the developing second and third pharyngeal pouches but on the dorsal portion of the first pouch , Foxg1 expression is detected at the ventral tip of the 3rd pouch endoderm .Our analysis revealed a dynamic pattern of endoderm . By 30–3al pouch , 2F. Thihe pouch . We alsoNkx2-5, Nkx2-6, Isl1, Gata3 and Foxg1 in the ventral portion of the third pharyngeal pouch suggested that these factors might be expressed in the thymic epithelial cells at later stages. To test this idea we examined the expression of these 5 transcription factors in E11.5 wild type embryos. In the E11.5 third pouch endoderm, the thymus domain can be identified by the expression of Foxn1 in the ventral posterior portion of the pouch, complimentary to the parathyroid domain which is marked by Gcm2 expression The expression of in situ hybridization analysis showed that Nkx2-5, Nkx2-6 and Gata3 are not expressed within the thymus domain of the third pouch at E11.5. In fact, at this stage Nkx2-5 and Nkx2-6 are no longer expressed in any of the pharyngeal pouches  protein, which has been shown to be expressed in medullary thymic epithelial cells and in a subset of the cortical epithelial cells at late fetal and postnatal stages In sections of E16.5 fetal thymus, ISL1 expression was detected in all FOXN1-expressing thymic epithelial cells . To confFoxg1 null embryo (data not shown) In contrast, we found that FOXG1 expression in E16.5 thymus completely co-localizes with FOXN1 in TECs . The antFoxn1 transcript and protein expression are at various levels in medullary thymic epithelial cells high; ISL1low; FOXG1low TECs and FOXN1high; ISL1high; FOXG1high TECs were widely present at these stages  embryos were dissected in DEPC-PBST  and somite number was determined. Embryos within a narrow range of somite stages were pooled in groups of 3–5 for processing. Therefore, we refer to individual embryos as being within a range of somite numbers since we cannot accurately count somites after the Nkx2-5, a cDNA plasmid clone was generated by RT-PCR using E11-day mouse total RNA (Clontech). Primers were Nkx2-5-3-F: CTACG GCGTG GGTCT CAATG C and Nkx2-5-3-R: GCGTT AGCGC ACTCA CTTTA ATGG. The transcription template was generated by PCR using the SP6 and T7 promoter primers and using our Nkx2-5 plasmid cDNA as template. The Nkx2-6 probe template was amplified from mouse genomic DNA using primers: T7-Nkx2-6-F: taata cgact cacta tagg ACTGGTACTGGACGGCAAGC and SP6-Nkx2-6-R: attta ggtga cacta taga GCACAGCATCTACGTGGCTA. Isl1 probe template was generated by PCR from an MGC cDNA (accession BC132263) using primers: Isl1F-T7: taata cgact cacta taggT CATCC GAGTG TGGTT TCAA and Isl1R-SP6: attta ggtga cacta tagaT GAATG TTCCT CATGC CTCA. Foxg1 probe template was generated by PCR from an MGC cDNA (accession BC046958) using primers: Foxg1F: AGTTACAACGGGACCACGTC and Foxg1R-T3: aatta accct cacta aagg CCCCT GATTT TGATG TGTGA. The Sox2 probe template was generated by PCR using mouse genomic DNA and primers: Sox2-F: GCCCA TGAAC GCCTT CATGG and T3-Sox2-R: aatta accct cacta aagg C ATGCT GATCA TGTCC CGGA. The Gata3 probe template was described previously Digoxygenin (DIG)-labeled antisense RNA probes were synthesized using standard procedures. All probe templates were generated by PCR reactions using either mouse genomic DNA or cDNA clones as templates. In all cases, probe templates were carefully designed to not include highly conserved sequences to eliminate the possibility of cross hybridization. In the following primer sequences the lower case letters indicate the phage promoters. For Comparisons of pouch morphology in 3D reconstructions from different embryos showed that pouch morphology is comparable between different embryos of the similar somite stage (data not shown). This similarity allowed us to make comparisons between reconstructions of different gene expression patterns. Digital images of serial sagittal paraffin sections from a single embryo were assembled into a three-dimensional (3D) image using the WinSurf 4.3 software. The gene expression positive areas and the third pharyngeal pouch endoderm were traced as separate objects.3Citrate pH 6, 0.05% Tween20) for 30 minutes. After cooling down, slides were washed once with 0.05% PBST (0.05% Triton X-100) and blocked in 10% serum in PBST at room temperature for at least 30 minutes. Primary antibodies were mixed in 1% serum/PBST and incubated at 4°C overnight. After 3 PBST washes, secondary antibodies diluted in PBST were added and incubated at room temperature for 30 minutes. Slides were then washed and mounted in FluoroGel (EMS). Images were acquired using a Zeiss LSM510 META confocal imaging system.Dissected embryos or postnatal thymus tissue were treated differently for ISL1 and FOXG1 antibody staining. For ISL1 staining, E16.5 embryos were fixed in 4% PFA for 4 hours or postnatal thymus for 1.5 hr on ice. Fixed embryos or tissue were then washed three times in PBS and dehydrated through methanol series and embedded into paraffin blocks. 8 µm sections were cut on a Leica RM2155 microtome and de-waxed and rehydrated into water. Antigen retrieval was done by boiling the slides in AR buffer . 10 µm frozen sections were then cut on a Leica CM3050 S cryostat. The sections were blocked and incubated with primary and secondary antibodies as described for ISL1 staining.Foxn1  The mouse anti-ISL monoclonal antibody was from the Developmental Studies Hybridoma Bank . This ISL1 monoclonal was developed by Dr. Thomas Jessell and has a long track record of use in mice


Current Cardiology Reviews. Their efforts have contributed greatly tothe high quality and continuous growth of the journal. Given below is the list of reviewers who reviewed articles for the Journalduring 2013.Bentham Science Publishers would like to thank and appreciate the co-operation from all reviewers for their constructivecomments and feedback on the manuscripts submitted to  S. Apostolakis (UK)K. Asrress (UK)A. Banerjee (UK)A. Baranchuk (Canada)H. Baumgartner (Germany)H. Baumgartner (Germany)A. Bobik R. Cemin G. J. Crystal (USA)P. M. Eckman (USA)M. Endoh (Japan)C. Foerch (Germany)S. Fujii (Japan)S. Fynn (UK)K. Guha (UK)K. Kallenbach (Germany)L .W. Klein (USA)L. H. Kuller (USA)J. L. Mehta (USA)P. Meier (UK)L. C. NazÁrio Scala (Brazil)P. Pagliaro M. A. Portman (USA)F. Recchia (USA)R. Sankaranarayanan (UK)R. J. Selvaraj (India)C. Shanahan (UK)E. Shantsila (UK)N. Turck (Switzerland)


