Despite the advent of targeted therapies and novel agents, allogeneic hematopoietic stem cell transplantation remains the only curative modality in the management of hematologic disorders. The necessity to find an HLA-matched related donor is a major obstacle that compromises the widespread application and development of this field. Matched unrelated donors and umbilical cord blood have emerged as alternative sources of donor stem cells; however, the cost of maintaining donor registries and cord blood banks is very high and even impractical in developing countries. Almost every patient has an HLA haploidentical relative in the family, meaning that haploidentical donors are potential sources of stem cells, especially in situations where cord blood or matched unrelated donors are not easily available. Due to the high rates of graft failure and graft-versus-host disease, haploidentical transplant was not considered a feasible option up until the late 20th century, when strategies such as “megadose stem cell infusions” and posttransplantation immunosuppression with cyclophosphamide showed the ability to overcome the HLA disparity barrier and significantly improve the rates of engraftment and reduce the incidence and severity of graft-versus-host disease. Newer technologies of graft manipulation have also yielded the same effects in addition to preserving the antileukemic cells in the donor graft. Today's age represents a truly unprecedented time in care of the patient with a hematologic malignancy with the advent of several targeted therapies and novel agents. These therapies have shown tremendous efficacy in treating several malignancies, yielding impressive response rates with limited toxicity profiles . DespiteUnrelated umbilical cord blood (UCB) represents another alternate source of stem cells, one which has been proven to be successful when performing allo-HSCT –10. Key On the other hand, haploidentical related donors offer several key advantages over MUDs and UCB. Almost all patients have at least one haploidentical related donor in the family and the best donor amongst all candidate family members can be selected. Haploidentical related donors are readily available and are highly motivated to donate to a family member , 19. DueThe HLA mismatch between the haploidentical donor and the recipient offers a potent graft-versus-tumor (GvT) effect to completely eradicate malignant cells and offer a permanent cure . Some stSeveral attempts to perform haplo-HSCT using T lymphocyte replete, unmanipulated grafts have been made. Since donor-derived T lymphocytes are the fundamental players in the pathogenesis of GvHD, these attempts have been associated with early and severe multiorgan failure and GvHD, leading to alarming rates of transplant-related mortality despite pharmacological GvHD prophylaxis . To reduThere is a key difference between positive selection of CD34+ progenitor cells and negative depletion of lymphocytes during graft manipulation. With CD34+ selection strategies, almost no cells other than the CD34+ stem cells are transplanted. On the other hand, negative depletion techniques (mainly CD3+ and CD19+ negative depletion) allows for other types of cells, such as dendritic cells, natural-killer (NK) cells, and monocytes, to be retained in the donor graft and transplanted with the CD34+ progenitors. Both graft manipulation techniques result in effective removal of B and T lymphocytes, leading to greatly reduced risks of developing posttransplant Epstein-Barr virus related lymphoproliferative disorders (B lymphocyte depletion) and GvHD (T lymphocyte depletion) , 33. TheNK cell activity is modulated by the interaction between killer cell immunoglobulin-like receptor (KIR) expressed on the surface of NK cells and cognate ligands  to KIRs . Engagemαβ+ T lymphocytes and CD19+ cells (TCRαβ+/CD19+ depletion) from donor grafts [γδ+ T lymphocytes in the graft. γδ+ T lymphocytes constitute approximately 5% of circulating T lymphocytes. These cells are nonalloreactive and exhibit potent antitumor and anti-infectious properties [γδ+ T lymphocyte counts after allo-HSCT show better survival with reduced incidence of relapse and GvHD [αβ+ T lymphocytes which act against alloantigens via MHC interactions and give rise to GvHD after allo-HSCT [αβ+/CD19+ depletion technique allows for satisfactory removal of αβ+ T lymphocytes to prevent GvHD while retaining γδ+ T lymphocytes to ensure timely immune reconstitution and a robust GvT effect, especially after haplo-HSCT [αβ+/CD19+ depletion in pediatric patients showed acceptable rates of GvHD (22%). In all but one patient, GvHD was grade 2 and responded to first-line therapy. Graft failure was observed in 27% of patients, all of whom were successfully retransplanted with different rescue protocols. The overall survival of patients from this study was 96.7%, advocating the efficacy of TCRαβ+/CD19+ depletion in improving outcomes after haplo-HSCT.Another fundamental difference between CD34+ selection and negative depletion of lymphocytes (CD3+/CD19+ depletion) is that the number of alloreactive lymphocytes “contaminating” the donor graft is approximately ten times higher in CD3+/CD19+ depletion. This necessitates the use of pharmacological GvHD prophylaxis after transplants using CD3+/CD19+ depleted grafts , 40. Howr grafts , 44. Thioperties , 46. Thiand GvHD . In contllo-HSCT . The TCRplo-HSCT , 49, 50.plo-HSCT  reportinα) and interferon gamma (IFNγ). These inflammatory mediators, and several others, play an integral role in the pathophysiology of GvHD [During the 1980s, outcomes of haplo-HSCT were discouraging due to severe GvHD and graft failure, often approaching rates as high as 90% , 53. The of GvHD , which m of GvHD , 63. The of GvHD , 57, 64.Nonmyeloablative conditioning (NMAC) regimens are an attractive alternative for patients requiring an allo-HSCT who are not considered suitable candidates for MAC regimens due to advanced age, comorbidities, and/or increased risks of therapy-related adverse effects . Using NCyclophosphamide is a highly immunosuppressive alkylating agent with an established role in anticancer chemotherapy and HSCT . HistoriA relatively newer strategy to increase the success rates of haplo-HSCT is the use of T regulatory cells (Tregs). Tregs are a specialized subset of CD4+ lymphocytes with concurrent expression of CD25 and FOXP3 genes which play crucial roles in antitumor immunity, posttransplant tolerance, and protection against autoimmunity –77. In a Aspergillus and Toxoplasma appeared significantly earlier in comparison to historical controls comprising standard haplo-HSCT recipients. At a median follow-up of 21 months, 46.1% were alive and in remission [Di Ianni et al.  performeemission .Tregs can be isolated in vitro using standard donor-derived leukapheresis products using double negative selection with anti-CD8 and anti-CD19 monoclonal antibodies and positive selection for CD25 . ExpressWith regard to Tregs, posttransplant GvHD prophylaxis may also play a role in selectively favoring the expansion of Tregs while suppressing mature effector T lymphocytes. Sirolimus is an immunosuppressant that demonstrates such properties . PeccatoIn conclusion, allo-HSCT remains the only treatment modality that offers a potential cure for some with hematologic disorders. HLA-matched donors are not always available, which has led to establishment of UCB and MUDs as alternative sources for stem cell transplantation. However, the high cost of maintaining donor registries and cord blood banks, limited size of the cord blood units, and high-risk nature of the ablative procedure limit the expansion of these donor sources as well. HLA haploidentical donors are an attractive choice for stem cells since nearly every patient has an available haploidentical donor from their family. In modern medicine, new strategies such as “megadose stem cell infusions” and posttransplantation immunosuppression with cyclophosphamide have shown the ability to overcome graft failure and GvHD, complications that occurred at overwhelmingly high rates and severely limited the application of haploidentical transplantation before the 21st century. Other strategies such as NMAC regimens prior to transplant and pretransplant graft manipulation have demonstrated similar survival rates when compared with HLA-matched allo-HSCT with MAC regimens, but with significantly reduced toxicities associated with HSCT and preservation of the antileukemic properties of immune cells in the donor graft. Future prospective studies to explore the biology of the GvT effects in this setting can provide more perspective on the advantages and drawbacks of haplo-HSCT over “traditional” allo-HSCT, suggest further enhancements of the process, and move this “now-controversial” modality into the standard of care for patients with limited options.


