Retinol binding protein 4 (RBP) enhances metabolic risk and atherogenesis. Whether RBP4 contributes to cardiovascular risk in rheumatoid arthritis (RA) is unknown.We assessed RBP4 concentrations and those of endothelial activation molecules including E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 by ELISA, and the common carotid artery intima-media thickness (cIMT) and carotid artery plaque by ultrasound in 217 (112 black and 105 white) patients with RA. Relationships were identified in potential confounder and mediator adjusted mixed regression models.RBP4 concentrations were associated with systolic and mean blood pressure, and those of glucose and E-selectin , −0.195 (p = 0.006), −0.155 (p = 0.03) and −0.191 (p = 0.007), respectively in all patients); these RBP4-cardiovascular risk relations were mostly reproduced in patients with but not without adverse traditional or non-traditional cardiovascular risk profiles. RBP4 concentrations were not associated with atherosclerosis in all patients, but related independently to cIMT  and plaque (OR (95%CI) = 2.95 (1.31–6.68), p = 0.008) in those with generalized obesity, as well as with plaque in those with abdominal obesity (OR (95%CI) = 1.95 (1.12–3.42), p = 0.01).In the present study, RBP4 concentrations were inversely associated with metabolic risk and endothelial activation in RA. This requires further investigation. RBP4 concentrations were related to enhanced atherosclerosis in patients with generalized or/and abdominal obesity. Retinol binding protein 4 (RBP4) was characterized in 1968 for its transporting role of retinol from storage sites in the liver to extrahepatic tissues A recent cellular study showed that RBP4 consistently stimulates the expression of inflammatory molecules in human retinal capillary and umbilical vein endothelial cells Several adipokines other than RBP4 and including adiponectin Patients with RA sustain a markedly enhanced risk of cardiovascular disease (CVD) that is effectuated by adverse conventional cardiovascular risk factors, high-grade inflammation and genetic determinants The presence of rheumatic disease can impact on the production as well as the relationships of adipokines with cardiometabolic risk and atherogenesis In the present study, we measured RBP4 concentrations, surrogate markers of enhanced early atherogenesis comprising E-selectin, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1), and common carotid artery intima-media thickness and carotid artery plaque by ultrasound in both black and white patients with RA. We examined the independent relationships of RBP4 concentrations with cardiometabolic risk, endothelial activation and carotid atherosclerosis.The present study was conducted according to the principles outlined in the Helsinki declaration. The Human Research Ethics Committee  from the University of the Witwatersrand in Johannesburg, South Africa approved the protocol . Participants gave informed, written consent. This investigation forms part of an ongoing study on cardiovascular risk in RA as previously reported We recorded demographic features and lifestyle factors. Height, weight and waist and hip circumference were measured using standard approaches. The body mass index (BMI) was calculated and abdominal obesity and fat distribution were estimated by waist circumference and waist-height ratio, and waist-hip ratio respectively. For the purpose of this study, generalized and abdominal obesity were diagnosed in patients with a BMI of >29.9 kg/m2 and National Cholesterol Education Program metabolic syndrome waist circumference criterion Recorded metabolic risk factors included systolic, diastolic and mean blood pressure, lipid concentrations and ratios, and glucose levels. Hypertension was defined as an average systolic blood pressure ≥140 or/and diastolic blood pressure ≥90 mmHg or/and current use of antihypertensive medications. Dyslipidemia was diagnosed when the atherogenic index, i.e. the cholesterol-HDL cholesterol ratio was >4 and proatherogenic non-HDL cholesterol concentrations were calculated by subtracting HDL cholesterol from total cholesterol concentrations We measured endothelial activation molecule concentrations including those of soluble E-selectin, VCAM-1, ICAM-1 and MCP-1 using a solid-phase sHS, R & D Systems, Inc., Minneapolis, MN, USA). Their lower detection limits were 0.009 ng/l, 0.6 ng/l, 0.096 ng/l and 5.0 pg/ml respectively; their inter- and intra-assay coefficients of variation were 7.9 and 5.8, 7.0 and 3.1, 5.5 and 4.6 and 5.7 and 5.8 respectively.BAS (see acknowledgement) and AS performed the carotid artery ultrasound measurements in private and public healthcare patients, respectively. Both operators obtained images of at least 1 cm length of the distal common carotid arteries for measurement of the intima-media thickness of the far wall from an optimal angle of incidence defined as the longitudinal angle of approach where both branches of the internal and external carotid artery are visualized simultaneously RBP4 concentrations were measured in serum using a solid-phase sandwich ELISA . The lower detection limit was 0.048 mM/l and the inter- and intra-assay coefficients of variation were 7.2 and 6.9% respectively.Dichotomous variables are expressed as proportions and continuous variables as mean (SD) or median (interquartile range) when non-normally distributed. Non-normally distributed characteristics were also logarithmically transformed prior to their inclusion in multivariable statistical analysis.Associations of age at disease onset or at the time of the study and sex and population grouping with RBP4 concentrations were assessed by entering the respective characteristics together in single mixed regression models. Associations of other baseline characteristics with RBP4 concentrations were evaluated in models with adjustment for demographic characteristics that included age at the time of the study and not age at disease onset since the latter was less strongly related to RBP4 concentrations.The independent relations of RBP4 concentrations with metabolic risk factors were assessed in demographic characteristic, alcohol use, glomerular filtration rate  and cardiovascular drug use adjusted mixed linear regression models; waist-hip ratio was additionally forced into these models. The independent relations of RBP4 concentrations with endothelial activation and cIMT and plaque were determined in Framingham score , race, alcohol use, waist-hip ratio glomerular filtration rate, and systemic inflammation (C-reactive protein and interleukin-6 concentrations) adjusted mixed (linear or logistic as appropriate) regression models.Patients with RA that experience conventional major risk factors or severe disease are reportedly at high risk of cardiovascular disease. Inflammation also directly increases cardiovascular risk and contributes to the atherogenic effects of conventional cardiovascular risk factors. Additionally, age is an established independent risk factor for RA as well as cardiovascular disease, and obesity contributes significantly to an increased 10-year cardiovascular disease event probability in RA patients Statistical computations were made using the GB Stat program  and SAS software, version 9.1 . Significance was set at p<0.05.The demographic features, lifestyle factors, anthropometric measures, RA characteristics, systemic inflammatory markers including C-reactive protein and interleukin-6 concentrations and leukocyte counts, glomerular filtration rate and use of antirheumatic and cardiovascular agents in the present cohort were previously reported. The median (interquartile range) erythrocyte sedimentation rate, and alanine transferase and aspartate transferase concentrations were 13 (5–28) mm/hr, and 22 (17–28) and 25 (20–30) u/l, respectively. Conventional metabolic risk factors, adipokine and endothelial activation molecule concentrations, and cIMT and plaque prevalence are shown in As given in As shown in As also shown in When RA characteristics were additionally adjusted for in the models in The mean (SD) RBP4 concentrations were numerically but not significantly higher in those with and without established cardiovascular disease (2.39 (1.28) versus 1.77 (0.94) uM/l, p = 0.1). In view of the small number of patients with the respective outcome characteristic, multivariable analysis was not performed.In this RA study, adiposity was not associated with RBP4 concentrations. However, obesity adversely influenced the impact of this adipokine on atherosclerosis. Indeed, RBP4 concentrations were independently related to increased carotid atherosclerosis only in patients with overall and abdominal obesity. Further, although RBP4 concentrations were lower in black compared to white patients, they were associated with atherosclerosis in the former but not the latter. This was possibly due to the far larger prevalence of excess adiposity in black compared to white patients. Consideration of RBP4 concentrations may improve cardiovascular risk stratification in RA Paradoxical adipokine - CVD risk associations as we found between RBP4 concentrations and metabolic risk and endothelial activation, were previously reported RBP4 induces insulin resistance Our finding of lower RBP4 concentrations in black compared to white patients with RA is congruent with an earlier investigation that showed disparities in RBP4 gene polymorphisms amongst black and white Americans We assessed the production of 4 endothelial activation molecules that mediate the initial stages of atherosclerosis. Endothelial activation is strongly up-regulated and associated with prevalent and incident atherosclerosis in RA The present study reveals that RBP4 concentrations are inversely related to metabolic risk and endothelial activation in RA. These findings require further elucidation. RBP4 concentrations were independently associated with enhanced atherosclerosis in RA patients with generalized or/and abdominal obesity.