Hazardous drinking among adolescents is a major public health concern. The purpose of this study was to examine the prevalence of binge drinking/alcohol consumption and its association with different types of friendship networks, gender and socioeconomic status among students in Belo Horizonte, Minas Gerais, Brazil.We conducted a cross-sectional study on a representative random sample of 891 adolescents  from public and private schools in 2009–2010. Information on friendship networks and binge drinking was collected using two validated self-administered questionnaires: the Integrated Questionnaire for the Measurement of Social Capital and the first 3 items in the Alcohol Use Disorders Identification Test (AUDIT C). We used the area-based Social Vulnerability Index (SVI), mother and father’s educational background, and the type of school to assess socioeconomic status. The chi-squared test was used to examine the associations between sample characteristics or the type of friends and binge drinking . Ordinal logistic regression was used to estimate the association between binge drinking and the independent variables.A total of 321 (36%) adolescents reported binge drinking (5 or more drinks in one occasion), and among them, 233 (26.2%) adolescents reported binge drinking less than monthly to monthly, and 88 (9.9%) weekly to daily. Binge drinking was associated with being male  and with living in a low vulnerability area  . Students who reported that their closest friends were from school (as opposed to friends from church) had an increased risk of binge drinking . In analyses stratified by gender, the association was significant only among the female students.The prevalence of binge drinking was high in this sample of Brazilian adolescents, and gender, low social vulnerability and friendship network were associated with binge drinking. Binge drinking among adolescents continues to be a significant public health concern due to its high prevalence in several countries, including Thailand (33.0.%), the United Kingdom 47.0%), Germany 26.9%), Sweden 30.8%), and Brazil 34.5%) -5. In Br% -5. In .9%, Swed.0%, Germ%, and BrBinge drinking is usually performed in groups; therefore, peers play a role in promoting binge drinking, perhaps due to peer selection or peer influence . Binge drinking has been associated with characteristics of the adolescents’ friendship networks -13. OverDespite the preponderance of research examining the influence of peer relationships on binge drinking, few studies have evaluated the link between drinking behavior and the specific types of social relationships represented by peers. In other words, not all friendship ties are equally influential in shaping adolescent behaviors, and the aim of our study was to distinguish between the different types of friendships in various contexts ,15,19-21Some of these studies ,11,13 exIn this paper, we examined the prevalence of binge drinking/alcohol consumption and its association with different types of friendship networks in different contexts  among male and female students in Belo Horizonte, Minas Gerais, Brazil.We designed a cross-sectional study to examine social and friendship networks and binge drinking among 936 adolescents aged 15 to 19 years attending public schools  and private schools  in the city of Belo Horizonte between August 2009 and February 2010. Belo Horizonte is the state capital of Minas Gerais, Brazil, and it has approximately two million inhabitants and is geographically divided into nine administrative districts (Brazilian Institute of Geography and Statistics/IBGE) . A samplThe minimum sample size needed to satisfy the requirements was estimated to be 600 individuals. To minimize the potential losses during data collection, the sample was increased by 20% (n = 720). A design effect of 1.3 was applied to increase the precision because a multistage sampling method rather than random sampling was adopted . Thus, tSelf-administered questionnaires were distributed in the classroom by a researcher and assistant who collected the response envelopes immediately after being the questionnaires were completed. They explained that the responses would remain anonymous and be treated confidentially. To guard against biases that might occur due to variability in reading proficiencies, the principal investigator (K.O.J.) read aloud each question. The students could refuse to participate and return the unfilled questionnaires inside the envelopes. Among those who submitted envelopes, 4.8% of the participants (45 students) had either refused to participate or had submitted incomplete questionnaires (with 891 adolescents remaining in the study)For this study, the AUDIT C  was used because it could be employed as a stand-alone screening measure to detect hazardous drinkers among adolescents ,27. The We assessed binge drinking using the question “How often do you have five or more drinks on one occasion?” The response options were: Never, Less than monthly, Monthly, Weekly, and Daily or almost daily. Those who answered “Never” were coded as 0 in the analysis, “Less than monthly” and “Monthly” were coded as 1, and those who reported “Weekly” and “Daily or almost daily” frequency were coded as 2. Because binge drinking may result in more negative consequences than low to moderate drinking , we usedThe remaining items on the AUDIT C instrument were as follows: a) “How often did you have a drink containing alcohol in the past year?” The response options for the frequency of alcohol consumption were: “Never”, “Monthly or less”, “2 to 4 times a month”, “2 to 3 times a week”, and “4 or more times a week”; and b) “How many drinks containing alcohol did you have on a typical day when you were drinking?” The response options for the amount of alcohol consumption were “1 or 2”, “3 or 4”, “5 or 6”, “7, 8, or 9”, and “10 or more”.The Integrated Questionnaire for the Measurement of Social Capital (CS-IQ) is a validated questionnaire developed by the World Bank in 2003 to assess social capital . The insThe Social Vulnerability Index (SVI) was used to assess the residential socioeconomic status. This index measures social exclusion in the city of Belo Horizonte. The City Hall database of SVI scores for each district was used based on the address of each student (derived from the survey) .According to the theoretical framework that supported the development of the SVI, this index has 20 variables grouped into five “dimensions of citizenship”: (a) access to housing and basic infrastructure, (b) access to education, (c) access to income and employment, (d) access to legal assistance and (e) access to health, food security and welfare 35). The index categorizes residential areas into 5 classes (class I to class V), with class I indicating the highest degree of social vulnerability  and class V indicating the lowest degree of social vulnerability (most advantageous conditions) ,35-41. T. The indThe mother and father’s educational attainment were chosen as an indicator of individual socioeconomic status because of its association to alcohol consumption/binge drinking in adolescents -44. The The type of school was also used as a socioeconomic indicator. Although type of school is a crude assessment, wealthy adolescents in Brazil are enrolled in private schools because most Brazilian public schools are known to have less educational resources than