Voluntary non-remunerated repeat blood donors are perceived to be safer than the first time blood donors. This study was planned for follow-up of previous hepatitis C virus (HCV) test results of anti-HCV enzyme-linked immunosorbent assay (ELISA) reactive repeat blood donors. The aim was to suggest a protocol for re-entry of the blood donors who are confirmed HCV negative by nucleic acid test (NAT) and recombinant immunoblot assay (RIBA). A group of repeat voluntary donors were followed retrospectively who became reactive on a cross sectional study and showed HCV reactivity while donating blood regularly.A total of 51,023 voluntary non remunerated blood donors were screened for anti-HCV ELISA routinely. If anybody showed positivity, they were tested by two ELISA kits (screening and confirmatory) and then confirmed infection status by NAT and or RIBA. The previous HCV test results of repeat donors reactive by anti-HCV ELISA were looked back from the records. Data of donors who were repeat reactive with single ELISA kit (in the present study) were analyzed separately from those reactive with two ELISA kits (in the present study).In this study, 140 (0.27%) donors who were reactive by anti HCV ELISA were included. Out of them, 35 were repeat voluntary donors and 16 (11.43%) were reactive with single ELISA kit. All 16 donors were reactive by single ELISA kit occasionally in previous donations. Their present ELISA positive donations were negative for HCV NAT and RIBA. A total of 19 (13.57%) donors were reactive with two ELISA kits. In their previous donations, the donors who were reactive even once with two ELISA kits were consistently reactive by the same two ELISA kits in their next donations also.Donor sample reactive by only single ELISA kit may not be considered as infectious for disposal as they were negative by NAT and or RIBA. One time ELISA positivity was found probably due to ELISA kit specificity and sensitivity. Donors reactive with two ELISA kit should be discarded as there is a high positivity with NAT/ RIBA. However, donors reactive by two ELISA kits and negative by NAT and RIBA should be followed up and may not be deferred permanently. Safety and adequacy remain the central goal of donor screening programs. IncidencThe present study was conducted at a regional blood transfusion center  in western India for a total period of 15 months from August 2007 to October 2008. A total of 51,023 donors were screened for anti-HCV by ELISA in the process of routine screening by a single kit. The donor samples which were initially reactive for anti-HCV were tested in duplicate by the same ELISA kit. When either of the samples was reactive in duplicate testing, the same was tested by a second ELISA kit. All the donor samples reactive by either first or the second ELISA kit were then tested by NAT and/or by RIBA. All samples were tested by NAT and NAT negative samples were again tested by RIBA. Test results of repeat donors were studied and the previous ELISA results of the same repeat donors were analyzed. As a policy, all TTI positive blood donors were informed with pre- donation counseling as per standard procedures. However, these blood donors donated blood inspite of them being informed of the TTI status. There was no system to detect TTI status of blood donors before collection especially during outdoor camps as the whole process was not computerized. However, all units showing reactive results in that particular donation were discarded as per standard procedures.All blood donors with discordant result were followed up prospectively for further analysis. The previous anti-HCV results of the repeat donors were looked back from the donation record and ELISA testing record. Data of donors who were repeat reactive in single ELISA kit (in the present study) were analyzed separately from those reactive in two ELISA kits (in the present study). During the study period, two approved ELISA kits were used (kit A and kit B). In a few instances, another kit (kit C) was also used in the case of non-availability of either kit. Each donation was tested either by kit A or B and once it was positive, the sample testing was repeated by the other ELSA kit. For each donor, the number of donation, testing kit in which the test was reactive, and results of previous donations when tested with kit A, kit B and or kit C were mentioned. ELISA kits used during the study period were Murex version 4, Hepanostika Ultra HCV and LG-HCV.In the study group, 105 (75%) donors were first time donors and 35 25%) donors were repeat donors . Among t5% donorsAmong the repeat donors, 16 (11.43%) donors were reactive with single ELISA kit in the present study. The results of their previous donations were analyzed. All these donors were at least once nonreactive by ELISA. In subsequent donations, they became reactive by ELISA. NAT and RIBA were negative in all repeat donors who were reactive only by single ELISA kits.th donation sample was included in the study and was negative by NAT and RIBA. After this 5th donation (study sample) also, the donor was followed prospectively and had donated twice. The results remained reactive by kit B (6th donation) and nonreactive by kit A (7th donation). One important point was that over a period of 27 months, the results of kit A and also kit B remained consistent throughout.Donor No. 14: It is evident from serial testing record that this donor was repeatedly reactive by kit B two times. The same donor was always nonreactive by the other kit (kit A) three times. The 5Donor no. 27: This donor’s sample was nonreactive twice with kit A. The same donor’s sample was nonreactive by kit B in the previous donation but reactive in the present donation by kit B (study sample). However, the sample was negative by NAT and RIBA.th donation) was reactive by ELISA (kit B), but was negative by NAT and RIBA. This donor donated once again (16th donation) and was tested again nonreactive by kit A.Donor no. 57: This donor had donated 15 times. This donor was nonreactive 14 times by all the three kits in different occasions. The present study sample . In the present donation, i.e. 3th donation sample was included in the study group and was negative by NAT and RIBA.Donor no. 88: This donor had donated total four times. In the first two donations, the sample was nonreactive by kit B. Third and fourth donation samples were reactive by kit A only. The 4th donation) was reactive by kit B but negative by NAT and RIBA.Donor no. 114: This donor was negative three times by two different kits (kit B or kit C). The present sample  samples were reactive with two ELISA kits in the study group sample. Few of these samples were reactive by both kit A and kit B in the previous donations also. The results of their previous donations were analyzed for all the 19 donors. Out of 19 donors, 8 (42.1%) were confirmed positive by NAT or RIBA.th donation. On 5th donation, sample (study group) was reactive by kit A. This donor was also negative by NAT and RIBA.Donor no.17: Donor was nonreactive by kit B or kit C till the 4st donation, but was reactive by both the kits in the 2nd donation (study sample). This sample was negative by NAT and indeterminate by RIBA. No history suggestive of HCV associated risk factor was revealed by the donor at the time of blood donation. Serious efforts were made to follow up this donor however; the donor was lost to follow up.Donor no. 20: Donor was nonreactive by kit B in the 1st and by kit B in the 2nd donation sample. The 3rd donation sample was reactive by kits A and B. The present donation (4th) was reactive by two kits and was included in the study. This sample was NAT negative but RIBA positive. The donor did not mention any HCV associated risk factor history at the time of donation and history of cirrhotic liver in the family.Donor no. 48: Sample was nonreactive by kit C in the 1st to 3rd donation. Third donation was included in the study group and was NAT positive. This donor was asymptomatic but revealed history of blood transfusion about 10 years back.Donor no. 51: Donor was reactive by kit A and kit B in the 1Donor no.73: This donor was negative by kit C once and by kit B the other time. In the third donation, the donor was reactive by both the kits. This sample was found to be NAT positive. This donor also did not reveal any suggestive history related to risk factor for HCV at the time of donation. This donor could not be followed up.th donation was reactive by both the kits, but negative by NAT and RIBA.Donor no. 115: Donor was nonreactive three times either by kit B or kit C. The 4rd sample (study group) was positive by NAT. No HCV risk factor associated history was revealed at the time of donation.Donor no. 123: This donor was reactive by both kits A and B in all the three donations. The 3st to 3rd and from 5th to 7th donation. The sample was nonreactive in the 4th and from 8th to 10th donation by kit A. In the 11th donation (study sample), it was reactive by kits A and B both. This sample was also NAT positive. No history of association of risk factor for HCV was known for this donor.Sample no. 131: Donor had donated blood 11 times. Sample was reactive by kit C from 1Donor no. 157: Reactive by both the kits in the first donation. The second donation was included in the study group and was reactive by both the kits. The sample was NAT negative and RIBA positive. This donor also had not mentioned any HCV risk associated history at the time of blood donation.In donors whose samples were reactive even once with two kits, were consistently reactive by the same two kits in their next donations also. All donors who were previously reactive by two ELISA kits were informed about their HCV status through a letter. These donors were counseled to consult a physician and not to donate again. This is done as a part of standard operating procedure for post-donation counseling of TTI reactive donors. In spite of this, these donors had donated again. Though the software had a provision of making these donor data inactive so that they were not called for blood donation, there was no identification system at donation sites and blood donation camps. Few of these donors had donated against the medical advice, without the knowledge of blood center.Data of donors who were repeat reactive with single ELISA kit (in the present study) were analyzed separately from those reactive with two ELISA kits (in the present study).th donation sample was reactive by kit B. This sample was negative by NAT and RIBA, but this blood unit would have been discarded on the basis of test done by kit B. Donor no. 88 donated blood totally four times. This donor’s sample was also consistently nonreactive two times by kit B and twice reactive by kit A. The sample was negative by NAT and RIBA. It was evident from the test results of above donors that samples which were reactive with only single ELISA kit may not be confirmed ELISA positive. These samples give consistent reactive result with a particular kit and nonreactive result with the other kit. These samples might have some cross-reactivity with some antigen in the kit. As stated by Sharma et al., false-positive test results in otherwise healthy blood donors have been attributed to the presence of cross-reacting circulating antigens and antibodies. Several factors may be associated with screening test reactive but confirmatory test negative and intermediate results. A history of allergy, acute illness, or alloimmunization, the presence of autoantibodies, or vaccination may result in false-reactive test results.[It was observed during follow-up of repeat blood donors that donors reactive by only single ELISA kit were repeatedly reactive by the same kit and nonreactive by the other kit. These donors were all NAT and RIBA negative. As observed in sample no. 14, this sample was consistently nonreactive by kit A and was reactive by kit B. Most probably, the same sample had some cross-reactivity with some antigens in the ELISA kit B and was consistently reactive with it. The donor of sample no. 57 had donated blood 16 times. The 15et al., in which 156 samples were tested by seven ELISA kits and those with discrepant results in the different kits were negative by both NAT and RIBA.