However, whether G-CSF could influence the T cell receptors (TCR) of gamma delta+ T cells (TRGV and TRDV repertoire) remains unclear. To further characterize this feature, we compared the distribution and clonality of TRGV and TRDV repertoire of T cells before and after G-CSF mobilization and investigated the association between the changes of TCR repertoire and GVHD in patients undergoing G-CSF mobilized allo-PBSCT.The immune modulatory effect of granulocyte colony-stimulating factor (G-CSF) on T cells resulted in an unexpected low incidence of graft-versus-host disease (GVHD) in allogeneic peripheral blood stem cell transplantation . Recent data indicated that gamma deltaTRGV and eight TRDV subfamily genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 20 donors before and after G-CSF mobilization, using RT-PCR and genescan technique. To determine the expression levels of TRGV subfamily genes, we performed quantitative analysis of TRGVI~III subfamilies by real-time PCR.The complementarity-determining region 3 (CDR3) sizes of three TRGV subfamilies were significantly decreased after G-CSF mobilization . The pattern of TRGV subfamily expression levels was TRGVII >TRGV I >TRGV III before mobilization, and changed to TRGV I >TRGV II >TRGV III after G-CSF mobilization. The expression frequencies of TRGV and TRDV subfamilies changed at different levels after G-CSF mobilization. Most TRGV and TRDV subfamilies revealed polyclonality from pre-G-CSF-mobilized and G-CSF-mobilized samples. Oligoclonality was detected in TRGV and TRDV subfamilies in 3 donors before mobilization and in another 4 donors after G-CSF mobilization, distributed in TRGVII, TRDV1, TRDV3 and TRDV6, respectively. Signiﬁcant positive association was observed between the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization and low incidence of GVHD in recipients .The expression levels of three TRGV and TRDV repertoire, but also changes the clonality of gamma delta+ T cells. This alteration of TRGV and TRDV repertoire might play a role in mediating GVHD in G-CSF mobilized allo-PBSCT.G-CSF mobilization not only influences the distribution and expression levels of  Recently, the peripheral blood stem cells (PBSCs) obtained from granulocyte colony-stimulating factor (G-CSF) mobilized donors has been used more frequently than bone marrow stem cells as the source of stem cells in allogeneic hematopoietic stem cell transplantation . The clinical advantages of G-CSF-mobilized peripheral blood stem cell transplantation (G-PBSCT) accelerate engraftment and shorten the neutropenic period compared with bone marrow transplantation (BMT),2. In G-TRG (γ chain) and TRD (δ chain), which are assembled by somatic recombination from variable (V), diversity , and joining (J) segments [TRG gene contains at least 14 functional variable (TRGV) segments belonging to four subgroups (TRGVI to IV), and the TRD contains at least 8 functional TRDV segments, which are subdivided into 8 TRDV subfamilies (TRDV1 toTRDV8)[+ T cells include cytokine production and potent cytotoxic effector activity [+ T cells might participate in regulation of autoimmune diseases and GVHD [+ T cells and thereby mediate GVHD. In the present study, to further investigate the immune modulatory effect of G-CSF on T cells, we characterized the distribution and clonality of TRGV and TRDV subfamilies of donor T cells before and after G-CSF mobilization.T cells recognize specific ligands by specific T cell receptors (TCR), which are heterodimers comprising either α/β or γ/δ chains. Genes encoding the variable domains of the TCR γ and δ heterodimer chains are segments -11. The  toTRDV8)-14. The activity -17. Receand GVHD ,18-21. HPeripheral blood was obtained from 20 healthy stem cell donors  before mobilization and on fifth day of mobilization with G-CSF . Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood samples by Ficoll-Hypaque gradient centrifugation. The twenty healthy stem cell donors were willing to accept the trial after being informed, and all samples were obtained with consent from them. All the procedures were conducted according to the guidelines of the local ethical review boards before study initiation.2 microglubin (β2M) gene amplification.RNA was extracted from the PBMCs of donors before and after G-CSF mobilization according to the manufacturer's protocol . The quality of RNA was analyzed in 0.8% agarose gel stained with ethidium bromide. Two μg RNA was reversely transcribed into the first single-stranded cDNA with random hexamer primers, using reverse transcriptase of the Superscript II Kit . The quality of cDNA was confirmed by RT-PCR for βTRGVIV is a pseudogene [TRGV repertoire was acquired in three TRGV subfamilies in the present study. Three sense TRGV primers and a single TRGC reverse primer, or 8 TRDV sense primers and a single TRDC primer were used in unlabeled PCR for amplification of the TRGV and TRDV subfamilies respectively. Subsequently, a runoff PCR was performed with fluorescent primers labeled at 5'end with the FAM fluorophore (Cγ-FAM or Cδ-FAM). Aliquots of the cDNA (1 μl) were amplified in 20 μl mixture with one of the 3 Vγ primers and a Cγ primer or one of 8 Vδ primers and a Cδ primer. The final mixture contained 0.5 μM sense primer and antisense primer, 0.1 mM dNTP, 1.5 mM MgCl2, 1 × PCR buffer and 1.25 U Taq polymerase . The amplification was performed on a DNA thermal cycler  with 3 min denaturation at 94°C and 40 PCR cycles. Each cycle consisted of 94°C for 1 min, 60°C for 1 min and 72°C for 1 min respectively, and a final 7 min elongation at 72°C. All PCR products were stored at 4°C and ready for genescan analysis [As eudogene , the anaanalysis ,22.2, PCR buffer and 0.25 U Taq polymerase . After a 3 min denaturation at 94°C, 35 cycles of amplification were carried out . The labeled runoff PCR products (2.5 μl) were heat denatured at 94°C for 4 min with 9.5 μl formamide  and 0.5 μl of Size Standards . The samples were then loaded on 3100 POP-4™ gel  and resolved by electrophoresis in 3100 DNA sequencer  for size and fluorescance intensity analysis using Genescan software [Aliquots of the unlabeled PCR products (2.5 μl) were separately added to a final 10 μl reaction system containing 0.1 μM Cγ-FAM or Cδ-FAM primer, 0.2 mM dNTP, 3 mM MgClsoftware ,22.TRGVI-III subfamily gene expression level in cDNA of PBMCs from 20 peripheral blood samples, the β2-microglobulin gene was used as an internal reference, the folds of change of TRGVI-III gene expression level were used by the 2-ΔCt method. Briefly, PCR in 20 μl total volume was performed with approximately 1 μl cDNA, 0.5 μM of each primer  and 2.5 × RealMasterMix 9 μl . After the initial denaturation at 95°C for 2 min, 45 cycles consisting of 95°C 15 s, 60°C 60 s and 82°C 1 s for plate reading were performed using MJ Research DNA Engine Opticon 2 PCR cycler . The relative mRNA expression level of TRGVI-III gene in each sample was calculated according to the comparative cycle time (Ct) method. Briefly, the target PCR Ct value, that is, the cycle number at which emitted fluorescence exceeds the 10 × SD of baseline emissions, is normalized by subtracting the β2M Ct value from the target PCR Ct value, which gives the ΔCt value. From this ΔCt value, the relative expression level to β2M for each target PCR can be calculated using the following equation: relative mRNA expression = 2-ΔCt × 100% (ΔCt = CtTRGV) (- Ct(β2M)) [Real-time PCR with SYBR Green I technique was used to examine Ct(β2M)) ,22.TRGV or TRDV subfamilies between pre-G-CSF-mobilized and G-CSF-mobilized groups. McNemar's test was used for comparison of the expression frequencies of TRGV or TRDV subfamilies between two groups. Differences in mRNA expression of TRGVI-III between two groups were analyzed using the Wilcoxon matched pair test. Kruskal-Wallis Test was used for comparison of different gene expression levels from three TRGV subfamilies before or after G-CSF mobilization, and bonferroni correction was used for pairwise comparisons. Binary logistic regression analysis was used to estimate the association between GVHD in recipients and the alteration of each TRGV and TRDV repertoire after G-CSF mobilization. GVHD was considered as the dichotomous dependent variable and the changes of all TRGV and TRDV repertoires after G-CSF mobilization were considered as the covariates. P < 0.05 was considered as statistically significant .Univariate analyses were performed using the Wilcoxon matched pair test to compare medians of TRGV subfamily genes in T cells were respectively analyzed by RT-PCR in 20 donors before and after G-CSF mobilization. The results showed that the expression frequencies of TRGVI, TRGVII and TRGVIII before mobilization were 95% (19/20), 65% (13/20) and 95% (19/20), respectively. After G-CSF mobilization, similar expression frequencies were found in TRGVI , TRGVII  and TRGVIII  (P > 0.05) . In addition, the expression levels of TRGVI, TRGVII and TRGVIII genes after G-CSF mobilization were significantly lower than that before mobilization , quantified by 2-ΔCt method . However, after G-CSF mobilization, it revealed the TRGV I >TRGV II >TRGV III pattern and there was also a significant difference among three TRGV subfamily groups  , but there were no signiﬁcant difference between TRGVII and TRGVIII , TRGVI and TRGVII  before and after G-CSF mobilization.The CDR3 sizes and expression levels of three were 95% 9/20, 65%TRDV subfamilies, the number of detectable subfamilies ranged from 0 to 6 (median 2.12) before mobilization, which was higher than that after mobilization . The frequently expressed members were TRDV1 , TRDV2 and TRDV5 , TRDV6  and TRDV3  before G-CSF mobilization, while TRDV1 , TRDV3 , TRDV2 , TRDV6  and TRDV5 after G-CSF mobilization. TRDV7 and TRDV8 were detected only in one case before mobilization, and were not identified in all samples after G-CSF mobilization  and TRDV5 , TRDV6 . However, there was no significant difference between pre-G-CSF and post-G-CSF expression frequencies of TRDV3, TRDV5 and TRDV6 .In median 2.2 before median 2.2 before TRGV and TRDV gene repertoire diversity before and after G-CSF mobilization, three TRGV and eight TRDV gene transcripts and profiles were examined using genescan analysis. Most PCR products of TRGV and TRDV subfamilies displayed a Gaussian distribution of CDR3 lengths (multi-peaks) before and after G-CSF mobilization, which corresponded to polyclonal rearrangement pattern, whereas three PCR products in TRDV6 subfamily from 20 samples displayed oligoclonality before mobilization , TRDV3 , TRDV1 , TRDV6 , TRDV5 , and TRDV2   Figure  and 3B.TRGV and TRDV repertoire after G-CSF mobilization. Simple effect analysis of binary logistic regression showed that the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization was associated with low incidence of GVHD , and the alteration of other TRDV or TRGV subfamilies had no significant association with GVHD. Multivariate analysis also showed that the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization indicated low incidence of GVHD  = 0.047, 95% confidence interval (CI): 0.004-0.552), and it was no signiﬁcant association between GVHD and the alteration of expression levels of three TRGV subgroups after G-CSF mobilization . Meanwhile, it could be seen from Figure TRDV1 clonality invariable group, whereas the incidence of GVHD reached 8/9 (88.9%) in the TRDV1 clonality variable group. In addition, the alteration of expression pattern of TRGV repertoire also was not significantly associated with GVHD (P = 0.120).From March 2010 to October 2010, 20 patients received G-CSF-mobilized allo-PBSCT from HLA-identical sibling donors. In total, 11 recipients experienced GVHD after transplantation, including 8 recipients with acute GVHD (grade I in 3 and gradeII in 5) and 7 recipients with chronic GVHD . Thereinto, 4 cases with chronic GVHD experienced acute GVHD. We analyzed the association between GVHD and the alteration of clonality of each + and CD8+ T cell could express the G-CSFR at the mRNA level after G-CSF stimulation in vivo or vitro [The immune modulatory effect of G-CSF on T cells resulted in an unexpected low incidence of GVHD in G-CSF mobilized allo-PBSCT. However, the underlying mechanism for the reduced reactivity or alloreactivity of T cells after G-CSF mobilization was not fully understood ,24. A gror vitro . These sor vitro ,24,26. H+ T cells are the predominant circulating population and can be subdivided into cells that express CD4+ or CD8+ antigens. γδ+ T cells, which represent approximately 5-10% of peripheral T cells and are predominantly CD3+ CD4- CD8- T cells [+ T cells respond rapidly and expand efficiently when stimulated by antigens or cytokines and may be a good target for modulation of immune responses in human diseases. Recently, the role of γδ+ T cells in mediating GVHD raised certain attention [+ T cells promoted or inhibited the occurrence of GVHD was still controversial. Several studies showed that γδ+ T cells might contribute to GVHD [+ T cells could inhibit GVHD [TRGV and TRDV repertoire of γδ+ T cells, thereby influencing the alloreactivity of T cells and mediating GVHD in G-CSF mobilized allo-PBSCT.T cells are comprised of two major subpopulations, identiﬁed by their expression of either the αβ or γδ TCR heterodimer. αβ T cells ,27, reco T cells ,29. As attention -36, but  to GVHD -34, whilbit GVHD ,36. The TRGV and TRDV repertoire from healthy donors before and after G-CSF mobilization. The results showed that G-CSF mobilization could influence the distribution and clonality of TRGV and TRDV repertoire of γδ+ T cells. The expression levels of three TRGV subfamilies were significantly decreased after G-CSF mobilization. In addition, the pattern of TRGV expression levels also changed after G-CSF mobilization, suggesting that G-CSF might influence the expression pattern of TRGV subfamilies. The expression frequencies of TRGV and TRDV subfamilies changed at different levels after G-CSF mobilization; however, there were no significant differences in the expression frequencies of all TRGV and TRDV subfamilies between pre-G-CSF and post-G-CSF group. Most TRGV and TRDV subfamilies revealed polyclonality from pre-G-CSF-mobilized and G-CSF-mobilized samples. Oligoclonality was detected in TRGV and TRDV subfamilies in 3 donors before mobilization and in another 4 donors after G-CSF mobilization, distributed in TRGVII, TRDV1, TRDV3 and TRDV6, respectively. Recent studies suggested that some clonal T cells might be a reaction to host alloantigen and related with GVHD activity [TRDV6 T cells appeared in two patients with aGVHD, while the expansion of the TRGVII T cells, as well as the expansion of the TRDV1 and TRDV3 T cells appeared in two patients without GVHD. Consequently, whether the change of clonality was due to the effect of G-CSF or it was related with GVHD needs further study. However, it was a pity that the donors were not followed up, as it took a long time to get the results.In the present study, we investigated the feature of distribution and clonality of activity . Meanwhiactivity ,38. Our + T cells produced a series of cytokines in pathology and played an indispensable role in pathogen elimination, immune regulation and autoimmunity [+ T cells modulated immune responses mainly by secreting cytokines and by regulating the function of other immune cells, such as αβ+ T cells, macrophages, NK cells and CD4+CD25+Foxp3+ regulatory T cells (Treg)and so on [+ T cells, their exact role in mediating GVHD remained unclear. Drobyski et al. postulated that transplantation with γδ+ T cells protected mice from GVHD [+ T cells induced lethal GVHD in mice [+ T cells and GVHD in mice [+ T cells dose and the cumulative incidence estimates of aGVHD [+ T cells recovery did not have an increased incidence of GVHD compared to those with normal or decreased numbers [Recently, researchers revealed that γδimmunity ,40. γδ+ nd so on -44. Desprom GVHD , whereas in mice . However in mice . Similarof aGVHD , whereas numbers .+ T cells, we studied from the perspective of different repertoire of γδ+ T cells (TRGV and TRDV repertoire), which was thought to have different functions for immune response. Positive association was observed between the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization and low incidence of GVHD, and the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization indicated low incidence of GVHD (OR = 0.047). Due to the small sample size, the association between GVHD and other TRDV and TRGV subfamilies needs further study. These results suggested that compared with other TRDV and TRGV repertoire, some donors' TRDV1 repertoire might be more sensitive to GVHD-associated antigens after G-CSF mobilization, causing the occurrence of GVHD; whereas TRDV1 repertoire of most donors maintained stability after G-CSF mobilization, resulting in low incidence of GVHD. Therefore, TRDV1 repertoire might be used as an observation index for GVHD in the future. In short, our studies observed that some repertoire of γδ+ T cells changed after G-CSF mobilization. Accordingly, the antigens recognized by them might change, thereby altering the immune responses of γδ+ T cells and mediating the occurrence of GVHD. However, the mechanisms are still not well understood and need further study.Different from most studies which analyzed from angle of quantity of γδTRGV and TRDV subfamilies of donor T cells before and after G-CSF mobilization. The results show that G-CSF mobilization not only influences the distribution and expression levels of TRGV and TRDV repertoire, but also changes the clonality of γδ+ T cells. This alteration of TRGV and TRDV repertoire might play a role in mediating GVHD in G-CSF mobilized allo-PBSCT.We characterized the distribution and clonality of The authors declare that they have no competing interests.LX and XLW performed research, analyzed data and wrote the paper; YZ, ZPF and FH analyzed data; YWL, FHZ and XZ performed research; QFL designed research and wrote the paper. The authors reported no potential conflicts of interest. All authors read and approved the final manuscript.