private ones . MoreoveUniversidade Federal de Minas Gerais. Authorization was obtained from the schools to perform the research. The participants and their parents/guardians signed the informed consent.This study was approved by the Ethics Committee of the The chi-squared test was used to study the associations of sample characteristics or the type of friends and binge drinking .We used binary logistic regression analysis to examine the association of binge drinking and the type of friends. The results were expressed as odds ratios (OR) and 95% confidence intervals (CI). Each type of most important group of friends was compared to friends from hobbies/other in relation to binge drinking at frequencies ranging from less than monthly to monthly and also from weekly to daily.p value less than 0.20 in the results of the bivariate analysis. The final model included all variables regardless of their p-values. Declines Homogeneity test and multi-colinearity test was performed with Pearson adjustments to analyze the validity of the final model. Because some studies have suggested that peer drinking has a greater impact on females than males [An ordinal logistic regression model was used because our dependent variable was ordinal . This model compares the likelihood of a response equal or less than a certain category with the likelihood of a highest response based on the odds ratio calculation ,52. The an males ,54, we cThe frequency of alcohol consumption was as follows: 212 (23.8%) adolescents reported consuming alcoholic beverages monthly or less frequently, 154 (17.3%) consumed alcohol 2 to 4 times a month, and 82 (9.2% ) adolescents reported drinking alcohol 2 or more times per week. Overall, the prevalence of any alcohol use during the past year was 50.3% (N = 448) in the sample of 16–19 year old adolescents.The amount of alcoholic beverages consumed in a typical day when drinking alcohol was as follows: 123 (13.8%) participants reported drinking 2 to 8 or more drinks. Binge drinking was reported by 321 (36%) adolescents; of these adolescents, 233 (26.2%) reported binge drinking less than monthly to monthly and 88 (9.9%) reported binge drinking weekly to daily.Table Table Table In a random sample of 891 Brazilian adolescents 15 to 19 years of age, half of the participants reported alcohol consumption during the past year. One-third of the participants reported binge drinking. Having their most important groups of friends from school was associated with a higher likelihood of binge drinking. Individual SES was not associated with binge drinking, but residential SES was associated with binge drinking.The prevalence of alcohol consumption in our study in Belo Horizonte, Brazil, was similar to previous studies conducted among adolescents in Brazil ,56,57. HSome studies of alcohol consumption and binge drinking in adolescents have shown a statistically significant association with socioeconomic status , but conflicting results have also been reported in the literature. Some studies confirmed that alcohol consumption is negatively associated with socioeconomic status ,60, whilIn this paper, we estimated the predictors of adolescent drinking behavior to identify the role of social friendship networks on binge drinking. Adolescents who reported that the majority of their groups of friends/close friends were from school rather than from church or hobbies/other activities had higher odds of binge drinking. This result corroborates finding in the National Longitudinal Study of Adolescent Health in the US, suggesting that an increase of 10% in the proportion of classmates who drink will increase the likelihood of drinking among peers by 4% . With siWhen we compared the adolescents who reported that the majority of their groups of close friends were from church with the adolescents who reported that the majority of their groups of close friends were from hobbies and other activities, we found that having friends from church was associated with less binge drinking. This result is similar to that found using other surveys, which suggest the association between attending church/religiosity and a lower likelihood of binge drinking ,20,63,64Studies developed by Valente et al. (2007) ; JamisonThroughout the course of a lifetime, friendships can direct development through support, modeling, and assistance, but their significance is heightened during adolescence . Peers hPublic health interventions targeted to the peer network might be more cost-effective than previous estimates have suggested because the health-promoting behavior of one person may spread to others via social networks ,12. The `Our results should be interpreted while considering some limitations. Because this study of the association between type of friendship network and binge drinking used cross-sectional data, two principal threats to causal inference should be considered: homophily and unobserved confounding. Homophily refers to the idea that individuals select different types of social network connections based partly on their own behavior preferences. For example, some students may enjoy associating with others based on their shared preference for observing religious proscriptions against drinking alcohol. In other words, the associations that we observed may have been driven by the tendency for “birds of a feather to flock together”, rather than by the network characteristics per se . Secondly, our finding may reflect the influence of unmeasured confounding factors that affect both patterns of friendship networks and drinking behavior. For example, the strictness of school policies against drinking may influence both the formation of different friendship connections as well as the prevalence of binge drinking. Because we did not collect information on variables such as school policies, we cannot rule out the possibility that unmeasured or unobserved factors could have led to the patterns we observed.We did not measure and control for variables such as “parental supervision” in our survey. Thus, if students who associated with peers from school were also those who were less likely to be supervised by their parents, the association between having school friends and binge drinking could have been confounded by such omitted variables.We assured the participants in this study that their responses would remain anonymous. However, there are some limitations associated with our measures of behaviors and negative outcomes because they rely on self-reports of alcohol consumption and binge drinking. It is possible that the actual amount of alcohol consumption and binge drinking could have been either under-reported or exaggerated. Furthermore, our sampling approach did not include individuals who were excluded from or had otherwise left school-based education, and the Vulnerability Index (SVI) was calculated on an ecologic level and not on an individual basis.The level