[These blood units which were reactive by single kit only were always discarded as per existing law. These were actually negative samples when tested by NAT or RIBA and were not infectious. These donations were unnecessarily discarded. Similar findings were reported in a study conducted in China by Ren rd and 4th donation. The 4th donation sample was negative by NAT but RIBA positive, which might be because the donor HCV RNA level was below the detection level but the antibody level remained elevated. This incident justifies that blood donor sample should be tested and reconfirmed by two ELISA kits before discarding the unit and not based on the result of single ELISA result. As observed by Seed et al., two assays applied sequentially can increase the positive predictive value of the process by selecting for true positive reaction because samples reactive in both the assays have a higher probability of representing true reaction rather than those reacting only in one. World Health Organization (WHO) guidelines also (2002) mentioned that if confirmatory testing is not available, use an alternate assay that is as sensitive as the primary assay, for use in confirming the status of the samples that are found to be repeatedly reactive by the primary assay.[3Out of 19 donors, 8 had confirmed HCV infection demonstrated by positive NAT or RIBA results. It was observed in sample no. 17 which was nonreactive by any of the kits (A or B or C) till the present donation. In the study group sample, the sample was reactive with both the kits, but was negative by NAT and RIBA. Sample no. 48 was reactive with two kits in the 3y assay.3Sample nos. 79, 145 and 157 were also negative by NAT but RIBA was positive. The reason may be that the circulating HCV RNA titer may vary considerably. While a single qualitative assay for HCV RNA confirms active viral replication, a single negative test does not exclude viremia and may reflect only a viral load below the detection limit of the assay.Donor no. 51 was reactive by both the kits in three donations and the last sample was NAT positive. This donor had history of blood transfusion in the past. In this donor, the results could be correlated with significant history of association of risk factor. Few other studies also have shown an association of history of blood transfusion with an increased risk of polymerase chain reaction (PCR) positivity or confirmed HCV cases.101110rd donation, ELISA was reactive with two kits and also NAT positive. Most probably, the donor acquired infection during this interval between the 2nd and 3rd donation. The donor did not reveal any history of risk behavior during this period. It was assumed that this donor was a dangerous type of donor harboring occult infection. The donor was nonreactive by kit B and kit C in previous donations. However, in the 3rd donation, the donor was reactive by both kits B and A. An attempt was made to trace the recipients of both these units without any success. Donor no. 115 was reactive by both the kits in the 4th donation, but was NAT and RIBA negative. Such donors should be dealt carefully and regular follow-up should be done. They may not be deferred permanently. Sample no. 131 was nonreactive 10 times with single kit. The 11th donation was reactive by kit A and B and this sample was also NAT positive. Again, this case was a highly dangerous case with occult infection. Due to unknown reason, probably due to low anti HCV antibody level, infection may not have detected for the last 10 times. and HCV viremia was confirmed by NAT on 11th donation. This donor might have acquired infection before this donation but no such history could be elicited. Though this donor is a repeat donor and had donated 11 times, still was not safe as a donor. So, it is very important to take proper medical and risk behavior associated history at the time of donation. In this type of cases, necessity of blood donor screening by using NAT is firmly established. NAT for HCV was implemented by blood centers in 1999 in the United States. In 3 years of NAT testing, 170 HCV NAT-positive, seronegative donations were identified in the United States among 39.7 million screened donations. NAT has probably reduced the residual risk for HCV transmission to less than 1 in 2,000,000 components transfused.[Donor no. 73 was nonreactive in first two donations. In the 3ansfused.13 In a sansfused.Donors who were ELISA reactive by two ELISA kits were informed about their HCV status and counseled not to donate again. In spite of this, these donors had donated again as there was no system of tracking by the software just before donation. The blood center did not have any method to check these donors and block them at the screening site before blood donation, especially at outdoor camp sites. So, these donors repeatedly donated blood without the knowledge of the blood center. If by chance, these donors’ sample is tested by a kit which is from different manufacturers, the blood unit may be labeled as nonreactive and issued to the patient. So, a provision in the blood bank software should be developed so that it can identify any donor whose sample is repeat reactive by two ELISA kits previously in the blood bank. There should be at least two identification marks for the donor i.e. date of birth or mother’s name or any other mark. The ideal situation will be, if with the help of this software, the donor is identified at the screening level only. But this may not be practical in most of the blood center setups in developing countries like India where most of Indian blood banks documentations are maintained manually. In such a case, the software should at least be able to identify the blood donor and block the blood unit and ensure that it is not issued to the patient.Discordant results on regular voluntary blood donors in this study is a matter of concern and justified employment of NAT test on routine blood donor screening. It is evident that only dependence of repeat voluntary blood donor as a source of safe blood may not be sufficient in this part of the world. It may be due to many reasons like occult infections, non-complaint donors or contacting infections in between donations. However, cost benefit issues to be examined before taking further steps in implementation in any services.et al.[et al.[Among the 19 donors who were reactive by two ELISA kits, 11 were negative or indeterminate by NAT and RIBA. These donors had not been reactive by two kits previously. These donors should be followed up at regular intervals. A protocol for re-entry of such donors is recommended. It was observed in a look-back study by Vrielink et al. that nonl.[et al., those dThis study demonstrated that blood units from repeat donors reactive by single ELISA kit only (and nonreactive by the second ELISA kit) may not be discarded though this policy needs regulatory approval in respective countries. Repeat donors are perceived to be safe donors. However, no safety features like proper detailed history, screening and follow up etc, should be lowered only being the repeat donors. Donors reactive by two ELISA kits and positive by NAT or RIBA should be informed and deferred permanently. These donors should be advised to take medical treatment and instructed never to donate again. Donors reactive by two ELISA kits but negative by NAT and RIBA should be properly counseled. A protocol for re-entry of these donors may be made after long term follow up. A safety features to be included in all blood bank software which identify a previously two ELISA kit reactive blood donor and blocks the blood unit from issue.


Mycobacterium tuberculosis.We find that genome-wide DNA transfer by conjugation in mycobacteria affords bacteria that reproduce by binary fission the same advantages of sexual reproduction, and may explain the genomic evolution of  oriT-based conjugation models, and whose potential evolutionary significance has not been evaluated. Here, we determined the genome sequences of 22 F1-generation transconjugants, providing the first genome-wide view of conjugal HGT in bacteria at the nucleotide level. Remarkably, mycobacterial recipients acquired multiple, large, unlinked segments of donor DNA, far exceeding expectations for any bacterial HGT event. Consequently, conjugal DNA transfer created extensive genome-wide mosaicism within individual transconjugants, which generated large-scale sibling diversity approaching that seen in meiotic recombination. We exploited these attributes to perform genome-wide mapping and introgression analyses to map a locus that determines conjugal mating identity in M. smegmatis. Distributive conjugal transfer offers a plausible mechanism for the predicted HGT events that created the genome mosaicism observed among extant Mycobacterium tuberculosis and Mycobacterium canettii species. Mycobacterial distributive conjugal transfer permits innovative genetic approaches to map phenotypic traits and confers the evolutionary benefits of sexual reproduction in an asexual organism.Horizontal gene transfer (HGT) in bacteria generates variation and drives evolution, and conjugation is considered a major contributor as it can mediate transfer of large segments of DNA between strains and species. We previously described a novel form of chromosomal conjugation in mycobacteria that does not conform to classic  Mycobacterium smegmatis. We find the resulting hybrid bacteria to be extremely diverse blends of the parental strains, reminiscent of the genetic mixing that occurs through meiotic recombination in sexual organisms. This novel mechanism of conjugation can create genome-wide mosaicism in a single event, generating segments of donor DNA that range from small (∼0.05 kb) to large (∼250 kb), widely distributed around the recipient chromosome. We exploit this mixing by using genetic tools originally developed for finding mammalian disease genes to locate the genes that confer a donor phenotype in M. smegmatis. We speculate that similar genomic mosaicism observed in pathogenic mycobacteria arose from conjugation between ancestral progenitor strains.Bacteria reproduce by binary fission, generating two clones of the original; this restricts the genomic diversity of the population, which brings with it inherent evolutionary drawbacks. This problem can be eased by conjugation, which transfers DNA from a donor to a recipient bacterium. Understanding the potential of conjugal DNA transfer for generating genetic diversity is necessary for estimating gene flow through populations and for predicting rates of bacterial evolution. The influence of chromosomal conjugal DNA transfer on mycobacterial diversity has not been previously addressed. Here, we determine and compare the complete genome sequences of independent progeny from bacterial matings between defined donor and recipient strains of  Sexual reproduction in eukaryotes promotes genetic diversity by increasing gene flow through a population, permitting both the loss of mutant genes and the acquisition of functionally distinct gene alleles. The diversifying potential is further enhanced by crossover events that create new mosaic recombinant meiotic products, which in turn may impart new functionalities not present in either parent. In contrast, bacterial fission provides rapid clonal expansion to fill an environmental niche, but lacks the evolutionary advantages of sexual reproduction. Horizontal gene transfer (HGT) mitigates the diversification constraints of asexual reproduction by mediating limited gene flow through the population. The fundamental forms of HGT include transformation, transduction, and conjugation. Conjugation is considered a major contributor to HGT, as it can transfer more extensive segments of DNA between different species and even kingdoms oriT) on the plasmid and guide the DNA into the recipient cell. oriT is cis-acting, and thus, when recombined into the chromosome, it can mediate transfer of chromosomal DNA, as first described for E. coli Hfr strains M. smegmatis displays all of the hallmarks of conjugation: it requires stable and extended contact between a donor and a recipient strain, it is DNase resistant, and the transferred DNA segments are incorporated into the recipient chromosome by homologous recombination E. coli Hfr system end there E. coli, Hfr transfer always initiates at the sole plasmid-encoded oriT site, and the DNA is transferred in a 5′ to 3′ direction, such that only genes proximal and 3′ to oriT are inherited at high frequencies M. smegmatis, all regions of the chromosome are transferred with comparable efficiencies as demonstrated by equivalent transfer of a