K, G, E, 3B, F, 2D, F, and 1E, carrying several mutations in antigenic and host determinant sites. Also, our findings show that the genome-wide genetic variations within the H3N2 CIVs are low; however, two antigenic protein (HA and NA) analysis demonstrates genetic diversification of the H3N2 CIVs, which evolves independently between Korea and China.Canine influenza A virus (CIV) causes a respiratory disease among dog populations and is prevalent in North America and Asia. Recently, Asian H3N2 CIV infection has been of particular concern, with recent reports related to reassortants with pandemic 2009 strains, direct transmission from a human H3N2, a possibility of H3N2 CIV transmission to other mammals, and even the first outbreak of H3N2 CIVs in North America in April 2015. However, despite these global concerns, our understanding of how influenza A virus transmission impacts the overall populations of H3N2 CIVs remains incomplete. Hence, we investigated the evolutionary history of the most recent two Korean CIV isolates, A/canine/Korea/BD-1/2013 and A/canine/Korea/DG1/2014, along with 57 worldwide CIVs, using comprehensive molecular analyses based on genomic genotyping. This study presents that the new Korean CIV isolates are closely related to the predominantly circulating H3N2 CIVs with genotypes The online version of this article (doi:10.1007/s11262-015-1274-x) contains supplementary material, which is available to authorized users. Influenza A virus (IAV) is highly contagious and causes respiratory diseases with symptoms ranging from minor to severe, depending on the host and the subtype of IAV  . Like a Since the first report of two different types of canine influenza virus (CIV)—H3N2 in the Republic of Korea as early as 2005  and a maUntil earlier this year, the geographic distribution of H3N2 CIV had been limited to Asian regions, such as the Republic of Korea, China, and Thailand , 19, 20;Further, the pandemic 2009 H1N1 and human H3N2 reassortants have occasionally jumped the host barrier, causing H3N2 CIV-pandemic mixed strains or complete transmission strains from human H3N2 –30. The To date, phylogenetic studies and genetic analyses of H3N2 CIV have been broadly conducted –20; howeThis study was performed with the following specific objectives: (1) to reveal the evolutionary history of two recent Korean H3N2 CIVs, and (2) to investigate the global distribution and prevalence of the extant H3N2 CIV genotypes using various molecular analyses.In this study, two Korean CIVs were collected from nasal swabs of pet dogs at animal hospitals in Seongnam in 2013 (A/canine/Korea/BD-1/2013) and in Daegu in 2014 (A/canine/Korea/DG1/2014), respectively. For the virus isolation, the IAVs were propagated in 10-day-old embryonated specific pathogen-free (SPF) chicken eggs , and 3 days later, the allantoic fluid was harvested. The procedure was conducted according to the instructions from World Organization for Animal Health (OIE) terrestrial manual  and viruTo amplify the eight viral segments of the two Korean CIV isolates, universal primers from Hoffmann et al. (2001)  were usehttp://www.fludb.org), with representatives of the different subtypes of CIVs  (Supporting Table S1). Each segment was aligned with two newly isolated H3N2 Korean CIVs, using MAFFT, version 7 . In particular, all eight of the segments of the two Korean CIVs were grouped together with the previous Korean CIVs isolated between 2010 and 2012. Moreover, the inferred phylogenetic trees from all of the segments exhibited distinct clades of variants separated from the H3N2 CIVs. The reassortants were clearly located outside the main H3N2 CIV clade, demonstrating that their segments are variants caused by reassortment events or direct transmission with different origins. Such differentiation was also observed in other segments according to the two events .The phylogenetic relationships between the 57 H3N2 CIVs and the two recent Korean CIV isolates are presented in S1 Fig. and Fig. Along with the results, inconsistent phylogenetic relationships related to genotype lineages were discovered in internal segments. This finding reflected a historical reassortant origin of each genotype lineage. As an illustration, A/canine/Korea/VC123578/2012, which is a reassortant with a pandemic 2009 strain, was clustered in the same genotype (F) with the main H3N2 CIV in M segment, whereas it was separated from the main H3N2 CIV by the 1A genotype in the NS segment  suggested by Partition Finder, produced similar topologies, except for the differences in support values in all of the analyses Fig. . As mentGenetic analysis of each segment from H3N2 CIVs was conducted using different numbers of sequences due to incomplete sequencing, and the results were described in Table The estimated genealogical relationships of haplotypes from two antigenic proteins (HA and NA) are shown in Fig. Φ value of AMOVA, close to 0 indicates no differentiation between the overall population and its subpopulations, whereas close to 1 indicates highly differentiated populations. First, little differentiation among populations was observed within the same geographical region: ΦSC = 0.06955 in HA and −0.01539 in NA. There was, however, substantial differentiation among regions: ΦCT = 0.19194 in HA and 0.24270 in NA. As a result, the differences among populations in different regions responsible for nearly all of the differences among populations produced significant values: ΦST = 0.24814 (p < 0.001) in HA and 0.23104 (p < 0.001) in NA. Thus, the results suggested that the independent evolution process is ongoing between the two countries.To estimate population differentiation between Korea and China including Thailand, AMOVA was conducted, and its results are described in the following. When considering the The amino acid residues of the eight segments of the two Korean isolates did not significantly differ from other H3N2 CIVs, based on the specific sites previously described in recent reports (data not shown). Only 11 amino acid changes were obviously observed as possible adaptation mutations for the two isolates Table . For detK, G, E, 3B, F, 2D, F, 1E), is predominantly circulating in Asian regions. Notably, the general genotype pattern of domestic poultry  previously described in Dong et al. (2011) [K, G, D, 3B, F, 2D, F, 1E) and A/duck/Korea/U4-1/2007 , determined in FluGenome, were almost same as the H3N2 CIVs.In this study, the analyzed genomic genotype of H3N2 CIVs showed that it was occasionally changed due to reassortment events with a pandemic 2009 strain since 2009, but one genotype, (. (2011) , is highUsing FluGenome, 10 % delimitation by p-distance to classify IAVs lineages seemed inappropriate for the current CIVs because the criterion could not fully classify the lineages caused by the recent interspecies transmission due to minor differences  complex, antigenic sites of NA, and a glycosylation site of HA. In addition, median-joining networking, based on HA and NA, showed diversification within the H3N2 CIVs processed independently between Korea and China since the first outbreak in 2006–2007. In general, H3N2 CIVs tended to cluster together according to geographic affinities, but intrasubtypic reassortment events were often observed in Guandong province in China, indicating the possibility of frequent animal trade.In this study, for an explicit and rapid approach to identifying each segment lineage of H3N2 CIVs, genotype classification suggested by FluGenome was attempted for the first time. For the approaches, comprehensive molecular analyses were conducted to reveal the evolutionary history of 57 CIVs. In addition, two newly isolated Korean CIVs, A/canine/Korea/BD-1/2013 and A/canine/Korea/DG1/2014, were characterized using complete genomic sequences. Phylogenetic relationships among the CIVs based on genotype profiling and genetic analysis for each segment within the H3N2 CIVs showed that the two Korean CIVs were closely related to the recent Korean isolates (2010–2012), which are predominantly circulating in Asia with genotype patterns Supplementary material 1 (PDF 118 kb)Supplementary material 2 (DOC 121 kb)Supplementary material 3 (DOCX 12 kb)


Bauhinia hookeri ethanol extract (BHE) against CCl4-induced liver injury was investigated in mice. BHE was administered (500 and 1000 mg/kg/day) along with CCl4 for 6 weeks. The hepatic marker enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were determined in the serum. The antioxidant parameters: glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST), and malondialdehyde (MDA) were estimated in the liver homogenate. BHE treatment significantly inhibited the CCl4-induced increase in ALT (44 and 64%), AST (36 and 46%), ALP (28 and 42%), and MDA (39 and 51%) levels at the tested doses, respectively. Moreover, BHE treatment markedly increased the activity of antioxidant parameters GSH, GPx, GR, GST, and SOD. Histological observations confirmed the strong hepatoprotective activity. These results suggest that a dietary supplement of BHE could exert a beneficial effect against oxidative stress and various liver diseases by enhancing the antioxidant defense status, reducing lipid peroxidation, and protecting against the pathological changes of the liver. The hepatoprotective activity of BHE is mediated, at least in part, by the antioxidant effect of its constituents. The active constituents of BHE were identified by HPLC-PDA-ESI/MS/MS.The hepatoprotective and antioxidant activity of  The liver is a vital organ that has a crucial role in the detoxification of various xenobiotics . ExcessiS-transferase (GST) and by direct scavenging of free radicals −. Compound 4 produced a [M-H]− peak at m/z 577.14 and fragment ions at m/z 425.09, 407.08, 289.07, 245.08, 125.03, which are typical for dimeric procyanidins − were observed, confirming the rutinoside (rhamnosyl (1→6) glucoside) interglycosidic linkage [− and fragment ion peaks as well as from the retention time and distinctive PDA data.In this study, 15 compounds were detected in BHE using the HPLC instrument coupled to a PDA detector and a mass spectrometer. The combination of the PDA and mass spectrometry (MS/MS) data provided a sensitive method for the characterization of the constituents of BHE . Compouncid unit . Compounescribed , 26, 27.50 > 5000 mg/kg). Based on these findings, BHE is considered safe in mice.No adverse behavioral changes, toxicity symptoms, or mortality were observed in mice at doses up to 5000 mg/kg (LDP < 0.001) was observed in the CCl4 group compared to the negative control group  was observed in CClol group . Adminised group . In addied group . The hig4 induced severe loss of the hepatic architecture, marked hydropic degeneration, fatty changes, central vein congestion, Kupffer cell hyperplasia, necrosis, and scattered lymphocytes in between the hepatocytes and in the sinusoids. The pathological changes induced by CCl4 were markedly ameliorated in the groups treated with BHE at the two treatment doses (http://dx.doi.org/10.1155/2014/245171). The liver architecture is preserved in 75 and 87.5% of animals treated with 500 and 1000 mg/kg of BHE, respectively. In addition, BHE conferred protection against liver damage as evidenced by the marked decrease in lymphocyte infiltration, hydropic degeneration, fatty changes, central vein congestion, Kupffer cell hyperplasia, and necrotic changes. It was clear that the lower dose of BHE is as effective as silymarin in reducing the Kupffer cell hyperplasia induced by CCl4 intoxication. Notably, treatment with BHE at doses of 500 and 1000 mg/kg reduced the fatty changes, central vein congestion, and necrosis more than in the silymarin-treated group. Moreover, the higher dose of BHE was more effective in restoring the hepatic architecture and in reducing the lymphocyte infiltration, Kupffer cell hyperplasia, and hydropic degeneration compared to the silymarin-treated group. Complete amelioration of the fatty changes was evident by the higher dose of BHE.CCl4 intoxication is a widely used experimental model for liver injury. The highly hepatotoxic metabolites, namely, trichloromethyl radicals (CCl3· and CCl3O2·) are generated during the metabolic activation of CCl4 by cytochrome P-450. These radicals have a central role in the initiation of lipid peroxidation, inflammation, and fatty changes of the liver [4 intoxication is associated with oxidative stress since the CCl3· and CCl3O2· radicals alter the antioxidant state of the liver by deactivating the hepatic antioxidant enzymes including SOD, GPx, GR, and GST [4 treatment markedly increased the levels of AST, ALT, and ALP. The leakage of the marker enzymes into the blood was associated with marked necrosis, loss of hepatic architecture, hydropic degeneration, fatty changes, Kupffer cell hyperplasia, central vein congestion, and infiltration of the liver by lymphocytes. The MDA level in the liver tissue was markedly increased in response to CCl4 intoxication, indicating oxidative damage of the liver. CCl4 administration also reduced the levels of GPx, SOD, GST, GSH, and GR in the liver tissue compared to the normal mice. The results of the present study demonstrated that treatment with BHE returned the increased MDA to its normal level. The inhibitory effect against lipid peroxidation suggested that BHE could prevent the liver injury induced by free radicals along with the subsequent pathological changes in the liver. The marked reduction in the leakage of liver enzymes into the serum also confirmed the inhibitory effect of BHE against lipid peroxidation. In contrast, the GSH, GPx, SOD, GST, and GR levels were markedly improved compared to the silymarin-treated group. Modulation of these antioxidant defenses clearly contributed to the antioxidant and hepatoprotective activity of BHE. In this study, the phytochemical composition of B. hookeri was identified for the first time using the HPLC-PDA-ESI/MS/MS analysis. The identified compounds include gallic acid, hydroxycinnamic acid derivatives, flavonoids, epigallocatechin gallate, and dimeric and trimeric procyanidins. The remarkable hepatoprotective and antioxidant effect of BHE may be attributed to a synergistic effect between these compounds [4-induced pathological changes in liver, along with the protective effect against oxidative stress, indicated that this plant has hepatoprotective and antifibrotic therapeutic potential.CClhe liver . Moreoveompounds . Experimompounds , 7. It iompounds . Flavonoompounds , 29. Flaompounds , 9, 29. ompounds , 30. Galompounds . Proanthompounds . Commercompounds . Proanthompounds . They alompounds . Grape sompounds , 33. Sufompounds , 33. The4 intoxication. The strong in vivo antioxidant activity also suggests that a dietary supplement of BHE may confer a beneficial effect against oxidative stress. The hepatoprotective activity of BHE is concluded to be partly mediated by the antioxidant effect of its constituents. This study represents the first report that employed the HPLC-PDA-ESI/MS/MS technique for the identification of the phytoconstituents of B. hookeri. The adopted HPLC-PDA-ESI/MS/MS method provided a useful tool to develop a characteristic chromatographic fingerprint for the authentication of BHE and for the identification of its composition.Based on the results of this study, the hepatoprotective effect of BHE is attributed to its ability to reduce the rate of lipid peroxidation, to enhance the antioxidant defense status, and to guard against the pathological changes of the liver induced by CClBauhinia hookeri ethanol extract (BHE) confered marked amelioration of the pathological changes induced by CCl4 at the two treatment doses (500 and 1000 mg/kg) as evidenced by the preserved hepatic architecture, the marked decrease in lymphocyte infiltration, hydropic degeneration, fatty changes, central vein congestion, Kupffer cell hyperplasia and necrotic changes. It was clear that the lower dose of BHE is as effective as silymarin in reducing the Kupffer cell hyperplasia induced by CCl4 intoxication. BHE treatment inhibited the fatty changes, central vein congestion and necrosis more than in the silymarin-treated group. Complete amelioration of the fatty changes was evident by the higher dose of BHE.Click here for additional data file.