of social capital to which an individual may gain access through their social networks depends on the structural characteristics of the networks as well as on the amount of social capital that other individuals in the network possess. Although they may be positive, friendship networks can also be viewed as negative, especially with regard to binge drinking during adolescence. Corroborating our results, at least three other studies have also found an association between peer influence and alcohol consumption and binge drinking ,11,13.We were unable to obtain information on the size, density, quality, or proximity of the friendship network or the normative drinking behaviors within those networks. Further studies are needed to investigate the importance of the detailed characteristics of friendship networks , parental control, religious beliefs and participation. Another important issue for future research is to measure the perceived norms among different types of friendships. Ideally, studies should measure the subjective norms surrounding drinking behavior within school-based versus church-based networks  approve/disapprove of drinking?”).The findings of this study highlight the association between social network (type of friends) and risky drinking behavior among adolescents, the importance of which is underscored in countries like Brazil that have a high prevalence of alcohol consumption and binge drinking among adolescents. This study revealed that the type of group of friends with which an adolescent associates and gender may determine the likelihood that an individual consumes large amounts of alcohol. Therefore, interventions to reduce alcohol consumption in adolescents should consider both the friendship network characteristics and gender.The authors declare that they have no competing interests.PZ, KJ, MV, and EF conceived of the study. KJ collected data. PZ conducted the analysis and wrote the first version. TO and IK contributed substantially to the interpretation of the results. KJ, TO, MV, EF and IK revised the manuscript for important intellectual content. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/257/prepub


In addition, fibroblast growth factors (FGFs), Wnt, and Notch signals are essential for establishment of functional thymic microenvironment. Transcription factors Foxn1 and autoimmune regulator (Aire) are powerful modulators of TEC development, differentiation, and self-tolerance. Dysfunction in thymic microenvironment including defects of TEC and thymocyte development would cause physiological disorders such as tumor, infectious diseases, and autoimmune diseases. In the present review, we will summarize our current understanding on TEC development and the underlying molecular signals pathways and the involvement of thymus dysfunction in human diseases.Thymic epithelial cells (TECs) are the key components in thymic microenvironment for T cells development. TECs, composed of cortical and medullary TECs, are derived from a common bipotent progenitor and undergo a stepwise development controlled by multiple levels of signals to be functionally mature for supporting thymocyte development. Tumor necrosis factor receptor (TNFR) family members including the receptor activator for NF Thymus, as a primary lymphoid organ for T lymphocyte development and maturation, plays an essential role in keeping host cellular immune tolerance to self-antigens. Thymic epithelial cells (TECs) forming a 3-dimentional network critically shape T cell repertoire in thymus, though other antigen-presenting cells in the thymus were also involved . Based o+K8+) are mainly located at the corticomedullary junction. They are part of cTECs or the immature progenitors for mTECs and cTECs. In addition, mTECs are positive for the expression of Ulex europaeus agglutinin-1 (UEA-1) on cell surface, but not Ly51 (UEA-1+Ly51−), while cTECs are UEA-1−Ly51+. With these markers, we can roughly distinguish mTECs and cTECs in immunofluorescence and flow cytometry assays. In addition, mature cTECs express high level of MHCII and protease β5t and thymus-specific serine protease (TSSP) participating in thymocyte positive selection, while mature mTECs express MHCII, CD80, autoimmune regulator (Aire) and tissue-restricted antigens (TRAs). Proteases Cathepsin-L and -S in mTECs mediate thymocyte negative selection. The major differences of cTECs and mTECs are briefly summarized in The rudimentary thymus arises from the endoderm of the third pharyngeal pouch around day 9 of embryonic development (E9) in mice. The thymic gland reaches its final anatomical location at about week 6 in the human fetus . TECs th+ mTECs to establish a functional thymic microenvironment. In addition, the individual progenitors for cTECs and mTECs exist [+Cld3,4hi) may represent the progenitors specifically for Aire+ mTECs [+CD205+CD40− [It has been reported that bipotent TEC precursors (TEPCs) could differentiate into both cTECs and mTECs –10. The Cs exist , 14. mTE05+CD40− .+MHCIIhiCD80hiAire+) as well as upregulation of Aire-dependent and Aire-independent TRAs participating in thymocyte negative selection [Generally, the development of mTECs is divided into 3 stages : bipotenelection . Eventuaelection .hiCD80hiAire+ mTEC subset was previously considered to be the postmitotic end stage of mTECs which will be removed by apoptosis. However, accumulating evidence has shown that mTECs may continually develop beyond Aire+ stage. First, Aire−/− mice have no Hassall's corpuscles (HCs) structure [+mTECs during ontogeny [+ cell stage [+CD80hiMHCIIhi mTECs developed into Aire−CD80intMHCIIlow end stage [+mTECs followed the Aire+mTECs during ontogeny [MHCIItructure  which isontogeny , and it ll stage . By usinnd stage . Recentlnd stage . The losnd stage . So the ontogeny .+CD205+CD40−MHCII−. Unlike the common bipotent progenitors, cTEPCs could self-renew after thymus injury is recovered [β5t thymoproteasome in cTECs is required for MHCI-restricted CD8+ T cells production, while cathepsin-L and TSSP are important for MHCII-restricted CD4+ T cells generation. Clearly, it is required to investigate the specific markers for cTEPCs and cTEC subsets in different developing stages, which will significantly help us to study cTEC development and the relevant mechanisms.In contrast to mTECs, the developing stages of cTECs remain poorly defined. It is proposed that TEPCs firstly develop into progenitors specific for cTECs (cTEPCs) phenotypically characterized as EpCAMecovered . Concomiecovered , 48–50. κB (RANK), CD40, and lymphotoxin β receptor (LTβR) are especially involved in determining mTEC formation and development, while fibroblast growth factor (FGF) and Wnt promote TEC expansion and functional maintenance. Transcription factors Foxn1 and Aire are essential for TEC development and functional maturation. The molecules involved in TEC development are summarized in TEC