Levels Of SCF Research Articles

Soluble mediators and clinical features predict transition to systemic lupus erythematosus classification in previously unaffected relatives of SLE patients (HUM3P.243)

Abstract Healthy relatives of lupus patients have an increased risk of developing SLE, but predictors and mechanisms of disease transition are unknown. Unaffected relatives of SLE patients (n=409) were enrolled in this follow-up study to identify factors associated with transition to SLE. Participants provided detailed demographic and clinical information, including the Connective Tissue Disease Screening Questionnaire (CSQ). Medical records were reviewed for ACR classification criteria. Plasma samples were tested for the presence of autoantibodies and soluble mediators. Forty-five relatives (11%) transitioned to classified SLE during follow-up (mean time = 6.4 years) who had more autoantibodies (p<0.0001) and higher pre-classification (baseline) CSQ scores (p<0.0001). Relatives who transitioned also had elevated baseline levels of inflammatory mediators, including BLyS, SCF, and IFN-associated chemokines (p<0.05), with concurrent decreased regulatory mediators, including TGF-β and IL-10 (p<0.05). Logistic regression analysis revealed that baseline ACR and CSQ scores (but not autoantibodies), and baseline plasma levels of SCF and TGF-β were significant and independent predictors of SLE transition. Thus, perturbations in immune-mediated processes precede SLE clinical classification and can help identify unaffected relatives at highest risk of future SLE classification for early intervention studies.

Prognostic Value of TREC, IL-7 and SCF Levels on Clinical Outcomes after Double Umbilical Cord Blood Transplantation in Adults

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells (HSC) for allogeneic HSC transplantation but reconstitution of T cell immunity remains problematic even with double unit UCB transplantation (dUCBT). We previously reported that reconstitution of thymopoiesis plays a critical role in the clearance of CMV viremia, and is associated with improved overall survival in dUCBT recipients. Furthermore, we determined that T cell subset recovery after dUCBT depends on the production of IL-7 and SCF. In the present study we investigated whether thymic reconstitution after dUCBT depends on IL-7 and SCF and examined the prognostic role of T-cell receptor excision circles (TRECs), IL-7 and SCF on clinical outcomes after dUCBT. We analyzed 52 adult patients with hematologic malignancies who enrolled in parallel Phase I/II clinical trials of dUCBT at the Dana-Farber/Harvard Cancer Center. Forty three patients received reduced intensity conditioning with fludarabine, melphalan and antithymocyte globulin; nine patients received myeloablative conditioning with fludarabine, cyclophosphamide and TBI. Thirteen of these patients received human PTH and 13 patients received one untreated and one ex-vivo PGE2 treated UCB unit, per study protocols. GvHD prophylaxis was tacrolimus in combination with sirolimus or mycophenolate mofetil. Assessments were performed at baseline (before conditioning) and at 1, 2, 3, 6 and 12 months after dUCBT. The median follow-up time among survivors was 71 months (range 24-96). The incidence of grade II-IV aGvHD was 15.4% and 14 out of 48 patients (29%) alive after 3 months developed cGvHD. The cumulative incidence of relapse at 5 years was 43%. The 5-year non-relapse mortality (NRM), progression-free survival (PFS) and overall survival (OS) were 31%, 26%, and 41% respectively. Plasma levels of IL-7 increased 3-fold from baseline at 1 month after dUCBT (median 26.4 pg/mL), remained elevated during the first three months, and gradually declined to pre-transplant levels by 1 year. SCF increased more slowly reaching peak levels at 2 months after dUCBT (median 1335.7 pg/mL), and gradually decreased thereafter. All but one evaluable patient had detectable TRECs at baseline with a median value of 3304 copies/ug DNA. TRECs were absent or extremely low during the first 3 months after dUCBT. At 6 months, 69.7% of patients had detectable TRECs, albeit below normal range (median value of 248 copies/ug DNA), but at 12 months 86.6% of the patients had detectable levels with a normal median value of 2404 copies/ug. We observed a statistically significant inverse correlation between IL-7 and TREC levels at 2 months after UCBT (r=-0.48, p=0.036). Similarly, SCF inversely correlated with TRECs at 6 months (r=-0.5, p=.004) and 12 months after dUCBT (r=-0.48, p=.02). We hypothesize that this inverse correlation is likely due to the uptake of IL-7 and SCF by thymocytes leading to their differentiation into TREC-containing Recent Thymic Emigrants (RTE). In support of this hypothesis, IL-7 and SCF inversely correlated with naïve CD4CD45RA+ T cells at various time points after dUCBT. Next, we examined whether TREC, IL-7 and SCF levels might have a prognostic value for clinical outcomes after UCBT. In multivariable models, higher TREC levels were found to be independently associated with improved progression-free survival (PFS) (p=0.02) and overall survival (OS) (p=0.02), and with less non-relapse mortality (NRM) (p=0.002). In contrast, higher levels of IL-7 and SCF were associated with lower OS (p=0.005 and p<.0001 respectively) and a trend towards lower PFS. Furthermore, higher levels of IL-7 and SCF were adverse prognostic factors for NRM (p=0.07 and p=0.007) but not for relapse. Taken together, our findings suggest that adult dUCBT recipients develop thymic reconstitution as early as 6 months after transplantation and TREC values correlate with improved clinical outcomes of dUCBT. In contrast, higher levels of IL-7 and SCF are associated with poor quantitative T cell and thymic reconstitution and they are negative prognostic factors for OS and NRM, which mainly reflects mortality due to infections and GvHD. These findings emphasize the link between cytokine-mediated immune reconstitution of post-thymic T cells and clinical outcomes of transplantation in adult dUCBT recipients. Disclosures No relevant conflicts of interest to declare.