Platymantisvitiana) are preyed upon by the introduced cane toads (Rhinellamarina), and we used ground frogs to test the hypothesis that the sight of a predator will induce a corticosterone stress response in an amphibian. Urinary corticosterone metabolite concentrations increased in male ground frogs exposed to the sight of a toad for 1, 3 or 6 h, whereas corticosterone did not change in frogs exposed to another male ground frog, a ball, or when no stimulus was present in the test compartment. The frogs exposed to a toad initially moved towards the stimulus then moved away, whereas frogs exposed to another frog moved towards the test frog and remained closer to the frog than at the start of the test. Tonic immobility (TI) was measured as an index of fearfulness immediately after the test exposure of the frogs to a stimulus. The duration of TI was longer in frogs exposed to a toad than to another frog or to a ball. The results provide novel evidence that the sight of a predator can induce a corticosterone response and lead to increased fearfulness in amphibians. In addition, they show that endemic frogs can recognise an introduced predator as a threat.Amphibians, like other animals, generate corticosterone or cortisol glucocorticoid responses to stimuli perceived to be threatening. It is generally assumed that the corticosterone response of animals to capture and handling reflects the corticosterone response to stimuli such as the sight of a predator that are thought to be natural stressors. Fijian ground frogs ( The hypothalamo-pituitary interrenal (HPI) axis is the central neuroendocrine pathway for physiological stress responses in amphibians. The glucocorticoid hormone corticosterone, the major stress hormone in amphibians , is secreted by the HPI-axis when individuals respond to a stressor –5. This Platymantisvitiana) is currently listed as Endangered (B1ab (v) ver 3.1) by the International Union for Conservation of Nature (IUCN) 2008 (see [Rhinellamarina) [Pseudechisporphyriacus) [TI tests to measure fearfulness in Fijian ground frogs exposed to the sight of cane toads. We tested the hypothesis that the sight of a cane toad will induce a corticosterone stress response and fearfulness in the Fijian ground frog. Furthermore, we also investigated whether the presence of cane toads altered the movement of the ground frogs to provide an insight into the potential effects of toads on the locomotory behaviour of ground frogs.The Fijian ground frog (008 (see :). The earina) –33. Evidarina) . The intarina) . It has iacus) . In thisiacus) , and tesiacus)  includiniacus) . We usedo00´S, 175o00´E), a small (60 ha) island located 900 m off the coast of mainland Viti Levu, Fiji. The traditional owner of the land  permitted us to undertake this research on Viwa through a traditional ceremony that was conducted prior to this study. The Griffith University Animal Ethics Committee (ENV/13/11/AEC) that approved the interventions with the amphibians in this study. All frogs were released back on into their natural habitat upon completion of the study in accordance with the Australian health check standards for amphibians [All experiments were conducted according to relevant national and international guidelines. Ethical clearances were obtained from the Department of Environment, Fiji Islands for the collection of the endangered Fijian ground frogs on Viwa Island  of 35 mm . Adult male cane toads weighed 55.0 + 1.5 g, and had mean SVL of 65 mm (n = 21 test stimulus toads). We used a urinary corticosterone metabolite enzyme-immunoassay (EIA) to measure urinary corticosterone metabolite concentrations (referred to hereafter as urinary corticosterone concentrations) using our established protocols for anuran amphibians [Achetusdomesticus) every second day and distilled water was sprinkled twice daily into the home container to keep it moist. The arena containing the home containers had a similar photoperiod to Viwa Island. Frogs were adjusted to captivity for 9 days before the experiments began. This adjustment period was selected based on our earlier results on the physiological adjustment of amphibians to captivity [We used adult male Fijian ground frogs for this study because of their easy accessibility compared to female ground frogs, and to reduce any confounding effects of reproductive condition on subsequent stress and behaviour measurements. Ground frogs and cane toads were caught at night (2000-2200h) on Viwa Island within shared terrestrial habitats in December 2011 during the annual breeding season of the ground frogs. Adult male frogs were identified by their distinct ‘stress call’ upon capture, as described earlier  while adphibians –46. Urinaptivity ,45. UrinThe responses of adult male ground frogs to three experimental treatments were examined 10 days after capture on Viwa Island. Individual frogs were introduced to either a cane toad, another adult male ground frog or to a ball, or not exposed to any stimulus. The home container, which was also used as the experimental chamber, was divided in half using plastic wire mesh (15 mm x 15 mm) through which frogs could perceive (see and smell) the test stimulus. This was confirmed by placing a live or a dead house cricket in the test compartment and noticing the frog’s movement in the test chamber.Male ground frogs (n = 21) and toads (n = 21) that were used as test stimuli were caught on Viwa Island on the same night in December 2011 as the experimental frogs, and were acclimated in separate home containers away from the arena where the experimental frogs were kept. On the day of the experiment, male frogs that were used as controls (n = 28) were sampled for urine 0, 1, 3 or 6 h (n = 7 frogs per time period) without any stimulus being present in the test compartment. Experimental male ground frogs (n = 63 frogs) were divided into groups and subjected to one of the three treatments , with a single urine sample collected 1, 3 or 6 h (n = 7 frogs sampled per time period for each treatment) after the experiment began. At the start of each experimental session a piece of black cardboard was placed in the middle of the test chamber and then a toad, frog or the ball was placed into the test compartment by the observer. The cardboard was removed so that the male ground frog could then see the test stimulus. The control frogs were also exposed to the placement and then urine was collected upon removal of the cardboard divider. Sampling time periods of 1, 3 or 6 h were used based on our previous works that showed that physiological stress responses of anurans (e.g. ground frogs) appeared in urinary corticosterone metabolites within 6 h after exposure to experimental stressor, such as adrenocorticotropic hormone (ACTH) injection .A rectangular sheet of white paper with lines drawn with a permanent marker across the sheet at 20, 40, 60 and 80% of the length of the area of each home container in which the ground frog was present was placed beneath the home container before the tests began so the observer could see the lines from a standard distance of 0.5 m. The lines divided the area in which the male frog was present in five locations which were termed 1, 2, 3, 4 and 5, with location 1 closest to the test compartment and location 5 furthest away from the test compartment at the start of the treatment. The location of the frog was recorded at the start of each test (0 min location). The location was then recorded 5, 15, 20, 40 and 60 min after the test began for frogs sampled for urine at 1 h; or 5, 15, 20, 40, 60, 120, 180 min after the test began for frogs sampled for urine at 3 h; or 5, 15, 20, 40, 60, 120, 180, 240 and 360 min after the test began for frogs sampled for urine at 6 h. Each frog was picked up for urine collection immediately after the final location observation was made for each treatment.TI, also known as the immobility reaction [The duration of tonic immobility . Cross reactivity of the CJM06 anti-corticosterone antiserum was reported earlier as 100% with corticosterone, 14.25% with desoxycorticosterone and 0.9% with tetrahydrocorticosterone [+ 0.27 pg/well (n = 24). Creatinine (Cr) was measured in each frog urine sample as an index of the urinary corticosterone metabolite concentrations because it is excreted at a constant rate and is therefore a good index of the amount of time over which hormones have been metabolized into the urine regardless of the volume of the sample [A urinary corticosterone enzyme-immunoassay (EIA) that was previously validated for measuring conjugated corticosterone metabolites in frog urine was used for this study ,43–45. Uosterone . Intra- e sample . UrinaryStatistical analyses were performed using Prism (GraphPad Software Inc.). Urinary corticosterone metabolite concentrations were transformed to logarithms, and D’Agostino & Pearson omnibus test confirmed normality and homogeneity of variances for log-transformed data. Urinary corticosterone metabolite concentrations in adult male ground frogs after they were captured from the wild and transferred into captivity were compared between days using one-way Repeated Measures ANOVA. Post-hoc comparisons were done between times  using Bonferroni’s Multiple Comparison Test.All of the physiological and behavioural data  at times 1, 3 or 6 h since the start of each treatment were compared between groups (control and treatments) using two-way ANOVA followed by post hoc comparisons.+ S.E.Comparisons of the physiological and behavioural data between times  within each frog group (control and treatments) were done using two-way ANOVA followed by post-hoc comparisons. Data are presented as individual points or mean F3,273 = 216.8, p < 0.0001, p < 0.05), decreased on day 5 (p < 0.05), and after 9 days in captivity did not differ from initial concentrations at the time of capture (p > 0.05).Urinary corticosterone concentrations in all male frogs (n = 91) had increased from 0.5 days after capture . CorticoF3,96 = 29.24, p < 0.001) and treatment , with a significant interaction between time and treatment . There were significant differences in mean urinary corticosterone metabolite concentrations between frogs in the control group and frogs that saw the cane toad 1, 3 and 6 h after exposure to the sight of the toad . Mean urinary corticosterone metabolite concentrations increased in frogs that saw a toad and were significantly higher compared with initial 0 h corticosterone concentrations at times 1, 3 and 6 h  for the frogs that were exposed to the cane toad. Corticosterone did not increase from initial concentrations in frogs that saw another frog or a ball or for the control group  and time  on the location of the male ground frogs, and a significant interaction between test stimulus and time . There was a significant difference in locations of frogs over 1, 3 or 6 h between frogs subjected to the toad or another frog .Adult male ground frogs were usually sitting in the middle of their home container (location number 3) before being subjected to the test stimulus. The locations of the ground frogs changed when the frogs were exposed to some of the test stimuli. Frogs exposed to cane toads moved towards the test compartment within the first 15-20 min and then moved away and stayed as far away as possible for the remainder of the test period . Frogs eF3,72 = 56.09, p < 0.0001, F3,72 = 11.78, p < 0.0001, F3,72 = 1.97, p = 0.080, TI at 1, 3 and 6 h (TI duration (75.0 + 13.2 seconds) was recorded at 3 h for the frog group that was subjected to the sight of a toad. There was no significant difference between time in mean TI durations for frogs that saw a frog or ball . There was a significant difference between the times in mean TI durations of the control frogs and frogs that had perceived the cane toad . The mean TI durations of the frogs that saw