kanamycin-resistance marker regardless of its chromosomal location Conjugation describes the unidirectional transfer of DNA from a donor to a recipient, and requires cell–cell contact. Conjugal processes are traditionally plasmid encoded, or encoded by a discrete genetic element integrated into the chromosome. Transfer proteins are generally classified into those that establish and maintain mating-pair formation or those responsible for DNA transfer Transposon mutagenesis screens provided initial insights into the genetic requirements of transfer M. smegmatis progeny. The genomic sequence of each of the M. smegmatis parental strains has been determined, and the abundant single nucleotide polymorphisms between the two strains indicated that the transferred segments comprising the transconjugant genomes could be mapped with precision. We found that the parental contributions to the transconjugants were much more complex than expected, indicating a surprisingly major role for conjugal DNA transfer in generating genomic diversity. The blending of the parental genomes is reminiscent of that seen in the meiotic products of sexual reproduction. This comparison is validated by our use here of genomic approaches previously developed and applied in sexual reproduction systems to define candidate genes for conjugal mating identity.Here, as an alternative approach, we examined the products of DNA transfer to better understand this process and its contributions to mycobacterial evolution. We used next-generation sequencing to determine the parental inheritance profiles in transconjugant rKm) was integrated in the chromosome of mc2155, the standard laboratory and conjugal donor strain of M. smegmatis. Donor mc2155 derivatives that differed in their rKm insertion site were mated to an apramycin-resistant (Apr) recipient strain, mc2874 . Our comparative sequence analyses of the parental strains had shown that the circular mc2155 and mc2874 genomes are collinear, and that they contained abundant single nucleotide polymorphisms  providing a clear distinction between parental DNA origins . Surprisingly, 5-fold more mc2155 DNA was co-inherited in segments that were not selected, and these segments were distributed around the genome with no obvious regional biases  associated with that phenotype. We have previously observed that a subset of transconjugants become donors, suggesting that these progeny acquired a donor-conferring locus 2155 DNA among those progeny encoding this trait. Transconjugants derived from crosses of the differentially marked donor strains were screened for donor ability, and 10 independent donor-proficient transconjugants were identified. We note that mating identity is a mutually exclusive phenotype, and transconjugants exhibit transfer efficiencies comparable to parental strains  that is sufficient to switch mating identity from recipient to donor in these transconjugants.The image in  strains . Genomic2155 DNA , with th2155 DNA . This re2155 DNA . The esx1 locus encodes a Type VII secretion system M. tuberculosis, are essential for pathogenicity esx1 locus and elsewhere in the genome are also secreted through ESX-1, some of which are co-dependent on EsxBA secretion. The functions of most of the proteins encoded by esx1 genes are unknown, but the overall composition of the esx1 loci between the parental mc2155 and mc2874 strains are similar (see below). Although our previous transposon mutagenesis studies have shown that ESX-1 plays an important role in the process of DNA transfer in both donor and recipient strains, mating-type identity is not reversed in ESX-1 mutants This region is not simply a hot spot for integration of acquired DNA, since the 12 recipient-proficient  transconjugants in esx1D locus, none of the recipient-proficient F1 strains did. Notably, four of the F1 recipient-proficient strains were derived from the Km0.1 parent, in which only 15 kb separate esx1D and the selected rKm gene. Despite this tight linkage, distributive conjugal transfer readily segregated the rKm gene and intact esx1D locus when appropriately screened, thereby augmenting the mapping resolution  further narrow the key conjugal mid gene region. Six F1 donor recombinants were backcrossed with mc2874 recipient derivatives that were marked with a different episomally encoded antibiotic resistance gene (rHyg or rApy) in successive generations. Introgression entailed co-selection for Kmr transfer and the recipient marker to identify transconjugants at each generation (Nx), and then screening progeny for donor proficiency  discard mcficiency . Comparat genome . In eachs 79 kb, .esx1D locus failed to transfer. One recipient strain, Km0.8BC, retained only 75 kb of mc2155 DNA of the 920 kb originally present in the F1 parent .As expected, backcross matings also resulted in recipient-proficient progeny, several of which were also sequenced . Coincid1 parent . Analyse72–0075D . Thus, tesx1 gene products are highly conserved among mycobacterial species, M. smegmatis proteins 0069, 0070, and the N-terminal two-thirds of 0071 have notably low amino acid identity between donor and recipient orthologs , but are not known to encode a mismatch repair system Every facet of the transfer process contributes to the genetic complexity of the transconjugants . The larmid candidate region of esx1 in transconjugants switched to donors  exhibit extremely low genetic variation, suggesting that they do not undergo HGT, are evolutionary young, and resulted from a recent clonal expansion M. canettii, and other smooth-colony MTBC strains, which display genome-wide mosaicism, although the precise mechanism(s) of HGT are unknown M. canettii strains are extant members of a genetically diverse MTBC progenitor species, M. prototuberculosis, whose members underwent frequent HGT M. prototuberculosis offers a plausible and parsimonious explanation for the remarkably similar mosaicism observed among the extant M. canettii. We could envision that distributive conjugal transfer in M. prototuberculosis rapidly incorporated the necessary blend of parental genotypes that drove the emergence of the pathogenic, rough-colony morphology species, like M. tuberculosis, allowing their subsequent clonal expansion. Moreover, if DCT drove these postulated HGT events, the evolutionary clock for M. tuberculosis is likely much shorter because of the capacity of DCT to generate genome-wide mosaicism in a single step. Given the widespread nature of conjugation, we speculate that distributive conjugal transfer also occurs in other bacteria, conferring similar evolutionary benefits.We have demonstrated conjugal DNA transfer in additional naturally derived mid) for mycobacterial conjugation , and still encode functional ESX-1 secretory apparatuses that secrete the major ESX-1 substrates  or protein motifs are necessary and sufficient to impart conjugal sex identity. Interestingly, orthologs for the mid candidate genes are found in the sequenced genomes of other environmental mycobacteria, suggesting a possible ongoing role for distributive conjugal transfer in gene flow between mycobacteria. Orthologs of these mid candidates are not apparent in the esx1 locus of M. tuberculosis, consistent with our speculative model that the MTBC represents a clonally expanded product of distributive conjugal transfer, not necessarily an active participant in this process. Nevertheless, recent evidence from genome sequencing comparisons indicates that some form of genetic exchange has occurred between M. tuberculosis and M. canettiiThe characteristics of mycobacterial distributive conjugation suggested to us that tools developed for mammalian genetics could be applied here. Using a eukaryotic-style genome-wide association mapping approach, we mapped the mating identity locus  in M. smegmatis donor strains were derivatives of the laboratory strain, mc2155 KmR gene inserted at a unique location in the chromosome 2155 genome sequence (http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org=gms), or the draft genome of the recipient (GenBank CM001762). The recipient strain mc2874 M. smegmatis strains were cultured at 37°C in Trypticase Soy Broth with 0.05% Tween80, or on Trypticase Soy Agar (TSA) plates. Antibiotics were added at 100 µg/ml (apramycin), 100 µg/ml (hygromycin), and 10 µg/ml (kanamycin). DNA transfer experiments were carried out as described previously selecting for dual-resistant transconjugants 8 cells 2155 reference sequence by IGS. Single nucleotide polymorphisms (SNPs) or sequence gaps were identified using the Integrative Genomics Viewer (IGV) sequence viewer http://www.ebi.ac.uk/ena/data/view/ERP002619.Transconjugants were colony purified, and genomic DNA was prepared and then subjected to whole-genome DNA sequence analysis at the Institute for Genome Sciences (IGS), U. Maryland, using paired-end Illumina technology. The sequence coverage for each genome was between 50-fold for F1 progeny and ∼1,000-fold for backcross strains. Sequence reads were mapped to the mcFigure S12155 (donor) and mc2874 (recipient). The circular genomes of the parental strains are depicted in linear form and aligned. Genome sequences for mc2874 were obtained by combining reads from one Illumina and two 454 paired-end libraries (GenBank CM001762). Sequence data are deposited in the EBI/ENA database at http://www.ebi.ac.uk/ena/data/view/ERP002619. A de novo build was assembled into a scaffold, and this nucleotide sequence was aligned (Mauve 2.3.1) with the GenBank/JCVI sequence for mc2155 (CP000480.1) Genome collinearity of the parental strains, mc(TIF)Click here for additional data file.Figure S22874 and a transconjugant are shown aligned to the mc2155 reference sequence. A gray bar indicates an individual Illumina sequence read, with the arrow indicating the direction of each read. For simplicity, a depth of 10 reads is shown here, but the average read depth was approximately 50- to 1,000-fold. Gray sequence indicates identity between the sequenced clone and the mc2155 reference genome. Single nucleotide polymorphisms (SNPs) present in sequenced strains appear as colored bases in each read that align vertically with the corresponding polymorphic mc2155 nucleotide. The total SNP content in this 649 bp region is revealed upon mc2874 recipient alignment with the mc2155 reference. Recipient sequence in the transconjugant is conservatively defined by the presence of two consecutive SNPs, and is indicated by the yellow bars below. The left boundary of the replacement donor sequence tract lies between the last recipient SNP present (green) and the next missing SNP (red); as intervening regions match the reference sequence (gray coloration), the donor segment is designated to extend from SNP to SNP, as indicated by the blue bar below. Note that to more clearly discern closely localized donor tracts on the lower resolution Circos plots, successive donor tracts were alternately colored blue or magenta.IGV image illustrating donor/recipient junction assignment in a transconjugant. Sequence reads from the recipient strain mc(TIF)Click here for additional data file.Figure S3Distribution of donor tract sizes identified in transconjugant genomes. The calculated donor-derived tract sizes for the initial 12 transconjugants are graphically displayed (blue bar represents donor segment length).