226P - Clinical Significance of Elevated Serum Levels of Cc Thymus and Activation-Related Chemokine in Primary Gastric Cancer: Potential for a Serum Diagnostic and Prognostic Biomarker

ABSTRACT Aim: Chemokines and their receptors are involved in various processes of tumor cell biology. CC chemokine receptor 4 (CCR4), a receptor for thymus and activation-related chemokine (TARC, CCL17), is overexpressed in solid tumors including gastric cancer (GC), and its ligand, TARC, is considered to be a potent stimulator of GC cell proliferation and migration. Here, we evaluate the clinical significance of elevated serum TARC in GC patients and validate its potential as a biomarker for GC. Methods: Serum levels of TARC, MDC (CCL22), MCP-1 (CCL2), and SCF were measured by chemiluminescent immunoassay, and compared among 4 disease groups; normal, high-risk, early GC (EGC), and advanced GC (AGC) groups (each, n = 25) along the gastric carcinogenic sequences (one-way ANOVA) in training set. Serum levels of TARC and SCF were re-evaluated in following independent validation set (90 normal, 30 high-risk, 50 EGC, 50 AGC) and compared with serum carcinoembryonic antigen (CEA) levels. To evaluate the diagnostic potential of serum TARC for GC, receiver operating characteristic (ROC) curve was generated and logistic regression analysis was performed. Correlations of serum TARC with clinicopathological parameters of GC were evaluated by Spearman's correlation. Results: In training set, serum TARC and SCF were significantly correlated with each other and different between cancer and non-cancer conditions (each, p Conclusions: Serum TARC is a potential serologic biomarker for GC superior to CEA. Disclosure: All authors have declared no conflicts of interest.

Phenotypic and Functional Characteristics of NK Cells Associated with Cytomegalovirus (CMV) Reactivation after Allogeneic Hematopoietic Stem Cell Transplantation (HCT)

s / Biol Blood Marrow Transplant 20 (2014) S128eS150 S146 virus (EBV) is a gammaherpes virus that is reactivated from latent to lytic cycle and can cause aggressive lymphomas known as post transplant lymphoproliferative disorder (PTLD) in immunocompromised hosts. We attempted to distinguish immune reconstitution profiles in double UCBT recipients who developed EBV viremia from those who did not. Thirty-one patients with hematologic malignancies received dUCBT with melphalan, fludarabine, ATG conditioning and tacrolimus plus sirolimus for GvHD prophylaxis. During the first 12 months after dUCBT, 14 of 31 (45%) patients developed EBV viremia and four (13%) developed PTLD. At one month after dUCBT, patients with EBV viremia displayed higher numbers of CD19+ B cells (p1⁄40.04) and CD4+CD25+ Treg cells (p1⁄40.03) compared with patients who never became viremic. Surprisingly, EBV viremia correlated with increased numbers of CD3+ (p1⁄40.04), CD4+ (p1⁄40.015) and CD8+ (p1⁄40.021) T cells, but this response was ineffective at controlling the virus. One explanation for this unexpected result might be skewing towards a late effector memory T cell phenotype, a stage in which T cells are incapable of mounting protective immune responses. We examined naive vs. memory T cell distribution and determined that patients who developed EBV reactivation had higher numbers of memory cell subsets (CD4+CD45RO+, p1⁄40.023; CD8+CD45RO+, p1⁄40.019) at two months after dUCBT. Analysis of repertoire diversity by deep-sequencing on PCR-amplified CDR3 regions of the TCRb gene using the ImmunoSEQ assay showed a more diverse TCR repertoire, as determined by higher entropy (p1⁄40.03) and lower clonality (p1⁄40.03), among patients who did not develop EBV reactivation. Assessment of SCF and IL-7, critical regulators of hematopoietic stem cell and naive T cell homeostasis, respectively, showed that patients without EBV viremia had higher levels of SCF (p1⁄40.0016) and IL-7 (p1⁄40.046) compared with patients who developed viremia and PTLD. Our findings suggest that control of EBV reactivation after dUCBT might be linked to the support of naive T cell homeostasis, which enables maintenance of a diverse TCR repertoire.

Reconstitution Of EBV-Specific Immunity In Adult Recipients Of Double Cord Blood Transplantation Depends On SCF and IL-7 Levels and Maintenance Of a Diverse TCR Repertoire