another frog were significantly shorter than those of frogs that saw a toad, and significantly longer than the mean TI durations of frogs that saw a ball 1, 3 or 6 h (p < 0.05).The mean duration of tonic immobility (TI) of the male ground frogs differed significantly between stimuli  immediately after exposure to the predator, than frogs that saw another frog or saw a ball. These results show that the Fijian ground frogs perceived the sight of a cane toad to be a threat, and experienced fearfulness at the same time as their hypothalamo-pituitary interrenal (HPI) axis was activated and corticosterone secretion increased.TI in the presence of cane toads than in the presence of another frog or a novel object . Tonic immobility has been demonstrated in a wide range of animals, including birds, fish, rabbits and guinea pigs, to reduce the probability of continued attacks by an intruder [Ranapipiens) escaped towards land while their aquatic dwelling congener, the green frog (Rclamitans. ), escaped towards the water when approached by snakes or humans around natural ponds. Seeking refuge following a stressful encounter could be beneficial if the intruder is a visual hunter, but may prove detrimental when the predator is able to enter these substrates. There is empirical evidence that the spatial distributions of the cane toads and Fijian ground frogs on Viwa Island overlap quite significantly [The adult male ground frogs experienced greater fearfulness, as measured by higher durations of intruder . Experimintruder . Furtherintruder . Cane tointruder . In theiintruder , demonstficantly . We studficantly ,54 and bficantly . Similarficantly .Herpestesjavanicus) have never been present, while rats (Rattusspp.) and feral cats (Felis catus) have already been eradicated from the island [Candoiasp.) as well as in the presence of invasive species . Certain native species that have evolved in the absence of any intruders could also display specific behaviours in response to the intruder. A classic example was an earlier study by [Amblyrhynchuscristatus) that has been isolated from mainland populations for over 15 million years was tested for behavioural and physiological stress hormone responses to some novel predators. Their study demonstrated that the ‘naïve’ marine iguana were able to generate anti-predator responses  in the presence of novel predators (dogs and cats) [Parusmajor) that saw an owl, which is a natural predator. In another study [Ficedulahypoleuca) that were exposed for 10 min to a stuffed weasel (Mustelasp.) model, a stuffed great spotted woodpecker (Dendrocoposmajor), or a stuffed flycatcher that was placed on or near their nest box to act as a territorial intruder. Exposure to a weasel or to a territorial intruder elevated plasma corticosterone concentrations in flycatchers caught 10-25 min after the beginning of a test stimulus.Behavioural and physiological stress responses are under some level of genetic control , and rese island . It coulstudy by  in whichnd cats) . Both thnd cats)  emphasiznd cats) , found aer study , studiedOverall, this study has provided novel evidence that amphibians can show a corticosterone response to the sight of a predator. Determining whether the physiological stress and behavioural responses of ground frogs towards cane toads are a result of natural selection or learnt responses could be tested by replicating our experimental protocols with a naïve population of ground frogs from neighbouring Gau Island (located at 18.00° S and 179.30 ° E), where there are currently no cane toads present ,60.


Amyloid-β (Aβ) plaque deposition plays a central role in the pathogenesis of Alzheimer’s disease (AD). Post-mortem analysis of plaque development in mouse models of AD revealed that plaques are initially small, but then increase in size and become more numerous with age. There is evidence that plaques can grow uniformly over time; however, a complementary hypothesis of plaque development is that small plaques cluster and grow together thereby forming larger plaques. To investigate the latter hypothesis, we studied plaque formation in APPPS1 mice using in vivo two-photon microscopy and immunohistochemical analysis. We used sequential pre- and post-mortem staining techniques to label plaques at different stages of development and to detect newly emerged plaques. Post-mortem analysis revealed that a subset (22 %) of newly formed plaques appeared very close (<40 μm) to pre-existing plaques and that many close plaques (25 %) that were initially separate merged over time to form one single large plaque. Our results suggest that small plaques can cluster together, thus forming larger plaques as a complementary mechanism to simple uniform plaque growth from a single initial plaque. This study deepens our understanding of Aβ deposition and demonstrates that there are multiple mechanisms at play in plaque development.The online version of this article (doi:10.1007/s00401-013-1137-2) contains supplementary material, which is available to authorized users. Amyloid-β (Aβ) plaques are a major pathological hallmark of AD. Although plaque deposition is not thought to correlate with disease severity , there aPrevious post-mortem analyses of brain sections from an AD mouse model at different time points showed that plaques become larger on average over time with increasing pathology progression . Post-moOur current study reexamines the question of how these large plaques form. In vivo two-photon imaging revealed that plaques can develop rapidly de novo within 24 h and remain relatively small and at a stable size for at least 2 subsequent weeks , 13. TheIn this study, we labeled plaques at multiple stages of development to test the hypothesis that new plaques preferentially deposit in clusters around pre-existing plaques and to evaluate the occurrence of large plaques formed from multiple precursors. Our data provide the first direct in vivo evidence of plaque growth resulting from consolidation of clusters of multiple plaques.KM670/671NL and PS1L166P under the control of the Thy-1 promoter [For this study, we used male heterozygous APPPS1 transgenic mice , expresspromoter . Mice wen = 4) that was injected at 2 months of age followed by a post-injection time of 2 weeks. A complete list with the number of animals used for each experiment is listed in Suppl. Table 1.For pre-mortem plaque labeling, we chose Methoxy-XO4 , a congo-red derivative, which has been shown to readily cross the blood brain barrier , bind toThe mice were anaesthetized with an i.p. injection of a mixture of Ketamine (10 mg/kg) and Xylazine (20 mg/kg). Under semi-sterile conditions, a cranial window was implanted as previously described , 31. Bri®-Histofix, Karlsruhe, Germany) for 48 h, followed by incubation in 30 % sucrose  for a further 48 h. The brains were frozen and 25 μm thick coronal frozen sections were cut with a sliding microtome . Immunofluorescent staining was performed on every tenth section with the anti-Aβ antibody 3552  was counted as plaques. Autofluorescent signal from particles of section edges were manually removed from the data set. Multicore plaques were classified as plaques bearing 2 or more, clearly separate, Methoxy-XO4 positive cores that were located within the boundaries of a single Aβ antibody labeled plaque. To determine the chance level of plaque deposition in close vicinity of a pre-existing plaque, we computer-generated new plaques in random locations using a custom-written program in MATLAB (Mathworks). A plaque-free region was chosen to estimate the level of the background noise. Detection stringency was set to nine times the standard deviation of the background noise and putative plaques had to consist of a minimum area of 20 pixels (5 μm2). All imagery was visually checked for correct plaque detection. For the simulation, the same number of newly deposited plaques that were counted in the original image was placed at random locations within the image, and their distances to the center of the closest pre-existing plaque determined. For each image, 1,000 iterations of the simulation were conducted, and the proportion of ‘new in vicinity’ plaques occurring by chance was compared to the original proportion of new plaques.Image analysis was done using Photoshop CS5 . Background signal was manually subtracted using the linear image histogram tool. A ‘new’ plaque was defined as a structure being Aβ antibody positive but Methoxy-XO4 negative. The distance between plaques was measured between the centers of two plaques using ImageJ software . Plaques with a core to core distance of less than 40 μm were categorized as ‘in the vicinity’ of each other and if multiple (more than 1) new plaques were in the vicinity of a pre-existing plaque, then these were denoted as ‘flower plaques’ due to their appearance as petals around a central core. To determine the size distribution of plaques, we analyzed images after smoothing, thresholding and background subtraction steps in ImageJ. Aβ antibody fluorescence ≥30 pixels .All data sets were tested for normality with the D’Agostino-Pearson omnibus K2 normality test with a significance level set to We used a two-stage staining technique with different dyes and antibodies to label plaques at multiple stages of plaque development. To label plaques at ‘day 0’, the fluorescent amyloid binding dye Methoxy-XO4 was used as it is a marker of amyloid fibrils and remains bound to plaques for at least 90 days in the living mouse brain . TherefoTo determine if the above-mentioned 22 % of all new plaques found very close to pre-existing ones was not simply due to chance, we generated computer-simulated ‘new’ plaques in random locations and measured the distance of these randomly placed new plaques to already existing ones. Indeed, the amount of original ‘new in vicinity’ plaques was significantly higher than the random, simulated new plaques  were multicored, indicating that this population of plaques developed from more than one initial plaque or a cluster of plaques within the post-Methoxy-XO4 injection interval. Moreover, we found a highly significant positive correlation between the number of Methoxy-XO4 positive plaque cores contained within a plaque and the size of that plaque, showing that greater number of plaque cores corresponds to a larger plaque  can fuse together over time, thereby giving rise to one large dense core plaque. To this end, we investigated the fate of multicore plaques after a 4 month post-Methoxy-XO4 injection period. We used Thiazin Red coupled with the Methoxy-XO4 and Aβ antibody staining to discern the nature of Aβ material. This triple labeling revealed that 75 % of multiple Methoxy-XO4 cores did not fuse together, but instead remained separate in the Thiazin Red staining Fig. a–g. We aIn summary, our data demonstrate that new plaques can deposit over time in the close vicinity of an initial plaque and that these multiple new plaques can cluster and give rise to the so-called ‘flower plaques’. Furthermore, large plaques can contain multiple pre-existing dense cores that can fuse over time supporting the novel hypothesis that multiple, individually small plaques cluster to ultimately build a larger plaque Fig. .Fig. 7ScIn this study, we used pre- and post-mortem staining techniques to label plaques at multiple stages of plaque development. With this two-stage staining technique, we were able to visualize new plaques that had formed over time and reliably distinguish them from pre-existing plaques. Thereby, we unraveled the spatial relationship of newly deposited plaques to pre-existing plaques in detail. We made two important observations: (1) newly developed plaques are more likely to appear in the vicinity of pre-existing plaques than can be attributed to pure random deposition and (2) large plaques