(TIF)Click here for additional data file.Figure S4M. smegmatis. A conceptual translation of the six open reading frames comprising the mid candidate regions defined by the combined mapping approaches were globally aligned using a Needleman-Wunsch algorithm (http://www.ebi.ac.uk/Tools/services/web_emboss_needle/). Immediately below each protein identifier (bold text) are input parameters and output statistics. In each alignment, the upper sequence represents the mc2155 (donor) sequence and the bottom is the mc2874 (recipient) sequence. The degree of amino acid conservation is indicated between paired residues: vertical line , dots (similar), or nothing (dissimilar). Horizontal lines represent gaps created by the algorithm to maintain alignment. Similarity statistics for the divergent N-terminus and conserved C-terminus of Msmeg_0071 are listed separately following the full-length alignment of this protein.Pairwise alignments of Mid candidate protein sequences between donor and recipient strains of (DOCX)Click here for additional data file.Figure S52155 and mc2874 parental strains by Mauve identified 694 in/dels of >18 bp. The 3′ end of the esx1 locus was identified by Mauve as having insertions in mc2155 . Sequence reads aligned to the donor reference viewed in IGV verified that no sequence reads from mc2874  mapped to the mc2155 reference sequence in this region, consistent with in/del status. This ∼9 kb region includes donor genes Ms0069 through Ms0071 and a cluster of defective IS elements (Ms0072–0075), displayed at the bottom of the IGV window. Donor sequences from a donor-proficient transconjugant  have replaced this recipient in/del region, showing that novel sequences can be acquired and incorporated by DCT. Note that reads spanning IS elements in this transconjugant have a lower mapping score (light-shaded reads) because they could map to multiple sites in the genome. Recombination events can occur close to in/del regions, as illustrated by the donor reads in Ms0068 derived from the recipient-proficient transconjugant at the bottom.In/dels are transferred in DCT. Whole genome alignment analysis of the mc(TIF)Click here for additional data file.Figure S6Ms0062, EsxB is not secreted (4) and is found exclusively in the pellet (5). In transconjugant Km0.1c  and the pellet (7) as for wild-type. Protein standards are shown in lane 1 and are listed in kilodaltons.A Western analysis shows that hybrid transconjugants, like their parents, still secrete EsxAB. Culture filtrates and cell pellets were collected as described previously t Km0.1c , which c(TIF)Click here for additional data file.Table S1Primers used to verify transferred donor SNPs in transconjugant genomes. The primers, their genome coordinates, used for each transconjugant are listed. Sanger sequencing of the PCR product verified the presence of uniquely transferred donor SNPs in transconjugant genomes. Multiple informative SNPs present in each amplicon to facilitated unambiguous parental origin identification. Two PCR clones were sequenced from each transconjugant strain to avoid potential complications from PCR errors.(DOCX)Click here for additional data file.Table S22155 derivative with an insertion at coordinate 114 kb. A lower case subscript indicates transconjugants derived from independent crosses using the same parental donor. Donor segments are mapped as where the last consecutive recipient SNP is present (donor begin) to where the next consecutive recipient SNP is detected (donor end). The length of the intervening donor tract (donor size), the total amount of donor DNA in each transconjugant , and the length of recipient DNA separating adjacent donor tracts are shown (separation). The segments containing the selected kanamycin resistance gene are highlighted in green. Totals for the cohort appear at the bottom. Note that donor regions separated by less than 1 kb are boxed in blue highlights to indicate they may be due to multiple, internal recombination events of a larger transferred segment, or a single recombination modified by mismatch repair.Donor and recipient boundary addresses as mapped to the reference sequence. Transconjugant clone name appears along the left margin. Strain nomenclature is based on the genomic location of the Km gene insertion (in Mb); thus, Km0.1 represents the mc(XLSX)Click here for additional data file.Table S38 donor or recipient parental cells.Transfer frequencies of F1 transconjugants. Transfer frequencies are the number of transconjugants divided by the number of donor cells. These frequencies are the average of at least two independent matings, which were carried out in parallel with a positive control Click here for additional data file.Table S4Km gene is also indicated (green shading), and a column listing the esx1 genes, if present in the BC strains, has been added.Donor and recipient boundary addresses as mapped to the reference sequence for donor-proficient F1 strains and their recipient-proficient backcross derivatives. Transconjugant strain nomenclature, and column headings are the same as in (XLS)Click here for additional data file.


Using shRNA-mediated downregulation of WASp in the murine monocyte/macrophage cell line RAW/LR5 (shWASp), we found that WASp was responsible for the initial wave of actin polymerization in response to global stimulation with CSF-1, which in Dictyostelium discoideum amoebae and carcinoma cells has been correlated with the ability to migrate towards chemoattractants. Real-time monitoring of shWASp cells, as well as WASp−/− bone marrow-derived macrophages (BMMs), in response to a CSF-1 gradient revealed that the protrusions from WASp-deficient cells were directional, showing intact directional sensing. However, the protrusions from WASp-deficient cells demonstrated reduced persistence compared to their respective control shRNA and wild-type cells. Further examination showed that tyrosine phosphorylation of WASp was required for both the first wave of actin polymerization following global CSF-1 stimulation and proper directional responses towards CSF-1. Importantly, the PI3K, Rac1 and WAVE2 proteins were incorporated normally in CSF-1 – elicited protrusions in the absence of WASp, suggesting that membrane protrusion driven by the WAVE2 complex signaling is intact. Collectively, these results suggest that WASp and its phosphorylation play critical roles in coordinating the actin cytoskeleton rearrangements necessary for the persistence of protrusions required for directional migration of macrophages towards CSF-1.Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor that is required for macrophages to directionally migrate towards various chemoattractants. The chemotaxis defect of WASp-deficient cells and its activation by Cdc42  Monocytes and macrophages circulate throughout the organism and their ability to be recruited to various sites is crucial for their function as important mediators of innate and adaptive immunity. Cellular migration can be mechanistically described as a sequence of events that repeats until the cell reaches its destination. The initial event in cell migration is polarization and extension of protrusions in the direction of migration de novo Arp2/3 complex dependent actin polymerization In response to migratory stimuli, macrophages polarize and extend F-actin rich protrusions in the direction of the chemotactic gradient in vivoMacrophage colony stimulating factor-1 (CSF-1) is a potent chemoattractant and a pleiotrophic growth factor that stimulates cell survival, proliferation and differentiation of myeloid-derived cells . Commercially available 129/svJ wild-type and WASp2 incubator. Murine RAW/LR5 monocyte/macrophages All cells were maintained at 37°C in a 5% COhttp://rsb.info.nih.gov/ij/) RAW 264.7 derived cell lines were plated on 12 mm glass coverslips and serum-starved for at least 3 hours in RPMI prior to stimulation with CSF-1 at either 37°C or 22°C, followed by fixation and staining according to The kinetics of total F-actin content in response to CSF-1 using a plate reader were quantified according to The micropipette assay was performed based on −/− BMMs (1×106) were deprived of CSF-1 for 14 hours before being loaded onto 3-µm pore size inserts, with media containing CSF-1 in the lower chamber, and incubated at 37°C for 30 min. Pseudopods from the lower surface of the inserts and total cell lysates were obtained by lysis in ice-cold buffer A . Lysates were resolved by SDS-PAGE, proteins were transferred onto PVDF membranes  followed by blocking and incubation in primary antibodies overnight at 4°C. Membranes were blotted with antibodies against β-actin , CD14 , p85 , Rac (C14), WASp , and WAVE2 . Membranes were then washed and incubated with horseradish peroxidase conjugated secondary antibodies against rat, mouse or rabbit IgG . Signals were visualized using the SuperSignal West Pico Chemiluminescent Substrate from Pierce . Images were acquired using a Kodak Image Station 440 and quantified using Kodak 1D Image Analysis Software. Relative protein levels in pseudopods were quantified by densitometry and normalized to CD14 protein levels. Reported values are the average of 3 independent experiments.WT or WASPResults were considered statistically different when analysis using a Student t-test resulted in differences between two means with a p value of less than 0.05. Error bars signify standard error of the mean.Dictyostelium discoideum have found enhanced temporal resolution of actin nucleation following equilibration at reduced temperature CSF-1R stimulation results in a transient and broad increase of newly synthesized F-actin in macrophages Since the observed kinetics of the first wave of increase in F-actin content coincided with our previously published kinetics of WASp activation status in response to CSF-1 Since tyrosine phosphorylation of WASp has been suggested to regulate WASp activity and is required for macrophage migration Since WASp is required for directional migration of macrophages in response to CSF-1 Since WASp phosphorylation is important for chemotaxis the protrusion dynamics in the absence of WASP phosphorylation was examined. Phosphodeficient Y291F cells demonstrated a significant delay in the onset of the protrusions compared to shControl cells as well as to shWASp cells  from wild-type or WASptype BMM  and S5 atype BMM . Of noteype BMMs , albeit −/− BMMs that were extending towards a gradient of CSF-1 . Lammermann and colleagues reported that symmetry breaking and actin polymerization rates in protrusions generated in response to chemotactic cues were comparable between WT and Cdc42−/− DCs. However, in the absence of Cdc42, the directional persistence was diminished and protrusions appeared to retract faster than those of WT DCs Directional sensing is a key response that enables the cells to migrate in the correct direction. While chemoattractant receptors, such as CSF-1R, are uniformly distributed on the cell surface Dictyostelium cells with reduced WASp expression 3 levels remained intact. Our results are consistent with the LEGI-biased excitable model since WASp-deficient macrophages respond to directional CSF-1 stimulation but the protrusions were not maintained.Several models of chemotaxis have been proposed to account for directional sensing and chemotaxis, such as those employing positive feedback loops or local excitation-global inhibition (LEGI) in vitroin vitroin vivo characterization revealed that actin polymerization by Y291F cells in response to CSF-1 was identical to shWASp cells, consistent with the inability of Y291F WASp expressing cells to migrate towards CSF-1 The role of phosphorylation of tyrosine residue 291 of WASp has been studied in detail Dictyostelium amoebae, carcinoma cells and neutrophils Alternatively, WASp may play a role in adhesion of chemotactic protrusions leading to their maintenance since protrusions will often retract if not stabilized by substrate adhesion even in the continued presence of a chemoattractant gradient, as reported in In conclusion, our data show that WASp is required for the first wave of actin assembly in response to CSF-1 that leads to the establishment of persistence of chemotactic protrusions of macrophages. We also show that tyrosine phosphorylation state of WASp plays a role in the initiation of chemotactic protrusion. A key question remains, which is how WASp activity is regulated and maintained in a temporally and spatially controlled manner for successful protrusion persistence and chemotaxis to take place. While further studies will be required to elucidate the molecular mechanism that regulate WASp activity during macrophage migration, the data presented herein highlight the critical role of WASp in the establishment of persistence of protrusion in macrophage chemotaxis.Movie S1Time lapse of shControl RAW/LR5 cells responding to a directional source of CSF-1. The movie corresponds to (MOV)Click here for additional data file.Movie S2Time lapse of shWASp RAW/LR5 cells responding to a directional source of CSF-1. The movie corresponds to (MOV)Click here for additional data file.Movie S3Time lapse of Y291F RAW/LR5 cells responding to a directional source of CSF-1. The movie corresponds to (MOV)Click here for additional data file.Movie S4Time lapse of WT BMMs responding to a directional source of CSF-1. The movie corresponds to (MOV)Click here for additional data file.Movie S5−/− BMMs responding to a directional source of CSF-1.Time lapse of WASp The movie corresponds to (MOV)Click here for additional data file.


Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells.CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry.in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ  and granzyme B  as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF.G-CSF treatment did not significantly influence frequency of antiviral T cells nor their To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production. Peripheral blood stem cells (PBSCs), which can be collected after granulocyte colony-stimulating factor (G-CSF) treatment, have been increasingly used for allogeneic stem cell transplantation in recent years Apart from GvHD, infectious complications of persistent viruses like cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (ADV) remain a problem in vivo and in vitro. To gain insight into the antiviral T-cell repertoire, we determined the frequencies of CMV-, EBV- and ADV-specific T cells in stem cell and HLA-typed platelet donors using overlapping peptide pools and multimer staining. Furthermore, we analyzed clinical data on recipients' and donors' CMV-serostatus and time of first CMV reactivation.To further clarify the effects of G-CSF on donor T cells, we addressed the questions of whether (1) multimer monitoring and detection of interferon-gamma (IFN-γ)-secreting cells by enzyme-linked immunospot (ELISpot)-based technique after short-term antigen stimulation are suitable tools to evaluate antiviral CTLs in various samples from stem cell donors, (2) G-CSF mobilization and apheresis influence antiviral T-cell levels, and (3) G-CSF treatment influences the functional activity of CTLs both Here, we analyze the influence of G-CSF stimulation on antiviral T-cells to obtain (1) better understanding of the kinetics of antiviral reconstitution, (2) determine the most effective subpopulation of antiviral T cells, and (3) select an optimal primary transplant or third-party donor for patients requiring antiviral T-cell therapy early after HSCT.Written informed consent was obtained from all donors and patients. Sample collection and analyses were part of an extended monitoring program conducted in the course of routine sampling for clinical follow-up. The study was approved by the Ethics Committee at the Hannover Medical School and is registered as #2906 for patients and as #1142-2011 for healthy donors. Sample material was obtained from 170 PBSC donors between February 2011 and December 2012. Each donor's HLA type, CMV and EBV serostatus were determined. ADV serology was not available. The following samples were collected: whole blood before G-CSF mobilization (WB), whole blood after G-CSF mobilization on the day of apheresis (WBM), blood from the apheresis tubing set (A) and an aliquot from the graft (G). All samples were processed within 48 h, as longer storage yielded no reliable results .G-CSF mobilization was performed by administering 10 µg/kg/day of G-CSF  for four consecutive days. Peripheral blood samples were collected via venipuncture into collection tubes containing EDTA as an anticoagulant . CD34All reagents/antigens used in this study are abbreviated as follows: multimer (m), peptide (p) or peptide pool (pp)-virus_viral protein_HLA restriction. For example, the multimer containing the HLA-A*02:01-restricted epitope NLVP of CMV protein pp65 is abbreviated “mCMV_pp65_A02”, and the EBNA1 peptide pool is abbreviated “ppEBV_EBNA1”. A nonsense peptide (mNeg) was used to determine unspecific background of multimer staining. A complete list of antigens/reagents is provided in Table S1 in 6 cells were used. Percentages of specific multimer staining were determined by subtracting the non-specific background value (mNeg). Flow cytometric analysis was performed on an FC500  as published previously 100 µl of blood  was stained with HLA-matched multimers (Table S1 in 7 cells per ml in 24-well plates (Sarstedt). G-CSF and/or antigens were added for the various read-out experiments detailed below.PBMCs were isolated from peripheral blood of platelet donors and from HSC donors before (WB) and on the day of donation . Cells were resuspended in T-cell culture medium (T-CM) consisting of RPMI1640  supplemented with 10% heat-inactivated human AB serum  plus 50 U/ml IL-2  and cultured overnight at a density of 1×10+ lymphocyte gate were analyzed, and the proportion of G-CSFR+/CD4+ and G-CSFR+/CD8+ cells was expressed as a percentage of all CD3+ lymphocytes analyzed. Upregulation of G-CSFR was three-fold on CD4+ T cells and 17-fold on CD8+ T cells after in vivo G-CSF treatment (data not shown).Flow cytometric analysis of G-CSF receptor (G-CSFR) expression was done on PBMCs  using peridinin chlorophyll (PercP)-conjugated anti-CD3, fluorescein isothiocyanate (FITC)-conjugated anti-CD4, allophycocyanin (APC)-conjugated anti-CD8 , and R-phycoerythrin (R-PE)-conjugated anti-G-CSFR monoclonal antibodies (mAbs) . Flow cytometric analyses were performed using a FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software (V6.1.2). At least 30,000 events in the CD36 cells per well. Cells were incubated with 10 µg/ml Brefeldin A (Sigma-Aldrich) for 12 hours and stained with CD3-PerCP and IFN-γ-R-PE (Beckmann Coulter). Intracellular staining was performed using the IntraPrep Kit (Beckmann Coulter) according to the manufacturer's instructions. At least 30,000 CD3+ cells were acquired in the lymphocyte gate. The proportion of IFN-γ+/CD3+ cells was analyzed and expressed as a percentage of total CD3+ lymphocytes. The IFN-γ concentration in untreated PBMCs (controls) was used as the reference value (100%). At 10 ng/ml G-CSF a reduction of IFN-γ-producing cells to 39% compared to control cells was detected  7 cells/ml were stimulated in 24-well plates with 10 µg/ml of the respective single HLA-matched peptides (Table S1 in + cells). Supernatants were analyzed for granzyme B secretion by ELISA (eBioscience). The percentages of CD8+ naïve (N), central memory (CM), effector memory (EM) and terminally differentiated effector memory (TEMRA) T-cell subpopulations were analyzed by additional staining with anti-CD62L-APC-Cy7 and anti-CD45RA-PerCP-Cy5.5 mAbs (both BioLegend).Baseline frequencies of CMV- and EBV-specific T cells in freshly isolated PBMCs were assessed by multimer staining prior to peptide stimulation. 1×105 PBMCs  or 1.25×105 PBMCs (after 7-day stimulation) were plated in 100 µl T-CM/well and incubated overnight with 10 µg/ml CEF pool , 10 µg/µl peptide, 10 µg/ml peptide pool or without antigen (negative control). Spots were counted using an ImmunoScan Core Analyzer and the results analyzed using ImmunoSpot 5.0 Academic software . Means of tests were calculated and expressed as spot-forming units (sfu).Virus-specific IFN-γ-producing T lymphocytes were enumerated by IFN-γ ELISpot assay as described previously In order to determine the influence of G-CSF treatment on cytotoxic activity of antiviral T cells, we performed a granzyme B ELISpot assay as described previously. 6 peptide- or peptide-pool-stimulated PBMCs were restimulated with pCMV_pp65_A02, pEBV_BZLF1_B08, ppCMV_pp65 and ppEBV_BZLF1, respectively. Phycoerythrin-Cyanin 7 (PCy7)-conjugated anti-CD107a antibody  was added and cells were cultured at 37°C, 5% CO2. After one hour, Monensin  was added and cells were further incubated for 4 hours before staining with anti-CD3 mAb  and anti-CD8 mAb .Degranulation of antiviral T cells as a surrogate marker for cytotoxicity Statistical analyses were performed using the Mann-Whitney test or Kruskal-Wallis test followed by Dunn's multiple comparison on GraphPad Prism v5.02 software . Levels of significance were expressed as p-values .Healthy G-CSF-mobilized stem donors (n = 170) were included in this study. Serology for CMV was available for 139 donors . EBV serology was available for 121 donors . Determination of ADV serostatus was not possible, as no fast routine assays for detection of various strains are available. Intermediate analysis showed comparable assay results for mobilized samples  differed significantly between the different sample types , mCMV_pp65_B07 (2.46%) and mCMV_pp50_A01 (0.67%) were significantly different between CMV-seropositive and CMV-seronegative individuals  . A similSurprisingly, the mCMV_IE1-A02 gave false positive staining results in CMV-seronegative individuals . In ELISpot analysis, ppBZLF1 yielded the highest spot counts in 32 of 101 donors (53/156 samples).The current study results for dominance of CMV- and EBV-specific epitopes from pp65 and BZLF1, respectively, are in concordance with those from our recent study n vivo G-CSF–mobilized stem cell donors (n = 81) were compared for their IFN-γ production upon stimulation with CMV-, EBV- and ADV-overlapping peptide pools. Analysis of the total number of sfu showed that G-CSF treatment led to a significant reduction in spots for all antigens tested   (T cells from untreated platelet donors (n = 24) and from i5.8 sfu) . This covs. 0.8) .Secretion of IFN-γ by cells from the same donor before and after G-CSF treatment (n = 7) was additionally compared . Again, in vitro with 10 ng/ml G-CSF for one week were evaluated by multimer staining, IFN-γ ELISpot, granzyme B ELISpot, granzyme B ELISA and CD107a degranulation assay. The frequency of multimer-positive T cells obtained after 7 days of peptide stimulation did not differ significantly between G-CSF-treated and untreated cells for the single HLA-restricted peptides used , central memory (CM), effector memory (EM) or terminally differentiated effector memory (TEMRA) CD8+ T cells was observed after peptide-specific stimulation in the presence or absence of G-CSF . While the frequency of multimer+ T cells remained stable, the percentage of IFN-γ secreting cells decreased after G-CSF treatment for pCMVpp65_A02 (63.7%) and pCMVpp65_B35 (45.7%). For EBV, the percentage of IFN-γ producing cells decreased to 83.1% (pEBV_EBNA1_B35) and 50.1% (pEBV_BZLF1_B08).T-cell function was evaluated by IFN-γ ELISpot on day 7 ; shown iThe immunosuppressive effects of G-CSF treatment on the cytotoxic function of T cells were further assessed by detection of granzyme B secretion and expression of lysosome-associated membrane protein CD107a on the cell surface. Granzyme B was detected .in a target-independent ELISA,  as well Follow-up data for seropositive family donors and patients (3/5) showed reconstitution of CMV-CTLs after HSCT, as expected  and donors (D) and the time of first CMV reactivation in order to determine the demand for adoptive therapies. The proportion of high-risk patients with an R+D+ and 103 R+D− patients between June 1998 and June 2012. The mean day of CMV reactivation was day +39 and +40 in R+D+ and R+D− patients, respectively. PBSC transplanted from G-CSF treated donors may still be impaired in their function at this early time of CMV-reactivation Early CMV reactivation (<day +100) occurred in 172 RHematopoietic stem cell transplantation is used to cure many malignant, benign and genetic bone marrow disorders, solid tumors, immunodeficiencies, metabolic diseases and autoimmune disorders It is well known that despite to a one-log increase in the number of T cells transplanted with G-CSF–mobilized PBSCs compared to bone marrow grafts no higher rates of severe aGvHD are associated whereas the incidence of chronic GvHD increases in vivo G-CSF-mobilized T–cell populations suggesting that this cytokine has a direct effect on T cells.G-CSF influences T cells by various mechanisms: (A) It inhibits the secretion of type I cytokines on the single-cell level as well as by reducing the population of cytokine-secreting cells in vitro and in vivo G-CSF treatment, we were able to show that G-CSF mobilization does not affect the frequency of antiviral T lymphocytes, but strongly impairs the functionality by reducing the capability to secrete IFN-γ (40% to 75% reduction) and cytolytic molecules like granzyme B (32% reduction). The reduced cytolytic function as determined by the reduction of granzyme B release in a target cell dependent manner and the decrease of CD107a expression on cell surface of CD8+ T cells further confirmed the negative impact of G-CSF on T-cell functionality (in vitro G-CSF treated PBMCs were not conclusive (data not shown). In order to gain insight into the underlying mechanism in G-CSF-induced impairment of T-cell function, subpopulations of T cells should be investigated separately in a larger cohort.By evaluating the effector function of antiviral T cells after ionality . Prelimiet al.