Hematopoietic stem cells (HSCs) and their transplantation has proved itself as an effective approach to treat different diseases. Since the first 3 cases of successful allo-HCT in 1968, the number of allo-HCTs performed annually has increased steadily over the past 3 decades [Traditionally, hematopoietic stem cells were harvested from the posterior iliac crests under general anaesthesia. More recently, mobilized peripheral blood stem cells have been increasingly used in both auto- and allo-HSCT.Hematopoietic stem cell transplantation presents a unique opportunity to study the complex and systemic effects of introducing a new genome in an individual. The biological perturbations caused depend on the genetic make-up of the donor and that of the recipient. Validation of the role of each individual HLA locus was established sequentially by comparing populations of patients of different but known genetic composition .The best donor remains an HLA-identical sibling donor. The best unrelated donor has been shown to be one who is matched, at high resolution for the major polymorphic HLA loci. HLA-A, -B, -C, -DRB1 (8/8) are all considered critical and many would consider an 8/8 matched donor as the gold standard. Genetic disparity in particular at the HLA loci, between patient and donor is a critical factor influencing transplantation outcome.One of the main factors influencing the clinical outcome of allogeneic hematopoietic stem cell transplantation is the restoration of a functional immune system. The post-transplant period is characterized by multiple immune defects that expose the patient to a high risk of opportunistic infections and eventually, disease relapse. The duration of this period may vary according to several variables, including patient’s age and immune status before transplant, the degree of donor compatibility, the intensity of the conditioning regimen, the source of stem cells, graft manipulation, and pharmacological immune suppression. Measurable immune biomarkers predicting the clinical outcome of HSCT await formal validation.In conclusion, hematopoietic stem cell transplantation has demonstrated well the importance and role of personalized approach in the implementation of this technology in the treatment of different severe diseases. However, in the era of predictive and molecular medicine the practice of HSCT is still characterized by many prognostic uncertainties. Therefore several questions will have to be addressed with the attempt to development approaches including the balance between the effectiveness of the therapy, safety, costs and socio-economic impact.


Oryza sativa) and Arabidopsis thaliana show that although these two species have distinct inflorescence architectures, cytokinins have a common effect on inflorescence branching. Based on these studies, we discuss how cytokinins regulate distinct types of inflorescence architecture through their effect on meristem activities.In flowering plants, the arrangement of flowers on a stem becomes an inflorescence, and a huge variety of inflorescence architecture occurs in nature. Inflorescence architecture also affects crop yield. In simple inflorescences, flowers form on a main stem; by contrast, in compound inflorescences, flowers form on branched stems and the branching pattern defines the architecture of the inflorescence. In this review, we highlight recent findings on the regulation of inflorescence architecture by cytokinin plant hormones. Results in rice ( Plants have an enormous, striking diversity of forms, with varying numbers and arrangements of organs in different sizes and shapes; this diversity derives from regulation of meristem activity. The aerial organs of a plant come from the shoot apical meristem (SAM) which gives rise to leaves, stem, and axillary meristems during the vegetative stage and transforms into the inflorescence meristem (IM) after the floral transition. The various developmental patterns of the IM in different species produce diverse inflorescence architectures, which not only attract artists and plant scientists, but also draw the attention of plant breeders, because inflorescence traits directly affect crop yields. Branching hierarchy and complexity depend on the species, but are also affected by environmental factors, including nutrition, light, and temperature .Arabidopsis), the cyme , and the panicle [complex, determinate, as in wheat (Triticum aestivum); or complex, indeterminate, as in maize (Zea mays), especially tassel; Figure 1; The enormous diversity of inflorescence architecture also leads to difficulties in defining consensus criteria to classify these structures. Following Arabidopsis, the IM directly initiates floral meristems  on its flanks; this forms a simple raceme . In maize and other Andropogoneae species, determinate spikelet-pair meristems (SPMs) are produced from the IM or BMs, and each SPM makes two SMs. The SM initiates one or more FMs  and TERMINAL FLOWER1 (TFL1), play a central role in meristem determinacy. LFY promotes determinate FM identity and termination of IMs, and TFL1 maintains the indeterminacy of IMs to prevent termination  catalyze the first step of cytokinin biosynthesis  encodes a cytokinin-activating enzyme catalyzing the final step of cytokinin biosynthesis and LOG is strongly expressed in BMs and FMs of developing panicles. The absence of LOG results in early termination of IM and BMs, which reduces branching complexity , AHK3, and AHK4. The ahk triple mutants have a smaller IM that terminates early, resulting in a simplified inflorescence with only a few flowers  plays a major role in the degradation of bioactive cytokinins . Arabido flowers . CKX oveerminate . Similarer yield .WUSCHEL (WUS) and suppressing the meristem inhibitors CLAVATA1 (CLV1) and CLV3. Plants ectopically treated with cytokinins show a clv-like phenotype with larger IMs and more floral organs , depending on the number of lateral branches, and can be considered a specialized cyme. Common examples include cauliflower and broccoli, which have a phenotype similar to that of the Arabidopsis apetala1-1 cauliflower-1  double mutants . In the ap1-1 single mutant , secondary flowers laterally initiate from sepal axils and from the pedicel. The ap1-1 cal-1 double mutants have the same but more complicated inflorescence branching pattern. This lateral inflorescence branching mechanism has many similarities to vegetative stage lateral shoot branching. In contrast to vegetative shoot branching, inflorescences like the raceme and panicle develop iteratively, similar to frond development in ferns  or FMs. In many determinate inflorescences, such as in wheat spikes, BM, SM, and FM can also initiate from the IM before its termination in a FM. In contrast to this initiation pattern, FM and BM can also initiate laterally from a terminal flower, either from the axil of a leaf-like organ  or can initiate without subtending lateral organs. These types of inflorescence are termed dichasium and pleiochasium . In addition to promoting FM formation and outer floral whorl specification of sepals and petals, AP1 inhibits sepal axil meristem activity . The inflorescence phenotype in ap1 is enhanced by cauliflower and fruitful mutants to form a cauliflower-like, highly elaborated pleiochasium inflorescence , AGAMOUS-LIKE 24 (AGL24), and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), to suppress secondary FM formation. Similar to IPT8 overexpression, overexpression of SVP, AGL24, or SOC1 leads to sepal axil secondary FM formation (A recent study has shown that ignaling  and duriignaling . The ap1henotype . This seormation . There aTaken together, the results described above show that cytokinins promote inflorescence complexity in different ways, by promoting meristem activity of IMs and BMs in inflorescences that branch iteratively, and by promoting indeterminate lateral meristem formation in inflorescences that branch laterally. Manipulating cytokinin levels directly or indirectly in crops is expected to change inflorescence complexity to increase yields .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genetically unrelated donors (486) were primarily partners. Genetically related donors (645) were siblings, parents, children, and others. Compared to genetically related couples, almost three times as many genetically unrelated couples were incompatible and participated in alternative programs (P < 0.001). 62% of couples were genetically related in the conventional donation program versus 32% in alternative programs (P < 0.001). Patient and graft survival were not significantly different between recipient programs. Alternative donation programs increase the number of transplantations by enabling genetically unrelated donors to donate.Donor-recipient ABO and/or HLA incompatibility used to lead to donor decline. Development of alternative transplantation programs enabled transplantation of incompatible couples. How did that influence couple characteristics? Between 2000 and 2014, 1232 living donor transplantations have been performed. In conventional and ABO-incompatible transplantation the willing donor becomes an actual donor for the intended recipient. In kidney-exchange and domino-donation the donor donates indirectly to the intended recipient. The relationship between the donor and intended recipient was studied. There were 935 conventional and 297 alternative program transplantations. There were 66 ABO-incompatible, 68 domino-paired, 62 kidney-exchange, and 104 altruistic donor transplantations. Waiting list recipients ( More than 1 out of 4 donor-recipient couples are incompatible, because of either blood type ABO incompatibility or a positive crossmatch. These couples cannot participate in the conventional living donation program. Worldwide, alternative donation programs were developed to enable these couples to pursue donation and transplantation.In kidney-exchange donation, a match is sought between 2 or more incompatible couples, so that each donor can donate to another couple's recipient –8. In 10For specified and unspecified altruistic donors there are several options. They can donate their kidney to a patient on the waiting list for deceased donor kidney transplantation, but they can also participate in the domino-paired donation program , 11. In The ABO-incompatible donation program is the most beneficial program for blood type O recipients , 12–17. Between January 1, 2000, and January 1, 2014, 1935 transplantations have been carried out in Rotterdam: 703 deceased donor and 1232 living donor (LD) transplantations. In this retrospective cohort study, all first and repeat kidney transplant recipients of both conventional and alternative program LD kidney transplantations were included. In conventional transplantation and ABO-incompatible transplantation the willing donor donates directly to the intended, specified recipient. In kidney-exchange and domino-donation the willing donor donates indirectly to the intended recipient. All recipients who received a LD kidney while on the waiting list were excluded from this study as they did not bring a willing living donor. The character of the relationship between the intended, specified recipient and the willing LD that enabled participation in living donation programs was studied. Relationships were divided into genetically related and genetically unrelated ones. Genetically related donors were parents, siblings, children, and other family members who were second-degree blood relatives. Genetically unrelated donors were partners and other nonblood relatives. The latter group consisted of family by marriage and friends. In addition, reasons for participation in alternative donation programs were studied. These reasons could be blood type ABO incompatibility, positive crossmatch, or both or on a voluntary basis.P values ≤0.05 were considered significant.We analysed the prevalence of different relationships between recipient-donor couples participating in conventional and alternative programs using chi-square tests. Univariate and multivariate Cox proportional hazards analyses were performed. Observation was until graft failure, until death, or until August 2015. The influence of program was tested as a variable with all programs separately and as a binary variable with conventional program versus all alternative programs pooled together. For multivariate analysis donor and recipient age and gender, pretransplant renal replacement treatment (yes or no), and preceding transplants (yes or no) were included. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) 21.0.0.1 . In n = 104) participated in the domino-paired program (n = 58) or donated to a recipient on the waiting list (n = 46). Domino donors (n = 65) donated to another domino recipient (n = 10) or to the waiting list (n = 55). There were 104 altruistic donors and only 101 waiting list recipients because 3 domino donors donated in other university hospitals in Netherlands.There were 297 recipients of LD kidney transplantation via alternative donation programs; 101 of them were waiting list patients that did not bring a donor. The reasons for participation in alternative donation programs of the remaining 196 recipients were as follows: ABO incompatibility in 149 couples (76%), a positive crossmatch in 41 couples (21%), both ABO incompatibility and a positive crossmatch in 2 couples, and voluntary participation in 4 couples. Sixty-three incompatible couples were genetically related and 133 were genetically unrelated. In the population of genetically related incompatible couples 73% was ABO-incompatible and 22% had a positive crossmatch, while, in the genetically unrelated incompatible couple population, 77% was ABO-incompatible and 21% had a positive crossmatch (ns). All couples with both a positive crossmatch and ABO incompatibility were genetically related. Three out of four couples that participated voluntarily were genetically unrelated.n = 1232), 48% of recipient and donor combinations were genetically unrelated and 52% were genetically related.The relationship between the intended, specified recipient and the willing LD that enabled participation in LD transplantation programs was studied. In the total population of donor-recipient combinations, including altruistic donor combinations (n = 101). 1131 couples remained of whom 196 couples participated in alternative living donation programs. There were 66 ABO-incompatible transplantations, 68 via domino-paired donation, and 62 via the local or national kidney-exchange programs  were partners (n = 359) or other nonblood relatives (n = 127). Genetically related donors (n = 645) were siblings (n = 264), parents (n = 217), children (n = 135), and other family members (n = 29). P < 0.001). Conversely, only 10% of genetically related couples participated in alternative donation programs and 90% in the conventional living donation program, while 27% of nongenetically related couples participated in alternative donation programs and 73% in the conventional donation program (P < 0.001).In the study on donor-recipient relationship, waiting list recipients were excluded, because they did not bring in a donor themselves  versus all alternative programs together (n = 196) did not have a significant influence on graft failure or patient death in univariate or multivariate analysis either.The binary variable containing conventional program (Graft survival according to the programs the patients participated in is shown in the Kaplan-Meier curve . In ordeIn 1995 the first report on living unrelated kidney donation showed that results were comparable to those of non-HLA identical living related transplants , 21. UnrAbout 50% of incompatible couples that participate in the donor-exchange program have a positive crossmatch. However, about 20% of the population transplanted via an alternative transplantation program was incompatible with the intended donor because of a positive crossmatch. This means that incompatible couples with a positive crossmatch have a smaller chance to get a transplant via one of the current alternative transplantation programs compared to ABO-incompatible couples. As a matter of fact, of all living and deceased donor kidney transplantation programs in our center, most highly sensitized patients are transplanted via the deceased donor acceptable mismatch program , 35. HowIn conclusion, alternative living donation programs increase the opportunity for nongenetically related couples to participate in living donation programs as they are more often incompatible with their intended recipient. Consequently, introduction and expansion of alternative living donation programs significantly expand the LD pool.


Cerebrospinal fluid (CSF) is a readily reachable body fluid that is reflective of the underlying pathological state of the central nervous system (CNS). Hence it has been targeted for biomarker discovery for a variety of neurological disorders. CSF is also the major route for seeding metastases of CNS malignancies and its analysis could be informative for diagnosis and risk stratification of brain cancers. Recently, modern high-throughput, microRNAs (miRNAs) measuring technology has enabled sensitive detection of distinct miRNAs that are bio-chemicallystable in the CSF and can distinguish between different types of CNS cancers. Owing to the fact that a CSF specimen can be obtained with relative ease, analysis of CSF miRNAs could be a promising contribution to clinical practice. In this review, we examine the current scientific knowledge on tumor associated CSF miRNAs that could guide diagnosis of different brain cancer types, or could be helpful in predicting disease progression and therapy response. Finally, we highlight their potential applications clinically as biomarkers and discuss limitations. The most frequently occurring brain malignancies in adults are metastatic brain cancers that have spread to the central nervous system (CNS) from other body parts, followed by glioblastomas ,2 while Several blood tumor markers are presently used for a wide range of cancer types including cancer antigen (CA)-125, α-fetoprotein (AFP), CA15-3/CA27.29 and prostate-specific antigen (PSA) for ovarian, liver, breast and prostate cancer, respectively, as well as β-2-microglobulin (B2M) for chronic lymphocytic leukemia, β-human chorionic gonadotropin (β-hCG) for testicular cancer; CA19-9 for gastric, gall bladder and pancreatic cancer; urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) for breast cancer; and thyroglobulin for thyroid cancer ,5.However, one of the challenges that contributes to the paucity of biomarkers in the serum of CNS malignancies is the blood–brain barrier, which is thought to prevent the release of tumor-specific molecules into the circulation. The Cerebrospinal fluid (CSF) has thus been investigated in the search for brain tumor markers.Profiling of circulating miRNA expression in the CSF has linked specific miRNAs to CNS malignancies. They can distinguish between different types of brain cancers, reflect disease activity and could be associated with drug resistance ,2,6,7. SIn this review, we provide a short overview on the diagnostic significance of miRNA circulating in CSF of patients with brain cancer disease and discuss limitations and challenges.et al. [et al. [et al. [et al. [While miRNAs are found mainly inside cells, a considerable number of miRNAs have recently been discovered circulating in a stable, extracellular form—in various body fluids, including plasma, serum, CSF, urine, and saliva ,13,14,15et al.  reportedet al. . In anot [et al.  showed t [et al.  reported [et al.  showed t [et al. . The sig [et al. ,24,25.CSF is formed mainly in the ventricular choroid plexus and distributed within the ventricular system and subarachnoid space. It contains 15 to 45 mg/dL protein, 50–80 mg/dL glucose and 0–5 mononuclear white blood cells/mL and serves as a medium for the nutrients delivery to the CNS as well as carrying away waste. Because CSF bathes the CNS and comes into close contact with any pathological brain tissue, it is considered an ideal source of molecules related to brain cancers as it can be accessed readily for diagnosis as well as for longitudinal disease monitoring during therapy. Approximately up to 20 mL of CSF is usually collected for testing through a lumbar puncture  where a needle is inserted, usually between the 3rd and 4th lumbar vertebrae. Such a laboratory smear poses only minimal risk to the patient. In a routine clinical setting, CSF analysis is widely used for diagnostic and prognostic purposes, and it provides critically important information for a number of neurological diseases ,64.Cancer cells infiltrate the CSF either directly from primary CNS cancers through direct spread from tumors that are in close contact with the CSF or through blood brain barrier penetration from cancers that have spread to the CNS from other body parts . CSF cytin vitro assessment of CSF looking for potential markers for CNS cancers including: proteochemical and immunophenotypic studies by flow cytometry (that provides information about cell surface protein expression), molecular genetic analyses of CSF [in-situ hybridization (FISH), DNA single cell cytometry, capillary electrophoresis and mass spectrometry reviewed in [3), elevated protein (>50 mg/dL), and decreased glucose (<60 mg/dL). Although indicative of brain cancer these CSF abnormalities are nonspecific [To maximize the chances of finding brain cancer markers in the CSF, it has been realized that it is necessary to detect changes at the molecular level rather than waiting for a macroscopic tumor to emerge . Variouss of CSF , immunociewed in ,65. Protiewed in ,69. Leveiewed in ,72,73,74iewed in . On the iewed in . Then agiewed in ,77. Abnospecific ,78,79,80Studying miRNAs as biomarkers in brain cancer tissue samples has created noticeable advances in cancer research. However, a key downside of the tissue-based approach is the need for surgical procedures in sample collection. Therefore, brain cancer biomarkers’ researchers have now turned their attention to the analysis of miRNAs present in biological fluids including CSF, which can be collected with minimal invasiveness and permit following the disease over time . It has et al. [Diagnosis of CNS involvement in neoplastic diseases, including both primary and metastatic cancers, is