can be composed of multiple dense cores, indicating that these plaques are formed from initially small, individual plaques that clustered together and fused to become one larger plaque. Our data provide direct in vivo evidence that plaques can enlarge by forming clusters of multiple plaques, implying a mechanism whereby the development of large plaques is attributed in part to the clustering of pre-deposited and newly deposited plaques. This clustering concept unites the heretofore seemingly conflicting observations of little or no growth following the first appearance of plaques , 24, groA first hint for plaque clustering came from computer simulations of plaque deposition, demonstrating that a mere random process could not account for the plaque deposition pattern observed in post-mortem brain sections. Rather, an influence of pre-existing plaques was necessary to successfully model the spatial pattern of plaque formation in AD pathology. Accordingly, simulated plaques formed preferentially in the vicinity of existing plaques . AdditioThe computer simulation of new plaque location we employed as a control analysis clearly showed the same effect—that new plaques formed in the vicinity of existing plaques at higher than chance levels. In older mice with higher plaque load, we observed the same trend. However, it did not reach statistical significance. We speculate that this is due to an underestimation of the number of ‘new in vicinity’ plaques in the older animals. There are three possible explanations to consider for this. First, the large, diffuse Aβ positive plaque ring-like structure that develops around the initial plaques could well obscure any very close newly appeared plaques. Second, we also observed clusters of plaques within the population of new plaques. These clusters of newly developed plaques suggest that once a new plaque has formed, subsequent new plaques cluster in the vicinity of this original new plaque. However, the temporal resolution of the method used here does not allow for this distinction. A third reason is the use of Methoxy-XO4. Although it has been shown that Methoxy-XO4 labels dense core plaques in vivo , 20, 31 Our finding that plaques form preferentially in the vicinity of pre-existing plaques raises the question which mechanism might cause the formation of new plaques in the peri-plaque region. There is evidence that plaques can be induced by a seeding agent  and so iAnother important outcome of our study is the identification of multicored plaques. We observed many multicored plaques that clearly descended from more than one initial core. This emphasizes the clustering concept of large plaque formation since these plaques had to originate from a cluster of smaller precursors. We also found that the more cores a plaque has, the larger it tends to be, indicating that large plaques can be formed from many neighboring small plaques that developed over time. After 4 months post-injection, we observed different fates for these multicored plaques: they either stay separate but embedded within diffuse Αβ material or merge together to form a single large dense core. The idea of dense core plaque fusion is supported by previous work using a different mouse model of AD that reported the merger of several Methoxy-XO4 positive plaque cores over time . Taken tAlthough the use of only one mouse model of AD is an important caveat to bear in mind, we believe that our results could pertain to a general mechanism of plaque growth. Our data suggest that a uniform growth of plaques from a single seed may provide only a partial explanation of how large plaques develop over time and rather complements a uniform growth model of plaque development by adding a new clustering dimension. We propose a multi-faceted clustering mechanism whereby new plaques can deposit close to existing plaques forming a cluster of small plaques. This cluster can then ultimately merge over time to give rise to the single, large plaques that we observe in aged mice and autopsy material of AD patients. The clustering hypothesis as a mechanism of plaque formation and growth suggests that the micro-environment becomes conducive to plaque deposition. Whether this is the first plaque leading to a greater chance of another plaque forming nearby or a reflection of something unique in that microenvironment that leads to both the first deposit and the increased chance of subsequent deposits remain uncertain. In either case, these observations of plaque development deepen our understanding of plaque formation and plaque growth in Alzheimer’s disease.J.F.M. performed all the experiments and data analyses (apart from chronic in vivo imaging and computer simulations). S.L. and M.H. carried out chronic in vivo two-photon imaging experiments and performed the computer simulation analysis. C.A.A. provide technical assistance. M.M.-L. together with B.T.H. initiated the project. J.F.M., S.L., T.B., B.T.H., C.H. and M.M.-L. discussed the results. J.F.M. and M.M.-L. wrote the manuscript and M.M.-L. coordinated the study. All authors edited the paper.Supplementary Figure 1 New dense core plaques. Premortem Methoxy-XO4 staining as shown in green (a) compared to subsequent postmortem Methoxy-XO4 staining and imaging as shown in red (b) reveals new Methoxy-XO4 positive plaques . Postmortem Thiazin Red staining (e in blue) shows a new dense core plaque  4 months after Methoxy-XO4 injection (d) (JPEG 385 kb)Supplementary Figure 2 Diffuse Aβ detected with 4G8 antibody. Immunhistochemical stainings of plaques with 4G8 antibody 1 day (a-d), 1 month (e–h) and 4 months (i-l) after Methoxy-XO4 injection revealed increasing amounts of diffuse amyloid surrounding the dense core plaque with time. Scale bar: 25 μm (JPEG 661 kb)Supplementary Figure 3 ‘New in the vicinity’ plaques compared to simulated new plaques. Computer-simulated plaque analysis revealed that the proportion of new plaques originally in the vicinity of pre-existing plaques was significantly higher than simulated new plaques in the young post injection group (a). This difference did not reach statistical significance in the 1 month and 4 months post injection groups (b). One data point represents one animal. Mean ± SEM, n = 5-9 per group; Wilcoxon matched-pairs signed rank test* p < 0.05, n.s. p > 0.05. The number of plaques per ROI increases with incubation time (c). Mean ± SEM, n = 5-6 per group, each symbol represents one animal (JPEG 374 kb)Supplementary Figure 4 Postmortem Methoxy-XO4 staining. Examples of brain sections stained postmortem with Methoxy-XO4 of an age matched naïve mouse (a-c). (a) A ‘flower plaque’ consisting of small plaques (white arrowheads) clustering around a larger plaque, (b) clusters of similar sized plaques and (c) seemingly fused plaque clusters. Scale bar: 25 μm (JPEG 382 kb)Supplementary Table 1 Table displaying the number of animals used in each experimental group (DOCX 15 kb)Below is the link to the electronic supplementary material.


Cytokines as molecular adjuvant play a critical role in differentiation of effector T cell subsets and in determination of the magnitude of the response after vaccination. In this study, we investigated the effects of GM-CSF and IL-2 as adjuvant on the immune responses of VP1 recombinant protein as a model antigen for foot and mouth disease.E. coli BL21. In guinea pigs, the immunological and molecular effects of the fusion proteins were determined by ELISA, LPA, DTH and semi-quantitative Reverse Transcriptase PCR (RT-PCR). The data revealed that IL-2 and GM-CSF as adjuvant of VP1 could stimulate both humoral and cell-mediated immune response. Interestingly, IL-2 and GM-CSF, either as a co-expressed protein or as a mixture of two single proteins, showed much better adjuvant effects than that of single one.Six expression plasmids were constructed and expressed in IL-2 and GM-CSF could be used as a potential adjuvant for VP1 and had synergistic effect when co-expressed or mixed with VP1. In recent years, there has been significant progress in the development of candidate vaccines against foot and mouth disease virus (FMDV), in the forms of both whole virus and recombinant proteins. Practical application of these vaccines, however, has often been limited by the lack of suitable adjuvant capable of stimulating an appropriate immune response in the absence of adverse reactions.Many compounds with adjuvant activity have been identified, but none has been emerged as being universally superior ,2. AlthoIL-2 is one of the most widely used adjuvants for vaccination to stimulate the proliferation and activation of various immune effector cells such as T cells, NK cells, B cells, and macrophages,13. GranIn this immunization study, we selected IL-2 and GM-CSF as adjuvant for the VP1 subunit vaccine, with an ultimate goal to verify whether these cytokines have the ability to stimulate humoral immune response and cellular immunity for FMDV.Bovine IL-2 (BoIL-2), Bovine GM-CSF (BoGM-CSF) and VP1 gene were amplified and cloned into pGEX-6P-1 vector by using the restriction enzymes as described before. Each construct was characterized by restriction mapping with one vector band and specific target bands at 405 bp, 450 bp, 378 bp and 669 bp, respectively, followed by DNA sequencing. The results showed that the plasmids of BoIL-2, BoGM-CSF and VP1 were correctly constructed with sequence integrity and right orientation.BoIL-2, BoGM-CSF and VP1 gene fragments were amplified and cloned into pGEX-6P-1 vector by using the restriction enzymes as described before. To construct fused products of BoIL-2/BoGM-CSF/VP1, BoIL-2/VP1, BoGM-CSF/VP1, These constructs were characterized by double digestion with the corresponding restriction enzymes and yielded fragments including one vector band and specific target bands, of which 669 bp was expected for the VP1, 405 bp for the BoIL-2, 378 bp for the BoGM-CSF, 1089 bp for the BoIL-2/VP1, 1062 bp for the BoGM-CSF/VP1 and 1482 bp for the BoIL-2/BoGM-CSF/VP1, respectively. It was further confirmed by PCR with respective primers.To analyze the expressed products, 20 μl samples from the supernatant and precipitation fractions of each culture were analyzed by SDS-PAGE. The result showed that all products were GST fusion proteins and expressed in inclusion body. 40 KDa, 51 KDa, 41 KDa, 65 KDa, 66 KDa, and 81 KDa were observed and represented the sizes of BoGM-CSF, VP1, BoIL-2, BoGM-CSF/VP1, BoIL-2/VP1, BoIL-2/BoGM-CSF/VP1, respectively . The second high level of IgG was observed in VP1+CFA group, and groups of single cytokine co-expressed or mixed with VP1 and group of vaccine only induced slightly lower level of IgG than VP1+CFA group, but not significantly different. The control groups immunized with BoIL-2 or BoGM-CSF alone induced the lowest level of IgG compared with PBS control group.To determine which cytokine induced better T cell responses, single suspensions of lymphocytes were prepared from guinea pig after the third immunization and assayed with MTT method. As shown in Figure The result indicated that VP1 plus BoIL-2 and BoGM-CSF could induce significant T cell response, and the combined use of two cytokines had better effect than that of single cytokine as adjuvant. It suggested that these cytokines enhanced the cell-mediated immunity, which was consistent with their known biological function.Delayed-type hypersensitivity (DTH) is a memory immune response and directly reflects the cellular immune response of body. All guinea pigs were treated as described before, and then the thicknesses of footpad were measured respectively at 24 h, 48 h and 72 h. The effects of DTH were assessed by the thickness of left footpad and right footpad ratio. As shown in Figure Cytokines play a dominant role in modulating immune response against infection or in the effectiveness of vaccination. Therefore, semi-quantitative RT-PCR was used to monitor the expression of the representative cytokines. hypoxanthine phosphoribosyl transferase (HPRT), a house-keeping gene, was used as a normalizing control after guinea pigs were immunized. As shown in Figure E.coli BL21 for the subsequent immunizations , BL21 expression vector, pGEX-6p-1, was purchased from Invitrogene, horseradish peroxidase(HRP)-conjugated goat anti-mouse IgG, MTT and TMB were from Sigma. Eight-week-old female guinea pigs were purchased from the Institute of Genetics of Chinese Academy of Sciences.RNA isolation and reverse transcription reagent Kits were purchased from Promega , FMDV O-serotype inactivated vaccine in oil emulsion was acquired from Zhongmu Ltd. , and the 146 S antigen component was obtained from the purified as described previously and stored at 4°C. The concentration of the 146 s antigen was determined by the Bradford protocol as described previously . 