development is a complex and continuous process under control of extrinsic and intrinsic signal regulatory network. Tumor necrosis factor receptor (TNFR) family members including the receptor activator for NFβR have been proven to cooperatively control the thymic medullary microenvironment and self-tolerance establishment.It is highly recognized that TECs development and maturation are definitely dependent on their interaction with other cells in thymus such as thymocytes, fibroblasts, and mesenchymal cells. TNFR superfamily members and their ligands play an essential role in TECs especially mTECs development . mTECs e+CD3− lymphoid tissue inducer (LTi) cells promote CD80−Aire−mTEC developing into CD80+Aire+mTECs [γ5+ dendritic epidermal T cells also made contribution to the development of Aire+ mTEC development through providing RANKL [+ T cells [+CD80hiMHCIIhi mTECs. In contrast, mice deficient for osteoprotegerin  developed thymic hyperplasia and had more mature mTECs [−/− thymus or transferring their splenocytes to immune deficient mice caused severe inflammatory cell infiltration and abundant production of autoimmune antibody [In the embryonic thymus, RANKL signals provided by CD4re+mTECs . Invariang RANKL . In the  T cells , 20. Disantibody , 19. So −/−RANKL−/− double deficient mice displayed a greater reduction in mature mTECs and more severe autoimmune disease, implying that RANK and CD40 act cooperatively in modulation of thymic medullary microenvironment and self-tolerance [+ and CD8+ T cells) promotes mTEC proliferation [CD40L-CD40 signaling pathway is also essential for mTEC development. CD40- or CD40L-deficient mice had obviously less mature mTECs and showed an autoimmune phenotype. Although the defects are less severe compared to RANK-deficient mice, CD40olerance –21. In tferation .βR is mainly expressed on thymic stromal cells other than T and B lymphocytes. Two ligands for LTβR are discovered: LTα1β2 and LIGHT, in which the former consists of LTα and LTβ subunits. The mature single positive thymocytes are the main source for LTβR ligands in the thymus [βR, its ligands, or downstream signal molecule nuclear factor-κB-inducing kinase (Nik) caused defects of thymic medulla development including disorganized medullary architecture, significant reduction in overall mTECs, and retention of T cell maturation with autoimmune disease [βR in Aire and TRAs expression. Previous work showed that lymphotoxin signaling is required for Aire and Aire-dependent as well as Aire-independent TRA expression [α- or LTβ-deficient mice showed normal CD80, CD40, and Aire as well as TRAs expression despite reduced medulla area. The distribution of regulatory T cells (Tregs) and DCs in the thymus was also not affected [βR signal upregulated RANK expression in the thymic stroma, thereby promoting RANK signaling and mTEC differentiation [+ stage also requires the activation of the LTα-LTβR signal provided by mature thymocytes [βR signals could indirectly influence mTEC development through regulating other stromal cells like MTS15+ fibroblasts which express the highest LTβRl than TECs [In the thymus, LTe thymus . Mice de disease –25. Howepression . The folpression . LTα- oraffected , 54. Theaffected , which nntiation . Continuymocytes . MeanwhiβR is usually NF-κB signal [κB1 and RelA are mainly localized in cortical areas, whereas NFκB2, c-Rel and RelB are in the medulla [κB signal pathways regulate mTEC development [κB pathways via TNFR-associated factor 6 (TRAF6). TRAF6-deficient mice showed severe destruction of medullary architecture and loss of UEA-1+mTECs [κB pathways, TRAF6 activates TGF-β activating kinase 1 (TAK1), which in turn activates the IKK complex composed of IKKα, IKKβ, and NEMO. The IKK complex phosphorylates IkBα for degradation, leading to translocation of the RelA/p50 complex to the nucleus. In addition, RANK, CD40, and LTβR signaling could elicit nonclassical NF-κB pathways via TRAF2/5 to activate p52/RelB [α is phosphorylated by NIK and in turn triggers p100 partial degradation to p52 and then translocation to the nucleus together with RelB. Mice deficient of genes in nonclassical NF-κB pathways including NIK, IKKα, and RelB had abnormal thymus development with reduced UEA-1+ and/or Aire+ mTECs [+ and CD80hi mTEC but with no obvious medullary architecture changes [The signaling pathway downstream of RANK, CD40, and LTB signal . In the  medulla . Both caelopment . RANK an-1+mTECs . In clasp52/RelB . IKKα ise+ mTECs –64. p52  changes . The effGcm2, Bmp4 and Foxn1 expression and thymus/parathyroid detachment [FGFs boost thymopoiesis and promote differentiation by working on both thymocytes and TECs. FGF8 influences TECs indirectly by regulating neural crest cells (NCCs) survival and differentiation; therefore, FGF8 deficiency and NCCs deletion result in similar manifestation . FGF7 antachment .+ and CD8+ thymocytes and fibroblasts are the main source for KGF in the thymus. KGF acts on both thymocytes and TECs, promoting their proliferation and function [RAG-deficient mice increased medullary compartment [BMP2, BMP4, Wnt5b, and Wnt10b via activation of p53 and NF-κB signal pathway [FGF7 is known as keratinocyte growth factor (KGF). Mature CD4function . Applyinpartment . Adminispartment –34. KGF partment , 36. KGF pathway .Wnt receptors are exclusively expressed on TECs and Wnt regulates Foxn1 expression in the thymus . Wnt4 isγδT cells [Development of both TECs relies on cell-cell interactions between the developing T-lymphocytes and the thymic epithelium. Such interdependency between thymocytes and TECs is often referred to as “thymic crosstalk.” Notch signaling represents one important molecular example for thymic crosstalk. In the thymus, both TECs and thymocytes express various Notch receptors and their ligands . It is wδT cells . In fetaδT cells . HoweverδT cells . AccordiδT cells . The molnu/nu, nude mice) have atrophic thymus and few T cells in the periphery, leading to severe immune deficiency [nu/nu mice, the earliest stage of TEC development was not impaired, in which the common progenitors could persist even in the postnatal thymus. However, thymus development was arrested after initial formation of the organ anlage (about E12.0 in mice) without hematopoietic precursor colonization [Δ/Δ mice with a hypomorphic Foxn1 allele, lacking exon 3 of Foxn1, had a highly cystic thymus, containing no discernible cortical or medullary regions [hiUEA-1hi mature population) than cTECs [Foxn1 transgenic mice, age-associated thymic atrophy was diminished, and the total number of EpCAM+ and MHCIIhi TECs was higher [+ endothelial cells are not detected in the epithelial region [Transcription factor Foxn1 plays a crucial role in TEC development. Mice deficient in Foxn1 s higher , 84 and s higher , 86; (4)l region , indicatFoxn1 gene in thymus are strictly under control [Pax1, expressed on the third pharyngeal