a major clinical intractability . To dateet al. ,62 inveset al. ,62.In a study by Teplyuk and colleagues , the autet al. demonstrated the presence of notably stable miRNAs in the CSF of PCNSL patients. The group identified specific miRNAs that are expressed with significant levels in the CSF of patients with PCNSL compared to control. Using candidate approach and miRNA quantification by qRT-PCR, the group discovered that miR-19, miR-21, and miR-92a in CSF accurately discriminates patients with primary central nervous system lymphoma from other neurologic disorders controls, indicating significant diagnostic value and increased diagnostic accuracy with 95.7% sensitivity and 96.7% specificity [et al. conducted a review of the literature on CNS lymphoma diagnosis (1966 to October 2011) and extracted data regarding the usefulness of CSF cytology in the diagnosis of CNS lymphoma. The authors reported low sensitivity for CSF cytology (2%–32%), which is increased when combined with flow cytometry. Studying β2-microglobulin, and immunoglobulin heavy chain rearrangement as well as lactate dehydrogenase isozyme 5 in the CSF has improved CSF cytology sensitivity but not specificity. Interestingly, miRNA analysis has more than 95% specificity in the diagnosis of CNS lymphoma [Primary central nervous system lymphoma (PCNSL) is another highly aggressive tumor that can lead to quick death if not diagnosed in time. The diagnosis of PCNSL can present a diagnostic challenge. It relies on histopathology of brain biopsies to the same extent as most brain tumors, while non-invasive tests to detect early tumor legions with sufficient diagnostic accuracy are not available yet. In an earlier work, Baraniskin cificity ,62,67. Olymphoma .Medulloblastoma (MB) is the most common malignant brain tumor in children. It includes various subtypes with group 3 and 4 subtypes being clinically distinct with regard to metastasis and prognosis, which may also manifest in a difference in their miRNA spectra. The presence and biological role of ex-miRNAs in MBs was unknown until our lab recently examined the existence of ex-miRNAs in MB extracellular environment and showed that the MB cells release miRNAs in their spent culture medium. We used microarray analysis to unveil the identity and level of expression of key miRNAs excreted in culture-medium of three cell lines representing different MB subtypes, D341 and D283 (metastasis-related group 3 and group 4 MB subtypes)  and DAOYversus CSF based miRNAs assays sensitivity for the diagnosis of CNS cancers. miRNAs in CSF showed higher levels in sensitivity of CNS cancers detection suggesting a relatively high diagnostic accuracy specially for PCNSL. By the end of the study, the authors concluded that CSF based miRNAs assays may be suitable as biomarkers for detection of CNS cancers and could be considered more reliable for clinical application [In an elegant work by Wei and colleagues , the autlication .et al. [in situ hybridization. Based on their results, the authors proposed a hypothetical diagnostic chart for CNS malignancies with a simple RT-PCR on patients’ CSF miRNAs. However, such miRNA signatures needs to be tested on more samples with additional pathology and prognostic classes to determine differentiating ranges of fold changes of expression among groups.At the time of writing this manuscript, Drusco et al.  analyzedAn accurate non-invasive diagnostic test for brain tumors is unavailable; moreover, the current diagnostic tools have limitations and under-diagnosis remains a major problem. Therefore, the goal is to search for biomarkers that are able to detect early indicative sign of CNS neoplastic presence in order to enable clinicians to react early with the most relevant therapy . Early hThe rapid growth of miRNAs’ identification techniques and measuring technology, such as qRT-PCR, next-generation sequencing or microarrays, gives us hope that the application of miRNAs in CSF as technically feasible biomarkers will soon become clinically practicable. There are great expectations that detecting miRNAs in CSF of brain tumor patients will achieve enhanced clinical utility in the near future and will lead to the identification of individuals in the “preclinical” stages of the illness. However, there are several concerns to consider before recommending the clinical use of CSF miRNAs as markers for brain tumors.The first concern is that little is known about the origin of c-miRNAs in the CSF of brain tumor patients, what factors influence their level of expression and what impact this will have on their specificity as biomarkers. The stability of circulating miRNAs in body fluids and culture media of some cell lines suggests that they are likely packaged in some manner that protects them against RNase digestion . StudiesThe ideal scenario is that CSF c-miRNAs are cancer related, originated from brain cancer cells in the primary tumor and accordingly reflect the magnitude of the disease. This will lead to the second concern that the chance is high that c-miRNAs in the CSF of brain tumor patients could be expressed from or secreted by a mixture of different cell types in the tumor microenvironment, such as inflammatory cells or other physiological response against the diseased tissue, jeopardizing their specificity and their ability to honestly represent tumor cells . The thiEstimates of miRNA expression levels are known to be significantly affected by methods of analysis and different miRNA profiles may be obtained depending on the sample analysis used. However, there are no universally implemented guidelines for CSF sample preparation, miRNAs extraction, measurements, normalization, or data analysis. On the contrary, there are various platforms/techniques which exist, each with specific biases that can influence the results of certain miRNA molecules expression measurements in the tested sample and may lead to foregone.It is not surprising that there are often low correlations of results between different labs and, hence, standardization is a challenge for the near future. The fourth concern is related to technical challenges associated with miRNAs analysis.Collection of CSF is the first step in sample preparation; however, CSF is not an easy specimen to obtain compared to other body fluids. Lumbar puncture (LP), although it poses a minimal risk to the patient compared to biopsy, major and minor complications can occur even when standard infection control measures and good techniques are used. These complications include: post-LP headache, infection, bleeding, cerebral herniation and back pain.Another worry is that CSF samples that are used as material for miRNA detection could be contaminated with blood iatrogenically. Blood contamination of CSF would result in significant increases in nucleated white blood cells (WBC) count that could contribute to miRNA content of the sample being analyzed. Skin cells can also contaminate CSF samples. In this situation, skin contains abundant epithelial miRNAs that can bias results of miRNAs profiles. Method that preserves the RNA expression profile during and after collection of CSF is another important factor for accurate analysis of miRNA expression . CentrifIn general, working with RNA requires very special precautions to prevent degradation and or contamination of the RNA sample. The practice for isolating miRNAs are, in principle, the same as for isolation of total RNA, except that miRNA isolation protocols are often slightly modified to retain the miRNAs. The first challenge is that CSF samples contain only small amounts of RNA and extraction of sufficient miRNA amounts can be difficult . SamplesThree major approaches are currently used for miRNAs detection in the CSF: qRT-PCR, hybridization-based methods, such as DNA microarrays, and high-throughput sequencing (RNA-seq). qRT-PCR assays are among the most frequently used high-throughput RT-PCR platforms for miRNA detection. The attractive aspect of this approach is the ease of incorporation into the workflow for laboratories that are familiar with real-time PCR. However, commercial assays that are commonly used for miRNA detection use a different strategy to reverse transcribe mature miRNA molecules and to amplify the cDNA, which might result in different detection biases. Another obstacle in performing qRT-PCR is that reaction conditions may vary as well as sequence-specific differences in primers .i.e., adjusting the data and removing technical variations across samples that are not related to the biological changes under investigation) is tricky and can be challenging. The optimal method to use for normalization is to identify stably expressed reference genes or commonly expressed miRNAs in a given sample as normalization factor. However, no constitutively expressed circulating miRNAs have been reported up till now in the CSF. Spiking samples with RNA controls, applying reference miRNA such as miR-16 or miR-26a or other non-coding RNA like U6 snRNA are some of the strategies used to standardize analysis, however some of the larger small RNA species such as U6 RNA are present in extremely low concentrations in body fluids. On the whole, the variability in normalization methods and analysis might be responsible for some of the discrepancies between miRNA-profiling studies and differences and overlapped miRNAs signatures reported in the literature.Finally, data normalization (In summary, miRNA investigation in CSF samples as a potential marker for brain cancers is a challenging procedure. The development of miRNAs as good biomarkers requires optimized and standardized procedures for CSF sample collection handling and preparation, highly sensitive and accurate miRNAs detection methods as well as reliable methods for data analysis .Tumor-associated miRNAs circulating in the CSF have recently shown promise as diagnostic indicators for some types of brain tumors owing to the substantial differences that have been described between their expression profiles in healthy individuals and patients. Their chemical stability in the CSF together with the recent advances in the sensitive mRNA measuring platforms have generated demand to apply CSF miRNAs investigation procedure in clinical practice. However, despite the promise, the available data on the suitability of the CSF miRNAs as diagnostic biomarkers for brain cancer are still very limited and critical issues need to be clarified before making recommendations for their clinical implementation. We need to have a better understanding about the mechanisms by which tumor-specific miRNAs are produced in the CSF, what factors influence their level of expression in the CSF of patients with brain tumors, and what impact will this have on their specificity as diagnostic markers. More studies are certainly necessary to establish miRNAs signatures for each brain cancer type and their different stages, and to determine which miRNAs meaningfully reflect the dynamics of tumor response to therapy. Undoubtedly, CSF miRNAs are a potential gold mine for identifying brain cancer biomarkers, however, much work remains to be done to harness findings from scientific research to advance clinical applications.