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells (HSC) for allogeneic HSC transplantation but reconstitution of T cell immunity remains problematic even with double UCB transplantation (dUCBT). This is related to the small T cell numbers of the UCB graft, the naïve nature of these T cells and often the use of ATG as part of the conditioning regimen. Consequently, UCBT recipients are susceptible to viral infections that require intact T cell immunity for their containment. Epstein Barr virus (EBV) is a gammaherpes virus that is transmitted orally. EBV initially replicates in a lytic cycle in the epithelium of the oropharynx and then establishes a latent state in memory B cells. Reactivation of EBV from latent to lytic cycle is mediated by physiologic stress of the memory B cell pool and by defects in antiviral T cell immunity, and can cause aggressive lymphomas known as post transplant lymphoproliferative disorder (PTLD) in immunocompromised hosts. The specific immunological correlates of EBV reactivation in adult UCBT recipients have not been examined. In the present study we attempted to distinguish immune reconstitution profiles in UCBT recipients who developed EBV viremia from those who did not, in order to better understand the immune profile of UCBT recipients who control gammaherpes virus reactivation. Thirty-one patients with hematologic malignancies received dUCBT with melphalan, fludarabine, ATG conditioning and tacrolimus plus sirolimus for GvHD prophylaxis. EBV viral load was determined on a weekly basis after dUCBT. Immunophenotype of peripheral blood lymphocytes, serum cytokine levels and T cell receptor excision circle analysis (TREC) values, were examined prior to and at 1, 2, 3, 6 and 12 months after UCBT and were compared between EBV viremic and non-viremic patients using the Wilcoxon-rank sum test. During the first 12 months after dUCBT, 14 of 31 (45%) patients developed EBV viremia and four (13%) developed PTLD. At one month after dUCBT, patients who developed EBV viremia displayed higher numbers of CD19+ B cell numbers (p=0.04) and CD4+CD25+ T regulatory cells (p=0.03) compared with patients who never became viremic. Surprisingly, development of EBV viremia correlated with increased numbers of CD3+ (p=0.04), CD4+ (p=0.015) and CD8+ (p=0.021) T cells. This finding was counterintuitive, as one might expect better quantitative peripheral T cell reconstitution in patients with recovering immunity and no EBV reactivation. One mechanism of impaired immune reconstitution after UCBT is skewing towards a late effector memory T cell phenotype, a stage in which T cells are incapable of mounting protective immune responses. We examined whether development of EBV viremia was associated with altered naïve versus memory T cell distribution. We determined that patients who developed EBV reactivation had higher numbers of memory cell subsets (CD4+CD45RO+, p=0.0023; CD8+CD45RO+, p=0.019) at two months after dUCBT. Neither development nor control of EBV viremia correlated with TREC recovery, which occurred after the time period of EBV viremia. We were unable to identify EBV-specific T cells in any patient group due to the very low T cell numbers during the time of viremia. However, analysis of repertoire diversity by deep-sequencing on PCR-amplified CDR3 regions of the TCRb gene using the ImmunoSEQ assay showed a more diverse TCR repertoire, as determined by higher entropy (p=0.03) and lower clonality (p=0.03), among patients who did not develop EBV reactivation, compared with those who developed EBV viremia and PTLD. Assessment of serum levels of SCF (a c-kit ligand), IL-7, thrombomodulin, VEGF and angiopoetin-1 showed that patients without EBV viremia had significantly higher levels of SCF (p=0.0001) and IL-7 (p=0.05), at 1 and 2 months after dUCBT, compared with patients who developed EBV reactivation and PTLD. SCF-mediated signaling via c-kit is integral to the homing and longevity of uncommitted hematopoietic stem cells whereas IL-7 is a critical factor for survival and homeostasis of naïve T cells. Together our findings suggest that control of EBV reactivation after dUCBT might be linked to the support of naïve T cell homeostasis, which enables maintenance of a diverse TCR repertoire.▪ Disclosures:No relevant conflicts of interest to declare.

Low level of baseline circulating VEGF-A is associated with better outcome in patients with vascular sarcomas receiving sorafenib: an ancillary study from a phase II trial

We have carried out a stratified phase II study of sorafenib (So) in patients with advanced angiosarcoma (n = 32) and epithelioid hemangioendothelioma (n = 13). This report concerns the correlative analysis of the predictive values of circulating pro/anti-angiogenetic biomarkers. Using the ELISA method (R&D Systems), circulating biomarkers (VEGF-A, in picograms per milliliter), thrombospondin-1 (TSP1, in micrograms per milliliter), stem cell factor (SCF, in picograms per milliliter), placental growth factor (PlGF, in picograms per milliliter), VEGF-C (in picograms per milliliter), and E-selectin (in nanograms per milliliter) were measured before So treatment and after 7days. VEGF-A (mean value 475 vs. 541, p = 0.002), TSP1 (16 vs. 24, p = 0.0002), and PlGF (20.9 vs. 40.7, p = 0.0001) significantly increased during the treatment. Treatment did not affect the levels of SCF, VEGF-C, and E-selectin. Only two biomarkers were associated with better outcome as follows: VEGF-A and PlGF. Best objective response and non-progression at 180days were associated with low level of VEGF-A at baseline (p = 0.04 and 0.03, respectively). There was a correlation between the circulating level of VEGF-A and time to progression (TTP) (r = -0.47, p = 0.001). Best objective response and non-progression at 180days were not associated with baseline level of PIGF, but there was a correlation between the circulating level of PIGF at baseline and TTP. Low level of VEGF-A at baseline (<500) was significantly associated with better outcome.

Different surgical techniques and L-carnitine supplementation in an experimental varicocele model

We aimed to investigate the impact of various varicocelectomy techniques and/or L-carnitine as an adjunct treatment, following the emergence of oxidative stress, on the expression levels of SCF/c-kit signalling pathways in spermatogenesis. Forty-two rats were divided into seven groups: group 1 (G1) control; group 2 (G2) sham; group 3 (G3) varicocele; group 4 (G4) varicocele + varicocelectomy with testicular nonartery sparing; group 5 (G5) same as G4 but with artery sparing; group 6 (G6) same as G4 but with L-carnitine and group 7 (G7) same as G5 with L-carnitine. mRNA expression levels of SCF and c-kit were measured quantitatively using real-time polymerase chain reaction. CASP-3 activity at protein level was determined, and histological evaluation was performed. mRNA expression level of SCF increased in G6 as compared to control group (3.52-folds change; P = 0.035), whereas mRNA expression level of c-kit gene remained the same. We found that in the left testis of G6 group, mRNA expression level of SCF increased 2.2-folds in comparison with the right testis (P < 0.05). There were no statistically significant differences in the CASP-3 protein expression levels between the control and other groups. When Cosentino Score analyses of immunostaining were conducted, we observed no significant differences among groups. Spermatogenic failure could be primarily due to a sertoli cell dysfunction. Although surgical treatment has been the best option for management of varicocele, auxiliary agents like L-carnitine may be considered as supportive treatment regimes in addition to conventional surgical treatments.