pouch at E9.5 and essentially regulating TECs differentiation and proliferation, is Foxn1-dependent [CCL25 and CXCL12, modulating hematopoietic stem cell localization in the thymus and stem cell factor (SCF), promoting T cell progenitor growth, were undetectable in Foxn1-deficient thymus [CathepsinL, CD40, and MHCII involved in TEC development and function are regulated by Foxn1 directly or indirectly [Foxn1 directly or indirectly regulates a series of genes involved in diverse aspects of thymus development and function . Meanwhi control . The regependent . CCL25 at thymus –92. Foxnt thymus , 93. In directly . ImportaFoxn1 gene. In the thymus, mostly Wnt4 and Wnt5b, produced by TECs and thymocytes, regulate Foxn1 expression in TECs through both autocrine and paracrine manners [Wnt and bone morphogenic proteins (BMPs) are two main regulators upstream of  manners . Overexp manners . In the  manners .Aire is not only a marker for mature mTECs but also regulates mTEC development and differentiation (reviewed in ). Aire-dRecent reports indicated that Aire also controlled the expression of microRNAs in mTECs, which in turn play a crucial role in maintaining thymic microenvironments , 103. GiThe major biological function of the thymus is to generate a diverse repertoire of T cells to constitute an important part in host adaptive immune system against foreign pathogens, while thymus also plays a critical role in self-tolerance via thymic negative selection and the production of Treg cells. TECs are the most important components in thymic microenvironment supporting thymocyte development and self-tolerance establishment. Since the thymus plays a key role in keeping balance between host immunity and tolerance, it is obvious that thymic dysfunction causes a diversity of diseases in humans .Thymus tumors are scarce. Thymomas and thymic carcinomas are two major epithelial tumors of the thymus. Thymomas are neoplasms arising from TECs, usually with organotypic features , numerous maturing thymocytes, and autoimmune syndromes such as myasthenia gravis (MG). Thymic carcinomas are malignant epithelial tumors with invariability and invasiveness and without organotypic feature and autoimmune disease .Abnormality of the thymus is always concomitant with lower production of functional T cells and leads to immunodeficiency. Immunodeficiency in hosts means higher susceptibility to pathogens infection including viruses, bacteria, and protozoa, as well as decreases in antitumor immunity. Abnormalities in TEC development lead to dysfunction of T cells which could cause chronic inflammatory disease. Targeted gp39 (CD40L) overexpression in thymocytes caused loss of cTECs and mTEC expansion, with decline in thymocyte numbers and morphologic features of chronic inflammatory bowel disease (IBD) .Central self-tolerance is established in the thymus by at least two main mechanisms: (1) negative selection—clonal deletion of self-antigen reactive T cells (2) generation of self-antigen-specific natural regulatory T cells (nTregs) to downregulate immune response. Impairment or breakdown of the thymic self-tolerance plays a primary role in the development of some autoimmune diseases. More and more evidence showed the correlation between thymus dysfunction in self-tolerance and autoimmune diseases.Aire mutation results in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), also known as autoimmune polyendocrinopathy syndrome type 1 (APS-1). APECED is a rare systemic autoimmune disease characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency. Different from many other autoimmune diseases, APECED is caused by a single gene mutation [In humans, mutation . APECED mutation . In matu+ T cells; overexpression of various cytokines and chemokines to recruit peripheral lymphocytes to the thymus leading to thymic hyperplasia, a hallmark of MG [Myasthenia gravis (MG) is a neuromuscular autoimmune disease characterized as muscle weakness and fatigability caused by T cell-dependent autoantibodies against neuromuscular junction. In MG patients, autoantibodies could directly attack muscle acetylcholine receptors (AChR), muscle-specific receptor tyrosine kinases (MuSK), and even muscles themselves. The exact trigger of MG is unclear; however, it is certain that alteration of the thymus and TECs is involved in MG pathogenesis. TEC dysfunction contributes to MG pathogenesis in several ways: defects in negative selection by producing AChR-reactive CD4rk of MG , 113.β cells. It is widely accepted that the absence or failure of immune tolerance to islet β cells is the primary cause for development of T1D. Previous results have demonstrated that all the members of insulin gene family were expressed in mTECs [β cells leading to the development of T1D [Type 1 diabetes (T1D) is an autoimmune disease resulting from destruction of pancreatic islet in mTECs . Insulint of T1D . Other a+CD25+Foxp3+ nTreg cells are developed in the thymus as negative regulation candidate to control peripheral self-tolerance. Dysfunction of the negative regulatory system mediated by nTreg cells could also play a crucial role in the development of autoimmune diseases. Loss of CD4+CD25+Foxp3+ nTreg cells alone is sufficient to induce autoimmune reaction. In humans, mutation of FOXP3, a specific transcription factor for nTreg cells, will cause a failure of nTreg cell development and will subsequently cause X-linked immunodeficiency syndrome IPEX  [+ T helper cells subsets  which is supposed to participate in other autoimmune diseases, such as MG [+CD25+ Treg cells in restraining B cell autoantibody production and defects in CD4+CD25+ Treg cells may be crucial to the development of primary biliary cirrhosis [CD4ropathy) . Dysfuncch as MG  and T1D ch as MG . It is rirrhosis . Defectiirrhosis .+CD25+Foxp3+ nTreg cells.In conclusion, thymus (TECs) dysfunction participates in autoimmune disease development mainly through the abnormality in the following two aspects: (1) self-tolerance established by Aire-mediated tissue-restricted antigens expression on mTECs; (2) negative regulatory system formed by CD4+CD8+ T cells). Thymic microenvironment of epithelial network is also affected by loss of mTECs and cTECs compartment, accumulation in extracellular matrix deposition. In HIV-infected children and adults, thymic dysfunction and involution including thymocyte apoptosis and severe TEC damage occur during disease progression [More and more observations have implied that thymus is very sensitive and fragile to many physiological disorders such as infection, autoimmune diseases, and aging . A variegression , 122. Thgression . In addigression  and MG [gression .2+ deficiencies could cause multiple thymic defects including thymic atrophy [Thymus involution remains a significant marker for senescence. In aged mice or humans, thymus size is reduced, as well as TECs, thymocytes, and peripheral T