Because the detailed mechanism has never been shown, we investigated the signal transduction pathway in granulocytic differentiation by G-CSF, alone or in combination with ATRA.Granulocyte colony-stimulating factor (G-CSF) promotes proliferation, survival, and differentiation of myeloid-linage leukemic cells, as well as normal hematopoietic cells. Terminal granulocytic differentiation can be induced in acute promyelocytic (APL) cell line HT93A by G-CSF and all-HT93A cell viability and growth were investigated by trypan blue exclusion assay. Cell differentiation was assessed by CD11b and CD34 expressions. Intracellular protein expressions were also evaluated by flow cytometry after fixation and permeabilization.ATRA (100 nM) induced granulocytic differentiation (upregulation of CD11b and downregulation of CD34) and the effect was potentiated by addition of G-CSF, while G-CSF alone had no effect on HT93A cells. The addition of G-CSF to ATRA had little or no effect on NB4 and THP-1 cells in comparison to ATRA alone. G-CSF receptor expression was reduced by ATRA treatment in a time-dependent manner. After 5 days’ incubation with ATRA, the expression levels of signal transducer and activator of transcription (STAT) 3, and phosphorylated STAT3 and STAT5, were significantly reduced. STAT5 was strongly activated by G-CSF stimulation in ATRA-pretreated cells in comparison to untreated cells. In contrast, STAT3 showed no response to G-CSF. Janus kinase (JAK) inhibitor ruxolitinib (320 nM) had little or no effect on ATRA-induced differentiation, but eliminated the enhancing effect of G-CSF, as evidenced by the levels of CD11b and CD34 expression. These results suggest G-CSF activates STAT5 through the JAK pathway in combination with ATRA, resulting in myeloid differentiation in HT93A cells.In conclusion, activation of the JAK-STAT pathway is likely essential for inducting differentiation in the APL cell line HT93A; thus, monitoring its expression and activation is important for predicting clinical efficacy and understanding the mechanisms of cytokine-dependent myelopoiesis, proliferation, and differentiation of acute myeloid leukemia. Cliniia (AML) -7.in vitro systems have been performed [Studies in various cell lines have revealed that G-CSF functions through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway -10. STATerformed -17, the Acute promyelocytic leukemia (APL) is characterized by a balanced reciprocal translocation between chromosome 15 and 17, resulting in fusion of the promyelocytic leukemia and retinoic acid receptor alpha genes -20. ATRAIn this study, we examined the differentiation mechanism of ATRA and G-CSF, alone or in combination, in HT93A cells. Our data suggest enhanced differentiation induction through the JAK-STAT pathway is induced by combination treatment with ATRA and G-CSF in AML.After treatment with 100 nM ATRA for 7 days, expression of major differentiation marker CD11b increased, while primitive marker CD34 decreased in HT93A cells  is likely essential for G-CSF-induced granulocytic differentiation in HT93A. This unique cell line mimics normal progenitor cells and may become a useful model for analyzing the effects of cytokine-dependent myelopoiesis, proliferation, and differentiation of hematopoietic cells.2α, fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34 IgG1, and PE-conjugated mouse anti-human G-CSF receptor IgG1 antibody were purchased from Becton-Dickinson . Non-binding mouse IgG-PE and IgG-FITC isotype antibodies (Becton-Dickinson) were used as controls. Antibodies for intracellular staining, rabbit anti-STAT3 (#GTX108630), anti-STAT5A (#GTX103750), anti-phosphorylated STAT3 , and anti-phosphorylated STAT5  polyclonal antibodies, were purchased from GeneTex ; anti-STAT5B polyclonal antibody (#bs-4254R) was from Bioss . FITC-labeled goat anti-rabbit IgG secondary antibody was obtained from Kirkegaard & Perry Laboratories . Non-binding rabbit IgG (GeneTex) was used instead of primary antibody as negative control.ATRA was purchased from Sigma , dissolved in ethanol to 2 mM, and stored at −20°C in the dark. The final concentration of ethanol (≤0.05%) did not affect cell viability or differentiation. Recombinant human G-CSF (Filgrastim) was obtained from Kyowa Hakko Kirin Co, Ltd. , dissolved in phosphate-buffered saline (PBS) to prepare the stock solution, and stored at −20°C. The JAK inhibitor ruxolitinib was purchased from ChemScene  and dissolved in dimethyl sulfoxide to 10 mM. Primary antibodies, phycoerythrin (PE)-conjugated mouse anti-human CD11b IgG2 in air).HT93A, a human APL cell line established from the peripheral blood of a patient with APL , was proHT93A cell viability was investigated by trypan blue exclusion assay. Cells that stained negative and positive with trypan blue were considered as viable and dead, respectively. Growth inhibition was expressed as a ratio of the number of viable cells in each treatment group to the control group.6 cells were washed with PBS containing 2.5% fetal bovine serum and 0.5% NaN3 (PBSF) and stained with PE-conjugated mouse anti-human CD11b IgG2a, PE-conjugated mouse anti-human G-CSF receptor IgG1, and FITC-conjugated mouse anti-human CD34 IgG1 for 30 min at 4°C in the dark. Cells were washed twice with PBSF and analyzed by flow cytometry with a minimum acquisition of 10,000 events. Non-binding mouse IgG-PE isotype antibodies or non-binding mouse IgG-FITC isotype antibodies were used as controls.Differentiation induction was confirmed by surface marker expression. Myeloid maturation was analyzed by FACSCalibur cytometry (Becton-Dickinson) with CD11b, CD34, and G-CSF receptor antibodies as described with minor modifications ,33,34. IFlow cytometry analysis was performed to analyze intracellular protein expression after fixation and permeabilization ,36. Cellt-test, and p < 0.05 was considered statistically significant.Experiments were independently repeated three times, and results shown are the mean ± standard deviation (SD). Data were analyzed using paired Student’s


Here we present a male patient with acute myeloid leukemia (AML) initially diagnosed as M5 and with karyotype 46,XY. After induction therapy, he underwent a HLA-matched allogeneic hematopoietic stem cell transplantation, and six years later he relapsed as AML M1 with an abnormal karyotype //47,XX,+10[2]/47,XX,+11[3]/48,XX,+10,+11[2]/46,XX[13]. Based on this, we tested the possibility of donor cell origin by FISH and molecular STR analysis. We found no evidence of Y chromosome presence by FISH and STR analysis consistent with the success of the allogeneic hematopoietic stem cell transplantation from the female donor. FISH studies confirmed trisomies and no evidence of MLL translocation either p53 or ATM deletion. Additionally 28 fusion common leukemia transcripts were evaluated by multiplex reverse transcriptase-polymerase chain reaction assay and were not rearranged. STR analysis showed a complete donor chimerism. Thus, donor cell leukemia (DCL) was concluded, being essential the use of cytological and molecular approaches. Pediatric DCL is uncommon, our patient seems to be the sixth case and additionally it presented a late donor cell leukemia appearance. Different extrinsic and intrinsic mechanisms have been considered to explain this uncommon finding as well as the implications to the patient. Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous hematological disease. Patient age, cytogenetic and minimal residual disease after induction therapy, play a major role in the classification and prognosis of AML, leading to stratification in risk categories ,2.et al. /47,XX,+11[3]/48,XX,+10,+11[2]/46,XX[13] , on bone marrow samples using centromere 10 , and MLL  FISH probes. The results were reported according to ISCN 2013 as follows. Centromere 10: //nucish(D10Z1x3)[92/200]. MLL: //nuc ish(5′MLL con 3′MLLx3)[150/200]. Cep 10 and MLL showed three copies, agreeing with the trisomy 10 and 11 found in karyotype  FISH probe, resulting as follows: //nucish(DXZ1x2)[192/200]. The presence of a pair of centromere X sequences supports a donor cell origin,  and TP53/ATM , resulted in normal fluorescence patterns, although ATM exhibited an additional signal showing the extra 11 chromosome  analysis were performed for 15 loci  and sex determination by amelogenin  on genomic DNA from bone marrow and mucosal swap of the post-transplant patient and in blood from the donor . The 28 fusion transcripts evaluated are shown in Table Written informed consent was obtained from the patient's parents for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Background: Early life stress (ELS) is cited as a risk for mood and anxiety disorders, potentially through altered serotonin neurotransmission. We examined the effects of ELS, utilizing the variable foraging demand (VFD) macaque model, on adolescent monoamine metabolites. We sought to replicate an increase in cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA) observed in two previous VFD cohorts. We hypothesized that elevated cisternal 5-HIAA was associated with reduced neurotrophic effects, conceivably due to excessive negative feedback at somatodendritic 5-HT1A autoreceptors. A putatively decreased serotonin neurotransmission would be reflected by reductions in hippocampal volume and white matter (WM) fractional anisotropy (FA).Methods: When infants were 2–6 months of age, bonnet macaque mothers were exposed to VFD. We employed cisternal CSF taps to measure monoamine metabolites in VFD (N = 22) and non-VFD (N = 14) offspring (mean age = 2.61 years). Metabolites were correlated with hippocampal volume obtained by MRI and WM FA by diffusion tensor imaging in young adulthood in 17 males [10 VFD (mean age = 4.57 years)].Results: VFD subjects exhibited increased CSF 5-HIAA compared to non-VFD controls. An inverse correlation between right hippocampal volume and 5-HIAA was noted in VFD- but not controls. CSF HVA and MHPG correlated inversely with hippocampal volume only in VFD. CSF 5-HIAA correlated inversely with FA of the WM tracts of the anterior limb of the internal capsule (ALIC) only in VFD.Conclusions: Elevated cisternal 5-HIAA in VFD may reflect increased dorsal raphe serotonin, potentially inducing excessive autoreceptor activation, inducing a putative serotonin deficit in terminal fields. Resultant reductions in neurotrophic activity are reflected by smaller right hippocampal volume. Convergent evidence of reduced neurotrophic activity in association with high CSF 5-HIAA in VFD was reflected by reduced FA of the ALIC. Early life stress (ELS) increases the risk of the development of depression and anxiety disorders in adulthood  model of ELS in bonnet macaques. In the VFD model, unpredictability of maternal food access compromises maternal care of the infant, producing an experimental homolog of human anxiety and mood disorders in the offspring   integrity as measured by FA have been observed throughout the brain across a range of psychiatric disorders  subjects reared under conditions of ELS (VFD) were compared to 14 subjects reared under normative conditions (non-VFD). For CSF monoamine adolescent studies, VFD subjects (N = 22) were older than non-VFD (N = 14) . Age was therefore used as a covariate. There were 10 males and four females in the non-VFD group whereas in the VFD group there were 