Regulation of APCCdh1 E3 ligase activity by the Fbw7/cyclin E signaling axis contributes to the tumor suppressor function of Fbw7

Fbw7 and Cdh1 are substrate-recognition subunits of the SCF- and APC-type E3 ubiquitin ligases, respectively. There is emerging evidence suggesting that both Fbw7 and Cdh1 function as tumor suppressors by targeting oncoproteins for destruction. Loss of Fbw7, but not Cdh1, is frequently observed in various human tumors. However, it remains largely unknown how Fbw7 mechanistically functions as a tumor suppressor and whether there is a signaling crosstalk between Fbw7 and Cdh1. Here, we report that Fbw7-deficient cells not only display elevated expression levels of SCF(Fbw7) substrates, including cyclin E, but also have increased expression of various APC(Cdh1) substrates. We further defined cyclin E as the critical signaling link by which Fbw7 governs APC(Cdh1) activity, as depletion of cyclin E in Fbw7-deficient cells results in decreased expression of APC(Cdh1) substrates to levels comparable to those in wild-type (WT) cells. Conversely, ectopic expression of cyclin E recapitulates the aberrant APC(Cdh1) substrate expression observed in Fbw7-deficient cells. More importantly, 4A-Cdh1 that is resistant to Cdk2/cyclin E-mediated phosphorylation, but not WT-Cdh1, reversed the elevated expression of various APC(Cdh1) substrates in Fbw7-deficient cells. Overexpression of 4A-Cdh1 also resulted in retarded cell growth and decreased anchorage-independent colony formation. Altogether, we have identified a novel regulatory mechanism by which Fbw7 governs Cdh1 activity in a cyclin E-dependent manner. As a result, loss of Fbw7 can lead to aberrant increase in the expression of both SCF(Fbw7) and APC(Cdh1) substrates. Our study provides a better understanding of the tumor suppressor function of Fbw7, and suggests that Cdk2/cyclin E inhibitors could serve as effective therapeutic agents for treating Fbw7-deficient tumors.

Role of bone marrow-derived stem cells, renal progenitor cells and stem cell factor in chronic renal allograft nephropathy

IntroductionChronic allograft nephropathy (CAN) is a poorly understood clinico-pathological entity associated with chronic allograft loss due to immunologic and non-immunologic causes. It remains the leading cause of late allograft loss. Bone marrow derived stem cells are undifferentiated cells typically characterized by their capacity for self renewal, ability to give rise to multiple differentiated cellular population, including hematopoietic (HSCs) and mesenchymal stem cells (MSCs). Characterization of HSCs includes their multipotency, expression of typical surface markers such as CD34 and CD45, while characterization of MSC includes their multipotency, expression of typical surface markers such as CD90 and CD105, and the absence of hemopoietic lineage markers. Aim & methodsThe aim of the present work was to study the role of bone marrow-derived HSCs and MSCs, renal progenitor cells and SCF in chronic renal allograft nephropathy in relation to renal hemodynamics and histopathological changes. We studied 30 patients with kidney transplantation for more than 6 months, divided into 15 patients with stable serum creatinine and 15 patients who developed CAN. Detection of HSCs and MSCs in the peripheral blood using flow cytometry via detection of CD34, CD45, CD117 and CD106, as well as immunohistochemical detection of CD34, CD133, VEGF and αSMA in transplanted kidney biopsies of patients with CAN were done. ResultsThere was a significant increase in the levels of SCF, number of peripheral blood HSCs and MSCs in both transplanted patient groups than the controls and they were higher in patients of group Ia than patients of group Ib, (F = 39.73, P < 0.001), (F = 13.28, P < 0.001), (F = 11.94, P < 0.001), respectively and this was accompanied by evident expression of markers of renal repair. ConclusionStem cells might have a role in renal regeneration in CAN and this may pave the way toward the use of stem cells in correction of CAN.

Panax Notoginseng Saponins Promote Endothelial Progenitor Cell Mobilization and Attenuate Atherosclerotic Lesions in Apolipoprotein E Knockout Mice

Background: Endothelial progenitor cells (EPCs) derived from the bone marrow (BM) play a key role in the homeostasis of vascular repair by enhanced reendothelialization. Panax notoginseng saponins (PNS), a highly valued traditional Chinese medicine, has been shown to reduce morbidity and mortality from coronary artery disease. The present research was designed to explore the contribution of progenitor cells to the progression of atherosclerotic plaques and the possible modulatory role of PNS in this process. Methods: PNS (60 or 120 mg/kg via intraperitoneal injection) was administered over 8 weeks in apolipoprotein E knockout mice on an atherogenic diet. The sizes and histochemical alteration of atherosclerotic lesions and numbers of EPCs in BM and peripheral blood were analyzed. The expression of chemokine stromal cell-derived factor 1α (SDF-1α) and its receptor, CXCR4, was monitored as well. Results: PNS significantly reduced the lesion area and intima-to-media ratio compared to vehicle treatment. PNS also augmented endothelialization and reduced the smooth muscle cell (SMCs) content of the lesions. The number of c-kit and sca-1 double-positive progenitor cells and flk-1 and sca-1 double-positive progenitor cells were significantly increased in the BM and the peripheral blood of the PNS-treated groups. PNS treatment increased the plasma levels of SDF-1α and SCF as well as the BM levels of matrix metalloproteinase-9 (MMP-9). Moreover, the mRNA levels of SDF-1α and protein levels of CXCR4 were both increased in the BM of mice treated with PNS, while SDF-1α expression decreased. Conclusion: PNS reduce the size of atherosclerotic plaques, and this effect appears to involve progenitor cell mobilization. SDF-1α-CXCR4 interactions and the possible modulatory role of PNS in this process may contribute to the increased progenitor cell mobilization.