cells. Aged thymus has disorganized thymic architecture, increased cavity and cysts, and more fibroblast and fatty cells , 126. Be atrophy . AccordiThe fundamental function of thymus is to establish host immunity with self-tolerance. TECs are the most important components in thymic microenvironment supporting thymocytes development and directing central tolerance. Multiple signals and cellular interactions are required for the maturation, expansion, and maintenance of thymic epithelial compartments. TNFR signals including RANKL, CD40L, and lymphotoxin cooperatively control the thymic medullary microenvironment and self-tolerance establishment, while FGFs, Wnt, and Notch signals are essential for TEC and thymocyte expansion and functional maintenance. Foxn1 is a powerful modulator of TECs lineage progression in fetal and adult thymus in a dose-dependent manner. Aire expression in mature mTECs drives mTEC development and directs self-tolerance establishment. Thymic dysfunction is associated with many diseases including tumors, infectious diseases, and autoimmune diseases. On the other hand, the thymus is a primary target organ for many physiological disorders such as pathogen infections, autoimmune diseases, aging, and malnutrition. Since the thymus plays an important role in immunity and diseases, understanding the mechanisms for TEC differentiation and function would offer the possibility for the clinical application of “modification of thymus function” to improve our cellular immunity in physiological and pathological conditions such as infections, autoimmune diseases, and aging.


Antibiotic consumption in hospitals is commonly measured using the accumulated amount of drugs delivered from the pharmacy to ward held stocks. The reliability of this method, particularly the impact of the length of the registration periods, has not been evaluated and such evaluation was aim of the study.- that is second- and third-generation cephalosporins, carbapenems, and quinolones - in five hospital wards and compared the data with corresponding pharmacy sales figures during the same period. Defined daily doses (DDDs) for antibiotics were used as measurement units (WHO ATC/DDD classification). Consumption figures obtained with the two methods for different registration intervals were compared by use of intraclass correlation analysis and Bland-Altman statistics.During 26 weeks, we performed a weekly ward stock count of use of broad-spectrum antibiotics Broad-spectrum antibiotics accounted for a quarter to one-fifth of all systemic antibiotics (ATC group J01) used in the hospital and varied between wards, from 12.8 DDDs per 100 bed days in a urological ward to 24.5 DDDs in a pulmonary diseases ward. For the entire study period of 26 weeks, the pharmacy and ward defined daily doses figures for all broad-spectrum antibiotics differed only by 0.2%; however, for single wards deviations varied from -4.3% to 6.9%. The intraclass correlation coefficient, pharmacy versus ward data, increased from 0.78 to 0.94 for parenteral broad-spectrum antibiotics with increasing registration periods (1-4 weeks), whereas the corresponding figures for oral broad-spectrum antibiotics (ciprofloxacin) were from 0.46 to 0.74. For all broad-spectrum antibiotics and for parenteral antibiotics, limits of agreement between the two methods showed, according to Bland-Altman statistics, a deviation of ± 5% or less from average mean DDDs at 3- and 4-weeks registration intervals. Corresponding deviation for oral antibiotics was ± 21% at a 4-weeks interval.There is a need for caution in interpreting pharmacy sales data aggregated over short registration intervals, especially so for oral formulations. Even a one-month registration period may be too short. Antibiotic use in hospitals accounts for 10% or less of total antibiotic consumption in most countries, but is characterized by the use of large quantities of broad-spectrum antibiotics (BSAs). Furthermore, hospital departments are densely populated with patients who are at particular risk of acquiring infections of resistant microorganisms . Thus, aThe most common way of measuring antibiotic use in hospitals is to apply sales data extracted from hospital pharmacy computer systems. However, until now in most European countries pharmacies have not dispensed antibiotics directly to the patients. A ward-held stock of antibiotics has been the routine. In a recent survey of hospital pharmacy practice in Europe it was found that 70% of hospitals hold antibiotic stocks at the wards . When waMoreover, the duration of the registration intervals may affect the results. In general, short registration intervals have been advocated, especially when the purpose has been to evaluate the effects of specific drug interventions -8. HowevAim of the study was to explore whether the number of defined daily doses (DDDs) reported from the pharmacy, that is to say the sales data to the wards, reflects actual patient consumption of BSAs. In particular, we wanted to evaluate the importance of the length of the registration intervals for the reliability of the pharmacy data.Oslo University Hospital Aker is a 350-bed tertiary hospital with adult surgical and medical specialities including regional functions for urology, vascular surgery, and endocrinology. In 2007, the number of somatic hospital beds was 356 and 20 060 patients were admitted for 116 251 days of in-patient treatment. Data on occupied bed-days were obtained from the hospital administration, where days of admission and discharge were counted together as one day.Five hospital wards were included in the study: a pulmonary diseases ward, a combined gastrointestinal and infectious diseases ward, a combined endocrinology and haematology ward, and two urological wards. These wards accounted for 28% of all patient treatment in terms of occupied bed days. The specialties were selected because of a historically relatively high per-ward total use of BSAs, which in our hospital are ciprofloxacin  and parenteral formulations of cefuroxime, cefotaxime, ceftazidime, ceftriaxone, meropenem, and imipenem/cilastatin. Ciprofloxacin is the only oral BSA registered in Norway. Piperacillin+tazobactam is the only registered penicillin with an enzyme inhibitor, but this agent was not used in our hospital during the study period.In Norway, a full assortment of antibiotics is normally stock-piled in the wards and administered by the ward nurses. Antibiotics are not ordered from the pharmacy on a per-patient basis. The ward nurses routinely prepare parenteral antibiotics, retrieved from the ward stock, just prior to administration. On rare occasions, when a drug is out of stock, it will be available from another ward or, in the daytime, from the hospital pharmacy. An emergency pharmacy service exists for essential and rarely used drugs; however, this does not apply for antibacterial agents.During 26 