146 S pAfter isolation of peripheral blood mononuclear cells(PBMC) from Holstein cow and stimulated with Con A (10 μg/mg) for 2 h in vitro, total RNA was extracted and reverse transcribed into cDNA by using RNA isolation kit and reverse transcription reagent kit(Promega Inc.) according to the manufacturer's instructions.The VP1 fragment was amplified from the plasmid PMD18-VP1 . The active mature peptide of BoIL-2 and BoGM-CSF were amplified from cDNA. PCR conditions and primers were indicated as Table E. coli, the VP1 fragment was amplified from the plasmid PMD18-VP1 with the upstream primer VP1 F1 and downstream primer VP1 R1 and digested with EcoRI and XhoI. The IL-2 fragment was subcloned from plasmids pGEX/BoIL-2 and digested with BamHI and EcoRI. The expression vector pGEX-6p-1 was also digested with BamHI and XhoI. All the digested fragments were ligated by T4 DNA ligase to yield three constructs, designated as pGEX/BoIL-2/VP1. Between fragments of BoIL-2 and VP1, they were divided by five glycine residues as linker.For the co-expression of BoIL-2 and VP1 in E. coli, IL-2, GM-CSF and VP1 were subcloned from plasmids pGEX/BoIL-2, pGEX/GM-CSF and pGEX/VP1. The PCR products of BoIL-2, BoGM-CSF and VP1 were purified and digested respectively. The expression vector pGEX-6p-1 was also digested with BamHI and XhoI. All the digested fragments were ligated by T4 DNA ligase respectively, designated as pGEX/IL-2/VP1, pGEX/GM-CSF/VP1, pGEX/BoIL-2/BoGM-CSF/VP1. Between fragments of BoIL-2 and VP1, BoIL-2 and BoGM-CSF, or BoGM-CSF and VP1, they were joined by five glycine residues as linkers.For the co-expression of VP1 and BoIL-2, BoGM-CSF in E. coli BL21 in LB plate with 50 μg/ml of Amp+ selection, followed by the identification procedures using both restriction enzyme digestions and PCR. The further confirmation was performed by sequencing analysis.These constructs were transformed into 600 value reached 0.5. The expression was induced for 6 h with addition of IPTG to achieve a final concentration of 1 mM.The confirmed colonies were cultured into LB liquid medium with 50 μg/ml of Amp+ at 37°C until the ODE. coli were homogenized by ultrasonic treatment at 0°C. The protein samples in supernatant and precipitation were subjected in a 15% SDS-PAGE.Sample of 100 μl cultures from each recombinant The inclusion bodies after ultrasonic treatment were washed three times in 10 mmol/L Tris-Cl buffer and subsequently washed three times in 10 mmol/L Tris-Cl buffer .Then the inclusion bodies were stepwise dialysed 6 h with Tris-Cl buffer and PBS. Purified proteins were collected for Western blot analysis and subsequent immunization.Purified protein samples were transferred onto the nitrocellulose membrane. The membrane was incubated overnight in 5% bovine serum albumin in Tris-buffered saline-Tween 20 at 4°C before washing for three times in TBS. Subsequently, the membrane was incubated at 37°C for 2 h with the sera of guinea pig anti- BoIL-2, guinea pig anti-BoGM-CSF and bovine FMDV positive sera, diluted 1:1000 in blocking solution. The membrane was washed in TBS and then incubated at 37°C for 2 h with HRP-labeled goat anti-mouse IgG(Sigma), diluted 1:500 in blocking solution. The membrane was washed again and the signals were developed with DAB substrate.th day post each immunization and subsequently analyzed for detection of FMDV antibody.Eighty four female guinea pigs were randomly divided into twelve groups (N = 7 per group) as Table 2SO4 and the OD reading was determined at 450 nm/655 nm with a plate reader .ELISA plates were used to detect anti-FMDV antibodies in guinea pigs as described previously ,18. 146 4 cells per well and cultured in triplicate wells for 48 h in presence of 10 μg/ml of 146 s antigen or alone. Culture supernatants were tested to quantify the T cell proliferation as described previously [570 nm of stimulated well (stimulated cell) to OD570 nm of unstimulated one [Guinea pigs were immunized as described earlier. Two weeks after final immunization, Guinea pigs were sacrificed and spleens were removed aseptically. Spleen cells were plated at 5 × 10eviously . T lymphated one .Two weeks after the last immunization, Guinea pigs were injected with the 146 S antigen into the right footpads and saline into the left as the negative control. Then the thicknesses of footpads were measured respectively at 24 h, 48 h and 72 h with micrometer to assess the effects of DTH .4 cells per well. The lymphocytes were cultured in triplicate wells with antigen stimulations for 1 h in RPMI-1640 containing 10% FCS. The total RNA was extracted from those cells and the cDNA was synthesized as described above. PCR conditions were optimized with specific primers for the housekeeping gene (HPRT) or cytokine genes indicated as Table Guinea pigs were immunized as described earlier. Two weeks after final immunization, Guinea pigs were sacrificed and spleens were removed aseptically. The lymphocytes were separated from spleens and plated in the 6-well microtiter plate at 5 × 10PCR parameters were performed with minor modifications. Briefly, the PCR mixtures contained 5 μl of PCR buffers, 4 μl of dNTP, 0.5 μl of ExTaq polymerase, 2 μg of cDNAs and 0.5 μl of each primer. The PCR was performed for 32 cycles with parameters of denaturation at 94°C for 1 min, annealing at 60°C for 30 s, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. cDNA from each group was first normalized with the house-keeping gene, HPRT as a reference, each adjusted cDNA was used as template to amplify IFN-γ, IL-2, and IL-4, respectively, according to the conditions described above. All these PCR products were subjected onto electrophoresis on 1.5% of agarose gel and photographed under the UV light . Densityt-test and P < 0.05 was considered statistically significant.Statistical significance between the treatment groups was calculated using One-sided Student's The authors declare that they have no competing interests.CZ carried out the experiments and wrote the manuscript. BW participated in experimental design and paper revise. MW conceived the studies and participated in experimental design and coordination. All authors read and approved the final manuscript.


Traumatic brain injury (TBI) is associated with neuro-inflammation, debilitating sensory-motor deficits, and learning and memory impairments. Cell-based therapies are currently being investigated in treating neurotrauma due to their ability to secrete neurotrophic factors and anti-inflammatory cytokines that can regulate the hostile milieu associated with chronic neuroinflammation found in TBI. In tandem, the stimulation and mobilization of endogenous stem/progenitor cells from the bone marrow through granulocyte colony stimulating factor (G-CSF) poses as an attractive therapeutic intervention for chronic TBI. Here, we tested the potential of a combined therapy of human umbilical cord blood cells (hUCB) and G-CSF at the acute stage of TBI to counteract the progressive secondary effects of chronic TBI using the controlled cortical impact model. Four different groups of adult Sprague Dawley rats were treated with saline alone, G-CSF+saline, hUCB+saline or hUCB+G-CSF, 7-days post CCI moderate TBI. Eight weeks after TBI, brains were harvested to analyze hippocampal cell loss, neuroinflammatory response, and neurogenesis by using immunohistochemical techniques.  Traumatic brain injury (TBI) produces debilitating conditions that affect millions worldwide Colony stimulating factors (CSF), also called haemopoietic growth factors, regulate the mobilization, proliferation, and differentiation of bone marrow cells. Growth factors such as granulocyte colony stimulating factor (G-CSF), granulocytes-macrophages colony stimulating factor (GM-CSF), colony stimulating factor-1(CSF-1), and erythropoietin are currently being investigated as therapeutics for cancer, certain autoimmune diseases, ischemic insults and neurodegenerative diseases in vivo study, we assessed if hUCBC transplantation combined with G-CSF treatment afforded enhanced neuroprotection in a rat CCI model of moderate TBI in the long term using validated TBI immunohistochemical parameters of hippocampal cell loss, neuroinflammatory response, and neurogenesis. Here we report synergistic effects of hUCB transplantation and G-CSF treatment as evidenced by highly effective sequestration of hippocampal cell loss, much more robust reduction in neuroinflammation and massive neurogenesis in the TBI brain compared to stand alone therapies.In the present Experimental procedures were approved by the University of South Florida Institutional Animal Care and Use Committee (IACUC). All male rats were housed under normal conditions . Normal light/dark cycle was employed. A separate cohort of animals, saline group, (n = 7); G-CSF group, (n = 8); hUCB group (n = 8); hUCB+G-CSF (n = 8) underwent the same experimental above and subjected to behavioral tests to determine the functional effects of hUCB and G-CSF. All behavioral testing was done during the light cycle at the same time across testing days. Necessary precautions were taken to reduce pain and suffering of animals throughout the study. All studies were performed by personnel blinded to the treatment condition.Ten-week old Sprague–Dawley rats (n = 55) were subjected to TBI using a controlled cortical impactor . Deep anesthesia was achieved using 1–2% isoflurane in nitrous oxide/oxygen (69/30%) using a nose mask. All animals were fixed in a stereotaxic frame . TBI injury surgeries consisted of animals subjected to scalp incision to expose the skull, and craniectomy. An electric drill was used to perform the craniectomy of about 2.5 mm radius with coordinates calculated from +0.2 anterior and −0.2 mm lateral right from bregma 6 viable cells supplied by Saneron CCEL Therapeutics, Inc., in 500 µl of sterile saline), or hUCB+G-CSF  at 7 days post TBI.One week post-TBI CCI surgery, rats were anesthetized with 1–2% isoflurane in nitrous oxide/oxygen (69/30%) using a nose mask. Four different groups, (n = 6), of TBI rats were treated intravenously through the jugular vein with either saline alone , G-CSF , hUCB+saline (4×10th (1/6) coronal section spanning the frontal cortex and the entire dorsal hippocampus.Under deep anesthesia, rats were sacrificed 8 weeks after TBI surgery. For transcardial perfusion, rats were placed on their backs, and blunt forceps were used to cut through the body cavity. The opening was extended laterally until reaching the rib cage. Rib cage was cut, and sternum was lifted to expose the heart. The heart was held gently, and the needle was inserted ¼ inch into the left ventricle. The right atrium was cut using the irridectomy scissors. Through the ascending aorta, approximately 200 ml of ice cold phosphate buffer saline (PBS) followed by 200 ml of 4% paraformaldehyde (PFA) in PBS were used