Recovery of Thymic Epithelial Cells and T Cell Immune Reconstitution After Cord Blood Transplantation in Adults Depends On Angiogenesis and Neovascularization

Abstract 4175Umbilical cord blood transplantation (UCBT) is increasingly used as alternative source for allogeneic hematopoietic stem cell transplantation. UCBT in adults results in delayed and insufficient immune reconstitution leading to high infection-related morbidity and mortality. A significant component of immune reconstitution after UCBT involves the thymic regeneration, which can be measured by T-cell receptor rearrangement excision circles (TRECs). TREC levels are a strong predictive factor for overall survival in adult recipients of UCBT. HSCT compromises thymopoiesis due to injury of the thymic microenvironment, particularly thymic epithelial cells (TEC). Human UCB is enriched in endothelial precursors that can sustain thymopoiesis in immunodeficient mice transplanted with human thymic grafts, where they engraft and promote neovascularization and wound healing. We hypothesized that thymic neovascularization induced by the UCB-derived endothelial progenitors might be a critical factor involved in the recovery of TEC, which subsequently support T cell development and expansion via production of interelukin-7 (IL-7) and stem cell factor (SCF; a kit ligand). In the present study we examined whether markers of angiogenesis and neovascularization ANG1 and VEGF were associated with TEC function as determined by assessment of IL-7 and SCF levels and with thymic regeneration as determined by TREC values and recovery of T cell subsets. 27 evaluable patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion with two sequential UCB grafts and GvHD prophylaxis with tacrolimus and sirolimus. Results are based on 27 evaluable patients. Assessments were done prior to transplantation and at 1, 2, 3, 6 and 12 months after UCBT. At various time points after UCBT, serum levels of IL-7 and SCF displayed a strong correlation (p<0.005) with the levels of ANG1 and VEGF indicating that functional recovery of TEC is associated and coincides with angiogenesis and neovascularization. In contrast, serum levels of IL-7 and SCF displayed a strong inverse correlation (p<0.001) with TREC, CD4+, CD8+ and Treg numbers suggesting that uptake of these cytokines by cognate IL-7R and Kit receptors expressed on immature T cell progenitors resulted in differentiation and expansion of these immature T cells. Similarly, IL-7 and SCF levels displayed a strong inverse correlation (p<0.001) with the ability of T cells to differentiate into pathogen-specific effectors as determined by CMV-specific, IFN-γ ELISpot, underlying the potential clinical implications of these findings. Conversely, VEGF and ANG1 levels positively correlated (p<0.001) with CD4+, CD4+CD25+ and CD4+CD45RA+ numbers at various time points after UCBT. Our results are consistent with a model of TEC recovery supported by VEGF and ANG1, leading to production of SCF and IL-7 by TEC, and uptake of these cytokines by T cell progenitors resulting in their differentiation and expansion. Thus, T cell immune reconstitution after UCBT depends on TEC and is associated with indicators of angiogenesis. These sequential steps in the process of thymic regeneration might represent novel therapeutic targets for improvement of immune reconstitution after UCBT. Disclosures:No relevant conflicts of interest to declare.

PDGFRα and CD51 Mark Human Nestin+ Sphere-Forming Mesenchymal Stem Cells Capable of Robust Hematopoietic Stem Cell Expansion

AbstractAbstract 505Few markers have thus far been identified on native mesenchymal stem cells (MSCs), both in the mouse and human systems. Most markers cited in the literature are indeed based on expression analyses on heterogeneous cultured cell populations, which may not have self-renewal properties if rigorously tested by transplantation assays. Previous studies using Nestin (Nes)-Gfp transgenic mice showed that Nes-GFP+ cells are self-renewing MSCs, a major constituent of the hematopoietic stem cell (HSC) niche in the bone marrow (BM) (Nature 2010; 466:829). However, the cytoplasmic location of Nestin precludes prospective live cell isolation outside of the transgenic mice. Hence, finding a combination of surface markers labeling Nestin+ cells in situ would be valuable to isolate bona fide MSCs and characterize niche cells. Screening analyses toward this end revealed that PDGFRα and CD51 expression among CD45− Ter119− CD31− BM stromal cells comprised a large fraction (∼60%) of Nes-GFP+ cells. Upon gating first on PDGFRα+ and CD51+, double-positive cells were also highly enriched in Nes-GFP+ cells (∼75%), and represented a rare fraction (∼2%) of the stromal population. Endogenous Nestin expression was also enriched in PDGFRα+ CD51+ cells, compared to single-positive or double-negative stromal cells (control subsets). Cell sorting of BM PDGFRα+ CD51+ and control subsets revealed that PDGFRα+ CD51+ significantly enriched (> 10-fold, p<0.05) for colony forming unit-fibroblastic (CFU-F) and multipotent clonal mesenspheres (> 7-fold, p<0.01) that differentiate robustly along the osteoblastic, chondrocytic and adipocytic lineages. To test in vivo self-renewal capacity, clonal spheres or polyclonal freshly sorted PDGFRα+ CD51+ cells and control subsets were transplanted into recipient mice by different approaches (renal capsule implants, collagen and/or HA/TCP carrier grafts). After 2 months, secondary sphere formation assays and histological analyses revealed the in vivo self-renewal and heterotopic BM niche regeneration capacity of PDGFRα+ CD51+ cells, but not the control subsets. In addition, the PDGFRα+ CD51+ fraction of Nestin+ cells was markedly enriched in major HSC regulatory genes (Cxcl12, Vcam1, Angpt1, Opn and Scf), supporting the notion that niche activity co-segregates with MSC activity in the BM. Next, we investigated whether PDGFRα+ CD51+ cells also labeled putative Nestin+ MSCs in the human BM. To this end, we analyzed the fetal human BM (13–19 gw), a period during which hematopoietic activity is nascent. At this stage, we found that PDGFRα+ CD51+ cells comprised ∼3% of stromal cells, contained most of the CFU-F activity (6.3 ± 0.8 CFU-Fs/102 cells) in the BM, and also expressed Nestin and HSC regulatory factors. PDGFRα+ CD51+ cells could also form mesenspheres that can self-renew in vivo after heterotopic transplantation. Furthermore, we found that human BM PDGFRα+ CD51+ cells represented a subset of CD146+ cells previously suggested to mark human MSCs (Cell 2007; 131:324), as ∼30% of the CD146high cells also expressed PDGFRα and CD51, and ∼65% of PDGFRα+ CD51+ cells were CD146high. To evaluate functionally the HSC niche properties of human PDGFRα+ CD51+ cells, we set up a co-culture system of human BM CD34+ cells with PDGFRα+ CD51+ mesenspheres. We found that mesenspheres were capable of expanding the number of human CD45+ Lin− CD38− CD34+ CD90+ CD49f+ cells (hHSCs) by 11-fold (p<0.05) compared to input (day 0). In addition, hHSC expansion was 2-fold greater (p<0.05) using mesenspheres compared to serum-free media alone with hematopoietic growth factors (SCF, TPO, Flt3L). Recent studies have suggested that SCF production in the BM niche is derived from perivascular and endothelial cells distinct from Nestin+ cells (Nature 2012; 481:457), although Nestin+ MSCs express high levels of SCF (Nature 2010; 466:829). Immunofluorescence analyses of human PDGFRα+ CD51+ mesenspheres showed that all cells forming the sphere uniformly expressed both Nestin and SCF. Moreover, in the absence of SCF from the media, PDGFRα+ CD51+ mesenspheres rescued hHSCs expansion, yielding a 46- and 5-fold (p<0.001) expansion, as compared to control media alone and input, respectively. These results thus indicate that the HSC niche is conserved between the murine and human species and suggest that highly purified non-adherent cultures of niche cells may represent a useful novel technology to expand hHSCs in vitro. Disclosures:No relevant conflicts of interest to declare.