+ G-CSF-mobilized T cells were functionally similar to G-CSF-untreated CD154+ T cells, making this isolation strategy more suitable. However, this finding is based on the comparison of a small number of mobilized and non-mobilized donors and not on a comparison of the same donors before and during G-CSF treatment, as done in this study.Recently, Samuel et al. described the successful isolation of functional CMV-specific T cells from G-CSF-mobilized donors after short-term stimulation using CMVpp65 overlapping peptide pool in vivo. Furthermore, seropositive patients with grafts from seronegative donors were the group at highest risk of (recurrent) CMV reactivation While avoiding additional apheresis by using surplus material from mobilized donors might be beneficial for the donor, it remains unclear whether the G-CSF-treated T cells will achieve control of viral reactivation The duration and characteristics of functional impairment of T-cell functions after G-CSF treatment were investigated in several studies. Toh and colleagues Apart from considerations for adoptive T-cell transfer, the duration of G-CSF influence in each donor, in particular the effects on T-cell function after HSC donation should be monitored to determine when T cells recover. This might be essential for donor safety. There are reports showing that donors suffer from side effects like bone pain, headache and flu-like symptoms in vivo G-CSF treatment. This suggests that cells of G-CSF-treated donors might not be the best choice in cases where donor lymphocyte infusion or adoptive T-cell transfer is required for treatment of viral reactivation early after transplantation. Further studies are needed to determine whether the adoptive transfer of G-CSF-mobilized T cells is comparable to that of cells from non-mobilized donors or mobilized donors in whom the effects of G-CSF have worn off. In order to increase donor safety, the possibility of higher susceptibility to infections after G-CSF treatment should be investigated and the time needed for T-cell recovery should be determined.Our results demonstrate that antiviral T-cell function is impaired after File S1Supporting Information files.Figure S1 in File S1. Effects of sample storage time and temperature. Multimer analysis is usually performed on whole blood samples stored at room temperature within 24 h. As apheresis and graft processing took longer, some samples were analyzed later. Thus, we evaluated the influence of storage time and temperature on the reliability of the results. Nine patient samples were analyzed consecutively to detect changes in CD3, mNeg (non-specific background) and total percentages of mCMV_pp65_A02 and/or mCMV_pp65_B07 over time. Representative examples are shown. Storage time is plotted on the X-axis, and percentages of total CD3 (A), mCMV_pp65_A02 (B) and mCMV_pp65_B07 (C) on the Y-axis. Staining results for fresh samples (unstored) are depicted as a reference line , and those for samples stored at room temperature (RT) and 4°C as filled circles (black line) and white circles (dashed line), respectively. Changes in CD3 and specific multimer percentages over time were less pronounced at 4°C in all sample types. Background staining did not change significantly at 24 h or 48 h (data not shown). After 48 h of storage at 4°C, a mean of 93%  and 96%  of the frequency in the fresh sample (baseline) was obtained. After 72 h, the variation from baseline was more pronounced, even in samples stored at 4°C. For example, in the 5 samples analyzed with mCMV_pp65_A02, only 82% and 30% (mean) of the multimer-positive population detected in fresh material was detected after 72 h of storage at 4°C or room temperature, respectively (Table S2 in File S1). Therefore, donor samples analyzed after more than 48 h (n = 2) were excluded from further analysis. As storage at 4°C led to less deviation from baseline (percentage change), it is preferable. Figure S2 in File S1. Gating strategy. (A) Gating strategy for one-platform quantification of percentage and total numbers of CD3CD8-double positive T cells. From left to right: A gate is set on the fluorescent beads for calculation of absolute cell numbers. Flow Count over time is recorded in order to detect changes in the flow rate. The 3rd plot from the left shows gating on lymphocytes for exclusion of debris and other mononuclear cells. CD4CD8-double-positive cells are excluded. The percentage of CD3+CD4+ and CD3+CD8+ T cells is determined in the lymphocytes (beads and CD4+CD8+ cells excluded) and the absolute number per µL blood can be calculated. For A and G samples, beads were added to confirm constant flow rates and signal intensity. (B) Gating strategy for quantification of multimer-positive T cells. From top to bottom, staining in all 3 sample types is shown. The percentage of multimer positive cells is given as percentage of CD3+CD8+ T cells. FSc – forward scatter, SSc – side scatter, FITC - fluorescein isothiocyanate, PE - Phycoerythrin, PCy7 – Phycoerythrin-Cyanin 7, WBM – whole blood mobilized, A – material from apharesis filter, G – material from graft quality control, Lymph – lymphocyte gate. For multimer abbreviations, please refer to Table S1 in File S1. Figure S3 in File S1. In vitro application of G-CSF reduces IFN-γ production in PBMCs. The optimal G-CSF concentration for stimulation was determined by treating PBMCs with different concentrations (5 to 50 ng/ml) of G-CSF for one week and subsequent stimulation with anti-CD3 monoclonal antibodies on day 7. IFN-γ production was measured by intracellular staining and flow cytometry. Results of four independent experiments are shown and expressed as mean ± SD. Asterisks indicate significant differences (**p<0.01). The IFN-γ production in untreated PBMCs (controls) was used as the reference value (100%). The percentage of IFN-γ-expressing cells decreased after G-CSF. A strong effect was achieved using 10 ng/ml G-CSF. At this dose, only 38.73% of IFN-γ-producing cells were retrieved on day 7. Because there was no significant difference after treatment with higher concentrations of G-CSF, 10 ng/ml G-CSF was used in further experiments. Figure S4 in File S1. Staining with specific multimers is not significantly influenced by sample type. (A) Unspecific background staining (mNeg): The background detected (mNeg) is shown for each donor sample and the sample type is indicated on the X-axis. Groups were compared by Kruskal-Wallis analysis followed by Dunn's multiple comparison. (B) Exemplary analysis of mCMV_pp65_A02 staining in different samples  in CMV-seropositive donors. (C–E) Blots C to E show the comparison of detection of multimer positive cells in the mobilized samples  to freshly isolated whole blood (WB). Percentages detected in the mobilized samples only differed slightly from those in WB samples. Sample sources: (A) apheresis tubing set, (G) graft, (WB) whole blood, (WBM) mobilized whole blood on day of apheresis. Figure S5 in File S1. Multimer staining examples. Examples for multimer staining in the different sample types  in 5 donors are depicted. The total sum of multimer positive CD3+CD8+ T cells after substraction of background (mNeg) for CMV (ΣmCMV) and EBV (ΣmEBV) is given. These values are also reported in +CD8+ T cells. Figure S6 in File S1. Multimer staining with mCMV_pp65_A02 (tetramer) and mCMV_IE1_A02 (pentamer). Staining examples in CMV-seropositive  and CMV-seronegative  donors are given, for each donor, all 3 samples types are shown . Staining with mCMV_pp65_A02 (tetramer) gives clear populations in CMV+ donors and low background in CMV-donors. In contrast, staining with mCMV_IE1_A02 (pentamer) results in less defined populations, furthermore positive staining results are found in CMV-donors . Figure S7 in File S1. Functional assays for in vitro G-CSF-treated pCMV_pp65_A02-stimulated cells on day 7. (A) IFN-γ ELISpot reveals a decrease in IFN-γ secretion after G-CSF treatment. Unstimulated cells served as control. Means of spots per well (spw) are indicated. (B) Granzyme B ELISpot shows a reduction in granzyme B secretion in a target cell dependent manner. Unloaded target cells served as a control. Means of spw are indicated. (C) Representative staining results from the CD107a degranulation assay show a reduced degranulation activity of CD8+ T cells after G-CSF treatment. +/D+ patients.Figure S8 in File S1. Follow up in R (A–C) Number of CMV-CTLs detected in the patient and donor before HSCT and post-HSCT follow-up in patients UPN2104 (A), UPN2131 (B) and UPN2145 (C). CMV reactivation led to expansion of CMV-CTL in UPN2145 (C). Figure S9 in File S1. Overview of CMV-serostatus of recipients and donors at Hannover Medical School (MHH). Number of CMV-seropositive patients with seropositive donors  and seronegative donors  per year. Table S1 in File S1. Overview of multimers and antigens used.Table S2 in File S1. Variation of total multimer-positive population from baseline (fresh samples) after 24, 48 and 72 hoursof storage at 4°C or room temperature (RT).Table S3 in File S1. Donors with no, low and high percentages of multimer-positive T cells.Table S4 in File S1. Sequence alignment for the A02-restricted IE-1 epitope VLAELVKQI.(DOC)Click here for additional data file.