for brain perfusion. Brains were removed and post-fixed in the same fixative at 4°C for 24 hours followed by 30% sucrose in phosphate buffer (PB) for 1 week. Brains were frozen at −24°C, mounted onto a 40 mm specimen disk using embedding medium. Coronal sectioning was carried out at a thickness of 40 µm by cryostat. H& E and Immunostainings were done on every 6Hematoxylin and eosin (H&E) staining was performed to confirm the core impact injury of our TBI model. As shown in our previous studies th coronal section throughout the entire striatum and dorsal hippocampus. In all animals, sections were anatomically matched. For the DCX staining normal horse serum was used. For the MHCII (OX6) staining normal goat serum was used. Sixteen free-floating coronal sections (40 µm) were washed 3 times in 0.1M phosphate-buffered saline (PBS) to clean the section from cryoprotectant. Thereafter, all section were incubated in 0.3% hydrogen peroxide (H2O2) solution for 20 minutes and washed 3 times with PBS for 10 minutes each wash. Next, all sections were incubated in blocking solution for 1 hour using PBS supplemented with 3% normal serum and 0.2% Triton X-100. Sections were then incubated overnight at 4°C with either goat anti rat DCX , or mouse anti rat MHCII (OX6)  antibody markers in PBS supplemented with 3% normal serum and 0.1% triton X-100. Sections were then washed 3 times with PBS and incubated in biotinylated horse anti-goat secondary antibody for the DCX staining, or goat anti-mouse goat secondary antibody  in PBS supplemented with normal serum, and 0.1% Triton X-100 for 1 hour. Next, the sections were incubated for 60 minutes in avidin–biotin substrate  and washed 3 times with PBS for 10 minute each wash. All sections were then incubated for 1 minute in 3,30-diaminobenzidine (DAB) solution (Vector Laboratories) and wash 3 times with PBS for 10 minutes each wash. Sections were then mounted onto glass slides, dehydrated in ascending ethanol concentration  for 2 minutes each and 2 minutes in xylenes, then cover-slipped using mounting medium.Staining for DCX and OX6 was conducted on separate sets of section of every 6Unbiased stereology was performed on brain sections immunostained with OX6, and DCX. Sets of 1/6 section, ∼16 systematically random sections, of about 240 µm apart, were taken from the brain spanning AP – 0.2 mm to AP – 3.8 mm in all 24 rats. Activated MHCII+ cells, and differentiation into immature neurons were visualized by staining with OX6, DCX, respectively. Positive stains were analyzed with a Nikon Eclipse 600 microscope and quantified using Stereo Investigator software, version 10 . The estimated volume of OX6-positive cells was examined using the Cavalieri estimator probe of the unbiased stereological cell technique revealing the volume of OX6 in the cortex, striatum, thalamus, fornix, cerebral peduncle, and corpus callosum. The literature supports the concept of the Cavalieri principle as a stereological technique used to estimate the volume of structures and regions with arbitrary shape and size, and not only to spherical structures or regions Elevated body swing test (EBST) involved handling the animal by its tail and recording the direction of the swings t-tests for each time point were used to evaluate statistical differences between treatment groups. For all analyses, differences were considered significant at p<0.05. All values are expressed as mean±SEM.For immunohistochemical data analyses, contralateral and ipsilateral corresponding brain areas were used as raw data providing 4 sets of data per treatment condition , therefore two-way and one-way analysis of variance (ANOVA) was used for group comparisons, followed by subsequent pairwise comparisons using post hoc Bonferonni test, which included analyses of any differences between treatments, as well as between contralateral and ipsilateral hemispheres. For behavioral data analyses, repeated measures of ANOVA and post hoc Bonferroni's 3, 20 = 4.913, p<0.0001; striatum F3,20 = 6.466, p<0.0001; thalamus F3,20 = 8.785, p<0.0001; SVZ F3,20 = 6.543, p<0.0001; DG F3,20 = 4.587, p<0.0001; corpus callosum, F3,20 = 14.6, p<0.0001; fornix, F3,20 = 9.017, p<0.0001; cerebral peduncle, F3,20 = 4.638, p<0.0001. Posthoc test analysis revealed a robust upregulation of activated microglia cells when comparing the total estimated volume of MHCII+ cells in the ipsilateral hemisphere of rats exposed to chronic TBI and treated with saline alone to their contralateral side across all gray and white matter areas analyzed (p<0.0001), except in the corpus callosum area (p>0.05).The brain regions examined for TBI-induced neuroinflammation included the gray matter areas of cortex, striatum, thalamus, SVZ, and DG of the hippocampus, while the white matter areas analyzed were corpus callosum, fornix, and cerebral peduncle   . G-CSF m(p<0.05) .3,20 = 6.506, p<0.0018; fornix, F3,20 = 8.324, p<0.0005; cerebral peduncle, F3,20 = 4.733, p<0.0088. Post hoc Bonferroni's test analysis revealed that combined therapy of hUCB+G-CSF decreased the TBI-associated upregulation of activated MHCII+ cells in corpus callosum, fornix, and cerebral peduncle ipsilateral to injury when compared to rats exposed to TBI treated with saline alone (p<0.05)  . hUCB ce(p<0.05) .3, 20 = 9.107, p<0.0005). Post hoc Bonferroni's test analysis revealed that hUCB and G-CSF monotherapy, and the combined therapy of hUCB+G-CSF significantly enhanced endogenous neurogenesis in the DG in our model of chronic TBI compared to injured rats treated with saline alone (p<0.05)   . Moreove<0.0001) . Post ho<0.0001) 3,20 = 159.3, p<0.0001). Treatment with hUCB cell monotherapy and the combined therapy of hUCB+G-CSF significantly rescued the neuronal cells in the CA3 region of the hippocampus  and rotorod test . Within-groups comparison across weeks revealed performance in EBST and rotorod test was impaired by moderate TBI immediately after the injury , and remained significantly deficient even up to the chronic stage  in TBI-saline treated animals  and time . ANOVA also revealed treatment by time after TBI interaction effects in both tasks EBST  may need to be optimized, such as repeated treatments especially during the chronic stage to achieve long-lasting benefits. Indeed, studies have shown modest behavioral effects with G-CSF treatments in animal models of neurological disorders A potential mechanism of action by which i.v. injected G-CSF and/or hUCBs influence diverse regions of the brain may be via receptor-mediated transport and paracrine mechanism. G-CSF is a cytokine able to readily mobilize stem cells from bone marrow to the peripheral blood. Previously, it has been shown that these mobilized cells are able to infiltrate injured tissues promoting self-repair of neurons, myocytes and other cells. Evidence suggests that G-CSF can cross the blood brain barrier (BBB) and act upon neurons and glial through G-CSF receptor. Using radioactive labeling, an experiment showed that G-CSF is able to pass through the blood brain barrier of intact animals. The capillaries associated with the BBB express G-CSF receptor and thus the entry of G-CSF could be mediated through this receptor Taken together, these results indicate that while stand-alone therapies of hUCB transplantation and G-CSF treatment demonstrated a moderate degree of efficacy, their combination afforded synergistic robust beneficial effects in neuroinflammation while decreasing neuronal cell death and stimulating endogenous neurogenesis in a chronic model of moderate TBI. The combined therapy also resulted in robust and long-lasting improvements of motor function. In the clinic, chronic TBI has been visualized as worsening histopathology with limited therapeutic manipulation


This feasibility study was designed to assess the ability to measure mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) cells that contributed to minimal disease/persistent or residual disease (MD/PRD) from children with acute lymphoblastic leukemia (ALL). Increase in mtDNA copies in cancer cells has been suggested to play a role in MD/PRD. CSF as well as blood specimens from 6 children were assayed for MD/PRD and mtDNA copy numbers by quantitative real-time polymerase chain reaction. Of 7 MD/PRD-positive specimens, 6 had increased mtDNA copy numbers; while 11 MD/PRD-negative specimens had no increase in mtDNA copy numbers, p < 0.003. This is the first proof-of-concept study to measure mtDNA copy numbers in MD/PRD-positive CSF specimens from children with ALL. Increase of mtDNA copy numbers in MD/PRD childhood ALL cells and its significance as a mechanism for recurrence requires further investigation.Minimal residual disease; Acute lymphoblastic leukemia; Central nervous system; Cerebrospinal fluid; Mitochondria The presence of minimal disease/persistent or residual disease (MD/PRD) is associated with poor prognosis in childhood acute lymphoblastic leukemia (ALL) . In idenAssessing MD/PRD in the cerebrospinal fluid (CSF) provides additional challenges due to the relatively small number of cells. However, when central nervous system (CNS) relapse is diagnosed from analyzing CSF cells, the percentages of CSF lymphoblasts is relatively high compared to analyses of peripheral blood lymphoblast percentages. Thus on a per-cell basis, recognizing the opportunity to study mtDNA copies in CSF lymphoblasts provided the mechanism for the proof-of-concept study to determine if mtDNA copy numbers could be measured in CSF lymphoblasts from clinical specimens positive for MD/PRD. We posit that CSF and peripheral blood specimens, which are positive for MD/PRD, would have increased mtDNA copy numbers compared to MD/PRD-negative specimens.CSF cells and peripheral blood mononuclear cells (PBMC) were obtained from a tissue repository which had previously processed and stored CSF and blood from pediatric ALL cases without patient identifiers as per guidelines by the University of Hawaii Institutional Review Board. Cases were selected from the repository from which diagnostic and follow-up specimens were available for CSF; and two consecutive follow-up specimens available for PBMC; all from children diagnosed with pre-B-cell ALL. Age, gender, and limited clinical data were available for each case.The PBMC and CSF samples were previously assessed for MD/PRD , 11. Bri2 copies to 106 copies containing a specific portion of mtDNA and the genomic sequence were prepared to establish the standard curves. The mitochondrial primers  amplified the mtDNA region encoding the NADH dehydrogenase subunit 2, while the genomic primers  amplified the encoding region for Fas ligand. DNA from the specimens and appropriate positive/negative controls were set up in either duplicate or triplicate with each reaction containing iQ SYBR Green Supermix, 10 pmol forward primer and 10 pmol reverse primer with cycling parameters: 95oC for 1 min; 40 cycles of 95oC for 30 sec; 58.9oC for 40 sec; 72oC for 4 min; and 50oC for 30 sec. At the conclusion of the PCR, melt curve data were collected from a starting temperature of 65oC, with 0.5oC incremental increases every 5 sec for duration of 70 cycles. Copy numbers of mtDNA were obtained as previously reported with the average mtDNA copy number in PBMC or non-malignant cells ranging from 60-100 copies per cell [All MD/PRD-positive and MD/PRD-negative specimens from the repository were repurified by ethanol precipitation, reconstituted in Tris-HCl-EDTA buffer, and found to be adequate in quantity and quality by UV spectrophotometry. Using techniques previously established in the laboratory, a multiplex real-time PCR assay was performed on each DNA specimen to amplify a Fas ligand nuclear region and a region of the mtDNA . Brieflyper cell .The Mann-Whitney test was used to determine the associations between the mtDNA copy numbers/cell