Mechanism and Efficacy of Mobilization of Granulocyte Colony-Stimulating Factor in the Treatment of Chronic Hepatic Failure

To explore the efficacy of G-CSF mobilization in the treatment of chronic liver failure (CLF) and the mechanism of its action. The proportions of cluster-of-differentiation (CD)-34+ cells and their receptor-CXCR4 were detected by flow cytometry in patients with different types of chronic HBV infection. The levels of chemokines and cytokines were measured by enzyme-linked immunosorbent assay. The proportion of CD34+ cells in patients with cirrhosis was significantly increased compared with the healthy controls (p<0.05) and was increased obviously after treatment by G-CSF mobilization (p<0.01). The expression levels of SDF-1, SCF and MMP-9 were significantly elevated in patients with chronic hepatitis B and liver cirrhosis (p<0.01). The expression levels of SCF and MMP-9 were significantly elevated after treatment with G-CSF (p<0.05). No significant differences were found in the levels of total bilirubin, albumin and prothrombin time between the treated and control groups; furthermore, no significant differences were observed in the cure and improvement rates between the two groups. The basal levels of stem cell mobilization in patients with liver cirrhosis might be associated with the repair of liver injury. G-CSF could promote hematopoietic stem cell mobilization through regulation of the expression levels of stem-cell-mobilization-related factors in patients with liver cirrhosis. No apparent effects of G-CSF therapy on both liver function and short-term prognosis in patients with liver cirrhosis were confirmed.

JCV+ Patients with Inflammatory Bowel Disease show elevated plasma levels of MIG and SCF

JCV+ Patients with Inflammatory Bowel Disease show elevated plasma levels of MIG and SCF

Human umbilical cord Wharton's jelly stem cells and its conditioned medium support hematopoietic stem cell expansion ex vivo

Bone marrow mesenchymal stromal cells (BMMSCs) have been used as feeder support for the ex vivo expansion of hematopoietic stem cells (HSCs) but have the limitations of painful harvest, morbidity, and risk of infection to the patient. This prompted us to explore the use of human umbilical cord Wharton's jelly MSCs (hWJSCs) and its conditioned medium (hWJSC-CM) for ex vivo expansion of HSCs in allogeneic and autologous settings because hWJSCs can be harvested in abundance painlessly, are proliferative, hypoimmunogenic, and secrete a variety of unique proteins. In the presence of hWJSCs and hWJSC-CM, HSCs put out pseudopodia-like outgrowths and became highly motile. Time lapse imaging showed that the outgrowths helped them to migrate towards and attach to the upper surfaces of hWJSCs and undergo proliferation. After 9 days of culture in the presence of hWJSCs and hWJSC-CM, MTT, and Trypan blue assays showed significant increases in HSC numbers, and FACS analysis generated significantly greater numbers of CD34(+) cells compared to controls. hWJSC-CM produced the highest number of colonies (CFU assay) and all six classifications of colony morphology typical of hematopoiesis were observed. Proteomic analysis of hWJSC-CM showed significantly greater levels of interleukins (IL-1a, IL-6, IL-7, and IL-8), SCF, HGF, and ICAM-1 compared to controls suggesting that they may be involved in the HSC multiplication. We propose that cord blood banks freeze autologous hWJSCs and umbilical cord blood (UCB) from the same umbilical cord at the same time for the patient for future ex vivo HSC expansion and cell-based therapies.

Knock-down of Kaiso induces proliferation and blocks granulocytic differentiation in blast crisis of chronic myeloid leukemia