10 males and 12 females . There were no significant statistically significant effects for weight . There was a trend for a rearing group × sex effect for weight . VFD females were not significantly heavier than Non-VFD females  although the effect neared a trend. VFD male weights were not distinguishable from non-VFD male weights . Thus, the significant age difference between VFD and non-VFD did not have any significant effect on body weight even when broken down by sexes. Nevertheless, given the near trend effect for VFD females to be heavier than non-VFD females, weight was included as a covariate.Twenty-two bonnet macaque ] or more difficult to obtain [high foraging demand (HFD)].Foraging carts were utilized to control the difficulty in obtaining food. In the HFD condition, mothers would reach through apertures in a foraging cart to dig through clean wood-chips in order to obtain food rations. Food deprivation was avoided in the high demand condition, illustrated by normal infant weight gain and adult maintenance of baseline weights throughout the study . Because subjects readily enter into the carrying cages, a routine procedure for weekly cleaning, physical capture of subjects was not necessary, and thus excludes the confound of “time” to capture, which has been shown to affect CSF 5-HIAA concentrations ; Turbo spin echo T2-weighted Axial .MRI data were acquired on a 3 T Siemens Allegra scanner. The protocol for the structural scans consisted of a three-plane sagittal localizer from which all other structural scans were prescribed. The following structural scans were acquired: axial 3D-MPRAGE . Briefly, a single subject raised under normative conditions was chosen as the initial template. All images were registered through linear  deformations to the single subject template. An average image, called template henceforth, was created with the obtained deformed images. Afterwards, the same original images were linearly deformed to the created template and this step was iterated 20 times to minimize the bias caused by utilization of a single subject as the initial template. On the 22nd step, original images were linearly and also non-linearly registered to the final template. A brain mask containing gray matter, white matter and CSF, was manually delineated for the template and used to eliminate skull and meninges from the final registered images. In order to preserve brain volume, images were scaled using the Jacobian matrix, so that the total amount of gray matter in the resulting images remains the same as it would be in the original images. The obtained images were finally smoothed with a Gaussian filter at Full Width at Height Maximum equal to 4 mm . Raw DTI data were transferred to an off-line workstation for post-processing. In-house software written in Matlab v6.5  was used to compute the anisotropy and vector maps. FA images were then converted to analyze format. MEDx v3.4.3 software  was used to inspect and define ROIs on the FA images. Regions of interest (ROI) included the left and right anterior limb of the internal capsule (ALIC).DTI . There were no significant relationships between the volume of total brain, left hippocampus and right hippocampus, and with any of the CSF monoamine metabolites, thus brain volume was not used as a covariate. Young adult age and weight were also not used as covariates as they were not associated with hippocampal volume or CSF monoamine metabolite levels. Within-rearing group Pearson's correlations were determined to test the hypothesis, outlined in the introduction, that monoamine metabolite concentrations, particularly CSF 5-HIAA, would be inversely related to hippocampal volume, particularly in VFD-reared subjects for whom early life stress was posited to accentuate the inverse relationship. For determination of the predictive value of rearing effects in the induction of the putative inverse relationship between hippocampal volume and each CSF monoamine metabolite concentration, general linear model (GLM) analyses were conducted separately using left and right hippocampal volume as the continuous predictor variable. Rearing group was used as a categorical independent variable and CSF monoamine metabolite as the dependent variable. The factorial interaction term of right or left hippocampal volume × rearing condition was also entered into the GLM to examine whether the relationship between hippocampal volume and each CSF monoamine metabolite differed as a function of ELS. The rationale of using both left and right hippocampal volume as independent predictor variables in separate GLM's is that VFD-rearing has previously been shown, in the current cohort, to be associated with reduced left, but not right, hippocampal volume in comparison to non-VFD subjects  of the ALIC. We examined the relationship between CSF monoamines and white matter FA of the ALIC. We have previously documented reduced FA in the ALIC bilaterally following ELS  D = 0.14, p > 0.20; Lilliefors p < 0.10] but not CSF 5-HIAA  was observed. Inspection of the histogram revealed an extended distribution on the right tail. Even though CSF 5-HIAA was both log10 and square-root transformed, distribution still remained non-normal. Therefore, parametric analyses, where age, weight or sex could be used as covariates, were performed followed by the non-parametric Mann-Whiney U. No outliers were noted for the combined male and female analyses.A normal distribution of CSF MHPG . There was no effect of age on CSF 5-HIAA , CSF HVA  but there was a significant inverse effect for CSF MHPG . There were no age effects in males for CSF 5-HIAA or HVA  but there was an inverse effect for CSF MHPG . There were no age effects for females . There were no sex × age effects for CSF 5-HIAA .Weight effects. There was no sex effect for weight . When males and females were combined, body weight bore no significant relationship to CSF 5-HIAA , HVA  or MHPG . There were no weight effects for CSF monoamines in males  or females . There were no sex × weight effects in the prediction of CSF 5-HIAA .F = 0.00; p = 0.93], VFD-reared subjects exhibited elevated CSF 5-HIAA concentrations (ng/ml) relative to non-VFD-reared counterparts  whereas no group effects were observed for CSF MHPG and CSF HVA concentrations (p > 0.1). Covarying for sex, CSF 5-HIAA remained elevated in VFD-reared subjects vs. controls . Covarying for weight, CSF 5-HIAA remained elevated in VFD-reared subjects vs. controls . The CSF 5-HIAA finding was confirmed using the non-parametric Mann Whitney U   providing modest support for an elevation of CSF HVA in VFD-reared subjects. No sex effects or sex by rearing group effects were noted for any of the CSF monoamines.Covarying for age, , CSF HVA was elevated in VFD , whereas MHPG was slightly yet significantly diminished in VFD . For CSF HVA, a right hippocampal volume × rearing effect was observed , adjusted for mean FA of the ALIC  = 6.16; p = 0.028]. A significant rearing group × mean FA interaction predicting CSF 5-HIAA was noted . The Pearson's correlation between CSF 5-HIAA and mean ALIC FA was inversely significant in VFD  but not in non-VFD subjects . A similar group effect was noted for CSF HVA concentrations on GLM analysis  when controlling for mean FA of ALIC, but no other CSF HVA effects were noted. No effects were noted for CSF MHPG.GLM analyses revealed a significant group effect only for CSF 5-HIAA [VFD mean   to maintain neuronal growth and survival (Duman et al., 1A autoreceptors, may conceivably attenuate serotonin outflow, thereby reducing serotonin-mediated inhibition of LC neuronal firing (Aston-Jones et al., That reduction of FA measures of white matter integrity in the ALIC inversely associated with elevated cisternal CSF 5-HIAA provide convergent validation of neurotrophic compromise secondary to a putative serotonin deficit state in downstream structures of the serotonergic pathway. Parental verbal abuse is linked to a decrease in FA in a variety of white matter tracts (Choi et al., Although our findings seemingly conflict with the CSF 5-HIAA deficit consistently observed in maternally-separated peer-reared vs. maternally-reared rhesus monkeys (Higley et al., Moreover, in vervet monkeys, reduced cisternal CSF 5HIAA was also found to be inversely associated with social impulsivity (Fairbanks et al., Post-hoc t-tests revealed that VFD females were not heavier than non-VFD females but because this comparison was a near trend, weight was used as a covariate when assessing rearing group differences for CSF 5-HIAA. Elevations in CSF 5-HIAA concentrations in VFD compared to non-VFD subjects remained significant. Finally, the inverse relationship between CSF 5-HIAA and right hippocampal volume is correlational but not causal. Other factors, such as HPA axis alterations, may represent an intermediary step between alterations in serotonin function and hippocampal volume. Moreover, without causal evidence, the directionality of the hypothesis stemming from altered serotonin function to adversely affect hippocampal remains an assumption. Evidence for the hypothesis, to the extent supported by convergent processes, is provided by the inverse relationship between CSF 5-HIAA concentrations and white matter FA. Studies directed at confirming the causality of CSF 5-HIAA elevations in the pathophysiology of hippocampal volume reductions are warranted.Limitations of the present study include the restriction of the imaging data to a subset of the males in the sample although the CSF 5-HIAA elevations following ELS were not sex-specific. The inverse association between CSF 5-HIAA, MHPG, and HVA in VFD-reared subjects and hippocampal volumes was restricted to the right side. It is interesting to note that we have previously reported in this same cohort reduced left hippocampal volume in VFD-reared vs. non-VFD-reared controls whereas differences for right hippocampal volume were not significant (Jackowski et al., Dr. Mann receives royalties for commercial use of the C-SSRS from the Research Foundation for Mental Hygiene and has stock options in Qualitas Health, a start-up company developing a PUFA food supplement. Dr. Mathew has been named as an inventor on a pending use-patent of ketamine for the treatment of depression. Dr. Mathew has relinquished his claim to any royalties and will not benefit financially if ketamine were approved for this use. Dr. Mathew has received consulting fees. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study we investigated the correlation between donor chimerism status and disease relapse following allogeneic hematopoietic stem cell transplantation . The chimerism of Fluorescence-activated cell sorter (FACS) sorted CD3+T lymphocytes of 153 cases, CD56+CD16+NK lymphocytes of 153 cases and CD19+B lymphocytes of 31 cases with acute B lymphoblastic leukemia (B-ALL) was analyzed post-transplant utilizing polymerase chain reaction amplification of short tandem repeats (PCR-STR). A total of 33 patients  had recurrent disease. The positive predictive values of declining donor chimerism for hematologic and isolated extramedullary relapse were 58.8% and 10% . The positive predictive values of declining donor chimerism in BMB, BMT, BMNK and PBB for hematologic relapse were 11.6%, 0%, 0% and 0% under close monitoring in patients with B-ALL. Only the donor chimerism in BMB significantly decreased in the group with hematologic relapse as compared with the group without hematologic relapse  in patients with B-ALL. The median drop of donor chimerism in PBT, BMT, PBNK and BMNK were 0%, 0%, 5.9% and 2.8% one or two weeks prior to hematologic relapse in patients with non-B-ALL. The donor chimerism in PBNK significantly decreased prior to hematologic relapse in the group with hematologic relapse as compared with the group without hematologic relapse .These data suggest donor chimerism of BMB can be used to predict the occurrence of hematologic relapse in patients with B-ALL. Donor chimerism decrease in PBNKwas associated with a somewhat increased risk of hematologic relapse in patients with non-B-ALL. Therefore, our results reveal a more effective path to individually predict for hematologic relapse by dynamic monitoring different cell lineages in different disease. Hematopoietic stem cell transplantation is now an established treatment for a number of non-malignant and malignant conditions ,2,3. HowThe microsatelite analysis by PCR of chimerism within specific leukocyte subsets isolated from peripheral blood or bone marrow samples by flow-sorting techniques provides more specific information on assessment of impending complications at a significantly higher sensitivity . DetectiOne hundred and fifty three consecutive patients received allo-HSCT at Shanghai First People’s Hospital. The study has been approved by ethical committee of Shanghai First People’s Hospital, Medical College, Shanghai Jiaotong University. Written informed consents from the patients were obtained for the use of this sample in research. Patients characteristics are shown in Peripheral blood from recipients and donor grafts were obtained before allo-HSCT. 4–10 ml peripheral blood (PB) or 2ml bone marrow (BM) from the recipients were collected at regular intervals of every two weeks until achieving complete donor chimerism (CDC) and then each month post-transplant, if the sample was available. If donor chimerism decrease of any patient was detected in any subset, the patient would be monitored every week till chimerism of all subsets reached CDC. PB and BM were collected into the EDTA tubes. Patients were systematically examined in parallel with the chimerism detection.3–1*106 cells of each subset. The purity of the sorted cells was more than 96%. Two days was the maximum time between specimen collection and FACS sorting.Peripheral blood mononuclear cells (PBMC) which extracted from PB or BM samples with ficoll stained for surface antigens. The antibodies were CD3+-PE, CD19+-FITC, CD56+CD16+-PE (BD). The CD3+ thymus derived cell (T cell) and CD56+CD16+ natural killer cell (NK cell) were sorted for all 153 patients by the Fluorescence-activated cell sorter (FACS) (FACSvantageSE). CD19+ bursa dependent lymphocyte (B cell) was sorted for 31 cases with acute B lymphoblastic leukemia (B-ALL) by the Fluorescence-activated cell sorter (FACS) (FACSvantageSE). The final quantity was 1*10We did operate according to the protocols of commercial kits. Genomic DNA which isolated from all cell subsets sorted by FACS was extracted by ReadyAmp Genomic DNA Purification System Kit (Promega). The final concentration of DNA was 0.5ng/ul. One day was the maximum time between FACS sorting and DNA extraction. Amplifications were performed with the AmpFlSTR Profiler Plus Kit  on an ABI 7300 Fast Real-Time PCR System. All Multiplex PCR reactions were completed in 20μl volume. The volume/quantity of DNA was 5ul/2.5ng. Cycle conditions were as follows: 95°C for 11 minutes followed by 28 cycles of 94°C for 1 minute,58°C for 1 minute,72°C for 1 minute, and a final step consisting of 45 minutes at 60°C. The PCR products were analyzed using capillary electrophoresis on ABI PRISM 3130 Genetic Analyzer. The GeneMapper v4.0 provided a complete STR genotyping. Serial samples were analyzed for degrees of donor-recipient chimerism using PCR of informative minisatellite regions . PCR proDisease relapse is defined as isolated donor chimerism decrease, hematologic and isolated extramedullary relapse. Hematologic relapse is according to the criteria of Williams Hematology. Isolated extramedullary relapse is meaning blasts at other sites outside of the bone marrow. Isolated donor chimerism decrease is diagnosed when the donor chimerism decreased without hematologic and extramedullary relapse. Complete donor chimerism (CDC) is 100% donor chimerism in all cell subsets of the sample which we examined.Statistical analysis was performed using SPSS 13.0 software. T test and Chi-Square were used. P values <0.05 were considered as significant.Of all patients, 144 achieved sustained complete donor chimerism (CDC). There were 7 patients who experienced graft rejection. We didn’t find the correlation between the conditioning regimen and the graft rejection. The other two patients had died of Cerebral Hemorrhage before their chimerism reached CDC. Decreased donor chimerism was observed in samples of 73 patients. A total of 33 patients  had hematologic and isolated extramedullary relapse, reaching a cumulative incidence of hematologic relapse of 15% (23/153) and isolated extramedullary relapse of 6.5% (10/153) from November 2004 to October 2013.Of the ten isolated extramedullary relapsed patients, one patient (No. 31) converted from CDC to mix chimerism (MC) in the BMT lineage with a drop of 10% one week prior to isolated extramedullary relapse, but no further decline was detected at the time of isolated extramedullary relapse. One patient (No. 33) was detected the drop of 11% and 8% in BMT and BMNK lineages at the moment of isolated extramedullary relapse. All of the remaining 8 cases sustained CDC in the whole study period . The posDecreased donor chimerism was detected in the samples of all of the 23 hematologic relapsed patients (100%). And there were 10 patients observed declining donor chimerism prior to the hematologic relapse in 17 patients under regular monitoring  except 6 patients  without regular monitoring.So the positive predictive values of declining donor chimerism for hematologic and isolated extramedullary relapse were 58.8% and 10% . The predict sensitivity of donor chimerism for hematologic relapse is higher than that for isolated extramedullary relapse.In 28 B-ALL patients, isolated donor chimerism decrease was detected in 3 patients and hematologic relapse occurred in 12 patients.The median drop of donor chimerism in B, T and NK cells of bone marrow  were 74.3%, 13.2% and 19.6% at the point of hematologic relapse. Donor chimerism in BMB significantly decreased as compared with those in BMT  and BMNK . The median levels of donor chimerism decreased in BMB, BMT and BMNK were 11.6%, 0% and 0% one or two weeks prior to hematologic relapse. The B cells, not T or NK cells, dropped as the earliest and deepest lineage of BM.The median drop of donor chimerism in B cells of bone marrow and peripheral blood (BMB and PBB) were 74.3% and 7.2% at the moment of hematologic relapse and 11.6% and 0% one or two weeks prior to hematologic relapse. The donor chimerism in BMB is significantly different from the donor chimerism in PBB at the moment of hematologic relapse  and at one or two weeks prior to hematologic relapse . The drop of donor chimerism in BMB was earlier and deeper than that in PBB.Of the 3 cases who underwent isolated donor chimerism decrease, the median drops of donor chimerism in BMB, BMT, BMNK and PBB were 0%, 1%, 0% and 0%. Only the donor chimerism in BMB significantly decreased in the group with hematologic relapse as compared with the group without hematologic relapse , while no significant difference was found in the donor chimerism in BMT, BMNK, PBB .As a result, the donor chimerism in BMB dropped as the earliest and deepest lineage in patients with B-ALL. The declining donor chimerism in BMB can be used as a sensitive marker to predict the hematologic relapse in patients with B-ALL. The B-cell donor chimerism showed having nothing to do with relapse in patients with non-B-ALL in our previous study, so only donor chimerism in T and NK cells were monitored in this study.In the group with hematologic relapse, the median drop of donor chimerism in NK cells of peripheral blood (PBNK) was 14.9% at the point of hematologic relapse and 5.9% one or two weeks prior to hematologic relapse. The median drop of donor chimerism in PBNK was 3.2% in the group with isolated donor chimerism decrease. The donor chimerism in PBNK significantly decreased prior to hematologic relapse in the group with hematologic relapse as compared with the group without hematologic relapse .In the group with hematologic relapse, the median drop of donor chimerism in NK cells of bone marrow (BMNK) was 50.85% at the point of hematologic relapse and 2.8% one or two weeks prior to hematologic relapse. The median drop of donor chimerism in BMNK was 1.97% in the group with isolated donor chimerism decrease. No significant difference was found in the donor chimerism decrease in BMNK prior to hematologic relapse in the group with hematologic relapse as compared with that in the group without hematologic relapse .The median drop of donor chimerism in PBT and BMT were 1.35% and 16.15% at the point of hematologic relapse and 0% and 0% one or two weeks prior to hematologic relapse.The drop of donor chimerism in PBNK and BMNK, not PBT or BMT, were detected prior to hematologic relapse. The declining PBNKdonor chimerism, can be used to predict the hematologic relapse in patients with non-B-ALL.Allogeneic hematopoietic stem cell transplantation  is a curative treatment for many patients suffering from malignant and non-malignant hematological disorders. One of the most useful tools for monitoring is the assessment of hematopoietic chimerism which occurs after alloHSCT and describes the percentage of donor hematopoietic and lymphoid cells in a transplant recipient ,16. STR-Maeng H et al.  report aIn recipients of allogeneic HSCT, identification of patients at high risk for hematologic relapse and use of interventions post-transplant to augment the graft-versus-tumor effect are possible areas of donor chimerism kinetics. Some investigations ,15 evaluActually it is useful in some disease to target selected sub-populations in order to have an earlier detection of hematologic relapse on cell fractions . In our Some studies suggest special cell subsets could predict for relapse, such as T-cell in ALL, AML and NHL , CD34 ceThe median drop of donor chimerism in PBNK and BMNK were 5.9% and 2.8% one or two weeks prior to hematologic relapse. The donor chimerism in PBNK significantly decreased prior to hematologic relapse in the group with hematologic relapse as compared with the group without hematologic relapse . Donor chimerism decrease in PBNK was associated with a somewhat increased risk of hematologic disease recurrence.In summary, decreasing donor chimerism of PBNK and BMB cells can be detected and predictive for hematologic relapse of patients with non-B-ALL and B-ALL by sequentially quantitative monitoring of their chimerism which sorted by FACS. Our results reveal a more effective path to individually predict for hematologic relapse by dynamic monitoring different cell lineages in different disease. Future studies are needed to address more samples to verify them.