weeks from October 2006 to April 2007, a pharmacist performed weekly counts of BSA stock in the five wards. The number of vials, infusion bags, and tablets were registered for each ward once a week, before daily orders of antibiotics were placed to the pharmacy, but after morning doses of antibiotics were administered to the patients. The milligram amounts for each antibiotic were converted to defined daily doses units in accordance with the latest WHO ATC/DDD version .The weekly amount of antibiotics consumed by patients (henceforth designated as "ward BSA") was calculated as the difference between the previous and the current week count, and also taking into account input and output to ward stock. Factors increasing the ward stock size were delivery from the pharmacy and loan from other wards. Factors reducing the ward stock size were loan to other wards, BSAs sent with patients for use after discharge, and discarding of old drugs. Discarded drugs were registered electronically in the pharmacy sales system while the other stock reducing factors were registered manually by ward nurses.Because of the lack of a ward stock count during holidays in study weeks 10 - 12 and 25 - 26, a weekly average was used for these weeks. Also, two weeks had to be omitted from a total of 26 weeks to establish complete 3- and 4-weeks registration intervals. We chose to omit the weeks 12 and 26, weeks with incomplete data acquisition, thus probably reducing the risk of error.For each ward, weekly sales figures for BSA DDDs for the same 26-week period were extracted from the hospital's pharmacy computer system . In addition to economic data, the system registers the number of DDDs for each antibiotic order placed by the separate wards, as well as total amounts of antibiotics returned by the same wards to the pharmacy provided that shelf-life and condition allow for further use. The resulting sales figures for different time intervals, designated as "pharmacy BSA", were compared with "ward BSA". We chose to assign a weekly average for the study weeks 10 - 12 and 25 - 26 also for the pharmacy sales data (see Discussion). The shortest registration interval was one week, and accumulated periods of two, three, and four weeks were also investigated.The numbers of DDDs for each BSA and for each ward obtained by the two methods were entered into a Microsoft Excel (version 2007) spreadsheet. All entries and subsequent calculations were double-checked by two of the investigators (JBH and RM).σs2+σe2, where σs2 is the between-subject variance and σe2 is the within-subject variance. The ICC will be high if the measures are in agreement , and the variation between ward data is large relative to the variation between pharmacy data measurements. A calculated ICC = 1 reflects perfect reliability of the method as evaluated against the assumed "gold standard". An ICC of 0.7 is commonly used as a threshold of sufficient reliability [All statistical analyses were performed using Stata software version 11 . The reliability or overall agreement of the pharmacy BSA with ward BSA was assessed by intraclass correlation coefficients (ICC) using a mixed model ANOVA. The theoretical formula for is ICC=iability .Using the method described by Bland and Altman ,12, the BSA use during the 26 study weeks ranged from 12.8 DDDs/100 bed days in one urological ward to 24.5 DDDs/100 bed days in the pulmonary diseases ward. BSA amounts ranged from 615.3 DDDs at one urological ward to 1144.8 DDDs at the pulmonary diseases ward  of the eight BSAs at the five wards. In 550 of these registrations, no use was registered by either of the two methods and this was primarily caused by a very low use of carbapenems . The number of DDDs that were discarded (10.5), sent with patients on discharge (18.0) or loaned to other wards (14.5) represented 1.1% of the total consumption. Only nine DDDs were borrowed from other wards.Total BSA use over 26 weeks measured as ward BSA was 3 970 DDDs with a weekly range of 104.7 - 257.2 DDDs. The corresponding total number for pharmacy sales data were almost identical at 3 977 DDDs, with a weekly range of 41.5 - 324.5 DDDs  as distinct from oral formulations (ciprofloxacin tablets) for which both larger bulks and a wider range of order size  were registered  and periods of registrations (one to four weeks), Bland-Altman statistics revealed mean differences between -0.136 to 0.158 DDDs. The limits of agreement for each of the above combinations were converted to corresponding DDD ranges  based electronic chart as source of data. The investigators speculated that transfer of antibiotics between wards, wastage, and data-entry errors may have been the reasons for the discrepancy . AnotherA limitation of our study is the relatively low BSA consumption in our institution, such that the number of registered DDDs was moderate. In hospitals with more extensive BSA consumption and a larger assortment of antibiotic substances, the findings may differ. Also of note, for two periods weekly ward data were missing because of holidays, and for the weeks in question, weekly averages were the basis for the analyses. However, since the corresponding pharmacy sales data were averaged accordingly, this deviation tend to introduce bias towards higher levels of agreement , particularly so for the short registration intervals.We propose that our method of weekly ward stock DDD accounting is an accurate method for indicating actual antibiotic use by patients. Only a much more labour-intensive patient chart review would be more accurate. Although also our method is demanding and therefore not practical for routine surveillance, we regard it as well suited for scientific purposes and for quality assessments of pharmacy sales figures. In some hospitals, electronic patient charts have been introduced and this allows for another method, probably with a high level of accuracy, for measuring drug consumption . HoweverPharmacy sales data for total BSA use were representative for the actual drug consumption when longer registration periods were used. For the data to be sufficiently reliable, a four-week registration period is required for parenteral formulations, whereas for oral medications one month is not sufficient. For analyses of BSA subclasses and separate hospital units, even these intervals are probably too short, at least in hospitals with a low-to-moderate consumption profile.The authors declare that they have no competing interests.JBH designed the study, analyzed and interpreted the data and drafted the manuscript. RM contributed to study design, acquired the ward stock data and helped in interpretation and writing of the manuscript. ÅR advised on design and interpretation of the study, and contributed significantly with critical revisions of the manuscript.All authors have read and approved of the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/11/166/prepub