BackgroundKaiso protein has been identified as a new member of the POZ-ZF subfamily of transcription factors that are involved in development and cancer. There is consistent evidence of the role of Kaiso and its involvement in human tumorigenesis but there is no evidence about its role in hematopoietic differentiation or establishment of chronic myeloid leukemia (CML). We used, normal K562 cell line, established from a CML patient in blast crisis, and imatinib-resistant K562 cell line, to investigate the specific distribution of Kaiso and their contribution to the cell differentiation status of the blast crisis of CML (CML-BP).ResultsWe found cytoplasmic expression of Kaiso, in K562 cells and patients, confirmed by immunofluorescence, immunohistochemistry and western blot of cytoplasmic protein fraction. Kaiso was weakly expressed in the imatinib-resistant K562 cell line confirmed by immunofluorescence and western blot. The cytoplasmic expression of Kaiso was not modified when the K562 cells were treated for 16 h with imatinib 0.1 and 1 μM. In our study, small interfering RNA (siRNA) was introduced to down regulate the expression of Kaiso and p120ctn in K562 cell line. Kaiso and p120ctn were down regulated individually (siRNA-Kaiso or siRNA-p120ctn) or in combination using a simultaneous co-transfection (siRNA-Kaiso/p120ctn). We next investigated whether knockdown either Kaiso or p120ctn alone or in combination affects the cell differentiation status in K562 cells. After down regulation we analyzed the expression of hematopoietic cell differentiation and proliferation genes: SCF, PU-1, c-MyB, C/EBPα, Gata-2 and maturation markers of hematopoietic cells expressed in the plasma membrane: CD15, CD11b, CD33, CD117. The levels of SCF and c-MyB were increased by 1000% and 65% respectively and PU-1, Gata-2 and C/EBPα were decreased by 66%, 50% and 80% respectively, when Kaiso levels were down regulated by siRNA. The results were similar when both Kaiso and p120ctn were down regulated by siRNA. The increased expression of SCF and decreased expression of GATA-2 could be responsible by the higher cell viability detected in K562 cells double knock-down of both Kaiso and p120ctn. Finally, we studied the effect of knock-down either Kaiso or p120ctn, alone or in combination on CD15, CD11b, CD33 and Cd117 expression. Using siRNA approach a reduction of 35%, 8% and 13% in CD15, CD33 and CD117 levels respectively, were achieved in all transfections, when compared to scrambled knock-down cells.ConclusionThese results suggest that both Kaiso and p120ctn, contributes to maintaining the differentiated state of the K562 cells and similar to other cancers, cytoplasmic localization of Kaiso is related to a poor prognosis in CML-BP. By the broad and profound effects on the expression of genes and markers of hematopoietic differentiation produced by Kaiso knock-down, these findings reveal Kaiso as a potential target for selective therapy of CML.

Immune Reconstitution After Cord Blood Transplantation in Adults Depends on Activity of Thymic Epithelial Cells and Vascular Endothelial Elements,

Abstract 4075Umbilical cord blood (UCB) grafts are increasingly used as sources of hematopoietic stem cells. UCBT in adults results in delayed and insufficient immune reconstitution leading to high infection-related morbidity and mortality. We recently determined that thymic regeneration as identified by T-cell receptor rearrangement excision circles (TREC) is a strong predictive factor for clearance of CMV viremia and for overall survival in adult UCBT recipients. We were able to detect TRECs by 100 days that was faster than in other adult UCBT studies reported previously, in which TRECs remained undetectable during the first year. A significant difference between our study and previous reports was the use of 2 UCB units in all our patients whereas only a single unit was used in the previous trials. Human UCB is enriched in endothelial precursors that sustain thymopoiesis in SCID mice transplanted with human thymic grafts where they engraft and promote neovascularization and wound healing. The loss of thymopoietic capacity after allogeneic stem cell transplantation results from injury of the thymic microenvironment, particularly of thymic epithelial cells (TEC). We hypothesized that thymic neovascularization induced by UCB-derived endothelial progenitors might be a critical factor promoting early recovery of TEC, which support T cell development and expansion via production of interelukin-7 (IL-7) and stem cell factor (SCF; a kit ligand). In the present study we examined whether thymic regeneration and immune reconstitution in adult recipients of double UCBT was associated with IL-7 and SCF levels and with activity of endothelial progenitors. Thirty-two patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion with two sequential UCB units. GvHD prophylaxis was with tacrolimus and sirolimus. Results are based on 27 evaluable patients. Assessments were done prior to and at 1, 2, 3, 6 and 12 months after UCBT. Serum levels of IL-7 displayed a 3-fold increase at 1 month after UCBT and gradually declined to the pre-transplant levels at 12 months. SCF peaked at 2 months after UCBT and gradually decreased to the pre-transplant levels by 12 months. Strikingly, IL-7 and SCF levels displayed a strong inverse correlation (p<0.001) with TREC, CD4, CD8 and Treg cell numbers at various time intervals after UCBT suggesting that uptake of these cytokines by cognate IL-7R and Kit receptors expressed on the surface of immature T cell progenitors resulted in differentiation and expansion of these T cell populations. Moreover, there was a strong inverse correlation (p<0.001) of IL-7 and SCF levels with the ability of T cells to differentiate into pathogen-specific effectors as determined by CMV-specific, IFN-γ ELISpot, underlying the potential clinical implications of these findings. To determine whether TEC activity correlated with endothelial lineage engraftment and neovascularization, we assessed levels of serum thrombomodulin (TM), which is expressed by metabolically active vascular endothelial cells. Serum TM at 1, 2, 3 and 6 months strongly correlated (p<0.005) with the levels of IL-7 and SCF at various time points after UCBT. Thus, quantitative and qualitative T cell immune reconstitution after UCBT depends on IL-7 and SCF and is associated with activity of vascular elements. These results support the intriguing hypothesis that infusion of 2 UCB units might have a cell dosage effect on thymic regeneration and immune reconstitution similar to that seen with improved rates of hematologic engraftment in double versus single UCB recipients. Disclosures:No relevant conflicts of interest to declare.

Prognostic cytokine markers in peripheral blood for oral cavity squamous cell carcinoma identified by multiplexed immunobead-based profiling

BackgroundOral cavity squamous cell carcinoma (OSCC) is one of the most common cancers worldwide and has been considered to be highly associated with altered biological processes, including immunocyte chemotaxis, inflammatory response, angiogenesis, and/or immune regulation, suggesting that the levels of the tumor-related cytokines and chemokines will be dysregulated in the tumor microenvironment as well as in the systemic circulation and might be associated with some OSCC phenotypes. MethodsTo profile cytokines in OSCC patients, the plasma levels of 48 proteins (26 cytokines, 10 chemokines, and 12 growth factors) were measured in 111 untreated OSCC patients, 112 healthy individuals, and 107 individuals with oral premalignant lesion (OPL). ResultCompared to the plasma levels in the healthy individuals and OPL group, the levels of 12 proteins were significantly dysregulated in the OSCC patients. Further analysis demonstrated that the levels of IFN-α2, IL-2RA, and SCF were significantly lower in patients with higher pT status. IFN-α2 levels also decreased in patients with higher tumor depths. Moreover, OSCC patients with greater levels of VEGF (>4.87pg/ml) before treatment had worse prognoses for overall survival after treatment (P=0.035). ConclusionsThis is the first report showing that the plasma VEGF levels may be a useful prognostic indicator of OSCC.