We have extended this analysis by comparing the relative hematopoietic potencies of G-CSF/IgG1-Fc to G-CSF in normal mice and to G-CSF and polyethylene glycol (PEG) - modified G-CSF in neutropenic rats. Mice were treated for 5 days using different doses and dosing regimens of G-CSF/IgG1-Fc or G-CSF and circulating neutrophil levels in the animals measured on Day 6. G-CSF/IgG1-Fc stimulated greater increases in blood neutrophils than comparable doses of G-CSF when administered using daily, every other day or every third day dosing regimens. In rats made neutropenic with cyclophosphamide, G-CSF/IgG1-Fc accelerated recovery of blood neutrophils to normal levels (from Day 9 to Day 5) when administered as 5 daily injections or as a single injection on Day 1. By contrast, G-CSF accelerated neutrophil recovery when administered as 5 daily injections, but not when administered as a single injection. G-CSF/IgG1-Fc was as effective as PEG-G-CSF at accelerating neutrophil recovery following a single injection in neutropenic rats. G-CSF/IgG1-Fc and G-CSF/IgG4-Fc fusion proteins in which the 7 amino acid linker was deleted also were effective at accelerating neutrophil recovery following a single injection in neutropenic rats. These studies confirm the enhanced in vivo hematopoietic properties of G-CSF/IgG-Fc fusion proteins.Previously we showed that granulocyte colony-stimulating factor (G-CSF)  Granulocyte colony-stimulating factor (G-CSF) is a 19 kDa protein that stimulates the proliferation, differentiation and functional activation of cells of the granulocyte lineage. Recombinant methionyl-G-CSF is widely used to ameliorate neutropenia following myelosuppressive chemotherapy and bone marrow transplantation, and to mobilize peripheral blood progenitor cells for transplantation and blood banking in vivo, which allows the protein to be effective when administered once per chemotherapy cycle  to cancer patients A number of technologies have been used to improve the pharmacokinetic properties of G-CSF, including modifying the protein with polyethylene glycol (PEG) and fusion of G-CSF to other proteins with long half-lives in vivo analysis of recombinant fusion proteins comprising G-CSF joined to the Fc (Hinge-CH2-CH3) domains of human IgG1 and IgG4 in vitro cell proliferation assay. The fusion proteins possessed ∼8-fold longer half-lives than G-CSF and stimulated greater and longer lasting increases in circulating neutrophils than G-CSF following a single injection in normal rats. Here we build upon these earlier studies by comparing relative in vivo potencies of G-CSF and G-CSF/IgG1-Fc in normal mice and G-CSF, PEG-G-CSF and G-CSF/IgG1-Fc in neutropenic rats. The effects of IgG isotype and the requirement for the flexible peptide linker on in vitro and in vivo bioactivities of the G-CSF fusion proteins also are explored.In a previous report we described the construction and preliminary 2 inhalation.All animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Protocols were approved by BolderPATH's Institutional Animal Care and Use Committee. To alleviate pain, rats and mice were anesthetized by inhalation of isoflurane prior to taking blood samples. Animals were euthanized by COIn vitro bioactivities of the fusion proteins were measured in a 3-day cell proliferation assay using the mouse NFS-60 cell line In vitro bioactivity experiments were performed in triplicate wells for each protein dilution and were repeated at least 3 times for each protein. Recombinant human methionyl-G-CSF  was used as the G-CSF standard in the bioassays.Plasmids encoding G-CSF/IgG1-Fc and G-CSF/IgG4-Fc fusion proteins containing a 7 amino acid peptide linker (SerGlyGlySerGlyGlySer) joining G-CSF to the IgG-Fc domains have been described Protein samples for purity analyses were prepared in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer with (reducing) or without (non-reducing) the addition of 1-5% (v/v) 2-mercaptoethanol, electrophoresed on precast Tris-glycine polyacrylamide gels  and stained with Coomassie Blue (Bio-Rad Laboratories).Female ICR mice, weighing 15–20 g at study initiation, were obtained from Harlan-Sprague Dawley . Animals were acclimated for at least 7 days before study initiation. One group of 5 mice was sacrificed on Day 0 to provide an untreated control group complete blood cell count (CBC). The remaining mice were randomized to different test groups, consisting of 5 mice each. Mice received subcutaneous injections of vehicle solution , methionyl-G-CSF  or G-CSF/IgG1-Fc according to different dosing schedules. The protein concentration of Neupogen and the fusion proteins were measured using the Bradford dye binding assay utilizing bovine serum albumin as a standard. Animals were treated using an every day dosing regimen (injections on Days 1–5), an every other day dosing schedule  or an every third day dosing schedule (injections on Days 1 and 4). On Day 6 the animals were sacrificed and a terminal blood sample was obtained using EDTA anticoagulant tubes. A portion of the blood samples was sent to Antech Diagnostics, Inc.  for CBC analyses. Statistical analyses between groups were done by comparing group means using the Student's t-test with significance set at p≤0.05. The spleen and sternum were removed, weighed, and fixed in 10% neutral buffered formalin for histopathologic evaluation. Relative spleen weight was calculated by multiplying the spleen weight by 100 and dividing by the body weight. The sternums were decalcified in 5% formic acid and processed along with the spleens for histopathologic evaluation of sections stained with hematoxylin and eosin. Bone marrow and spleen sections were examined without prior knowledge of the treatment. Spleen sections were examined qualitatively. Bone marrow sections were examined under low and high power for qualitative differences in numbers of myeloid and erythroid precursor cells in order to obtain an estimate of the myeloid:erythroid cell ratio (M:E ratio). In some experiments, M:E ratios in bone marrow sections were determined quantitatively by performing an approximate 200 cell differential count. Cells were counted at 400× magnification along a linear guide in the ocular field of view. Approximately 3–5 random fields were counted from different vertebrate levels to ensure random sampling until 200 cells were obtained. Statistical analyses between groups were done by comparing group means using the Student's t-test with significance set at p≤0.05.Sprague-Dawley rats, weighing ∼200 g, were obtained from Harlan-Sprague Dawley. Rats were allowed to acclimate for at least 7 days before study initiation. Rats were randomized to different test groups based upon their Day 0 neutrophil counts. There were 5 rats per test group. All but one of the test groups received an intraperitoneal dose of 100 mg/kg of cyclophosphamide (CPA) on Day 0 to induce neutropenia. One group of control rats did not receive CPA. During the next 1–5 days the different test groups received subcutaneous injections of vehicle solution , 100 μg/kg methionyl-G-CSF , 100 μg/kg of various G-CSF/IgG-Fc fusion proteins, or 100 μg/kg PEG-methionyl-G-CSF , using different dosing schedules depending upon the study design. The protein concentration of Neulasta, Neupogen and the fusion proteins were measured using the Bradford dye binding assay utilizing bovine serum albumin as a standard. Protein samples were prepared in vehicle solution. Blood samples (0.4 mL) were collected from the rats using EDTA anticoagulant tubes on Day 0, Days 1–10 and Day 12 and sent to Antech Diagnostics to perform CBC analyses. Statistical analyses between groups were done by comparing group means using the Student's t-test with significance set at p≤0.05in vitro bioactivities as G-CSF, as measured by proliferation of the G-CSF–dependent mouse NFS-60 cell line. Because the peptide linkers are non-natural amino acids and could potentially be immunogenic in humans new genes encoding fusion proteins in which the carboy-terminal amino acid of G-CSF (Proline-174) is joined directly to the amino-terminal amino acid (Glutamic acid-1) of the IgG1-Fc and IgG4-Fc domains were constructed in vitro bioactivities, as measured by their ability to stimulate proliferation of NFS-60 cells. The EC50s for the G-CSF/IgG1-FcD and G-CSF/IgG4-FcD fusion proteins in this bioassay were 45±12 and 47±12 pg/mL, respectively, similar to the values reported previously for G-CSF/IgG1-FcL (38.3±4.0 pg/mL) and G-CSF/IgG4-FcL (56.7±5.9 pg/mL) 50 of 22±1 pg/mL in this bioassay, similar to the EC50 value of 17.7±0.6 ng/mL reported previously by us in the NFS60 cell assay in vitro bioactivities, since G-CSF contributes about 40% of the mass of the fusion proteins. These data indicated that G-CSF/IgG-Fc in vitro bioactivity is not appreciably affected by the presence or absence of the peptide linker joining G-CSF to the IgG-Fc domains.The construction of genes encoding G-CSF/IgG-Fc fusion proteins in which the carboxy-terminus of human G-CSF is joined via a 7 amino acid peptide linker (SerGlyGlySerGlyGlySer) to the amino-terminus of the Fc domain of human IgG1 or IgG4 was described previously in vivo potencies of G-CSF and G-CSF/IgG-Fc fusion proteins, we compared the hematopoietic properties of recombinant G-CSF  and a representative G-CSF/IgG-Fc fusion protein, G-CSF/IgG1-FcL, in normal mice, employing methods similar to those used by others to measure relative potencies of various G-CSF analogs To gain a better understanding of the relative Mice treated every day (Days 1–5) with 0.8, 4, 20 or 100 μg/kg of G-CSF/IgG1-FcL showed dose-dependent increases in circulating neutrophils, white blood cells and lymphocytes . Mice trMice receiving every other day  injections of G-CSF/IgG1-FcL also showed dose-dependent increases in circulating neutrophils, white blood cells and lymphocytes on Day 6 . Mice reEvery third day (Days 1 and 4) administration of 100 μg/kg of G-CSF/IgG1-FcL or G-CSF to mice did not significantly increase blood neutrophil levels above those seen in animals receiving vehicle solution, although bone marrow M:E ratios were mildly elevated in the animals on Day 6 . HoweverThe primary clinical use for G-CSF is the acceleration of neutrophil recovery following myelosuppressive chemotherapy in cancer patients in vivo bioactivities of the different G-CSF/IgG-Fc fusion proteins in this clinically relevant model. The design of this study was identical to that described above except that the rats received only a single injection of the test proteins (100 μg protein/kg) on Day 1 following CPA treatment. Control rats received a single injection of vehicle solution. As shown in . A single injection