in the MD/PRD-negative and MD/PRD-positive specimens. Statistical analyses were performed using SPSS, Version 16 .Fourteen CSF samples from four children diagnosed with B-cell ALL and four additional PBMC specimens from two children with the same diagnoses were available from the tissue repository which comprised of diagnostic and follow-up specimens. Gender, age, and clinical course of the children are summarized in Analysis of the clinical course and CSF specimens from Patient 1 showed that the child remained in clinical remission with no clinical CNS relapse; and this was reflected in the MD/PRD-negative status of the CSF diagnostic and follow-up specimens. The diagnostic and follow-up mtDNA assessment of the MD/PRD-negative CSF specimens showed that the mtDNA copy number remained relatively stable and within the normal range measured in non-malignant cells (60 - 100 copies/cell) . SimilarThe CSF analyses from the next two patients showed parallel changes in MD/PRD status and mtDNA copy numbers/cell. Patients 3 and 4 both had clinical CNS disease at diagnoses; and both CSF specimens at diagnoses were MD/PRD-positive. While the diagnostic CSF for Patient 3 had high mtDNA copies/cell at diagnosis (191 copies/cell), in contrast, the diagnostic CSF from Patient 4 was low (63 copies/cell); even though both children had clinical CNS disease initially. Both Patients 3 and 4 developed CNS relapse within one year of diagnosis; and both patients had CSF specimens during the first year that were MD/PRD-positive and had high mtDNA copies/cell, 173 copies/cell at Visit 2 and 143 copies/cell at Visit 3, respectively.To assess the relationship of mtDNA copy numbers/cell in PBMC in children with ALL, Patients 5 and 6 were selected because both children developed relapse of their leukemia within one year of diagnoses . From boThe overall objective of this study was to determine the ability and feasibility of measuring mitochondrial DNA (mtDNA) copies per cell in CSF cells from children diagnosed with ALL in the context of the presence or absence of MD/PRD. The rationale for looking at CSF cells in childhood ALL cases in the context of mtDNA copy numbers for the current study focused on the fact that CSF lymphoblasts, when present, typically make up a higher percentage of cells in the CSF . The desIn conclusion, this study was designed as a proof-of-concept to test the feasibility of measuring mtDNA copy numbers from CSF specimens. Limitations of the study design center around the number of cases analyzed. Expanded studies are necessary to address the hypothesis that children with ALL who have MD/PRD-positive CSF specimens with high mtDNA copy numbers per cell may be at risk for CNS relapse. CNS relapse remains a major cause of morbidity in childhood ALL with treatment failure attributable to de novo or acquired resistance to a wide variety of cytotoxic drugs such as multi drug resistance , 16. Add


Tacrolimus is the first-line immunosuppressant after organ transplantation. It is mainly metabolized by cytochrome P450, family 3, subfamily A (CYP3A) enzymes, but there are large individual differences in metabolism. Interleukin 6 (IL6) has been shown to cause a pan-suppression of mRNA levels of ten major CYP enzymes in human hepatocyte cultures. IL6 has been shown to provide hepatoprotection in various models of liver injury. Rs1800796 is a locus in the IL6 gene promoter region which regulates cytokine production. We speculated that IL6 rs1800796 polymorphisms may lead to individual differences in tacrolimus metabolism by affecting CYP3A enzymes levels and liver function after liver transplantation.Ninety-six liver transplant patients receiving tacrolimus were enrolled in the study. Two single nucleotide polymorphisms (SNP), CYP3A5 rs776746 and IL6 rs1800796, were genotyped in both donors and recipients. The effects of SNPs on tacrolimus concentration/dose (C/D ratio) at four weeks after transplantation were studied, as well as the effects of donor IL6 rs1800796 polymorphisms on liver function. Both donor and recipient CYP3A5 rs776746 allele A showed association with lower C/D ratios, while donor IL6 rs1800796 allele G showed an association with higher C/D ratios. Donor CYP3A5 rs776746 allele A, IL6 rs1800796 allele C, and recipient CYP3A5 rs776746 allele A were associated with fast tacrolimus metabolism. With increasing numbers of these alleles, patients were found to have increasingly lower tacrolimus C/D ratios at time points after transplantation. Donor IL6 rs1800796 allele G carriers showed an association with higher glutamic-pyruvic transaminase (GPT) levels.Combined analysis of donor CYP3A5 rs776746, IL6 rs1800796, and recipient CYP3A5 rs776746 polymorphisms may distinguish tacrolimus metabolism better than CYP3A5 rs776746 alone. IL6 may lead to individual differences in tacrolimus metabolism mainly by affecting liver function. Tacrolimus is the first-line immunosuppressant after organ transplantation, reducing rejection and improving graft and recipient survival. However, it is also characterized by a narrow therapeutic window, and large individual differences in metabolism CYP3A enzymes, which are mainly expressed in liver and intestine, are the major metabolic enzymes of tacrolimus IL6 has been demonstrated to cause a pan-suppression of mRNA of ten major CYP enzymes in human hepatocyte cultures The aim of the study was to investigate the relationship between tacrolimus metabolism and IL6 rs1800796 in a large liver transplant cohort to evaluate the possibility of individualizing tacrolimus treatment in the early postoperative period of liver transplantation.A total of 96 patients  who underwent liver transplantation at Shanghai Jiao Tong University Affiliated First People's Hospital between July 2007 and February 2011 were enrolled in this study. One patient was excluded from further analysis because the genotyping failed. The patients were all Han Chinese. The average age of the patients was 47.8±8.3 years, and the average weight was 63.0±10.4 kg. All of the patients received tacrolimus-based immunosuppressive regimens. Tacrolimus  was administered orally twice daily at an initial dose of 0.06 mg/kg/d. The dose of tacrolimus was adjusted according to the target blood concentration of 7 to10 ng/ml during the first month after transplantation.This research was approved by the Ethics Committee of Shanghai Jiao Tong University, and informed written consent was obtained according to the Declaration of Helsinki and its amendments.Blood samples were collected half an hour before tacrolimus was administered, and trough concentrations (ng/ml) were then detected by PRO-Trac™ II Tacrolimus ELISA kit  with microparticle enzyme immunoassay . C/D ratio was calculated with trough concentration and weight standardized 24-hour tacrolimus dose (mg/kg/d). We calculated C/D ratios at four weeks after transplantation, and we used the median of C/D ratios at weeks 1 to 4 to measure weekly changes in tacrolimus metabolism. We also used the median of glutamic-pyruvic transaminase levels (U/L) at weeks 1 to 4 after transplantation to measure weekly liver function.Using an AllPrep DNA/RNA Mini Kit  according to the manufacturer’s instructions, genomic DNA was isolated from both donor and recipient liver tissue, where were previously stored at −80°C. Genotyping of SNPs was conducted by the Sequenom MassARRAY SNP genotyping platform  http://pngu.mgh.harvard.edu/purcell/plink/). Quantitative data between two groups were compared using Mann-Whitney U tests, and among several groups by Kruskal-Wallis. Non-parametric tests were performed in SPSS v17.0 . Two-sided tests were used in all analysis, and P<0.05 was considered statistically significant.Hardy-Weinberg equilibrium and allele frequency were analyzed using PLINK v1.07 (P>0.05). There were no differences in allele frequencies of the two SNPs between donors and recipients. Genotype frequencies of the two SNPs are shown in For CYP3A5, rs776746 allele A (28.4%) was found to be the minor allele, while allele G (71.6%) was the major allele. For IL6, rs1800796 allele G (30.3%) was found to be the minor allele, while allele C (69.7%) was the major allele. Both SNP frequencies were in accordance with Hardy-Weinberg equilibrium . Tacrolimus C/D ratios of donor IL6 rs1800796 allele G carriers at weeks 2 and 3 were 132.3 and 127.4, respectively, while C/D ratios of non-carriers were 105.2 and 97.4, respectively, and the differences were significant . Thus, donor CYP3A5 rs776746 allele A and IL6 rs1800796 allele C are associated with fast tacrolimus metabolism.The effects of donor CYP3A5 rs776746 and IL6 rs1800796 polymorphisms on tacrolimus C/D ratios at four weeks after transplantation are shown in P = 0.003, 0.018, 0.030, 0.017, respectively). There was no significant difference in tacrolimus C/D ratio between recipient IL6 rs1800796 allele G carriers and non-carriers. Thus, recipient CYP3A5 rs776746 allele A is also associated with fast tacrolimus metabolism.The effects of recipient CYP3A5 rs776746 and IL6 rs1800796 polymorphisms on tacrolimus C/D ratios at four weeks after transplantation are shown in P = 0.001, 0.001, <0.001, <0.001, respectively).Donor CYP3A5 rs776746 allele A, IL6 rs1800796 allele C, and recipient CYP3A5 rs776746 allele A were shown to be associated with fast tacrolimus metabolism as stated above. Therefore, these three alleles were further investigated in a combination analysis. The associations between the number of alleles associated with fast metabolism and tacrolimus C/D ratios are shown in P = 0.004, 0.002, 0.006, respectively), but not at week 1 (P = 0.141).The effects of donor IL6 rs1800796 polymorphisms on GPT at four weeks after transplantation were studied. GPT of donor IL6 rs1800796 allele G carriers at weeks 1, 2, 3, and 4 were 167.0, 45.5, 30.0, and 34.5, respectively, while GPT of non-carriers were 142.5, 29.0, 20.0, and 20.0, respectively. The differences between donor IL6 rs1800796 allele G carriers and non-carriers at weeks 2, 3, and 4 were significant (The CYP3A enzymes including three functional enzymes as CYP3A4, CYP3A5, and CYP3A7 are responsible for the oxidative metabolism of over 50% of the drugs in widespread use It has been shown previously that CYP3A5 rs776746 could predict tacrolimus metabolism to a certain extent, but could not completely explain the individual differences Previous studies have shown that cytokines affect pharmacokinetic and pharmacodynamic behaviors of drugs The current study demonstrated that donor IL6 gene polymorphisms were associated with tacrolimus metabolism, not the recipient. Donor IL10 gene polymorphisms have been reported to be associated with tacrolimus metabolism At present, genotyping of CYP3A5 rs776746 polymorphisms has been used in clinical applications, however, it could not predict tacrolimus metabolism accurately. Our study showed that genotyping of donor IL6 rs1800796 polymorphisms could assist CYP3A5 rs776746 to predict tacrolimus metabolism more effectively. Various populations in other regions of the world may differ in certain features of genetic architecture. Whether IL6 rs1800796 is applicable to other populations with regards to tacrolimus metabolism requires further investigation. The gene-drug observations in the current study suggest that genetic factors may also affect liver transplant patient outcomes including graft and recipient survival rates. Proof of such associations requires further study.Donor gene polymorphisms of IL6 play a more important role than those of recipient. Combined polymorphisms of donor CYPA5 rs776746, IL6 rs1800796, and recipient CYP3A5 rs776746 have a greater effect on tacrolimus metabolism than CYP3A5 rs776746 alone. IL6 levels may lead to individual differences in tacrolimus metabolism mainly by affecting liver function.