Clinical and Functional Relevance of Interactions Between the Bone Marrow Stormal Microenvironment and Primary Human Acute Myeloid Leukemia (AML) Cells

AbstractAbstract 2756 Objectives:Adhesion of acute myeloid leukemia (AML) blasts in the bone marrow (BM) microenvironment confers protection from chemotherapy cytotoxicity. We sought to extend our knowledge of the molecular mechanisms by which the BM stroma support AML by identifying all functionally relevant adhesion receptors that were associated with clinical outcomes, identifying new functional AML-stromal interactions, and examining for aberrant cytokine production by stroma derived from patient AML BM. Methods:We prospectively analyzed by multicolor flow cytometry the expression of 17 different adhesion receptors by AML blasts derived from peripheral blood or BM of 42 adult patients, ages 19–80. Analyses included percent expression, mean fluorescence intensity (MFI), and in 7 patients, the adhesion receptors expressed by the CD34+CD38−CD123+ putative leukemia stem cell (LSC) population. We correlated percent expression and MFI with the likelihood of complete remission (CR) using Wilcoxon two-sample testing with Monte Carlo estimation of the exact p value. Guided by data from the AML blast adhesion receptor analyses, we designed a functional screen to assay primary AML blast adhesion to a normal BM stromal cell line (HS27a). For 10 patients, we assessed the capacity of function blocking monoclonal antibodies directed against a panel of 11 adhesion molecules to inhibit adhesion of purified primary AML blasts to HS27a, allowing functional identification of previously undescribed AML-stromal adhesion determinants not only on AML cells but also on BM stroma. Lastly, we successfully grew primary BM stroma from 5 AML patients, assessed production of 10 cytokines important for normal hematopoiesis, and measured the same cytokines directly in plasma of AML patient BM aspirate specimens. Results:Adhesion receptor analysis demonstrates that the mean expression by all AML patients was >90% for CD11a, β1, PECAM-1, CD44, α4, and α5. Comparison of the adhesion receptor profile of the LSC population to the blast population from which they derived showed a 23.1% +/− 7.7% (mean ± standard error) increase in the expression of α6, and a decrease by 0.4% ± 2.5% for CD34+ blasts compared with all blasts (p = 0.04, Student t-test). We found that in our sample of 18 pretreatment BM samples the 15 patients having CR had a higher MFI for αL (p = 0.021), a higher % blasts expressing α4 (p = 0.039) and PECAM1 (p = 0.012), and a lower MFI for α2 (p = 0.029), α6 (p = 0.028), and CXCR4 (p = 0.005). The data for α4 and CXCR4 agree with prior publications demonstrating improved outcomes for high α4β1 expression and poorer outcomes with high CXCR4 expression (Blood 2009; 113:866–74, J Clin Oncol 2010; 28:2831–8, Blood 2007; 109:786–91). Analyzing the functional importance of adhesion receptors for AML-stromal interactions, we found that function blocking antibodies to β1 (p = 0.0003, Student two-sided t-test), CXCR4 (p < 0.0001), and E-cadherin (p < 0.00002) most strongly inhibited AML blast adhesion to HS27a, with mean inhibitions of 71%, 67%, and 63% respectively (Fig 1). [Display omitted] While β1 and CXCR4 represent known factors in AML-stromal adhesion, we propose a novel role for E-cadherin in AML as a potentially novel therapeutic target whose known biology in other malignancies spans cell-cell adhesion and canonical β-catenin/Wnt signaling. The magnitude of the β1 blocking effect proved consistent with its partnering role with multiple adhesion receptors (e.g. α2, α4, α5, and α6). Analyzing the microenvironment cytokine milieu we found normal BM produced G-CSF, SDF-1, and SCF (6, 3001, and 4857 pg/mL respectively) as expected while some AML specimens produced undetectable levels of G-CSF (4/5 plasmas, 4/5 stromas), SCF (4/5 plasmas), or SDF-1 (2/5 plasmas, 3/5 stromas). Conclusions:Among the 6 receptors correlated with response to induction chemotherapy, high α6 (VLA6 laminin receptor) and CXCR4 MFI correlated with poor outcome. The putative LSC subpopulation showed significantly more α6 expression compared with the entire AML blast population.Antibodies against β1, CXCR4, and E-cadherin exhibited the highest function blocking (60–75% range) of AML-stromal adhesion and our AML-stroma functional assay offers a tool to screen for more novel interactions.Cytokine profiling of the AML microenvironment showed deficiencies in cytokines necessary for normal hematopoiesis which may contribute to the clinical cytopenias in AML. Disclosures:No relevant conflicts of interest to declare.

Stem Cell Factor, Interleukin-16, and Interleukin-2 Receptor Alpha are Predictive Biomarkers for Delayed and Slow Graft Function

Introduction Delayed graft function (DGF) and slow graft function (SGF) due to ischemic and reperfusion injury (IRI) are common complications of deceased donor kidney transplantation. We tested whether a panel of serum and urine cytokines represent early biomarkers for DGF and SGF. Methods We collected serum and urine samples from 61 patients 48 hours posttransplantation and used a multiplex enzyme-linked immunosorbent assay (ELISA) technique to measure levels of 23 cytokines. Fourteen patients developed poor graft function (PGF), with 6 having DGF and 8 with SGF. Results Area under receiver operation characteristics curve (AUC) demonstrated the following: serum levels of SCF (0.88) and interleukin (IL) 16 (0.74). Conclusions This study showed that a select panel of cytokines measured early post kidney transplantation may predict poor graft function.