of any of G-CSF/IgG-Fc fusion proteins also caused neutrophil counts to return to normal levels by about Day 5, similar to PEG-G-CSF. Animals treated with G-CSF/IgG4-FcD had the highest neutrophil counts on Days 5-8. Neutrophil levels were significantly (p<0.05) higher on Days 5-8 in animals treated with G-CSF/IgG4-FcD compared to animals treated with PEG-G-CSF. White blood cell levels in the PEG-G-CSF treatment group and all of the G-CSF/IgG-Fc treatment groups began to increase by Day 5 and the increase was more rapid than in vehicle- or G-CSF-treated animals. White blood cell counts in the PEG-G-CSF treatment group and all of the G-CSF/IgG-Fc treatment groups were significantly higher than white blood cell levels in vehicle-treated animals on days 5-8. Only animals treated with the G-CSF/IgG4-FcD fusion protein recovered white blood cell levels to Day 0 values. By Day 10 white blood cell levels were similar in all test groups treated with CPA, and this level was below the Day 0 level, largely reflecting a deficit in lymphocytes  in cancer patients in vitro bioactivity, but does significantly increase G-CSF potency in vivoin vitro or in vivo bioactivities of the fusion proteins. G-CSF has a 4-helix bundle structure shared by many cytokines and growth factors in vitro bioactivities, indicating that the linker is superfluous and not necessary to preserve G-CSF bioactivity. These data support the notion that the extreme carboxy-terminus of G-CSF is dispensable for G-CSF bioactivity. This conclusion is also supported by the finding that large PEGs can be attached to the C-terminus of G-CSF without significantly altering G-CSF in vitro bioactivity in vitro or in vivo bioactivity.These studies confirm and extend our previous finding that the fusion of G-CSF to human IgG-Fc domains does not appreciably affect G-CSF in vivo potencies of G-CSF/IgG-Fc fusions with G-CSF in normal mice and neutropenic rats. Titration experiments in mice using different doses of G-CSF/IgG1-FcL and a fixed dose of G-CSF suggested that only about 20% as much G-CSF/IgG1-FcL by weight was required to produce equivalent increases in circulating neutrophils and white blood cells as G-CSF using an every-day dosing regimen. G-CSF/IgG1-FcL was more effective than G-CSF at stimulating granulopoiesis in mice when less frequent dosing regimens were employed, presumably due to its longer half-life. Whereas G-CSF was able to stimulate increases in Day 6 neutrophil levels in mice when administered every day (on Days 1-5) it was not effective when administered every other day  or every third day (on Days 1 and 4). In contrast, G-CSF/IgG-FcL stimulated Day 6 neutrophil levels when administered every day, every other day or every third day.G-CSF/IgG1-FcL was used as a representative G-CSF/IgG-Fc fusion protein to compare relative G-CSF/IgG1-FcL also proved significantly more potent than G-CSF at accelerating recovery from neutropenia in CPA-treated rats, which is a model for chemotherapy-induced neutropenia in cancer patients, the largest clinical indication for G-CSF. G-CSF typically is administered to cancer patients by daily sc injection beginning 24 hr following chemotherapy and continuing for 10–14 days until neutrophil counts have returned to normal levels PEG-G-CSF has captured a dominant share of the cancer neutropenia market due to its more convenient once per chemotherapy cycle dosing regimen in vivo potency compared to G-CSF in vivo as G-CSF/IgG1-FcL in the single injection rat neutropenia study. The G-CSF/IgG-Fc direct fusion proteins contain only human protein sequences and thus, at least theoretically, have less potential for being immunogenic in humans than the fusion proteins containing the non-natural linker sequences. For this reason, the direct fusion proteins may be preferred human clinical development candidates. Testing immunogenicity of the different fusion proteins is beyond the scope of this study, as it would require performing human clinical trials with multiple G-CSF/IgG-Fc fusion proteins. Although immunogenicity of the different G-CSF/IgG-Fc fusion proteins could be compared in animals, such studies are of limited value because animals typically develop antibodies to human proteins and the results are not always predictive of immunogenicity of the proteins in humans in vivo and may lead to the development of a new class of long-acting G-CSF analogs for treating human diseases.The G-CSF/IgG1-FcL protein has a 5- to 8-fold longer half-life than G-CSF, which accounts for its greater


Acute lymphoblastic leukemia (ALL) is a pediatric malignancy associated with remissions and relapses. Common relapsing sitesare meninges, testis and ovary. Testicular scintigraphy is a highly specific modality used mainly in the differential diagnosis of testicular torsion and epidydimitis/epidydimo-orchitis. There is only one interesting image on leukemic infiltration with scrotal scintigraphy in the literature. The aim of this case presentation is to report that although the scintigraphic appearance of testicular torsion was observed in a patient with the diagnosis of ALL, testicular ALL infiltration was revealed in pathologic examination. Conflict of interest:None declared. Scrotal mass lesions may present with or without pain. Testicular and extra-testicular painless non-tumoral lesions are benign pathologies such as varicocele and spermatocele. Those pathologies account for more than half of the scrotal pathologies in pediatric age. Diagnosis of these diseases is made with physical examination and simple radiological imaging studies. Painful testicular mass lesions may result from many various causes ranging from torsion of spermatic cord to trauma and intra-tumoral hemorrhage ,2.Acute lymphoblastic leukemia (ALL) accounts for approximately 30% of pediatric tumors . In ALL,Ultrasonography is among the first-line diagnostic methods in scrotal lesions associated with pain. Moreover, testicular scintigraphy and sometimes magnetic resonance imaging (MRI) can be used in differential diagnosis of testicular torsion, epidydimitis, abscess and tumoral lesions in case of clinical necessity.In this current case report, we are presenting a patient with ALL who experienced severe pain in his testis. He had a scintigraphic appearance of testicular torsion, but histopathology revealed testicular ALL infiltration.A 14-year old male patient was followed up with the diagnosis of ALL since 2007 and had bone marrow transplantation after central nervous system (CNS) and bone marrow relapse. He was referred to pediatric emergency department because of severe pain in his left testis for 2 days.3 μL, PLT:185x103 μL, Na:133 mmol/L, K:2,6 mmol/L, Ca:5.4 mg/dL and Mg:0.3 mg/dL and significant electrolyte imbalance was found. Fluid replacement therapy was started in the emergency department. Clinically, cutaneous and gastrointestinal graft-versus-host-disease (GVHD) and testis involvement/testis torsion were considered. In the physical examination of the patient, diffuse rashes throughout the body and diffuse tenderness in abdominal supra-pubic region were observed. He also had abdominal pain and diarrhea. Laboratory values were Hgb:12.9 g/dL, WBC:7.3x10US examination was performed for the pain in his left testis. Right testis was 28x26 mm in size and parenchymal echogenity was normal. Left testis was 35x30 mm with decreased and heterogeneous parenchymal echogenity. Increased vascularity was observed in structures surrounding the left testis and at the left epidydimis. Radiological diagnoses of ALL infiltration and/or inflammation-epidydimo-orchitis in left testis were considered, but definite diagnosis could not be made.The patient was referred to the nuclear medicine department to have a testicular scintigraphy in order to eliminate testicular torsion.In the physical examination of the patient in nuclear medicine department, apparent swelling and hyperemia were observed in the left testis. Palpation was associated with severe pain and rigidity was noted in left testis.Penis was elevated upwards and fixed to pelvic bone with adhesive band. Imaging was performed with Low Energy General Purpose (LEGP) collimator. Following intravenous administration of 10 mCi Tc-99m pertechnetate, dynamic perfusion images were obtained in order to examine blood supply of testis, followed by pinhole and static images. In dynamic images, perfusion in the area suggestive of left testis was decreased. In static images, the left testicular area was hypoactive and a hyperactive halo surrounding this area was noted . Those fTesticular leukemic infiltration is diagnosed with the presence of leukemic cells in testicular interstitial tissue. Wakasa et al. histopathologically demonstrated that leukemic infiltration can be diffuse or in the form of patches in perivascular area and interstitium of the testis . In theiCalama reported in a series of 653 cases aged 0-18 years that testicular hematopoiesis could be at the rate of 26.5 percent . In the Testicular torsion is among emergencies of nuclear medicine and this condition is not rare. Occlusion occurs in vascular structures supplying blood to the testis when spermatic cord twists, resulting in testicular ischemia.Testicular torsion can be differentiated from other testicular pathologies using testicular scintigraphy at the rate of 90 percent . In a noUltrasonography is among the first-line imaging methods in patients under follow-up for scrotal lesions ,2. TestiFor patients presented with suspected testicular torsion, there are publications recommending that ultrasonography and radionuclide scintigraphy is performed as soon as possible after patient is examined .US may sometimes be inadequate to differentiate vascular diseases, inflammatory process and solid lesions. In such cases, MRI is used. MRI not only evaluates extent of existent disease, but it may also more clearly differentiate solid and cystic lesions and vascular abnormalities .There is only one interesting image on leukemic infiltration with scrotal scintigraphy in the literature. They reported that in scrotal scintigraphy, the appearance of ALL infiltration may be similar to that of epidydimo-orchitis .Since there was occlusion secondary to leukemic infiltration originating from ALL in testicular vessels in the case presented, vascular block has occurred in vessels supplying blood to testis and therefore, decrease in blood supply was observed in testicular scintigraphy.In this case report, we reported that an appearance resembling testicular torsion may occur as a result of thrombosis in vascular structures due to testicular ALL infiltration, which should be kept in mind while evaluating testicular scintigraphy.